cyclic-gmp and bis(3--5-)-cyclic-diguanylic-acid

In natural environments, bacteria constantly encounter various stressful conditions, including nutrient starvation, toxic chemicals, and oxidative stress. The ability to adapt to these adverse conditions is crucial for bacterial survival. Frequently, bacteria utilize nucleotide signaling molecules such as cyclic diguanylate (c-di-GMP) to regulate their behaviors when encounter stress conditions. c-di-GMP is a ubiquitous bacterial second messenger regulating the transition between the planktonic state and biofilm state. An essential feature of biofilms is the production of extracellular matrix that covers bacterial cells and offers a physical barrier protecting the cells from environmental assaults. Beyond that, accumulating evidences have demonstrated that changes in the environment, including stress stimuli, cause the alteration of intracellular levels of c-di-GMP in bacterial cells, which is immediately sensed by a variety of downstream effectors that induce an appropriate stress response. In this review, we summarize recent research on the role of c-di-GMP signaling in bacterial responses to diverse stress conditions.

Topics: Bacteria; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Second Messenger Systems

The widespread bacterial second messenger c-di-GMP is responsible for regulating many important physiological functions such as biofilm formation, motility, cell differentiation, and virulence. The synthesis and degradation of c-di-GMP in bacterial cells depend, respectively, on diguanylate cyclases and c-di-GMP-specific phosphodiesterases. Since c-di-GMP metabolic enzymes (CMEs) are often fused to sensory domains, their activities are likely controlled by environmental signals, thereby altering cellular c-di-GMP levels and regulating bacterial adaptive behaviors. Previous studies on c-di-GMP-mediated regulation mainly focused on downstream signaling pathways, including the identification of CMEs, cellular c-di-GMP receptors, and c-di-GMP-regulated processes. The mechanisms of CME regulation by upstream signaling modules received less attention, resulting in a limited understanding of the c-di-GMP regulatory networks. We review here the diversity of sensory domains related to bacterial CME regulation. We specifically discuss those domains that are capable of sensing gaseous or light signals and the mechanisms they use for regulating cellular c-di-GMP levels. It is hoped that this review would help refine the complete c-di-GMP regulatory networks and improve our understanding of bacterial behaviors in changing environments. In practical terms, this may eventually provide a way to control c-di-GMP-mediated bacterial biofilm formation and pathogenesis in general.

Topics: Bacteria; Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Signal Transduction

The Pseudomonas syringae Complex is one of the model phytopathogenic bacteria for exploring plant-microbe interactions, causing devastating plant diseases and economic losses worldwide. The ubiquitous second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays an important role in the 'lifestyle switch' from single motile cells to biofilm formation and modulates bacterial behavior, thus influencing virulence in Pseudomonas and other bacterial species. However, less is known about the role of c-di-GMP in the P. syringae complex, in which c-di-GMP levels are controlled by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), such as Chp8, BifA and WspR. Deletion the chemotaxis receptor PscA also influences c-di-GMP levels, suggesting a cross-talk between chemotaxis and c-di-GMP pathways. Another transcription factor, FleQ, plays a dual role (positive or negative) in regulating cellulose synthesis as a c-di-GMP effector, whereas the transcription factor AmrZ regulates local c-di-GMP levels by inhibiting the DGC enzyme AdcA and the PDE enzyme MorA. Our recent research demonstrated that an increase in the c-di-GMP concentration increased biofilm development, siderophore biosynthesis and oxidative stress tolerance, while it decreased the siderophore content, bacterial motility and type III secretion system activity in P. syringae complex. These findings show that c-di-GMP intricately controls virulence in P. syringae complex, indicating that adjusting c-di-GMP levels may be a valuable tactic for defending plants against pathogens. This review highlights recent research on metabolic enzymes, regulatory mechanisms and the phenotypic consequences of c-di-GMP signaling in the P. syringae.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphoric Diester Hydrolases; Polymers; Pseudomonas syringae; Siderophores; Transcription Factors

Cyclic bis-(3', 5')-dimeric guanosine monophosphate (c-di-GMP) is ubiquitous in many bacterial species, where it functions as a nucleotide-based secondary messenger and is a vital regulator of numerous biological processes. Due to its ubiquity, most bacterial species possess a wide range of downstream receptors that has a binding affinity to c-di-GMP and elicit output responses. In eukaryotes, several enzymes and riboswitches operate as receptors that interact with c-di-GMP and transduce cellular or environmental signals. This review examines the functional variety of receptors in prokaryotic and eukaryotic systems that exhibit distinct biological responses after interacting with c-di-GMP. Evolutionary relationships and similarities in distance among the c-di-GMP receptors in various bacterial species were evaluated to understand their specificities. Furthermore, residues of receptors involved in c-di-GMP binding are summarized. This review facilitates the understanding of how distinct receptors from different origins bind c-di-GMP equally well, yet fulfill diverse biological roles at the interspecies, intraspecies, and interkingdom levels. Furthermore, it also highlights c-di-GMP receptors as potential therapeutic targets, particularly those found in pathogenic microorganisms. Video Abstract.

Topics: Cyclic GMP; Eukaryota; Phosphorylation; Polymers

Cyclic diguanylate (c-di-GMP) signal transduction systems provide bacteria with the ability to sense changing cell status or environmental conditions and then execute suitable physiological and social behaviors in response. In this review, we provide a comprehensive census of the stimuli and receptors that are linked to the modulation of intracellular c-di-GMP. Emerging evidence indicates that c-di-GMP networks sense light, surfaces, energy, redox potential, respiratory electron acceptors, temperature, and structurally diverse biotic and abiotic chemicals. Bioinformatic analysis of sensory domains in diguanylate cyclases and c-di-GMP-specific phosphodiesterases as well as the receptor complexes associated with them reveals that these functions are linked to a diverse repertoire of protein domain families. We describe the principles of stimulus perception learned from studying these modular sensory devices, illustrate how they are assembled in varied combinations with output domains, and summarize a system for classifying these sensor proteins based on their complexity. Biological information processing via c-di-GMP signal transduction not only is fundamental to bacterial survival in dynamic environments but also is being used to engineer gene expression circuitry and synthetic proteins with à la carte biochemical functionalities.

Topics: Bacterial Proteins; Computational Biology; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Protein Domains; Signal Transduction

The HD-GYP domain, named after two of its conserved sequence motifs, was first described in 1999 as a specialized version of the widespread HD phosphohydrolase domain that had additional highly conserved amino acid residues. Domain associations of HD-GYP indicated its involvement in bacterial signal transduction and distribution patterns of this domain suggested that it could serve as a hydrolase of the bacterial second messenger c-di-GMP, in addition to or instead of the EAL domain. Subsequent studies confirmed the ability of various HD-GYP domains to hydrolyze c-di-GMP to linear pGpG and/or GMP. Certain HD-GYP-containing proteins hydrolyze another second messenger, cGAMP, and some HD-GYP domains participate in regulatory protein-protein interactions. The recently solved structures of HD-GYP domains from four distinct organisms clarified the mechanisms of c-di-GMP binding and metal-assisted hydrolysis. However, the HD-GYP domain is poorly represented in public domain databases, which causes certain confusion about its phylogenetic distribution, functions, and domain architectures. Here, we present a refined sequence model for the HD-GYP domain and describe the roles of its most conserved residues in metal and/or substrate binding. We also calculate the numbers of HD-GYPs encoded in various genomes and list the most common domain combinations involving HD-GYP, such as the RpfG (REC-HD-GYP), Bd1817 (DUF3391-HD-GYP), and PmGH (GAF-HD-GYP) protein families. We also provide the descriptions of six HD-GYP-associated domains, including four novel integral membrane sensor domains. This work is expected to stimulate studies of diverse HD-GYP-containing proteins, their N-terminal sensor domains and the signals to which they respond.

Topics: Bacterial Proteins; Cyclic GMP; Phosphoric Diester Hydrolases; Phylogeny

Typical second messengers include cyclic AMP (cAMP), cyclic GMP (cGMP), and inositol phosphate. In bacteria, cyclic diguanylate (c-di-GMP), which is not used in animals, is widely used as a second messenger for environmental responses. Initially found as a regulator of cellulose synthesis, this small molecule is known to be widely present in bacteria. A wide variety of synthesis and degradation enzymes for c-di-GMP exist, and the activities of effector proteins are regulated by changing the cellular c-di-GMP concentration in response to the environment. It has been shown well that c-di-GMP plays an essential role in pathogenic cycle and is involved in flagellar motility in Vibrio cholerae. In this review, we aim to explain the direct or indirect regulatory mechanisms of c-di-GMP in bacteria, focusing on the study of c-di-GMP in Vibrio spp. and in flagella, which are our research subjects.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Second Messenger Systems; Vibrio cholerae

The striking multiplicity, signal input diversity, and output specificity of c-di-GMP signaling proteins in many bacteria has brought second messenger signaling back onto the agenda of contemporary microbiology. How can several signaling pathways act in parallel in a specific manner if all of them use the same diffusible second messenger present at a certain global cellular concentration? Recent research has now shown that bacteria achieve this by flexibly combining modes of local and global c-di-GMP signaling in complex signaling networks. Three criteria have to be met to define local c-di-GMP signaling: specific knockout phenotypes, direct interactions between proteins involved, and actual cellular c-di-GMP levels remaining below the K

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases

The treatment of lung infection in the context of cystic fibrosis (CF) is limited by a biofilm mode of growth of pathogenic organisms. When compared to planktonically grown bacteria, bacterial biofilms can survive extremely high levels of antimicrobials. Within the lung, bacterial biofilms are aggregates of microorganisms suspended in a matrix of self-secreted proteins within the sputum. These structures offer both physical protection from antibiotics as well as a heterogeneous population of metabolically and phenotypically distinct bacteria. The bacteria themselves and the components of the extracellular matrix, in addition to the signaling pathways that direct their behaviour, are all potential targets for therapeutic intervention discussed in this review. This review touches on the successes and failures of current anti-biofilm strategies, before looking at emerging therapies and the mechanisms by which it is hoped they will overcome current limitations.

Topics: Alginates; Anti-Bacterial Agents; Bacteriophages; Biofilms; Cell Communication; Cyclic GMP; Cystic Fibrosis; Humans; Iron; Lung Diseases; Quorum Sensing

Bacterial infections lead to high morbidity and mortality globally. While current therapies against bacteria often employ antibiotics, most bacterial pathogens can form biofilms and prevent effective treatment of infections. Biofilm cells can aggregate and encased themselves in a self-secreted protective exopolymeric matrix, to reduce the penetration by antibiotics. Biofilm formation is mediated by c-di-GMP signaling, the ubiquitous secondary messenger in bacteria. Synthesis of c-di-GMP by diguanylate cyclases leads to biofilm formation via the loss of motility, increased surface attachment, and production of biofilm matrix, whereas c-di-GMP degradation by phosphodiesterases causes biofilm dispersal to new sites via increased bacterial motility and matrix breakdown. The highly variable nature of biofilm development and antimicrobial tolerance imposes tremendous challenges in conventional antimicrobial therapies, indicating an imperative need to develop anti-biofilm drugs against biofilm infections. In this review, we focus on two main emergent approaches-active dispersal and disruption. While both approaches aim to demolish biofilms, we will discuss their fundamental differences and associated methods. Active dispersal of biofilms involves signaling the bacterial cells to leave the biofilm, where resident cells ditch their sessile lifestyle, gain motility and self-degrade their matrix. Biofilm disruption leads to direct matrix degradation that forcibly releases embedded biofilm cells. Without the protection of biofilm matrix, released bacterial cells are highly exposed to antimicrobials, leading to their eradication in biofilm infections. Understanding the advantages and disadvantages of both approaches will allow optimized utility with antimicrobials in clinical settings.

Topics: Animals; Anti-Infective Agents; Antibodies; Antineoplastic Agents; Biofilms; Chemistry, Pharmaceutical; Cyclic GMP; Drug Design; Gram-Negative Bacteria; Humans; Phosphoric Diester Hydrolases; Polymers; Signal Transduction

Most bacteria form biofilms, which are thick multicellular communities covered in extracellular matrix. Biofilms can become thick enough to be even observed by the naked eye, and biofilm formation is a tightly regulated process. Paracoccus denitrificans is a non-motile, Gram-negative bacterium that forms a very thin, unique biofilm. A key factor in the biofilm formed by this bacterium is a large surface protein named biofilm-associated protein A (BapA), which was recently reported to be regulated by cyclic diguanosine monophosphate (cyclic-di-GMP or c-di-GMP). Cyclic-di-GMP is a major second messenger involved in biofilm formation in many bacteria. Though cyclic-di-GMP is generally reported as a positive regulatory factor in biofilm formation, it represses biofilm formation in P. denitrificans. Furthermore, quorum sensing (QS) represses biofilm formation in this bacterium, which is also reported as a positive regulator of biofilm formation in most bacteria. The QS signal used in P. denitrificans is hydrophobic and is delivered through membrane vesicles. Studies on QS show that P. denitrificans can potentially form a thick biofilm but maintains a thin biofilm under normal growth conditions. In this review, we discuss the peculiarities of biofilm formation by P. denitrificans with the aim of deepening the overall understanding of bacterial biofilm formation and functions.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Membrane Proteins; Paracoccus denitrificans; Quorum Sensing

Bacterial gene regulation occurs through complex networks, wherein linear systems respond to intracellular or extracellular cues and engage on vivid crosstalk. The ubiquitous water-borne bacterium Legionella pneumophila colonizes various distinct environmental niches ranging from biofilms to protozoa, and - as an 'accidental' pathogen - the human lung. Consequently, L. pneumophila gene regulation evolved to integrate a broad spectrum of different endogenous and exogenous signals. Endogenous signals produced and detected by L. pneumophila comprise the quorum sensing autoinducer LAI-1 (3-hydroxypentadecane-4-one) and c-di-GMP. As an exogenous cue, nitric oxide controls the c-di-GMP regulatory network of L. pneumophila. The Legionella quorum sensing (Lqs) system regulates virulence, motility and natural competence of L. pneumophila. The Lqs system is linked to c-di-GMP signaling through the pleiotropic transcription factor LvbR, which also regulates the architecture of L. pneumophila biofilms. In this review, we highlight recent insights into the crosstalk of Legionella quorum sensing and c-di-GMP signaling.

Topics: 4-Butyrolactone; Alkanes; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Ketones; Legionella pneumophila; Quorum Sensing; Signal Transduction; Transcription Factors; Virulence

Pseudomonads rely on the flagellar motor to rotate a polar flagellum for swimming and swarming, and to sense surfaces for initiating the motile-to-sessile transition to adopt a surface-dwelling lifestyle. Deciphering the function and regulation of the flagellar motor is of paramount importance for understanding the behaviours of environmental and pathogenic pseudomonads. Recent studies disclosed the preeminent role played by the messenger c-di-GMP in controlling the real-time performance of the flagellar motor in pseudomonads. The studies revealed that c-di-GMP controls the dynamic exchange of flagellar stator units to regulate motor torque/speed and modulates the frequency of flagellar motor switching via the chemosensory signalling pathways. Apart from being a rotary motor, the flagellar motor is emerging as a mechanosensor that transduces surface-induced mechanical signals into an increase of cellular c-di-GMP concentration to initiate the cellular programs required for long-term colonization. Collectively, the studies generate long-awaited mechanistic insights into how c-di-GMP regulates bacterial motility and the motile-to-sessile transition. The new findings also raise the fundamental questions of how cellular c-di-GMP concentrations are dynamically coupled to flagellar output and the proton-motive force, and how c-di-GMP signalling is coordinated spatiotemporally to fine-tune flagellar response and the behaviour of pseudomonads in solutions and on surfaces.

Topics: Bacterial Proteins; Cyclic GMP; Flagella; Gene Expression Regulation, Bacterial; Locomotion; Molecular Motor Proteins; Pseudomonas aeruginosa; Pseudomonas syringae; Signal Transduction

The regulation of multiple bacterial phenotypes was found to depend on different cyclic dinucleotides (CDNs) that constitute intracellular signaling second messenger systems. Most notably, c-di-GMP, along with proteins related to its synthesis, sensing, and degradation, was identified as playing a central role in the switching from biofilm to planktonic modes of growth. Recently, this research topic has been under expansion, with the discoveries of new CDNs, novel classes of CDN receptors, and the numerous functions regulated by these molecules. In this review, we comprehensively describe the three main bacterial enzymes involved in the synthesis of c-di-GMP, c-di-AMP, and cGAMP focusing on description of their three-dimensional structures and their structural similarities with other protein families, as well as the essential residues for catalysis. The diversity of CDN receptors is described in detail along with the residues important for the interaction with the ligand. Interestingly, genomic data strongly suggest that there is a tendency for bacterial cells to use both c-di-AMP and c-di-GMP signaling networks simultaneously, raising the question of whether there is crosstalk between different signaling systems. In summary, the large amount of sequence and structural data available allows a broad view of the complexity and the importance of these CDNs in the regulation of different bacterial behaviors. Nevertheless, how cells coordinate the different CDN signaling networks to ensure adaptation to changing environmental conditions is still open for much further exploration.

Topics: Bacteria; Bacterial Proteins; Binding Sites; Biofilms; Cyclic GMP; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Models, Molecular; Nucleotides, Cyclic; Plankton; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Signal Transduction

The formation of microbial biofilms enables single planktonic cells to assume a multicellular mode of growth. During dispersion, the final step of the biofilm life cycle, single cells egress from the biofilm to resume a planktonic lifestyle. As the planktonic state is considered to be more vulnerable to antimicrobial agents and immune responses, dispersion is being considered a promising avenue for biofilm control. In this Review, we discuss conditions that lead to dispersion and the mechanisms by which native and environmental cues contribute to dispersion. We also explore recent findings on the role of matrix degradation in the dispersion process, and the distinct phenotype of dispersed cells. Last, we discuss the translational and therapeutic potential of dispersing bacteria during infection.

Topics: Animals; Bacteria; Bacterial Proteins; Biofilms; Cyclic GMP; Endonucleases; Gene Expression Regulation, Bacterial; Glycoside Hydrolases; Mice; Peptide Hydrolases; Phosphoric Diester Hydrolases; Plankton; Quorum Sensing; Signal Transduction; Transcriptome

Biofilms are the dominant bacterial lifestyle. The regulation of the formation and dispersal of bacterial biofilms has been the subject of study in many organisms. Over the last two decades, the mechanisms of

Topics: Adhesins, Bacterial; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Pseudomonas fluorescens

The guanosine-based second messengers (p)ppGpp and c-di-GMP are key players of the physiological regulation of the Gram-positive model organism Bacillus subtilis. Their regulatory spectrum ranges from key metabolic processes over motility to biofilm formation. Here we review our mechanistic knowledge on their synthesis and degradation in response to environmental and stress signals as well as what is known on their cellular effectors and targets. Moreover, we discuss open questions and our gaps in knowledge on these two important second messengers.

Topics: Bacillus subtilis; Cyclic GMP; Guanosine; Signal Transduction

Bacteria thrive both in liquids and attached to surfaces. The concentration of bacteria on surfaces is generally much higher than in the surrounding environment, offering bacteria ample opportunity for mutualistic, symbiotic, and pathogenic interactions. To efficiently populate surfaces, they have evolved mechanisms to sense mechanical or chemical cues upon contact with solid substrata. This is of particular importance for pathogens that interact with host tissue surfaces. In this review we discuss how bacteria are able to sense surfaces and how they use this information to adapt their physiology and behavior to this new environment. We first survey mechanosensing and chemosensing mechanisms and outline how specific macromolecular structures can inform bacteria about surfaces. We then discuss how mechanical cues are converted to biochemical signals to activate specific cellular processes in a defined chronological order and describe the role of two key second messengers, c-di-GMP and cAMP, in this process.

Topics: Adaptation, Physiological; Bacteria; Bacterial Physiological Phenomena; Biofilms; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Signal Transduction; Surface Properties; Symbiosis

The second messenger cyclic di-GMP regulates a variety of processes in bacteria, many of which are centered around the decision whether to adopt a sessile or a motile life style. Regulatory circuits include pathogenicity, biofilm formation, and motility in a wide variety of bacteria, and play a key role in cell cycle progression in Caulobacter crescentus. Interestingly, multiple, seemingly independent c-di-GMP pathways have been found in several species, where deletions of individual c-di-GMP synthetases (DGCs) or hydrolases (PDEs) have resulted in distinct phenotypes that would not be expected based on a freely diffusible second messenger. Several recent studies have shown that individual signaling nodes exist, and additionally, that protein/protein interactions between DGCs, PDEs and c-di-GMP receptors play an important role in signaling specificity. Additionally, subcellular clustering has been shown to be employed by bacteria to likely generate local signaling of second messenger, and/or to increase signaling specificity. This review highlights recent findings that reveal how bacteria employ spatial cues to increase the versatility of second messenger signaling.

Topics: Caulobacter crescentus; Cyclic GMP; Signal Transduction

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger which has been associated with a motile to sessile lifestyle switch in many bacteria. Here, we review recent insights into c-di-GMP regulated processes related to environmental adaptations in alphaproteobacterial rhizobia, which are diazotrophic bacteria capable of fixing nitrogen in symbiosis with their leguminous host plants. The review centers on Sinorhizobium meliloti, which in the recent years was intensively studied for its c-di-GMP regulatory network.

Topics: Cyclic GMP; Sinorhizobium meliloti

The intracellular signaling molecule cyclic di-GMP (c-di-GMP) regulates the lifestyle of bacteria and controls many key functions and mechanisms. In the case of bacterial pathogens, a wide variety of virulence lifestyle factors have been shown to be regulated by c-di-GMP. Evidence of the importance of this molecule for bacterial pathogenesis has become so great that new antimicrobial agents are tested for their capacity of targeting c-di-GMP signaling. This review summarizes the current knowledge on this topic and reveals its application for the development of new antivirulence intervention strategies.

Topics: Bacteria; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Signal Transduction; Virulence; Virulence Factors

The Escherichia coli operon dosCP, also called yddV-yddU, co-expresses two heme proteins, DosC and DosP, both of which are direct oxygen sensors but paradoxically have opposite effects on the levels of the second messenger c-di-GMP. DosC is a diguanylate cyclase that synthesizes c-di-GMP from GTP, whereas DosP is a phosphodiesterase that linearizes c-di-GMP to pGpG. Both proteins are associated with the large degradosome enzyme complex that regulates many bacterial genes post-transcriptionally by processing or degrading the corresponding RNAs. Moreover, the c-di-GMP directly binds to PNPase, a key degradosome enzyme, and enhances its activity. This review combines biochemical, biophysical, and genetic findings on DosC and DosP, a task that has not been undertaken until now, partly because of the varied nomenclature. The DosC and DosP system is examined in the context of the current knowledge of degradosomes and considered as a possible prototype for the compartmentalization of sensing by E. coli.

Topics: Cyclic GMP; Endoribonucleases; Escherichia coli; Escherichia coli Proteins; Multienzyme Complexes; Nucleotidyltransferases; Phosphoric Diester Hydrolases; Polyribonucleotide Nucleotidyltransferase; RNA Helicases; Second Messenger Systems

The environment exerts strong influence on microbes. Adaptation of microbes to changing conditions is a dynamic process regulated by complex networks. Pseudomonas aeruginosa is a life-threating, versatile opportunistic and multi drug resistant pathogen that provides a model to investigate adaptation mechanisms to environmental changes. The ability of P. aeruginosa to form biofilms and to modify virulence in response to environmental changes is coordinated by various mechanisms including two-component systems (TCS), and secondary messengers involved in quorum sensing (QS) and c-di-GMP networks (diguanylate cyclase systems, DGC). In this review, we focus on the role of c-di-GMP during biofilm formation. We describe TCS and QS signal cascades regulated by c-di-GMP in response to changes in the external environment. We present a complex signaling network dynamically changing during the transition of P. aeruginosa from the free-living to sessile mode of growth.

Topics: Biofilms; Cyclic GMP; Pseudomonas aeruginosa; Quorum Sensing; Signal Transduction

Cyclic dimeric guanosine monophosphate (c-di-GMP) is a universally conserved second messenger that contributes to the pathogenicity of numerous bacterial species. In recent years, growing evidence has shown that bacterial extracellular c-di-GMP can interact with the innate immune system and regulate host immune responses. This review summarizes our current understanding on the dual roles of bacterial c-di-GMP in pathogen-host interaction: activation of the antibacterial innate immune response through the cytosolic surveillance pathway and inhibition of innate immune defense for iron restriction.

Topics: Cyclic GMP; DEAD-box RNA Helicases; Host-Pathogen Interactions; Humans; Immunity, Innate; Lipocalin-2; Membrane Proteins

Pathogenic bacteria have many strategies for causing disease in humans. One such strategy is the ability to live both as single-celled motile organisms or as part of a community of bacteria called a biofilm. Biofilms are frequently adhered to biotic or abiotic surfaces and are extremely antibiotic resistant. Upon biofilm dispersal, bacteria become more antibiotic susceptible but are also able to readily infect another host. Various studies have shown that low, nontoxic levels of nitric oxide (NO) may induce biofilm dispersal in many bacterial species. While the molecular details of this phenotype remain largely unknown, in several species, NO has been implicated in biofilm-to-planktonic cell transitions via ligation to 1 of 2 characterized NO sensors, NosP or H-NOX. Based on the data available to date, it appears that NO binding to H-NOX or NosP triggers a downstream response based on changes in cellular cyclic di-GMP concentrations and/or the modulation of quorum sensing. In order to develop applications for control of biofilm infections, the identification and characterization of biofilm dispersal mechanisms is vital. This review focuses on the efforts made to understand NO-mediated control of H-NOX and NosP pathways in the 3 pathogenic bacteria Legionella pneumophila, Vibrio cholerae, and Pseudomonas aeruginosa.

Topics: Biofilms; Cyclic GMP; Legionella pneumophila; Nitric Oxide; Pseudomonas aeruginosa; Quorum Sensing; Signal Transduction; Vibrio cholerae

Iron is necessary for the survival of almost all aerobic organisms. In the mammalian host, iron is a required cofactor for the assembly of functional iron-sulfur (Fe-S) cluster proteins, heme-binding proteins and ribonucleotide reductases that regulate various functions, including heme synthesis, oxygen transport and DNA synthesis. However, the bioavailability of iron is low due to its insolubility under aerobic conditions. Moreover, the host coordinates a nutritional immune response to restrict the accessibility of iron against potential pathogens. To counter nutritional immunity, most commensal and pathogenic bacteria synthesize and secrete small iron chelators termed siderophores. Siderophores have potent affinity for iron, which allows them to seize the essential metal from the host iron-binding proteins. To safeguard against iron thievery, the host relies upon the innate immune protein, lipocalin 2 (Lcn2), which could sequester catecholate-type siderophores and thus impede bacterial growth. However, certain bacteria are capable of outmaneuvering the host by either producing "stealth" siderophores or by expressing competitive antagonists that bind Lcn2 in lieu of siderophores. In this review, we summarize the mechanisms underlying the complex iron tug-of-war between host and bacteria with an emphasis on how host innate immunity responds to siderophores.

Topics: Cyclic GMP; Ferrous Compounds; Host Microbial Interactions; Humans; Immunity, Innate; Inflammation; Iron; Lipocalin-2; Neutrophils; Peptides; Reactive Oxygen Species; Siderophores

PilZ domain-containing proteins constitute a large family of bacterial signaling proteins. As a widely distributed protein domain for the binding of the second messenger c-di-GMP, the canonical PilZ domain contains a set of motifs that define the binding site for c-di-GMP and an allosteric switch for propagating local conformational changes. Here, we summarize some new insights gathered from recent studies on the commonly occurring single-domain PilZ proteins, YcgR-like proteins and PilZ domain-containing cellulose synthases. The studies collectively illuminate how PilZ domains function as

Topics: Allosteric Regulation; Bacteria; Bacterial Proteins; Cyclic GMP; Protein Domains; Regulatory Sequences, Nucleic Acid; Signal Transduction

The BvgAS two-component system of Bordetella pertussis directly activates the expression of a large number of virulence genes in an environmentally responsive manner. The Bvg

Topics: Bacterial Proteins; Bordetella pertussis; Cyclic GMP; Gene Expression Regulation, Bacterial; Phosphoric Diester Hydrolases; Receptors, Cell Surface; Regulon; Transcription Factors; Virulence Factors

Since the discovery of cyclic dimeric guanosine 3',5'-monophosphate (c-di-GMP) in 1987, the role of cyclic dinucleotides in signal pathways has been extensively studied. Many receptors and effectors of cyclic dinucleotides have been identified which play important roles in cellular processes. Example of such effectors include cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP)-binding proteins and endoplasmic reticulum membrane adaptor. Accumulating evidence indicate that cyclic dinucleotides act as second messengers that not only regulate the bacterial physiological processes but also affect host immune responses during infections. Streptococci species, which produce cyclic dinucleotides, are responsible for many human diseases. Numerous studies suggest that the cyclic dinucleotides are vital in signal transduction pathways as second messengers and influence the progression of infectious diseases. Here, we provide an overview of the molecular principles of cyclic dinucleotides synthesis and degradation and discuss recent progress on streptococcal signal transduction pathways by cyclic dinucleotide second messengers and their role in regulating host immune reaction. This review will provide a better understanding of the molecular mechanisms of streptococcal cyclic dinucleotide second messengers thereby revealing novel targets for preventing infections.

Topics: Bacterial Adhesion; Bacterial Proteins; Carrier Proteins; Cyclic AMP Response Element-Binding Protein; Cyclic GMP; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Host-Pathogen Interactions; Humans; Intracellular Signaling Peptides and Proteins; Phenotype; Second Messenger Systems; Streptococcal Infections; Streptococcus pneumoniae; Streptococcus pyogenes; Virulence

Bacterial biofilms (BFs) are membrane-like structures formed by the secretion of extracellular polymeric substances (EPS) by bacteria. The formation of BFs contributes to bacterial survival and drug resistance. When bacteria proliferate, they produce secondary metabolites that act as signaling molecules in bacterial communities that regulate intracellular and cell-to-cell communication. This communication can directly affect the physiological behavior of bacteria, including the production and emission of light (bioluminescence), the expression of virulence factors, the resistance to antibiotics, and the shift between planktonic and biofilm lifestyles. We review the major signaling molecules that regulate BF formation, with a focus on quorum-sensing systems (QS), cyclic diguanylate (c-di-GMP), two-component systems (TCS), and small RNA (sRNA). Understanding these processes will lead to new approaches for treating chronic diseases and preventing bacterial resistance.

Topics: Bacteria; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Quorum Sensing; RNA, Bacterial; RNA, Small Untranslated; Signal Transduction

Plant pathogenic bacteria are responsible for the loss of hundreds of millions of dollars each year, impacting a wide range of economically relevant agricultural crops. The plant immune system detects conserved bacterial molecules and deploys an arsenal of effective defense measures at different levels; however, during compatible interactions, some pathogenic bacteria suppress and manipulate the host immunity and colonize and infect the plant host. Different bacteria employ similar strategies to circumvent plant innate immunity, while other tactics are specific to certain bacterial species. Recent studies have highlighted the secondary messenger c-di-GMP as a key molecule in the transmission of environmental cues in an intracellular regulatory network that controls virulence traits in many plant pathogenic bacteria. In this review, we focus on the recent knowledge of the molecular basis of c-di-GMP signaling mechanisms that promote or prevent the evasion of bacterial phytopathogens from the plant immune system. This review will highlight the considerable diversity of mechanisms evolved in plant-associated bacteria to elude plant immunity.

Topics: Crops, Agricultural; Cyclic GMP; Defensins; Erwinia amylovora; Gene Expression Regulation; Immune Evasion; Oryza; Oxylipins; Phytoalexins; Plant Immunity; Pseudomonas syringae; Receptors, Pattern Recognition; Sesquiterpenes; Signal Transduction; Type III Secretion Systems; Virulence; Xanthomonas; Xylella

Signaling pathways allow bacteria to adapt to changing environments. For pathogenic bacteria, signaling pathways allow for timely expression of virulence factors and the repression of antivirulence factors within the mammalian host. As the bacteria exit the mammalian host, signaling pathways enable the expression of factors promoting survival in the environment and/or nonmammalian hosts. One such signaling pathway uses the dinucleotide cyclic-di-GMP (c-di-GMP), and many bacterial genomes encode numerous proteins that are responsible for synthesizing and degrading c-di-GMP. Once made, c-di-GMP binds to individual protein and RNA receptors to allosterically alter the macromolecule function to drive phenotypic changes. Each bacterial genome encodes unique sets of genes for c-di-GMP signaling and virulence factors so the regulation by c-di-GMP is organism specific. Recent works have pointed to evidence that c-di-GMP regulates virulence in different bacterial pathogens of mammalian hosts. In this review, we discuss the criteria for determining the contribution of signaling nucleotides to pathogenesis using a well-characterized signaling nucleotide, cyclic AMP (cAMP), in Pseudomonas aeruginosa. Using these criteria, we review the roles of c-di-GMP in mediating virulence and highlight common themes that exist among eight diverse pathogens that cause different diseases through different routes of infection and transmission. WIREs RNA 2018, 9:e1454. doi: 10.1002/wrna.1454 This article is categorized under: RNA in Disease and Development > RNA in Disease.

Topics: Allosteric Regulation; Animals; Clostridioides difficile; Cyclic GMP; Gene Expression Regulation, Bacterial; Gram-Negative Bacteria; Humans; Protein Binding; Signal Transduction; Virulence; Virulence Factors

The nitrogen cycle pathways are responsible for the circulation of inorganic and organic N-containing molecules in nature. Among these pathways, those involving amino acids, N-oxides and in particular nitric oxide (NO) play strategic roles in the metabolism of microorganisms in natural environments and in host-pathogen interactions. Beyond their role in the N-cycle, amino acids and NO are also signalling molecules able to influence group behaviour in microorganisms and cell-cell communication in multicellular organisms, including humans. In this minireview, we summarise the role of these compounds in the homeostasis of the bacterial communities called biofilms, commonly found in environmental, industrial and medical settings. Biofilms are difficult to eradicate since they are highly resistant to antimicrobials and to the host immune system. We highlight the effect of amino acids such as glutamate, glutamine and arginine and of NO on the signalling pathways involved in the metabolism of 3',5'-cyclic diguanylic acid (c-di-GMP), a master regulator of motility, attachment and group behaviour in bacteria. The study of the metabolic routes involving these N-containing compounds represents an attractive topic to identify targets for biofilm control in both natural and medical settings.

Topics: Animals; Arginine; Bacteria; Biofilms; Cyclic GMP; Energy Metabolism; Environmental Microbiology; Humans; Nitric Oxide; Nitrogen; Signal Transduction

A biofilm is a complex assemblage of microbial communities adhered to a biotic or an abiotic surface which is embedded within a self-produced matrix of extracellular polymeric substances. Many transcriptional regulators play a role in triggering a motile-sessile switch and in consequently producing the biofilm matrix. This review is aimed at highlighting the role of two nucleotide signaling molecules (c-di-GMP and c-di-AMP), toxin antitoxin modules and a novel transcriptional regulator BolA in biofilm formation in various bacteria. In addition, it highlights the common themes that have appeared in recent research regarding the key regulatory components and signal transduction pathways that help Bacillus subtilis and Pseudomonas aeruginosa to acquire the biofilm mode of life.

Topics: Bacillus subtilis; Biofilms; Cyclic GMP; Dinucleoside Phosphates; Extracellular Matrix; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa; Signal Transduction

Pseudomonas aeruginosa is a versatile opportunistic pathogen capable of infecting a broad range of hosts, in addition to thriving in a broad range of environmental conditions outside of hosts. With this versatility comes the need to tightly regulate its genome to optimise its gene expression and behaviour to the prevailing conditions. Two-component systems (TCSs) comprising sensor kinases and response regulators play a major role in this regulation. This minireview discusses the growing number of TCSs that have been implicated in the virulence of P. aeruginosa, with a special focus on the emerging theme of multikinase networks, which are networks comprising multiple sensor kinases working together, sensing and integrating multiple signals to decide upon the best response. The networks covered in depth regulate processes such as the switch between acute and chronic virulence (GacS network), the Cup fimbriae (Roc network and Rcs/Pvr network), the aminoarabinose modification of lipopolysaccharide (a network involving the PhoQP and PmrBA TCSs), twitching motility and virulence (a network formed from the Chp chemosensory pathway and the FimS/AlgR TCS), and biofilm formation (Wsp chemosensory pathway). In addition, we highlight the important interfaces between these systems and secondary messenger signals such as cAMP and c-di-GMP.

Topics: Arabinose; Bacterial Proteins; Cyclic AMP; Cyclic GMP; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Genes, Bacterial; Lipopolysaccharides; Pseudomonas aeruginosa; Virulence Factors

The Gram-negative pathogen Pseudomonas aeruginosa is found ubiquitously within the environment and is recognised as an opportunistic human pathogen that commonly infects burn wounds and immunocompromised individuals, or patients suffering from the autosomal recessive disorder cystic fibrosis (CF). During chronic infection, P. aeruginosa is thought to form structured aggregates known as biofilms characterised by a self-produced matrix which encases the bacteria, protecting them from antimicrobial attack and the host immune response. In many cases, antibiotics are ineffective at eradicating P. aeruginosa from chronically infected CF airways. Cyclic-di-GMP has been identified as a key regulator of biofilm formation; however, the way in which its effector proteins elicit a change in biofilm formation remains unclear. Identifying regulators of biofilm formation is a key theme of current research and understanding the factors that activate biofilm formation may help to expose potential new drug targets that slow the onset of chronic infection. This minireview outlines the contribution made by exopolysaccharides to biofilm formation, and describes the current understanding of biofilm regulation in P. aeruginosa with a particular focus on CF airway-associated infections.

Topics: Alginates; Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Cyclic GMP; Cystic Fibrosis; Drug Resistance, Bacterial; Escherichia coli Proteins; Glucuronic Acid; Hexuronic Acids; Humans; Phosphorus-Oxygen Lyases; Polysaccharides, Bacterial; Pseudomonas aeruginosa

In the past decade, Clostridium difficile has emerged as an important gut pathogen. This anaerobic, Gram-positive bacterium is the main cause of infectious nosocomial diarrhea. Whereas much is known about the mechanism through which the C. difficile toxins cause diarrhea, relatively little is known about the dynamics of adhesion and motility, which is mediated by cell surface proteins. This review will discuss the recent advances in our understanding of the sortase-mediated covalent attachment of cell surface (adhesion) proteins to the peptidoglycan layer of C. difficile and their release through the action of a highly specific secreted metalloprotease (Pro-Pro endopeptidase 1, PPEP-1). Specific emphasis will be on a model in which PPEP-1 and its substrates control the switch from a sessile to motile phenotype in C. difficile, and how this is regulated by the cyclic dinucleotide c-di-GMP (3'-5' cyclic dimeric guanosine monophosphate).

Topics: Bacterial Proteins; Biofilms; Cell Adhesion; Clostridioides difficile; Cross Infection; Cyclic GMP; Dipeptides; Endopeptidases; Gene Expression Regulation, Bacterial; Humans; Membrane Proteins; Metalloproteases; Peptidoglycan

Cyclic diguanylate (c-di-GMP) is a near universal signaling molecule produced by diguanylate cyclases that can direct a variety of bacterial behaviors. A major area of research over the last several years has been aimed at understanding how a cell with dozens of diguanylate cyclases can deploy a given subset of them to produce a desired phenotypic outcome without undesired cross talk between c-di-GMP-dependent systems. Several models have been put forward to address this question, including specificity of cyclase activation, tuned binding constants of effector proteins, and physical interaction between cyclases and effectors. Additionally, recent evidence has suggested that there may be a link between the catalytic state of a cyclase and its physical contact with an effector. This review highlights several key studies, examines the proposed global and local models of c-di-GMP signaling specificity in bacteria, and attempts to identify the most fruitful steps that can be taken to better understand how dynamic networks of sibling cyclases and effector proteins result in sensible outputs that govern cellular behavior.

Topics: Bacteria; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Models, Biological; Phosphorus-Oxygen Lyases; Protein Binding; Signal Transduction; Substrate Specificity

The nearly ubiquitous bacterial second messenger cyclic di-GMP is involved in a multitude of fundamental physiological processes such as sessility/motility transition and the switch between the acute and chronic infection status, combined with cell cycle control. The discovery of cyclic di-GMP, though, has been an example par excellence of scientific serendipity. We recapitulate here its years-long discovery process as an activator of the cellulose synthase of the environmental bacterium Komagataeibacter xylinus and its consequences for follow-up research. Indeed, the discovery of cyclic di-GMP as a ubiquitous second messenger contributed to the change in perception of bacteria as simple unicellular organisms just randomly building-up multicellular communities. Subsequently, cyclic di-GMP also paved the way to the identification of other pro- and eukaryotic cyclic dinucleotide second messengers.

Topics: Bacteria; Bacterial Physiological Phenomena; Biofilms; Cyclic GMP; Eukaryotic Cells; Glucosyltransferases; History, 20th Century; History, 21st Century; Research; Second Messenger Systems

Bacteria possess several signaling molecules that regulate distinct phenotypes. Cyclic di-GMP (c-di-GMP) has emerged as a ubiquitous second messenger that regulates bacterial virulence, cell cycle, motility, and biofilm formation. The link between c-di-GMP signaling and biofilm formation affords novel strategies for treatment of biofilm-associated infections, which is a major public health problem. The complex c-di-GMP signaling pathway creates a hurdle in the development of small molecule modulators. Nonetheless, some progress has been made in this regard and inhibitors of c-di-GMP metabolizing enzymes that affect biofilm formation and motility have been documented. Herein we discuss the components of c-di-GMP signaling, their correlation with biofilm formation as well as motility and reported small molecule inhibitors of c-di-GMP signaling.

Topics: Bacteria; Bacterial Physiological Phenomena; Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Intracellular Space; Membrane Proteins; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Binding; Protein Interaction Domains and Motifs; Proteolysis; Second Messenger Systems

The development of effective strategies to combat biofilm infections by means of either mechanical or chemical approaches could dramatically change today's treatment procedures for the benefit of thousands of patients. Remarkably, considering the increased focus on biofilms in general, there has still not been invented and/or developed any simple, efficient and reliable methods with which to "chemically" eradicate biofilm infections. This underlines the resilience of infective agents present as biofilms and it further emphasizes the insufficiency of today's approaches used to combat chronic infections. A potential method for biofilm dismantling is chemical interception of regulatory processes that are specifically involved in the biofilm mode of life. In particular, bacterial cell to cell signaling called "Quorum Sensing" together with intracellular signaling by bis-(3'-5')-cyclic-dimeric guanosine monophosphate (cyclic-di-GMP) have gained a lot of attention over the last two decades. More recently, regulatory processes governed by two component regulatory systems and small non-coding RNAs have been increasingly investigated. Here, we review novel findings and potentials of using small molecules to target and modulate these regulatory processes in the bacterium Pseudomonas aeruginosa to decrease its pathogenic potential.

Topics: Bacteria; Bacterial Physiological Phenomena; Biofilms; Cyclic GMP; Humans; Quorum Sensing; Signal Transduction

Inflammatory bowel disease (IBD) symbolizes a group of intestinal disorders in which prolonged inflammation occur in the digestive tract (esophagus, large intestine, small intestine mouth, stomach). Both genetic and environmental factors (infections, stress, diet) are involved in the development of IBD. As we know that bacteria are found in the intestinal mucosa of human and clinical observations revealed bacterial biofilms associated with patients of IBD. Various factors and microbes are found to play an essential role in biofilm formation and mucosal colonization during IBD. Biofilm formation in the digestive tract is dependent on an extracellular matrix synthesized by the bacteria and it has an adverse effect on the immune response of the host. There is no satisfactory and safe treatment option for IBD. Therefore, the current research aims to disrupt biofilm in IBD and concentrates predominantly on improving the drug. Here, we review the literature on bacterial biofilm and IBD to gather new knowledge on the current understanding of biofilm formation in IBD, host immune deregulation and dysbiosis in IBD, molecular mechanism, bacteria involved in biofilm formation, current and future regimen. It is urgently required to plan new ways to control and eradicate bacteria in biofilms that will open up novel diagnostic and therapeutic avenues for IBD. This article includes the mechanism of signaling molecules with respect to the biofilm-related genes as well as the diagnostic methods and new technologies involved in the treatment of IBD.

Topics: Anti-Inflammatory Agents; Bacteria; Biofilms; Cyclic GMP; Dysbiosis; Gastrointestinal Microbiome; Gastrointestinal Tract; Humans; Immunosuppressive Agents; Inflammation; Inflammatory Bowel Diseases; Intestinal Mucosa; Intestines; Quorum Sensing; Stomach

cyclic di-GMP (c-di-GMP) is a universal second messenger in bacterial cells. It regulates various biological processes such as biofilm development, pathogenicity, motility, exopolysaccharide (EPS) production and cell cycle. The second messenger exerts its function by binding to effectors, such as riboswitches and proteins. However, due to the diverse conformations of c-di-GMP, its effectors are hardly to be predicted by homology search. Identification of c-di-GMP effectors is the initial step to investigate its regulatory function in bacterial signal transduction, however, it remains to be a technically difficult task. Here we reviewed the mechanism of biofilm development controlled by c-di-GMP through binding to various types of protein effectors, and summarized the screening strategies, including genetics analysis, protein pull-down combined with LC/MS/MS identification, DRaCALA systematic screening and molecular docking-based prediction. We also summarized experimental methods for verifying protein-c-di-GMP interaction, including isothermal titration calorimetry, surface plasmon resonance, microscale thermophoresis etc. In addition, we discussed the advantages and disadvantages of these strategies and methods. The present review aims to facilitate the future investigations that are focused on regulatory role of novel c-di-GMP effectors.. 环二鸟苷单磷酸 (cyclic di-GMP 或c-di-GMP) 是细菌细胞中广泛存在的第二信使，调控细菌生物被膜发育、致病力、运动性、胞外多糖产生及细胞周期在内的诸多重要生理表型。c-di-GMP 通过结合多种类型的效应子 (包括核糖开关或效应蛋白) 来发挥调控功能。由于c-di-GMP 分子在构象上具有多变性，其结合的效应子同样具有多样性。新型效应蛋白的筛选、鉴定是当前细菌信号转导领域的研究热点和难点，也是解析c-di-GMP 调控机制的首要环节。本文在阐述c-di-GMP 结合不同类型的效应蛋白并调控细菌生物被膜发育的基础上，综述了目前筛选c-di-GMP 效应蛋白的方法，包括遗传筛选、亲和色谱结合质谱鉴定、DRaCALA 系统鉴定以及基于分子对接的预测等。同时，对验证c-di-GMP 效应蛋白的技术，如等温微量热滴定、表面等离子共振、微量热泳动在内的多种验证方法进行了总结，对比了这些策略和方法在应用上的优、缺点，为在细菌及其真核宿主基因组水平鉴定c-di-GMP 效应蛋白的研究提供参考。.

Topics: Bacterial Proteins; Biofilms; Carrier Proteins; Chromatography, Liquid; Cyclic GMP; High-Throughput Screening Assays; Intracellular Signaling Peptides and Proteins; Molecular Docking Simulation; Second Messenger Systems; Tandem Mass Spectrometry

The cyclic dinucleotide c-di-GMP is known as an important second messenger in bacteria, which controls various important cellular processes, such as cell differentiation, biofilm formation and virulence factors production. It is extremely vital for the development of new antibacterial agents by virtue of blocking c-di-GMP signal conduction. Current research indicates that there are three potential targets for discovering new antibacterial agents based on c-di-GMP regulated signal pathway, which are c-di-GMP synthases, c-di-GMP degrading enzymes and c-di-GMP receptors. Herein, we review small molecules that have been developed to inhibit c-di-GMP related enzymes and indicate perspectives of c-di-GMP inhibitors.. 环二鸟苷酸 (Bis-(3′-5′) cyclic diguanylic acid, c-di-GMP) 是细菌所特有的一类核酸类第二信使，参与并调节细菌多种生理功能，包括细胞分化、生物被膜的形成以及致病因子的产生等。阻断c-di-GMP 信号的传导对于发展新型抗菌药物具有重要的意义。现有研究结果表明，基于c-di-GMP 调控的信号通路开发新型抗菌药物具有3 类潜在的靶点，分别是c-di-GMP 合成酶 (DGCs)、c-di-GMP 降解酶 (PDEs) 以及c-di-GMP 受体。文中根据上述3 类关键靶点，介绍了相关小分子抑制剂的研究进展，并展望了c-di-GMP 信号分子抑制剂的发展方向。.

Topics: Anti-Bacterial Agents; Bacteria; Cyclic GMP; Second Messenger Systems

Yersinia pestis, the cause of plague, is transmitted by flea bite. Y. pestis forms a biofilm in the proventriculus of its flea vector to enhance transmission. Biofilm formation in Y. pestis is positively regulated by the intracellular levels of the second messenger cyclic diguanylate (c-di-GMP). The c-di-GMP in Y. pestis is synthesized by two diguanylate cyclases (DGC), HmsT and HmsD, and degraded by phosphodiesterase (PDE), HmsP. Here we summarized the regulators that modulate c-di-GMP metabolism and biofilm formation in Y. pestis and discussed their regulatory mechanism.. 鼠疫耶尔森氏菌 (Yersinia pestis，以下简称“鼠疫菌”) 是烈性传染病鼠疫的病原菌，以鼠蚤作为传播媒介。鼠疫菌在其传播媒介鼠蚤的前胃中形成生物被膜从而促进其在宿主间传播。鼠疫菌生物被膜的形成受第二信使分子环二鸟苷酸 (c-di-GMP) 的正向调控。鼠疫菌中c-di-GMP由二鸟苷酸环化酶 (DGC) HmsT 和HmsD合成，由磷酸二酯酶 (PDE) HmsP 降解。文中主要介绍影响鼠疫菌环二鸟苷酸代谢及生物被膜形成的调控因子，并对其作用机制进行讨论和总结。.

Topics: Animals; Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Phosphorus-Oxygen Lyases; Second Messenger Systems; Siphonaptera; Yersinia pestis

RNA helicases are involved in almost every aspect of RNA, from transcription to RNA decay. DExD/H-box helicases comprise the largest SF2 helicase superfamily, which are characterized by two conserved RecA-like domains. In recent years, an increasing number of unexpected functions of these proteins have been discovered. They play important roles not only in innate immune response but also in diseases like cancers and chronic hepatitis C. In this review, we summarize the recent literatures on one member of the SF2 superfamily, the DEAD-box protein DDX41. After bacterial or viral infection, DNA or cyclic-di-GMP is released to cells. After phosphorylation of Tyr414 by BTK kinase, DDX41 will act as a sensor to recognize the invaders, followed by induction of type I interferons (IFN). After the immune response, DDX41 is degraded by the E3 ligase TRIM21, using Lys9 and Lys115 of DDX41 as the ubiquitination sites. Besides the roles in innate immunity, DDX41 is also related to diseases. An increasing number of both inherited and acquired mutations in DDX41 gene are identified from myelodysplastic syndrome and/or acute myeloid leukemia (MDS/AML) patients. The review focuses on DDX41, as well as its homolog Abstrakt in Drosophila, which is important for survival at all stages throughout the life cycle of the fly.

Topics: Agammaglobulinaemia Tyrosine Kinase; Animals; Bacterial Infections; Cyclic GMP; DEAD-box RNA Helicases; Drosophila melanogaster; Drosophila Proteins; Humans; Leukemia, Myeloid, Acute; Mutation; Myelodysplastic Syndromes; Nuclear Proteins; Protein-Tyrosine Kinases; Virus Diseases

Cyclic di-GMP was the first cyclic dinucleotide second messenger described, presaging the discovery of additional cyclic dinucleotide messengers in bacteria and eukaryotes. The GGDEF diguanylate cyclase (DGC) and EAL and HD-GYP phosphodiesterase (PDE) domains conduct the turnover of cyclic di-GMP. These three unrelated domains belong to superfamilies that exhibit significant variations in function, and they include both enzymatically active and inactive members, with a subset involved in synthesis and degradation of other cyclic dinucleotides. Here, we summarize current knowledge of sequence and structural variations that underpin the functional diversification of cyclic di-GMP turnover proteins. Moreover, we highlight that superfamily diversification is not restricted to cyclic di-GMP signaling domains, as particular DHH/DHHA1 domain and HD domain proteins have been shown to act as cyclic di-AMP phosphodiesterases. We conclude with a consideration of the current limitations that such diversity of action places on bioinformatic prediction of the roles of GGDEF, EAL, and HD-GYP domain proteins.

Topics: Bacteria; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Pharmacogenomic Variants

The class Alphaproteobacteria includes Gram-negative free-living, symbiotic and obligate intracellular bacteria, as well as important plant, animal and human pathogens. Recent work has established the key antagonistic roles that phosphorylated guanosines, cyclic-di-GMP (c-di-GMP) and the alarmones guanosine tetraphosphate and guanosine pentaphosphate (collectively referred to as (p)ppGpp), have in the regulation of the cell cycle in these bacteria. In this Review, we discuss the insights that have been gained into the regulation of the initiation of DNA replication and cytokinesis by these second messengers, with a particular focus on the cell cycle of Caulobacter crescentus. We explore how the fluctuating levels of c-di-GMP and (p)ppGpp during the progression of the cell cycle and under conditions of stress control the synthesis and proteolysis of key regulators of the cell cycle. As these signals also promote bacterial interactions with host cells, the enzymes that control (p)ppGpp and c-di-GMP are attractive antibacterial targets.

Topics: Caulobacter crescentus; Cell Cycle; Cell Cycle Checkpoints; Cell Division; Cyclic GMP; Cytokinesis; DNA Replication; Gene Expression Regulation, Bacterial; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Phosphorylation; Sinorhizobium meliloti

Cyclic dinucleotides (CDNs) are highly versatile signalling molecules that control various important biological processes in bacteria. The best-studied example is cyclic di-GMP (c-di-GMP). Known since the late 1980s, it is now recognized as a near-ubiquitous second messenger that coordinates diverse aspects of bacterial growth and behaviour, including motility, virulence, biofilm formation and cell cycle progression. In this Review, we discuss important new insights that have been gained into the molecular principles of c-di-GMP synthesis and degradation, which are mediated by diguanylate cyclases and c-di-GMP-specific phosphodiesterases, respectively, and the cellular functions that are exerted by c-di-GMP-binding effectors and their diverse targets. Finally, we provide a short overview of the signalling versatility of other CDNs, including c-di-AMP and cGMP-AMP (cGAMP).

Topics: Bacteria; Biofilms; Cyclic GMP; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Nucleotides, Cyclic; Second Messenger Systems; Signal Transduction

The second messenger nucleotide cyclic dimeric guanosine monophosphate (c-di-GMP) governs many cellular processes in the facultative human pathogen Vibrio cholerae. This organism copes with changing environmental conditions in aquatic environments and during transitions to and from human hosts. Modulation of c-di-GMP allows V. cholerae to shift between motile and sessile stages of life, thus allowing adaptation to stressors and environmental conditions during its transmission cycle. The V. cholerae genome encodes a large set of proteins predicted to degrade and produce c-di-GMP. A subset of these enzymes has been demonstrated to control cellular processes - particularly motility, biofilm formation, and virulence - through transcriptional, post-transcriptional, and translational mechanisms. Recent studies have identified and characterized enzymes that modulate or sense c-di-GMP levels and have led towards mechanistic understanding of c-di-GMP regulatory circuits in V. cholerae.

Topics: Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Genome, Bacterial; Humans; Second Messenger Systems; Signal Transduction; Vibrio cholerae; Virulence

Clostridium difficile (CD)-associated diarrhoea is currently the most prevalent nosocomial diarrhoea worldwide. Many characteristics of CD pathogenicity remain poorly understood. Recent data strongly indicate the importance of an RNA network for the control of gene expression in CD. More than 200 regulatory RNAs have been identified by deep sequencing and targeted approaches, including Hfq-dependent trans riboregulators, cis-antisense RNAs, CRISPR RNAs, and c-di-GMP-responsive riboswitches. These regulatory RNAs are involved in the control of major processes in the CD infection cycle, for example motility, biofilm formation, adhesion, sporulation, stress response, and defence against bacteriophages. We will discuss recent advances in elucidation of the original features of RNA-based mechanisms in this important enteropathogen. This knowledge may pave the way for further discoveries in this emergent field.

Topics: Clostridioides difficile; Clostridium Infections; Clustered Regularly Interspaced Short Palindromic Repeats; Cyclic GMP; Gene Expression Regulation, Bacterial; Host Factor 1 Protein; Humans; Riboswitch; RNA; Signal Transduction; Virulence

Cyclic di-GMP (c-di-GMP) is currently recognized as the most widely used intracellular signal molecule in prokaryotes, but roles in eukaryotes were only recently discovered. In the social amoeba Dictyostelium discoideum, c-di-GMP, produced by a prokaryote-type diguanylate cyclase, induces the differentiation of stalk cells, thereby enabling the formation of spore-bearing fruiting bodies. In this review, we summarize the currently known mechanisms that control the major life cycle transitions of Dictyostelium and focus particularly on the role of c-di-GMP in stalk formation. Stalk cell differentiation has characteristics of autophagic cell death, a process that also occurs in higher eukaryotes. We discuss the respective roles of c-di-GMP and of another signal molecule, differentiation-inducing factor 1, in autophagic cell death in vitro and in stalk formation in vivo.

Topics: Cyclic GMP; Dictyostelium; Signal Transduction; Spores, Protozoan

The first International Symposium on c-Di-GMP Signaling in Bacteria (22 to 25 March 2015, Harnack-Haus, Berlin, Germany)brought together 131 molecular microbiologists from 17 countries to discuss recent progress in our knowledge of bacterial nucleotide second messenger signaling. While the focus was on signal input, synthesis, degradation, and the striking diversity of the modes of action of the current second messenger paradigm, i.e., cyclic di-GMP (c-di-GMP), “classics” like cAMP and (p)ppGpp were also presented, in novel facets, and more recent “newcomers,” such as c-di-AMP and c-AMP-GMP, made an impressive appearance. A number of clear trends emerged during the 30 talks, on the 71 posters, and in the lively discussions, including (i)c-di-GMP control of the activities of various ATPases and phosphorylation cascades, (ii) extensive cross talk between c-di-GMP and other nucleotide second messenger signaling pathways, and (iii) a stunning number of novel effectors for nucleotide second messengers that surprisingly include some long-known master regulators of developmental pathways. Overall, the conference made it amply clear that second messenger signaling is currently one of the most dynamic fields within molecular microbiology,with major impacts in research fields ranging from human health to microbial ecology.

Topics: Bacteria; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Second Messenger Systems; Signal Transduction

Cyclic di-GMP (c-di-GMP) synthetases and hydrolases (GGDEF, EAL, and HD-GYP domains) can be readily identified in bacterial genome sequences by using standard bioinformatic tools. In contrast, identification of c-di-GMP receptors remains a difficult task, and the current list of experimentally characterized c-di-GMP-binding proteins is likely incomplete. Several classes of c-di-GMP-binding proteins have been structurally characterized; for some others, the binding sites have been identified; and for several potential c-di-GMP receptors, the binding sites remain to be determined. We present here a comparative structural analysis of c-di-GMP-protein complexes that aims to discern the common themes in the binding mechanisms that allow c-di-GMP receptors to bind it with (sub)micromolar affinities despite the 1,000-fold excess of GTP. The available structures show that most receptors use their Arg and Asp/Glu residues to bind c-di-GMP monomers, dimers, or tetramers with stacked guanine bases. The only exception is the EAL domains that bind c-di-GMP monomers in an extended conformation. We show that in c-di-GMP-binding signature motifs, Arg residues bind to the O-6 and N-7 atoms at the Hoogsteen edge of the guanine base, while Asp/Glu residues bind the N-1 and N-2 atoms at its Watson-Crick edge. In addition, Arg residues participate in stacking interactions with the guanine bases of c-di-GMP and the aromatic rings of Tyr and Phe residues. This may account for the presence of Arg residues in the active sites of every receptor protein that binds stacked c-di-GMP. We also discuss the implications of these structural data for the improved understanding of the c-di-GMP signaling mechanisms.

Topics: Bacteria; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Protein Binding; Signal Transduction

Cells respond to the environment and alter gene expression. Recent studies have revealed the social aspects of bacterial life, such as biofilm formation. Biofilm formation is largely affected by the environment, and the mechanisms by which the gene expression of individual cells affects biofilm development have attracted interest. Environmental factors determine the cell's decision to form or leave a biofilm. In addition, the biofilm structure largely depends on the environment, implying that biofilms are shaped to adapt to local conditions. Second messengers such as cAMP and c-di-GMP are key factors that link environmental factors with gene regulation. Cell-to-cell communication is also an important factor in shaping the biofilm. In this short review, we will introduce the basics of biofilm formation and further discuss environmental factors that shape biofilm formation. Finally, the state-of-the-art tools that allow us investigate biofilms under various conditions are discussed.

Topics: Bacillus subtilis; Bacterial Adhesion; Bacterial Proteins; Biofilms; Clostridium perfringens; Cyclic AMP; Cyclic GMP; Gene Expression Regulation, Bacterial; Gene-Environment Interaction; Microscopy, Electron, Scanning; Pseudomonas aeruginosa; Quorum Sensing; Second Messenger Systems; Species Specificity; Vibrio cholerae

The cyclic dinucleotides cyclic 3′,5′-diguanylate (c-di-GMP) and cyclic 3′,5′-diadenylate (c-di-AMP) have emerged as key components of bacterial signal transduction networks. These closely related second messengers follow the classical general principles of nucleotide signaling by integrating diverse signals into regulatory pathways that control cellular responses to changing environments. They impact distinct cellular processes, with c-di-GMP having an established role in promoting bacterial adhesion and inhibiting motility and c-di-AMP being involved in cell wall metabolism, potassium homeostasis, and DNA repair. The involvement of c-dinucleotides in the physiology of the filamentous, nonmotile streptomycetes remained obscure until recent discoveries showed that c-di-GMP controls the activity of the developmental master regulator BldD and that c-di-AMP determines the level of the resuscitation-promoting factor A(RpfA) cell wall-remodelling enzyme. Here, I summarize our current knowledge of c-dinucleotide signaling in Streptomyces species and highlight the important roles of c-di-GMP and c-di-AMP in the biology of these antibiotic-producing, multicellular bacteria.

Topics: Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Signal Transduction; Species Specificity; Streptomyces

Nearly all bacterial species, including pathogens, have the ability to form biofilms. Biofilms are defined as structured ecosystems in which microbes are attached to surfaces and embedded in a matrix composed of polysaccharides, eDNA, and proteins, and their development is a multistep process. Bacterial biofilms constitute a large medical problem due to their extremely high resistance to various types of therapeutics, including conventional antibiotics. Several environmental and genetic signals control every step of biofilm development and dispersal. From among the latter, quorum sensing, cyclic diguanosine-5'-monophosphate, and small RNAs are considered as the main regulators. The present review describes the control role of these three regulators in the life cycles of biofilms built by Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella enterica serovar Typhimurium, and Vibrio cholerae. The interconnections between their activities are shown. Compounds and strategies which target the activity of these regulators, mainly quorum sensing inhibitors, and their potential role in therapy are also assessed.

Topics: Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa; Quorum Sensing; RNA, Bacterial; RNA, Small Untranslated; Salmonella typhimurium; Staphylococcus aureus; Vibrio cholerae

Nowadays, there is increasing interest for bacterial polysaccharides in a wide variety of industrial sectors. This is due to their chemical and reological properties, and also the possibility to be obtained by fermentation processes. Biosynthesis of a growing number of exopolysaccharides (EPS) has been reported to be regulated by the ubiquitous second messenger c-di-GMP in a limited number of bacterial species. Since most bacteria are yet unexplored, it is likely that an unsuspected number and variety of EPS structures activated by c-di-GMP await to be uncovered. In the search of new EPS, manipulation of bacterial c-di-GMP metabolism can be combined with high throughput approaches for screening of large collections of bacteria. In addition, c-di-GMP activation of EPS production and promotion of cell aggregation may have direct applications in environmental industries related with biofuel production or wastewater treatments.

Topics: Bacteria; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Polysaccharides, Bacterial

Alphaproteobacteria include many medically and environmentally important organisms. Despite the diversity of their niches and lifestyles, from free-living to host-associated, they usually rely on very similar mechanisms to control their cell cycles. Studies on Caulobacter crescentus still lay the foundation for understanding the molecular details of pathways regulating DNA replication and cell division and coordinating these two processes with other events of the cell cycle. This review highlights recent discoveries on the regulation and the mode of action of conserved global regulators and small molecules like c-di-GMP and (p)ppGpp, which play key roles in cell cycle control. It also describes several newly identified mechanisms that modulate cell cycle progression in response to stresses or environmental conditions.

Topics: Alphaproteobacteria; Bacterial Proteins; Cell Cycle; Cyclic GMP; Gene Expression Regulation, Bacterial

The ability to precisely regulate gene expression is one of the most important features of the living cells as it enables the adaptation and survival in different environmental conditions. The majority of regulatory mechanisms involve protein action, however, multiple genes are controlled by nucleic acids. Among RNA-based regulators, the riboswitches present a large group of specific domains within messenger RNAs able to respond to small metabolites, tRNA, secondary messengers, ions, vitamins or amino acids. A simple, accurate, and efficient mechanism of action as well as easiness in handling and engineering make the riboswitches a potent practical tool in industry, medicine, pharmacy or environmental protection. Hereby, we summarize the current achievements and challenges in designing and practical employment of the riboswitch-based tools.

Topics: Bacteria; Biosensing Techniques; Cyclic GMP; Gene Expression Regulation; Lysine; Protein Biosynthesis; Purines; Riboswitch; RNA; RNA Splicing; Thiamine Pyrophosphate; Transcription, Genetic; Viruses

The cyclic di-GMP (c-di-GMP) second messenger represents a signaling system that regulates many bacterial behaviors and is of key importance for driving the lifestyle switch between motile loner cells and biofilm formers. This review provides an up-to-date compendium of c-di-GMP pathways connected to biofilm formation, biofilm-associated motilities, and other functionalities in the ubiquitous and opportunistic human pathogen Pseudomonas aeruginosa This bacterium is frequently adopted as a model organism to study bacterial biofilm formation. Importantly, its versatility and adaptation capabilities are linked with a broad range of complex regulatory networks, including a large set of genes involved in c-di-GMP biosynthesis, degradation, and transmission.

Topics: Bacteria; Bacterial Proteins; Biofilms; Biosynthetic Pathways; Cyclic GMP; Humans; Models, Biological; Pseudomonas aeruginosa; Second Messenger Systems; Signal Transduction

Borrelia burgdorferi, the causative agent of Lyme disease (along with closely related genospecies), is in the deeply branching spirochete phylum. The bacterium is maintained in nature in an enzootic cycle that involves transmission from a tick vector to a vertebrate host and acquisition from a vertebrate host to a tick vector. During its arthropod sojourn, B. burgdorferi faces a variety of stresses, including nutrient deprivation. Here, we review some of the spirochetal factors that promote persistence, maintenance and dissemination of B. burgdorferi in the tick, and then focus on the utilization of available carbohydrates as well as the exquisite regulatory systems invoked to adapt to the austere environment between blood meals and to signal species transitions as the bacteria traverse their enzootic cycle. The spirochetes shift their source of carbon and energy from glucose in the vertebrate to glycerol in the tick. Regulation of survival under limiting nutrients requires the classic stringent response in which RelBbu controls the levels of the alarmones guanosine tetraphosphate and guanosine pentaphosphate (collectively termed (p)ppGpp), while regulation at the tick-vertebrate interface as well as regulation of protective responses to the blood meal require the two-component system Hk1/Rrp1 to activate production of the second messenger cyclic-dimeric-GMP (c-di-GMP).

Topics: Animals; Arachnid Vectors; Bacterial Proteins; Borrelia burgdorferi; Carbon; Cyclic GMP; Gene Expression Regulation, Bacterial; Guanosine Pentaphosphate; Host-Pathogen Interactions; Lyme Disease; Ticks

Microorganisms growing in a biofilm state are very resilient in the face of treatment by many antimicrobial agents. Biofilm infections are a significant problem in chronic and long-term infections, including those colonizing medical devices and implants. Anti-biofilm peptides represent a very promising approach to treat biofilm-related infections and have an extraordinary ability to interfere with various stages of the biofilm growth mode. Anti-biofilm peptides possess promising broad-spectrum activity in killing both Gram-positive and Gram-negative bacteria in biofilms, show strong synergy with conventional antibiotics, and act by targeting a universal stringent stress response. Understanding downstream processes at the molecular level will help to develop and design peptides with increased activity. Anti-biofilm peptides represent a novel, exciting approach to treating recalcitrant bacterial infections.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacterial Infections; Biofilms; Cyclic GMP; Drug Synergism; Escherichia coli; Microbial Sensitivity Tests; Prostheses and Implants; Prosthesis-Related Infections; Quorum Sensing; Staphylococcus aureus

The nucleotide second messenger 3'-5' cyclic diguanylate monophosphate (c-di-GMP) is a central regulator of the transition between motile and non-motile lifestyles in bacteria, favoring sessility. Most research investigating the functions of c-di-GMP has focused on Gram-negative species, especially pathogens. Recent work in Gram-positive species has revealed that c-di-GMP plays similar roles in Gram-positives, though the precise targets and mechanisms of regulation may differ. The majority of bacterial life exists in a surface-associated state, with motility allowing bacteria to disseminate and colonize new environments. c-di-GMP signaling regulates flagellum biosynthesis and production of adherence factors and appears to be a primary mechanism by which bacteria sense and respond to surfaces. Ultimately, c-di-GMP influences the ability of a bacterium to alter its transcriptional program, physiology and behavior upon surface contact. This review discusses how bacteria are able to sense a surface via flagella and type IV pili, and the role of c-di-GMP in regulating the response to surfaces, with emphasis on studies of Gram-positive bacteria.

Topics: Bacterial Adhesion; Biofilms; Cyclic GMP; Fimbriae, Bacterial; Flagella; Gram-Positive Bacteria; Microbial Interactions; Signal Transduction

Cellular levels of the second messenger cyclic di-guanosine monophosphate (c-di-GMP) are determined by the antagonistic activities of diguanylate cyclases and specific phosphodiesterases. In a given bacterial organism, there are often multiple variants of the two enzymes, which are tightly regulated by a variety of external and internal cues due to the presence of specialized sensory or regulatory domains. Dependent on the second messenger level, specific c-di-GMP receptors then control fundamental cellular processes, such as bacterial life style, biofilm formation, and cell cycle control. Here, I review the large body of data on structure-function relationships in diguanylate cyclases. Although the catalytic GGDEF domain is related to the respective domain of adenylate cyclases, the catalyzed intermolecular condensation reaction of two GTP molecules requires the formation of a competent GGDEF dimer with the two substrate molecules juxtaposed. This prerequisite appears to constitute the basis for GGDEF regulation with signal-induced changes within the homotypic dimer of the input domain (PAS, GAF, HAMP, etc.), which are structurally coupled with the arrangement of the GGDEF domains via a rigid coiled-coil linker. Alternatively, phosphorylation of a Rec input domain can drive GGDEF dimerization. Both mechanisms allow modular combination of input and output function that appears advantageous for evolution and rationalizes the striking similarities in domain architecture found in diguanylate cyclases and histidine kinases.

Topics: Bacteria; Cyclic GMP; Escherichia coli Proteins; Evolution, Molecular; Gene Expression Regulation; Guanosine Triphosphate; Metabolic Networks and Pathways; Models, Molecular; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Conformation; Protein Multimerization

The bacterial second messenger c-di-GMP controls bacterial biofilm formation, motility, cell cycle progression, development and virulence. It is synthesized by diguanylate cyclases (with GGDEF domains), degraded by specific phosphodiesterases (PDEs, with EAL of HD-GYP domains) and sensed by a wide variety of c-di-GMP-binding effectors that control diverse targets. c-di-GMP-binding effectors can be riboswitches as well as proteins with highly diverse structures and functions. The latter include 'degenerate' GGDEF/EAL domain proteins that are enzymatically inactive but still able to bind c-di-GMP. Surprisingly, two enzymatically active 'trigger PDEs', the Escherichia coli proteins PdeR and PdeL, have recently been added to this list of c-di-GMP-sensing effectors. Mechanistically, trigger PDEs are multifunctional. They directly and specifically interact with a macromolecular target (e.g. with a transcription factor or directly with a promoter region), whose activity they control by their binding and degradation of c-di-GMP-their PDE activity thus represents the c-di-GMP sensor or effector function. In this process, c-di-GMP serves as a regulatory ligand, but in contrast to classical allosteric control, this ligand is also degraded. The resulting kinetics and circuitry of control are ideally suited for trigger PDEs to serve as key components in regulatory switches.This article is part of the themed issue 'The new bacteriology'.

Topics: Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Phosphoric Diester Hydrolases

Bacteria utilize a diverse set of nucleotide second messengers to regulate cellular responses by binding macromolecular receptors (RNAs and proteins). Recent studies on cyclic di-GMP (c-di-GMP) have shown that this signaling molecule binds multiple receptors to regulate different steps in the same biological process. We propose this property of the same molecule regulating multiple steps in the same process is biologically meaningful and have termed this phenomenon 'sustained sensing'. Here, we discuss the recent findings that support the concept of sustained sensing of c-di-GMP levels and provide additional examples that support the utilization of sustained sensing by other second messengers. Sustained sensing may be widespread in bacteria and provides an additional level of complexity in prokaryotic signal transduction networks.

Topics: Bacteria; Bacterial Proteins; Biofilms; Cyclic GMP; Second Messenger Systems

Delivery systems are a powerful technology for enhancing the effect of cancer immunotherapy. We have been in the process of developing lipid-based delivery systems for controlling the physical properties and dynamics of immunofunctional molecules such as antigens and adjuvants. The lipid nanoparticulation of these molecules improves their physical properties, resulting in a good water dispensability, greater stability, and small size. The cell wall skeleton of bacille Calmette-Guerin (BCG-CWS) could be used to replace live BCG as a drug for treating bladder cancer, but problems associated with the physical properties of BCG-CWS have prevented its use. To overcome such problems, we developed a novel packaging method that permits BCG-CWS to be encapsulated into lipid nanoparticles, which induce antitumor responses against bladder cancer. Lipid nanoparticulation also improves the intracellular trafficking and biodistribution of immunofunctional molecules. Cyclic di-GMP (c-di-GMP) is an adjuvant that is recognized by the cytosolic sensor. However, c-di-GMP cannot pass through the cell membrane. We encapsulated c-di-GMP into lipid nanoparticles containing a pH-responsive lipid that was developed in our laboratory and achieved efficient cytosolic delivery and the induction of antitumor immunity. Furthermore, we are attempting to control the functions of immune cells by RNA interference. We have recently succeeded in the efficient delivery of small interfering RNA into mouse dendritic cells (DCs), which led to the enhancement of antitumor activity of DCs. In this review, our recent efforts regarding cancer immunotherapy using lipid-based nanoparticles are reviewed.

Topics: Adjuvants, Immunologic; Animals; Cell Wall Skeleton; Cyclic GMP; Dendritic Cells; Drug Delivery Systems; Drug Design; Humans; Immunotherapy; Lipids; Mice; Mycobacterium bovis; Nanoparticles; Neoplasms; RNA Interference; Urinary Bladder Neoplasms

Cyclic diguanylate monophosphate (c-di-GMP) is a ubiquitous second messenger in bacteria. In this issue of Science Signaling, Xu et al show that c-di-GMP regulates chemotaxis by binding to the PilZ domain protein MapZ to alter the methyltransferase activity of its protein partner CheR, fleshing out the c-di-GMP signaling network of the opportunistic pathogen Pseudomonas aeruginosa.

Topics: Bacterial Proteins; Chemotaxis; Cyclic GMP; Flagella; Movement; Pseudomonas aeruginosa; Second Messenger Systems

Cyclic di-GMP is a second messenger found in almost all eubacteria that acts to regulate a wide range of functions including developmental transitions, adhesion and biofilm formation. Cyclic di-GMP is synthesised from two GTP molecules by diguanylate cyclases that have a GGDEF domain and is degraded by phosphodiesterases with either an EAL or an HD-GYP domain. Proteins with these domains often contain additional signal input domains, suggesting that their enzymatic activity may be modulated as a response to different environmental or cellular cues. Cyclic di-GMP exerts a regulatory action through binding to diverse receptors that include a small protein domain called PilZ, enzymatically inactive GGDEF, EAL or HD-GYP domains, transcription factors and riboswitches. In many bacteria, high cellular levels of cyclic di-GMP are associated with a sessile, biofilm lifestyle, whereas low levels of the nucleotide promote motility and virulence factor synthesis in pathogens. Elucidation of the roles of cyclic di-GMP signalling in biofilm formation has suggested strategies whereby modulation of the levels of the nucleotide or interference with signalling pathways may lead to inhibition of biofilm formation or promotion of biofilm dispersal. In this review we consider these approaches for the control of biofilm formation, beginning with an overview of cyclic di-GMP signalling and the different ways that it can act in regulation of biofilm dynamics.

Topics: Bacteria; Biofilms; Cyclic GMP; Humans; Protein Structure, Tertiary; Signal Transduction

The cyclic dinucleotide c-di-GMP has emerged in the last decade as a prevalent intracellular messenger that orchestrates the transition between the motile and sessile lifestyles of many bacterial species. The motile-to-sessile transition is often associated with the formation of extracellular matrix-encased biofilm, an organized community of bacterial cells that often contributes to antibiotic resistance and host-pathogen interaction. It is increasingly clear that c-di-GMP controls motility, biofilm formation and bacterial pathogenicity partially through regulating the production of exopolysaccharides (EPS) and small-molecule secondary metabolites. This review summarizes our current understanding of the regulation of EPS biosynthesis by c-di-GMP in a diversity of bacterial species and highlights the emerging role of c-di-GMP in the biosynthesis of small-molecule secondary metabolites.

Topics: Biofilms; Cyclic GMP; Molecular Structure; Polysaccharides, Bacterial

Pseudomonas aeruginosa is the most common pathogen that colonizes the lungs of patients with cystic fibrosis. Isolates from sputum are typically all derived from the same strain of bacterium but show extensive phenotypic heterogeneity. One of these variants is the so-called small colony variant, which also shows increased ability to form a biofilm and is frequently resistant to multiple antibiotics. The presence of small colony variants in the sputum of patients with cystic fibrosis is associated with a worse clinical condition. The underlying mechanism responsible for generation of the small colony phenotype remains unclear, but a final common pathway would appear to be elevation of intracellular levels of cyclic di-GMP. This phenotypic variant is thus not just a laboratory curiosity, but a significant bacterial adaptation that favors survival within the lung of patients with cystic fibrosis and contributes to the pulmonary damage caused by P. aeruginosa.

Topics: Anti-Bacterial Agents; Biofilms; Chronic Disease; Cyclic GMP; Cystic Fibrosis; Drug Resistance, Bacterial; Humans; Lung; Phenotype; Pseudomonas aeruginosa; Pseudomonas Infections

The anaerobic Gram-positive bacterium Clostridium difficile causes intestinal infections responsible for symptoms ranging from mild diarrhea to fulminant colitis. Like other bacteria, C. difficile needs to sense and integrate environmental signals in order to adapt to changes and thrive in its environment. The second messenger cyclic diguanosine monophosphate (c-di-GMP) was recently recognized as a quasi-ubiquitous phenotype coordinator in bacteria. Mostly known to be involved in the transition from motile to sessile and multicellular behaviors in Gammaproteobacteria, c-di-GMP is now known to regulate many other phenotypes from cell morphogenesis to virulence, in many Gram-negative and a few Gram-positive bacteria. Herein, we review recent advances in our understanding of c-di-GMP signaling in the lifecycle of C. difficile.

Topics: Bacterial Proteins; Bacterial Toxins; Biofilms; Clostridioides difficile; Cyclic GMP; Enterotoxins; Gene Expression Regulation, Bacterial; Phenotype; Riboswitch; Second Messenger Systems; Virulence

Since its initial discovery as an allosteric factor regulating cellulose biosynthesis in Gluconacetobacter xylinus, the list of functional outputs regulated by c-di-GMP has grown. We have focused this article on one of these c-di-GMP-regulated processes, namely, biofilm formation in the organism Pseudomonas aeruginosa. The majority of diguanylate cyclases and phosphodiesterases encoded in the P. aeruginosa genome still remain uncharacterized; thus, there is still a great deal to be learned about the link between c-di-GMP and biofilm formation in this microbe. In particular, while a number of c-di-GMP metabolizing enzymes have been identified that participate in reversible and irreversible attachment and biofilm maturation, there is a still a significant knowledge gap regarding the c-di-GMP output systems in this organism. Even for the well-characterized Pel system, where c-di-GMP-mediated transcriptional regulation is now well documented, how binding of c-di-GMP by PelD stimulates Pel production is not understood in any detail. Similarly, c-di-GMP-mediated control of swimming, swarming and twitching also remains to be elucidated. Thus, despite terrific advances in our understanding of P. aeruginosa biofilm formation and the role of c-di-GMP in this process since the last version of this book (indeed there was no chapter on c-di-GMP!) there is still much to learn.

Topics: Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa

Aggregative multicellularity, resulting in formation of a spore-bearing fruiting body, evolved at least six times independently amongst both eukaryotes and prokaryotes. Amongst eukaryotes, this form of multicellularity is mainly studied in the social amoeba Dictyostelium discoideum. In this review, we summarise trends in the evolution of cell-type specialisation and behavioural complexity in the four major groups of Dictyostelia. We describe the cell-cell communication systems that control the developmental programme of D. discoideum, highlighting the central role of cAMP in the regulation of cell movement and cell differentiation. Comparative genomic studies showed that the proteins involved in cAMP signalling are deeply conserved across Dictyostelia and their unicellular amoebozoan ancestors. Comparative functional analysis revealed that cAMP signalling in D. discoideum originated from a second messenger role in amoebozoan encystation. We highlight some molecular changes in cAMP signalling genes that were responsible for the novel roles of cAMP in multicellular development.

Topics: Biological Evolution; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Dictyostelium; Genome, Protozoan; Genomics; Hexanones; Histidine Kinase; Protein Kinases; Protozoan Proteins; Quorum Sensing; Signal Transduction

Signal sensing in bacteria has traditionally been attributed to protein-based factors. It is however becoming increasingly clear that bacteria also exploit RNAs to serve this role. This review discusses how key developmental processes in bacteria, such as community formation, choice of a sessile versus motile lifestyle, or vegetative growth versus dormant spore formation may be governed by signal sensing RNAs. The signaling molecules that affect these processes, the RNAs that sense these molecules and the underlying molecular basis for specific signal-response are discussed here.

Topics: Bacteria; Binding Sites; Cyclic GMP; Dinucleoside Phosphates; Molecular Structure; Nucleic Acid Conformation; Riboswitch; RNA, Bacterial; Second Messenger Systems; Signal Transduction

The complex life cycle of streptomycetes involves two distinct filamentous cell forms: the growing (or vegetative) hyphae and the reproductive (or aerial) hyphae, which differentiate into long chains of spores. Until recently, little was known about the signalling pathways that regulate the developmental transitions leading to sporulation. In this Review, we discuss important new insights into these pathways that have led to the emergence of a coherent regulatory network, focusing on the erection of aerial hyphae and the synchronous cell division event that produces dozens of unigenomic spores. In particular, we highlight the role of cyclic di-GMP (c-di-GMP) in controlling the initiation of development, and the role of the master regulator BldD in mediating c-di-GMP signalling.

Topics: Cyclic GMP; Gene Expression Regulation, Bacterial; Models, Biological; Signal Transduction; Spores, Bacterial; Streptomycetaceae; Transcription Factors

In nature, the Lyme disease spirochete Borrelia burgdorferi cycles between the unrelated environments of the Ixodes tick vector and mammalian host. In order to survive transmission between hosts, B. burgdorferi must be able to not only detect changes in its environment, but also rapidly and appropriately respond to these changes. One manner in which this obligate parasite regulates and adapts to its changing environment is through cyclic-di-GMP (c-di-GMP) signaling. c-di-GMP has been shown to be instrumental in orchestrating the adaptation of B. burgdorferi to the tick environment. B. burgdorferi possesses only one set of c-di-GMP-metabolizing genes (one diguanylate cyclase and two distinct phosphodiesterases) and one c-di-GMP-binding PilZ-domain protein designated as PlzA. While studies in the realm of c-di-GMP signaling in B. burgdorferi have exploded in the last few years, there are still many more questions than answers. Elucidation of the importance of c-di-GMP signaling to B. burgdorferi may lead to the identification of mechanisms that are critical for the survival of B. burgdorferi in the tick phase of the enzootic cycle as well as potentially delineate a role (if any) c-di-GMP may play in the transmission and virulence of B. burgdorferi during the enzootic cycle, thereby enabling the development of effective drugs for the prevention and/or treatment of Lyme disease.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Bacterial Proteins; Borrelia burgdorferi; Carrier Proteins; Cyclic GMP; Escherichia coli Proteins; Humans; Lyme Disease; Phosphorus-Oxygen Lyases; Protein Binding; Signal Transduction

Bacteria have evolved to survive the ever-changing environment using intriguing mechanisms of quorum sensing (QS). Very often, QS facilitates formation of biofilm to help bacteria to persist longer and the formation of such biofilms is regulated by c-di-GMP. It is a well-known second messenger also found in mycobacteria. Several methods have been developed to study c-di-GMP signaling pathways in a variety of bacteria. In this review, we have attempted to highlight a connection between c-di-GMP and biofilm formation and QS in mycobacteria and several methods that have helped in better understanding of c-di-GMP signaling.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Humans; Mycobacterium; Quorum Sensing; Second Messenger Systems; Signal Transduction; Virulence

Twenty-five years have passed since the discovery of cyclic dimeric (3'→5') GMP (cyclic di-GMP or c-di-GMP). From the relative obscurity of an allosteric activator of a bacterial cellulose synthase, c-di-GMP has emerged as one of the most common and important bacterial second messengers. Cyclic di-GMP has been shown to regulate biofilm formation, motility, virulence, the cell cycle, differentiation, and other processes. Most c-di-GMP-dependent signaling pathways control the ability of bacteria to interact with abiotic surfaces or with other bacterial and eukaryotic cells. Cyclic di-GMP plays key roles in lifestyle changes of many bacteria, including transition from the motile to the sessile state, which aids in the establishment of multicellular biofilm communities, and from the virulent state in acute infections to the less virulent but more resilient state characteristic of chronic infectious diseases. From a practical standpoint, modulating c-di-GMP signaling pathways in bacteria could represent a new way of controlling formation and dispersal of biofilms in medical and industrial settings. Cyclic di-GMP participates in interkingdom signaling. It is recognized by mammalian immune systems as a uniquely bacterial molecule and therefore is considered a promising vaccine adjuvant. The purpose of this review is not to overview the whole body of data in the burgeoning field of c-di-GMP-dependent signaling. Instead, we provide a historic perspective on the development of the field, emphasize common trends, and illustrate them with the best available examples. We also identify unresolved questions and highlight new directions in c-di-GMP research that will give us a deeper understanding of this truly universal bacterial second messenger.

Topics: Amino Acid Sequence; Animals; Bacteria; Bacterial Physiological Phenomena; Cyclic GMP; Eukaryotic Cells; Gene Expression Regulation, Bacterial; Humans; Models, Molecular; Molecular Sequence Data; Phylogeny; Second Messenger Systems

Signal transduction pathways involving the second messenger cyclic di-GMP [bis-(3'-5')-cyclic di-guanosine monophosphate] occur widely in bacteria where they act to link perception of environmental or intracellular cues and signals to specific alterations in cellular function. Such alterations can contribute to bacterial lifestyle transitions including biofilm formation and virulence. The cellular levels of the nucleotide are controlled through the opposing activities of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs). The GGDEF domain of DGCs catalyses the synthesis of cyclic di-GMP from GTP, whereas EAL or HD-GYP domains in different classes of PDE catalyse cyclic di-GMP degradation to pGpG and GMP. We are now beginning to understand how alterations in cyclic di-GMP exert a regulatory action through binding to diverse receptors or effectors that include a small 'adaptor' protein domain called PilZ, transcription factors and riboswitches. The regulatory action of enzymically active cyclic di-GMP signalling proteins is, however, not restricted to an influence on the level of nucleotide. Here, I will discuss our recent findings that highlight the role that protein-protein interactions involving these signalling proteins have in regulating functions that contribute to bacterial virulence.

Topics: Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Protein Interaction Maps; Signal Transduction; Virulence; Xanthomonas campestris

The Burkholderia cepacia complex (Bcc) consists of 17 closely related species that are problematic opportunistic bacterial pathogens for cystic fibrosis patients and immunocompromised individuals. These bacteria are capable of utilizing two different chemical languages: N-acyl homoserine lactones (AHLs) and cis-2-unsaturated fatty acids. Here we summarize the current knowledge of the underlying molecular architectures of these communication systems, showing how they are interlinked and discussing how they regulate overlapping as well as specific sets of genes. A particular focus is laid on the role of these signaling systems in the formation of biofilms, which are believed to be highly important for chronic infections. We review genes that have been implicated in the sessile lifestyle of this group of bacteria. The new emerging role of the intracellular second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) as a downstream regulator of the fatty acid signaling cascade and as a key factor in biofilm formation is also discussed.

Topics: Acyl-Butyrolactones; Biofilms; Burkholderia cepacia complex; Cyclic GMP; Fatty Acids, Unsaturated; Humans; Quorum Sensing; Signal Transduction; Virulence; Virulence Factors

Cyclic (c-di-GMP) is the prevalent intracellular signaling intermediate in bacteria. It triggers a spectrum of responses that cause bacteria to shift from a swarming motile phase to sessile biofilm formation. However, additional functions for c-di-GMP and roles for related molecules, such as c-di-AMP and c-AMP-GMP continue to be uncovered. The first usage of cyclic-di-nucleotide (c-di-NMP) signaling in the eukaryote domain emerged only recently. In dictyostelid social amoebas, c-di-GMP is a secreted signal that induces motile amoebas to differentiate into sessile stalk cells. In humans, c-di-NMPs, which are either produced endogenously in response to foreign DNA or by invading bacterial pathogens, trigger the innate immune system by activating the expression of interferon genes. STING, the human c-di-NMP receptor, is conserved throughout metazoa and their closest unicellular relatives, suggesting protist origins for human c-di-NMP signaling. Compared to the limited number of conserved protein domains that detect the second messengers cAMP and cGMP, the domains that detect the c-di-NMPs are surprisingly varied.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Dictyostelium; Dinucleoside Phosphates; Humans; Immunity, Innate; Membrane Proteins; Nucleotides, Cyclic; Nucleotidyltransferases; Phylogeny; Protein Structure, Tertiary; Second Messenger Systems; Signal Transduction

Movement over an agar surface via swarming motility is subject to formidable challenges not encountered during swimming. Bacteria display a great deal of flexibility in coping with these challenges, which include attracting water to the surface, overcoming frictional forces, and reducing surface tension. Bacteria that swarm on "hard" agar surfaces (robust swarmers) display a hyperflagellated and hyperelongated morphology. Bacteria requiring a "softer" agar surface (temperate swarmers) do not exhibit such a dramatic morphology. For polarly flagellated robust swarmers, there is good evidence that restriction of flagellar rotation somehow signals the induction of a large number of lateral flagella, but this scenario is apparently not relevant to temperate swarmers. Swarming bacteria can be further subdivided by their requirement for multiple stators (Mot proteins) or a stator-associated protein (FliL), secretion of essential polysaccharides, cell density-dependent gene regulation including surfactant synthesis, a functional chemotaxis signaling pathway, appropriate cyclic (c)-di-GMP levels, induction of virulence determinants, and various nutritional requirements such as iron limitation or nitrate availability. Swarming strategies are as diverse as the bacteria that utilize them. The strength of these numerous designs stems from the vantage point they offer for understanding mechanisms for effective colonization of surface niches, acquisition of pathogenic potential, and identification of environmental signals that regulate swarming. The signature swirling and streaming motion within a swarm is an interesting phenomenon in and of itself, an emergent behavior with properties similar to flocking behavior in diverse systems, including birds and fish, providing a convenient new avenue for modeling such behavior.

Topics: Agar; Bacteria; Bacterial Physiological Phenomena; Bacterial Proteins; Chemotaxis; Culture Media; Cyclic GMP; Flagella; Friction; Membrane Proteins; Quorum Sensing; Surface Tension; Virulence

The secondary messenger cyclic di-GMP coordinately regulates the transition between motility/sessility/virulence in bacterial populations and upon adaptation to novel habitats. Thereby, multiple independent regulatory circuits regulate a diversity of targets. This specific output response is surprising considering the diverse physiological processes regulated by this signalling molecule, which range from transcription to proteolysis and clearly demonstrates the presence of sophisticated developmental programmes in these so-called simple organisms.

Topics: Animals; Bacteria; Bacterial Physiological Phenomena; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Humans; Second Messenger Systems; Signal Transduction

Cyclic dimeric guanosine monophosphate (c-di-GMP) is a common, bacterial second messenger that regulates diverse cellular processes in bacteria. Opposing activities of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) control c-di-GMP homeostasis in the cell. Many microbes have a large number of genes encoding DGCs and PDEs that are predicted to be part of c-di-GMP signaling networks. Other building blocks of these networks are c-di-GMP receptors which sense the cellular levels of the dinucleotide. C-di-GMP receptors form a more diverse family, including various transcription factors, PilZ domains, degenerate DGCs or PDEs, and riboswitches. Recent studies revealing the molecular basis of c-di-GMP signaling mechanisms enhanced our understanding of how this molecule controls downstream biological processes and how c-di-GMP signaling specificity is achieved.

Topics: Bacteria; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Riboswitch; Second Messenger Systems; Signal Transduction

Most bacteria can exist in either a planktonic-motile single-cell state or an adhesive multicellular state known as a biofilm. Biofilms cause medical problems and technical damage since they are resistant against antibiotics, disinfectants or the attacks of the immune system. In recent years it has become clear that most bacteria use cyclic diguanylate (c-di-GMP) as a biofilm-promoting second messenger molecule. C-di-GMP is produced by GGDEF-domain-containing diguanylate cyclases and is degraded by phosphodiesterases featuring EAL or HD-GYP domains. Many bacterial species possess multiple proteins with GGDEF and EAL domains, which actually belong to the most abundant protein families in genomic data bases. Via an unprecedented variety of effector components, which include c-di-GMP-binding proteins as well as RNAs, c-di-GMP controls a wide range of targets that down-regulate motility, stimulate adhesin and biofilm matrix formation or even control virulence gene expression. Moreover, local c-di-GMP signaling in macromolecular complexes seems to allow the independent and parallel control of different output reactions. In this review, we use Escherichia coli as a paradigm for c-di-GMP signaling. Despite the huge diversity of components and molecular processes involved in biofilm formation throughout the bacterial kingdom, c-di-GMP signaling represents a unifying principle, which suggests that the enzymes that make and break c-di-GMP may be promising targets for anti-biofilm drugs.

Topics: Biofilms; Cyclic GMP; Escherichia coli; Second Messenger Systems

An intracellular second messenger unique to bacteria, c-di-GMP, has gained appreciation as a key player in adaptation and virulence strategies, such as biofilm formation, persistence, and cytotoxicity. Diguanylate cyclases containing GGDEF domains and phosphodiesterases containing either EAL or HD-GYP domains have been identified as the enzymes controlling intracellular c-di-GMP levels, yet little is known regarding signal transmission and the sensory targets for this signaling molecule. Although limited in number, identified c-di-GMP receptors in bacteria are characterized by prominent diversity and multilevel impact. In addition, c-di-GMP has been shown to have immunomodulatory effects in mammals and several eukaryotic c-di-GMP sensors have been proposed. The structural biology of c-di-GMP receptors is a rapidly developing field of research, which holds promise for the development of novel therapeutics against bacterial infections. In this review, we highlight recent advances in identifying bacterial and eukaryotic c-di-GMP signaling mechanisms and emphasize the need for mechanistic structure-function studies on confirmed signaling targets.

Topics: Bacteria; Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Models, Molecular; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Conformation; Signal Transduction

Cyclic diguanylate (c-di-GMP) is a ubiquitous second messenger present in a wide variety of bacteria, which is responsible for cell differentiation, biofilm formation, pathogenic factor generation, and so on. The level of c-di-GMP in bacteria is regulated by two opposing active domains, diguanylate cyclase (DGC) and phosphodiesterase (PDE), which are present in the same bifunctional protein, and in charge of the synthesis and the degradation of c-di-GMP, respectively. The target of c-di-GMP in the bacterial cell consists of PilZ domain and GEMM riboswitch, the only riboswitch that involved in signal transduction. This article gives an overview of c-di-GMP, focusing on its metabolic pathway, regulatory mechanism, biological function of c-di-GMP, and the synthesis of c-di-GMP analogues and their biological activity.

Topics: Bacteria; Cyclic GMP; Escherichia coli Proteins; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Riboswitch; Second Messenger Systems; Signal Transduction

Bacteria sense and respond to environmental cues to control important developmental processes. Two widely conserved and important strategies that bacteria employ to sense changes in population density and local environmental conditions are quorum sensing (QS) and cyclic di-GMP (c-di-GMP) signaling, respectively. The importance of these pathways in controlling a broad variety of functions, including virulence, biofilm formation, and motility, has been recognized in many species. Recent research has shown that these pathways are intricately intertwined. Here we review the regulatory connections between QS and c-di-GMP signaling. We propose that the integration of QS with c-di-GMP allows bacteria to assimilate information about the local bacterial population density with other physicochemical environmental signals within the broader c-di-GMP signaling network.

Topics: Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Quorum Sensing; Signal Transduction; Vibrio cholerae; Vibrio parahaemolyticus; Xanthomonas campestris

The Gram-negative opportunistic pathogen Klebsiella pneumoniae is responsible for causing a spectrum of nosocomial and community-acquired infections. Globally, K. pneumoniae is a frequently encountered hospital-acquired opportunistic pathogen that typically infects patients with indwelling medical devices. Biofilm formation on these devices is important in the pathogenesis of these bacteria, and in K. pneumoniae, type 3 fimbriae have been identified as appendages mediating the formation of biofilms on biotic and abiotic surfaces. The factors influencing the regulation of type 3 fimbrial gene expression are largely unknown but recent investigations have indicated that gene expression is regulated, at least in part, by the intracellular levels of cyclic di-GMP. In this review, we have highlighted the recent studies that have worked to elucidate the mechanism by which type 3 fimbrial expression is controlled and the studies that have established the importance of type 3 fimbriae for biofilm formation and nosocomial infection by K. pneumoniae.

Topics: Biofilms; Catheter-Related Infections; Community-Acquired Infections; Cross Infection; Cyclic GMP; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Humans; Klebsiella Infections; Klebsiella pneumoniae; Virulence Factors

Biofilm formation of microorganisms causes persistent tissue and foreign body infections resistant to treatment with antimicrobial agents. Up to 80% of human bacterial infections are biofilm associated; such infections are most frequently caused by Staphylococcus epidermidis, Pseudomonas aeruginosa, Staphylococcus aureus and Enterobacteria such as Escherichia coli. The accurate diagnosis of biofilm infections is often difficult, which prevents the appropriate choice of treatment. As biofilm infections significantly contribute to patient morbidity and substantial healthcare costs, novel strategies to treat these infections are urgently required. Nucleotide second messengers, c-di-GMP, (p)ppGpp and potentially c-di-AMP, are major regulators of biofilm formation and associated antibiotic tolerance. Consequently, different components of these signalling networks might be appropriate targets for antibiofilm therapy in combination with antibiotic treatment strategies. In addition, cyclic di-nucleotides are microbial-associated molecular patterns with an almost universal presence. Their conserved structures sensed by the eukaryotic host have a widespread effect on the immune system. Thus, cyclic di-nucleotides are also potential immunotherapeutic agents to treat antibiotic-resistant bacterial infections.

Topics: Anti-Bacterial Agents; Bacterial Infections; Bacterial Physiological Phenomena; Biofilms; Cyclic GMP; Drug Resistance, Bacterial; Escherichia coli; Humans; Pseudomonas aeruginosa; Second Messenger Systems; Staphylococcus; Therapies, Investigational

The ability of bacteria to adapt to a changing environment is essential for their survival. One mechanism used to facilitate behavioral adaptations is the second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). c-di-GMP is widespread throughout the bacterial domain and plays a vital role in regulating the transition between the motile planktonic lifestyle and the sessile biofilm forming state. This second messenger also controls the virulence response of pathogenic organisms and is thought to be connected to quorum sensing, the process by which bacteria communicate with each other. The intracellular concentration of c-di-GMP is tightly regulated by the opposing enzymatic activities of diguanlyate cyclases and phosphodiesterases, which synthesize and degrade the second messenger, respectively. The change in the intracellular concentration of c-di-GMP is directly sensed by downstream targets of the second messenger, both protein and RNA, which induce the appropriate phenotypic response. This review will summarize our current state of knowledge of c-di-GMP signaling in bacteria with a focus on protein and RNA binding partners of the second messenger. Efforts towards the synthesis of c-di-GMP and its analogs are discussed as well as studies aimed at targeting these macromolecular effectors with chemically synthesized cyclic dinucleotide analogs.

Topics: Bacteria; Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Models, Molecular; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Plankton; Protein Binding; Quorum Sensing; Riboswitch; RNA, Bacterial; Second Messenger Systems; Transcription Factors; Virulence

The c-di-GMP [bis-(3'-5')-cyclic dimeric guanosine monophosphate] riboswitch is a macromolecular target in the c-di-GMP second messenger signalling pathway. It regulates many genes related to c-di-GMP metabolism as well as genes involved in bacterial motility, virulence and biofilm formation. The riboswitch makes asymmetric contacts to the bases and phosphate backbone of this symmetric dinucleotide. The phylogenetics suggested and mutagenesis has confirmed that this is a flexible motif where variants can make alternative interactions with each of the guanine bases of c-di-GMP. A mutant riboswitch has been designed that can bind a related molecule, c-di-AMP, confirming the most important contacts made to the ligand. The binding kinetics reveal that this is a kinetically controlled riboswitch and mutations to the riboswitch lead to increases in the off-rate. This riboswitch is therefore flexible in sequence as well as kinetic properties.

Topics: Animals; Base Sequence; Cyclic GMP; Humans; Ligands; Models, Molecular; Nucleic Acid Conformation; Riboswitch; Second Messenger Systems

In the healthy subgingiva, oral treponemes account for a small percentage of the total bacteria. However, in diseased periodontal pockets, treponemes thrive and become a dominant component of the bacterial population. Oral treponemes are uniquely adept at capitalizing on the environmental conditions that develop with periodontal disease. The molecular basis of adaptive responses of oral treponemes is just beginning to be investigated and defined. The completion of several treponeme genome sequences and the characterization of global regulatory systems provide an important starting point in the analysis of signaling and adaptive responses. In this review, we discuss existing literature focused on the genetic regulatory mechanisms of Treponema denticola and present an overview of the possible roles of regulatory proteins identified through genome analyses. This information provides insight into the possible molecular mechanisms utilized by oral spirochetes to survive in the periodontal pocket and transition from a minor to a dominant organism.

Topics: Adaptation, Physiological; Animals; Bacterial Proteins; Base Sequence; Cyclic GMP; DNA-Directed RNA Polymerases; Gene Expression Regulation, Bacterial; Histidine Kinase; Humans; Periodontal Pocket; Protein Kinases; Sigma Factor; Signal Transduction; Treponema denticola

Genome sequencing has produced thousands of nonprotein coding (nc)RNA sequences including new ribozymes and riboswitches. Such RNAs are notable for their extraordinary functionality, which entails exquisite folding that culminates in biocatalytic or ligand-binding capabilities. Here we discuss advances in relating ncRNA form to function with an emphasis on base pK(a) shifting by the hairpin and hepatitis delta virus ribozymes. We then describe ligand binding by the two smallest riboswitches, which target preQ(1) and S-adenosyl-(l)-homocysteine, followed by an analysis of a second-messenger riboswitch that binds cyclic-di-GMP. Each riboswitch is then compared to a protein that binds the same ligand to contrast binding properties. The results showcase the breadth of functionality attainable from ncRNAs, as well as molecular features notable for antibacterial design.

Topics: Cyclic GMP; Gene Expression Regulation; Hydrogen Bonding; Ligands; Metals; Riboswitch; RNA, Catalytic; Second Messenger Systems; Substrate Specificity

Cyclic-di-GMP (c-di-GMP) regulates many important bacterial processes. Freely diffusible intracellular c-di-GMP is determined by the action of metabolizing enzymes that allow integration of numerous input signals. c-di-GMP specifically regulates multiple cellular processes by binding to diverse target molecules. This review highlights important questions in research into the mechanisms of c-di-GMP signalling and its role in bacterial physiology.

Topics: Adaptation, Physiological; Bacterial Physiological Phenomena; Cyclic GMP; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Models, Biological; Second Messenger Systems

Topics: Bacterial Proteins; Binding Sites; Cyclic GMP; Energy Transfer; Flagella; Ions; Molecular Motor Proteins; Multiprotein Complexes; Protein Binding; Torque

Bacteria can switch between planktonic forms (single cells) and biofilms, i.e., bacterial communities growing on solid surfaces and embedded in a matrix of extracellular polymeric substance. Biofilm formation by pathogenic bacteria often results in lower susceptibility to antibiotic treatments and in the development of chronic infections; thus, biofilm formation can be considered an important virulence factor. In recent years, much attention has been directed towards understanding the biology of biofilms and towards searching for inhibitors of biofilm development and of biofilm-related cellular processes. In this report, we review selected examples of target-based screening for anti-biofilm agents: We focus on inhibitors of quorum sensing, possibly the most characterized target for molecules with anti-biofilm activity, and on compounds interfering with the metabolism of the signal molecule cyclic di-GMP metabolism and on inhibitors of DNA and nucleotide biosynthesis, which represent a novel and promising class of biofilm inhibitors. Finally, we discuss the activation of biofilm dispersal as a novel mode of action for anti-biofilm compounds.

Topics: Anti-Bacterial Agents; Bacteria; Biofilms; Cyclic GMP; DNA; Nucleotides; Quorum Sensing

3', 5'-Cyclic diguanylic acid (c-di-GMP) is a bacterial intracellular signaling molecule that plays a crucial role in the regulation of bacterial motility, adhesion, cell-to-cell communication, exopolysaccharide synthesis, biofilm formation and virulence. The recent finding that c-di-GMP can act as a danger signal on eukaryotic cells has prompted the study of the immunostimulatory and immunomodulatory properties of c-di-GMP in an effort to determine whether c-di-GMP might be further developed as a potential vaccine adjuvant. In this review, we discussed the recent in vitro and in vivo studies of the immunostimulatory properties of c-di-GMP and the progress that has been made in the preclinical development of c-di-GMP as a potential vaccine adjuvant for systemic and mucosal vaccination.

Topics: Adjuvants, Immunologic; Animals; Cyclic GMP; Humans; Vaccines

3',5'-Cyclic diguanylic acid (c-di-GMP) is a naturally occurring small cyclic dinucleotide found in bacteria. There has been a recent surge of interest in the two-component signalling networks involving this molecule. This tutorial review introduces the biosynthesis of c-di-GMP, particularly the conserved domain features involved in its enzymatic synthesis and degradation, cellular functions and phenotypes regulated by c-di-GMP through c-di-GMP-binding proteins. The chemical synthesis and structural studies of c-di-GMP are also summarized. Two potential applications of c-di-GMP, i.e. bacterial biofilm formation and immunostimulation, are surveyed. Recent observations on c-di-GMP-binding riboswitches are also introduced.

Topics: Animals; Bacterial Proteins; Biofilms; Cyclic GMP; Humans; Signal Transduction

Small molecules that can attenuate bacterial toxin production or biofilm formation have the potential to solve the bacteria resistance problem. Although several molecules, which inhibit bacterial cell-to-cell communication (quorum sensing), biofilm formation and toxin production, have been discovered, there is a paucity of US FDA-approved drugs that target these processes. Here, we review the current understanding of quorum sensing in important pathogens such as Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus and provide examples of experimental molecules that can inhibit both known and unknown targets in bacterial virulence factor production and biofilm formation. Structural data for protein targets that are involved in both quorum sensing and cyclic diguanylic acid signaling are needed to aid the development of molecules with drug-like properties in order to target bacterial virulence factors production and biofilm formation.

Topics: Animals; Anti-Bacterial Agents; Bacteria; Bacterial Infections; Bacterial Physiological Phenomena; Biofilms; Cyclic GMP; Drug Discovery; Humans; Quorum Sensing; Signal Transduction; Small Molecule Libraries

On the stage of bacterial signal transduction and regulation, bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) has long played the part of Sleeping Beauty. c-di-GMP was first described in 1987, but only recently was it recognized that the enzymes that 'make and break' it are not only ubiquitous in the bacterial world, but are found in many species in huge numbers. As a key player in the decision between the motile planktonic and sedentary biofilm-associated bacterial 'lifestyles', c-di-GMP binds to an unprecedented range of effector components and controls diverse targets, including transcription, the activities of enzymes and larger cellular structures. This Review focuses on emerging principles of c-di-GMP signalling using selected systems in different bacteria as examples.

Topics: Bacteria; Cyclic GMP; Escherichia coli Proteins; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Signal Transduction

In all domains of life nucleotide-based second messengers transduce signals originating from changes in the environment or in intracellular conditions into appropriate cellular responses. In prokaryotes cyclic di-GMP has emerged as an important and ubiquitous second messenger regulating bacterial life-style transitions relevant for biofilm formation, virulence, and many other bacterial functions. This review describes similarities and differences in the architecture of the cAMP, (p)ppGpp, and c-di-GMP signaling systems and their underlying signaling principles. Moreover, recent advances in c-di-GMP-mediated signaling will be presented and the integration of c-di-GMP signaling with other nucleotide-based signaling systems will be discussed.

Topics: Bacterial Physiological Phenomena; Cyclic AMP; Cyclic GMP; Guanosine Pentaphosphate; Second Messenger Systems

Cyclic (5 to 3 )-diguanosine monophosphate (c-di-GMP) is a small molecule that regulates the transition between the sessile and motile lifestyle, an integrative part of biofilm formation and other multicellular behavior, in many bacteria. The recognition of c-di-GMP as a novel secondary messenger soon raised the question about the specificity of the signaling system, as individual bacterial genomes frequently encode numerous c-di-GMP metabolizing proteins. Recent work has demonstrated that several global regulators concertedly modify the expression of selected panels of c-di-GMP metabolizing proteins, which act on targets with physiological functions. Within complex feed-forward arrangements, the global regulators commonly combine the control of c-di-GMP metabolism with the direct regulation of proteins with functions in motility or biofilm formation, leading to precise and fine-tuned output responses that determine bacterial behavior. c-di-GMP metabolizing proteins are also controlled at the post-translational level by mechanisms including phosphorylation, localization, protein-protein interactions or protein stability. A detailed understanding of such complex regulatory mechanisms will not only help to explain the specificity in c-di-GMP signaling systems, but will also be necessary to understand the high phenotypic diversity within bacterial biofilms at the single cell level.

Topics: Biofilms; Cyclic GMP; Enzyme Activation; Enzymes; Flagella; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Phosphorylation; Protein Processing, Post-Translational; Quorum Sensing; RNA Processing, Post-Transcriptional; RNA Stability; Second Messenger Systems

Recently, the list of ubiquitous bacterial secondary messengers which include cAMP and ppGpp has been extended by 3',5'-cyclic diguanylic acid (c-di-GMP). C-di-GMP metabolism is tuned by the tightly controlled activity of diguanylate cyclases and c-di-GMP-specific phosphodiesterases. As c-di-GMP-metabolizing enzymes are not only found frequently in bacterial genomes, but also are often numerous in individual genomes, the c-di-GMP metabolic network is highly complex whereby signaling specificity is adjusted on the level of expression, enzymatic activity, protein localization and, most likely, receptor affinity. The targets of c-di-GMP, which include protein and RNA receptors, are subsequently being unraveled. Besides the transition between sessility and motility, probably the most ancient regulatory control of bacterial behavior by c-di-GMP, many more phenotypes such as virulence are affected by c-di-GMP. However, the exact molecular mechanisms of c-di-GMP action remain to be discovered.

Topics: Bacterial Physiological Phenomena; Binding Sites; Cyclic GMP; Genome, Bacterial; Phenotype; Signal Transduction; Virulence

Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a ubiquitous second messenger that regulates cell surface-associated traits in bacteria. Components of this regulatory network include GGDEF and EAL domain-containing proteins that determine the cellular concentrations of c-di-GMP by mediating its synthesis and degradation, respectively. Crystal structure analyses in combination with functional studies have revealed the catalytic mechanisms and regulatory principles involved. Downstream, c-di-GMP is recognized by PilZ domain-containing receptors that can undergo large-scale domain rearrangements on ligand binding. Here, we review recent data on the structure and functional properties of the protein families that are involved in c-di-GMP signalling and discuss the mechanistic implications.

Topics: Bacteria; Bacterial Proteins; Cyclic GMP; Phosphoric Diester Hydrolases; Protein Binding; Signal Transduction

It is now clear that cell-cell communication, often referred to as quorum sensing (QS), is the norm in the prokaryotic kingdom and this community-wide genetic regulatory mechanism has been adopted for regulation of many important biological functions. Since the 1980s, several types of QS signals have been identified, which are associated commonly with different types of QS mechanisms. Among them, the diffusible signal factor (DSF)-dependent QS system, originally discovered from bacterial pathogen Xanthomonas campestris pv. campestris, is a relatively new regulatory mechanism. The rapid research progress over the last few years has identified the chemical structure of the QS signal DSF, established the DSF regulon, and unveiled the general signaling pathways and mechanisms. Particular noteworthy are that DSF biosynthesis is modulated by a novel posttranslational autoinduction mechanism involving protein-protein interaction between the DSF synthase RpfF and the sensor RpfC, and that QS signal sensing is coupled to intracellular regulatory networks through a second messenger cyclic-di-GMP and a global regulator Clp. Genomic and genetic analyses show that the DSF QS-signaling pathway regulates diverse biological functions including virulence, biofilm dispersal, and ecological competence. Moreover, evidence is emerging that the DSF QS system is conserved in a range of plant and human bacterial pathogens.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Cyclic GMP; Quorum Sensing; Virulence; Virulence Factors; Xanthomonas campestris

Topics: Bacterial Physiological Phenomena; Cyclic GMP; Flagella; Gene Expression Regulation, Bacterial; Locomotion

It is now apparent that the signaling molecule 3',5'-cyclic diguanylic acid (c-di-GMP) is a central regulator of the prokaryote biofilm lifestyle and recent evidence also links this molecule to virulence. Environmentally responsive signal transduction systems that control expression and/or activity of the enzymes (GGDEF and EAL domain containing proteins) that are responsible for synthesis and degradation of c-di-GMP have recently been identified. Members of the phosphorelay family feature prominently amongst these systems, which include several with hybrid polydomain sensors and one that is similar to well-characterized chemotaxis-controlling pathways. These findings support the hypothesis that c-di-GMP levels are tightly controlled in response to a broad range, in terms of both diversity and intensity, of extracellular signals. Insight into how c-di-GMP affects changes in gene expression and/or protein activity has come from the demonstration that proteins containing the PilZ domain can bind c-di-GMP and control phenotypes involved in biofilm formation and virulence. These recent developments should pave the way for researchers to answer the important question of how a vast array of extracellular signals that are sensed by multiple sensory transduction pathways which all lead to the production or destruction of c-di-GMP are coordinated such that the appropriate phenotypic response is produced.

Topics: Adaptation, Physiological; Bacteria; Bacterial Physiological Phenomena; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Signal Transduction; Virulence

Cyclic diguanylate (c-di-GMP) is a bacterial second messenger of growing recognition involved in the regulation of a number of complex physiological processes. This review describes the biosynthesis and hydrolysis of c-di-GMP and several mechanisms of regulation of c-di-GMP metabolism. The contribution of c-di-GMP to regulating biofilm formation and motility, processes that affect pathogenesis of many bacteria, is described, as is c-di-GMP regulation of virulence gene expression. Finally, ways in which c-di-GMP may mediate these regulatory effects are proposed.

Topics: Bacteria; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Hydrolysis; Movement; Protein Structure, Tertiary; Virulence

Bacteria use diverse small molecules for extra- and intracellular signaling. They scan small-molecule mixtures to access information about both their extracellular environment and their intracellular physiological status, and based on this information, they continuously interpret their circumstances and react rapidly to changes. Bacteria must integrate extra- and intracellular signaling information to mount appropriate responses to changes in their environment. We review recent research into two fundamental bacterial small-molecule signaling pathways: extracellular quorum-sensing signaling and intracellular cyclic dinucleotide signaling. We suggest how these two pathways may converge to control complex processes including multicellularity, biofilm formation, and virulence. We also outline new questions that have arisen from recent studies in these fields.

Topics: 4-Butyrolactone; Bacterial Physiological Phenomena; Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genes, Bacterial; Homoserine; Lactones; Models, Biological; Oligopeptides; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Purine Nucleotides; Quinolones; Second Messenger Systems; Signal Transduction; Virulence

Topics: Biofilms; Cyclic AMP; Cyclic GMP; Escherichia coli; Flagella; Gene Expression Regulation, Bacterial; Transcription, Genetic

Cyclic-di-GMP is a ubiquitous second messenger in bacteria. The recent discovery that c-di-GMP antagonistically controls motility and virulence of single, planktonic cells on one hand and cell adhesion and persistence of multicellular communities on the other has spurred interest in this regulatory compound. Cellular levels of c-di-GMP are controlled through the opposing activities of diguanylate cyclases and phosphodiesterases, which represent two large families of output domains found in bacterial one- and two-component systems. This review concentrates on structural and functional aspects of diguanylate cyclases and phosphodiesterases, and on their role in transmitting environmental stimuli into a range of different cellular functions. In addition, we examine several well-established model systems for c-di-GMP signaling, including Pseudomonas, Vibrio, Caulobacter, and Salmonella.

Topics: Bacteria; Caulobacter crescentus; Cyclic GMP; Escherichia coli Proteins; Gluconacetobacter xylinus; Glucose; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Structure, Tertiary; Pseudomonas; Second Messenger Systems; Structure-Activity Relationship; Vibrio cholerae

The synthesis of virulence factors in the plant pathogen Xanthomonas campestris pathovar campestris is regulated by cell-cell signaling mediated by a diffusible signal factor (DSF), and by the RpfC/RpfG two-component regulatory system. Recent findings have indicated that the perception of the DSF signal requires the RpfC sensor and is linked to the degradation of the intracellular second messenger cyclic di-GMP by the HD-GYP domain regulator RpfG.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Bacterial Proteins; Cyclic GMP; Plants; Protein Structure, Tertiary; Quorum Sensing; Second Messenger Systems; Signal Transduction; Virulence Factors; Xanthomonas campestris

Cyclic di-GMP is an almost ubiquitous second messenger in bacteria that was first described as an allosteric activator of cellulose synthase but is now known to regulate a range of functions, including virulence in human and animal pathogens. Two protein domains, GGDEF and EAL, are implicated in the synthesis and degradation, respectively, of cyclic di-GMP. These domains are widely distributed in bacteria, including plant pathogens. The majority of proteins with GGDEF and EAL domains contain additional signal input domains, suggesting that their activities are responsive to environmental cues. Recent studies have demonstrated that a third domain, HD-GYP, is also active in cyclic di-GMP degradation. In the plant pathogen Xanthomonas campestris pv. campestris, a two-component signal transduction system comprising the HD-GYP domain regulatory protein RpfG and cognate sensor RpfC positively controls virulence. The signals recognized by RpfC may include the cell-cell signal DSF, which also acts to regulate virulence in X. campestris pv. campestris. Here, we review these recent advances in our understanding of cyclic di-GMP signaling with particular reference to one or more roles in the bacterial pathogenesis of plants.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Bacterial Proteins; Cyclic GMP; Plants; Protein Structure, Tertiary; Signal Transduction; Xanthomonas campestris; Xylella

The rdar morphotype, a multicellular behaviour of Salmonella enterica and Escherichia coli is characterized by the expression of the adhesive extracellular matrix components cellulose and curli fimbriae. The response regulator CsgD, which transcriptionally activates the biosynthesis of the exopolysaccharide cellulose and curli, also transforms cell physiology to the multicellular state. However, the only role of CsgD in cellulose biosynthesis is the activation of AdrA, a GGDEF domain protein that mediates production of the allosteric activator cyclic-di-(3'-5')guanylic acid (c-di-GMP). In S. enterica serovar Typhimurium a regulatory network consisting of 19 GGDEF/EAL domain-containing proteins tightly controls the concentration of c-di-GMP. c-di-GMP not only regulates the expression of cellulose, but also stimulates expression of adhesive curli and represses various modes of motility. Functions of characterized GGDEF and EAL domain proteins, as well as database searches, point to a global role for c-di-GMP as a novel secondary messenger that regulates a variety of cellular functions in response to diverse environmental stimuli already in the deepest roots of the prokaryotes.

Topics: Animals; Biofilms; Cellulose; Cyclic GMP; Enterobacteriaceae; Escherichia coli Proteins; Humans; Models, Biological; Phylogeny; Trans-Activators

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) has come to the limelight as a result of the recent advances in microbial genomics and increased interest in multicellular microbial behaviour. Known for more than 15 years as an activator of cellulose synthase in Gluconacetobacter xylinus, c-di-GMP is emerging as a novel global second messenger in bacteria. The GGDEF and EAL domain proteins involved in c-di-GMP synthesis and degradation, respectively, are (almost) ubiquitous in bacterial genomes. These proteins affect cell differentiation and multicellular behaviour as well as interactions between the microorganisms and their eukaryotic hosts and other phenotypes. While the role of GGDEF and EAL domain proteins in bacterial physiology and behaviour has gained appreciation, and significant progress has been achieved in understanding the enzymology of c-di-GMP turnover, many questions regarding c-di-GMP-dependent signalling remain unanswered. Among these, the key questions are the identity of targets of c-di-GMP action and mechanisms of c-di-GMP-dependent regulation. This review discusses phylogenetic distribution of the c-di-GMP signalling pathway in bacteria, recent developments in biochemical and structural characterization of proteins involved in its metabolism, and biological processes affected by c-di-GMP. The accumulated data clearly indicate that a novel ubiquitous signalling system in bacteria has been discovered.

Topics: Bacteria; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Second Messenger Systems

The cyclic nucleotide cyclic di-guanosine-monophosphate (c-diGMP) was recognized in the 1980s as a signaling compound that is involved in controlling the condensation of glucose moieties into cellulose polymers. More recent data from several different bacterial species now suggest that c-diGMP might have a general role as secondary messenger in modulating bacterial growth on surfaces by regulating cellular adhesion components and preparing cells for cell-cell and cell-surface interactions.

Topics: Bacteria; Bacterial Adhesion; Bacterial Proteins; Cyclic GMP; Membrane Proteins; Second Messenger Systems

Pseudomonas aeruginosa is an ubiquitous environmental bacterium and an opportunistic human pathogen. Not only in most natural habitats but also within the human host, e.g. within the chronically infected cystic fibrosis lung, P. aeruginosa is associated with surfaces in structures known as biofilms. These functional communities represent a unique mode of bacterial growth where bacteria display particular phenotypes that are fundamentally different from planktonic cells. In this review the issue of the molecular mechanisms underlying the emergence of small colony variant (SCV) P. aeruginosa morphotypes that are especially capable of forming biofilms is addressed. It is assumed that the expression of the chaperone usher pathway (cup) genes encoding putative fimbrial adhesins is responsible for the phenotypic switch to an autoaggregative SCV phenotype. The elucidation of phenotypic switching in response to environmental stimuli will significantly increase our understanding of regulatory processes during bacterial adaptation and might be the basis for the initiation of the development of new antimicrobial treatment strategies.

Topics: Adhesins, Bacterial; Biofilms; Cyclic GMP; Environment; Gene Expression; Models, Biological; Phenotype; Pseudomonas aeruginosa; Species Specificity

Bacterial signalling network includes an array of numerous interacting components that monitor environmental and intracellular parameters and effect cellular response to changes in these parameters. The complexity of bacterial signalling systems makes comparative genome analysis a particularly valuable tool for their studies. Comparative studies revealed certain general trends in the organization of diverse signalling systems. These include (i) modular structure of signalling proteins; (ii) common organization of signalling components with the flow of information from N-terminal sensory domains to the C-terminal transmitter or signal output domains (N-to-C flow); (iii) use of common conserved sensory domains by different membrane receptors; (iv) ability of some organisms to respond to one environmental signal by activating several regulatory circuits; (v) abundance of intracellular signalling proteins, typically consisting of a PAS or GAF sensor domains and various output domains; (vi) importance of secondary messengers, cAMP and cyclic diguanylate; and (vii) crosstalk between components of different signalling pathways. Experimental characterization of the novel domains and domain combinations would be needed for achieving a better understanding of the mechanisms of signalling response and the intracellular hierarchy of different signalling pathways.

Topics: Adenylyl Cyclases; Bacteria; Bacterial Physiological Phenomena; Cyclic AMP; Cyclic GMP; Environment; Escherichia coli Proteins; Genome, Bacterial; Histidine Kinase; Phosphoprotein Phosphatases; Phosphorus-Oxygen Lyases; Protein Kinases; Protein Structure, Tertiary; Second Messenger Systems; Signal Transduction

Environmental signals trigger changes in the bacterial cell surface, including changes in exopolysaccharides and proteinaceous appendages that ultimately favour bacterial persistence and proliferation. Such adaptations are regulated in diverse bacteria by proteins with GGDEF and EAL domains. These proteins are predicted to regulate cell surface adhesiveness by controlling the level of a second messenger, the cyclic dinucleotide c-di-GMP. Genetic evidence suggests that the GGDEF domain acts as a nucleotide cyclase for c-di-GMP synthesis while the EAL domain is a good candidate for the opposing activity, a phosphodiesterase for c-di-GMP degradation.

Topics: Animals; Bacteria; Bacterial Adhesion; Bacterial Proteins; Cell Communication; Cyclic GMP; Gluconacetobacter xylinus; Plants; Protein Structure, Tertiary; Second Messenger Systems

In an effort to explore strategies to control Clostridium perfringens, we investigated the synergistic effect of a ubiquitous bacterial second messenger 3',5'-cyclic diguanylic acid (c-di-GMP) with penicillin G in a broiler challenge model. All chicks were inoculated in the crop by gavage on d 14, 15, and 16 with a mixture of 4 C. perfringens strains. Birds were treated with saline (control group) or 20 nmol of c-di-GMP by gavage or intramuscularly (IM) on d 24, all in conjunction with penicillin G in water for 5 d. Weekly samplings of ceca and ileum were performed on d 21 to 35 for C. perfringens and Lactobacillus enumeration. On d 35 of age, the IM treatment significantly (P < 0.05) reduced C. perfringens in the ceca, suggesting possible synergistic activity between penicillin G and c-di-GMP against C. perfringens in broiler ceca. Moreover, analysis of ceca DNA for the presence of a series of C. perfringens virulence genes showed a prevalence of 30% for the Clostridium perfringens alpha-toxin gene (cpa) from d 21 to 35 in the IM-treated group, whereas the occurrence of the cpa gene increased from 10 to 60% in the other 2 groups (control and gavage) from d 21 to 35. Detection of β-lactamase genes (blaCMY-2, blaSHV, and blaTEM) indicative of gram-negative bacteria in the same samples from d 21 to 35 did not show significant treatment effects. Amplified fragment-length polymorphism showed a predominant 92% similarity between the ceca of 21-d-old control birds and the 35-d-old IM-treated c-di-GMP group. This suggests that c-di-GMP IM treatment might be effective at restoring the normal microflora of the host on d 35 after being challenged by C. perfringens. Our results suggest that c-di-GMP can reduce the colonization of C. perfringens in the gut without increasing the selection pressure for some β-lactamase genes or altering the commensal bacterial population.

Topics: Adjuvants, Immunologic; Animal Feed; Animals; Anti-Bacterial Agents; Bacterial Toxins; Cecum; Chickens; Clostridium Infections; Clostridium perfringens; Colony Count, Microbial; Cyclic GMP; Enteral Nutrition; Enteritis; Injections, Intramuscular; Male; Penicillin G; Polymerase Chain Reaction; Poultry Diseases

The ability of Pseudomonas aeruginosa to establish chronic infections is associated with an effective switch from a motile to a sessile lifestyle. This proficiency is controlled by intracellular levels of the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Targeting the c-di-GMP network could be a strategy to interfere with P. aeruginosa pathogenicity. Therefore, the development of tools to profile c-di-GMP intracellular levels is crucial. Here, we describe a protocol for the in vivo measurement of c-di-GMP levels in P. aeruginosa.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Fluorescence; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa; Second Messenger Systems

C-di-GMP is a bacterial second messenger implicated in the regulation of many key functions including antibiotic tolerance and biofilm formation. Our understanding of how c-di-GMP exerts its action via receptors to modulate different biological functions is still limited. Here we used a c-di-GMP affinity pull-down assay coupled to LC-MS/MS to identify c-di-GMP-binding proteins in the opportunistic pathogen Stenotrophomonas maltophilia. This analysis identified Smlt3238 (SodA), a protein of the superoxide dismutase family, as a c-di-GMP-binding protein. Microscale thermophoresis showed that purified SodA protein bound c-di-GMP with an estimated dissociation constant (Kd) value of 141.5 μM. Using various in vivo and in vitro experiments, we demonstrated that c-di-GMP modulates the enzyme activity of SodA directly. Circular dichroism experiments revealed that SodA protein gradually altered its basic structure with increasing levels of c-di-GMP. Phenotypic experiments conducted in the presence of a range of intracellular c-di-GMP levels showed that SodA function is modulated by c-di-GMP. The findings thus identify a novel c-di-GMP binding protein that governs oxidative stress tolerance in S. maltophilia.

Topics: Bacterial Proteins; Biofilms; Carrier Proteins; Chromatography, Liquid; Cyclic GMP; Gene Expression Regulation, Bacterial; Oxidative Stress; Stenotrophomonas maltophilia; Superoxide Dismutase; Tandem Mass Spectrometry

As a central signaling molecule, c-di-GMP (bis-(3,5)-cyclic diguanosine monophosphate) is becoming the focus for research in bacteria physiology. Pseudomonas aeruginosa PAO1 genome contains highly complicated c-di-GMP metabolizing genes and a number of these proteins have been identified and investigated. Especially, a sophisticated network of these proteins is emerging. In current study, mainly through Bacteria-2-Hybrid assay, we found PA0575 (RmcA), a GGDEF-EAL dual protein, to interact with two other dual proteins of PA4601 (MorA) and PA4959 (FimX). These observations imply the intricacy of c-di-GMP metabolizing protein interactions. Our work thus provides one piece of data to increase the understandings to c-di-GMP signaling.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa

Vibrio splendidus, a gram-negative bacterium that is ubiquitously present in marine environments, has been increasingly deemed an important opportunistic pathogen of marine animals. In this study, the biofilm formation of V. splendidus was quantitatively determined and morphologically characterized. Three stages of biofilm formation, including adhesion, aggregation and maturation were observed in the biofilm formed by V. splendidus. The inhibitory effect of exogenous bis (3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) on the biofilm formation from the scratch and preformed established biofilms of V. splendidus was determined. When 200 μmol/L c-di-GMP was added, the quantity of biofilm decreased by 88.1% or 66.7% under the two conditions. To explore the preliminary mechanism of exogenous c-di-GMP on the biofilm formed by V. splendidus, proteomic analysis was performed. GO enrichment analysis showed that exogenous c-di-GMP upregulated biological processes, including the tricarboxylic acid cycle, oxidation‒reduction reactions and organonitrogen compound catabolism and significantly downregulated tRNA threonylcarbamoyladenosine modification, protein dephosphorylation, and lactate transmembrane transporter activity. Sequence-specific DNA binding activity was the most markedly downregulated molecular function. KEGG analysis showed that the valine, leucine and isoleucine degradation pathway was the most enriched pathway, followed by nitrogen metabolism, among the 20 upregulated pathways. Among the downregulated pathways, a nonribosomal peptide structure pathway and the streptomycine, polyketide sugar unit, acarbose and validamycin biosynthesis pathways were significantly enriched. Our present study provides basic data for the biofilm formation of V. splendidus and the preliminary inhibitory mechanism of exogenous c-di-GMP on the biofilm formation of V. splendidus.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Proteomics; Vibrio cholerae

The opportunistic pathogen Pseudomonas aeruginosa is an environmental microorganism and is a model organism for biofilm research. Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that plays critical roles in biofilm formation. P. aeruginosa contains approximately 40 genes that encode enzymes that participate in the metabolism of c-di-GMP (biosynthesis or degradation), yet it lacks tools that aid investigation of the systematic expression pattern of those genes. In this study, we constructed a promoter-

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli; Gene Expression Regulation, Bacterial; Humans; Promoter Regions, Genetic; Pseudomonas aeruginosa

Biofilms consist of single or multiple species of bacteria embedded in extracellular polymeric substances (EPSs), which affect the increase in antibiotic resistance by restricting the transport of antibiotics to the bacterial cells. An alternative approach to treatment with antimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In this study, we found that citrus peel extract from Jeju Island (CPEJ) can inhibit bacterial biofilm formation. According to the results, CPEJ concentration-dependently reduces biofilm formation without affecting bacterial growth. Additionally, CPEJ decreased the production of extracellular polymeric substances but increased bacterial swarming motility. These results led to the hypothesis that CPEJ can reduce intracellular bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) concentration. The results showed that CPEJ significantly reduced the c-di-GMP level through increased phosphodiesterase activity. Altogether, these findings suggest that CPEJ as a biofilm inhibitor has new potential for pharmacological (e.g. drug and medication) and industrial applications (e.g. ship hulls, water pipes, and membrane processes biofouling control).

Topics: Bacteria; Bacterial Proteins; Biofilms; Cyclic GMP

Bacterial second messengers c-di-GMP and (p)ppGpp have broad functional repertoires ranging from growth and cell cycle control to the regulation of biofilm formation and virulence. The recent identification of SmbA, an effector protein from Caulobacter crescentus that is jointly targeted by both signaling molecules, has opened up studies on how these global bacterial networks interact. C-di-GMP and (p)ppGpp compete for the same SmbA binding site, with a dimer of c-di-GMP inducing a conformational change that involves loop 7 of the protein that leads to downstream signaling. Here, we report a crystal structure of a partial loop 7 deletion mutant, SmbA

Topics: Bacterial Proteins; Cyclic GMP; Dimerization; Guanosine Pentaphosphate; Ligands

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule. It is also a critical player in the regulation of cell size and cell behaviors such as cell aggregation and phototaxis in cyanobacteria, which constitute an important group of prokaryotes for their roles in the ecology and evolution of the Earth. However, c-di-GMP receptors have never been revealed in cyanobacteria. Here, we report the identification of a c-di-GMP receptor, CdgR, from the filamentous cyanobacterium

Topics: Bacterial Proteins; Cyanobacteria; Cyclic GMP; Gene Expression Regulation; Gene Expression Regulation, Bacterial; Signal Transduction

c-di-GMP primarily controls motile to sessile transitions in bacteria. Diguanylate cyclases (DGCs) catalyze the synthesis of c-di-GMP from two GTP molecules. Typically, bacteria encode multiple DGCs that are activated by specific environmental signals. Their catalytic activity is modulated by c-di-GMP binding to autoinhibitory sites (I-sites). YfiN is a conserved inner membrane DGC that lacks these sites. Instead, YfiN activity is directly repressed by periplasmic YfiR, which is inactivated by redox stress. In Escherichia coli, an additional envelope stress causes YfiN to relocate to the mid-cell to inhibit cell division by interacting with the division machinery. Here, we report a third activity for YfiN in E. coli, where cell growth is inhibited without YfiN relocating to the division site. This action of YfiN is only observed when the bacteria are cultured on gluconeogenic carbon sources, and is dependent on absence of the autoinhibitory sites. Restoration of I-site function relieves the growth-arrest phenotype, and disabling this function in a heterologous DGC causes acquisition of this phenotype. Arrested cells are tolerant to a wide range of antibiotics. We show that the likely cause of growth arrest is depletion of cellular GTP from run-away synthesis of c-di-GMP, explaining the dependence of growth arrest on gluconeogenic carbon sources that exhaust more GTP during production of glucose. This is the first report of c-di-GMP-mediated growth arrest by altering metabolic flow.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Guanosine Triphosphate; Phosphorus-Oxygen Lyases; Second Messenger Systems

The bacterial signaling molecule cyclic diguanosine monophosphate (c-di-GMP) plays a key role in controlling biofilm formation and pathogenic virulence, among many other functions. It has widespread consequences for human health, and current research is actively exploring its molecular mechanisms. The convenient one-flask, gram-scale synthesis of c-di-GMP described here has facilitated these efforts and has been applied to a variety of analogs. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Topics: Bacteria; Bacterial Proteins; Cyclic GMP; Humans; Signal Transduction

A spectrophotometric method to measure hydrolysis of the bacterial second messenger cyclic dimeric guanosine monophosphate is described for characterization of enzymes under aerobic and anaerobic conditions. The method allows for obtaining all necessary data to calculate K

Topics: Cyclic GMP; Hydrolysis; Oxygen; Second Messenger Systems

Curli amyloid fibers are a major constituent of the extracellular biofilm matrix formed by bacteria of the Enterobacteriaceae family. Within Escherichia coli biofilms, curli gene expression is limited to a subpopulation of bacteria, leading to heterogeneity of extracellular matrix synthesis. Here we show that bimodal activation of curli gene expression also occurs in well-mixed planktonic cultures of E. coli, resulting in all-or-none stochastic differentiation into distinct subpopulations of curli-positive and curli-negative cells at the entry into the stationary phase of growth. Stochastic curli activation in individual E. coli cells could further be observed during continuous growth in a conditioned medium in a microfluidic device, which further revealed that the curli-positive state is only metastable. In agreement with previous reports, regulation of curli gene expression by the second messenger c-di-GMP via two pairs of diguanylate cyclase and phosphodiesterase enzymes, DgcE/PdeH and DgcM/PdeR, modulates the fraction of curli-positive cells. Unexpectedly, removal of this regulatory network does not abolish the bimodality of curli gene expression, although it affects dynamics of activation and increases heterogeneity of expression levels among individual cells. Moreover, the fraction of curli-positive cells within an E. coli population shows stronger dependence on growth conditions in the absence of regulation by DgcE/PdeH and DgcM/PdeR pairs. We thus conclude that, while not required for the emergence of bimodal curli gene expression in E. coli, this c-di-GMP regulatory network attenuates the frequency and dynamics of gene activation and increases its robustness to cellular heterogeneity and environmental variation.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Second Messenger Systems; Transcriptional Activation

The second messenger cyclic dimeric GMP (c-di-GMP) plays a central role in controlling decision-making processes that are vitally important for the environmental survival of the human pathogen Vibrio parahaemolyticus. The mechanisms by which c-di-GMP levels and biofilm formation are dynamically controlled in V. parahaemolyticus are poorly understood. Here, we report the involvement of OpaR in controlling c-di-GMP metabolism and its effects on the expression of the trigger phosphodiesterase (PDE) TpdA and the biofilm-matrix related gene

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Homeostasis; Humans; Phosphoric Diester Hydrolases; Vibrio parahaemolyticus

The opportunistic human pathogen Pseudomonas aeruginosa causes chronic infections that involve multicellular aggregates called biofilms. Biofilm formation is modulated by the host environment and the presence of cues and/or signals, likely affecting the pool of the bacterial second messenger cyclic diguanylate monophosphate (c-di-GMP). The manganese ion Mn

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Humans; Manganese; Polysaccharides; Pseudomonas aeruginosa

Actinobacteria are ubiquitous bacteria undergoing complex developmental transitions coinciding with antibiotic production in response to stress or nutrient starvation. This transition is mainly controlled by the interaction between the second messenger c-di-GMP and the master repressor BldD. To date, the upstream factors and the global signal networks that regulate these intriguing cell biological processes remain unknown. In Saccharopolyspora erythraea, we found that acetyl phosphate (AcP) accumulation resulting from environmental nitrogen stress participated in the regulation of BldD activity through cooperation with c-di-GMP. AcP-induced acetylation of BldD at K11 caused the BldD dimer to fall apart and dissociate from the target DNA and disrupted the signal transduction of c-di-GMP, thus governing both developmental transition and antibiotic production. Additionally, practical mutation of BldDK11R bypassing acetylation regulation could enhance the positive effect of BldD on antibiotic production. The study of AcP-dependent acetylation is usually confined to the control of enzyme activity. Our finding represents an entirely different role of the covalent modification caused by AcP, which integrated with c-di-GMP signal in modulating the activity of BldD for development and antibiotic production, coping with environmental stress. This coherent regulatory network might be widespread across actinobacteria, thus has broad implications.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Saccharopolyspora

A number of multidrug-resistant bacterial pathogens code for S1-P1 nucleases with a poorly understood role. We have characterized a recombinant form of S1-P1 nuclease from Stenotrophomonas maltophilia, an opportunistic pathogen. S. maltophilia nuclease 1 (SmNuc1) acts predominantly as an RNase and is active in a wide range of temperatures and pH. It retains a notable level of activity towards RNA and ssDNA at pH 5 and 9 and about 10% of activity towards RNA at 10 °C. SmNuc1 with very high catalytic rates outperforms S1 nuclease from Aspergillus oryzae and other similar nucleases on all types of substrates. SmNuc1 degrades second messenger c-di-GMP, which has potential implications for its role in the pathogenicity of S. maltophilia.

Topics: Cyclic GMP; Endonucleases; RNA; Stenotrophomonas maltophilia

Pseudomonas plecoglossicida (P. plecoglossicida) is the causative agent of visceral granulomas disease in large yellow croaker (Larimichthys crocea) and it causes severe economic loss to its industry. Biofilm formation, related to intracellular cyclic bis (3'-5') diguanylic acid (c-di-GMP) levels, is essential for the lifestyle of P. plecoglossicida. This research aims to investigate the role of YfiR-a key regulator of the diguanylate cyclase YfiN to regulate c-di-GMP levels and reveal its regulatory function of bacterial virulence expression in P. plecoglossicida.. A genetic analysis was carried out to identify the yfiBNR operon for c-di-GMP regulation in P. plecoglossicida. Then, we constructed a yfiR mutant and observed increased c-di-GMP levels, enhanced biofilm formation, increased exopolysaccharides, and diminished swimming and swarming motility in this strain. Moreover, through establishing a yolk sac microinjection infection model in zebrafish larvae, an attenuated phenotype of yfiR mutant that manifested as restored survival and lower bacterial colonization was found.. YfiR is the key regulator of virulence in P. plecoglossicida, which contributes to c-di-GMP level, biofilm formation, exopolysaccharides production, swimming, swarming motility, and bacterial colonization in zebrafish model.

Topics: Animals; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Phenotype; Virulence; Zebrafish

The L-arabinose inducible pBAD vectors are commonly used to turn on and off the expression of specific genes in bacteria. The utilization of certain carbohydrates can influence bacterial growth, virulence factor production, and biofilm formation.

Topics: Arabinose; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression; Gene Expression Regulation, Bacterial; Vibrio parahaemolyticus; Virulence Factors

In mammals, the enzyme cGAS senses the presence of cytosolic DNA and synthesizes the cyclic dinucleotide (CDN) 2'3'-cGAMP, which triggers STING-dependent immunity. In Drosophila melanogaster, two cGAS-like receptors (cGLRs) produce 3'2'-cGAMP and 2'3'-cGAMP to activate STING. We explored CDN-mediated immunity in 14 Drosophila species covering 50 million years of evolution and found that 2'3'-cGAMP and 3'2'-cGAMP failed to control infection by Drosophila C virus in D. serrata and two other species. We discovered diverse CDNs produced in a cGLR-dependent manner in response to viral infection in D. melanogaster, including 2'3'-c-di-GMP. This CDN was a more potent STING agonist than cGAMP in D. melanogaster and it also activated a strong antiviral transcriptional response in D. serrata. Our results shed light on the evolution of cGLRs in flies and provide a basis for understanding the function and regulation of this emerging family of pattern recognition receptors in animal innate immunity.

Topics: Animals; Antiviral Agents; Cyclic GMP; Drosophila; Drosophila melanogaster; Mammals

Topics: Bacteria; Cyclic GMP

The regulation of biofilm and motile states as alternate bacterial lifestyles has been studied extensively in flagellated bacteria, where the second messenger cyclic-di-GMP (cdG) plays a crucial role. However, much less is known about the mechanisms of such regulation in motile bacteria without flagella. The bacterial type IV pilus (T4P) serves as a motility apparatus that enables

Topics: Adenosine Triphosphatases; Biofilms; Cyclic GMP; Myxococcus xanthus

The ability of many bacteria to form biofilms contributes to their resilience and makes infections more difficult to treat. Biofilm growth leads to the formation of internal oxygen gradients, creating hypoxic subzones where cellular reducing power accumulates, and metabolic activities can be limited. The pathogen Pseudomonas aeruginosa counteracts the redox imbalance in the hypoxic biofilm subzones by producing redox-active electron shuttles (phenazines) and by secreting extracellular matrix, leading to an increased surface area-to-volume ratio, which favors gas exchange. Matrix production is regulated by the second messenger bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) in response to different environmental cues. RmcA (Redox modulator of c-di-GMP) from P. aeruginosa is a multidomain phosphodiesterase (PDE) that modulates c-di-GMP levels in response to phenazine availability. RmcA can also sense the fermentable carbon source arginine via a periplasmic domain, which is linked via a transmembrane domain to four cytoplasmic Per-Arnt-Sim (PAS) domains followed by a diguanylate cyclase (DGC) and a PDE domain. The biochemical characterization of the cytoplasmic portion of RmcA reported in this work shows that the PAS domain adjacent to the catalytic domain tunes RmcA PDE activity in a redox-dependent manner, by differentially controlling protein conformation in response to FAD or FADH2. This redox-dependent mechanism likely links the redox state of phenazines (via FAD/FADH2 ratio) to matrix production as indicated by a hyperwrinkling phenotype in a macrocolony biofilm assay. This study provides insights into the role of RmcA in transducing cellular redox information into a structural response of the biofilm at the population level. Conditions of resource (i.e. oxygen and nutrient) limitation arise during chronic infection, affecting the cellular redox state and promoting antibiotic tolerance. An understanding of the molecular linkages between condition sensing and biofilm structure is therefore of crucial importance from both biological and engineering standpoints.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Oxygen; Phenazines; Polymers; Pseudomonas aeruginosa

Attachment of bacteria onto a surface, consequent signaling, and accumulation and growth of the surface-bound bacterial population are key initial steps in the formation of pathogenic biofilms. While recent reports have hinted that surface mechanics may affect the accumulation of bacteria on that surface, the processes that underlie bacterial perception of surface mechanics and modulation of accumulation in response to surface mechanics remain largely unknown. We use thin and thick hydrogels coated on glass to create composite materials with different mechanics (higher elasticity for thin composites; lower elasticity for thick composites) but with the same surface adhesivity and chemistry. The mechanical cue stemming from surface mechanics is elucidated using experiments with the opportunistic human pathogen Pseudomonas aeruginosa combined with finite-element modeling. Adhesion to thin composites results in greater changes in mechanical stress and strain in the bacterial envelope than does adhesion to thick composites with identical surface chemistry. Using quantitative microscopy, we find that adhesion to thin composites also results in higher cyclic-di-GMP levels, which in turn result in lower motility and less detachment, and thus greater accumulation of bacteria on the surface than does adhesion to thick composites. Mechanics-dependent c-di-GMP production is mediated by the cell-surface-exposed protein PilY1. The biofilm lag phase, which is longer for bacterial populations on thin composites than on thick composites, is also mediated by PilY1. This study shows clear evidence that bacteria actively regulate differential accumulation on surfaces of different stiffnesses via perceiving varied mechanical stress and strain upon surface engagement.

Topics: Biofilms; Cyclic GMP; Humans; Pseudomonas aeruginosa; Signal Transduction

Cyclic di-GMP (c-di-GMP) is a second messenger that transduces extracellular stimuli into cellular responses and regulates various biological processes in bacteria. H-NS is a global regulatory protein that represses expression of many genes, but how H-NS activity is modulated by environmental signals remains largely unclear. Here, we show that high intracellular c-di-GMP levels, induced by environmental cues, relieve H-NS-mediated transcriptional silencing in Salmonella enterica serovar Typhimurium. We find that c-di-GMP binds to the H-NS protein to inhibit its binding to DNA, thus derepressing genes silenced by H-NS. However, c-di-GMP is unable to displace H-NS from DNA. In addition, a K107A mutation in H-NS abolishes response to c-di-GMP but leaves its DNA binding activity unaffected in vivo. Our results thus suggest a mechanism by which H-NS acts as an environment-sensing regulator in Gram-negative bacteria.

Topics: Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Salmonella typhimurium; Second Messenger Systems

Pseudomonas aeruginosa, an opportunistic bacterial pathogen, can synthesize and catabolize several small cationic molecules known as polyamines. In several clades of bacteria, polyamines regulate biofilm formation, a lifestyle-switching process that confers resistance to environmental stress. The polyamine putrescine and its biosynthetic precursors, l-arginine and agmatine, promote biofilm formation in Pseudomonas spp. However, it remains unclear whether the effect is a direct effect of polyamines or occurs through a metabolic derivative. Here, we used a genetic approach to demonstrate that putrescine accumulation, either through disruption of the spermidine biosynthesis pathway or the catabolic putrescine aminotransferase pathway, promoted biofilm formation in P. aeruginosa. Consistent with this observation, exogenous putrescine robustly induced biofilm formation in P. aeruginosa that was dependent on putrescine uptake and biosynthesis pathways. Additionally, we show that l-arginine, the biosynthetic precursor of putrescine, also promoted biofilm formation but did so by a mechanism independent of putrescine or agmatine conversion. We found that both putrescine and l-arginine induced a significant increase in the intracellular level of bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) (c-di-GMP), a bacterial second messenger widely found in

Topics: Arginine; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa; Putrescine; Up-Regulation

To alter the immunosuppressive tumor microenvironment (TME), we developed an immunostimulatory nanoparticle (NP) to reprogram a tumor's dysfunctional and inhibitory antigen-presenting cells (APCs) into properly activated APCs that stimulate tumor-reactive cytotoxic T cells. Importantly, systemic delivery allowed NPs to efficiently utilize the entire microvasculature and gain access into the majority of the perivascular TME, which coincided with the APC-rich tumor areas leading to uptake of the NPs predominantly by APCs. In this work, a 60 nm NP was loaded with a STING agonist, which triggered robust production of interferon β, resulting in activation of APCs. In addition to untargeted NPs, we employed 'mainstream' ligands targeting fibronectin, α

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Breast Neoplasms; Cell Line, Tumor; Cyclic GMP; Dendritic Cells; Immunity, Innate; Immunologic Factors; Ligands; Macrophages; Mice, Inbred BALB C; Nanoparticles; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Polyethylene Glycols; T-Lymphocytes; Tumor Microenvironment

Many bacteria use cyclic dimeric guanosine monophosphate (c-di-GMP) to control changes in lifestyle. The molecule, synthesized by proteins having diguanylate cyclase activity, is often a signal to transition from motile to sedentary behaviour. In Vibrio cholerae, c-di-GMP can exert its effects via the transcription factors VpsT and VpsR. Together, these proteins activate genes needed for V. cholerae to form biofilms. In this work, we have mapped the genome-wide distribution of VpsT in a search for further regulatory roles. We show that VpsT binds 23 loci and recognises a degenerate DNA palindrome having the consensus 5'-W-5R-4[CG]-3Y-2W-1W+1R+2[GC]+3Y+4W+5-3'. Most genes targeted by VpsT encode functions related to motility, biofilm formation, or c-di-GMP metabolism. Most notably, VpsT activates expression of the vpvABC operon that encodes a diguanylate cyclase. This creates a positive feedback loop needed to maintain intracellular levels of c-di-GMP. Mutation of the key VpsT binding site, upstream of vpvABC, severs the loop and c-di-GMP levels fall accordingly. Hence, as well as relaying the c-di-GMP signal, VpsT impacts c-di-GMP homeostasis.

Topics: Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Homeostasis; Operon; Phosphorus-Oxygen Lyases; Protein Binding; Regulatory Sequences, Nucleic Acid; Transcription Factors; Vibrio cholerae

The widespread bacterial second messenger bis-(3'-5')-cyclic diguanosine monophosphate (c-di-GMP) is an important regulator of biofilm formation, virulence and cell differentiation. C-di-GMP-specific biosensors that allow detection and visualization of c-di-GMP levels in living cells are key to our understanding of how c-di-GMP fluctuations drive cellular responses. Here, we describe a novel c-di-GMP biosensor, CensYBL, that is based on c-di-GMP-induced dimerization of the effector protein BldD from Streptomyces resulting in bimolecular fluorescence complementation of split-YPet fusion proteins. As a proof-of-principle, we demonstrate that CensYBL is functional in detecting fluctuations in intracellular c-di-GMP levels in the Gram-negative model bacteria Escherichia coli and Salmonella enterica serovar Typhimurium. Using deletion mutants of c-di-GMP diguanylate cyclases and phosphodiesterases, we show that c-di-GMP dependent dimerization of CBldD-YPet results in fluorescence complementation reflecting intracellular c-di-GMP levels. Overall, we demonstrate that the CensYBL biosensor is a user-friendly and versatile tool that allows to investigate c-di-GMP variations using single-cell and population-wide experimental set-ups.

Topics: Cyclic GMP; Escherichia coli; Fluorescence; Salmonella typhimurium; Second Messenger Systems

The exopolysaccharide (EPS) Pea is essential for wrinkly colony morphology, pellicle formation, and robust biofilm production in Pseudomonas putida. The second messenger cyclic diguanylate monophosphate (c-di-GMP) induces wrinkly colony morphology in P. putida through an unknown mechanism(s). Herein, we found that c-di-GMP modulates wrinkly colony morphology via the regulation of expression of

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Phenotype; Pisum sativum; Promoter Regions, Genetic; Pseudomonas putida; Second Messenger Systems

An effective method to identify c-di-GMP may significantly facilitate the exploration of its signaling pathways and bacterial pathogenesis. Herein, we have developed the first conjugated polymer-amplified RNA aptamer NanoKit with a unique core-shell-shell architecture, which combines the advantages of high selectivity of RNA aptamers and high sensitivity of strong fluorescence resonance energy transfer (FRET) effect, for precisely detecting c-di-GMP. We identified that NanoKit could selectively detect c-di-GMP with a low detection limit of 50 pM. Importantly, NanoKit could identify bacterial species and physiological states, such as planktonic, biofilm, and even antibiotic-resistance, on the basis of their different c-di-GMP expression patterns. Particularly, NanoKit could distinguish bacterial infection and inflammation and identify

Topics: Aptamers, Nucleotide; Bacterial Proteins; Biofilms; Cyclic GMP; Fluorescence Resonance Energy Transfer

In Pseudomonas putida KT2440, cfcR encodes an orphan multidomain response regulator with diguanylate cyclase activity, which is responsible for the synthesis of c-di-GMP, a second messenger key in the transition from planktonic to sessile bacterial lifestyles. When overexpressed, cfcR enhances biofilm formation and causes other phenotype alterations. The cfcA gene, encoding a membrane-anchored multisensory CHASE3/GAF hybrid histidine kinase (HK), is required to develop this pleiotropic phenotype. Here we show autophosphorylation of CfcA through HisKA/HATPase_c domains and then transfer of the phosphoryl group to an internal receiver (REC) domain. CfcA REC domains are nonessential for phosphotransfer from CfcA~P to the REC domain of CfcR. CfcA~P also phosphorylates the REC domain of CfcD, a second HK encoded in the same gene cluster as CfcA, which negatively regulates the CfcA/CfcR pathway. To evaluate the impact of CfcA domains on CfcR activity, a battery of mutants with in-frame domain deletions was generated, whose CfcA protein locations were also examined. CfcA membrane anchorage contributes to protein stability and CfcR activation. Salt enhances c-di-GMP levels through CfcR, a response which is hampered by alteration of a presumed ligand-binding motif in the CHASE3 sensor domain. Thus, in P. putida, c-di-GMP is salt-regulated through the CfcA/CfcR/CfcD system.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Pseudomonas putida; Salts

Mammalian innate immune sensor STING (STimulator of INterferon Gene) was recently found to originate from bacteria. During phage infection, bacterial STING sense c-di-GMP generated by the CD-NTase (cGAS/DncV-like nucleotidyltransferase) encoded in the same operon and signal suicide commitment as a defense strategy that restricts phage propagation. However, the precise binding mode of c-di-GMP to bacterial STING and the specific recognition mechanism are still elusive. Here, we determine two complex crystal structures of bacterial STING/c-di-GMP, which provide a clear picture of how c-di-GMP is distinguished from other cyclic dinucleotides. The protein-protein interactions further reveal the driving force behind filament formation of bacterial STING. Finally, we group the bacterial STING into two classes based on the conserved motif in β-strand lid, which dictate their ligand specificity and oligomerization mechanism, and propose an evolution-based model that describes the transition from c-di-GMP-dependent signaling in bacteria to 2'3'-cGAMP-dependent signaling in eukaryotes.

Topics: Bacteria; Crystallography, X-Ray; Cyclic GMP; Dinucleoside Phosphates; Humans; Immunity, Innate; Interferons; Ligands; Membrane Proteins; Nucleotidyltransferases; Prevotella

Work on surface sensing in bacterial biofilms has focused on how cells transduce sensory input into cyclic diguanylate (c-di-GMP) signaling, low and high levels of which generally correlate with high-motility planktonic cells and low-motility biofilm cells, respectively. Using Granger causal inference methods, however, we find that single-cell c-di-GMP increases are not sufficient to imply surface commitment. Tracking entire lineages of cells from the progenitor cell onward reveals that c-di-GMP levels can exhibit increases but also undergo oscillations that can propagate across 10 to 20 generations, thereby encoding more complex instructions for community behavior. Principal component and factor analysis of lineage c-di-GMP data shows that surface commitment behavior correlates with three statistically independent composite features, which roughly correspond to mean c-di-GMP levels, c-di-GMP oscillation period, and surface motility. Surface commitment in young biofilms does not correlate to c-di-GMP increases alone but also to the emergence of high-frequency and small-amplitude modulation of elevated c-di-GMP signal along a lineage of cells. Using this framework, we dissect how increasing or decreasing signal transduction from wild-type levels, by varying the interaction strength between PilO, a component of a principal surface sensing appendage system, and SadC, a key hub diguanylate cyclase that synthesizes c-di-GMP, impacts frequency and amplitude modulation of c-di-GMP signals and cooperative surface commitment.

Topics: Bacterial Physiological Phenomena; Bacterial Proteins; Cyclic GMP; Mutation; Protein Binding; Pseudomonas aeruginosa; Signal Transduction

The bacterial second messenger bis-(3'-5')-cyclic diguanylate monophosphate (c-di-GMP) controls various cellular processes, including motility, toxin production, and biofilm formation. c-di-GMP is enzymatically synthesized by GGDEF domain-containing diguanylate cyclases and degraded by HD-GYP domain-containing phosphodiesterases (PDEs) to 2 GMP or by EAL domain-containing PDE-As to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG). Since excess pGpG feedback inhibits PDE-A activity and thereby can lead to the uncontrolled accumulation of c-di-GMP, a PDE that degrades pGpG to 2 GMP (PDE-B) has been presumed to exist. To date, the only enzyme known to hydrolyze pGpG is oligoribonuclease Orn, which degrades all kinds of oligoribonucleotides. Here, we identified a pGpG-specific PDE, which we named PggH, using biochemical approaches in the gram-negative bacteria Vibrio cholerae. Biochemical experiments revealed that PggH exhibited specific PDE activity only toward pGpG, thus differing from the previously reported Orn. Furthermore, the high-resolution structure of PggH revealed the basis for its PDE activity and narrow substrate specificity. Finally, we propose that PggH could modulate the activities of PDE-As and the intracellular concentration of c-di-GMP, resulting in phenotypic changes including in biofilm formation.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Phosphoric Diester Hydrolases; Signal Transduction; Substrate Specificity; Vibrio cholerae

Programmed cell death plays an important role in modulating host immune defense and pathogen infection. Ferroptosis is a type of inflammatory cell death induced by intracellular iron-dependent accumulation of toxic lipid peroxides. Although ferroptosis has been associated with cancer and other sterile diseases, very little is known about the role of ferroptosis in modulating host-pathogen interactions. We show that accumulation of the secondary messenger bis-(3',5')-cyclic dimeric GMP (c-di-GMP) in the pathogenic bacterium

Topics: Bacterial Proteins; Cyclic GMP; Edwardsiella; Enterobacteriaceae Infections; Ferroptosis; HeLa Cells; Humans; Virulence

The second messenger cyclic di-GMP (c-di-GMP) is a key molecule that controls different physiological and behavioral processes in many bacteria, including motile-to-sessile lifestyle transitions. Although the external stimuli that modulate cellular c-di-GMP contents are not fully characterized, there is growing evidence that certain amino acids act as environmental cues for c-di-GMP turnover. In the plant-beneficial bacterium Pseudomonas putida KT2440, both arginine biosynthesis and uptake influence second messenger contents and the associated phenotypes. To further understand this connection, we have analyzed the role of ArgR, which in different bacteria is the master transcriptional regulator of arginine metabolism but had not been characterized in P. putida. The results show that ArgR controls arginine biosynthesis and transport, and an

Topics: Arginine; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Pseudomonas putida

Dickeya oryzae is a bacterial pathogen causing the severe rice stem rot disease in China and other rice-growing countries. We showed recently that the universal bacterial second messenger c-di-GMP plays an important role in modulation of bacterial motility and pathogenicity, but the mechanism of regulation remains unknown. In this study, bioinformatics analysis of the D. oryzae EC1 genome led to the identification of two proteins, YcgR and BcsA, both of which contain a conserved c-di-GMP receptor domain, known as the PilZ-domain. By deleting all the genes encoding c-di-GMP-degrading enzymes in D. oryzae EC1, the resultant mutant 7ΔPDE with high c-di-GMP levels became nonmotile, formed hyperbiofilm, and lost the ability to colonize and invade rice seeds. These phenotypes were partially reversed by deletion of ycgR in the mutant 7ΔPDE, whereas deletion of bcsA only reversed the hyperbiofilm phenotype of mutant 7ΔPDE. Significantly, double deletion of ycgR and bcsA in mutant 7ΔPDE rescued its motility, biofilm formation, and virulence to levels of wild-type EC1. In vitro biochemical experiments and in vivo phenotypic assays further validated that YcgR and BcsA proteins are the receptors for c-di-GMP, which together play a critical role in regulating the c-di-GMP-associated functionality. The findings from this study fill a gap in our understanding of how c-di-GMP modulates bacterial motility and biofilm formation, and provide useful clues for further elucidation of sophisticated virulence regulatory mechanisms in this important plant pathogen.

Topics: Bacterial Proteins; Carrier Proteins; Cyclic GMP; Dickeya; Gene Expression Regulation, Bacterial; Oryza; Phenotype; Virulence

Bacterial biofilms are multicellular communities that collectively overcome environmental threats and clinical treatments. To regulate the biofilm lifecycle, bacteria commonly transduce sensory information via the second messenger molecule cyclic diguanylate (c-di-GMP). Using experimental and modeling approaches, we quantitatively capture c-di-GMP signal transmission via the bifunctional polyamine receptor NspS-MbaA, from ligand binding to output, in the pathogen Vibrio cholerae. Upon binding of norspermidine or spermidine, NspS-MbaA synthesizes or degrades c-di-GMP, respectively, which, in turn, drives alterations specifically to biofilm gene expression. A long-standing question is how output specificity is achieved via c-di-GMP, a diffusible molecule that regulates dozens of effectors. We show that NspS-MbaA signals locally to specific effectors, sensitizing V. cholerae to polyamines. However, local signaling is not required for specificity, as changes to global cytoplasmic c-di-GMP levels can selectively regulate biofilm genes. This work establishes the input-output dynamics underlying c-di-GMP signaling, which could be useful for developing bacterial manipulation strategies.

Topics: Biofilms; Cyclic GMP; Signal Transduction; Vibrio cholerae

Nucleotide second messengers, such as cAMP and c-di-GMP, regulate many physiological processes in bacteria, including biofilm formation. There is evidence of cross-talk between pathways mediated by c-di-GMP and those mediated by the cAMP receptor protein (CRP), but the mechanisms are often unclear. Here, we show that cAMP-CRP modulates biofilm maintenance in Shewanella putrefaciens not only via its known effects on gene transcription, but also through direct interaction with a putative c-di-GMP effector on the inner membrane, BpfD. Binding of cAMP-CRP to BpfD enhances the known interaction of BpfD with protease BpfG, which prevents proteolytic processing and release of a cell surface-associated adhesin, BpfA, thus contributing to biofilm maintenance. Our results provide evidence of cross-talk between cAMP and c-di-GMP pathways through direct interaction of their effectors, and indicate that cAMP-CRP can play regulatory roles at the post-translational level.

Topics: Bacterial Proteins; Biofilms; Cyclic AMP Receptor Protein; Cyclic GMP; Gene Expression Regulation, Bacterial; Signal Transduction

As a key bacterial second messenger, cyclic di-GMP (c-di-GMP) regulates various physiological processes, such as motility, biofilm formation, and virulence. Cellular c-di-GMP levels are regulated by the opposing activities of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs). Beyond that, the enzymatic activities of c-di-GMP metabolizing proteins are controlled by a variety of extracellular signals and intracellular physiological conditions. Here, we report that

Topics: Bacterial Proteins; Biofilms; Burkholderia; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Virulence

During infection, intracellular pathogens inevitably face the pressure of hypoxia. Mycobacterium tuberculosis and Mycobacterium bovis represent two typical intracellular bacteria, but the signalling pathway of their adaptation to hypoxia remains unclear. Here, we report a new mechanism of the hypoxic adaptation in M. bovis driven by the second messenger molecule c-di-GMP. We found that c-di-GMP was significantly accumulated in bacterial cells under hypoxic stress and blocked the inhibitory activity of ArgR, an arginine metabolism gene cluster regulator, which increased arginine synthesis and slowed tricarboxylic acid cycle (TCA cycle) and aerobic respiration. Meanwhile, c-di-GMP relieved the self-inhibition of argR expression, and ArgR could interact with the nitrite metabolic gene regulator Cmr, promoting the positive regulation of Cmr and, thereafter, the nitrite respiration. Consistently, c-di-GMP significantly induced the expression of arginine and nitrite metabolism gene clusters and increased the mycobacterial survival ability under hypoxia. Therefore, we found a new function of the second messenger molecule c-di-GMP and characterized ArgR as a metabolic switching regulator that can coordinate the c-di-GMP signal to trigger hypoxic adaptation in mycobacteria. Our findings provide a potential new target for blocking the life cycle of M. tuberculosis infection.

Topics: Arginine; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Humans; Hypoxia; Mycobacterium bovis; Mycobacterium tuberculosis; Nitrites

Dickeya dadantii is a phytopathogenic bacterium that causes diseases on a wide range of host plants. The pathogen secretes pectate lyases (Pel) through the type II secretion system (T2SS) that degrades the cell wall in host plants. The virulence of

Topics: Bacterial Proteins; Cyclic GMP; Dickeya; Enterobacteriaceae; Gene Expression Regulation, Bacterial; Polysaccharide-Lyases

C-di-GMP signaling can directly influence bacterial behavior by affecting the functionality of c-di-GMP-binding proteins. In addition, c-di-GMP can exert a global effect on gene transcription or translation, for example, via riboswitches or by binding to transcription factors. In this study, we investigated the effects of changes in intracellular c-di-GMP levels on gene expression and protein production in the opportunistic pathogen Pseudomonas aeruginosa. We induced c-di-GMP production via an ectopically introduced diguanylate cyclase and recorded the transcriptional, translational as well as proteomic profile of the cells. We demonstrate that rising levels of c-di-GMP under growth conditions otherwise characterized by low c-di-GMP levels caused a switch to a non-motile, auto-aggregative P. aeruginosa phenotype. This phenotypic switch became apparent before any c-di-GMP-dependent role on transcription, translation, or protein abundance was observed. Our results suggest that rising global c-di-GMP pools first affects the motility phenotype of P. aeruginosa by altering protein functionality and only then global gene transcription.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Proteomics; Pseudomonas aeruginosa

Erwinia amylovora is an economically devastating plant pathogen that causes fire blight disease in members of the Rosaceae family, most notably in apple and pear. The exopolysaccharide amylovoran is a pathogenicity determinant in E. amylovora and a major component of the extracellular matrix of biofilms formed within the xylem vasculature of the host plant. The second messenger cyclic-di-GMP (c-di-GMP) has been reported to positively regulate the transcription of amsG (the first gene in the 12-gene amylovoran [ams] biosynthetic operon), thus impacting amylovoran production. However, the regulatory mechanism by which this interaction occurs is largely unknown. Here, we report that c-di-GMP can bind to specific residues in the EAL domain of the E. amylovora protein CsrD. CsrD and RNase E regulate the degradation of the sRNA CsrB in E. amylovora. When CsrD is bound to c-di-GMP, there is an enhancement in the level of RNase E-mediated degradation of CsrB, which then alters amsG transcription. Additionally, csrD was also found to positively contribute to virulence and biofilm formation. We thus present a pathway of conditional regulation of amylovoran production mediated by changing intracellular levels of c-di-GMP, which impacts disease progression.

Topics: Bacterial Proteins; Cyclic GMP; Erwinia amylovora; Plant Diseases; Polysaccharides, Bacterial; Virulence

Pathogenic bacteria causing human rickettsioses, transmitted in nature by arthropod vectors, primarily infect vascular endothelial cells lining the blood vessels, resulting in 'endothelial activation' and onset of innate immune responses. Nucleotide second messengers are long presumed to be the stimulators of type I interferons, of which bacterial cyclic-di-GMP (c-di-GMP) has been implicated in multiple signaling pathways governing communication with other bacteria and host cells, yet its importance in the context of rickettsial interactions with the host has not been investigated. Here, we report that all rickettsial genomes encode a putative diguanylate cyclase

Topics: Bacterial Proteins; Cyclic GMP; Endothelial Cells; Gene Expression Regulation, Bacterial; Humans; Rickettsia; Rickettsia rickettsii; Virulence

C-di-GMP is a ubiquitous second messenger in bacterium, which regulates cellular functions such as the formation of biofilm membrane, cell mobility, virulence, cell adhesion, cell cycle et al. These functions are associated with an increasing number of c-di-GMP effector proteins and/or riboswitchs. In the study, CEP1 (c-di-GMP effector protein 1), a novel c-di-GMP binding protein, was screened with a combination of affinity pull-down and LC/MS/MS methods. The binding of CEP1 and c-di-GMP was demonstrated by surface plasmon resonance, with the dissociation constants of 127 ± 1.03 μM. Quantitative real time PCR assay showed the mRNA levels of cep1 gene in Rhodococcus ruber SD3 increased to 63.29 times and 71.18 times after toluene and phenol stress, respectively. Furthermore, cep1 gene enhanced strain was constructed using shuttle plasmid pNV18, which showed improved growth compared to the wild-type strain in the presence of different organic solvents. The study provided an insight into a mechanism, by which c-di-GMP was connected with organic solvent tolerance of Rhodococcus ruber SD3.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Rhodococcus; Tandem Mass Spectrometry

Cyclic dimeric guanosine monophosphate (c-di-GMP) is a bacterial second messenger with immunomodulatory activities in mice, suggesting potential applications as a vaccine immunopotentiator or therapeutic agent. In this study, we evaluated the efficacy of c-di-GMP as an immunopotentiator for pseudorabies virus (PRV) inactivated vaccine in a murine model. We found that c-di-GMP improved the humoral and cellular immune responses induced by PRV inactivated vaccine and its effects on immunity reached the level comparable to that of a live attenuated vaccine. Furthermore, c-di-GMP enhanced the murine antibody response against the viral glycoprotein gB up to 120 days after immunization. The c-di-GMP-adjuvanted PRV inactivated vaccine induced long-term humoral immunity by promoting a potent T follicular helper cell response, which is known to directly control the magnitude of the germinal center B cell response. Furthermore, the c-di-GMP enhanced the response of bone marrow plasma cells and upregulated the expression of

Topics: Adjuvants, Immunologic; Animals; Antibodies, Viral; Cyclic GMP; Herpesvirus 1, Suid; Immunity, Humoral; Mice; Vaccines, Attenuated; Vaccines, Inactivated

Vibrio cholerae biofilm formation/maintenance is controlled by myriad factors; chief among these are the regulator VpsR and cyclic di-guanosine monophosphate (c-di-GMP). VpsR has strong sequence similarity to enhancer binding proteins (EBPs) that activate RNA polymerase containing sigma factor σ54. However, we have previously shown that transcription from promoters within the biofilm biogenesis/maintenance pathways uses VpsR, c-di-GMP and RNA polymerase containing the primary sigma factor (σ70). Previous work suggested that phosphorylation of VpsR at a highly conserved aspartate, which is phosphorylated in other EBPs, might also contribute to activation. Using the biofilm biogenesis promoter PvpsL, we show that in the presence of c-di-GMP, either wild type or the phospho-mimic VpsR D59E activates PvpsL transcription, while the phospho-defective D59A variant does not. Furthermore, when c-di-GMP levels are low, acetyl phosphate (Ac∼P) is required for significant VpsR activity in vivo and in vitro. Although these findings argue that VpsR phosphorylation is needed for activation, we show that VpsR is not phosphorylated or acetylated by Ac∼P and either sodium phosphate or potassium phosphate, which are not phosphate donors, fully substitutes for Ac∼P. We conclude that VpsR is an unusual regulator that senses phosphate directly, rather than through phosphorylation, to aid in the decision to form/maintain biofilm.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; DNA-Binding Proteins; DNA-Directed RNA Polymerases; Gene Expression Regulation, Bacterial; Phosphates; Sigma Factor; Vibrio cholerae

Cyclic di-guanosine monophosphate (c-di-GMP) is a second messenger found ubiquitously in bacteria. This signaling molecule regulates a variety of physiological activities such as phototaxis and flocculation in cyanobacteria and is critical for their environmental adaptation. Although genes encoding the enzymes for synthesis and/or degradation of c-di-GMP are found in the genomes of both multicellular and unicellular cyanobacteria, little is known about the biological functions of these enzymes in cyanobacterial cells. Here we have established a robust and highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method for c-di-GMP quantification using a cost-effective solvent, methanol. Quantification methods were validated by measuring c-di-GMP in the cyanobacterium Synechococcus elongatus PCC 7942 through spiking and recovery assays after which the method was applied to examine short-term changes in cellular levels of c-di-GMP in response to a transition from light to dark or from dark to light in S. elongatus. Results showed that a transient increase in c-di-GMP upon transitioning from light to dark was occurring which resembled responses involving cyclic adenosine monophosphate and other second messengers in cyanobacteria. These findings demonstrated that our method enabled relatively specific and sensitive quantification of c-di-GMP in cyanobacteria at lower cost.

Topics: Bacterial Proteins; Chromatography, Liquid; Cyanobacteria; Cyclic GMP; Guanosine Monophosphate; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

Bacteria use signal transduction systems to sense and respond to their external environment. The two-component system CpxA/CpxR senses misfolded envelope protein stress and responds by up-regulating envelope protein factors and down-regulating virulence factors in several animal pathogens. Dickeya dadantii is a phytopathogen equipped with a type III secretion system (T3SS) for manipulating the host immune response. We found that deletion of cpxR enhanced the expression of the T3SS marker gene hrpA in a designated T3SS-inducing minimal medium (MM). In the ∆cpxR mutant, multiple T3SS and c-di-GMP regulators were also up-regulated. Subsequent analysis revealed that deletion of the phosphodiesterase gene egcpB in ∆cpxR abolished the enhanced T3SS expression. This suggested that CpxR suppresses EGcpB levels, causing low T3SS expression in MM. Furthermore, we found that the ∆cpxR mutant displayed low c-di-GMP phenotypes in biofilm formation and swimming. Increased production of cellular c-di-GMP by in trans expression of the diguanylate cyclase gene gcpA was negated in the ∆cpxR mutant. Here, we propose that CpxA/CpxR regulates T3SS expression by manipulating the c-di-GMP network, in turn modifying the multiple physiological activities involved in the response to environmental stresses in D. dadantii.

Topics: Bacterial Proteins; Cyclic GMP; Dickeya; Enterobacteriaceae; Gene Expression Regulation, Bacterial; Virulence

c-di-GMP is a ubiquitous bacterial second messenger that plays a central regulatory role in diverse biological processes. c-di-GMP was known to regulate chemotaxis in multiple bacterial species, but its effect on Escherichia coli chemotaxis remained unclear. As an effector of c-di-GMP in E. coli, YcgR when bound with c-di-GMP interacts with the flagellar motor to reduce its speed and its probability of rotating clockwise (CW bias). Here, we found that a significant fraction of the c-di-GMP::YcgR dynamically exchange between the motor and the cytosol. Through fluorescent measurements, we found that there was no competitive binding between the chemotaxis response regulator CheY-P and c-di-GMP::YcgR to the motor. To test the influence of elevated c-di-GMP levels on the chemotaxis pathway, we measured the chemotactic responses of E. coli cells using a FRET assay, finding that elevated c-di-GMP levels had no effect on the upstream part of chemotaxis pathway down to the level of CheY-P concentration. This suggested that the possible effect of elevated c-di-GMP levels on chemotactic motion was through regulation of motor speed and CW bias. Using stochastic simulations of chemotactic swimming, we showed that the effects of reducing motor speed and decreasing CW bias on chemotactic drift velocity are compensating for each other, resulting in minimal effect of elevated c-di-GMP levels on E. coli chemotaxis. Therefore, elevated c-di-GMP levels promote the transition from motile to sedentary forms of bacterial life by reducing the bacterial swimming speed and CW bias, while still maintaining a nearly intact chemotaxis capability in E. coli.

Topics: Bacterial Proteins; Chemotaxis; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Flagella; Second Messenger Systems

Pertussis, caused by the gram-negative bacterium

Topics: Adjuvants, Immunologic; Animals; Bordetella pertussis; Cyclic GMP; Disease Models, Animal; Immunization; Mammals; Mice; Pertussis Vaccine; Vaccination; Whooping Cough

Cyanobacteria, like plants, grow by capturing energy from sunlight. But they have an advantage over their leafy counterparts: they can explore their environment to find the type of light that best suits their needs. These movements rely on hook-like structures, called type IV pili, which allow the cells to pull themselves forward. The pili are usually located at the opposite poles of a rod-shaped cell, allowing the bacteria to move along their longer axis. Yet, the molecular mechanisms that allow cyanobacteria to react to the light are poorly understood. To explore these processes in more detail, Nakane, Enomoto et al. started by shining coloured lights on the rod-shaped cyanobacteria

Topics: Bacterial Proteins; Cyanobacteria; Cyclic GMP; Phototaxis; Thermosynechococcus

The second messenger cyclic di-GMP (c-di-GMP) is a ubiquitous molecule in bacteria that regulates diverse phenotypes. Among them, motility and biofilm formation are the most studied. Furthermore, c-di-GMP has been suggested to regulate virulence factors, making it important for pathogenesis. Previously, we reported that c-di-GMP regulates biofilm formation and swimming motility in Bordetella bronchiseptica. Here, we present a multi-omics approach for the study of B. bronchiseptica strains expressing different cytoplasmic c-di-GMP levels, including transcriptome sequencing (RNA-seq) and shotgun proteomics with label-free quantification. We detected 64 proteins significantly up- or downregulated in either low or high c-di-GMP levels and 358 genes differentially expressed between strains with high c-di-GMP levels and the wild-type strain. Among them, we found genes for stress-related proteins, genes for nitrogen metabolism enzymes, phage-related genes, and virulence factor genes. Interestingly, we observed that a virulence factor like the type III secretion system (TTSS) was regulated by c-di-GMP. B. bronchiseptica with high c-di-GMP levels showed significantly lower levels of TTSS components like Bsp22, BopN, and Bcr4. These findings were confirmed by independent methods, such as quantitative reverse transcription-PCR (q-RT-PCR) and Western blotting. Higher intracellular levels of c-di-GMP correlated with an impaired capacity to induce cytotoxicity in a eukaryotic cell

Topics: Animals; Bacterial Proteins; Biofilms; Bordetella bronchiseptica; Cyclic GMP; Gene Expression Regulation, Bacterial; Mice; Type III Secretion Systems; Virulence; Virulence Factors

In Pseudomonas aeruginosa PAO1, 41 genes encode proteins predicted to be involved in the production or degradation of c-di-GMP, a ubiquitous secondary messenger that regulates a variety of physiological behaviors closely related to biofilm and aggregate formation. Despite extensive effort, the entire picture of this important signaling network is still unclear, with one-third of these proteins remaining uncharacterized. Here, we show that the deletion of

Topics: Bacterial Proteins; Bacteriophages; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa

Stimulator of Interferon Genes (STING) is a type of endoplasmic reticulum (ER)-membrane receptor. STING is activated by a ligand binding, which leads to an enhancement of the immune-system response. Therefore, a STING ligand can be used to regulate the immune system in therapeutic strategies. However, the natural (or native) STING ligand, cyclic-di-nucleotide (CDN), is unsuitable for pharmaceutical use because of its susceptibility to degradation by enzymes and its low cell-membrane permeability. In this study, we designed and synthesized CDN derivatives by replacing the sugar-phosphodiester moiety, which is responsible for various problems of natural CDNs, with an amine skeleton. As a result, we identified novel STING ligands that activate or inhibit STING. The cyclic ligand

Topics: Amines; Cyclic GMP; Ligands; Membrane Proteins; Nucleotides, Cyclic; Skeleton

The symbiont Vibrio fischeri uses motility to colonize its host. In numerous bacterial species, motility is negatively controlled by cyclic-di-GMP (c-di-GMP), which is produced by diguanylate cyclases (DGCs) with GGDEF domains and degraded by phosphodiesterases with either EAL or HD-GYP domains. To begin to decode the functions of the 50 Vibrio fischeri genes with GGDEF, EAL, and/or HD-GYP domains, we deleted each gene and assessed each mutant's migration through tryptone broth salt (TBS) soft agar medium containing or lacking magnesium (Mg) and calcium (Ca), which are known to influence V. fischeri motility. We identified 6, 13, and 16 mutants with altered migration in TBS-Mg, TBS, and TBS-Ca soft agar, respectively, a result that underscores the importance of medium conditions in assessing gene function. A biosensor-based assay revealed that Mg and Ca affected c-di-GMP levels negatively and positively, respectively; the severe decrease in c-di-GMP caused by Mg addition correlates with its strong positive impact on bacterial migration. A mutant defective for

Topics: Agar; Aliivibrio fischeri; Bacterial Proteins; Biofilms; Calcium; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Magnesium; Vibrio cholerae

The opportunistic nosocomial pathogen Clostridioides difficile exhibits phenotypic heterogeneity through phase variation, a stochastic, reversible process that modulates expression. In C. difficile, multiple sequences in the genome undergo inversion through site-specific recombination. Two such loci lie upstream of pdcB and pdcC, which encode phosphodiesterases (PDEs) that degrade the signaling molecule c-di-GMP. Numerous phenotypes are influenced by c-di-GMP in C. difficile including cell and colony morphology, motility, colonization, and virulence. In this study, we aimed to assess whether PdcB phase varies, identify the mechanism of regulation, and determine the effects on intracellular c-di-GMP levels and regulated phenotypes. We found that expression of pdcB is heterogeneous and the orientation of the invertible sequence, or 'pdcB switch', determines expression. The pdcB switch contains a promoter that when properly oriented promotes pdcB expression. Expression is augmented by an additional promoter upstream of the pdcB switch. Mutation of nucleotides at the site of recombination resulted in phase-locked strains with significant differences in pdcB expression. Characterization of these mutants showed that the pdcB locked-ON mutant has reduced intracellular c-di-GMP compared to the locked-OFF mutant, consistent with increased and decreased PdcB activity, respectively. These alterations in c-di-GMP had concomitant effects on multiple known c-di-GMP regulated processes, indicating that phase variation of PdcB allows C. difficile to coordinately diversify multiple phenotypes in the population to enhance survival.

Topics: Bacterial Proteins; Biofilms; Clostridioides difficile; Cyclic GMP; Gene Expression Regulation, Bacterial; Phase Variation; Phosphoric Diester Hydrolases

Staphylococcus aureus is a leading cause for morbidity and mortality associated with skin and burn wound infections. Therapeutic options for methicillin-resistant S. aureus (MRSA) have dwindled and therefore alternative treatments are urgently needed. In this study, the immuno-stimulating and anti-MRSA effects of cyclic di-guanosine monophosphate (c-di-GMP), a uniquely bacterial second messenger and immuno-modulator, were investigated in HaCaT human epidermal keratinocytes and a murine skin wound infection model.. Stimulation of HaCaT cells with 125 μM c-di-GMP for 12 h prior to MRSA challenge resulted in a 20-fold reduction in bacterial colonization compared with untreated control cells, which was not the result of a direct c-di-GMP toxic effect, since bacterial viability was not affected by this dose in the absence of HaCaT cells. C-di-GMP-stimulated or MRSA-challenged HaCaT cells displayed enhanced secretion of the antimicrobial peptides human β-defensin 1 (hBD-1), hBD-2, hBD-3 and LL-37, but for hBD1 and LL-37 the responses were additive in a c-di-GMP-dose-dependent manner. Secretion of the chemokines CXCL1 and CXCL8 was also elevated after stimulation of HaCaT cells with lower c-di-GMP doses and peaked at a dose of 5 μM. Finally, pre-treatment of mice with a 200 nmol dose of c-di-GMP 24 h before a challenge with MRSA in skin wound infection model resulted in a major reduction (up to 1,100-fold by day 2) in bacterial CFU counts recovered from challenged skin tissue sections compared PBS-treated control animals. Tissue sections displayed inflammatory cell infiltration and enhanced neutrophil influx in the c-di-GMP pre-treated animals, which might account for the reduced ability of MRSA to colonize c-di-GMP pre-treated mice.. These results demonstrate that c-di-GMP is a potent immuno-modulator that can stimulate anti-MRSA immune responses in vivo and might therefore be a suitable alternative prophylactic or therapeutic agent for MRSA skin or burn wound infections.

Topics: Adjuvants, Immunologic; Animals; Burns; Cyclic GMP; Disease Models, Animal; Humans; Immunity, Innate; Keratinocytes; Methicillin-Resistant Staphylococcus aureus; Mice; Staphylococcal Skin Infections

The AmrZ/FleQ hub has been identified as a central node in the regulation of environmental adaption in the plant growth-promoting rhizobacterium and model for rhizosphere colonization Pseudomonas ogarae F113. AmrZ is involved in the regulation of motility, biofilm formation, and bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) turnover, among others, in this bacterium. The mutants in amrZ have a pleiotropic phenotype with distinguishable colony morphology, reduced biofilm formation, increased motility, and are severely impaired in competitive rhizosphere colonization. Here, RNA-Seq and qRT-PCR gene expression analyses revealed that AmrZ regulates many genes related to the production of extracellular matrix (ECM) components at the transcriptional level. Furthermore, overproduction of c-di-GMP in an amrZ mutant, by ectopic production of the Caulobacter crescentus constitutive diguanylate cyclase PleD*, resulted in increased expression of many genes implicated in the synthesis of ECM components. The overproduction of c-di-GMP in the amrZ mutant also suppressed the biofilm formation and motility phenotypes, but not the defect in competitive rhizosphere colonization. These results indicate that although biofilm formation and motility are mainly regulated indirectly by AmrZ, through the modulation of c-di-GMP levels, the implication of AmrZ in rhizosphere competitive colonization occurs in a c-di-GMP-independent manner.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Extracellular Matrix; Gene Expression Regulation, Bacterial; Pseudomonas

Overproduction of the exopolysaccharide alginate contributes to the pathogenicity and antibiotic tolerance of Pseudomonas aeruginosa in chronic infections. The second messenger, c-di-GMP, is a positive regulator of the production of various biofilm matrix components and is known to regulate alginate synthesis at the posttranslational level in P. aeruginosa. We provide evidence that c-di-GMP also regulates transcription of the alginate operon in P. aeruginosa. Previous work has shown that transcription of the alginate operon is regulated by nine different proteins, AmrZ, AlgP, IHFα, IHFβ, CysB, Vfr, AlgR, AlgB, and AlgQ, and we investigated if some of these proteins function as a c-di-GMP effector. We found that deletion of

Topics: Alginates; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Humans; Operon; Pseudomonas aeruginosa

Cyclic di-guanosine monophosphate (c-di-GMP) is a ubiquitous second messenger, i.e. essential to bacterial adaptation to environments. Cellular c-di-GMP level is regulated by the diguanylate cyclases and the phosphodiesterases, and the signal transduction depends on its receptors. In Xanthomonas oryzae pv. oryzae strain PXO99A, 37 genes were predicted to encode GGDEF, EAL, GGDEF/EAL, HD-GYP, FleQ, MshE, PilZ, CuxR, Clp, and YajQ proteins that may be involved in c-di-GMP turnover or function as c-di-GMP receptors. Although the functions of some of these genes have been studied, but the rest have not been extensively studied. Here, we deleted these 37 genes from PXO99A and analyzed the virulence, motility, biofilm, and EPS production of these mutants. Our results show that most of these genes are required for PXO99A virulence, motility, biofilm formation, or exopolysaccharide production. Although some of them have been reported in previous studies, we found four novel genes (gedpX8, gdpX11, pliZX4, and yajQ) are implicated in X. oryzae pv. oryzae virulence. Our data demonstrate that c-di-GMP signaling is vital for X. oryzae pv. oryzae virulence and some virulence-related factors production, but there is no positive correlation between them in most cases. Taken together, our systematic research provides a new light to understand the c-di-GMP signaling network in X. oryzae pv. oryzae.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Oryza; Signal Transduction; Virulence; Xanthomonas

Pseudomonas aeruginosa is generally believed to establish biofilm-associated infections under the regulation of the secondary messenger c-di-GMP. To evaluate P. aeruginosa biofilm physiology during ocular infections, comparative transcriptomic analysis was performed on wild-type P. aeruginosa PAO1, a Δ

Topics: Animals; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Mice; Pseudomonas aeruginosa; Type III Secretion Systems

Cyclic-di-GMP (c-di-GMP) is an essential bacterial second messenger that regulates biofilm formation and pathogenicity. To study the global regulatory effect of individual components of the c-di-GMP metabolic system, we deleted all 12 diguanylate cyclase (dgc) and phosphodiesterase (pde)-encoding genes in E. amylovora Ea1189 (Ea1189Δ12). Ea1189Δ12 was impaired in surface attachment due to a transcriptional dysregulation of the type IV pilus and the flagellar filament. A transcriptomic analysis of surface-exposed WT Ea1189 and Ea1189Δ12 cells indicated that genes involved in metabolism, appendage generation and global transcriptional/post-transcriptional regulation were differentially regulated in Ea1189Δ12. Biofilm formation was regulated by all 5 Dgcs, whereas type III secretion and disease development were differentially regulated by specific Dgcs. A comparative transcriptomic analysis of Ea1189Δ8 (lacks all five enzymatically active dgc and 3 pde genes) against Ea1189Δ8 expressing specific dgcs, revealed the presence of a dual modality of spatial and global regulatory frameworks in the c-di-GMP signaling network.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Erwinia amylovora; Escherichia coli Proteins; Gene Deletion; Gene Expression Regulation, Bacterial; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases

Pseudomonas aeruginosa uses multiple protein regulators that work in tandem to control the production of a wide range of virulence factors and facilitate rapid adaptation to diverse environmental conditions. In this opportunistic pathogen, ToxR was known to positively regulate the production of the major virulence factor exotoxin A and now, through analysis of genetic changes between two sublines of P. aeruginosa PAO1 and functional complementation of swarming, we have identified a previously unknown role of ToxR in surface-associated motility in P. aeruginosa. Further analysis revealed that ToxR had an impact on swarming motility by regulating the Rhl quorum sensing system and subsequent production of rhamnolipid surfactants. Additionally, ToxR was found to tightly bind cyclic diguanylate (c-di-GMP) and negatively affect traits controlled by this second messenger including reducing biofilm formation and the expression of Psl and Pel exopolysaccharides, necessary for attachment and sessile communities matrix scaffolding, in P. aeruginosa. Moreover, a link between the post-transcriptional regulator RsmA and toxR expression via the alternative sigma factor PvdS, induced under iron-limiting conditions, is established. This study reveals the importance of ToxR in a sophisticated regulation of free-living and biofilm-associated lifestyles, appropriate for establishing acute or chronic P. aeruginosa infections.

Topics: Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphoric Diester Hydrolases; Pseudomonas aeruginosa

A subpopulation of small-colony variants (SCVs) is a frequently observed feature of Pseudomonas aeruginosa isolates obtained from colonized cystic fibrosis lungs. Since most SCVs have until now been isolated from clinical samples, it remains unclear how widespread the ability of P. aeruginosa strains to develop this phenotype is and what the genetic mechanism(s) behind the emergence of SCVs are according to the origin of the isolate. In the present work, we investigated the ability of 22 P. aeruginosa isolates from various environmental origins to spontaneously adopt an SCV-like smaller alternative morphotype distinguishable from that of the ancestral parent strain under laboratory culture conditions. We found that all the P. aeruginosa strains tested could adopt an SCV phenotype, regardless of their origin. Whole-genome sequencing of SCVs obtained from clinical and environmental sources revealed single mutations exclusively in two distinct c-di-GMP signaling pathways, the Wsp and YfiBNR pathways. We conclude that the ability to switch to an SCV phenotype is a conserved feature of P. aeruginosa and results from the acquisition of a stable genetic mutation, regardless of the origin of the strain.

Topics: Cyclic GMP; Cystic Fibrosis; Humans; Mutation; Phenotype; Pseudomonas aeruginosa; Pseudomonas Infections

Transitions between individual and communal lifestyles allow bacteria to adapt to changing environments. Bacteria must integrate information encoded in multiple sensory cues to appropriately undertake these transitions. Here, we investigate how two prevalent sensory inputs converge on biofilm morphogenesis: quorum sensing, which endows bacteria with the ability to communicate and coordinate group behaviors, and second messenger c-di-GMP signaling, which allows bacteria to detect and respond to environmental stimuli. We use Vibrio cholerae as our model system, the autoinducer AI-2 to modulate quorum sensing, and the polyamine norspermidine to modulate NspS-MbaA-mediated c-di-GMP production. Individually, AI-2 and norspermidine drive opposing biofilm phenotypes, with AI-2 repressing and norspermidine inducing biofilm formation. Surprisingly, however, when AI-2 and norspermidine are simultaneously detected, they act synergistically to increase biofilm biomass and biofilm cell density. We show that this effect is caused by quorum-sensing-mediated activation of

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Morphogenesis; Quorum Sensing; Spermidine; Transcription Factors; Vibrio cholerae

This study aimed to investigate the regulation of histone-like nucleoid structuring protein (H-NS) on biofilm formation and cyclic diguanylate (c-di-GMP) synthesis in. Regulatory mechanisms were analyzed by the combined utilization of crystal violet staining, quantification of c-di-GMP, quantitative real-time polymerase chain reaction, LacZ fusion, and electrophoretic-mobility shift assay.. One of the mechanisms by which H-NS represses the biofilm formation by

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Gentian Violet; Histones; Vibrio parahaemolyticus

Modulation of the intracellular cyclic di-GMP (c-di-GMP) pool is central to the formation of structured bacterial communities. Genome annotations predict the presence of dozens of conserved c-di-GMP catalytic enzymes in many bacterial species, but the functionality and regulatory control of the vast majority remain underexplored. Here, we begin to fill this gap by utilizing an experimental evolution system in Pseudomonas fluorescens Pf0-1, which repeatedly produces a unique social behavior through bidirectional transitions between two distinct phenotypes converging on c-di-GMP modulation. Parallel evolution of 33 lineages captured 147 unique mutations among 191 evolved isolates in genes that are empirically demonstrated, bioinformatically predicted, or previously unknown to impact the intracellular pool of c-di-GMP. Quantitative chemistry confirmed that each mutation causing the phenotypic shift either amplifies or reduces c-di-GMP production. We identify missense or in-frame deletion mutations in numerous diguanylate cyclase genes that largely fall outside the conserved catalytic domain. We also describe a novel relationship between a regulatory component of branched-chain amino acid biosynthesis and c-di-GMP production, and predict functions of several other unexpected proteins that clearly impact c-di-GMP production. Sequential mutations that continuously disrupt or recover c-di-GMP production across discrete functional elements suggest a complex and underappreciated interconnectivity within the c-di-GMP regulome of P. fluorescens.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Pseudomonas fluorescens

Streptomyces are our principal source of antibiotics, which they generate concomitant with a complex developmental transition from vegetative hyphae to spores. c-di-GMP acts as a linchpin in this transition by binding and regulating the key developmental regulators, BldD and WhiG. Here we show that c-di-GMP also binds the glycogen-debranching-enzyme, GlgX, uncovering a direct link between c-di-GMP and glycogen metabolism in bacteria. Further, we show c-di-GMP binding is required for GlgX activity. We describe structures of apo and c-di-GMP-bound GlgX and, strikingly, their comparison shows c-di-GMP induces long-range conformational changes, reorganizing the catalytic pocket to an active state. Glycogen is an important glucose storage compound that enables animals to cope with starvation and stress. Our in vivo studies reveal the important biological role of GlgX in Streptomyces glucose availability control. Overall, we identify a function of c-di-GMP in controlling energy storage metabolism in bacteria, which is widespread in Actinobacteria.

Topics: Allosteric Regulation; Animals; Anti-Bacterial Agents; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Glucose; Glycogen; Second Messenger Systems; Streptomyces

Biofilm dispersion can be triggered by the application of dispersing agents such as nitric oxide (NO)-donors, resulting in the release of biofilm-dispersed cells into the environment. In this work, biofilm-dispersed cells were obtained by adding different concentrations of NO-donor sodium nitroprusside (0.5, 5, 50 µM, and 2.5 mM of SNP) to batch cultures of pre-formed

Topics: Bacterial Proteins; Batch Cell Culture Techniques; Biofilms; Cyclic GMP; Escherichia coli; Gene Expression Regulation, Bacterial; Nitroprusside; Plankton

Bacteria in biofilms are embedded in extracellular matrix and display low metabolic activity, partly due to insufficient diffusive exchange of metabolic substrate. The extracellular matrix and low metabolic activity both contribute to the high antibiotic tolerance-the hallmark of biofilm bacteria. The second messenger molecule, c-di-GMP, regulates biofilm development in Pseudomonas aeruginosa, where high internal levels lead to biofilm formation and low levels are associated with planktonic bacteria. Using a microcalorimetric approach, we show that c-di-GMP signaling is a major determinant of the metabolic activity of P. aeruginosa, both in planktonic culture and in two biofilm models. The high c-di-GMP content of biofilm bacteria forces them to rapidly spend a large amount of energy on the production of exopolysaccharides, resulting in a subsequent low metabolic state. This suggests that the low metabolic state of bacteria in mature biofilms, to some extent, is a consequence of a c-di-GMP-regulated survival strategy.

Topics: Anti-Bacterial Agents; Bacteria; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa

Cyclic di-GMP (c-di-GMP) transduces extracellular stimuli into intracellular responses, coordinating a plethora of important biological processes. Low levels of c-di-GMP are often associated with highly virulent behavior that depends on the type III secretion system (T3SS) effectors encoded, whereas elevated levels of c-di-GMP lead to the repression of T3SSs. However, extracellular signals that modulate c-di-GMP metabolism to control T3SSs and c-di-GMP effectors that relay environmental stimuli to changes in T3SS activity remain largely obscure. Here, we show that the quorum sensing signal autoinducer-2 (AI-2) induces c-di-GMP synthesis via a GAPES1 domain-containing diguanylate cyclase (DGC) YeaJ to repress T3SS-1 gene expression in Salmonella enterica serovar Typhimurium. YeaJ homologs capable of sensing AI-2 are present in many other species belonging to Enterobacterales. We also reveal that taurocholate and taurodeoxycholate bind to the sensory domain of the DGC YedQ to induce intracellular accumulation of c-di-GMP, thus repressing the expression of T3SS-1 genes. Further, we find that c-di-GMP negatively controls the function of T3SSs through binding to the widely conserved CesD/SycD/LcrH family of T3SS chaperones. Our results support a model in which bacteria sense changes in population density and host-derived cues to regulate c-di-GMP synthesis, thereby modulating the activity of T3SSs via a c-di-GMP-responsive T3SS chaperone.

Topics: Bacterial Proteins; Bile Acids and Salts; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Molecular Chaperones; Salmonella typhimurium

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger required for normal physiology as well as survival under hypoxic and reductive stress conditions of mycobacterial cells. Complete degradation of c-di-GMP is necessary for signal termination and maintaining its homeostasis inside the cells. Homeostasis of c-di-GMP in mycobacteria is brought about by the bifunctional diguanylate cyclase (DGC) that synthesizes c-di-GMP from two molecules of GTP and also catalyses the asymmetric cleavage of c-di-GMP to linear pGpG through its phosphodiesterase activity. However, the mycobacterial enzyme for the last step of degradation from pGpG to GMP has not been characterized thus far. Here, we present the identification of oligoribonuclease (Orn) as the most likely phosphodiesterase to degrade pGpG to GMP through AlphaFold-empowered structural homology that exhibited in vitro phosphodiesterase activity on pGpG substrates. In order to understand the physiological role of Orn in mycobacteria, we created a deletion mutant of orn in M. smegmatis and analysed the phenotypes that are associated with c-di-GMP signaling. We find that orn plays important roles in vivo and is required not only for proper growth of M. smegmatis in normal and stress conditions but also for biofilm formation.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Deletion; Gene Expression Regulation, Bacterial; Phosphoric Diester Hydrolases

Many bacterial species use the secondary messenger, c-di-GMP, to promote the production of biofilm matrix components. In Pseudomonas aeruginosa, c-di-GMP production is stimulated upon initial surface contact and generally remains high throughout biofilm growth. Transcription of several gene clusters, including the Sia signal transduction system, are induced in response to high cellular levels of c-di-GMP. The output of this system is SiaD, a diguanylate cyclase whose activity is induced in the presence of the detergent SDS. Previous studies demonstrated that Sia-mediated cellular aggregation is a key feature of P. aeruginosa growth in the presence of SDS. Here, we show that the Sia system is important for producing low levels of c-di-GMP when P. aeruginosa is growing planktonically. In addition, we show that Sia activity is important for maintaining cell-associated Psl in planktonic populations. We also demonstrate that Sia mutant strains have reduced cell-associated Psl and a surface attachment-deficient phenotype. The Sia system also appears to posttranslationally impact cell-associated Psl levels. Collectively, our findings suggest a novel role for the Sia system and c-di-GMP in planktonic populations by regulating levels of cell-associated Psl.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a bacterial second messenger that affects diverse processes in different bacteria, including the cell cycle, motility, and biofilm formation. Its cellular levels are controlled by the opposing activities of two types of enzymes, with synthesis by diguanylate cyclases containing a GGDEF domain and degradation by phosphodiesterases containing either an HD-GYP or an EAL domain. These enzymes are ubiquitous in bacteria with up to 50 encoded in some genomes, the specific functions of which are mostly unknown.. We used comparative analyses to identify genomic patterns among genes encoding proteins with GGDEF, EAL, and HD-GYP domains in five orders of the class Alphaproteobacteria. GGDEF-containing sequences and GGDEF-EAL hybrids were the most abundant and had the highest diversity of co-occurring auxiliary domains while EAL and HD-GYP containing sequences were less abundant and less diverse with respect to auxiliary domains. There were striking patterns in the chromosomal localizations of the genes found in two of the orders. The Rhodobacterales' EAL-encoding genes and Rhizobiales' GGDEF-EAL-encoding genes showed opposing patterns of distribution compared to the GGDEF-encoding genes. In the Rhodobacterales, the GGDEF-encoding genes showed a tri-modal distribution with peaks mid-way between the origin (ori) and terminus (ter) of replication and at ter while the EAL-encoding genes peaked near ori. The patterns were more complex in the Rhizobiales, but the GGDEF-encoding genes were biased for localization near ter.. The observed patterns in the chromosomal localizations of these genes suggest a coupling of synthesis and hydrolysis of c-di-GMP with the cell cycle. Moreover, the higher proportions and diversities of auxiliary domains associated with GGDEF domains and GGDEF-EAL hybrids compared to EAL or HD-GYP domains could indicate that more stimuli affect synthesis compared to hydrolysis of c-di-GMP.

Topics: Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genomics; Phosphoric Diester Hydrolases; Signal Transduction

Cyclic dinucleotides (CDNs) are key secondary messenger molecules produced by cyclic dinucleotide synthases that trigger various cellular signaling cascades from bacteria to vertebrates. In mammals, cyclic GMP-AMP synthase (cGAS) has been shown to bind to intracellular DNA and catalyze the production of the dinucleotide 2'3' cGAMP, which signals downstream effectors to regulate immune function, interferon signaling, and the antiviral response. Despite the importance of CDNs, sensitive and accurate methods to measure their levels in vivo are lacking. Here, we report a novel LC-MS/MS method to quantify CDNs in vivo. We characterized the mass spectrometric behavior of four different biologically relevant CDNs (c-di-AMP, c-di-GMP, 3'3' cGAMP, 2'3' cGAMP) and provided a means of visually representing fragmentation resulting from collision-induced dissociation at different energies using collision energy breakdown graphs. We then validated the method and quantified CDNs in two in vivo systems, the bacteria Escherichia coli OP50 and the killifish Nothobranchius furzeri. We found that optimization of LC-MS/MS parameters is crucial to sensitivity and accuracy. These technical advances should help illuminate physiological and pathological roles of these CDNs in in vivo settings. Graphical abstract.

Topics: Animals; Chromatography, Liquid; Cyclic GMP; Dinucleoside Phosphates; Escherichia coli; Fundulidae; Nucleotides, Cyclic; Tandem Mass Spectrometry

Many pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) and lipoteichoic acid, are potent immunostimulatory molecules and promote the expression of cyclooxygenase 2 (COX-2). While the production of COX-2, and ultimately prostaglandin E

Topics: Animals; Beclin-1; Cell Line; Cyclic GMP; Cyclooxygenase 2; Dinucleoside Phosphates; Gene Expression Regulation; Guanine; Immunity, Innate; Interferon Regulatory Factor-3; Interferon Type I; Macrophages; Mice; NF-kappa B; Oligonucleotides; Phosphorylation; Prostaglandins; Signal Transduction

Soil bacteria often harbour various toxins to against eukaryotic or prokaryotic. Diffusible signal factors (DSFs) represent a unique group of quorum sensing (QS) chemicals that modulate interspecies competition in bacteria that do not produce antibiotic-like molecules. However, the molecular mechanism by which DSF-mediated QS systems regulate antibiotic production for interspecies competition remains largely unknown in soil biocontrol bacteria. In this study, we find that the necessary QS system component protein RpfG from Lysobacter, in addition to being a cyclic dimeric GMP (c-di-GMP) phosphodiesterase (PDE), regulates the biosynthesis of an antifungal factor (heat-stable antifungal factor, HSAF), which does not appear to depend on the enzymatic activity. Interestingly, we show that RpfG interacts with three hybrid two-component system (HyTCS) proteins, HtsH1, HtsH2, and HtsH3, to regulate HSAF production in Lysobacter. In vitro studies show that each of these proteins interacted with RpfG, which reduced the PDE activity of RpfG. Finally, we show that the cytoplasmic proportions of these proteins depended on their phosphorylation activity and binding to the promoter controlling the genes implicated in HSAF synthesis. These findings reveal a previously uncharacterized mechanism of DSF signalling in antibiotic production in soil bacteria.

Topics: Antifungal Agents; Cyclic GMP; Lysobacter; Quorum Sensing; Soil Microbiology

ExlA is a highly virulent pore-forming toxin that has been recently discovered in outlier strains from

Topics: Animals; Bacterial Proteins; Bacterial Toxins; Cell Membrane; Cyclic GMP; Enzyme-Linked Immunosorbent Assay; Mice; Pseudomonas aeruginosa; Pseudomonas Infections

Clostridioides difficile is the causative agent of antibiotic-associated diarrhea and is the leading cause of nosocomial infection in developed countries. An increasing number of C. difficile infections are attributed to epidemic strains that produce more toxins and spores. C. difficile spores are the major factor for the transmission and persistence of the organism. Previous studies have identified global regulators that influence sporulation in C. difficile. This study discovers that PdcB, a phosphodiesterase, enhances sporulation in C. difficile strain UK1. Through genetic and biochemical assays, we show that phase-variable expression of pdcB results in hypo- and hyper-sporulation phenotypes. In the "ON" orientation, the identified promotor is in the right orientation to drive the expression of pdcB. Production of the PdcB phosphodiesterase reduces the intracellular cyclic-di-GMP (c-di-GMP) concentration, resulting in a hyper-sporulation phenotype. Loss of PdcB due to the pdcB promoter being in the OFF orientation or mutation of pdcB results in increased c-di-GMP levels and a hypo-sporulation phenotype. Additionally, we demonstrate that CodY binds to the upstream region of pdcB. DNA inversion reorients the CodY binding site so that in the OFF orientation, CodY binds a site that is upstream of the pdcB promoter and can further repress gene expression.

Topics: Bacterial Proteins; Clostridioides difficile; Clostridium Infections; Cyclic GMP; Gene Expression Regulation, Bacterial; Mutation; Phosphoric Diester Hydrolases; Promoter Regions, Genetic; Spores, Bacterial; Transcription Factors

Cyclic-di-GMP plays crucial role in the cell cycle regulation of the α-Proteobacterium Caulobacter crescentus. Here we investigated its role in the α-Proteobacterium Brucella abortus, a zoonotic intracellular pathogen. Surprisingly, deletion of all predicted cyclic-di-GMP synthesizing or degrading enzymes did not drastically impair the growth of B. abortus, nor its ability to grow inside cell lines. As other Rhizobiales, B. abortus displays unipolar growth from the new cell pole generated by cell division. We found that the phosphodiesterase PdeA, the ortholog of the essential polar growth factor RgsP of the Rhizobiale Sinorhizobium meliloti, is required for rod shape integrity but is not essential for B. abortus growth. Indeed, the radius of the pole is increased by 31 ± 1.7% in a ΔpdeA mutant, generating a coccoid morphology. A mutation in the cyclic-di-GMP phosphodiesterase catalytic site of PdeA does not generate the coccoid morphology and the ΔpdeA mutant kept the ability to recruit markers of new and old poles. However, the presence of PdeA is required in an intra-nasal mouse model of infection. In conclusion, we propose that PdeA contributes to bacterial morphology and virulence in B. abortus, but it is not crucial for polarity and asymmetric growth.

Topics: Animals; Bacterial Proteins; Brucella abortus; Brucellosis; Cyclic GMP; Female; Gene Expression Regulation, Bacterial; Humans; Mice; Mice, Inbred C57BL; Phosphoric Diester Hydrolases

The marine bacterium Vibrio fischeri colonizes its host, the Hawaiian bobtail squid, in a manner requiring both bacterial biofilm formation and motility. The decision to switch between sessile and motile states is often triggered by environmental signals and regulated by the widespread signaling molecule c-di-GMP. Calcium is an environmental signal previously shown to affect both biofilm formation and motility by V. fischeri. In this study, we investigated the link between calcium and c-di-GMP, determining that calcium increases intracellular c-di-GMP dependent on a specific diguanylate cyclase,

Topics: Aliivibrio fischeri; Bacterial Proteins; Biofilms; Calcium; Calcium Signaling; Cellulose; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Hawaii; Phosphorus-Oxygen Lyases; Transcription Factors; Vibrio cholerae

Bifunctional enzymes, which contain two domains with opposing enzymatic activities, are widely distributed in bacteria, but the regulatory mechanism(s) that prevent futile cycling are still poorly understood. The recently described bifunctional enzyme, DcpG, exhibits unusual heme properties and is surprisingly able to differentially regulate its two cyclic dimeric guanosine monophosphate (c-di-GMP) metabolic domains in response to heme gaseous ligands. Mutagenesis of heme-edge residues was used to probe the heme pocket and resulted in decreased O

Topics: Amino Acid Sequence; Amino Acid Substitution; Bacterial Proteins; Binding Sites; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression; Heme; Hemeproteins; Kinetics; Models, Molecular; Oxygen; Paenibacillus; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Multimerization; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Signal Transduction; Static Electricity; Structure-Activity Relationship; Substrate Specificity

The formation of dormant spores is essential for the anaerobic pathogen Clostridioides difficile to survive outside the host gastrointestinal tract. The regulatory pathways and environmental signals that initiate C. difficile spore formation within the host are not well understood. One second-messenger signaling molecule, cyclic diguanylate (c-di-GMP), modulates several physiological processes important for C. difficile pathogenesis and colonization, but the impact of c-di-GMP on sporulation is unknown. In this study, we investigated the contribution of c-di-GMP to C. difficile sporulation. The overexpression of a gene encoding a diguanylate cyclase,

Topics: Bacterial Proteins; Bacterial Toxins; Clostridioides difficile; Cyclic GMP; Gene Expression Regulation, Bacterial; Spores, Bacterial

Bacteriophages are ubiquitous parasites of bacteria and major drivers of bacterial ecology and evolution. Despite an ever-growing interest in their biotechnological and therapeutic applications, detailed knowledge of the molecular mechanisms underlying phage-host interactions remains scarce. Here, we show that bacteriophage N4 exploits a novel surface glycan (NGR) as a receptor to infect its host Escherichia coli. We demonstrate that this process is regulated by the second messenger c-di-GMP and that N4 infection is specifically stimulated by the diguanylate cyclase DgcJ, while the phosphodiesterase PdeL effectively protects E. coli from N4-mediated killing. PdeL-mediated protection requires its catalytic activity to reduce c-di-GMP and includes a secondary role as a transcriptional repressor. We demonstrate that PdeL binds to and represses the promoter of the

Topics: Bacteriophage N4; Carbohydrate Epimerases; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Glucans; Nucleotidyltransferases; Operon; Polysaccharides, Bacterial

Vibrio parahaemolyticus is a moderately halophilic, salt-requiring organism that exhibits optimal growth at approximately 3% salt. Thus, salinity stress is one of the most important stimuli during its lifecycle. The bacterium possesses a strong ability to form biofilms on surfaces, which are thought to be involved in protecting it from adverse environmental conditions. In the present study, salinity-dependent biofilm formation by V. parahaemolyticus was investigated by combining crystal violet staining, colony morphology, intracellular c-di-GMP quantification and quantitative PCR. The results showed that biofilm formation by V. parahaemolyticus was significantly enhanced in low salinity growth conditions and was affected by incubation time. In addition, low salinity reduced intracellular c-di-GMP degradation in V. parahaemolyticus. Transcription of genes encoding ScrABC and ScrG proteins, which are involved in intracellular c-di-GMP metabolism, was inhibited by low salinity growth conditions. Thus, reduced intracellular c-di-GMP degradation in V. parahaemolyticus in low salinity growth conditions may be mediated by repression of scrG and scrABC transcription. Taken together, these results demonstrated for the first time that salinity regulates biofilm formation and c-di-GMP production in V. parahaemolyticus.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Salinity; Vibrio parahaemolyticus

Curli, a major component of the bacterial biofilms in the intestinal tract, activates pattern recognition receptors and triggers joint inflammation after infection with Salmonella enterica serovar Typhimurium. The factors that allow

Topics: Bacterial Proteins; Biofilms; Cues; Cyclic GMP; Flagella; Gene Expression Regulation, Bacterial; Humans; Inflammation; Nitrates; Salmonella enterica; Salmonella typhimurium; Serogroup

Myxococcus xanthus has a nutrient-regulated biphasic life cycle forming predatory swarms in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. The second messenger 3'-5', 3'-5 cyclic di-GMP (c-di-GMP) is essential during both stages of the life cycle; however, different enzymes involved in c-di-GMP synthesis and degradation as well as several c-di-GMP receptors are important during distinct life cycle stages. To address this stage specificity, we determined transcript levels using transcriptome sequencing (RNA-seq) and transcription start sites using Cappable sequencing (Cappable-seq) during growth and development genome wide. All 70 genes encoding c-di-GMP-associated proteins were expressed, with 28 upregulated and 10 downregulated during development. Specifically, the three genes encoding enzymatically active proteins with a stage-specific function were expressed stage specifically. By combining operon mapping with published chromatin immunoprecipitation sequencing (ChIP-seq) data for MrpC (M. Robinson, B. Son, D. Kroos, L. Kroos, BMC Genomics 15:1123, 2014, http://dx.doi.org/10.1186/1471-2164-15-1123), the cAMP receptor protein (CRP)-like master regulator of development, we identified nine developmentally regulated genes as regulated by MrpC. In particular, MrpC directly represses the expression of

Topics: Bacterial Proteins; Cyclic AMP Receptor Protein; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Myxococcus xanthus; Nucleotides; Phosphoric Diester Hydrolases

The resistance of bacteria to antibiotics is a major public health issue. Klebsiella pneumoniae is a type exemplification of multi-resistant enterobacteria. Its high biofilm forming capacity is a major factor in the recurrent infection of the intestinal tract. In this study, the intrinsic mechanism of secondary growth of K. pneumoniae in response to antibiotics and the inhibition effect of probiotic supernatant on biofilm formation after antibiotic treatment were investigated in a polyester nonwoven chemostat bioreactor. The experimental results showed that the c-di-GMP content in the cells increased after treatment with levofloxacin, leading to the formation of a thick biofilm due to an increase in the production of extracellular polymer substance (EPS) and type 3 fimbriae. Biofilm prevents the mass transfer of levofloxacin and protects K. pneumoniae cells from being killed by levofloxacin. Under suitable conditions, K. pneumoniae cells on the biofilm enter into the suspension for secondary growth. Moreover, the inhibition of probiotic supernatant on the biofilm formation was mainly due to the reduced expression of yfiN and mrkJ genes, and the decreased concentration of c-di-GMP in cells, as well as the less secretion of EPS. At the same time, the decrease in the concentration of c-di-GMP also reduced the expression of the mrkABCDF gene and prevented the synthesis of the type 3 fimbriae. The results would help to understand the mechanism of antibiotic resistance of pathogenic bacteria and to provide evidence to address this problem through the use of probiotics.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Klebsiella pneumoniae; Probiotics

Bacterial biofilms provide high cell density and a superior adaptation and protection from stress conditions compared to planktonic cultures, making them a very promising approach for bioremediation. Several Rhodococcus strains can desulfurize dibenzothiophene (DBT), a major sulphur pollutant in fuels, reducing air pollution from fuel combustion. Despite multiple efforts to increase Rhodococcus biodesulfurization activity, there is still an urgent need to develop better biocatalysts. Here, we implemented a new approach that consisted in promoting Rhodococcus erythropolis biofilm formation through the heterologous expression of a diguanylate cyclase that led to the synthesis of the biofilm trigger molecule cyclic di-GMP (c-di-GMP). R. erythropolis biofilm cells displayed a significantly increased DBT desulfurization activity when compared to their planktonic counterparts. The improved biocatalyst formed a biofilm both under batch and continuous flow conditions which turns it into a promising candidate for the development of an efficient bioreactor for the removal of sulphur heterocycles present in fossil fuels.

Topics: Biofilms; Cyclic GMP; Rhodococcus

The bacterial secondary messenger bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) has been implicated in the pathogenesis of Vibrio cholerae, due to its significant role in regulating the virulence, biofilm formation and motility of the host organism. The VC0395_0300 protein from V. cholerae, possessing a GGEEF sequence has been established as a diguanylate cyclase (DGC) capable of catalyzing the conversion of two GTP molecules to form cyclic-di-GMP. This in turn, plays a crucial role in allowing the organism to adopt a dual lifestyle, thriving both in human and aquatic systems. The difficulty in procuring sufficient amounts of homogenous soluble protein for structural assessment of the GGDEF domain in VC0395_0300 and the lack of soluble protein yield, prompted the truncation into smaller constructs (Sebox31 and Sebox32) carrying the GGDEF domain. The truncates retained their diguanylate cyclase activity comparable to the wild type, and were able to form biofilms as well. Fluorescence and circular dichroism spectroscopy measurements revealed that the basic structural elements do not show significant changes in the truncated proteins as compared to the full-length. This has also been confirmed using homology modeling and molecular docking of the wild type and truncates. This led us to conclude that the truncated constructs retain their activity in spite of the deletions in the N terminal region. This is supportive of the fact that DGC activity in GGDEF proteins is predominantly dependent on the presence of the conserved GGD(/E)EF domain and its interaction with GTP.

Topics: Amino Acid Sequence; Bacterial Proteins; Cholera; Cyclic GMP; Escherichia coli Proteins; Guanosine Triphosphate; Humans; Models, Molecular; Phosphorus-Oxygen Lyases; Vibrio cholerae

Bacteria use small signalling molecules such as (p)ppGpp or c-di-GMP to tune their physiology in response to environmental changes. It remains unclear whether these regulatory networks operate independently or whether they interact to optimize bacterial growth and survival. We report that (p)ppGpp and c-di-GMP reciprocally regulate the growth of Caulobacter crescentus by converging on a single small-molecule-binding protein, SmbA. While c-di-GMP binding inhibits SmbA, (p)ppGpp competes for the same binding site to sustain SmbA activity. We demonstrate that (p)ppGpp specifically promotes Caulobacter growth on glucose, whereas c-di-GMP inhibits glucose consumption. We find that SmbA contributes to this metabolic switch and promotes growth on glucose by quenching the associated redox stress. The identification of an effector protein that acts as a central regulatory hub for two global second messengers opens up future studies on specific crosstalk between small-molecule-based regulatory networks.

Topics: Binding Sites; Binding, Competitive; Caulobacter crescentus; Cyclic GMP; Gene Expression Regulation, Bacterial; Glucose; Guanosine Pentaphosphate; Oxidation-Reduction; Second Messenger Systems; Signal Transduction; Transferases

Bacteria can form a biofilm composed of diverse bacterial microorganism, which work as a barrier to protect from threats, such as antibiotics and host immunity system. The formation of biofilms significantly impairs the efficacy of antibiotics against pathogenic bacteria. It is also a serious problem to be solved that the emergence of multidrug-resistant bacteria (such as methicillin-resistant Staphylococcus aureus, MRSA) accelerated by the overuse of antibiotics. Therefore, the usage of biofilm inhibition agents has attracted immense interest as a novel strategy for treatment of diseases related to bacterial infection. From the difference of mode of action against bacterial cells, biofilm inhibition agents are expected to circumvent the emergence of multidrug-resistant bacteria. In this study, we have developed the derivatives of c-di-GMP, a kind of cyclic dinucleotide that is expected to have the effect of inhibiting bacterial biofilm formation. Some of the synthesized derivatives were found to inhibit biofilm formation of Gram-positive bacteria.

Topics: Amines; Anti-Bacterial Agents; Biofilms; Cyclic GMP; Gram-Negative Bacteria; Gram-Positive Bacteria

Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, the histidine phosphotransferase ChpT, and the response regulator CtrA are conserved throughout the alphaproteobacteria. In

Topics: Amino Acid Substitution; Bacterial Proteins; Cyclic GMP; Gene Transfer Techniques; Gene Transfer, Horizontal; Histidine Kinase; Phosphorylation; Phosphotransferases; Recombinant Proteins; Rhodobacter capsulatus; Signal Transduction; Transcription Factors

C-di-GMP is a key signalling molecule which impacts bacterial motility and biofilm formation and is formed by the condensation of two GTP molecules by a diguanylate cyclase. We here describe the identification and characterization of a family of bacteriophage-encoded peptides that directly impact c-di-GMP signalling in Pseudomonas aeruginosa. These phage proteins target Pseudomonas diguanylate cyclase YfiN by direct protein interaction (termed YIPs, YfiN Interacting Peptides). YIPs induce an increase of c-di-GMP production in the host cell, resulting in a decrease in motility and an increase in biofilm mass in P. aeruginosa. A dynamic analysis of the biofilm morphology indicates a denser biofilm structure after induction of the phage protein. This intracellular signalling interference strategy by a lytic phage constitutes an unexplored phage-based mechanism of metabolic regulation and could potentially serve as inspiration for the development of molecules that interfere with biofilm formation in P. aeruginosa and other pathogens.

Topics: Bacterial Proteins; Bacteriophages; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa

Probiotics (Bacillus subtilis 04178) were entrapped in alginate-chitosan microcapsules by high-voltage electrostatic process. The encapsulation pattern was established as entrapped low density cells with culture (ELDCwc). The performance of ELDCwc cells was investigated against stress environments of simulated digestive fluids.. After incubation in simulated gastric (pH 2.5) and intestinal fluids (4% bile salt) for 2 h, the survival rate of ELDCwc cells (18.19% and 27.54%) was significantly higher than that of the free cells (0.0000009% and 0.0005%). The reason why B. subtilis embedded in microcapsules can resist the stress environments was that the mass production of extracellular proteins and polysaccharides prompted B. subtilis to form cell aggregates. The production of extracellular proteins and polysaccharides were regulated by the concentration of c-di-GMP and the expression of ydaJKLMN operon, abbA, sinI, slrA, slrB, abrR and sinR.. c-di-GMP is important for the production of extracellular polymer substance to enhance probiotic viability in stress environments.

Topics: Alginates; Bacillus subtilis; Cell Encapsulation; Chitosan; Cyclic GMP; Models, Biological; Probiotics; Stress, Physiological

The high mortality associated with glioblastoma multiforme (GBM) is attributed to its invasive nature, hypoxic core, resistant cell subpopulations and a highly immunosuppressive tumor microenvironment (TME). To support adaptive immune function and establish a more robust antitumor immune response, we boosted the local innate immune compartment of GBM using an immunostimulatory mesoporous silica nanoparticle, termed immuno-MSN. The immuno-MSN was specifically designed for systemic and proficient delivery of a potent innate immune agonist to dysfunctional antigen-presenting cells (APCs) in the brain TME. The cargo of the immuno-MSN was cyclic diguanylate monophosphate (cdGMP), a Stimulator of Interferon Gene (STING) agonist. Studies showed the immuno-MSN promoted the uptake of STING agonist by APCs in vitro and the subsequent release of the pro-inflammatory cytokine interferon β, 6-fold greater than free agonist. In an orthotopic GBM mouse model, systemically administered immuno-MSN particles were taken up by APCs in the near-perivascular regions of the brain tumor with striking efficiency. The immuno-MSNs facilitated the recruitment of dendritic cells and macrophages to the TME while sparing healthy brain tissue and peripheral organs, resulting in elevated circulating CD8

Topics: Animals; Antigen-Presenting Cells; Antineoplastic Agents; Brain Neoplasms; CD8-Positive T-Lymphocytes; Cyclic GMP; Dendritic Cells; Female; Glioblastoma; Immunity, Innate; Immunologic Factors; Immunotherapy; Interferon Type I; Macrophages; Mice; Mice, Inbred C57BL; Nanoparticles; Porosity; RAW 264.7 Cells; Silicon Dioxide; Tumor Microenvironment

Topics: Acidithiobacillus thiooxidans; Acyl-Butyrolactones; Biofilms; Biosynthetic Pathways; Cyclic GMP; Extremophiles; Gene Expression Regulation, Bacterial; Operon; Polysaccharide-Lyases; Polysaccharides, Bacterial; Quorum Sensing

Periodontal disease (PD) is a progressive inflammatory condition characterized by degradation of the gingival epithelium, periodontal ligament, and alveolar bone ultimately resulting in tooth loss. Treponema denticola is a keystone periopathogen that contributes to immune dysregulation and direct tissue destruction. As periodontal disease develops, T. denticola must adapt to environmental, immunological and physiochemical changes in the subgingival crevice. Treponema denticola produces bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), an important regulatory nucleotide. While T. denticola encodes several putative diguanylate cyclases (DGCs), none have been studied and hence the biological role of c-di-GMP in oral treponemes remains largely unexplored. Here, we demonstrate that the T. denticola open reading frame, TDE0125, encodes a functional DGC designated as DgcA (Diguanylate cyclase A). The dgcA gene is universal among T. denticola isolates, highly conserved and is a stand-alone GGEEF protein with a GAF domain. Recombinant DgcA converts GTP to c-di-GMP using either manganese or magnesium under aerobic and anaerobic reaction conditions. Size exclusion chromatography revealed that DgcA exists as a homodimer and in larger oligomers. Site-directed mutagenesis of residues that define the putative inhibitory site of DgcA suggest that c-di-GMP production is allosterically regulated. This report is the first to characterize a DGC of an oral treponeme.

Topics: Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Guanosine Triphosphate; Humans; Mutagenesis, Site-Directed; Periodontal Diseases; Phosphorus-Oxygen Lyases; Phylogeny; Protein Domains; Recombinant Proteins; Sequence Analysis, DNA; Treponema denticola

A decade of research has shown that the molecule c-di-GMP functions as a central second messenger in many bacteria. A high level of c-di-GMP is associated with biofilm formation, whereas a low level of c-di-GMP is associated with a planktonic single-cell bacterial lifestyle. c-di-GMP is formed by diguanylate cyclases and is degraded by specific phosphodiesterases. We previously presented evidence that the ectopic expression of the

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphoric Diester Hydrolases; Pseudomonas aeruginosa

Shewanella spp. possess a broad respiratory versatility, which contributes to the occupation of hypoxic and anoxic environmental or host-associated niches. Here, we observe a strain-specific induction of biofilm formation in response to supplementation with the anaerobic electron acceptors dimethyl sulfoxide (DMSO) and nitrate in a panel of Shewanella algae isolates. The respiration-driven biofilm response is not observed in DMSO and nitrate reductase deletion mutants of the type strain S. algae CECT 5071, and can be restored upon complementation with the corresponding reductase operon(s) but not by an operon containing a catalytically inactive nitrate reductase. The distinct transcriptional changes, proportional to the effect of these compounds on biofilm formation, include cyclic di-GMP (c-di-GMP) turnover genes. In support, ectopic expression of the c-di-GMP phosphodiesterase YhjH of Salmonella Typhimurium but not its catalytically inactive variant decreased biofilm formation. The respiration-dependent biofilm response of S. algae may permit differential colonization of environmental or host niches.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Dimethyl Sulfoxide; Electrons; Gene Expression Regulation, Bacterial; Mutation; Nitrates; Oxidation-Reduction; Oxidoreductases; Shewanella; Signal Transduction

Clostridioides difficile is the leading cause of nosocomial antibiotic-associated diarrhoea worldwide, yet there is little insight into intestinal tract colonisation and relapse. In many bacterial species, the secondary messenger cyclic-di-GMP mediates switching between planktonic phase, sessile growth and biofilm formation. We demonstrate that c-di-GMP promotes early biofilm formation in C. difficile and that four cell surface proteins contribute to biofilm formation, including two c-di-GMP regulated; CD2831 and CD3246, and two c-di-GMP-independent; CD3392 and CD0183. We demonstrate that C. difficile biofilms are composed of extracellular DNA (eDNA), cell surface and intracellular proteins, which form a protective matrix around C. difficile vegetative cells and spores, as shown by a protective effect against the antibiotic vancomycin. We demonstrate a positive correlation between biofilm biomass, sporulation frequency and eDNA abundance in all five C. difficile lineages. Strains 630 (RT012), CD305 (RT023) and M120 (RT078) contain significantly more eDNA in their biofilm matrix than strains R20291 (RT027) and M68 (RT017). DNase has a profound effect on biofilm integrity, resulting in complete disassembly of the biofilm matrix, inhibition of biofilm formation and reduced spore germination. The addition of exogenous DNase could be exploited in treatment of C. difficile infection and relapse, to improve antibiotic efficacy.

Topics: Bacterial Proteins; Biofilms; Clostridioides difficile; Clostridium Infections; Cyclic GMP; DNA, Bacterial; Humans

Topics: Anti-Bacterial Agents; Azabicyclo Compounds; Bacterial Proteins; Biofilms; Ceftazidime; Cell Wall; Ciprofloxacin; Cyclic GMP; Drug Combinations; Drug Resistance, Multiple, Bacterial; Gene Expression Regulation, Bacterial; Green Fluorescent Proteins; In Vitro Techniques; Microbial Sensitivity Tests; Pseudomonas aeruginosa; Tobramycin; Transformation, Bacterial

In

Topics: Bacterial Adhesion; Bacterial Proteins; Biofilms; Cyclic GMP; Glucose-6-Phosphate; Pseudomonas aeruginosa; Signal Transduction

In many bacteria, cyclic diguanosine monophosphate (c-di-GMP), synthesized by diguanylate cyclase (DGC), serves as a second messenger involved in the regulation of biofilm formation. Although studies have suggested that c-di-GMP also regulates the formation of electrochemically active biofilms (EABFs) by

Topics: Bacterial Proteins; Bioelectric Energy Sources; Biofilms; Cyclic GMP; Electrodes; Escherichia coli Proteins; Phosphorus-Oxygen Lyases; Shewanella

Bacteria in general can develop a wide range of phenotypes under different conditions and external stresses. The phenotypes that reside in biofilms, overproduce exopolymers, and show increased motility often exhibit drug tolerance and drug persistence. In this work, we describe a class of small molecules that delay and inhibit the overproduction of alginate by a non-swarming mucoid Pseudomonas aeruginosa. Among these molecules, selected benzophenone-derived alkyl disaccharides cause the mucoid bacteria to swarm on hydrated soft agar gel and revert the mucoid to a nonmucoid phenotype. The sessile (biofilm) and motile (swarming) phenotypes are controlled by opposing signaling pathways with high and low intracellular levels of bis-(3',5')-cyclic diguanosine monophosphate (cdG), respectively. As our molecules control several of these phenotypes, we explored a protein receptor, pilin of the pili appendages, that is consistent with controlling these bioactivities and signaling pathways. To test this binding hypothesis, we developed a bacterial motility-enabled binding assay that uses the interfacial properties of hydrated gels and bacterial motility to conduct label-free ligand-receptor binding studies. The structure-activity correlation and receptor identification reveal a plausible mechanism for reverting mucoid to nonmucoid phenotypes by binding pili appendages with ligands capable of sequestering and neutralizing reactive oxygen species.

Topics: Alginates; Binding Sites; Cyclic GMP; Fimbriae Proteins; Ligands; Oxidation-Reduction; Phenotype; Pseudomonas aeruginosa; Structure-Activity Relationship

Topics: Alternaria; Alternariosis; Animals; Cyclic GMP; Cytokines; Immunity, Innate; Inflammation; Lung; Membrane Proteins; Mice; Mice, Knockout; Pneumonia; Signal Transduction

In the soil gammaproteobacterium Lysobacter enzymogenes, a natural fungal predator, the response regulator PilR controls type IV pili (T4P)-mediated twitching motility as well as synthesis of the heat-stable antifungal factor (HSAF). Earlier we showed that PilR acts via the second messenger, c-di-GMP; however, the mechanism remained unknown. Here, we describe how PilR, c-di-GMP signalling, and HSAF synthesis are connected. We screened genes for putative diguanylate cyclases (c-di-GMP synthases) and found that PilR binds to the promoter region of lchD and down-regulates its transcription. The DNA-binding affinity of PilR, and therefore its repressor function, are enhanced by phosphorylation by its cognate histidine kinase, PilS. The lchD gene product is a diguanylate cyclase, and the decrease in LchD levels shifts the ratio of c-di-GMP-bound and c-di-GMP-free transcription factor Clp, a key activator of the HSAF biosynthesis operon expression. Furthermore, Clp directly interacts with LchD and enhances its diguanylate cyclase activity. Therefore, the PilS-PilR two-component system activates T4P-motility while simultaneously decreasing c-di-GMP levels and promoting HSAF production via the highly specific LchD-c-di-GMP-Clp pathway. Coordinated increase in motility and secretion of the "long-distance" antifungal weapon HSAF is expected to ensure safer grazing of L. enzymogenes on soil or plant surfaces, unimpeded by fungal competitors, or to facilitate bacterial preying on killed fungal cells. This study uncovered the mechanism of coregulated pili-based motility and production of an antifungal antibiotic in L. enzymogenes, showcased the expanded range of functions of the PilS-PilR system, and highlighted exquisite specificity in c-di-GMP-mediated circuits.

Topics: Antifungal Agents; Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Fimbriae, Bacterial; Lysobacter; Models, Biological; Phosphorus-Oxygen Lyases; Phosphorylation; Promoter Regions, Genetic; Signal Transduction; Transcription Factors

Asymmetric cell division generates two daughter cells with distinct characteristics and fates. Positioning different regulatory and signaling proteins at the opposing ends of the predivisional cell produces molecularly distinct daughter cells. Here, we report a strategy deployed by the asymmetrically dividing bacterium

Topics: Adaptor Proteins, Signal Transducing; Asymmetric Cell Division; Bacterial Proteins; Caulobacter crescentus; Cyclic GMP; Endopeptidase Clp; Protein Multimerization; Proteolysis

Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria.

Topics: Algorithms; Bacterial Proteins; Biofilms; Chromatography, Liquid; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Mass Spectrometry; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa; Second Messenger Systems; Signal Transduction; Temperature

Secretion of bacterial effector proteins into host cells plays a key role in bacterial virulence. Yet, the dynamics of the secretion systems activity remains poorly understood, especially when machineries deal with the export of numerous effectors. We address the question of multi-effector secretion by focusing on the Legionella pneumophila Icm/Dot T4SS that translocates a record number of 300 effectors. We set up a kinetic translocation assay, based on the β-lactamase translocation reporter system combined with the effect of the protonophore CCCP. When used for translocation analysis of Icm/Dot substrates constitutively produced by L. pneumophila, this assay allows a fine monitoring of the secretion activity of the T4SS, independently of the expression control of the effectors. We observed that effectors are translocated with a specific timing, suggesting a control of their docking/translocation by the T4SS. Their delivery is accurately organized to allow effective manipulation of the host cell, as exemplified by the sequential translocation of effectors targeting Rab1, namely SidM/DrrA, LidA, LepB. Remarkably, the timed delivery of effectors does not depend only on their interaction with chaperone proteins but implies cyclic-di-GMP signaling, as the diguanylate cyclase Lpl0780/Lpp0809, contributes to the timing of translocation.

Topics: Bacterial Secretion Systems; Cyclic GMP; Kinetics; Legionella; Molecular Chaperones; Protein Processing, Post-Translational; Protein Transport; Signal Transduction

Vibrio parahaemolyticus cells transit from free-swimming to surface adapted lifestyles, such as swarming colonies and three-dimensional biofilms. These transitions are regulated by sensory modules and regulatory networks that involve the second messenger cyclic diguanylate monophosphate (c-di-GMP). In this work, we show that a previously uncharacterized c-di-GMP phosphodiesterase (VP1881) from V. parahaemolyticus plays an important role in modulating the c-di-GMP pool. We found that the product of VP1881 promotes its own expression when the levels of c-di-GMP are low or when the phosphodiesterase (PDE) is catalytically inactive. This behavior has been observed in a class of c-di-GMP receptors called trigger phosphodiesterases, and hence we named the product of VP1881 TpdA, for

Topics: Amino Acid Sequence; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Genes, Bacterial; Phosphoric Diester Hydrolases; Second Messenger Systems; Vibrio parahaemolyticus

In bacteria, the nucleotide-based second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus, c-di-GMP regulates type IV pilus-dependent motility and the starvation-induced developmental program that results in formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP effectors. We confirm that the stand-alone PilZ domain protein PlpA is important for regulation of motility independently of the Frz chemosensory system and that Pkn1, which is composed of a Ser/Thr kinase domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP

Topics: Bacterial Proteins; Cyclic GMP; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Myxococcus xanthus; Protein Binding; Protein Domains

Induction of lung mucosal immune responses is highly desirable for vaccines against respiratory infections. We recently showed that monocyte-derived dendritic cells (moDCs) are responsible for lung IgA induction. However, the dendritic cell subset inducing lung memory T

Topics: Adjuvants, Immunologic; Animals; Cell Differentiation; Cyclic GMP; Dendritic Cells; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Monocytes; Proto-Oncogene Proteins c-bcl-6; Th1 Cells; Vaccines

Two-component systems (TCSs) are predominant means by which bacteria sense and respond to environment signals. Genome of Pseudomonas putida contains dozens of putative TCS-encoding genes, but phenotypical-genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterized function and transcriptional regulation of a conserved P. putida TCS, named TarR-TarS. TarS (PP_0769) encodes a potential histidine kinase, and tarR (PP_0768) encodes a potential response regulator. Protein-protein interaction assay and phosphorylation assay confirmed that TarR-TarS was a functional TCS. Growth assay under antibiotics revealed that TarR-TarS positively regulated bacterial resistance to multiple antibiotics. Pull-down assay revealed that TarR directly interacted with PP_0800 (a hypothetical protein) and GroEL (the chaperonin). GroEL played a positive role in antibiotic resistance, while PP_0800 seemed to have no effect on antibiotic resistance. The regulator FleQ indirectly activated tarR-tarS transcription. However, the second messenger c-di-GMP antagonized FleQ activation to inhibit tarR-tarS transcription. The sigma factor FliA directly activated tarR-tarS transcription via a consensus motif. These findings reveal function and transcriptional regulation of TarR-TarS, and enrich knowledge regarding the relationship between c-di-GMP and antibiotic susceptibility in P. putida.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Pseudomonas putida

The development of fluorescent biosensors is motivated by the desire to monitor cellular metabolite levels in real time. Most genetically encodable fluorescent biosensors are based on receptor proteins fused to fluorescent protein domains. More recently, small molecule-binding riboswitches have been adapted for use as fluorescent biosensors through fusion to the in vitro selected Spinach aptamer, which binds a profluorescent, cell-permeable small molecule mimic of the GFP chromophore, DFHBI. Here we describe methods to prepare and analyze riboswitch-Spinach tRNA fusions for ligand-dependent activation of fluorescence in vivo. Example procedures describe the use of the Vc2-Spinach tRNA biosensor to monitor perturbations in cellular levels of cyclic di-GMP using either fluorescence microscopy or flow cytometry. In this updated chapter, we have added procedures on using biosensors in flow cytometry to detect exogenously added compounds. The relative ease of cloning and imaging of these biosensors, as well as their modular nature, should make this method appealing to other researchers interested in utilizing riboswitch-based biosensors for metabolite sensing.

Topics: Aptamers, Nucleotide; Bacterial Proteins; Benzyl Compounds; Biosensing Techniques; Cloning, Molecular; Cyclic GMP; Escherichia coli Proteins; Flow Cytometry; Fluorescent Dyes; Imidazolines; Intravital Microscopy; Isopropyl Thiogalactoside; Microscopy, Fluorescence; Nucleic Acid Conformation; Phosphorus-Oxygen Lyases; Plasmids; Riboswitch; RNA; RNA, Transfer

Riboswitches are a class of noncoding RNAs that regulate gene expression in response to changes in intracellular metabolite concentrations. When riboswitches are placed upstream of genetic reporters, the degree of reporter activity reflects the relative abundance of the metabolite that is sensed by the riboswitch. This method describes how reporters for live cell imaging, such as yellow fluorescent protein (YFP), can be placed under genetic control by metabolite-sensing riboswitches in the bacterium Bacillus subtilis. Specifically, a protocol for generating a fluorescent YFP reporter, based on a c-di-GMP responsive riboswitch, is outlined below.

Topics: 5' Untranslated Regions; Aptamers, Nucleotide; Bacillus subtilis; Bacterial Proteins; Base Sequence; Cyclic GMP; Gene Expression Regulation, Bacterial; Genes, Reporter; Intravital Microscopy; Ligands; Luminescent Proteins; Nucleic Acid Conformation; Plasmids; Riboswitch; Transformation, Bacterial

In the tick-borne pathogens, Borreliella burgdorferi and Borrelia hermsii, c-di-GMP is produced by a single diguanylate cyclase (Rrp1). In these pathogens, the Plz proteins (PlzA, B and C) are the only c-di-GMP receptors identified to date and PlzA is the sole c-di-GMP receptor found in all Borreliella isolates. Bioinformatic analyses suggest that PlzA has a unique PilZN3-PilZ architecture with the relatively uncommon xPilZ domain. Here, we present the crystal structure of PlzA in complex with c-di-GMP (1.6 Å resolution). This is the first structure of a xPilz domain in complex with c-di-GMP to be determined. PlzA has a two-domain structure, where each domain comprises topologically equivalent PilZ domains with minimal sequence identity but remarkable structural similarity. The c-di-GMP binding site is formed by the linker connecting the two domains. While the structure of apo PlzA could not be determined, previous fluorescence resonance energy transfer data suggest that apo and holo forms of the protein are structurally distinct. The information obtained from this study will facilitate ongoing efforts to identify the molecular mechanisms of PlzA-mediated regulation in ticks and mammals.

Topics: Bacterial Proteins; Borrelia burgdorferi; Crystallization; Cyclic GMP; Models, Molecular; Protein Domains

Bacteria that colonize animals must overcome, or coexist, with the reactive oxygen species products of inflammation, a front-line defense of innate immunity. Among these is the neutrophilic oxidant bleach, hypochlorous acid (HOCl), a potent antimicrobial that plays a primary role in killing bacteria through nonspecific oxidation of proteins, lipids, and DNA. Here, we report that in response to increasing HOCl levels, Escherichia coli regulates biofilm production via activation of the diguanylate cyclase DgcZ. We identify the mechanism of DgcZ sensing of HOCl to be direct oxidation of its regulatory chemoreceptor zinc-binding (CZB) domain. Dissection of CZB signal transduction reveals that oxidation of the conserved zinc-binding cysteine controls CZB Zn

Topics: Bacteria; Bacterial Adhesion; Biofilms; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genome, Bacterial; Hypochlorous Acid; Inflammation; Signal Transduction

To initiate biofilm formation, it is critical for bacteria to sense a surface and respond precisely to activate downstream components of the biofilm program. Type 4 pili (T4P) and increasing levels of c-di-GMP have been shown to be important for surface sensing and biofilm formation, respectively; however, mechanisms important in modulating the levels of this dinucleotide molecule to define a precise output response are unknown. Here, using macroscopic bulk assays and single-cell tracking analyses of

Topics: Amino Acid Motifs; Biofilms; Conserved Sequence; Cyclic GMP; Escherichia coli Proteins; Fimbriae, Bacterial; Models, Biological; Mutation; Phosphorus-Oxygen Lyases; Protein Binding; Protein Domains; Pseudomonas aeruginosa; Signal Transduction; Single-Cell Analysis; Type IV Secretion Systems

Animal microbiomes are assembled predominantly from environmental microbes, yet the mechanisms by which individual symbionts regulate their transmission into hosts remain underexplored. By tracking the experimental evolution of Aeromonas veronii in gnotobiotic zebrafish, we identify bacterial traits promoting host colonization. Multiple independently evolved isolates with increased immigration harbored mutations in a gene we named sensor of proline diguanylate cyclase enzyme (SpdE) based on structural, biochemical, and phenotypic evidence that SpdE encodes an amino-acid-sensing diguanylate cyclase. SpdE detects free proline and to a lesser extent valine and isoleucine, resulting in reduced production of intracellular c-di-GMP, a second messenger controlling bacterial motility. Indeed, SpdE binding to amino acids increased bacterial motility and host colonization. Hosts serve as sources of SpdE-detected amino acids, with levels varying based on microbial colonization status. Our work demonstrates that bacteria use chemically regulated motility, or chemokinesis, to sense host-emitted cues that trigger active immigration into hosts.

Topics: Amino Acids; Animals; Bacteria; Bacterial Proteins; Biofilms; Chemokines; Cues; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Host Microbial Interactions; Phosphorus-Oxygen Lyases; Symbiosis; Zebrafish

Xanthomonas oryzae pv. oryzae (Xoo) causes a serious disease of rice known as bacterial leaf blight. Several virulence-associated functions have been characterized in Xoo. However, the role of important second messenger c-di-GMP signalling in the regulation of virulence-associated functions still remains elusive in this phytopathogen. In this study we have performed an investigation of 13 c-di-GMP modulating deletion mutants to understand their contribution in Xoo virulence and lifestyle transition. We show that four Xoo proteins, Xoo2331, Xoo2563, Xoo2860 and Xoo2616, are involved in fine-tuning the in vivo c-di-GMP abundance and also play a role in the regulation of virulence-associated functions. We have further established the importance of the GGDEF domain of Xoo2563, a previously characterized c-di-GMP phosphodiesterase, in the virulence-associated functions of Xoo. Interestingly the strain harbouring the GGDEF domain deletion (ΔXoo2563

Topics: Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Plant Diseases; Virulence; Xanthomonas

Microbial biofilms are involved in a number of infections that cannot be cured, as microbes in biofilms resist host immune defenses and antibiotic therapies. With no strict biofilm-antibiotic in the current pipelines, there is an unmet need for drug candidates that enable the current antibiotics to eradicate bacteria in biofilms. We used high-throughput screening to identify chemical compounds that reduce the intracellular c-di-GMP content in Pseudomonas aeruginosa. This led to the identification of a small molecule that efficiently depletes P. aeruginosa for c-di-GMP, inhibits biofilm formation, and disperses established biofilm. A combination of our lead compound with standard of care antibiotics showed improved eradication of an implant-associated infection established in mice. Genetic analyses provided evidence that the anti-biofilm compound stimulates the activity of the c-di-GMP phosphodiesterase BifA in P. aeruginosa. Our work constitutes a proof of concept for c-di-GMP phosphodiesterase-activating drugs administered in combination with antibiotics as a viable treatment strategy for otherwise recalcitrant infections.

Topics: Animals; Anti-Bacterial Agents; Biofilms; Chromatography, High Pressure Liquid; Cyclic GMP; Dose-Response Relationship, Drug; Drug Discovery; Gene Expression Profiling; Gene Expression Regulation, Bacterial; High-Throughput Nucleotide Sequencing; Mice; Pseudomonas aeruginosa; Signal Transduction; Tandem Mass Spectrometry; Transcriptome

In this study, we examined the relationship between c-di-GMP and its only known effector protein, PlzA, in Borrelia burgdorferi during the arthropod and mammalian phases of the enzootic cycle. Using a B. burgdorferi strain expressing a plzA point mutant (plzA-R145D) unable to bind c-di-GMP, we confirmed that the protective function of PlzA in ticks is c-di-GMP-dependent. Unlike ΔplzA spirochetes, which are severely attenuated in mice, the plzA-R145D strain was fully infectious, firmly establishing that PlzA serves a c-di-GMP-independent function in mammals. Contrary to prior reports, loss of PlzA did not affect expression of RpoS or RpoS-dependent genes, which are essential for transmission, mammalian host-adaptation and murine infection. To ascertain the nature of PlzA's c-di-GMP-independent function(s), we employed infection models using (i) host-adapted mutant spirochetes for needle inoculation of immunocompetent mice and (ii) infection of scid mice with in vitro-grown organisms. Both approaches substantially restored ΔplzA infectivity, suggesting that PlzA enables B. burgdorferi to overcome an early bottleneck to infection. Furthermore, using a Borrelia strain expressing a heterologous, constitutively active diguanylate cyclase, we demonstrate that 'ectopic' production of c-di-GMP in mammals abrogates spirochete virulence and interferes with RpoS function at the post-translational level in a PlzA-dependent manner. Structural modeling and SAXS analysis of liganded- and unliganded-PlzA revealed marked conformational changes that underlie its biphasic functionality. This structural plasticity likely enables PlzA to serve as a c-di-GMP biosensor that in its respective liganded and unliganded states promote vector- and host-adaptation by the Lyme disease spirochete.

Topics: Adaptation, Physiological; Animals; Bacterial Proteins; Borrelia burgdorferi; Cyclic GMP; Female; Host-Pathogen Interactions; Immune Evasion; Ixodes; Lyme Disease; Mice; Virulence

Filamentous actinobacteria of the genus

Topics: Actinobacteria; Crystallography, X-Ray; Cyclic GMP; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Models, Molecular; Protein Binding; Protein Conformation; Protein Domains; Sigma Factor; Streptomyces

The formation of biofilms by clinical pathogens typically leads to chronic and recurring antibiotic-resistant infections. High cellular levels of cyclic diguanylate (c-di-GMP), a ubiquitous secondary messenger of bacteria, have been proven to be associated with a sessile biofilm lifestyle of pathogens. A promising antibiofilm strategy involving the induction of c-di-GMP to form dysfunctional G-quadruplexes, thereby blocking the c-di-GMP-mediated biofilm regulatory pathway, was proposed in this study. In this new strategy, a series of novel c-di-GMP G-quadruplex inducers were designed and synthesized for development of therapeutic biofilm inhibitors. Compound

Topics: Anti-Bacterial Agents; Benzothiazoles; Biofilms; Cyclic GMP; Dose-Response Relationship, Drug; Drug Design; G-Quadruplexes; Microbial Sensitivity Tests; Molecular Structure; Pseudomonas aeruginosa; Quinolines; Structure-Activity Relationship

Plague-causing Yersinia pestis is transmitted through regurgitation when it forms a biofilm-mediated blockage in the foregut of its flea vector. This biofilm is composed of an extracellular polysaccharide substance (EPS) produced when cyclic-di-GMP (c-di-GMP) levels are elevated. The Y. pestis diguanylate cyclase enzymes HmsD and HmsT synthesize c-di-GMP. HmsD is required for biofilm blockage formation but contributes minimally to

Topics: Animals; Biofilms; Cyclic GMP; Gastrointestinal Tract; Host Factor 1 Protein; Host-Pathogen Interactions; RNA, Messenger; Siphonaptera; Yersinia pestis

The ubiquitous bacterial second messenger c-di-GMP is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). Pseudomonas putida has dozens of DGC/PDE-encoding genes in its genome, but the phenotypical-genotypical correlation and transcriptional regulation of these genes are largely unknown. Herein, we characterize function and transcriptional regulation of a P. putida c-di-GMP-metabolizing enzyme, GcsA. GcsA consists of two per-ARNT-sim (PAS) domains, followed by a canonical conserved central sequence pattern (GGDEF) domain and a truncated EAL domain. In vitro analysis confirmed the DGC activity of GcsA. The phenotypic observation revealed that GcsA inhibited swimming motility in an FlgZ-dependent manner. In terms of transcriptional regulation, gcsA was found to be cooperatively regulated by c-di-GMP and cAMP via their effectors, FleQ and Crp respectively. The transcription of gcsA was promoted by c-di-GMP and inhibited by cAMP. In vitro binding analysis revealed that FleQ indirectly regulated the transcription of gcsA, while Crp directly regulated the transcription of gcsA by binding to its promoter. Besides, an inverse relationship between the cellular c-di-GMP and cAMP levels in P. putida was confirmed. These findings provide basic knowledge regarding the function and transcriptional regulation of GcsA and demonstrate a crosstalk between c-di-GMP and cAMP in the regulation of the expression of GcsA in P. putida.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Bacterial Proteins; Conserved Sequence; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Promoter Regions, Genetic; Pseudomonas putida; Second Messenger Systems

Gene transfer agents (GTAs) are bacteriophage-like particles produced by several bacterial and archaeal lineages that contain small pieces of the producing cells' genomes that can be transferred to other cells in a process similar to transduction. One well-studied GTA is RcGTA, produced by the alphaproteobacterium

Topics: Amino Acid Sequence; Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Gene Transfer, Horizontal; Molecular Sequence Data; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Rhodobacter capsulatus; Signal Transduction

Cyclic diguanylate (c-di-GMP) is a broadly conserved bacterial signalling molecule that modulates diverse cellular processes, such as biofilm formation, colony morphology and swimming motility. The intracellular level of c-di-GMP is controlled by diguanylate cyclases (DGCs) with GGDEF domain and phosphodiesterases (PDEs) with either EAL or HD-GYP domain. Pseudomonas putida KT2440 has a large group of genes on its genome encoding proteins with GGDEF/EAL/HD-GYP domains. However, phenotypic-genotypic correlation and c-di-GMP metabolism of these genes were largely unknown. Herein, by systematically constructing deletion mutants/overexpression strains of the 42 predicted c-di-GMP metabolism-related genes and analysing the phenotypes, we preliminarily revealed the role of each gene in biofilm formation, colony morphology and swimming motility. Subsequent results from protein sequence alignments and cellular c-di-GMP assessment indicated that 25 out of the 42 genes were likely to encode DGCs, nine genes were predicted to encode PDEs, four genes encoded bifunctional enzymes and the other four genes encoded enzymatically inactive proteins. This study offers a basic understanding of the roles of these 42 genes and can serve as a toolkit for investigators to further elucidate the functions of these GGDEF and EAL/HD-GYP domain-containing proteins.

Topics: Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phenotype; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Domains; Pseudomonas putida

YajQ, a binding protein of the universal bacterial second messenger cyclic di-GMP (c-di-GMP), affects virulence in several bacterial pathogens, including Xanthomonas campestris. In this bacterium, YajQ interacts with the transcription factor LysR. Upon c-di-GMP binding, the whole c-di-GMP-YajQ-LysR complex is found to dissociate from DNA, resulting in virulence gene regulation. Here, we identify a YajQ-LysR-like system in the bacterial biocontrol agent Lysobacter enzymogenes OH11 that secretes an antifungal antibiotic, heat-stable antifungal factor (HSAF) against crop fungal pathogens. We show that the YajQ homologue, CdgL (c-di-GMP receptor interacting with LysR) affects expression of the HSAF biosynthesis operon by interacting with the transcription activator LysR. The CdgL-LysR interaction enhances the apparent affinity of LysR to the promoter region upstream of the HSAF biosynthesis operon, which increases operon expression. Unlike the homologues CdgL (YajQ)-LysR system in X. campestris, we show that c-di-GMP binding to CdgL seems to weaken CdgL-LysR interactions and promote the release of CdgL from the LysR-DNA complex, which leads to decreased expression. Together, this study takes the YajQ-LysR-like system from bacterial pathogens to a crop-protecting bacterium that is able to regulate antifungal HSAF biosynthesis via disassembly of the c-di-GMP receptor-transcription activator complex.

Topics: Antifungal Agents; Cyclic GMP; Gene Expression Regulation, Bacterial; Lysobacter; Xanthomonas campestris

Attachment to surfaces is an important early step during bacterial infection and during formation of submerged biofilms. Although flagella-mediated motility is known to be important for attachment of Escherichia coli and other bacteria, implications of motility regulation by cellular signalling remain to be understood. Here, we show that motility largely promotes attachment of E. coli, including that mediated by type 1 fimbriae, by allowing cells to reach, get hydrodynamically trapped at and explore the surface. Inactivation or inhibition of the chemotaxis signalling pathway improves attachment by suppressing cell reorientations and thereby increasing surface residence times. The attachment is further enhanced by deletion of genes encoding the cyclic diguanosine monophosphate (c-di-GMP)-dependent flagellar brake YcgR or the diguanylate cyclase DgcE. Such increased attachment in absence of c-di-GMP signalling is in contrast to its commonly accepted function as a positive regulator of the sessile state. It is apparently due to the increased swimming speed of E. coli in absence of YcgR-mediated motor control, which strengthens adhesion mediated by the type 1 fimbriae. Thus, both signalling networks that regulate motility of E. coli also control its engagement with both biotic and abiotic surfaces, which has likely implications for infection and biofilm formation.

Topics: Bacterial Adhesion; Bacterial Proteins; Biofilms; Chemotaxis; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Fimbriae, Bacterial; Signal Transduction

Sensing of red and far-red light by bacteriophytochromes involves intricate interactions between their bilin chromophore and the protein environment. The light-triggered rearrangements of the cofactor configuration and eventually the protein conformation enable bacteriophytochromes to interact with various protein effector domains for biological modulation of diverse physiological functions. Excitation of the holoproteins by red or far-red light promotes the photoconversion to their far-red light-absorbing Pfr state or the red light-absorbing Pr state, respectively. Because prototypical bacteriophytochromes have a parallel dimer architecture, it is generally assumed that symmetric activation with two Pfr state protomers constitutes the signaling-active species. However, the bacteriophytochrome from

Topics: Allosteric Regulation; Alteromonadaceae; Bacterial Proteins; Crystallography, X-Ray; Cyclic GMP; Enzyme Activation; Escherichia coli Proteins; Models, Molecular; Phosphorus-Oxygen Lyases; Phytochrome; Protein Conformation; Protein Multimerization

Streptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σ

Topics: Amino Acid Sequence; Bacterial Proteins; Cyclic GMP; DNA-Binding Proteins; Gene Expression Regulation, Bacterial; Protein Domains; RNA, Bacterial; Sigma Factor; Spores, Bacterial; Streptomyces

Topics: Bacterial Proteins; Cell Wall; Cyclic GMP; Dickeya; Gammaproteobacteria; Gene Expression Regulation, Bacterial; Mutation

Cyclic-di-GMP (c-di-GMP) synthesized by diguanylate cyclases has been an important and ubiquitous secondary messenger in almost all bacterial systems. In Vibrio cholerae, c-di-GMP plays an intricate role in the production of the exopolysaccharide matrix, and thereby, in biofilm formation. The formation of the surface biofilm enables the bacteria to survive in aquatic bodies, when not infecting a human host. Diguanylate cyclases are the class of enzymes which synthesize c-di-GMP from two molecules of GTP and are endowed with a GGDEF or, a GGEEF signature domain. The VC0395_0300 protein from V. cholerae, has been established as a diguanylate cyclase with a necessary role in biofilm formation. Here we present the structure of an N-terminally truncated form of VC0395_0300, which retains the active GGEEF domain for diguanylate cyclase activity but lacks 160 residues from the poorly organized N-terminal domain. X-ray diffraction data was collected from a crystal of VC0395_0300

Topics: Bacterial Proteins; Biofilms; Catalytic Domain; Crystallography, X-Ray; Cyclic GMP; Escherichia coli Proteins; Humans; Models, Molecular; Phosphorus-Oxygen Lyases; Protein Domains; Second Messenger Systems; Solubility; Static Electricity; Vibrio cholerae

PilB is the assembly ATPase for the bacterial type IV pilus (T4P), and as a consequence, it is essential for T4P-mediated bacterial motility. In some cases, PilB has been demonstrated to regulate the production of exopolysaccharide (EPS) during bacterial biofilm development independently of or in addition to its function in pilus assembly. While the ATPase activity of PilB resides at its C-terminal region, the N terminus of a subset of PilBs forms a novel cyclic-di-GMP (cdG)-binding domain. This multi-domain structure suggests that PilB binds cdG and adenine nucleotides through separate domains which may influence the functionality of PilB in both motility and biofilm development. Here, Chloracidobacterium thermophilum PilB is used to investigate ligand binding by its separate domains and by the full-length protein. Our results confirm the specificity of these individual domains for their respective ligands and demonstrate communications between these domains in the full-length protein. It is clear that when the N- and the C-terminal domains of PilB bind to cdG and ADP, respectively, they mutually influence each other in conformation and in their binding to ligands. We propose that the interactions between these domains in response to their ligands play critical roles in modulating or controlling the functions of PilB as a regulator of EPS production and as the T4P assembly ATPase.

Topics: Acidobacteria; Adenosine Diphosphate; Allosteric Regulation; Bacterial Proteins; Cyclic GMP; Models, Molecular; Oxidoreductases; Protein Binding; Protein Domains

Cytosolic hybrid histidine kinases (HHKs) constitute major signaling nodes that control various biological processes, but their input signals and how these are processed are largely unknown. In

Topics: Bacterial Proteins; Caulobacter crescentus; Cell Cycle; Crystallography, X-Ray; Cyclic GMP; Histidine Kinase; Models, Molecular; Molecular Dynamics Simulation; Phosphorylation; Protein Binding; Protein Conformation; Protein Domains; Second Messenger Systems

Salmonellosis is the second most common food-borne zoonosis in the European Union, with pigs being a major reservoir of this pathogen. Salmonella control in pig production requires multiple measures amongst which vaccination may be used to reduce subclinical carriage and shedding of prevalent serovars, such as Salmonella enterica serovar Typhimurium. Live attenuated vaccine strains offer advantages in terms of enhancing cell mediated immunity and allowing inoculation by the oral route. However, main failures of these vaccines are the limited cross-protection achieved against heterologous serovars and interference with serological monitoring for infection. We have recently shown that an attenuated S. Enteritidis strain (ΔXIII) is protective against S. Typhimurium in a murine infection model. ΔXIII strain harbours 13 chromosomal deletions that make it unable to produce the sigma factor RpoS and synthesize cyclic-di-GMP (c-di-GMP). In this study, our objectives were to test the protective effects of ΔXIII strain in swine and to investigate if the use of ΔXIII permits the discrimination of vaccinated from infected pigs. Results show that oral vaccination of pre-weaned piglets with ΔXIII cross-protected against a challenge with S. Typhimurium by reducing faecal shedding and ileocaecal lymph nodes colonization, both at the time of weaning and slaughter. Vaccinated pigs showed neither faecal shedding nor tissue persistence of the vaccine strain at weaning, ensuring the absence of ΔXIII strain by the time of slaughter. Moreover, lack of the SEN4316 protein in ΔXIII strain allowed the development of a serological test that enabled the differentiation of infected from vaccinated animals (DIVA).

Topics: Animals; Bacterial Proteins; Cyclic GMP; Salmonella enteritidis; Salmonella Infections, Animal; Salmonella Vaccines; Sigma Factor; Swine; Swine Diseases

Cyclic diguanylate (c-di-GMP) is a second messenger involved in the regulation of various physiological processes in bacteria. However, its function in spoilage bacteria has not yet been addressed. Here, we studied the function of a tandem GGDEF-EAL domain protein, Sbal_3235, in the spoilage bacterium Shewanella baltica OS155. The deletion of sbal_3235 significantly reduced the c-di-GMP level, biofilm formation, and exopolysaccharide, trimethylamine (TMA), and putrescine production; sbal_3235 deletion also downregulated the expression of the torS and speF genes and affected membrane fatty acid composition. Site-directed mutagenesis in conserved GGDEF and EAL motifs abolished diguanylate cyclase (DGC) and phosphodiesterase (PDE) activity, respectively. These data indicate that Sbal_3235 is an essential contributor to the c-di-GMP pool with bifunctional DGC and PDE activity, which is involved in the biofilm formation and spoilage activity of S. baltica OS155. Our findings expand the biochemical role of c-di-GMP and uncover its link to spoilage activities, providing novel targets for food quality and safety controlling.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Fatty Acids; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Protein Domains; Shewanella

Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, is a ubiquitous second messenger controlling diverse cellular processes in bacteria. In streptomycetes, c-di-GMP plays a crucial role in a complex morphological differentiation by modulating an activity of the pleiotropic regulator BldD. Here we report that c-di-GMP plays a key role in regulating secondary metabolite production in streptomycetes by altering the expression levels of bldD. Deletion of cdgB encoding a diguanylate cyclase in Streptomycesghanaensis reduced c-di-GMP levels and the production of the peptidoglycan glycosyltransferase inhibitor moenomycin A. In contrast to the cdgB mutant, inactivation of rmdB, encoding a phosphodiesterase for the c-di-GMP hydrolysis, positively correlated with the c-di-GMP and moenomycin A accumulation. Deletion of bldD adversely affected the synthesis of secondary metabolites in S. ghanaensis, including the production of moenomycin A. The bldD-deficient phenotype is partly mediated by an increase in expression of the pleiotropic regulatory gene wblA. Genetic and biochemical analyses demonstrate that a complex of c-di-GMP and BldD effectively represses transcription of wblA, thus preventing sporogenesis and sustaining antibiotic synthesis. These results show that manipulation of the expression of genes controlling c-di-GMP pool has the potential to improve antibiotic production as well as activate the expression of silent gene clusters.

Topics: Bacterial Proteins; Bambermycins; Biological Products; Cyclic GMP; DNA-Binding Proteins; Escherichia coli Proteins; Gene Deletion; Gene Expression Regulation, Bacterial; Nucleotides; Peptidoglycan Glycosyltransferase; Phosphorus-Oxygen Lyases; Second Messenger Systems; Streptomycetaceae; Transcription Factors

Heat-stable antifungal factor (HSAF) is a broad-spectrum antifungal antibiotic produced by the biological control agent, Lysobacter enzymogenes. In our earlier works, we have applied HSAF to effectively control wheat and pear fungal disease. However, a major bottleneck in its practical application is the low HSAF production level; therefore, boosting its production is essential for its wide application. In the past, we find that c-di-GMP, a universal bacterial second messenger, is inhibitory to HSAF production. In this work, we further identified eight active diguanylate cyclases (DGCs) responsible for c-di-GMP synthesis in Lysobacter enzymogenes via both bioinformatics and genetic analyses. We generated a strain lacking seven active DGC genes and found that this DGC-modified strain, OH11LC, produced a higher HSAF amount in a c-di-GMP concentration-dependent manner. Subsequently, by employing OH11LC as the host fermentation strain, we could even produce a much higher HSAF amount (> 200-fold). After improving the HSAF production, we further developed a technique of seed coating method with HSAF, which turned out to be effective in fighting against the maize seed-borne filamentous pathogen, Pythium gramineacola. Overall, via combining strain modification and fermentation optimization, we demonstrated a good example of translating fundamental knowledge of bacterial c-di-GMP signaling into biological control application in which we relieved the inhibitory effect of c-di-GMP on HSAF biosynthesis by deleting a bunch of potentially active L. enzymogenes DGC genes to improve HSAF yield and to expand its usage in antifungal seed coating.

Topics: Antifungal Agents; Cyclic GMP; Escherichia coli Proteins; Fermentation; Gene Knockout Techniques; Lysobacter; Phosphorus-Oxygen Lyases; Plant Diseases; Pythium; Seeds; Zea mays

Opportunistic pathogen Pseudomonas aeruginosa can cause acute and chronic infections in humans. It is notorious for its resistance to antibiotics due to the formation of biofilms. Cyclic-di-GMP is a bacterial second messenger that plays important roles during biofilm development. There are 40 genes in P. aeruginosa predicted to participate in c-di-GMP biosynthesis or degradation. It is time-consuming for the functional characterization of these genes. Here, we cloned 16 genes from P. aeruginosa PAO1 that are predicted to encode diguanylate cyclases (DGCs, responsible for c-di-GMP biosynthesis) and constructed their corresponding in-frame deletion mutants. We evaluated the methods to measure the intracellular c-di-GMP concentration by using deletion mutants and PAO1 strains containing a plasmid expressing one of the 16 genes, respectively. Functional outputs of all PAO1-derived stains were also detected and evaluated, including biofilm formation, production of exopolysaccharide, swimming and swarming motilities. Our data showed that measuring the c-di-GMP level only characterized a few DGC by using either pCdrA::gfp as a reporter or LC/MS/MS. Functional output results indicated that overexpression of a DGC gave more pronounced phenotypes than the corresponding deletion mutant and suggested that the swimming motility assay could be a quick way to briefly estimate a predicted DGC for further studies. The overall evaluation suggested 15 out of 16 predicted DGCs were functional DGCs, wherein six were characterized to encode DGCs previously. Altogether, we have provided not only a cloning library of 16 DGC-encoding genes and their corresponding in-frame deletion mutants but also paved ways to briefly characterize a predicted DGC.

Topics: Bacterial Proteins; Biofilms; Biomarkers; Chromatography, Liquid; Cyclic GMP; Enzyme Activation; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa; Sequence Deletion; Tandem Mass Spectrometry

Acinetobacter baumannii has emerged as an increasing multidrug-resistant threat in hospitals and a common opportunistic nosocomial pathogen worldwide. However, molecular details of the pathogenesis and physiology of this bacterium largely remain to be elucidated. Here we identify and characterize the c-di-GMP signalling network and assess its role in biofilm formation and surface associated motility. Bioinformatic analysis revealed eleven candidate genes for c-di-GMP metabolizing proteins (GGDEF/EAL domain proteins) in the genome of A. baumannii strain 17978. Enzymatic activity of the encoded proteins was assessed by molecular cloning and expression in the model organisms Salmonella typhimurium and Vibrio cholerae. Ten of the eleven GGDEF/EAL proteins altered the rdar morphotype of S. typhimurium and the rugose morphotype of V. cholerae. The over expression of three GGDEF proteins exerted a pronounced effect on colony formation of A. baumannii on Congo Red agar plates. Distinct panels of GGDEF/EAL proteins were found to alter biofilm formation and surface associated motility of A. baumannii upon over expression. The GGDEF protein A1S_3296 appeared as a major diguanylate cyclase regulating macro-colony formation, biofilm formation and the surface associated motility. AIS_3296 promotes Csu pili mediated biofilm formation. We conclude that a functional c-di-GMP signalling network in A. baumannii regulates biofilm formation and surface associated motility of this increasingly important opportunistic bacterial pathogen.

Topics: Acinetobacter baumannii; Bacterial Proteins; Biofilms; Cloning, Molecular; Computational Biology; Cyclic GMP; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Humans; Opportunistic Infections; Recombinant Proteins; Salmonella typhimurium; Signal Transduction; Vibrio cholerae

A recent paper by Gallagher et al. (2020) demonstrates that c-di-GMP controls spore formation in Streptomyces venezuelae through sequestering the sporulation sigma factor σ

Topics: Bacterial Proteins; Cell Differentiation; Cyclic GMP; Sigma Factor; Streptomyces

The ability of organisms to sense and adapt to oxygen levels in their environment leads to changes in cellular phenotypes, including biofilm formation and virulence. Globin coupled sensors (GCSs) are a family of heme proteins that regulate diverse functions in response to O2 levels, including modulating synthesis of cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial second messenger that regulates biofilm formation. While GCS proteins have been demonstrated to regulate O2-dependent pathways, the mechanism by which the O2 binding event is transmitted from the globin domain to the cyclase domain is unknown. Using chemical cross-linking and subsequent liquid chromatography-tandem mass spectrometry, diguanylate cyclase (DGC)-containing GCS proteins from Bordetella pertussis (BpeGReg) and Pectobacterium carotovorum (PccGCS) have been demonstrated to form direct interactions between the globin domain and a middle domain π-helix. Additionally, mutation of the π-helix caused major changes in oligomerization and loss of DGC activity. Furthermore, results from assays with isolated globin and DGC domains found that DGC activity is affected by the cognate globin domain, indicating unique interactions between output domain and cognate globin sensor. Based on these studies a compact GCS structure, which depends on the middle domain π-helix for orienting the three domains, is needed for DGC activity and allows for direct sensor domain interactions with both middle and output domains to transmit the O2 binding signal. The insights from the present study improve our understanding of DGC regulation and provide insight into GCS signaling that may lead to the ability to rationally control O2-dependent GCS activity.

Topics: Bacterial Proteins; Binding Sites; Bordetella pertussis; Cyclic GMP; Kinetics; Oxygen; Pectobacterium carotovorum; Phosphorus-Oxygen Lyases; Protein Interaction Domains and Motifs; Protein Multimerization; Protein Structure, Quaternary; Protein Structure, Secondary; Structure-Activity Relationship

Bacteria adapt their growth rate to their metabolic status and environmental conditions by modulating the length of their G1 period. Here we demonstrate that a gradual increase in the concentration of the second messenger c-di-GMP determines precise gene expression during G1/S transition in Caulobacter crescentus. We show that c-di-GMP stimulates the kinase ShkA by binding to its central pseudo-receiver domain, activates the TacA transcription factor, and initiates a G1/S-specific transcription program leading to cell morphogenesis and S-phase entry. Activation of the ShkA-dependent genetic program causes c-di-GMP to reach peak levels, which triggers S-phase entry and promotes proteolysis of ShkA and TacA. Thus, a gradual increase of c-di-GMP results in precise control of ShkA-TacA activity, enabling G1/S-specific gene expression that coordinates cell cycle and morphogenesis.

Topics: Bacterial Proteins; Caulobacter crescentus; Cell Cycle; Cyclic GMP; Gene Expression Regulation, Bacterial; Histidine Kinase; Morphogenesis; Phosphorylation; Protein Binding; Protein Domains; Proteolysis; Signal Transduction; Trans-Activators

The second messenger bis(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates numerous important physiological functions in bacteria. In this study, we identified and characterized the first dimeric, full-length, non-heme iron-bound phosphodiesterase (PDE) containing bacterial hemerythrin and HD-GYP domains (Bhr-HD-GYP). We found that the amino acid sequence encoded by the

Topics: Amino Acid Sequence; Bacterial Proteins; Betaproteobacteria; Catalysis; Cyclic GMP; Enzyme Assays; Hemerythrin; Hydrolysis; Iron; Oxidation-Reduction; Phosphoric Diester Hydrolases; Protein Domains; Sequence Alignment

c-di-GMP is a major player in the switch between biofilm and motile lifestyles. Several bacteria exhibit a large number of c-di-GMP metabolizing proteins, thus a fine-tuning of this nucleotide levels may occur. It is hypothesized that some c-di-GMP metabolizing proteins would provide the global c-di-GMP levels inside the cell whereas others would maintain a localized pool, with the resulting c-di-GMP acting at the vicinity of its production. Although attractive, this hypothesis has yet to be demonstrated in Pseudomonas aeruginosa. We found that the diguanylate cyclase DgcP interacts with the cytosolic region of FimV, a polar peptidoglycan-binding protein involved in type IV pilus assembly. Moreover, DgcP is located at the cell poles in wild type cells but scattered in the cytoplasm of cells lacking FimV. Overexpression of dgcP leads to the classical phenotypes of high c-di-GMP levels (increased biofilm and impaired motilities) in the wild-type strain, but not in a ΔfimV background. Therefore, our findings suggest that DgcP activity is regulated by FimV. The polar localization of DgcP might contribute to a local c-di-GMP pool that can be sensed by other proteins at the cell pole, bringing to light a specialized function for a specific diguanylate cyclase.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Fimbriae, Bacterial; Models, Biological; Mutation; Phenotype; Phosphorus-Oxygen Lyases; Protein Binding; Protein Domains; Pseudomonas aeruginosa

Bacterial cyclic-di-GMP (c-di-GMP) production is associated with biofilm development and the switch from acute to chronic infections. In Pseudomonas aeruginosa, the diguanylate cyclase (DGC) SiaD and phosphatase SiaA, which are co-transcribed as part of a siaABCD operon, are essential for cellular aggregation. However, the detailed functions of this operon and the relationships among its constituent genes are unknown. Here, we demonstrate that the siaABCD operon encodes for a signaling network that regulates SiaD enzymatic activity to control biofilm and aggregates formation. Through protein-protein interaction, SiaC promotes SiaD diguanylate cyclase activity. Biochemical and structural data revealed that SiaB is an unusual protein kinase that phosphorylates SiaC, whereas SiaA phosphatase can dephosphorylate SiaC. The phosphorylation state of SiaC is critical for its interaction with SiaD, which will switch on or off the DGC activity of SiaD and regulate c-di-GMP levels and subsequent virulence phenotypes. Collectively, our data provide insights into the molecular mechanisms underlying the modulation of DGC activity associated with chronic infections, which may facilitate the development of antimicrobial drugs.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phenotype; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa; Signal Transduction; Virulence

Pectobacterium is a diverse genus of phytopathogenic species from soil and water that cause infection either to restricted or multiple plant hosts. Phylogenetic analysis and metabolic fingerprinting of large numbers of genomes have expanded classification of Pectobacterium members. Pectobacterium brasiliense sp. nov has been elevated to the species level having detached from P. carotovorum. Here we present two P. brasiliense strains BF20 and BF45 isolated in Mexico from Opuntia and tobacco, respectively, which cluster into two different groups in whole genome comparisons with other Pectobacterium. We found that BF20 and BF45 strains are phenotypically different as BF45 showed more severe and rapid symptoms in comparison to BF20 in the host models celery and broccoli. Both strains produced similar levels of the main autoinducers, but BF45 shows an additional low abundant autoinducer compared to strain BF20. The two strains had different levels of c-di-GMP, which regulates the transition from motile to sessile lifestyle. In contrast to BF45, BF20 had the highest levels of c-di-GMP, was more motile (swarming), non-flocculant and less proficient in biofilm formation and exopolysaccharide production. Genomic comparisons revealed that differences in c-di-GMP accumulation and perhaps the associated phenotypes might be due to unique c-di-GMP metabolic genes in these two strains. Our results improve our understanding of the associations between phenotype and genotype and how this has shaped the physiology of Pectobacterium strains.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Genome, Bacterial; Genomics; Mexico; Movement; Nicotiana; Opuntia; Pectobacterium; Phenotype; Phylogeny; Polysaccharides, Bacterial

A microneedle is a biomedical device which consists of multiple micron scale needles. It is widely used in various fields to deliver drugs and vaccines to the skin effectively. However, when considering improved vaccine efficacy in microneedle vaccination, it is important to find an appropriate adjuvant that is able to be used in transdermal delivery. Herein, we demonstrated the applicability of c-di-GMP, which is a stimulator of interferon genes (STING) agonist, as an adjuvant for influenza microneedle vaccination. Thus, 2 and 10 μg of GMP with the influenza vaccine were coated onto a microneedle, and then, BALB/c mice were immunized with the coated microneedle to investigate the immunogenicity and protection efficacy of the influenza microneedle vaccination. As a result, the adjuvant groups had an enhanced IgG response, IgG subtypes and HI titer compared to the vaccine only group. In addition to the humoral immunity, the use of an adjuvant has also been shown to improve the cellular immune response. In a challenge study, adjuvant groups had a 100% survival rate and rapid weight recovery. Taken together, this study confirms that GMP is an effective adjuvant for influenza microneedle vaccination. Graphical abstract.

Topics: Adjuvants, Immunologic; Administration, Cutaneous; Animals; Cyclic GMP; Female; Immunity, Cellular; Immunoglobulin G; Influenza A Virus, H1N1 Subtype; Influenza Vaccines; Mice; Mice, Inbred BALB C; Microinjections; Needles; Orthomyxoviridae Infections; Skin; Vaccination

The assembly status of the V. cholerae flagellum regulates biofilm formation, suggesting that the bacterium senses a lack of movement to commit to a sessile lifestyle. Motility and biofilm formation are inversely regulated by the second messenger molecule cyclic dimeric guanosine monophosphate (c-di-GMP). Therefore, we sought to define the flagellum-associated c-di-GMP-mediated signaling pathways that regulate the transition from a motile to a sessile state. Here we report that elimination of the flagellum, via loss of the FlaA flagellin, results in a flagellum-dependent biofilm regulatory (FDBR) response, which elevates cellular c-di-GMP levels, increases biofilm gene expression, and enhances biofilm formation. The strength of the FDBR response is linked with status of the flagellar stator: it can be reversed by deletion of the T ring component MotX, and reduced by mutations altering either the Na+ binding ability of the stator or the Na+ motive force. Absence of the stator also results in reduction of mannose-sensitive hemagglutinin (MSHA) pilus levels on the cell surface, suggesting interconnectivity of signal transduction pathways involved in biofilm formation. Strains lacking flagellar rotor components similarly launched an FDBR response, however this was independent of the status of assembly of the flagellar stator. We found that the FDBR response requires at least three specific diguanylate cyclases that contribute to increased c-di-GMP levels, and propose that activation of biofilm formation during this response relies on c-di-GMP-dependent activation of positive regulators of biofilm production. Together our results dissect how flagellum assembly activates c-di-GMP signaling circuits, and how V. cholerae utilizes these signals to transition from a motile to a sessile state.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Fimbriae, Bacterial; Flagella; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Second Messenger Systems; Signal Transduction; Vibrio cholerae

Second messenger signaling networks allow cells to sense and adapt to changing environmental conditions. In bacteria, the nearly ubiquitous second messenger molecule cyclic di-GMP coordinates diverse processes such as motility, biofilm formation, and virulence. In bacterial pathogens, these signaling networks allow the bacteria to survive changing environmental conditions that are experienced during infection of a mammalian host. While studies have examined the effects of cyclic di-GMP levels on virulence in these pathogens, it has not been possible to visualize cyclic di-GMP levels in real time during the stages of host infection. Toward this goal, we generate the first ratiometric, chemiluminescent biosensor scaffold that selectively responds to c-di-GMP. By engineering the biosensor scaffold, a suite of Venus-YcgR-NLuc (VYN) biosensors is generated that provide extremely high sensitivity (

Topics: Bacterial Proteins; Biosensing Techniques; Cyclic GMP; Energy Transfer; Escherichia coli; Escherichia coli Proteins; Limit of Detection; Luciferases; Luminescent Agents; Luminescent Measurements; Luminescent Proteins; Proof of Concept Study; Protein Binding; Protein Engineering; Signal Transduction; Vibrio cholerae

Biofilm formation by Vibrio cholerae facilitates environmental persistence, and hyperinfectivity within the host. Biofilm formation is regulated by 3',5'-cyclic diguanylate (c-di-GMP) and requires production of the type IV mannose-sensitive hemagglutinin (MSHA) pilus. Here, we show that the MSHA pilus is a dynamic extendable and retractable system, and its activity is directly controlled by c-di-GMP. The interaction between c-di-GMP and the ATPase MshE promotes pilus extension, whereas low levels of c-di-GMP correlate with enhanced retraction. Loss of retraction facilitated by the ATPase PilT increases near-surface roaming motility, and impairs initial surface attachment. However, prolonged retraction upon surface attachment results in reduced MSHA-mediated surface anchoring and increased levels of detachment. Our results indicate that c-di-GMP directly controls MshE activity, thus regulating MSHA pilus extension and retraction dynamics, and modulating V. cholerae surface attachment and colonization.

Topics: Adenosine Triphosphatases; Bacterial Adhesion; Biofilms; Cell Tracking; Cyclic GMP; Fimbriae Proteins; Fimbriae, Bacterial; Movement; Vibrio cholerae

Second messenger molecules are crucial components of environmental signaling systems to integrate multiple inputs and elicit physiological responses. Among various kinds of second messengers, cyclic nucleotides cAMP and cyclic di-GMP (c-di-GMP) play pivotal roles in bacterial environmental responses. However, how these signaling systems are interconnected for a concerted regulation of cellular physiology remains elusive. In a thermophilic cyanobacterium Thermosynechococcus vulcanus strain RKN, incident light color is sensed by cyanobacteriochrome photoreceptors to transduce the light information to the levels of c-di-GMP, which induces cellular aggregation probably via cellulose synthase activation. Herein, we identified that Tlr0485, which is composed of a cGMP-specific phosphodiesterases, adenylate cyclases, and FhlA (GAF) domain and an HD-GYP domain, is a cAMP-activated c-di-GMP phosphodiesterase. We also show biochemical evidence that the two class-III nucleotide cyclases, Cya1 and Cya2, are both adenylate cyclases to produce cAMP in T. vulcanus. The prevalence of cAMP-activated c-di-GMP phosphodiesterase genes in cyanobacterial genomes suggests that the direct crosstalk between cAMP and c-di-GMP signaling systems may be crucial for cyanobacterial environmental responses.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adenylyl Cyclases; Bacterial Proteins; Chromatography, High Pressure Liquid; Cyclic AMP; Cyclic GMP; Electrophoresis, Polyacrylamide Gel; Mutation; Protein Domains; Recombinant Proteins; Second Messenger Systems; Signal Transduction; Thermosynechococcus

Our understanding of the biofilm matrix components utilized by Gram-positive bacteria, and the signalling pathways that regulate their production are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the analysis of synthase-dependent exopolysaccharide biosynthetic systems. Here, we explore the finding that many species of Gram-positive bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B. cereus PelD ortholog binds cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the production of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signalling in Gram-positive organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of bacilli, clostridia, streptococci, and actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-positive bacteria.

Topics: Bacillus cereus; Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Multigene Family; Operon; Phosphorus-Oxygen Lyases; Phylogeny; Polysaccharides; Protein Conformation

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Mycobacterium bovis; Mycolic Acids; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases

The cyanobacterial phytochrome Cph2 is a light-dependent diguanylate cyclase of the cyanobacterium Synechocystis 6803. Under blue light, Cph2-dependent increase in the cellular c-di-GMP concentration leads to inhibition of surface motility and enhanced flocculation of cells in liquid culture. However, the targets of second messenger signalling in this cyanobacterium and its mechanism of action remained unclear. Here, we determined the cellular concentrations of cAMP and c-di-GMP in wild-type and Δcph2 cells after exposure to blue and green light. Inactivation of cph2 completely abolished the blue-light dependent increase in c-di-GMP content. Therefore, a microarray analysis with blue-light grown wild-type and Δcph2 mutant cells was used to identify c-di-GMP dependent alterations in transcript accumulation. The increase in the c-di-GMP content alters expression of genes encoding putative cell appendages, minor pilins and components of chemotaxis systems. The mRNA encoding the minor pilins pilA5-pilA6 was negatively affected by high c-di-GMP content under blue light, whereas the minor pilin encoding operon pilA9-slr2019 accumulates under these conditions, suggesting opposing functions of the respective gene sets. Artificial overproduction of c-di-GMP leads to similar changes in minor pilin gene expression and supports previous findings that c-di-GMP is important for flocculation via the function of minor pilins. Mutational and gene expression analysis further suggest that SyCRP2, a CRP-like transcription factor, is involved in regulation of minor pilin and putative chaperone usher pili gene expression.

Topics: Bacterial Proteins; Cyclic GMP; Fimbriae Proteins; Fimbriae, Bacterial; Light; Mutation; Phytochrome; Second Messenger Systems; Synechocystis

Cyclic di-GMP (c-di-GMP) is a second messenger that modulates multiple responses to environmental and cellular signals in bacteria. Here we identify CdbA, a DNA-binding protein of the ribbon-helix-helix family that binds c-di-GMP in Myxococcus xanthus. CdbA is essential for viability, and its depletion causes defects in chromosome organization and segregation leading to a block in cell division. The protein binds to the M. xanthus genome at multiple sites, with moderate sequence specificity; however, its depletion causes only modest changes in transcription. The interactions of CdbA with c-di-GMP and DNA appear to be mutually exclusive and residue substitutions in CdbA regions important for c-di-GMP binding abolish binding to both c-di-GMP and DNA, rendering these protein variants non-functional in vivo. We propose that CdbA acts as a nucleoid-associated protein that contributes to chromosome organization and is modulated by c-di-GMP, thus revealing a link between c-di-GMP signaling and chromosome biology.

Topics: Bacterial Proteins; Base Sequence; Cell Nucleus; Chromosome Segregation; Chromosomes, Bacterial; Cyclic GMP; DNA-Binding Proteins; DNA, Bacterial; Genetic Loci; Models, Molecular; Myxococcus xanthus; Protein Multimerization; Protein Structure, Secondary; Transcription, Genetic

Light is known to trigger regulatory responses in diverse organisms, including slime molds, animals, plants, and phototrophic bacteria. However, light-dependent processes in nonphototrophic bacteria, and those of pathogens in particular, have received comparatively little research attention. In this study, we examined the impact of light on multicellular development in

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Light; Oxidation-Reduction; Phenazines; Pseudomonas aeruginosa

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger known to control a variety of bacterial processes. The model cyanobacterium,

Topics: Amino Acid Motifs; Amino Acid Sequence; Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genome, Bacterial; Loss of Function Mutation; Phosphorus-Oxygen Lyases; Protein Domains; Salt Stress; Synechocystis

Pseudomonas putida is a microorganism of biotechnological interest that-similar to many other environmental bacteria-adheres to surfaces and forms biofilms. Although various mechanisms contributing to the swim-attach decision have been studied in this species, the role of a 7-gene operon homologous to the wsp cluster of Pseudomonas aeruginosa-which regulates cyclic di-GMP (cdGMP) levels upon surface contact-remained to be investigated. In this work, the function of the wsp operon of P. putida KT2440 has been characterized through inspection of single and multiple wsp deletion variants, complementation with Pseudomonas aeruginosa's homologues, combined with mutations of regulatory genes fleQ and fleN and removal of the flagellar regulator fglZ. The ability of the resulting strains to form biofilms at 6 and 24 h under three different carbon regimes (citrate, glucose and fructose) revealed that the Wsp complex delivers a similar function to both Pseudomonas species. In P. putida, the key components include WspR, a protein that harbours the domain for producing cdGMP, and WspF, which controls its activity. These results not only contribute to a deeper understanding of the network that regulates the sessile-planktonic decision of P. putida but also suggest strategies to exogenously control such a lifestyle switch.

Topics: Bacterial Adhesion; Bacterial Outer Membrane Proteins; Bacterial Proteins; Biofilms; Cyclic GMP; Flagella; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa; Pseudomonas putida

The zoonotic bacterium Leptospira interrogans is the aetiological agent of leptospirosis, a re-emerging infectious disease that is a growing public health concern. Most human cases of leptospirosis result from environmental infection. Biofilm formation and its contribution to the persistence of virulent leptospires in the environment or in the host have scarcely been addressed. Here, we examined spatial and time-domain changes in biofilm production by L. interrogans. Our observations showed that biofilm formation in L. interrogans is a highly dynamic process and leads to a polarized architecture. We notably found that the biofilm matrix is composed of extracellular DNA, which enhances the biofilm's cohesiveness. By studying L. interrogans mutants with defective diguanylate cyclase and phosphodiesterase genes, we show that biofilm production is regulated by intracellular levels of bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) and underpins the bacterium's ability to withstand a wide variety of simulated environmental stresses. Our present results show how the c-di-GMP pathway regulates biofilm formation by L. interrogans, provide insights into the environmental persistence of L. interrogans and, more generally, highlight leptospirosis as an environment-borne threat to human health.

Topics: Animals; Bacterial Proteins; Bacterial Zoonoses; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Humans; Leptospira interrogans; Mutation; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Spatio-Temporal Analysis; Stress, Physiological

In many bacteria, the biofilm-promoting second messenger c-di-GMP is produced and degraded by multiple diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively. High target specificity of some of these enzymes has led to theoretical concepts of "local" c-di-GMP signaling. In Escherichia coli K-12, which has 12 DGCs and 13 PDEs, a single DGC, DgcC, is specifically required for the biosynthesis of the biofilm exopolysaccharide pEtN-cellulose without affecting the cellular c-di-GMP pool, but the mechanistic basis of this target specificity has remained obscure. DGC activity of membrane-associated DgcC, which is demonstrated in vitro in nanodiscs, is shown to be necessary and sufficient to specifically activate cellulose biosynthesis in vivo. DgcC and a particular PDE, PdeK (encoded right next to the cellulose operon), directly interact with cellulose synthase subunit BcsB and with each other, thus establishing physical proximity between cellulose synthase and a local source and sink of c-di-GMP. This arrangement provides a localized, yet open source of c-di-GMP right next to cellulose synthase subunit BcsA, which needs allosteric activation by c-di-GMP. Through mathematical modeling and simulation, we demonstrate that BcsA binding from the low cytosolic c-di-GMP pool in E. coli is negligible, whereas a single c-di-GMP molecule that is produced and released in direct proximity to cellulose synthase increases the probability of c-di-GMP binding to BcsA several hundred-fold. This local c-di-GMP signaling could provide a blueprint for target-specific second messenger signaling also in other bacteria where multiple second messenger producing and degrading enzymes exist.

Topics: Biofilms; Cellulose; Cyclic GMP; Escherichia coli K12; Escherichia coli Proteins; Glucosyltransferases; Intracellular Signaling Peptides and Proteins; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Polysaccharides, Bacterial; Signal Transduction

Interspecies bacterial competition may occur via cell-associated or secreted determinants and is key to successful niche colonization. We previously evolved Pseudomonas aeruginosa in the presence of Staphylococcus aureus and identified mutations in the Wsp surface-sensing signalling system. Surprisingly, a ΔwspF mutant, characterized by increased c-di-GMP levels and biofilm formation capacity, showed potent killing activity towards S. aureus in its culture supernatant. Here, we used an unbiased metabolomic analysis of culture supernatants to identify rhamnolipids, alkyl quinoline N-oxides and two siderophores as members of four chemical clusters, which were more abundant in the ΔwspF mutant supernatants. Killing activities were quorum-sensing controlled but independent of c-di-GMP levels. Based on the metabolomic analysis, we formulated a synthetic cocktail of four compounds, showing broad-spectrum anti-bacterial killing, including both Gram-positive and Gram-negative bacteria. The combination of quorum-sensing-controlled killing and Wsp-system mediated biofilm formation endows P. aeruginosa with capacities essential for niche establishment and host colonization.

Topics: Anti-Bacterial Agents; Antibiosis; Biofilms; Cyclic GMP; Glycolipids; Oligopeptides; Phenols; Pseudomonas aeruginosa; Quinolines; Quorum Sensing; Siderophores; Staphylococcus aureus; Thiazoles

The LonA (or Lon) protease is a central post-translational regulator in diverse bacterial species. In Vibrio cholerae, LonA regulates a broad range of behaviors including cell division, biofilm formation, flagellar motility, c-di-GMP levels, the type VI secretion system (T6SS), virulence gene expression, and host colonization. Despite LonA's role in cellular processes critical for V. cholerae's aquatic and infectious life cycles, relatively few LonA substrates have been identified. LonA protease substrates were therefore identified through comparison of the proteomes of wild-type and ΔlonA strains following translational inhibition. The most significantly enriched LonA-dependent protein was TfoY, a known regulator of motility and the T6SS in V. cholerae. Experiments showed that TfoY was required for LonA-mediated repression of motility and T6SS-dependent killing. In addition, TfoY was stabilized under high c-di-GMP conditions and biochemical analysis determined direct binding of c-di-GMP to LonA results in inhibition of its protease activity. The work presented here adds to the list of LonA substrates, identifies LonA as a c-di-GMP receptor, demonstrates that c-di-GMP regulates LonA activity and TfoY protein stability, and helps elucidate the mechanisms by which LonA controls important V. cholerae behaviors.

Topics: Animals; Bacterial Proteins; Biofilms; Cholera; Cyclic GMP; Disease Models, Animal; Humans; Mice; Mutation; Protease La; Protein Processing, Post-Translational; Protein Stability; Proteolysis; Proteomics; Recombinant Proteins; Type VI Secretion Systems; Vibrio cholerae; Virulence

The bacterial second messenger cyclic diguanylate (c-di-GMP) regulates a wide range of cellular functions from biofilm formation to growth and survival. Targeting a second-messenger network is challenging because the system involves a multitude of components with often overlapping functions. Here, we present a strategy to intercept c-di-GMP signaling pathways by directly targeting the second messenger. For this, we developed a c-di-GMP-sequestering peptide (CSP) that was derived from a CheY-like c-di-GMP effector protein. CSP binds c-di-GMP with submicromolar affinity. The elucidation of the CSP⋅c-di-GMP complex structure by NMR identified a linear c-di-GMP-binding motif, in which a self-intercalated c-di-GMP dimer is tightly bound by a network of H bonds and π-stacking interactions involving arginine and aromatic residues. Structure-based mutagenesis yielded a variant with considerably higher, low-nanomolar affinity, which subsequently was shortened to 19 residues with almost uncompromised affinity. We demonstrate that endogenously expressed CSP intercepts c-di-GMP signaling and effectively inhibits biofilm formation in

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Models, Molecular; Mutagenesis; Peptides; Point Mutation; Protein Conformation; Protein Domains; Protein Interaction Domains and Motifs; Pseudomonas aeruginosa; Second Messenger Systems; Signal Transduction

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is an important intracellular signaling molecule that affects diverse physiological processes in bacteria. The intracellular levels of c-di-GMP are controlled by proteins acting as diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes that synthesize and degrade c-di-GMP, respectively. In the alphaproteobacterium Rhodobacter capsulatus, flagellar motility and gene exchange via production of the gene transfer agent RcGTA are regulated by c-di-GMP. One of the R. capsulatus proteins involved in this regulation is Rcc00620, which contains an N-terminal two-component system response regulator receiver (REC) domain and C-terminal DGC and PDE domains. We demonstrate that the enzymatic activity of Rcc00620 is regulated through the phosphorylation status of its REC domain, which is controlled by a cognate histidine kinase protein, Rcc00621. In this system, the phosphorylated form of Rcc00620 is active as a PDE enzyme and stimulates gene transfer and motility. In addition, we discovered that the rcc00620 and rcc00621 genes are present in only one lineage within the genus Rhodobacter and were acquired via horizontal gene transfer from a distantly related alphaproteobacterium in the order Sphingomonadales. Therefore, a horizontally acquired regulatory system regulates gene transfer in the recipient organism.

Topics: Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Gene Transfer, Horizontal; Histidine Kinase; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Phosphorylation; Protein Domains; Rhodobacter capsulatus

Cellular differentiation is a fundamental strategy used by cells to generate specialized functions at specific stages of development. The bacterium

Topics: Bacterial Physiological Phenomena; Caulobacter crescentus; Cell Cycle; Cyclic GMP; DNA Replication; Fimbriae, Bacterial; Gram-Negative Bacterial Infections; Models, Biological; Mutation

Streptomycetes are filamentous bacteria famous for their ability to produce a vast majority of clinically important secondary metabolites. Both complex morphogenesis and onset of antibiotic biosynthesis are tightly linked in streptomycetes and require series of specific signals for initiation. Cyclic dimeric 3'-5' guanosine monophosphate, c-di-GMP, one of the well-known bacterial second messengers, has been recently shown to govern morphogenesis and natural product synthesis in Streptomyces by altering the activity of the pleiotropic regulator BldD. Here we report a role of the heme-binding diguanylate cyclase SSFG_02181 from Streptomyces ghanaensis in the regulation of the peptidoglycan glycosyltransferase inhibitor moenomycin A biosynthesis. Deletion of ssfg_02181 reduced the moenomycin A accumulation and led to a precocious sporulation, while the overexpression of the gene blocked sporogenesis and remarkably improved antibiotic titer. We also demonstrate that BldD negatively controls the expression of ssfg_02181, which stems from direct binding of BldD to the ssfg_02181 promoter. Notably, the heterologous expression of ssfg_02181 in model Streptomyces spp. arrested morphological progression at aerial mycelium level and strongly altered the production of secondary metabolites. Altogether, our work underscores the significance of c-di-GMP-mediated signaling in natural product biosynthesis and pointed to extensively applicable approach to increase antibiotic production levels in streptomycetes.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Bambermycins; Cyclic GMP; DNA-Binding Proteins; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Developmental; Heme-Binding Proteins; Metabolic Engineering; Morphogenesis; Phosphorus-Oxygen Lyases; Promoter Regions, Genetic; Second Messenger Systems; Streptomyces; Transcription Factors

Electrochemically active biofilms are capable of exchanging electrons with solid electron acceptors and have many energy and environmental applications such as bioelectricity generation and environmental remediation. The performance of electrochemically active biofilms is usually dependent on c-type cytochromes, while biofilm development is controlled by a signal cascade mediated by the intracellular secondary messenger bis-(3'-5') cyclic dimeric guanosine monophosphate (c-di-GMP). However, it is unclear whether there are any links between the c-di-GMP regulatory system and the expression of c-type cytochromes. In this study, we constructed a S. oneidensis MR-1 strain with a higher cytoplasmic c-di-GMP level by constitutively expressing a c-di-GMP synthase and it exhibited expected c-di-GMP-influenced traits, such as lowered motility and increased biofilm formation. Compared to MR-1 wild-type strain, the high c-di-GMP strain had a higher Fe(III) reduction rate (21.58 vs 11.88 pM of Fe(III)/h cell) and greater expression of genes that code for the proteins involved in the Mtr pathway, including CymA, MtrA, MtrB, MtrC and OmcA. Furthermore, single-cell Raman microspectroscopy (SCRM) revealed a great increase of c-type cytochromes in the high c-di-GMP strain as compared to MR-1 wild-type strain. Our results reveal for the first time that the c-di-GMP regulation system indirectly or directly positively regulates the expression of cytochromes involved in the extracellular electron transport (EET) in S. oneidensis, which would help to understand the regulatory mechanism of c-di-GMP on electricity production in bacteria.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Cytochromes; Ferric Compounds; Gene Expression Regulation, Bacterial; Shewanella

The ubiquitous cyclic di-GMP (c-di-GMP) network is highly redundant with numerous GGDEF domain proteins as diguanylate cyclases and EAL domain proteins as c-di-GMP specific phosphodiesterases comprising those domains as two of the most abundant bacterial domain superfamilies. One hallmark of the c-di-GMP network is its exalted plasticity as c-di-GMP turnover proteins can rapidly vanish from species within a genus and possess an above average transmissibility. To address the evolutionary forces of c-di-GMP turnover protein maintenance, conservation, and diversity, we investigated a Gram-positive and a Gram-negative species, which preserved only one single clearly identifiable GGDEF domain protein. Species of the family Morganellaceae of the order Enterobacterales exceptionally show disappearance of the c-di-GMP signaling network, but

Topics: Cyclic GMP; Gene Expression Regulation, Bacterial; Proteus; Signal Transduction; Streptococcus

Most bacteria respond to surfaces by biogenesis of intracellular c-di-GMP, which inhibits motility and induces secretion of biofilm-promoting adherence factors. Bacterial cellulose is a widespread biofilm component whose secretion in Gram-negative species requires an inner membrane, c-di-GMP-dependent synthase tandem (BcsAB), an outer membrane porin (BcsC), and various accessory subunits that regulate synthase assembly and function as well as the exopolysaccharide's chemical composition and mechanical properties. We recently showed that in

Topics: Biofilms; Cellulose; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases

Cyclic diguanylate (c-di-GMP) is a broadly conserved intracellular second messenger that influences different bacterial processes, including virulence, stress tolerance or social behaviours and biofilm development. Although in most cases the environmental cue that initiates the signal transduction cascade leading to changes in cellular c-di-GMP levels remains unknown, certain L- and D-amino acids have been described to modulate c-di-GMP turnover in some bacteria. In this work, we have analysed the influence of L-amino acids on c-di-GMP levels in the plant-beneficial bacterium Pseudomonas putida KT2440, identifying L-arginine as the main one causing a significant increase in c-di-GMP. Both exogenous (environmental) and endogenous (biosynthetic) L-arginine influence biofilm formation by P. putida through changes in c-di-GMP content and altered expression of structural elements of the biofilm extracellular matrix. The contribution of periplasmic binding proteins forming part of amino acid transport systems to the response to environmental L-arginine was also studied. Contrary to what has been described in other bacteria, in P. putida these proteins seem not to be directly responsible for signal transduction. Rather, their contribution to global L-arginine pools appears to determine changes in c-di-GMP turnover. We propose that arginine plays a connecting role between cellular metabolism and c-di-GMP signalling in P. putida.

Topics: Arginine; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Pseudomonas Infections; Pseudomonas putida

Many bacteria cycle between sessile and motile forms in which they must sense and respond to internal and external signals to coordinate appropriate physiology. Maintaining fitness requires genetic networks that have been honed in variable environments to integrate these signals. The identity of the major regulators and how their control mechanisms evolved remain largely unknown in most organisms. During four different evolution experiments with the opportunist betaproteobacterium

Topics: Bacterial Proteins; Biofilms; Burkholderia cenocepacia; Cyclic GMP; Directed Molecular Evolution; Gene Expression Regulation, Bacterial; Mutation; Phenotype; Quorum Sensing; Signal Transduction; Virulence

Cyclic dinucleotides (CDNs) are promising vaccine adjuvants inducing balanced, potent humoral, and cellular immune responses. How aging influences CDN efficacy is unclear. We examined the vaccine efficacy of 3',5'-cyclic diguanylic acid (cyclic di-GMP, CDG), the founding member of CDNs, in 1-year-old (middle-aged) and 2-year-old (aged) C57BL/6J mice. We found that 1- and 2-year-old C57BL/6J mice are defective in CDG-induced memory T helper (Th)1 and Th17 responses and high-affinity serum immunoglobulin (Ig)G, mucosal IgA production. Next, we generated two novel tumor necrosis factor (TNF) fusion proteins that target soluble TNF (solTNF) and transmembrane TNF (tmTNF) to monocyte-derived dendritic cells (moDCs) to enhance CDG vaccine efficacy in 1- and 2-year-old mice. The moDC-targeting TNF fusion proteins restored CDG-induced memory Th1, Th17, and high-affinity IgG, IgA responses in the 1- and 2-year-old mice. Together, the data suggested that aging negatively impacts CDG vaccine adjuvanticity. MoDC-targeting TNF fusion proteins enhanced CDG adjuvanticity in the aging mice.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Age Factors; Aging; Animals; Cells, Cultured; Cyclic GMP; Dendritic Cells; Immunity, Mucosal; Immunization; Immunogenicity, Vaccine; Immunoglobulin A; Immunoglobulin Fc Fragments; Lung; Mice, Inbred C57BL; Recombinant Fusion Proteins; Th1 Cells; Th17 Cells; Tumor Necrosis Factor-alpha

We investigated the effect of temperature on the biofilm formation of

Topics: Biofilms; Body Temperature Regulation; Cyclic GMP; Extracellular Polymeric Substance Matrix; Pseudomonas aeruginosa

Topics: Alginates; Azotobacter vinelandii; Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Molecular Weight; Oxygen; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Polysaccharides, Bacterial

The genus

Topics: Alginates; Azotobacter vinelandii; Bacterial Proteins; Carbohydrate Epimerases; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa

Bacteriophytochromes are the most abundant and ubiquitous light-sensing receptors in bacteria and are involved in time-of-day behavior or responses. However, their biological and regulatory role in non-photosynthetic bacteria is poorly understood, and even less is known about how they regulate diverse cellular processes. Here, we show that a bacteriophytochrome (XooBphP) from the plant pathogen Xanthomonas oryzae pv. oryzae perceives light signals and transduces a signal through its EAL-mediated phosphodiesterase activity, modulating the intracellular level of the ubiquitous bacterial second messenger c-di-GMP. We discover that light-mediated fine-tuning of intracellular c-di-GMP levels by XooBphP regulates production of virulence functions, iron metabolism, and transition from a sessile to a free-swimming motile lifestyle, contributing to its colonization of the host and virulence. XooBphP thus plays a crucial role in integrating photo-sensing with intracellular signaling to control the pathogenic lifestyle and social behavior.

Topics: Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Signal Transduction; Virulence

Second messengers, c-di-GMP and (p)ppGpp, are vital regulatory molecules in bacteria, influencing cellular processes such as biofilm formation, transcription, virulence, quorum sensing, and proliferation. While c-di-GMP and (p)ppGpp are both synthesized from GTP molecules, they play antagonistic roles in regulating the cell cycle. In C. crescentus, c-di-GMP works as a major regulator of pole morphogenesis and cell development. It inhibits cell motility and promotes S-phase entry by inhibiting the activity of the master regulator, CtrA. Intracellular (p)ppGpp accumulates under starvation, which helps bacteria to survive under stressful conditions through regulating nucleotide levels and halting proliferation. (p)ppGpp responds to nitrogen levels through RelA-SpoT homolog enzymes, detecting glutamine concentration using a nitrogen phosphotransferase system (PTS. We propose a mathematical model for the dynamics of c-di-GMP and (p)ppGpp in C. crescentus and analyze how the guanine nucleotide-based second messenger system responds to certain environmental changes communicated through the PTS. In this work, we integrate current knowledge and experimental observations from the literature to formulate a novel mathematical model. We analyze the model and demonstrate how the PTS

Topics: Caulobacter crescentus; Cell Cycle Checkpoints; Cyclic GMP; Models, Theoretical; Nitrogen; Phosphotransferases; Second Messenger Systems

The bacterial enhancer-binding protein (bEBP) FlrC, controls motility and colonization of

Topics: Adenosine Triphosphatases; Bacterial Proteins; Cholera; Crystallography, X-Ray; Cyclic GMP; DNA-Binding Proteins; Flagella; Gene Expression Regulation, Bacterial; Phylogeny; Protein Structure, Tertiary; Vibrio cholerae

Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage-specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and high-throughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phage-mediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.

Topics: Bacterial Proteins; Bacteriophages; Biosynthetic Pathways; CRISPR-Cas Systems; Cyclic GMP; DNA; Down-Regulation; Escherichia coli; Gene Expression Regulation, Bacterial; Genes, Essential; Genome, Bacterial; Mutation; Phenotype; Reproducibility of Results; Suppression, Genetic

The cell morphology of rod-shaped bacteria is determined by the rigid net of peptidoglycan forming the cell wall. Alterations to the rod shape, such as the curved rod, occur through manipulating the process of cell wall synthesis. The human pathogen

Topics: Bacterial Proteins; Biofilms; Cell Shape; Cyclic GMP; Gene Expression Regulation, Bacterial; Humans; Life Style; Second Messenger Systems; Vibrio cholerae

Cyclic di-nucleotides are important secondary signaling molecules in bacteria that regulate a wide range of processes. In this study, we found that Caenorhabditis elegans can detect and are attracted to multiple signal molecules produced by Vibrio cholerae, specifically the 3',5'-cyclic diguanylate (c-di-GMP), even though this bacterium kills the host at a high rate. C-di-GMP is sensed through C. elegans olfactory AWC neurons, which then evokes a series of signal transduction pathways that lead to reduced activity of two key stress response transcription factors, SKN-1 and HSF-1, and weakened innate immunity. Taken together, our study elucidates the role of c-di-GMP in interkingdom communication. For C. elegans, bacterial c-di-GMP may serve as a cue that they can use to detect food. On the other hand, preexposure to low concentrations of c-di-GMP may impair their immune response, which could facilitate bacterial invasion and survival.

Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Communication; Cyclic GMP; Second Messenger Systems; Signal Transduction; Transcription Factors; Vibrio cholerae

Non-typhoid Salmonella infection may present as acute gastroenteritis or chronic infection, primarily in the bile-rich gallbladder. Biofilm formation is a mechanism of bile resistance in Salmonella. Our aim was to determine how Salmonella utilizes bile as a signal, and to study the relevance of the interaction between the PhoP-PhoQ two-component system and cyclic diguanosine monophosphate (c-di-GMP) signaling to biofilm formation.. Two-dimensional (2-D) gel electrophoresis was used to identify genes required for Salmonella biofilm formation in bile. Quantitative real-time PCR (qRT-PCR) was used to clarify the role of the PhoP-PhoQ two-component system and its interaction with genes involved in the c-di-GMP network during biofilm formation.. Our result revealed that Salmonella mutants with incomplete outer membrane (△ompA), defective flagella (△flgE), or incomplete PhoP-PhoQ two-component system (△phoP), were unable to develop complete biofilms in the presence of bile. Moreover, PhoP-PhoQ two-component system-related Salmonella mutants (△phoP, △phoQ, △phoP△phoQ) had lower expression of c-di-GMP related genes (csgD, adrA) than the wild-type Salmonella strain had in the bile environment.. Salmonella may sense and respond to bile through the PhoP-PhoQ two-component system during biofilm formation. Furthermore, the PhoP-PhoQ two-component system might activate regulators of the c-di-GMP signaling network.

Topics: Bacterial Proteins; Bile; Biofilms; Cyclic GMP; Humans; Salmonella Infections; Salmonella typhimurium; Signal Transduction

The opportunistic human pathogen Vibrio vulnificus inhabits warm coastal waters and asymptomatically colonizes seafood, most commonly oysters. We previously characterized an isolate that exhibited greater biofilm formation, aggregation and oyster colonization than its parent. This was due, in part, to the production of a Type IV Tad pilus (Iam). However, the locus lacked key processing and regulatory genes required for pilus production. Here, we identify a pilin peptidase iamP, and LysR-type regulator (LRTR) iamR, that fulfil these roles and show that environmental calcium, which oysters enrich for shell repair and growth, regulates iam expression. The architecture of the iam locus differs from the classical LRTR paradigm and requires an additional promoter to be integrated into the regulatory network. IamR specifically recognized the iamR promoter (P

Topics: Animals; Biofilms; Calcium; Cyclic GMP; Fimbriae Proteins; Genetic Loci; Ostreidae; Promoter Regions, Genetic; Vibrio vulnificus

Cyclic di-GMP (c-di-GMP) is a bacterial second messenger molecule that is important in the biology of

Topics: Cyclic GMP; Gene Expression Regulation, Bacterial; Riboswitch; RNA Stability; RNA, Bacterial; Second Messenger Systems; Transcription, Genetic; Vibrio cholerae

The two-component signalling system (TCS) comprising a histidine kinase (HK) and a response regulator (RR) is the predominant bacterial sense-and-response machinery. Because bacterial cells usually encode a number of TCSs to adapt to various ecological niches, the specificity of a TCS is in the centre of regulation. Specificity of TCS is defined by the capability and velocity of phosphoryl transfer between a cognate HK and a RR. Here, we provide genetic, enzymology and structural data demonstrating that the second messenger cyclic-di-GMP physically and specifically binds to RavS, a HK of the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. The [c-di-GMP]-RavS interaction substantially promotes specificity between RavS and RavR, a GGDEF-EAL domain-containing RR, by reinforcing the kinetic preference of RavS to phosphorylate RavR. [c-di-GMP]-RavS binding effectively decreases the phosphorylation level of RavS and negatively regulates bacterial swimming. Intriguingly, the EAL domain of RavR counteracts the above regulation by degrading c-di-GMP and then increasing the level of phosphorylated RavS. Therefore, RavR acts as a bifunctional phosphate sink that finely controls the level of phosphorylated RavS. These biochemical processes interactively modulate the phosphoryl flux between RavS-RavR and bacterial lifestyle transition. Our results revealed that c-di-GMP acts as an allosteric effector to dynamically modulate specificity between HK and RR.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Flagella; Gene Expression Regulation, Bacterial; Histidine Kinase; Phosphorylation; Signal Transduction; Virulence; Xanthomonas campestris

As a ubiquitous bacterial secondary messenger, c-di-GMP plays key regulatory roles in processes such as bacterial motility and transcription regulation. CobB is the Sir2 family protein deacetylase that controls energy metabolism, chemotaxis, and DNA supercoiling in many bacteria. Using an Escherichia coli proteome microarray, we found that c-di-GMP strongly binds to CobB. Further, protein deacetylation assays showed that c-di-GMP inhibits the activity of CobB and thereby modulates the biogenesis of acetyl-CoA. Interestingly, we also found that one of the key enzymes directly involved in c-di-GMP production, DgcZ, is a substrate of CobB. Deacetylation of DgcZ by CobB enhances its activity and thus the production of c-di-GMP. Our work establishes a novel negative feedback loop linking c-di-GMP biogenesis and CobB-mediated protein deacetylation.

Topics: Acetyl Coenzyme A; Acetylation; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Feedback, Physiological; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Protein Array Analysis; Proteomics; Second Messenger Systems; Sirtuins

Effective cancer immunotherapy depends on the robust activation of tumor-specific antigen-presenting cells (APC). Immune agonists encapsulated within nanoparticles (NP) can be delivered to tumor sites to generate powerful antitumor immune responses with minimal off-target dissemination. Systemic delivery enables widespread access to the microvasculature and draining to the APC-rich perivasculature. We developed an immuno-nanoparticle (immuno-NP) coloaded with cyclic diguanylate monophosphate, an agonist of the stimulator of interferon genes pathway, and monophosphoryl lipid A, and a Toll-like receptor 4 agonist, which synergize to produce high levels of type I IFNβ. Using a murine model of metastatic triple-negative breast cancer, systemic delivery of these immuno-NPs resulted in significant therapeutic outcomes due to extensive upregulation of APCs and natural killer cells in the blood and tumor compared with control treatments. These results indicate that NPs can facilitate systemic delivery of multiple immune-potentiating cargoes for effective APC-driven local and systemic antitumor immunity. SIGNIFICANCE: Systemic administration of an immuno-nanoparticle in a murine breast tumor model drives a robust tumor site-specific APC response by delivering two synergistic immune-potentiating molecules, highlighting the potential of nanoparticles for immunotherapy.

Topics: Animals; Antigen-Presenting Cells; Cyclic GMP; Drug Delivery Systems; Drug Screening Assays, Antitumor; Drug Synergism; Female; Interferon-beta; Killer Cells, Natural; Lipid A; Lymphocytes, Tumor-Infiltrating; Mammary Neoplasms, Experimental; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Microcirculation; Nanocapsules; Toll-Like Receptor 4; Triple Negative Breast Neoplasms

Cyclic diguanosine monophosphate (c-di-GMP) is an important second messenger involved in bacterial switching from motile to sessile lifestyles. In the opportunistic pathogen

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genome, Bacterial; Meta-Analysis as Topic; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Phylogeny; Pseudomonas aeruginosa; Pseudomonas Infections; Transcriptome; Virulence

Reagents that activate the signaling adaptor stimulator of interferon genes (STING) suppress experimentally induced autoimmunity in murine models of multiple sclerosis and arthritis. In this study, we evaluated STING agonists as potential reagents to inhibit spontaneous autoimmune type I diabetes (T1D) onset in non-obese diabetic (NOD) female mice. Treatments with DNA nanoparticles (DNPs), which activate STING when cargo DNA is sensed, delayed T1D onset and reduced T1D incidence when administered before T1D onset. DNP treatment elevated indoleamine 2,3 dioxygenase (IDO) activity, which regulates T-cell immunity, in spleen, pancreatic lymph nodes and pancreas of NOD mice. Therapeutic responses to DNPs were partially reversed by inhibiting IDO and DNP treatment synergized with insulin therapy to further delay T1D onset and reduce T1D incidence. Treating pre-diabetic NOD mice with cyclic guanyl-adenyl dinucleotide (cGAMP) to activate STING directly delayed T1D onset and stimulated interferon-αβ (IFN-αβ), while treatment with cyclic diguanyl nucleotide (cdiGMP) did not delay T1D onset or induce IFN-αβ in NOD mice. DNA sequence analyses revealed that NOD mice possess a STING polymorphism that may explain differential responses to cGAMP and cdiGMP. In summary, STING agonists attenuate T1D progression and DNPs enhance therapeutic responses to insulin therapy.

Topics: Animals; Cyclic GMP; Diabetes Mellitus, Type 1; Disease Models, Animal; DNA; Drug Synergism; Female; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Insulin; Membrane Proteins; Mice; Mice, Inbred NOD; Nanoparticles; Nucleotides, Cyclic; Polymorphism, Genetic; T-Lymphocytes; Up-Regulation

Biofilms form when bacteria adhere to a surface and secrete an extracellular polymeric substance. Bacteria embedded within a biofilm benefit from increased resistance to antibiotics, host immune responses, and harsh environmental factors. Nitric oxide (NO) is a signaling molecule that can modulate communal behavior, including biofilm formation, in many bacteria. In many cases, NO-induced biofilm dispersal is accomplished through signal transduction pathways that ultimately lead to a decrease in intracellular cyclic-di-GMP levels. H-NOX (heme nitric oxide/oxygen binding domain) proteins are the best characterized bacterial NO sensors and have been implicated in NO-mediated cyclic-di-GMP signaling, but we have recently discovered a second family of NO-sensitive proteins in bacteria named NosP (NO sensing protein); to date, a clear link between NosP signaling and cyclic-di-GMP metabolism has not been established. Here we present evidence that NosP (Lpg0279) binds to NO and directly affects cyclic-di-GMP production from two-component signaling proteins Lpg0278 and Lpg0277 encoded within the NosP operon. Lpg0278 and Lpg0277 are a histidine kinase and cyclic-di-GMP synthase/phosphodiesterase, respectively, that have already been established as being important in regulating

Topics: Adenosine Triphosphate; Biofilms; Cyclic GMP; Escherichia coli; Genetic Vectors; Hemeproteins; Histidine Kinase; Hydrolysis; Legionella pneumophila; Nitric Oxide; Operon; Phosphoric Diester Hydrolases; Phosphorylation

Precisely determining the intracellular concentrations of metabolites and signaling molecules is critical in studying cell biology. Fluorogenic RNA-based sensors have emerged to detect various targets in living cells. However, it is still challenging to apply these genetically encoded sensors to quantify the cellular concentrations and distributions of targets. Herein, using a pair of orthogonal fluorogenic RNA aptamers, DNB and Broccoli, we engineered a modular sensor system to apply the DNB-to-Broccoli fluorescence ratio to quantify the cell-to-cell variations of target concentrations. These ratiometric sensors can be broadly applied for live-cell imaging and quantification of metabolites, signaling molecules, and other synthetic compounds.

Topics: Adenine; Aniline Compounds; Aptamers, Nucleotide; Biosensing Techniques; Cyclic GMP; Escherichia coli; Fluorescence; Fluorescent Dyes; Molecular Imaging; Tetracycline

A thorough understanding of the mechanisms of Rcs and EnvZ/OmpR phosphorelay systems that allow Yersinia enterocolitica to thrive in various environments is crucial to prevent and control Y. enterocolitica infections. In this study, we showed that RcsB and OmpR have the ability to function differently in modulating a diverse array of physiological processes in Y. enterocolitica. The rcsB mutant stimulated flagella biosynthesis and increased motility, biofilm formation and c-di-GMP production by upregulating flhDC, hmsHFRS and hmsT. However, mutation in ompR exhibited a non-motile phenotype due to the lack of flagella. Biofilm formation was reduced and less c-di-GMP was produced through the downregulation of flhDC, hmsHFRS and hmsT expression when Y. enterocolitica was exposed to low osmolarity conditions. Furthermore, OmpR was identified to be important for Y. enterocolitica to grow in extreme temperature conditions. Importantly, ompR mutations in Y. enterocolitica were more sensitive to polymyxin B and sodium dodecyl sulfate than rcsB mutations. Since motility, biofilm formation and environmental tolerance are critical for bacterial colonization of the host, these findings indicated that OmpR is more critical than RcsB in shaping the pathogenic phenotype of Y. enterocolitica.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Cyclic GMP; Drug Resistance, Bacterial; Gene Expression Regulation, Bacterial; Microbial Viability; Mutation; Osmolar Concentration; Polymyxin B; Temperature; Trans-Activators; Yersinia enterocolitica; Yersinia Infections

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3'3'-cyclic GMP-AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5'-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5'-pGpG-Ca2+ structure, β5-α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5'-pGpG-Ca2+ structure quite different from other 5'-pGpG bound structures reported earlier.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Nucleotides, Cyclic; Phosphoric Diester Hydrolases; Vibrio cholerae

Oxidative stress is an unavoidable consequence of interactions with various reactive oxygen species (ROS)-inducing agents that would damage cells or even cause cell death. Bacteria have developed defensive systems, including induction of stress-sensing proteins and detoxification enzymes, to handle oxidative stress. Cyclic diguanylate (c-di-GMP) is a ubiquitous intracellular bacterial second messenger that coordinates diverse aspects of bacterial growth and behavior. In this study, we revealed a mechanism by which c-di-GMP regulated bacterial oxidative stress resistance in Pseudomonas putida KT2440. High c-di-GMP level was found to enhance bacterial resistance towards hydrogen peroxide. Transcription assay showed that expression of two oxidative stress resistance genes, fpr-1 and katE, was promoted under high c-di-GMP level. Deletion of fpr-1 and katE both decreased bacterial tolerance to hydrogen peroxide and weakened the effect of c-di-GMP on oxidative stress resistance. The promoted expression of fpr-1 under high c-di-GMP level was caused by increased cellular ROS via a transcriptional regulator FinR. We further demonstrated that the influence of high c-di-GMP on cellular ROS depend on the existence of FleQ, a transcriptional regulatory c-di-GMP effector. Besides, the regulation of katE by c-di-GMP was also FleQ dependent in an indirect way. Our results proved a connection between c-di-GMP and oxidative stress resistance and revealed a mechanism by which c-di-GMP regulated expression of fpr-1 and katE in P. putida KT2440.

Topics: Bacterial Proteins; Catalase; Cyclic GMP; Gene Expression Regulation, Bacterial; Hydrogen Peroxide; Intracellular Signaling Peptides and Proteins; Oxidative Stress; Pseudomonas putida

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Heme; Nitric Oxide; Shewanella; Signal Transduction

Biofilm formation protects bacteria from stresses including antibiotics and host immune responses. Carbon sources can modulate biofilm formation and host colonization in Vibrio cholerae, but the underlying mechanisms remain unclear. Here, we show that EIIA

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Glucose; Phosphoenolpyruvate Sugar Phosphotransferase System; Sugars; Vibrio cholerae

The dinucleotide second messenger c-di-GMP has emerged as a central regulator of reversible cell attachment during bacterial biofilm formation. A prominent cell adhesion mechanism first identified in pseudomonads combines two c-di-GMP-mediated processes: transcription of a large adhesin and its cell surface display via posttranslational proteolytic control. Here, we characterize an orthologous c-di-GMP effector system and show that it is operational in

Topics: Adhesins, Bacterial; Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli; Gene Expression Regulation, Bacterial; Signal Transduction; Vibrio cholerae

Riboswitches are RNA molecules that regulate gene expression using conformation change, affected by binding of small molecule ligands. Although a number of ligand-bound aptamer complex structures have been solved, it is important to know ligand-free conformations of the aptamers in order to understand the mechanism of specific binding by ligands. In this paper, we use dynamics simulations on a series of models to characterize the ligand-free and ligand-bound aptamer domain of the c-di-GMP class I (GEMM-I) riboswitch. The results revealed that the ligand-free aptamer has a stable state with a folded P2 and P3 helix, an unfolded P1 helix and open binding pocket. The first Mg ions binding to the aptamer is structurally favorable for the successive c-di-GMP binding. The P1 helix forms when c-di-GMP is successive bound. Three key junctions J1/2, J2/3 and J1/3 in the GEMM-I riboswitch contributing to the formation of P1 helix have been found. The binding of the c-di-GMP ligand to the GEMM-I riboswitch induces the riboswitch's regulation through the direct allosteric communication network in GEMM-I riboswitch from the c-di-GMP binding sites in the J1/2 and J1/3 junctions to the P1 helix, the indirect ones from those in the J2/3 and P2 communicating to P1 helix via the J1/2 and J1/3 media.

Topics: Allosteric Regulation; Allosteric Site; Aptamers, Nucleotide; Cyclic GMP; Ligands; Models, Molecular; Molecular Dynamics Simulation; Nucleic Acid Conformation; Riboswitch

Shewanella oneidensis is an aquatic proteobacterium with remarkable respiratory and chemotactic abilities. It is also capable of forming biofilms either associated to surfaces (SSA-biofilm) or at the air-liquid interface (pellicle). We have previously shown that pellicle biogenesis in S. oneidensis requires the flagellum and the chemotaxis regulatory system including CheA3 kinase and CheY3 response regulator. Here we searched for additional factors involved in pellicle development. Using a multicopy library of S. oneidensis chromosomal fragments, we identified two genes encoding putative diguanylate cyclases (pdgA and pdgB) and allowing pellicle formation in the non-pellicle-forming cheY3-deleted mutant. A mutant deleted of both pdgA and pdgB is affected during pellicle development. By overexpressing phosphodiesterase encoding genes, we confirmed the key role of c-di-GMP in pellicle biogenesis. The mxd operon, previously proposed to encode proteins involved in exopolysaccharide biosynthesis, is also essential for pellicle formation. In addition, we showed that the MxdA protein, containing a degenerate GGDEF motif, binds c-di-GMP and interacts with both CheY3 and PdgA. Therefore, we propose that pellicle biogenesis in S. oneidensis is controlled by a complex pathway that involves the chemotaxis response regulator CheY3, the two putative diguanylate cyclases PdgA and PdgB, and the c-di-GMP binding protein MxdA.

Topics: Bacterial Proteins; Biofilms; Chemotaxis; Cyclic GMP; Escherichia coli Proteins; Flagella; Gene Expression Regulation, Bacterial; Operon; Phosphorus-Oxygen Lyases; Shewanella; Signal Transduction

In Pseudomonas putida KT2440, the exopolysaccharide Pea is associated with biofilm stability and pellicle formation; however, little is known about its regulatory pathway. In this study, we identified that the gene cluster pea was transcribed from 25 bp upstream of the operon and the stationary phase alternative sigma factor RpoS regulated the transcription of pea. When RpoS was absent, another sigma factor, likely the housekeeping sigma factor RpoD, could also mediate pea transcription but at a low level. The function of Pea polysaccharide was further confirmed to be necessary for full production of biofilm, formation of pellicle and c-di-GMP-dependent wrinkly colony morphology. Additionally, evidences were provided to demonstrate that the transcriptional regulator AmrZ was a negative regulator for pea expression. DNase I footprinting studies verified that AmrZ bound directly to the site overlapping the pea promoter, which might interfere with the binding of RNA polymerase to the promoter and resulted in inhibition of transcription initiation.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Multigene Family; Polysaccharides, Bacterial; Promoter Regions, Genetic; Pseudomonas putida; Sigma Factor; Transcriptional Activation

The opportunistic human pathogen Pseudomonas aeruginosa effectively colonizes host epithelia using pili as primary adhesins. Here we uncover a surface-specific asymmetric virulence program that enhances P. aeruginosa host colonization. We show that when P. aeruginosa encounters surfaces, the concentration of the second messenger c-di-GMP increases within a few seconds. This leads to surface adherence and virulence induction by stimulating pili assembly through activation of the c-di-GMP receptor FimW. Surface-attached bacteria divide asymmetrically to generate a piliated, surface-committed progeny (striker) and a flagellated, motile offspring that leaves the surface to colonize distant sites (spreader). Cell differentiation is driven by a phosphodiesterase that asymmetrically positions to the flagellated pole, thereby maintaining c-di-GMP levels low in the motile offspring. Infection experiments demonstrate that cellular asymmetry strongly boosts infection spread and tissue damage. Thus, P. aeruginosa promotes surface colonization and infection transmission through a cooperative virulence program that we termed Touch-Seed-and-Go.

Topics: A549 Cells; Adhesins, Bacterial; Apoptosis; Bacterial Proteins; Biofilms; Carrier Proteins; Cell Differentiation; Cyclic GMP; DNA-Binding Proteins; Fimbriae, Bacterial; Gene Deletion; Gene Expression Regulation, Bacterial; Homologous Recombination; Humans; Mutagenesis, Site-Directed; Phosphoric Diester Hydrolases; Pseudomonas aeruginosa; Pseudomonas Infections; Virulence

A core regulatory pathway that directs developmental transitions and cellular asymmetries in Agrobacterium tumefaciens involves two overlapping, integrated phosphorelays. One of these phosphorelays putatively includes four histidine sensor kinase homologues, DivJ, PleC, PdhS1 and PdhS2, and two response regulators, DivK and PleD. In several different alphaproteobacteria, this pathway influences a conserved downstream phosphorelay that ultimately controls the phosphorylation state of the CtrA master response regulator. The PdhS2 sensor kinase reciprocally regulates biofilm formation and swimming motility. In the current study, the mechanisms by which the A. tumefaciens sensor kinase PdhS2 directs this regulation are delineated. PdhS2 lacking a key residue implicated in phosphatase activity is markedly deficient in proper control of attachment and motility phenotypes, whereas a kinase-deficient PdhS2 mutant is only modestly affected. A genetic interaction between DivK and PdhS2 is revealed, unmasking one of several connections between PdhS2-dependent phenotypes and transcriptional control by CtrA. Epistasis experiments suggest that PdhS2 may function independently of the CckA sensor kinase, the cognate sensor kinase for CtrA, which is inhibited by DivK. Global expression analysis of the pdhS2 mutant reveals a restricted regulon, most likely functioning through CtrA to separately control motility and regulate the levels of the intracellular signal cyclic diguanylate monophosphate (cdGMP), thereby affecting the production of adhesive polysaccharides and attachment. We hypothesize that in A. tumefaciens the CtrA regulatory circuit has expanded to include additional inputs through the addition of PdhS-type sensor kinases, likely fine-tuning the response of this organism to the soil microenvironment.

Topics: Agrobacterium tumefaciens; Bacterial Adhesion; Bacterial Proteins; Biofilms; Cyclic GMP; Epistasis, Genetic; Gene Expression Regulation, Bacterial; Histidine Kinase; Locomotion; Mutation; Phosphorylation; Polysaccharides, Bacterial; Signal Transduction; Transcription Factors

The causative agent of Legionnaires' disease, Legionella pneumophila, colonizes amoebae and biofilms in the environment. The opportunistic pathogen employs the Lqs (Legionella quorum sensing) system and the signalling molecule LAI-1 (Legionella autoinducer-1) to regulate virulence, motility, natural competence and expression of a 133 kb genomic "fitness island", including a putative novel regulator. Here, we show that the regulator termed LvbR is an LqsS-regulated transcription factor that binds to the promoter of lpg1056/hnox1 (encoding an inhibitor of the diguanylate cyclase Lpg1057), and thus, regulates proteins involved in c-di-GMP metabolism. LvbR determines biofilm architecture, since L. pneumophila lacking lvbR accumulates less sessile biomass and forms homogeneous mat-like structures, while the parental strain develops more compact bacterial aggregates. Comparative transcriptomics of sessile and planktonic ΔlvbR or ΔlqsR mutant strains revealed concerted (virulence, fitness island, metabolism) and reciprocally (motility) regulated genes in biofilm and broth respectively. Moreover, ΔlvbR is hyper-competent for DNA uptake, defective for phagocyte infection, outcompeted by the parental strain in amoebae co-infections and impaired for cell migration inhibition. Taken together, our results indicate that L. pneumophila LvbR is a novel pleiotropic transcription factor, which links the Lqs and c-di-GMP regulatory networks to control biofilm architecture and pathogen-host cell interactions.

Topics: 4-Butyrolactone; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Gene Regulatory Networks; Legionella pneumophila; Legionnaires' Disease; Quorum Sensing; Transcription Factors; Virulence

Secondary messengers (such as (p)ppGpp and c-di-GMP) were proved to play important roles in antibiotic biosynthesis in actinobacteria. In this study, we found that transcription levels of erythromycin-biosynthetic ( ery) genes were upregulated in nutrient limitation, which depended on (p)ppGpp in Saccharopolyspora erythraea. Further study demonstrated that the expression of ery genes and intracellular concentrations of (p)ppGpp showed synchronization during culture process. The erythromycin yield was significantly improved (about 200%) by increasing intracellular concentration of (p)ppGpp through introduction of C-terminally truncated (p)ppGpp synthetase RelA (1.43 kb of the N-terminal segment) from Streptomyces coelicolor into S. erythraea strain NRRL2338 (named as WT/pIB-P

Topics: Cyclic GMP; Erythromycin; Gene Expression Regulation, Bacterial; Metabolic Engineering; Promoter Regions, Genetic; Saccharopolyspora

Flagellar motility is critical for surface attachment and biofilm formation in many bacteria. A key regulator of flagellar motility in

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Flagella; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa; Second Messenger Systems

The human pathogen Pseudomonas aeruginosa can cause both acute infections and chronic biofilm-based infections. Expression of acute virulence factors is positively regulated by cAMP, whereas biofilm formation is positively regulated by c-di-GMP. We provide evidence that increased levels of cAMP, caused by either a lack of degradation or increased production, inhibit P. aeruginosa biofilm formation. cAMP-mediated inhibition of P. aeruginosa biofilm formation required Vfr, and involved a reduction of the level of c-di-GMP, as well as reduced production of biofilm matrix components. A mutant screen and characterization of defined knockout mutants suggested that a subset of c-di-GMP-degrading phosphodiesterases is involved in cAMP-Vfr-mediated biofilm inhibition in P. aeruginosa.

Topics: Bacterial Proteins; Biofilms; Cyclic AMP; Cyclic AMP Receptor Protein; Cyclic GMP; Extracellular Polymeric Substance Matrix; Mutation; Phosphoric Diester Hydrolases; Pseudomonas aeruginosa

Topics: Allosteric Regulation; Alteromonadaceae; Bacterial Proteins; Cyclic GMP; Kinetics; Light; Light Signal Transduction; Photoreceptors, Microbial; Spectrophotometry, Ultraviolet

Quorum sensing (QS) signals are widely used by bacterial pathogens to control biological functions and virulence in response to changes in cell population densities.

Topics: Acyl-Butyrolactones; Bacterial Proteins; Biofilms; Burkholderia cenocepacia; Burkholderia Infections; Cyclic GMP; Fatty Acids, Monounsaturated; Gene Expression Regulation, Bacterial; Microbial Sensitivity Tests; Phenotype; Quorum Sensing; Signal Transduction; Virulence

Alcanivorax borkumensis is a ubiquitous marine bacterium that utilizes alkanes as a sole carbon source. We observed two phenotypes in the A. borkumensis SK2 type strain: rough (R) and smooth (S) types. The S type exhibited lower motility and higher polysaccharide production than the R type. Full genome sequencing revealed a mutation in the S type involved in cyclic-di-GMP production. The present results suggest that higher c-di-GMP levels in the S type control the biofilm forming behavior of this bacterium in a manner commensurate with other Gram-negative bacteria.

Topics: Alcanivoraceae; Alkanes; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Genome, Bacterial; Phenotype; Point Mutation; Polysaccharides, Bacterial

YcgR, a cyclic diguanylate (c-di-GMP)-binding protein expressed in Escherichia coli, brakes flagellar rotation by binding to the motor in a c-di-GMP dependent manner and has been implicated in triggering biofilm formation. Vibrio alginolyticus has a single polar flagellum and encodes YcgR homologue, PlzD. When PlzD or PlzD-GFP was highly over-produced in nutrient-poor condition, the polar flagellar motility of V. alginolyticus was reduced. This inhibitory effect is c-di-GMP independent as mutants substituting putative c-di-GMP-binding residues retain the effect. Moderate over-expression of PlzD-GFP allowed its localization at the flagellated cell pole. Truncation of the N-terminal 12 or 35 residues of PlzD abolished the inhibitory effect and polar localization, and no inhibitory effect was observed by deleting plzD or expressing an endogenous level of PlzD-GFP. Subcellular fractionation showed that PlzD, but not its N-terminally truncated variants, was precipitated when over-produced. Moreover, immunoblotting and N-terminal sequencing revealed that endogenous PlzD is synthesized from Met33. These results suggest that an N-terminal extension allows PlzD to localize at the cell pole but causes aggregation and leads to inhibition of motility. In V. alginolyticus, PlzD has a potential property to associate with the polar flagellar motor but this interaction is too weak to inhibit rotation.

Topics: Bacterial Proteins; Cyclic GMP; Flagella; Movement; Vibrio alginolyticus

Pathoadaptive mutations linked to c-di-GMP signalling were investigated in neonatal meningitis-causing Escherichia coli (NMEC). The results indicated that NMEC strains deficient in RpoS (the global stress regulator) maintained remarkably low levels of c-di-GMP, a major bacterial sessility-motility switch. Deletion of ycgG2, shown here to encode a YcgG allozyme with c-di-GMP phosphodiesterase activity, and the restoration of RpoS led to a decrease in S-fimbriae, robustly produced in artificial urine, hinting that the urinary tract could serve as a habitat for NMEC. We showed that NMEC were skilled in aerobic citrate utilization in the presence of glucose, a property that normally does not exist in E. coli. Our data suggest that this metabolic novelty is a property of extraintestinal pathogenic E. coli since we reconstituted this ability in E. coli UTI89 (a cystitis isolate) via deactivation rpoS; additionally, a set of pyelonephritis E. coli isolates were shown here to aerobically use citrate in the presence of glucose. We found that the main reason for this metabolic capability is RpoS inactivation leading to the production of the citrate transporter CitT, exploited by NMEC for ferric citrate uptake dependent on YcgG2 (an allozyme with c-di-GMP phosphodiesterase activity).

Topics: Bacterial Proteins; Citric Acid; Cyclic GMP; Escherichia coli Proteins; Extraintestinal Pathogenic Escherichia coli; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Glucose; Meningitis, Escherichia coli; Organic Anion Transporters; Sigma Factor

Cellulose, whose production is controlled by c-di-GMP, is a commonly found exopolysaccharide in bacterial biofilms. Pseudomonas syringae pv. tomato (Pto) DC3000, a model organism for molecular studies of plant-pathogen interactions, carries the wssABCDEFGHI operon for the synthesis of acetylated cellulose. The high intracellular levels of the second messenger c-di-GMP induced by the overexpression of the heterologous diguanylate cyclase PleD stimulate cellulose production and enhance air-liquid biofilm (pellicle) formation. To characterize the mechanisms involved in Pto DC3000 pellicle formation, we studied this process using mutants lacking flagella, biosurfactant or different extracellular matrix components, and compared the pellicles produced in the absence and in the presence of PleD. We have discovered that neither alginate nor the biosurfactant syringafactin are needed for their formation, whereas cellulose and flagella are important but not essential. We have also observed that the high c-di-GMP levels conferred more cohesion to Pto cells within the pellicle and induced the formation of intracellular inclusion bodies and extracellular fibres and vesicles. Since the pellicles were very labile and this greatly hindered their handling and processing for microscopy, we have also developed new methods to collect and process them for scanning and transmission electron microscopy. These techniques open up new perspectives for the analysis of fragile biofilms in other bacterial strains.

Topics: Bacterial Adhesion; Cellulose; Cyclic GMP; Metabolic Engineering; Mutation; Pseudomonas syringae; Recombinant Proteins

Although the ubiquitous bacterial secondary messenger cyclic diguanylate (c-di-GMP) has important cellular functions in a wide range of bacteria, its function in the model plant pathogen

Topics: Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Genetic Pleiotropy; Microorganisms, Genetically-Modified; Pseudomonas syringae; Virulence

Topics: Animals; Biosensing Techniques; Cellulose; Cyclic GMP; Cytoplasm; Disease Models, Animal; Fluorescence; Glucosyltransferases; Host-Pathogen Interactions; Macrophages; Mice; Mice, Inbred BALB C; Microbial Viability; Phagocytosis; Phosphoric Diester Hydrolases; Salmonella typhimurium; Virulence

The c-di-GMP-binding effector protein FlgZ has been demonstrated to control motility in the opportunistic pathogen Pseudomonas aeruginosa and it was suggested that c-di-GMP-bound FlgZ impedes motility via its interaction with the MotCD stator. To further understand how motility is downregulated in P. aeruginosa and to elucidate the general control mechanisms operating during bacterial growth, we examined the spatiotemporal activity of FlgZ. We re-annotated the P. aeruginosaflgZ open reading frame and demonstrated that FlgZ-mediated downregulation of motility is fine-tuned via three independent mechanisms. First, we found that flgZ gene is transcribed independently from flgMN in stationary growth phase to increase FlgZ protein levels in the cell. Second, FlgZ localizes to the cell pole upon c-di-GMP binding and third, we describe that FimV, a cell pole anchor protein, is involved in increasing the polar localized c-di-GMP bound FlgZ to inhibit both, swimming and swarming motility. Our results shed light on the complex dynamics and spatiotemporal control of c-di-GMP-dependent bacterial motility phenotypes and on how the polar anchor protein FimV, the motor brake FlgZ and the stator proteins function to repress flagella-driven swimming and swarming motility.

Topics: Bacterial Proteins; Cyclic GMP; Flagella; Gene Expression Regulation, Bacterial; Movement; Phenotype; Protein Binding; Pseudomonas aeruginosa; Signal Transduction

The human stimulator of interferon genes protein (hSTING) can bind cyclic dinucleotides (CDNs) to activate the production of type I interferons and inflammatory cytokines. These CDNs can be either bacterial secondary messengers, 3'3'-CDNs, or endogenous 2'3'-cGAMP. cGAMP, with a unique 2'-5' bond, is the most potent activator of hSTING among all CDNs. However, current understanding of the molecular principles underlying the unique ability of 2'3'-cGAMP to potently activate hSTINGs other than 3'3'-CDNs remains incomplete. In this work, molecular dynamics simulations were used to provide an atomistic picture of the binding of 2'3'-cGAMP and one 3'3'-CDN (c-di-GMP) to hSTING. The results suggest that hSTING binds more strongly to 2'3'-cGAMP than to c-di-GMP, which prefers to bind with a more open and flexible state of hSTING. Finally, a potential "dock-lock-anchor" mechanism is proposed for the activation of hSTING upon the binding of a potent ligand. It is believed that deep insights into understanding the binding of hSTING with 3'3'-CDNs and the endogenous 2'3'-cGAMP would help to establish the principles underlying powerful 2'3'-cGAMP signaling and the nature of hSTING activation, as well as related drug design.

Topics: Binding Sites; Cyclic GMP; Guanine Nucleotides; Humans; Ligands; Membrane Proteins; Molecular Dynamics Simulation; Mutation; Principal Component Analysis; Protein Binding; Protein Conformation

Dickeya dadantii is a plant pathogen that causes soft rot disease on vegetable and potato crops. To successfully cause infection, this pathogen needs to coordinately modulate the expression of genes encoding several virulence determinants, including plant cell wall degrading enzymes (PCWDEs), type III secretion system (T3SS) and flagellar motility. Here, we uncover a novel feed-forward signalling circuit for controlling virulence. Global RNA chaperone Hfq interacts with an Hfq-dependent sRNA ArcZ and represses the translation of pecT, encoding a LysR-type transcriptional regulator. We demonstrate that the ability of ArcZ to be processed to a 50 nt 3'- end fragment is essential for its regulation of pecT. PecT down-regulates PCWDE and the T3SS by repressing the expression of a global post-transcriptional regulator- (RsmA-) associated sRNA encoding gene rsmB. In addition, we show that the protein levels of two cyclic di-GMP (c-di-GMP) diguanylate cyclases (DGCs), GcpA and GcpL, are repressed by Hfq. Further studies show that both DGCs are essential for the Hfq-mediated post-transcriptional regulation on RsmB. Overall, our report provides new insights into the interplays between ubiquitous signalling transduction systems that were most studied independently and sheds light on multitiered regulatory mechanisms for a precise disease regulation in bacteria.

Topics: Bacterial Proteins; Cell Wall; Cyclic GMP; Enterobacteriaceae; Gene Expression Regulation, Bacterial; Plant Diseases; RNA-Binding Proteins; RNA, Bacterial; RNA, Small Untranslated; Signal Transduction; Type III Secretion Systems; Virulence; Virulence Factors

Clostridium difficile is a Gram-positive, anaerobic bacterium and the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis. C. difficile modulates its transition from a motile to a sessile lifestyle through a mechanism of riboswitches regulated by cyclic diguanosine monophosphate (c-di-GMP). Previously described as a sortase substrate positively regulated by c-di-GMP, CD2831 was predicted to be a collagen-binding protein and thus potentially involved in sessility. By overexpressing CD2831 in C. difficile and heterologously expressing it on the surface of Lactococcus lactis, here we further demonstrated that CD2831 is a collagen-binding protein, able to bind to immobilized collagen types I, III and V as well as native collagen produced by human fibroblasts. We also observed that the overexpression of CD2831 raises the ability to form biofilm on abiotic surface in both C. difficile and L. lactis. Notably, we showed that CD2831 binds to the collagen-like domain of the human complement component C1q, suggesting a role in preventing complement cascade activation via the classical pathway. This functional characterization places CD2831 in the Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMMs) family, a class of virulence factors with a dual role in adhesion to collagen-rich tissues and in host immune evasion by binding to human complement components.

Topics: Bacterial Adhesion; Bacterial Proteins; Biofilms; Clostridioides difficile; Clostridium Infections; Collagen; Complement C1q; Cyclic GMP; Extracellular Matrix; Host-Pathogen Interactions; Humans; Immune Evasion; Lactococcus lactis; Protein Domains; Recombinant Proteins

Topics: Bacterial Proteins; Biofilms; Biological Variation, Population; Cyclic GMP; Escherichia coli; Escherichia coli Infections; Extracellular Matrix; Fluorescent Antibody Technique; Gene Expression Regulation, Bacterial; Models, Biological; Mutation; Phenotype; Signal Transduction

The bacterial secondary metabolite cyclic di-GMP is a widespread, cytoplasmic signal that promotes a physiological transition in which motility is inhibited and biofilm formation is activated. A paper published in this issue (A. E. Baker, S. S. Webster, A. Diepold, S. L. Kuchma, E. Bordeleau, et al., J Bacteriol 201:e00741-18, 2019, https://doi.org/10.1128/JB.00741-18) makes an important connection between cyclic di-GMP and flagellar components. They show that stator units, which normally interact with the flagellum to power rotation, can alternatively interact with and activate an enzyme that synthesizes cyclic di-GMP in

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Flagella; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa

The physiological functions of c-di-GMP and its involvement in many key processes led to its recognition as a major and ubiquitous bacterial second messenger. Aside from being a bacterial signaling molecule, c-di-GMP is also an immunostimulatory molecule capable of inducing innate and adaptive immune responses through maturation of immune mammalian cells. Given the broad biological functions of c-di-GMP and its potential applications as a nucleic-acid-based drug, the chemical synthesis of c-di-GMP has drawn considerable interest. An improved phosphoramidite approach to the synthesis of c-di-GMP is reported herein. The synthetic approach is based on the use of a 5'-O-formyl protecting group, which can be rapidly and chemoselectively cleaved from a key dinucleotide phosphoramidite intermediate to enable a cyclocondensation reaction leading to a fully protected c-di-GMP product in a yield ∼80%. The native c-di-GMP is isolated, after complete deprotection, in an overall yield of 36% based on the commercial ribonucleoside used as starting material. © 2019 by John Wiley & Sons, Inc.

Topics: Amides; Cyclic GMP; Esters; Phosphoric Acids; Ribonucleosides

The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers effector proteins into prokaryotic and eukaryotic preys. This secretion system has emerged as a key player in regulating the microbial diversity in a population. In the plant pathogen Agrobacterium tumefaciens, the signalling cascades regulating the activity of this secretion system are poorly understood. Here, we outline how the universal eubacterial second messenger cyclic di-GMP impacts the production of T6SS toxins and T6SS structural components. We demonstrate that this has a significant impact on the ability of the phytopathogen to compete with other bacterial species in vitro and in planta. Our results suggest that, as opposed to other bacteria, c-di-GMP turns down the T6SS in A. tumefaciens thus impacting its ability to compete with other bacterial species within the rhizosphere. We also demonstrate that elevated levels of c-di-GMP within the cell decrease the activity of the Type IV secretion system (T4SS) and subsequently the capacity of A. tumefaciens to transform plant cells. We propose that such peculiar control reflects on c-di-GMP being a key second messenger that silences energy-costing systems during early colonization phase and biofilm formation, while low c-di-GMP levels unleash T6SS and T4SS to advance plant colonization.

Topics: Agrobacterium tumefaciens; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Type IV Secretion Systems; Type VI Secretion Systems

The synthesis of signaling molecules is one strategy bacteria employ to sense alterations in their environment and rapidly adjust to those changes. In Gram-negative bacteria, bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulates the transition from a unicellular motile state to a multicellular sessile state. However, c-di-GMP signaling has been less intensively studied in Gram-positive organisms. To that end, we constructed a fluorescent

Topics: Bacillus subtilis; Bacterial Proteins; Cyclic GMP; Genes, Reporter; Luminescent Proteins; Microscopy; Single-Cell Analysis

Flagellum mediated motility is an essential trait for rhizosphere colonization by pseudomonads. Flagella synthesis is a complex and energetically expensive process that is tightly regulated. In Pseudomonas fluorescens, the regulatory cascade starts with the master regulatory protein FleQ that is in turn regulated by environmental signals through the Gac/Rsm and SadB pathways, which converge in the sigma factor AlgU. AlgU is required for the expression of amrZ, encoding a FleQ repressor. AmrZ itself has been shown to modulate c-di-GMP levels through the control of many genes encoding enzymes implicated in c-di-GMP turnover. This cyclic nucleotide regulates flagellar function and besides, the master regulator of the flagellar synthesis signaling pathway, FleQ, has been shown to bind c-di-GMP. Here we show that AdrA, a diguanylate cyclase regulated by AmrZ participates in this signaling pathway. Epistasis analysis has shown that AdrA acts upstream of SadB, linking SadB with environmental signaling. We also show that SadB binds c-di-GMP with higher affinity than FleQ and propose that c-di-GMP produced by AdrA modulates flagella synthesis through SadB.

Topics: Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Flagella; Gene Expression Regulation, Bacterial; Organelle Biogenesis; Phosphorus-Oxygen Lyases; Pseudomonas fluorescens; Sigma Factor; Signal Transduction; Trans-Activators

The second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP) drives the transition between planktonic and biofilm growth in many bacterial species.. Bacteria can adopt different lifestyles, depending on the environment in which they grow. They can exist as single cells that are free to explore their environment or group together to form ‘biofilms’. The bacteria in biofilms stick to a surface, and produce a slimy ‘matrix’ that covers and thereby protects them. Biofilms have been found in lung infections that affect people with the genetic disorder cystic fibrosis, and can also form on the surface of medical implants. Because the biofilm lifestyle protects bacteria from the immune system and antimicrobial drugs, learning about how biofilms form could help researchers to discover ways to prevent and treat such infections. Many bacteria switch between the free-living and biofilm lifestyles by altering their levels of a signaling molecule called cyclic diguanylate monophosphate (called c-di-GMP for short). Bacteria living in biofilms have much higher levels of c-di-GMP than their free-living counterparts, and bacteria that have high levels of c-di-GMP produce more biofilm matrix. Bacteria called

Topics: Bacterial Adhesion; Bacterial Proteins; Biofilms; Cell Membrane; Cyclic GMP; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa; Quorum Sensing

During its life cycle, the environmental pathogen

Topics: Amino Acids; Bacterial Proteins; Culture Media; Cyclic GMP; Gene Expression Regulation, Bacterial; Hydroxybutyrates; Legionella pneumophila; Microbial Viability; Polyesters; Signal Transduction

How the central innate immune protein, STING, is activated by its ligands remains unknown. Here, using structural biology and biochemistry, we report that the metazoan second messenger 2'3'-cGAMP induces closing of the human STING homodimer and release of the STING C-terminal tail, which exposes a polymerization interface on the STING dimer and leads to the formation of disulfide-linked polymers via cysteine residue 148. Disease-causing hyperactive STING mutations either flank C148 and depend on disulfide formation or reside in the C-terminal tail binding site and cause constitutive C-terminal tail release and polymerization. Finally, bacterial cyclic-di-GMP induces an alternative active STING conformation, activates STING in a cooperative manner, and acts as a partial antagonist of 2'3'-cGAMP signaling. Our insights explain the tight control of STING signaling given varying background activation signals and provide a therapeutic hypothesis for autoimmune syndrome treatment.

Topics: Binding Sites; Cyclic GMP; Dimerization; Endoplasmic Reticulum; HEK293 Cells; Humans; Ligands; Membrane Proteins; Models, Molecular; Mutagenesis, Site-Directed; Nucleotides, Cyclic; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins; Signal Transduction

Cyclic di-GMP (c-di-GMP) is an important second messenger in bacteria, and its regulatory network has been extensively studied. However, information regarding the activation mechanisms of its receptors remains limited. In this study, we characterized the two-component regulator DevR as a new c-di-GMP receptor and further uncovered a novel co-activation mechanism for effective regulation of DevR in mycobacteria. We show that high c-di-GMP levels induce the expression of the

Topics: Bacterial Proteins; Cyclic GMP; Mycobacterium smegmatis; Oxidative Stress; Recombinant Proteins

Topics: Bacterial Proteins; Biofilms; Catalase; Cyclic GMP; Hydrogen Peroxide; Signal Transduction; Vibrio cholerae

The second messenger cyclic diguanylate (c-di-GMP) plays a prominent role in regulating flagellum-dependent motility in the single-flagellated pathogenic bacterium

Topics: Bacterial Proteins; Biofilms; Chemotaxis; Cyclic GMP; Escherichia coli Proteins; Flagella; Gene Expression Regulation, Bacterial; Methyltransferases; Molecular Motor Proteins; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa; Second Messenger Systems; Signal Transduction

Antimicrobial photodynamic inactivation (APDI) is a new therapeutic modality which needs more precision during application due to the possibility of exposure of bacteria to sub-lethal doses (sAPDI). In this study, we aimed to evaluate the effect of sAPDI on Pseudomonas aeruginosa quorum sensing (QS) and c-di-GMP signaling which are important virulence factor regulatory systems.. Biofilm formation, pyoverdine, pyocyanin and protease production of P. aeruginosa was evaluated before and after a single sAPDI treatment with 0.8 mM methylene blue (MB) plus 1, 2, and 5-min irradiation with red laser light. Fluorescent lasB, rhlA, pqsA, and cdrA reporters of P. aeruginosa PAO1 and P. aeruginosa ΔmexAB-oprM were treated individually with sAPDI and the regulatory signals were detected. The gene expressions were also assessed after sAPDI using quantitative real-time PCR analysis.. Morphological observations and molecular assessments indicated that sAPDI with 0.8 mM MB along with 2- and 5-min irradiation led to an increase in the expression of the Las QS system and c-di-GMP signaling, while 1 min irradiation revealed dissimilar results (increase in lasB expression and decrease in c-di-GMP levels). Expression of rhlA and pqsA did not change in response to sAPDI. Further, a severe lethal effect of sAPDI was observed in P. aeruginosa ΔmexAB-oprM as compared with the wild type strain, whilst there was no difference in QS and c-di-GMP levels as detected by reporters between treated and untreated samples.. The results suggest that sAPDI affects QS and c-di-GMP signaling inP. aeruginosa in a time-dependent manner.

Topics: Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Genes, Bacterial; Methylene Blue; Photochemotherapy; Photosensitizing Agents; Pseudomonas aeruginosa; Quorum Sensing; Real-Time Polymerase Chain Reaction; Virulence Factors

The DivJ-DivK-PleC signaling system of

Topics: Bacterial Adhesion; Bacterial Proteins; Caulobacteraceae; Cyclic GMP; Escherichia coli Proteins; Mutation; Phenotype; Phosphorus-Oxygen Lyases; Signal Transduction

Intracellular signaling enzymes drive critical changes in cellular physiology and gene expression, but their endogenous activities in vivo remain highly challenging to study in real time and for individual cells. Here we show that flow cytometry can be performed in complex media to monitor single-cell population distributions and dynamics of cyclic di-GMP signaling, which controls the bacterial colonization program. These in vivo biochemistry experiments are enabled by our second-generation RNA-based fluorescent (RBF) biosensors, which exhibit high fluorescence turn-on in response to cyclic di-GMP. Specifically, we demonstrate that intracellular levels of cyclic di-GMP in Escherichia coli are repressed with excess zinc, but not with other divalent metals. Furthermore, in both flow cytometry and fluorescence microscopy setups, we monitor the dynamic increase in cellular cyclic di-GMP levels upon zinc depletion and show that this response is due to de-repression of the endogenous diguanylate cyclase DgcZ. In the presence of zinc, cells exhibit enhanced cell motility and increased sensitivity to antibiotics due to inhibited biofilm formation. Taken together, these results showcase the application of RBF biosensors in visualizing single-cell dynamic changes in cyclic di-GMP signaling in direct response to environmental cues such as zinc and highlight our ability to assess whether observed phenotypes are related to specific signaling enzymes and pathways.

Topics: Biosensing Techniques; Cyclic GMP; Escherichia coli; Flow Cytometry; Microscopy, Fluorescence; RNA; Signal Transduction; Single-Cell Analysis; Zinc

Synthetic biologists have attempted to solve real-world problems, such as those of bacterial biofilms, that are involved in the pathogenesis of many clinical infections and difficult to eliminate. To address this, we employed a blue light responding system and integrated it into the chromosomes of Pseudomonas aeruginosa. With making rational adaptions and improvements of the light-activated system, we provided a robust and convenient means to spatiotemporally control gene expression and manipulate biological processes with minimal perturbation in P. aeruginosa. It increased the light-induced gene expression up to 20-fold. Moreover, we deliberately introduced a functional protein gene PA2133 containing an EAL domain to degrade c-di-GMP into the modified system, and showed that the optimally engineered optogenetic tool inhibited the formation of P. aeruginosa biofilms through the induction of blue light, resulting in much sparser and thinner biofilms. Our approach establishes a methodology for leveraging the tools of synthetic biology to guide biofilm formation and engineer biofilm patterns with unprecedented spatiotemporal resolution. Furthermore, the findings suggest that the synthetic optogenetic system may provide a promising strategy that could be applied to control and fight biofilms.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Green Fluorescent Proteins; Histidine Kinase; Light; Microscopy, Fluorescence; Optogenetics; Plasmids; Pseudomonas aeruginosa; Time-Lapse Imaging

Topics: Bacterial Proteins; Chemotaxis; Crystallography, X-Ray; Cyclic GMP; Flagella; Humans; Models, Molecular; Protein Binding; Protein Conformation; Pseudomonas aeruginosa; Pseudomonas Infections

The bacterial second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) has been shown to influence the expression of virulence factors in certain pathogenic bacteria, but little is known about its activity in the increasingly antibiotic-resistant pathogen

Topics: Animals; Cyclic GMP; Disease Models, Animal; Female; Fimbriae, Bacterial; Gene Knockdown Techniques; Klebsiella pneumoniae; Lung; Mice, Inbred C57BL; Pneumonia, Bacterial; Up-Regulation; Virulence

Immunity against microbes depends on recognition of pathogen-associated molecular patterns by innate receptors. Signaling pathways triggered by

Topics: Animals; Brucella abortus; Brucellosis; Cyclic GMP; Cytokines; Gene Expression; Gene Expression Profiling; Granuloma; GTP-Binding Proteins; Host-Pathogen Interactions; Immunity, Innate; Inflammasomes; Inflammation Mediators; Interferon Regulatory Factor-3; Interferon Type I; Macrophages; Membrane Proteins; Mice; Mice, Knockout; Models, Biological; NF-kappa B; Signal Transduction

The Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterases (PDEs) that catalyze degradation of cyclic di-adenosine monophosphate (c-di-AMP) could be subdivided into two subfamilies based on the final product [5'-phosphadenylyl-adenosine (5'-pApA) or AMP]. In a previous study, we revealed that Rv2837c, a stand-alone DHH/DHHA1 PDE, employs a 5'-pApA internal flipping mechanism to produce AMPs. However, why the membrane-bound DHH/DHHA1 PDE can only degrade c-di-AMP to 5'-pApA remains obscure. Here, we report the crystal structure of the DHH/DHHA1 domain of GdpP (GdpP-C), and structures in complex with c-di-AMP, cyclic di-guanosine monophosphate (c-di-GMP), and 5'-pApA. Structural analysis reveals that GdpP-C binds nucleotide substrates quite differently from how Rv2837c does in terms of substrate-binding position. Accordingly, the nucleotide-binding site of the DHH/DHHA1 PDEs is organized into three (C, G, and R) subsites. For GdpP-C, in the C and G sites c-di-AMP binds and degrades into 5'-pApA, and its G site determines nucleotide specificity. To further degrade into AMPs, 5'-pApA must slide into the C and R sites for flipping and hydrolysis as in Rv2837c. Subsequent mutagenesis and enzymatic studies of GdpP-C and Rv2837c uncover the complete flipping process and reveal a unified catalytic mechanism for members of both DHH/DHHA1 PDE subfamilies.

Topics: Amino Acid Motifs; Bacterial Proteins; Binding Sites; Cloning, Molecular; Crystallography, X-Ray; Cyclic GMP; Dinucleoside Phosphates; Escherichia coli; Gene Expression; Genetic Vectors; Kinetics; Manganese; Models, Molecular; Mycobacterium tuberculosis; Phosphoric Diester Hydrolases; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Recombinant Proteins; Staphylococcus aureus; Substrate Specificity

Elevated levels of the second messenger c-di-GMP suppress virulence in diverse pathogenic bacteria, yet mechanisms are poorly characterized. In the foodborne pathogen

Topics: Amino Acids, Branched-Chain; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Guanosine Triphosphate; Host-Pathogen Interactions; HT29 Cells; Humans; Listeria monocytogenes; Listeriosis; Peptide Termination Factors; Promoter Regions, Genetic; Regulon; Transcription Factors; Virulence

The transcriptional regulator BrlR from Pseudomonas aeruginosa is a member of the MerR family of multidrug transport activators. Studies have shown that BrlR plays an important role in the drug tolerance of P. aeruginosa in biofilms. The tolerance to drugs can be enhanced by 3',5'-cyclic diguanylic acid (c-di-GMP). In the present study, we analyze the apo structure of BrlR and the direct binding between GyrI-like domain of BrlR and P. aeruginosa toxin pyocyanin. Furthermore, we show that pyocyanin can enhance the binding between BrlR and DNA in vitro. These findings suggest that BrlR can serve as the binding partner for both c-di-GMP and pyocyanin.

Topics: Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Cyclic GMP; DNA, Bacterial; Models, Molecular; Protein Domains; Pseudomonas aeruginosa; Pyocyanine; Transcription Factors

Bacterial exopolysaccharides (EPS) often confer a survival advantage by protecting the cell against abiotic and biotic stresses, including host defensive factors. They are also main components of the extracellular matrix involved in cell-cell recognition, surface adhesion and biofilm formation. Biosynthesis of a growing number of EPS has been reported to be regulated by the ubiquitous second messenger c-di-GMP, which promotes the transition to a biofilm mode of growth in an intimate association with the eukaryotic host. Here we describe a strategy based on the combination of an approach to artificially increase the intracellular level of c-di-GMP in virtually any gram-negative bacteria with a high throughput screening (HTS) for the identification of monosaccharide composition and carbohydrate fingerprinting of novel EPS, or modified variants, that can be involved in host-bacteria interactions.

Topics: Bacteria; Bacterial Physiological Phenomena; Biofilms; Carbohydrate Metabolism; Chromatography, High Pressure Liquid; Cyclic GMP; Genetic Vectors; Gram-Negative Bacteria; Host-Pathogen Interactions; Mass Spectrometry; Metabolome; Metabolomics; Polysaccharides, Bacterial

3',5'-Cyclic diguanylic acid (c-di-GMP) is a bacterial second messenger molecule that is a key global regulator in

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Movement; Promoter Regions, Genetic; Protein Processing, Post-Translational; Riboswitch; Signal Transduction; Transcription Factors; Transcription, Genetic; Vibrio cholerae

Bacterial cyclic di-GMP signaling networks often consist of dozens of components, and the majority of these components have no observable function. Dahlstrom et al. (J. Bacteriol. 200:e00703-17, 2018, https://doi.org/10.1128/JB.00703-17) explored the function of every component of the

Topics: Biofilms; Cyclic GMP; Pseudomonas fluorescens

The transcriptional regulator AmrZ is a global regulatory protein conserved within the pseudomonads. AmrZ can act both as a positive and a negative regulator of gene expression, controlling many genes implicated in environmental adaption. Regulated traits include motility, iron homeostasis, exopolysaccharides production and the ability to form biofilms. In Pseudomonas fluorescens F113, an amrZ mutant presents a pleiotropic phenotype, showing increased swimming motility, decreased biofilm formation and very limited ability for competitive colonization of rhizosphere, its natural habitat. It also shows different colony morphology and binding of the dye Congo Red. The amrZ mutant presents severely reduced levels of the messenger molecule cyclic-di-GMP (c-di-GMP), which is consistent with the motility and biofilm formation phenotypes. Most of the genes encoding proteins with diguanylate cyclase (DGCs) or phosphodiesterase (PDEs) domains, implicated in c-di-GMP turnover in this bacterium, appear to be regulated by AmrZ. Phenotypic analysis of eight mutants in genes shown to be directly regulated by AmrZ and encoding c-di-GMP related enzymes, showed that seven of them were altered in motility and/or biofilm formation. The results presented here show that in P. fluorescens, AmrZ determines c-di-GMP levels through the regulation of a complex network of genes encoding DGCs and PDEs.

Topics: Bacterial Proteins; Biofilms; Colony Count, Microbial; Cyclic GMP; Gene Expression Regulation, Bacterial; Genes, Bacterial; Movement; Polysaccharides; Pseudomonas fluorescens; Rhizosphere; Transcription, Genetic

Topics: Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Phosphoric Diester Hydrolases; Pseudomonas aeruginosa

Bacteria colonize highly diverse and complex environments, from gastrointestinal tracts to soil and plant surfaces. This colonization process is controlled in part by the intracellular signal cyclic di-GMP, which regulates bacterial motility and biofilm formation. To interrogate cyclic di-GMP signaling networks, a variety of fluorescent biosensors for live cell imaging of cyclic di-GMP have been developed. However, the need for external illumination precludes the use of these tools for imaging bacteria in their natural environments, including in deep tissues of whole organisms and in samples that are highly autofluorescent or photosensitive. The need for genetic encoding also complicates the analysis of clinical isolates and environmental samples. Toward expanding the study of bacterial signaling to these systems, we have developed the first chemiluminescent biosensors for cyclic di-GMP. The biosensor design combines the complementation of split luciferase (CSL) and bioluminescence resonance energy transfer (BRET) approaches. Furthermore, we developed a lysate-based assay for biosensor activity that enabled reliable high-throughput screening of a phylogenetic library of 92 biosensor variants. The screen identified biosensors with very large signal changes (∼40- and 90-fold) as well as biosensors with high affinities for cyclic di-GMP ( K

Topics: Amino Acid Sequence; Base Sequence; Biosensing Techniques; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Fluorescence; Luminescent Proteins; Mutation; Phylogeny; Second Messenger Systems

Cyclic di-GMP (c-di-GMP) is a global signaling molecule that widely modulates diverse cellular processes. However, whether or not the c-di-GMP signal participates in regulation of bacterial antioxidant defense is unclear, and the involved regulators remain to be explored. In this study, we characterized HpoR as a novel c-di-GMP effective transcription factor and found a link between the c-di-GMP signal and the antioxidant regulation in Mycobacterium smegmatis. H2O2 stress induces c-di-GMP accumulation in M. smegmatis. High level of c-di-GMP triggers expression of a redox gene cluster, designated as hpoR operon, which is required for the mycobacterial H2O2 resistance. HpoR acts as an inhibitor of the hpoR operon and recognizes a 12-bp motif sequence within the upstream regulatory region of the operon. c-di-GMP specifically binds with HpoR at a ratio of 1:1. Low concentrations of c-di-GMP stimulate the DNA-binding activity of HpoR, whereas high concentrations of the signal molecule inhibit the activity. Strikingly, high level of c-di-GMP de-represses the intracellular association of HpoR with the regulatory region of the hpoR operon in M. smegmatis and enhances the mycobacterial H2O2 resistance. Therefore, we report a novel c-di-GMP effective regulator in mycobacteria, which extends the second messenger's function to bacterial antioxidant defense.

Topics: Antioxidants; Biofilms; Cyclic GMP; DNA-Binding Proteins; Gene Expression Regulation, Bacterial; Hydrogen Peroxide; Mycobacterium smegmatis; Operon; Promoter Regions, Genetic; Second Messenger Systems; Signal Transduction

Cyclic dinucleotides are second messenger molecules produced by both prokaryotes and eukaryotes in response to external stimuli. In bacteria, these molecules bind to RNA riboswitches and several protein receptors ultimately leading to phenotypic changes such as biofilm formation, ion transport and secretion of virulence factors. Some cyclic dinucleotide analogs bind differentially to biological receptors and can therefore be used to better understand cyclic dinucleotide mechanisms in vitro and in vivo. However, production of some of these analogs involves lengthy, multistep syntheses. Here, we describe a new, simple method for enzymatic synthesis of several 3', 5' linked cyclic dinucleotide analogs of c-di-GMP, c-di-AMP and c-AMP-GMP using the cyclic-AMP-GMP synthetase, DncV. The enzymatic reaction efficiently produced most cyclic dinucleotide analogs, such as 2'-amino sugar substitutions and phosphorothioate backbone modifications, for all three types of cyclic dinucleotides without the use of protecting groups or organic solvents. We used these novel analogs to explore differences in phosphate backbone and 2'-hydroxyl recognition between GEMM-I and GEMM-Ib riboswitches.

Topics: Algorithms; Bacterial Proteins; Cyclic GMP; Dinucleoside Phosphates; Kinetics; Ligases; Magnesium; Molecular Structure; Nucleotides, Cyclic; Protein Binding; Vibrio cholerae

Riboswitches are RNA elements found in non-coding regions of messenger RNAs that regulate gene expression through a ligand-triggered conformational change. Riboswitches typically bind tightly and specifically to their ligands, so they have the potential to serve as highly effective sensors in vitro. In B. subtilis and other gram-positive bacteria, purine nucleotide synthesis is regulated by riboswitches that bind to guanine. We modified the xpt-pbuX guanine riboswitch for use in a fluorescence quenching assay that allowed us to specifically detect and quantify guanine in vitro. Using this assay, we reproducibly detected as little as 5 nM guanine. We then produced sensors for 2'-deoxyguanosine and cyclic diguanylate (c-diGMP) by appending the P1 stem of the guanine riboswitch to the ligand-binding domains of a 2'-deoxyguanosine riboswitch and a c-diGMP riboswitch. These hybrid sensors could detect 15 nM 2'-deoxyguanosine and 3 nM c-diGMP, respectively. Each sensor retained the ligand specificity of its corresponding natural riboswitch. In order to extend the utility of our approach, we developed a strategy for the in vitro selection of sensors with novel ligand specificity. Here we report a proof-of-principle experiment that demonstrated the feasibility of our selection strategy.

Topics: Bacillus subtilis; Biosensing Techniques; Cyclic GMP; Fluorescence; Guanine; Guanosine; Ligands; Riboswitch; RNA, Bacterial

c-di-GMP is an attractive target in the fight against bacterial infections since it is a near ubiquitous second messenger that regulates important cellular processes of pathogens, including biofilm formation and virulence. Screening of a combinatorial peptide library enabled the identification of the proline-rich tetrapeptide Gup-Gup-Nap-Arg, which binds c-di-GMP selectively over other nucleotides in water. Computational and CD spectroscopic studies provided a possible binding mode of the complex and enabled the design of a pentapeptide with even higher binding strength towards c-di-GMP. Biological studies showed that the tetrapeptide inhibits biofilm growth by the opportunistic pathogen P. aeruginosa.

Topics: Biofilms; Circular Dichroism; Cyclic GMP; Peptides; Proline; Protein Binding; Pseudomonas aeruginosa; Second Messenger Systems; Thermodynamics

The cyclic-di-GMP (c-di-GMP) second messenger exemplifies a signaling system that regulates many bacterial behaviors of key importance; among them, c-di-GMP controls the transition between motile and sessile life-styles in bacteria. Cellular c-di-GMP levels in bacteria are regulated by the opposite enzymatic activities of diguanylate cyclases and phosphodiesterases, which are proteins that have GGDEF and EAL domains, respectively. Azospirillum is a genus of plant-growth-promoting bacteria, and members of this genus have beneficial effects in many agronomically and ecologically essential plants. These bacteria also inhabit aquatic ecosystems, and have been isolated from humus-reducing habitats. Bioinformatic and structural approaches were used to identify genes predicted to encode GG[D/E]EF, EAL and GG[D/E]EF-EAL domain proteins from nine genome sequences.. The analyzed sequences revealed that the genomes of A. humicireducens SgZ-5. Bacteria of the Azospirillum genus cope with diverse environmental conditions to survive in soil and aquatic habitats and, in certain cases, to colonize and benefit their host plant. Gaining information on the structures of proteins involved in c-di-GMP metabolism in Azospirillum appears to be an important step in determining the c-di-GMP signaling pathways, involved in the transition of a motile cell towards a biofilm life-style, as an example of microbial genome plasticity under diverse in situ environments.

Topics: Adaptation, Biological; Azospirillum; Bacterial Proteins; Biofilms; Computational Biology; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Models, Molecular; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Conformation; Protein Domains; Second Messenger Systems; Signal Transduction

Pellicles, a type of biofilm, have gathered a renewed interest in the field of tuberculosis as a structure that mimics some characteristics occurring during M. tuberculosis infection, such as antibiotic recalcitrance and chronicity of infection, and as a source of antigens for humoral response in infected guinea pigs. In other bacteria, it has been well documented that the second messenger c-di-GMP modulates the transition from planktonic cells to biofilm formation. In this work, we used the live vaccine Mycobacterium bovis BCG to determine whether deletion of genes involved in c-di-GMP metabolism would affect interaction with macrophages, capacity to induce immune response in a murine cell line and mice, and how the protein profile was modified when grown as surface pellicles. We found that deletion of the BCG1419c (Delta c-di-GMP phosphodiesterase, ΔPDE) gene, or deletion of the BCG1416c (Delta c-di-GMP diguanylate cyclase, ΔDGC) gene, altered production of TNF-α, IL-6, and IL-1β, in murine macrophages, and resulted in attenuation in intra-macrophage replication. Moreover, in addition to the improved immunogenicity of the BCGΔBCG1419c mutant already reported, deletion of the BCG1416c gene leads to increased T CD4

Topics: Animals; BCG Vaccine; Cyclic GMP; Cytokines; Gene Order; Genetic Vectors; Immunity; Lung; Lymphocyte Activation; Macrophages; Mice; Mycobacterium tuberculosis; T-Lymphocyte Subsets; Tuberculosis; Vaccination; Virulence

Topics: Bacterial Proteins; Cyanobacteria; Cyclic GMP; Gene Expression Regulation, Bacterial; Glucosyltransferases; Models, Biological; Signal Transduction; Transcription, Genetic

In this paper, we present a new strategy for microprinting dense bacterial communities with a prescribed organization on a substrate. Unlike conventional bioprinting techniques that require bioinks, through optogenetic manipulation, we directly manipulated the behaviors of Pseudomonas aeruginosa to allow these living bacteria to autonomically form patterned biofilms following prescribed illumination. The results showed that through optogenetic manipulation, patterned bacterial communities with high spatial resolution (approximately 10 μm) could be constructed in 6 h. Thus, optogenetic manipulation greatly increases the range of available bioprinting techniques.

Topics: Biofilms; Bioprinting; Cyclic GMP; Light; Microorganisms, Genetically-Modified; Optogenetics; Pseudomonas aeruginosa

The riboswitch is a class of RNA-based gene regulatory machinery that is dependent on recognition of its target ligand by RNA tertiary structures. Ligand recognition is achieved by the aptamer domain, and ligand-dependent structural changes of the expression platform then usually mediate termination of transcription or translational initiation. Ligand-dependent structural changes of the aptamer domain and expression platform have been reported for several riboswitches with short (<40 nucleotides) expression platforms. In this study, we characterized structural changes of the Vc2 c-di-GMP riboswitch that represses translation of downstream open reading frames in a ligand-dependent manner. The Vc2 riboswitch has a long (97 nucleotides) expression platform, but its structure and function are largely unknown. Through mutational analysis and chemical probing, we identified its secondary structures that are possibly responsible for switch-OFF and switch-ON states of translational initiation.

Topics: Aptamers, Nucleotide; Base Sequence; Binding Sites; Cyclic GMP; Escherichia coli; Models, Molecular; Nucleic Acid Conformation; Protein Biosynthesis; Ribosomes; Riboswitch; RNA, Bacterial

Pseudomonads typically carry multiple non-identical alleles of the post-transcriptional regulator rsmA. In Pseudomonas aeruginosa, RsmN is notable in that its structural rearrangement confers distinct and overlapping functions with RsmA. However, little is known about the specificities of RsmN for its target RNAs and overall impact on the biology of this pathogen. We purified and mapped 503 transcripts directly bound by RsmN in P. aeruginosa. About 200 of the mRNAs identified encode proteins of demonstrated function including some determining acute and chronic virulence traits. For example, RsmN reduces biofilm development both directly and indirectly via multiple pathways, involving control of Pel exopolysaccharide biosynthesis and c-di-GMP levels. The RsmN targets identified are also shared with RsmA, although deletion of rsmN generally results in less pronounced phenotypes than those observed for ΔrsmA or ΔrsmArsmNind mutants, probably as a consequence of different binding affinities. Targets newly identified for the Rsm system include the small non-coding RNA CrcZ involved in carbon catabolite repression, for which differential binding of RsmN and RsmA to specific CrcZ regions is demonstrated. The results presented here provide new insights into the intricacy of riboregulatory networks involving multiple but distinct RsmA homologues.

Topics: Alginates; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Genome, Bacterial; Polysaccharides, Bacterial; Pseudomonas aeruginosa; Regulon; Repressor Proteins; RNA-Binding Proteins; RNA, Messenger; RNA, Small Untranslated; Type VI Secretion Systems

Nanotechnology has demonstrated tremendous clinical utility, with potential applications in cancer immunotherapy. Although nanoparticles with intrinsic cytotoxicity are often considered unsuitable for clinical applications, such toxicity may be harnessed in the fight against cancer. Nanoparticle-associated toxicity can induce acute necrotic cell death, releasing tumor-associated antigens which may be captured by antigen-presenting cells to initiate or amplify tumor immunity. To test this hypothesis, cytotoxic cationic silica nanoparticles (CSiNPs) were directly administered into B16F10 melanoma implanted in C57BL/6 mice. CSiNPs caused plasma membrane rupture and oxidative stress of tumor cells, inducing local inflammation, tumor cell death and the release of tumor-associated antigens. The CSiNPs were further complexed with bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a molecular adjuvant which activates the stimulator of interferon genes (STING) in antigen-presenting cells. Compared with unformulated c-di-GMP, the delivery of c-di-GMP with CSiNPs markedly prolonged its local retention within the tumor microenvironment and activated tumor-infiltrating antigen-presenting cells. The combination of CSiNPs and a STING agonist showed dramatically increased expansion of antigen-specific CD8+ T cells, and potent tumor growth inhibition in murine melanoma. These results demonstrate that cationic nanoparticles can be used as an effective in situ vaccine platform which simultaneously causes tumor destruction and immune activation.

Topics: Animals; CD8-Positive T-Lymphocytes; Cell Death; Cyclic GMP; Female; Melanoma, Experimental; Membrane Proteins; Mice; Mice, Inbred C57BL; Nanoparticles; Oxidative Stress; Silicon Dioxide; Tumor Microenvironment

To address the drug-resistance of bacterial pathogens without imposing a selective survival pressure, virulence and biofilms are highly attractive targets. Here, we show that terrein, which was isolated from Aspergillus terreus, reduced virulence factors (elastase, pyocyanin, and rhamnolipid) and biofilm formation via antagonizing quorum sensing (QS) receptors without affecting Pseudomonas aeruginosa cell growth. Additionally, the effects of terrein on the production of QS signaling molecules and expression of QS-related genes were verified. Interestingly, terrein also reduced intracellular 3,5-cyclic diguanylic acid (c-di-GMP) levels by decreasing the activity of a diguanylate cyclase (DGC). Importantly, the inhibition of c-di-GMP levels by terrein was reversed by exogenous QS ligands, suggesting a regulation of c-di-GMP levels by QS; this regulation was confirmed using P. aeruginosa QS mutants. This is the first report to demonstrate a connection between QS signaling and c-di-GMP metabolism in P. aeruginosa, and terrein was identified as the first dual inhibitor of QS and c-di-GMP signaling.

Topics: Aspergillus; Biofilms; Cyclic GMP; Cyclopentanes; Pseudomonas aeruginosa; Quorum Sensing; Virulence

The second messenger cyclic diguanylate (c-di-GMP) is ubiquitously used by bacteria to modulate and shift between different phenotypes including motility, biofilm formation and virulence. Here we show that c-di-GMP-associated genes are widespread on plasmids and that enzymes that synthesize or degrade c-di-GMP are preferentially encoded on transmissible plasmids. Additionally, expression of enzymes that synthesize c-di-GMP was found to increase both biofilm formation and, interestingly, conjugative plasmid transfer rates.

Topics: Bacteria; Bacterial Physiological Phenomena; Biofilms; Cyclic GMP; Gene Transfer, Horizontal; Plasmids; Signal Transduction; Virulence

The virulence factor pyocyanin and the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP) play key roles in regulating biofilm formation and multi-drug efflux pump expression in Pseudomonas aeruginosa. However, the crosstalk between these two signaling pathways remains unclear. Here we show that BrlR (PA4878), previously identified as a c-di-GMP responsive transcriptional regulator, acts also as a receptor for pyocyanin. Crystal structures of free BrlR and c-di-GMP-bound BrlR reveal that the DNA-binding domain of BrlR contains two separate c-di-GMP binding sites, both of which are involved in promoting brlR expression. In addition, we identify a pyocyanin-binding site on the C-terminal multidrug-binding domain based on the structure of the BrlR-C domain in complex with a pyocyanin analog. Biochemical analysis indicates that pyocyanin enhances BrlR-DNA binding and brlR expression in a concentration-dependent manner.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Binding Sites; Biofilms; Crystallography, X-Ray; Cyclic GMP; Drug Resistance, Multiple, Bacterial; Gene Expression Regulation, Bacterial; Humans; Microbial Sensitivity Tests; Molecular Docking Simulation; Protein Domains; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Recombinant Proteins; Transcription Factors; Virulence Factors

Microbial communities primarily consist of multiple species that affect one another's fitness both directly and indirectly. This study showed that the cocultivation of Paenibacillus amylolyticus and Xanthomonas retroflexus exhibited facultative mutualistic interactions in a static environment, during the course of which a new adapted phenotypic variant of X. retroflexus appeared. Although the emergence of this variant was not directly linked to the presence of P. amylolyticus, its establishment in the coculture enhanced the productivity of both species due to mutations that stimulated biofilm formation. The mutations were detected in genes encoding a diguanylate cyclase predicted to synthesise cyclic-di-GMP. Examinations of the biofilm formed in cocultures of P. amylolyticus and the new variant of X. retroflexus revealed a distinct spatial organisation: P. amylolyticus only resided in biofilms in association with X. retroflexus and occupied the outer layers. The X. retroflexus variant therefore facilitated increased P. amylolyticus growth as it produced more biofilm biomass. The increase in X. retroflexus biomass was thus not at the expense of P. amylolyticus, demonstrating that interspecies interactions can shape diversification in a mutualistic coculture and reinforce these interactions, ultimately resulting in enhanced communal performance.

Topics: Biofilms; Cyclic GMP; Escherichia coli Proteins; Paenibacillus; Phenotype; Phosphorus-Oxygen Lyases; Symbiosis; Xanthomonas

In eubacteria, cyclic di-GMP (c-di-GMP) signaling is involved in virulence, persistence, motility and generally orchestrates multicellular behavior in bacterial biofilms. Intracellular c-di-GMP levels are maintained by the opposing activities of diguanylate cyclases (DGCs) and cognate phosphodiesterases (PDEs). The c-di-GMP homeostasis in

Topics: Amino Acid Sequence; Bacterial Proteins; Conserved Sequence; Cyclic GMP; Escherichia coli Proteins; Homeostasis; Mycobacterium smegmatis; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Binding; Protein Conformation; Protein Domains

Cyclic diguanylate monophosphate (c-di-GMP) is a global signaling molecule that modulates diverse cellular processes through its downstream receptors. However, no study has fully clarified the mechanisms by which c-di-GMP organizes functionally divergent regulators to drive the gene expression for coping with environmental stress. Here, we reported that c-di-GMP can integrate two functionally opposite receptor transcription factors, namely, LtmA and HpoR, into a pathway to regulate the antioxidant processes in Mycobacterium smegmatis. In contrast to HpoR, LtmA is an activator that positively regulates the expression of redox gene clusters and the mycobacterial H2O2 resistance. LtmA can physically interact with HpoR. A high level of c-di-GMP stimulates the positive regulation of LtmA and boosts the physical interaction between the two regulators, further enhancing the DNA-binding ability of LtmA and reducing the inhibitory activity of HpoR. Therefore, upon exposure to oxidative stress, c-di-GMP can orchestrate functionally divergent transcription factors to trigger antioxidant defense in mycobacteria. This finding presents a noteworthy example of how a bacterium remodels its transcriptional network via c-di-GMP in response to environmental stress.

Topics: Antioxidants; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Hydrogen Peroxide; Mycobacterium smegmatis; Oxidants; Protein Binding; Signal Transduction; Transcription Factors

The bacterial intracellular second messenger, cyclic dimeric GMP (c-di-GMP), regulates biofilm formation for many bacteria. The binding of c-di-GMP by the inner membrane protein LapD controls biofilm formation, and the LapD receptor is central to a complex network of c-di-GMP-mediated biofilm formation. In this study, we examine how c-di-GMP signaling specificity by a diguanylate cyclase (DGC), GcbC, is achieved via interactions with the LapD receptor and by small ligand sensing via GcbC's

Topics: Biofilms; Cyclic GMP; Escherichia coli Proteins; Phosphorus-Oxygen Lyases; Pseudomonas fluorescens; Signal Transduction

In certain structural or functional contexts, RNA structures can contain protonated nucleotides. However, a direct role for stably protonated nucleotides in ligand binding and ligand recognition has not yet been demonstrated unambiguously. Previous X-ray structures of c-GAMP binding riboswitch aptamer domains in complex with their near-cognate ligand c-di-GMP suggest that an adenine of the riboswitch either forms two hydrogen bonds to a G nucleotide of the ligand in the unusual enol tautomeric form or that the adenine in its N1 protonated form binds the G nucleotide of the ligand in its canonical keto tautomeric state. By using NMR spectroscopy we demonstrate that the c-GAMP riboswitches bind c-di-GMP using a stably protonated adenine in the ligand binding pocket. Thereby, we provide novel insights into the putative biological functions of protonated nucleotides in RNA, which in this case influence the ligand selectivity in a riboswitch.

Topics: Adenine; Cyclic GMP; Ligands; Magnetic Resonance Spectroscopy; Nucleotides, Cyclic; Protein Binding; Riboswitch; RNA; RNA, Bacterial; Vibrio cholerae

The small molecule cyclic di-GMP (c-di-GMP) is known to affect bacterial gene expression in myriad ways. In Vibrio cholerae in vivo, the presence of c-di-GMP together with the response regulator VpsR results in transcription from PvpsL, a promoter of biofilm biosynthesis genes. VpsR shares homology with enhancer binding proteins that activate σ54-RNA polymerase (RNAP), but it lacks conserved residues needed to bind to σ54-RNAP and to hydrolyze adenosine triphosphate, and PvpsL transcription does not require σ54 in vivo. Consequently, the mechanism of this activation has not been clear. Using an in vitro transcription system, we demonstrate activation of PvspL in the presence of VpsR, c-di-GMP and σ70-RNAP. c-di-GMP does not significantly change the affinity of VpsR for PvpsL DNA or the DNase I footprint of VpsR on the DNA, and it is not required for VpsR to dimerize. However, DNase I and KMnO4 footprints reveal that the σ70-RNAP/VpsR/c-di-GMP complex on PvpsL adopts a different conformation from that formed by σ70-RNAP alone, with c-di-GMP or with VpsR. Our results suggest that c-di-GMP is required for VpsR to generate the specific protein-DNA architecture needed for activated transcription, a previously unrecognized role for c-di-GMP in gene expression.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; DNA Footprinting; DNA-Binding Proteins; DNA-Directed RNA Polymerases; DNA, Bacterial; Enzyme Activation; Gene Expression Regulation, Bacterial; Promoter Regions, Genetic; Protein Binding; Sigma Factor; Structure-Activity Relationship; Transcription Initiation, Genetic; Vibrio cholerae

Stenotrophomonas maltophilia as one of increasing food spoilage bacteria and fish pathogens has become a threat to aquiculture industry. A major factor contributing to the success of bacterium is its outstanding ability to secrete protease at low temperatures. Here, a cAMP receptor like protein (Clp) shows a positive regulation on this protease, named S. maltophilia temperature-response protease (SmtP). Interestingly, a two-component system, comprising of LotS sensor and LotR regulator, for low-temperature response is also confirmed to modulate SmtP expression with similar effect to Clp. Evidence is presented that LotS/LotR modulates smtP (coding SmtP) expression via Clp: clp promoter activity was reduced significantly at low temperatures and protease activity was partially restored by Clp overexpressed in lotS or lotR deletion strain. Furthermore, as a Clp negative effector, the binding ability of c-di-GMP with Clp is not impacted by temperature. c-di-GMP level was increased in S. maltophilia growing at high temperature, but not exhibited significantly in lotR deleted strain, these indicate that LotR is required for temperature modulating c-di-GMP level, although the synthesis or degradation activity of c-di-GMP by LotR was not detected. These findings suggest that LotS/LotR/Clp play an important role in responding to temperature stimuli via c-di-GMP mediated manner.

Topics: Cyclic GMP; Gene Expression Regulation, Bacterial; Peptide Hydrolases; Signal Transduction; Stenotrophomonas maltophilia; Temperature

Nutrients such as amino acids play key roles in shaping the metabolism of microorganisms in natural environments and in host-pathogen interactions. Beyond taking part to cellular metabolism and to protein synthesis, amino acids are also signaling molecules able to influence group behavior in microorganisms, such as biofilm formation. This lifestyle switch involves complex metabolic reprogramming controlled by local variation of the second messenger 3', 5'-cyclic diguanylic acid (c-di-GMP). The intracellular levels of this dinucleotide are finely tuned by the opposite activity of dedicated diguanylate cyclases (GGDEF signature) and phosphodiesterases (EAL and HD-GYP signatures), which are usually allosterically controlled by a plethora of environmental and metabolic clues. Among the genes putatively involved in controlling c-di-GMP levels in P. aeruginosa, we found that the multidomain transmembrane protein PA0575, bearing the tandem signature GGDEF-EAL, is an l-arginine sensor able to hydrolyse c-di-GMP. Here, we investigate the basis of arginine recognition by integrating bioinformatics, molecular biophysics and microbiology. Although the role of nutrients such as l-arginine in controlling the cellular fate in P. aeruginosa (including biofilm, pathogenicity and virulence) is already well established, we identified the first l-arginine sensor able to link environment sensing, c-di-GMP signaling and biofilm formation in this bacterium.

Topics: Amino Acid Sequence; Arginine; Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Humans; Hydrolysis; Models, Molecular; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Binding; Protein Domains; Pseudomonas aeruginosa; Pseudomonas Infections; Sequence Alignment

Cyclic-di-GMP (c-di-GMP) contributes to the regulation of processes required by the Lyme disease (LD) spirochetes to complete the tick-mammal enzootic cycle. Our understanding of the effector mechanisms of c-di-GMP in the

Topics: Animals; Bacterial Proteins; Borrelia burgdorferi; Cyclic GMP; Disease Models, Animal; Gene Deletion; Genetic Complementation Test; Ixodes; Larva; Locomotion; Lyme Disease; Mice; Microbial Viability; Protein Binding

To permanently attach to surfaces, Caulobacter crescentusproduces a strong adhesive, the holdfast. The timing of holdfast synthesis is developmentally regulated by cell cycle cues. When C. crescentusis grown in a complex medium, holdfast synthesis can also be stimulated by surface sensing, in which swarmer cells rapidly synthesize holdfast in direct response to surface contact. In contrast to growth in complex medium, here we show that when cells are grown in a defined medium, surface contact does not trigger holdfast synthesis. Moreover, we show that in a defined medium, flagellum synthesis and regulation of holdfast production are linked. In these conditions, mutants lacking a flagellum attach to surfaces over time more efficiently than either wild-type strains or strains harboring a paralyzed flagellum. Enhanced adhesion in mutants lacking flagellar components is due to premature holdfast synthesis during the cell cycle and is regulated by the holdfast synthesis inhibitor HfiA. hfiA transcription is reduced in flagellar mutants and this reduction is modulated by the diguanylate cyclase developmental regulator PleD. We also show that, in contrast to previous predictions, flagella are not necessarily required for C. crescentus surface sensing in the absence of flow, and that arrest of flagellar rotation does not stimulate holdfast synthesis. Rather, our data support a model in which flagellum assembly feeds back to control holdfast synthesis via HfiA expression in a c-di-GMP-dependent manner under defined nutrient conditions.

Topics: Bacterial Adhesion; Bacterial Proteins; Biofilms; Caulobacter crescentus; Cell Cycle; Culture Media; Cyclic GMP; Feedback, Physiological; Flagella; Gene Expression Regulation, Bacterial; Mutation; Statistics, Nonparametric

Cyclic di-GMP (CDG) was applied to MUC1 glycopeptide-based cancer vaccines with physical mixing and built-in (at 2'-OH of CDG) strategies for activating the STING pathway. CDG in both strategies behaved as a potent immunostimulant and contributed to high titers of IgG antibodies and the expression of multiple cytokines.

Topics: Adjuvants, Immunologic; Amino Acid Sequence; Animals; B7-2 Antigen; Cancer Vaccines; Cyclic GMP; Cytokines; Female; Glycopeptides; Humans; Immunity, Cellular; Immunity, Humoral; Immunoglobulin G; Immunoglobulin M; Macrophage Activation; MCF-7 Cells; Membrane Proteins; Mice; Mice, Inbred BALB C; Mucin-1; Peptide Fragments; RAW 264.7 Cells; Signal Transduction

During growth on chitinous surfaces in its natural aquatic environment Vibrio cholerae develops natural competence for transformation and kills neighboring non-immune bacteria using a type VI secretion system (T6SS). Activation of these two phenotypes requires the chitin-induced regulator TfoX, but also integrates signals from quorum sensing via the intermediate regulator QstR, which belongs to the LuxR-type family of regulators. Here, we define the QstR regulon using RNA sequencing. Moreover, by mapping QstR binding sites using chromatin immunoprecipitation coupled with deep sequencing we demonstrate that QstR is a transcription factor that binds upstream of the up- and down-regulated genes. Like other LuxR-type family transcriptional regulators we show that QstR function is dependent on dimerization. However, in contrast to the well-studied LuxR-type biofilm regulator VpsT of V. cholerae, which requires the second messenger c-di-GMP, we show that QstR dimerization and function is c-di-GMP independent. Surprisingly, although ComEA, which is a periplasmic DNA-binding protein essential for transformation, is produced in a QstR-dependent manner, QstR-binding was not detected upstream of comEA suggesting the existence of a further regulatory pathway. Overall, these results provide detailed insights into the function of a key regulator of natural competence and type VI secretion in V. cholerae.

Topics: Amino Acid Sequence; Bacterial Proteins; Bacterial Secretion Systems; Biofilms; Chitin; Cyclic GMP; Dimerization; DNA, Bacterial; Gene Expression Regulation, Bacterial; Periplasmic Binding Proteins; Protein Binding; Quorum Sensing; Regulon; Repressor Proteins; Sequence Alignment; Sequence Analysis, RNA; Sequence Homology, Amino Acid; Trans-Activators; Transcription Factors; Transformation, Bacterial; Vibrio cholerae

Bis-(3'-5')-cyclic diguanylic acid (c-di-GMP) belongs to the class of cyclic dinucleotides, key carriers of cellular information in prokaryotic and eukaryotic signal transduction pathways. In bacteria, the intracellular levels of c-di-GMP and their complex physiological outputs are dynamically regulated by environmental and internal stimuli, which control the antagonistic activities of diguanylate cyclases (DGCs) and c-di-GMP specific phosphodiesterases (PDEs). Allostery is one of the major modulators of the c-di-GMP-dependent response. Both the c-di-GMP molecule and the proteins interacting with this second messenger are characterized by an extraordinary structural plasticity, which has to be taken into account when defining and possibly predicting c-di-GMP-related processes. Here, we report a structure-function relationship study on the catalytic portion of the PA0575 protein from Pseudomonas aeruginosa, bearing both putative DGC and PDE domains. The kinetic and structural studies indicate that the GGDEF-EAL portion is a GTP-dependent PDE. Moreover, the crystal structure confirms the high degree of conformational flexibility of this module. We combined structural analysis and protein engineering studies to propose the possible molecular mechanism guiding the nucleotide-dependent allosteric control of catalysis; we propose that the role exerted by GTP via the GGDEF domain is to allow the two EAL domains to form a dimer, the species competent to enter PDE catalysis.

Topics: Allosteric Regulation; Bacterial Proteins; Crystallography, X-Ray; Cyclic GMP; Guanosine Triphosphate; Hydrolysis; Phosphoric Diester Hydrolases; Protein Conformation; Protein Multimerization; Pseudomonas aeruginosa

Bacteria swim and swarm using rotating flagella that are driven by a membrane-spanning motor complex. Performance of the flagella motility apparatus is modulated by the chemosensory signal transduction system to allow navigation through physico-chemical gradients - a process that can be fine-tuned by the bacterial second messenger c-di-GMP. We have previously analysed the Pseudomonas putida signalling protein PP2258 that has the capacity to both synthesize and degrade c-di-GMP. A PP2258 null mutant displays reduced motility, implicating the c-di-GMP signal originating from this protein in control of P. putida motility. In Escherichia coli and Salmonella, the PilZ-domain protein YcgR mediates c-di-GMP responsive control of motility through interaction with the flagellar motors. Here we provide genetic evidence that the P. putida protein PP4397 (also known as FlgZ), despite low sequence homology and a different genomic context to YcgR, functions as a c-di-GMP responsive link between the signal arising from PP2258 and alterations in swimming and swarming motility in P. putida.

Topics: Bacterial Proteins; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Flagella; Gene Expression Regulation, Bacterial; Protein Binding; Protein Domains; Pseudomonas putida; Second Messenger Systems; Signal Transduction

Bacterial biofilms are large aggregates of cells embedded in an extracellular matrix of self-produced polymers. In macrocolony biofilms of

Topics: Biofilms; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Green Fluorescent Proteins; Sigma Factor; Signal Transduction

Poor clinical outcomes (disfigurement, amputation, and death) and significant economic losses in the aquaculture industry can be attributed to the potent opportunistic human pathogen

Topics: Bacterial Proteins; Biofilms; Calcium; Cyclic GMP; Environment; Gene Expression Regulation, Bacterial; Phenotype; Phosphoadenosine Phosphosulfate; Signal Transduction; Vibrio vulnificus

Enzymes controlling intracellular second messengers in bacteria, such as c-di-GMP, often affect some but not other targets. How such specificity is achieved is understood only partially. Here, we present a novel mechanism that enables specific c-di-GMP-dependent inhibition of the antifungal antibiotic production. Expression of the biosynthesis operon for Heat-Stable Antifungal Factor, HSAF, in Lysobacter enzymogenes occurs when the transcription activator Clp binds to two upstream sites. At high c-di-GMP levels, Clp binding to the lower-affinity site is compromised, which is sufficient to decrease gene expression. We identified a weak c-di-GMP phosphodiesterase, LchP, that plays a disproportionately high role in HSAF synthesis due to its ability to bind Clp. Further, Clp binding stimulates phosphodiesterase activity of LchP. An observation of a signaling complex formed by a c-di-GMP phosphodiesterase and a c-di-GMP-binding transcription factor lends support to the emerging paradigm that such signaling complexes are common in bacteria, and that bacteria and eukaryotes employ similar solutions to the specificity problem in second messenger-based signaling systems.

Topics: Anti-Bacterial Agents; Antifungal Agents; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Lysobacter; Models, Genetic; Phosphoric Diester Hydrolases; Protein Binding; Protein Interaction Maps; Signal Transduction

In

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Mutation; Oryza; Phosphoric Diester Hydrolases; Plant Diseases; Polysaccharides, Bacterial; Protein Domains; Signal Transduction; Virulence; Xanthomonas

Topics: Cyclic GMP; Escherichia coli Proteins; Genome, Bacterial; Microbial Interactions; Nitric Oxide; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Rhodobacteraceae

Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that regulates processes, such as biofilm formation and virulence. During degradation, c-di-GMP is first linearized to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG) and subsequently hydrolyzed to two GMPs by a previously unknown enzyme, which was recently identified in

Topics: Bacillus anthracis; Bacterial Proteins; Cyclic GMP; Exoribonucleases; Hydrolysis; Mutation; Pseudomonas aeruginosa; Second Messenger Systems; Signal Transduction; Vibrio cholerae

Initial attachment to a surface is a key and highly regulated step in biofilm formation. In this study, we present a platform for reversibly functionalizing bacterial cell surfaces with an emphasis on designing biofilms. We engineered the Lap system of Pseudomonas fluorescens Pf0-1, which is normally used to regulate initial cell surface attachment, to display various protein cargo at the bacterial cell surface and control extracellular release of the cargo in response to changing levels of the second messenger c-di-GMP. To accomplish this goal, we fused the protein cargo between the N-terminal retention module and C-terminal secretion signal of LapA and controlled surface localization of the cargo with natural signals known to stimulate or deplete c-di-GMP levels in P. fluorescens Pf0-1. We show this system can tolerate large cargo in excess of 500 amino acids, direct P. fluorescens Pf0-1 to surfaces it does not typically colonize, and program this microbe to sequester the toxic medal cadmium.

Topics: Adhesins, Bacterial; Biofilms; Cell Wall; Cyclic GMP; Plasmids; Pseudomonas fluorescens

Microneedle-based skin allergen-specific immunotherapy (AIT) can benefit from adjuvants that can stimulate a stronger Th1 response against the allergen. We evaluated two stimulator of interferon genes (STING) agonists, namely, cyclic diguanylate monophosphate (c-di-GMP) and cyclic diadenylate monophosphate (c-di-AMP), as skin adjuvants using coated microneedles (MNs). For comparison, the approved subcutaneous (SC) hypodermic injection containing alum was used. Ovalbumin (Ova) was used as a model allergen. Ova-specific IgG2a antibody in serum, which is a surrogate marker for Th1 type immune response was significantly higher when STING agonists were used with coated MNs as compared to SC injection of Ova+alum in mice. In contrast, IgG1 antibody, a surrogate marker for Th2 type immune response, was at comparable levels in the MN and SC groups. Restimulation of splenocytes with Ova produced higher levels of Th1 cytokines (IFN-γ and IL-2) in the STING agonists MN groups as compared to the SC group. In conclusion, delivery of STING agonists into the skin using coated MNs activated the Th1 pathway better than SC- and MN-based delivery of alum. Thus, STING agonists could fulfill the role of adjuvants for skin AIT and even for infectious disease vaccines, where stimulation of the Th1 pathway is of interest.

Topics: Adjuvants, Immunologic; Administration, Cutaneous; Allergens; Alum Compounds; Animals; Cyclic GMP; Desensitization, Immunologic; Dinucleoside Phosphates; Female; Membrane Proteins; Mice; Mice, Inbred BALB C; Models, Animal; Needles; Ovalbumin; Th1 Cells; Th2 Cells

Pseudomonas putida KT2442, a natural producer of polyhydroxyalkanoate, spends a lot of energy and carbon sources to form flagella and pili; therefore, deleting the genes involved in the biosynthesis and assembly of flagella and pili might improve PHA productivity. In this study, two novel deletion systems were constructed in order to efficiently remove the 76 genes involved in the biosynthesis and assembly of flagella and pili in P. putida KT2442. Both systems combine suicide-plasmid-based homologous recombination and mutant lox site-specific recombination and involve three plasmids. The first includes pK18mobsacB, pWJW101, and pWJW102; and the second includes pZJD29c, pDTW202, and pWJW103. These newly constructed systems were successfully used to remove different gene clusters in P. putida KT2442 and showed a high deletion efficiency (above 90%) whether for the second-round or the third-round recombination. Both systems could efficiently delete the gene PP4378 encoding flagellin in putida KT2442, resulting in the mutant strain WJPP01. The second system was used to remove the pili-forming gene cluster PP2357-PP2363 in putida KT2442, resulting in the mutant strain WJPP02, and also used to remove the flagella-forming gene cluster PP4329-PP4397 in WJPP02, resulting in the mutant strain WJPP03. Compared with the wild-type KT2442, the 1.2% genome reduction mutant WJPP03 grew faster, lacked flagella and motility, showed sharply decreased biofilm and 3',5'-cyclic diguanylic acid (c-di-GMP), but accumulated more polyhydroxyalkanoate. The biomass, polyhydroxyalkanoate yield, and content of WJPP03 increased 19.1, 73.4, and 45.6%, respectively, with sodium hexanoate supplementation, and also increased 11.4, 53.6, and 37.9%, respectively, with lauric acid supplementation.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Fimbriae, Bacterial; Flagella; Gene Deletion; Genetic Engineering; Genome, Bacterial; Homologous Recombination; Microorganisms, Genetically-Modified; Multigene Family; Mutation; Plasmids; Polyhydroxyalkanoates; Pseudomonas putida

The intracellular signaling molecule cyclic diguanylate (c-di-GMP) regulates many processes in bacteria, with a central role in controlling the switch between motile and nonmotile lifestyles. Recent work has shown that in

Topics: Bacterial Proteins; Clostridioides difficile; Cyclic GMP; Gene Expression Regulation, Bacterial; Riboswitch; Virulence; Virulence Factors

Toxin-antitoxin (TA) systems play important roles in bacteria persister formation. Increasing evidence demonstrate the roles of TA systems in regulating virulence factors in pathogenic bacteria. The toxin HigB in

Topics: Bacterial Proteins; Bacterial Toxins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa; Type III Secretion Systems

Quorum sensing (QS) and nucleotide-based second messengers are vital signaling systems that regulate bacterial physiology in response to changing environments. Disrupting bacterial signal transduction is a promising direction to combat infectious diseases, and QS and the second messengers are undoubtedly potential targets. In Vibrio cholerae, both QS and the second messenger 3', 5'-cyclic diguanylate (c-di-GMP) play a central role in controlling motility, motile-to-sessile life transition, and virulence. In this study, we found that water-soluble extract from the North American cranberry could significantly inhibit V. cholerae biofilm formation during the development/maturation stage by reducing the biofilm matrix production and secretion. The anti-biofilm effect by water-soluble cranberry extract was possibly through modulating the intracellular c-di-GMP level and was independent of QS and the QS master regulator HapR. Our results suggest an opportunity to explore more functional foods to fight stubborn infections through interference with the bacterial signaling systems.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Plant Extracts; Quorum Sensing; Vaccinium macrocarpon; Vibrio cholerae; Water

Riboswtich RNAs can control gene expression through the structural change induced by the corresponding small-molecule ligands. Molecular dynamics simulations and free energy calculations on the aptamer domain of the 3',3'-cGAMP riboswitch in the ligand-free, cognate-bound and noncognate-bound states were performed to investigate the structural features of the 3',3'-cGAMP riboswitch induced by the 3',3'-cGAMP ligand and the specificity of ligand recognition. The results revealed that the aptamer of the 3',3'-cGAMP riboswitch in the ligand-free state has a smaller binding pocket and a relatively compact structure versus that in the 3',3'-cGAMP-bound state. The binding of the 3',3'-cGAMP molecule to the 3',3'-cGAMP riboswitch induces the rotation of P1 helix through the allosteric communication from the binding sites pocket containing the J1/2, J1/3 and J2/3 junction to the P1 helix. Simultaneously, these simulations also revealed that the preferential binding of the 3',3'-cGAMP riboswitch to its cognate ligand, 3',3'-cGAMP, over its noncognate ligand, c-di-GMP and c-di-AMP. The J1/2 junction in the 3',3'-cGAMP riboswitch contributing to the specificity of ligand recognition have also been found.

Topics: Allosteric Regulation; Binding Sites; Cyclic GMP; Hydrogen Bonding; Ligands; Molecular Dynamics Simulation; Nucleic Acid Conformation; Nucleotides, Cyclic; Principal Component Analysis; Riboswitch; Thermodynamics; Time Factors

Bacteria can use the second messenger c-di-GMP to adjust their motility in response to environmental cues. The protein YcgR, upon binding of c-di-GMP, interacts with the flagellar motor to affect the motor behavior. However, the full feature of the effects of c-di-GMP::YcgR on the flagellar motor remains unclear, and its interacting partners on the motor is still controversial. Here, we characterized the effects of c-di-GMP::YcgR on the torque-speed curve of the flagellar motor, one of the most important properties of the motor, finding that it affects the motor behavior throughout the full range of load conditions from zero to high loads by shifting the motor torque-speed curve downward. We also investigated the interacting partner on the motor through dynamical fluorescent studies, finding that c-di-GMP::YcgR mainly interacts with the motor-switch complex instead of the torque-generating units (stators). To directly test the behavioral consequence of elevated c-di-GMP levels, we measured the distribution of bacteria swimming near a surface, finding that elevated c-di-GMP levels promote bacterial aggregation on surfaces. The effects of c-di-GMP on bacterial motile behavior that we characterized here are consistent with the key role that c-di-GMP plays in the transition between motile and sedentary forms of bacterial life.

Topics: Bacterial Adhesion; Cell Membrane; Cell Movement; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Flagella; Models, Molecular; Second Messenger Systems; Signal Transduction

Bacterial biofilms are multicellular aggregates encased in an extracellular matrix mainly composed of exopolysaccharides (EPSs), protein and nucleic acids, which determines the architecture of the biofilm. Erwinia amylovora Ea1189 forms a biofilm inside the xylem of its host, which results in vessel plugging and water transport impairment. The production of the EPSs amylovoran and levan is critical for the formation of a mature biofilm. In addition, cyclic dimeric GMP (c-di-GMP) has been reported to positively regulate amylovoran biosynthesis and biofilm formation in E. amylovora Ea1189. In this study, we demonstrate that cellulose is synthesized by E. amylovora Ea1189 and is a major modulator of the three-dimensional characteristics of biofilms formed by this bacterium, and also contributes to virulence during systemic host invasion. In addition, we demonstrate that the activation of cellulose biosynthesis in E. amylovora is a c-di-GMP-dependent process, through allosteric binding to the cellulose catalytic subunit BcsA. We also report that the endoglucanase BcsZ is a key player in c-di-GMP activation of cellulose biosynthesis. Our results provide evidence of the complex composition of the extracellular matrix produced by E. amylovora and the implications of cellulose biosynthesis in shaping the architecture of the biofilm and in the expression of one of the main virulence phenotypes of this pathogen.

Topics: Bacterial Proteins; Biofilms; Cellulose; Cyclic GMP; Erwinia amylovora; Extracellular Matrix; Glucosyltransferases; Operon; Virulence; Virulence Factors

When encountering surfaces, many bacteria produce adhesins to facilitate their initial attachment and to irreversibly glue themselves to the solid substrate. A central molecule regulating the processes of this motile-sessile transition is the second messenger c-di-GMP, which stimulates the production of a variety of exopolysaccharide adhesins in different bacterial model organisms. In

Topics: Adhesins, Bacterial; Arylamine N-Acetyltransferase; Bacterial Adhesion; Caulobacter crescentus; Cyclic GMP; Gene Deletion; Gene Expression Regulation, Bacterial

Knowledge about the molecular mechanisms that are involved in the regulation of biofilm formation is essential for the development of biofilm-control measures. It is well established that the nucleotide second messenger cyclic diguanosine monophosphate (c-di-GMP) is a positive regulator of biofilm formation in many bacteria, but more knowledge about c-di-GMP effectors is needed. We provide evidence that c-di-GMP, the alternative sigma factor RpoN (σ54), and the enhancer-binding protein BerB play a role in biofilm formation of Burkholderia cenocepacia by regulating the production of a biofilm-stabilizing exopolysaccharide. Our findings suggest that BerB binds c-di-GMP, and activates RpoN-dependent transcription of the berA gene coding for a c-di-GMP-responsive transcriptional regulator. An increased level of the BerA protein in turn induces the production of biofilm-stabilizing exopolysaccharide in response to high c-di-GMP levels. Our findings imply that the production of biofilm exopolysaccharide in B. cenocepacia is regulated through a cascade involving two consecutive transcription events that are both activated by c-di-GMP. This type of regulation may allow tight control of the expenditure of cellular resources.

Topics: Biofilms; Burkholderia cenocepacia; Cyclic GMP; Gene Expression Regulation, Bacterial; Polysaccharides, Bacterial; Sigma Factor; Transcription Factors

Therapies using T cells that are programmed to express chimeric antigen receptors (CAR T cells) consistently produce positive results in patients with hematologic malignancies. However, CAR T cell treatments are less effective in solid tumors for several reasons. First, lymphocytes do not efficiently target CAR T cells; second, solid tumors create an immunosuppressive microenvironment that inactivates T cell responses; and third, solid cancers are typified by phenotypic diversity and thus include cells that do not express proteins targeted by the engineered receptors, enabling the formation of escape variants that elude CAR T cell targeting. Here, we have tested implantable biopolymer devices that deliver CAR T cells directly to the surfaces of solid tumors, thereby exposing them to high concentrations of immune cells for a substantial time period. In immunocompetent orthotopic mouse models of pancreatic cancer and melanoma, we found that CAR T cells can migrate from biopolymer scaffolds and eradicate tumors more effectively than does systemic delivery of the same cells. We have also demonstrated that codelivery of stimulator of IFN genes (STING) agonists stimulates immune responses to eliminate tumor cells that are not recognized by the adoptively transferred lymphocytes. Thus, these devices may improve the effectiveness of CAR T cell therapy in solid tumors and help protect against the emergence of escape variants.

Topics: Adoptive Transfer; Animals; Antigen-Presenting Cells; Antineoplastic Agents; Biopolymers; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cyclic GMP; Drug Carriers; Female; Implants, Experimental; Melanoma, Experimental; Membrane Proteins; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Transplantation; Pancreatic Neoplasms; T-Lymphocytes

Streptomyces are ubiquitous soil bacteria that undergo a complex developmental transition coinciding with their production of antibiotics. This transition is controlled by binding of a novel tetrameric form of the second messenger, 3΄-5΄ cyclic diguanylic acid (c-di-GMP) to the master repressor, BldD. In all domains of life, nucleotide-based second messengers allow a rapid integration of external and internal signals into regulatory pathways that control cellular responses to changing conditions. c-di-GMP can assume alternative oligomeric states to effect different functions, binding to effector proteins as monomers, intercalated dimers or, uniquely in the case of BldD, as a tetramer. However, at physiological concentrations c-di-GMP is a monomer and little is known about how higher oligomeric complexes assemble on effector proteins and if intermediates in assembly pathways have regulatory significance. Here, we show that c-di-GMP binds BldD using an ordered, sequential mechanism and that BldD function necessitates the assembly of the BldD2-(c-di-GMP)4 complex.

Topics: Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Cyclic GMP; Hydrogen Bonding; Models, Molecular; Protein Binding; Protein Domains; Protein Stability; Protein Structure, Quaternary; Repressor Proteins; Streptomyces

The opportunistic human pathogen Burkholderia cenocepacia H111 uses two chemically distinct signal molecules for controlling gene expression in a cell density-dependent manner: N-acyl-homoserine lactones (AHLs) and cis-2-dodecenoic acid (BDSF). Binding of BDSF to its cognate receptor RpfR lowers the intracellular c-di-GMP level, which in turn leads to differential expression of target genes. In this study we analysed the transcriptional profile of B. cenocepacia H111 upon artificially altering the cellular c-di-GMP level. One hundred and eleven genes were shown to be differentially expressed, 96 of which were downregulated at a high c-di-GMP concentration. Our analysis revealed that the BDSF, AHL and c-di-GMP regulons overlap for the regulation of 24 genes and that a high c-di-GMP level suppresses expression of AHL-regulated genes. Phenotypic analyses confirmed changes in the expression of virulence factors, the production of AHL signal molecules and the biosynthesis of different biofilm matrix components upon altered c-di-GMP levels. We also demonstrate that the intracellular c-di-GMP level determines the virulence of B. cenocepacia to Caenorhabditis elegans and Galleria mellonella.

Topics: Acyl-Butyrolactones; Animals; Burkholderia cenocepacia; Caenorhabditis elegans; Cyclic GMP; Fatty Acids, Monounsaturated; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Moths; Quorum Sensing; Signal Transduction; Virulence; Virulence Factors

Biofilms are communities of sessile microbes that are phenotypically distinct from their genetically identical, free-swimming counterparts. Biofilms initiate when bacteria attach to a solid surface. Attachment triggers intracellular signaling to change gene expression from the planktonic to the biofilm phenotype. For

Topics: Bacterial Adhesion; Biofilms; Cyclic GMP; Mechanotransduction, Cellular; Pseudomonas aeruginosa; Stress, Physiological

Cyclic dimeric GMP (c-di-GMP) has emerged as a key regulatory player in the transition between planktonic and sedentary biofilm-associated bacterial lifestyles. It controls a multitude of processes including production of extracellular polysaccharides (EPSs). The PilZ domain, consisting of an N-terminal "RxxxR" motif and a β-barrel domain, represents a prototype c-di-GMP receptor. We identified a class of c-di-GMP-responsive proteins, represented by the AraC-like transcription factor CuxR in plant symbiotic α-proteobacteria. In

Topics: Amino Acid Motifs; Amino Acid Sequence; AraC Transcription Factor; Bacterial Proteins; Conserved Sequence; Crystallography, X-Ray; Cyclic GMP; Models, Molecular; Polysaccharides, Bacterial; Promoter Regions, Genetic; Protein Binding; Protein Domains; Protein Structure, Quaternary; Sinorhizobium meliloti; Trans-Activators

Macrolides have been reported to exert a variety of effects on both host immunomodulation and repression of bacterial pathogenicity. In this study, we report that the 3',5'-cyclic diguanylic acid (c-di-GMP) signaling system, which regulates virulence in Pseudomonas aeruginosa, is affected by the macrolide azithromycin. Using DNA microarray analysis, we selected a gene encoding PA2567 related to c-di-GMP metabolism that was significantly affected by azithromycin treatment. Expression of the PA2567 gene was significantly repressed by azithromycin in a time- and dose-dependent manner, whereas no difference in PA2567 gene expression was observed in the absence of azithromycin. In-frame deletion of the PA2567 gene affected both virulence factors and the quorum-sensing system, and significantly decreased total bacteria in a mouse pneumonia model compared to the wild-type strain (P < 0.05). These results suggest that macrolides possess the ability to modulate c-di-GMP intracellular signaling in P. aeruginosa.

Topics: Animals; Anti-Bacterial Agents; Azithromycin; Bacterial Proteins; Colony Count, Microbial; Cyclic GMP; Disease Models, Animal; Female; Mice; Mice, Inbred C57BL; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Signal Transduction; Virulence Factors

Topics: Alteromonas; Bacterial Proteins; Biofilms; Copper; Cyclic GMP; Disinfectants; Drug Resistance, Bacterial; Escherichia coli Proteins; Genes, Bacterial; Models, Theoretical; Mutation; Phosphorus-Oxygen Lyases; Seawater

Diverse organisms secrete redox-active antibiotics, which can be used as extracellular electron shuttles by resistant microbes. Shuttle-mediated metabolism can support survival when substrates are available not locally but rather at a distance. Such conditions arise in multicellular communities, where the formation of chemical gradients leads to resource limitation for cells at depth. In the pathogenic bacterium

Topics: Anti-Bacterial Agents; Biofilms; Cyclic GMP; Microbial Consortia; Phenazines; Pseudomonas aeruginosa; Second Messenger Systems

Two-component signal transduction systems (TCSs), typically composed of a sensor histidine kinase (HK) and a response regulator (RR), are the primary mechanism by which pathogenic bacteria sense and respond to extracellular signals. The pathogenic bacterium

Topics: Biofilms; Cyclic GMP; DNA Mutational Analysis; Gene Deletion; Histidine Kinase; Signal Transduction; Transcription Factors; Vibrio cholerae

The expression of the Gloeobacter rhodopsin (GR) in a chemotrophic Escherichia coli enables the light-driven phototrophic energy generation. Adaptive laboratory evolution has been used for acquiring desired phenotype of microbial cells and for the elucidation of basic mechanism of molecular evolution. To develop an optimized strain for the artificially acquired phototrophic metabolism, an ancestral E. coli expressing GR was adaptively evolved in a chemostat reactor with constant illumination and limited glucose conditions. This study was emphasized at an unexpected genomic mutation contributed to the improvement of microbial performance.. During the chemostat culture, increase of cell size was observed, which were distinguished from that of the typical rod-shaped ancestral cells. A descendant ET5 strain was randomly isolated from the chemostat culture at 88-days. The phototrophic growth and the light-induced proton pumping of the ET5 strain were twofold and eightfold greater, respectively, than those of the ancestral E. coli strain. Single point mutation of C1082A at dgcQ gene (encoding diguanylate cyclase, also known as the yedQ gene) in the chromosome of ET5 strain was identified from whole genome sequencing analysis. An ancestral E. coli complemented with the same dgcQ mutation from the ET5 was repeated the subsequently enhancements of light-driven phototrophic growth and proton pumping. Intracellular c-di-GMP, the product of the diguanylate cyclase (dgcQ), of the descendant ET5 strain was suddenly increased while that of the ancestral strain was negligible.. Newly acquired phototrophic metabolism of E. coli was further improved via adaptive laboratory evolution by the rise of a point mutation on a transmembrane cell signaling protein followed by increase of signal molecule that eventually led an increase proton pumping and phototrophic growth.

Topics: Cyanobacteria; Cyclic GMP; Directed Molecular Evolution; Escherichia coli; Escherichia coli Proteins; Genome, Bacterial; Glucose; High-Throughput Nucleotide Sequencing; Light; Mutation; Phenotype; Phosphorus-Oxygen Lyases; Phototrophic Processes; Proton Pumps; Rhodopsins, Microbial

The transcriptional regulator BrlR is a member of the MerR family of multidrug transport activators in Pseudomonas aeruginosa. Recent study indicates that BrlR is a novel 3',5'-cyclic diguanylic acid (c-di-GMP) receptor and can be activated by c-di-GMP. To gain insight into BrlR function, we determined the structure of BrlR with c-di-GMP complex structure to 2.5 Å. The structure and size exclusion chromatography (SEC) data revealed BrlR forms a tetramer and each BrlR protomer consists of three parts, DNA-binding domain, a coiled-coil region and GyrI-like domain. There are two different c-di-GMP binding sites located mainly at the DNA binding domain of each BrlR protomer and do not overlap with the GyrI-like domain. The drug-binding pocket in GyrI-like domain is much conserved indicating it can also bind flat-shaped molecules like other multidrug resistance (MDR) proteins.

Topics: Crystallography, X-Ray; Cyclic GMP; Models, Molecular; Pseudomonas aeruginosa; Transcription Factors

Because of ubiquity of thioesters, thioesterases play a critical role in metabolism, membrane biosynthesis, signal transduction, and gene regulation. In many bacteria, YbgC is such an enzyme, whose coding gene mostly resides in the tol-pal cluster. Although all other proteins encoded in the tol-pal cluster are clearly involved in maintaining cell envelope integrity and cell division, little is known about the physiological role of YbgC. In this study, we identify in Shewanella oneidensis, a γ-proteobacterium used as a research model for environmental microbes, YbgC as a motility regulator. The loss of YbgC results in enhanced motility, which is likely due to the increased rotation rate of the flagellum. The regulatory function of YbgC requires its thioesterase activity but could not be replaced by YbgC homologues of other bacteria. We further show that the regulation of YbgC is mediated by the second message c-di-GMP.

Topics: Bacterial Proteins; Cell Movement; Cyclic GMP; Esterases; Flagella; Shewanella

The hallmark of the lifecycle of Vibrio cholerae is its ability to switch between two lifestyles - the sessile, non-pathogenic form and the motile, infectious form in human hosts. One of these changes is in the formation of surface biofilms, when in sessile aquatic habitats. The cell-cell interactions within a V. cholerae biofilm are stabilized by the production of an exopolysachharide (EPS) matrix, which in turn is regulated by the ubiquitous secondary messenger, cyclic di-GMP (c-di-GMP), synthesized by proteins containing GGD(/E)EF domains in all prokaryotic systems. Here, we report the functional role of the VC0395_0300 protein (Sebox3) encoded by the chromosome I of V. cholerae, with a GGEEF signature sequence, in the formation of surface biofilms. In our study, we have shown that Escherichia coli containing the full-length Sebox3 displays enhanced biofilm forming ability with cellulose production as quantified and visualized by multiple assays, most notably using FEG-SEM. This has also been corroborated with the lack of motility of host containing Sebox3 in semi-solid media. Searching for the reasons for this biofilm formation, we have demonstrated in vitro that Sebox3 can synthesize c-di-GMP from GTP. The homology derived model of Sebox3 displayed significant conservation of the GGD(/E)EF architecture as well. Hence, we propose that the putative protein VC0395_0300 from V. cholerae is a diguanylate cyclase which has an active role in biofilm formation.

Topics: Bacterial Proteins; Base Sequence; Biofilms; Cellulose; Cloning, Molecular; Cyclic GMP; DNA, Bacterial; Enzyme Assays; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genome, Bacterial; Guanosine Triphosphate; Locomotion; Microscopy, Electron, Scanning; Models, Molecular; Molecular Structure; Phosphorus-Oxygen Lyases; Recombinant Proteins; Sequence Homology; Vibrio cholerae

The signaling molecule cyclic diguanylate (c-di-GMP) mediates physiological adaptation to extracellular stimuli in a wide range of bacteria. The complex metabolic pathways governing c-di-GMP synthesis and degradation are highly regulated, but the specific cues that impact c-di-GMP signaling are largely unknown. In the intestinal pathogen

Topics: Bacterial Toxins; Biofilms; Clostridioides difficile; Cyclic GMP; Gene Expression Regulation, Bacterial; Gene Knockout Techniques; Locomotion; Phosphoric Diester Hydrolases

Expression of cfcR, encoding the only GGDEF/EAL response regulator in Pseudomonas putida, is transcriptionally regulated by RpoS, ANR and FleQ, and the functionality of CfcR as a diguanylate cyclase requires the multisensor CHASE3/GAF hybrid histidine kinase named CfcA. Here an additional level of cfcR control, operating post-transcriptionally via the RNA-binding proteins RsmA, RsmE and RsmI, is unraveled. Specific binding of the three proteins to an Rsm-binding motif (5'CANGGANG3') encompassing the translational start codon of cfcR was confirmed. Although RsmA exhibited the highest binding affinity to the cfcR transcript, single deletions of rsmA, rsmE or rsmI caused minor derepression in CfcR translation compared to a ΔrsmIEA triple mutant. RsmA also showed a negative impact on c-di-GMP levels in a double mutant ΔrsmIE through the control of cfcR, which is responsible for most of the free c-di-GMP during stationary phase in static conditions. In addition, a CfcR-dependent c-di-GMP boost was observed during this stage in ΔrsmIEA confirming the negative effect of Rsm proteins on CfcR translation and explaining the increased biofilm formation in this mutant compared to the wild type. Overall, these results suggest that CfcR is a key player in biofilm formation regulation by the Rsm proteins in P. putida.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Pseudomonas putida; Repressor Proteins; RNA-Binding Proteins

Chapalote is a maize (corn) landrace grown continuously by subsistence farmers in the Americas since 1000 BC, valued in part for its broad-spectrum pathogen resistance. Previously, we showed that Chapalote possesses a bacterial endophyte, Burkholderia gladioli strain 3A12, which suppresses growth of Sclerotinia homoeocarpa, a fungal pathogen of a maize relative, used as a model system. Ten mutants that lost the anti-pathogen activities were identified, corresponding to five genes. However, S. homoeocarpa is not a known maize pathogen; hence, the relevance of these anti-fungal mechanisms to its ancient host has not been clear. Here, the strain 3A12 mutants were tested against a known pathogen of maize and many crops, Rhizoctonia solani. Microscopy established that wild-type 3A12 swarms towards, and attaches onto, the pathogen, forming microcolonies, resulting in hyphal cleavage. Analysis of the mutants revealed that 3A12 uses common downstream gene products (e.g. fungicides) to suppress the growth of both S. homoeocarpa and R. solani, but apparently different upstream regulatory machinery, with the former, but not latter pathogen, requiring YajQ, a receptor for the secondary messenger c-di-GMP. We conclude that B. gladioli strain 3A12, an endophyte of an ancient maize, employs both c-di-GMP-dependent and independent signaling to target diverse fungal pathogens.

Topics: Amino Acyl-tRNA Synthetases; Ascomycota; Burkholderia; Carboxy-Lyases; Cyclic GMP; Endophytes; Host-Pathogen Interactions; Mutation; Plant Diseases; Rhizoctonia; Signal Transduction; Zea mays

c-di-GMP is a cellular second messenger that regulates diverse bacterial processes, including swimming, biofilm formation and virulence. However, in Stenotrophomonas maltophilia, a nosocomial pathogen that frequently infects immunodeficient or immunoincompetent patients, the regulatory function of c-di-GMP remains unclear. Here we show that BsmR is a negative regulator of biofilm development that degrades c-di-GMP through its EAL domain. Increasing BsmR expression resulted in significant increase in bacterial swimming and decrease in cell aggregation. BsmR regulates the expression of at least 349 genes. Among them, 34 involved in flagellar assembly and a flagellar-assembly-related transcription factor (fsnR) are positively regulated. Although BsmR is a response regulator of the two-component signaling system, its role in biofilm formation depends on the expression level of its respective gene (bsmR), not on the protein's phosphorylation level. A transcription factor, BsmT, whose coding gene is located in the same tetra-cistronic operon as bsmR, was shown to directly bind to the promoter region of the operon and, through a positive regulatory loop, modulate bsmR transcription. Thus, our results revealed that the c-di-GMP signaling pathway controls biofilm formation and swimming in S. maltophilia, suggesting c-di-GMP signaling as a target in the development of novel antibacterial agents to resist this pathogen.

Topics: Bacterial Proteins; Binding Sites; Biofilms; Cross Infection; Cyclic GMP; Gene Expression Profiling; Gene Expression Regulation, Bacterial; High-Throughput Nucleotide Sequencing; Operon; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Sequence Analysis, RNA; Signal Transduction; Stenotrophomonas maltophilia

Oral cavity acts as a reservoir of bacterial pathogens for systemic infections and several oral microorganisms have been linked to systemic diseases. Quorum sensing and cyclic dinucleotides, two "decision-making" signaling systems, communicate to regulate physiological process in bacteria. Discovery of cyclic dinucleotides has a long history, but the progress in our understanding of how cyclic dinucleotides regulate bacterial lifestyle is relatively new. Oral microorganisms form some of the most intricate biofilms, yet c-di-GMP, and c-di-AMP signaling have been rarely studied in oral biofilms. Recent studies demonstrated that, with the aid of bacterial messenger molecules and their analogs, it is possible to activate host innate and adaptive immune responses and epithelial integrity with a dose that is relevant to inhibit bacterial virulence mechanisms, such as fimbriae and exopolysaccharide production, biofilm formation, and host cell invasion. The aim of this perspective article is to present available information on cyclic dinucleotides in oral bacteria and in oral biofilms. Moreover, technologies that can be used to detect cyclic dinucleotides in oral biofilms are described. Finally, directions for future research are highlighted.

Topics: Adaptive Immunity; Bacteria; Bacterial Physiological Phenomena; Biofilms; Cyclic GMP; Dinucleoside Phosphates; Immunity, Innate; Mouth; Porphyromonas gingivalis; Quorum Sensing; Signal Transduction; Streptococcus mutans; Treponema denticola; Virulence

Tuberculosis is still on the top of infectious diseases list on both mobility and mortality, especially due to drug-resistance of Mycobacterium tuberculosis (M.tb). Ethionamide (ETH) is one of effective second line anti-TB drugs, a synthetic compound similar to isoniazid (INH) structurally, with existing severe problem of ETH resistance. ETH is a prodrug, which is activated by Etha inside M.tb, and etha is transcriptionally repressed by Ethr. We found that c-di-GMP could bind Ethr, enhanced the binding of Ethr to the promoter of etha, and then repressed the transcription of etha, thus caused resistance of M.tb to ETH. Through docking analysis and in vitro validation, we identified that c-di-GMP binds 3 amino acids of Ethr, i.e., Q125, R181 and E190, while the first 2 were the major binding sites. Homology analysis showed that Ethr was highly conservative among mycobacteria. Further docking analysis showed that c-di-GMP preferentially bound proteins of TetR family at the junction hole of symmetric dimer or tetramer proteins. Our results suggest a possible drug-resistance mechanism of ETH through the regulation of Ethr by c-di-GMP.

Topics: Amino Acid Sequence; Bacterial Proteins; Cyclic GMP; Dimerization; Drug Resistance, Bacterial; Ethionamide; Molecular Docking Simulation; Mycobacterium tuberculosis; Promoter Regions, Genetic

Substrate concentrations generally fluctuate in wastewaters. However, how anammox biomass behaves to overcome the stress of alternating feed loadings remains unclear. Here, we combined long-term reactor operation, batch tests, 16S rRNA transcript sequencing, and metabolomics analysis to investigate the aggregation of anammox biomass under the regulation of c-di-GMP, a key second messenger, in response to alternating feed loadings. We demonstrated that the aggregation process was significantly faster under alternating loadings and was significantly correlated with higher levels of c-di-GMP and extracellular polymeric substances (EPS) production. The increase in c-di-GMP was positively correlated with a higher relative transcript expression level in the c-di-GMP pathway-dependent community. The targeted metabolomics results indicated that the increased production of fructose 6-phosphate and UDP-N-acetyl-d-glucosamine, the precursor substances for the synthesis of exopolysaccharides, was induced by higher levels of c-di-GMP. Consequently, the granulation process was accelerated via EPS production. Higher levels of intracellular hydrophobic amino acids were also positively correlated with increased extracellular protein levels, considering the significant increase in peptides under alternating loadings. On the basis of our findings, we believe that c-di-GMP regulation and EPS production of the anammox biomass are important mechanisms to enhance its tolerance against unfavorable feed stress. These results highlight the role of c-di-GMP in anammox biomass as it works to survive in unfavorable niches.

Topics: Acetylglucosamine; Cyclic GMP; RNA, Ribosomal, 16S; Wastewater

We have engineered

Topics: Cell Cycle; Chromatin Assembly and Disassembly; Cyclic AMP; Cyclic GMP; Epistasis, Genetic; Fermentation; Gene Expression Profiling; Genes, Synthetic; Genetic Engineering; Guanosine Diphosphate; Host-Pathogen Interactions; Humans; Mitochondria; Mutation; Oligonucleotide Array Sequence Analysis; Pyrophosphatases; Saccharomyces cerevisiae; Signal Transduction

Bacteria of many species rely on a simple molecule, the intracellular secondary messenger c-di-GMP (Bis-(3'-5')-cyclic dimeric guanosine monophosphate), to make a vital choice: whether to stay in one place and form a biofilm, or to leave it in search of better conditions. The c-di-GMP network has a bow-tie shaped architecture that integrates many signals from the outside world-the input stimuli-into intracellular c-di-GMP levels that then regulate genes for biofilm formation or for swarming motility-the output phenotypes. How does the 'uninformed' process of evolution produce a network with the right input/output association and enable bacteria to make the right choice? Inspired by new data from 28 clinical isolates of Pseudomonas aeruginosa and strains evolved in laboratory experiments we propose a mathematical model where the c-di-GMP network is analogous to a machine learning classifier. The analogy immediately suggests a mechanism for learning through evolution: adaptation though incremental changes in c-di-GMP network proteins acquires knowledge from past experiences and enables bacteria to use it to direct future behaviors. Our model clarifies the elusive function of the ubiquitous c-di-GMP network, a key regulator of bacterial social traits associated with virulence. More broadly, the link between evolution and machine learning can help explain how natural selection across fluctuating environments produces networks that enable living organisms to make sophisticated decisions.

Topics: Biofilms; Cell Movement; Computational Biology; Cyclic GMP; Machine Learning; Models, Biological; Phenotype; Pseudomonas aeruginosa; Signal Transduction

The bacterial second messenger cyclic diguanylate monophosphate (c-di-GMP) mediates multiple aspects of bacterial physiology through binding to various effectors. In some cases, these effectors are single-domain proteins which only contain a PilZ domain. It remains largely unknown how single-domain PilZ proteins function and regulate their downstream targets. Recently, a single-domain PilZ protein, MapZ (PA4608), was identified to inhibit the activity of the methyltransferase CheR1. Here, crystal structures of the C-terminal domain of CheR1 containing SAH and of CheR1 in complex with c-di-GMP-bound MapZ are reported. It was observed that the binding site of MapZ in CheR1 partially overlaps with the SAH/SAM-binding pocket. Consequently, binding of MapZ blocks SAH/SAM binding. This provides direct structural evidence on the mechanism of inhibition of CheR1 by MapZ in the presence of c-di-GMP.

Topics: Bacterial Proteins; Chemotaxis; Crystallography, X-Ray; Cyclic GMP; Humans; Methyltransferases; Models, Molecular; Protein Binding; Protein Conformation; Protein Domains; Pseudomonas aeruginosa; Pseudomonas Infections

Reducing activity of commensal bacteria in inocula may enhance anammox bacteria proliferation and realization of anammox process. Fast start-up of anammox process in an UASB reactor was successfully achieved by using autoclaved sludge (anaerobic granular sludge pretreated by autoclaving) and 0.3% active anammox sludge as inoculum. Continuous experiments indicated that R2 (autoclaved sludge addition) could shorten the start-up period from 72days to 63days. The first 50days anammox population specific growth rates (μ) of R1 (the control) and R2 were determined to be 0.014d

Topics: Bacteria; Bioreactors; Cyclic GMP; Nitrogen; Oxidation-Reduction; Sewage

Anthranilate, one of tryptophan degradation products has been reported to interfere with biofilm formation by Pseudomonas aeruginosa. Here, we investigated the effects of anthranilate on biofilm formation by various bacteria and the mechanisms responsible. Anthranilate commonly inhibited biofilm formation by P. aeruginosa, Vibrio vulnificus, Bacillus subtilis, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus, and disrupted biofilms preformed by these bacteria. Because anthranilate reduced intracellular c-di-GMP and enhanced swimming and swarming motilities in P. aeruginosa, V. vulnificus, B. subtilis, and S. enterica, it is likely that anthranilate disrupts biofilms by inducing the dispersion of these bacteria. On the other hand, in S. aureus, a non-flagellate bacterium that has no c-di-GMP signaling, anthranilate probably inhibits biofilm formation by reducing slime production. These results suggest that anthranilate has multiple ways for biofilm inhibition. Furthermore, because of its good biofilm inhibitory effects and lack of cytotoxicity to human cells even at high concentration, anthranilate appears to be a promising agent for inhibiting biofilm formation by a broad range of bacteria.

Topics: Bacteria; Biofilms; Cyclic GMP; Hep G2 Cells; Humans; Microbial Sensitivity Tests; Movement; ortho-Aminobenzoates; Tryptophan

The aquatic bacterium and human intestinal pathogen,

Topics: Biofilms; Cyclic GMP; Humans; Intestinal Mucosa; Intestines; Models, Biological; Periplasmic Proteins; Phosphoric Diester Hydrolases; Signal Transduction; Spermine; Vibrio cholerae

BgsA is the glycosyltransferase (GT) involved in the synthesis of a linear mixed-linkage β-glucan (MLG), a recently described exopolysaccharide activated by c-di-GMP in Sinorhizobium meliloti and other Rhizobiales. Although BgsA displays sequence and structural homology with bacterial cellulose synthases (CS), it does not contain any predictable c-di-GMP binding domain. In this work we demonstrate that the cytoplasmic C-terminal domain of BgsA (C-BgsA) binds c-di-GMP with both high affinity (K

Topics: beta-Glucans; Catalytic Domain; Cyclic GMP; DNA Mutational Analysis; Glycosyltransferases; Kinetics; Mutant Proteins; Protein Binding; Sinorhizobium meliloti; Surface Plasmon Resonance

Topics: Bacterial Proteins; Biofilms; Copper Sulfate; Cyclic GMP; Oxidation-Reduction; Periplasmic Proteins; Plague; Protein Stability; Yersinia pestis

Type IVa pili (T4P) are bacterial surface structures that enable motility, adhesion, biofilm formation and virulence. T4P are assembled by nanomachines that span the bacterial cell envelope. Cycles of T4P assembly and retraction, powered by the ATPases PilB and PilT, allow bacteria to attach to and pull themselves along surfaces, so-called "twitching motility". These opposing ATPase activities must be coordinated and T4P assembly limited to one pole for bacteria to show directional movement. How this occurs is still incompletely understood. Herein, we show that the c-di-GMP binding protein FimX, which is required for T4P assembly in Pseudomonas aeruginosa, localizes to the leading pole of twitching bacteria. Polar FimX localization requires both the presence of T4P assembly machine proteins and the assembly ATPase PilB. PilB itself loses its polar localization pattern when FimX is absent. We use two different approaches to confirm that FimX and PilB interact in vivo and in vitro, and further show that point mutant alleles of FimX that do not bind c-di-GMP also do not interact with PilB. Lastly, we demonstrate that FimX positively regulates T4P assembly and twitching motility by promoting the activity of the PilB ATPase, and not by stabilizing assembled pili or by preventing PilT-mediated retraction. Mutated alleles of FimX that no longer bind c-di-GMP do not allow rapid T4P assembly in these assays. We propose that by virtue of its high-affinity for c-di-GMP, FimX can promote T4P assembly when intracellular levels of this cyclic nucleotide are low. As P. aeruginosa PilB is not itself a high-affinity c-di-GMP receptor, unlike many other assembly ATPases, FimX may play a key role in coupling T4P mediated motility and adhesion to levels of this second messenger.

Topics: Bacterial Proteins; Carrier Proteins; Chromatography, Gel; Cyclic GMP; Fimbriae, Bacterial; Image Processing, Computer-Assisted; Intracellular Signaling Peptides and Proteins; Microscopy, Electron, Transmission; Oxidoreductases; Polymerase Chain Reaction; Pseudomonas aeruginosa; Surface Plasmon Resonance; Virulence; Virulence Factors

The second messenger c-di-GMP regulates the switch between motile and sessile bacterial lifestyles. A general feature of c-di-GMP metabolism is the presence of a surprisingly large number of genes coding for diguanylate cyclases and phosphodiesterases, the enzymes responsible for its synthesis and degradation respectively. However, the physiological relevance of this apparent redundancy is not clear, emphasizing the need for investigating the functions of each of these enzymes. Here we focused on the phosphodiesterase PA2133 from Pseudomonas aeruginosa, an important opportunistic pathogen. We phenotypically characterized P. aeruginosa strain K overexpressing PA2133 or its inactive mutant. We showed that biofilm formation and motility are severely impaired by overexpression of PA2133. Our quantitative proteomic approach applied to the membrane and exoprotein fractions revealed that proteins involved in three processes were mostly affected: flagellar motility, type III secretion system and chemotaxis. While inhibition of biofilm formation can be ascribed to the phosphodiesterase activity of PA2133, down-regulation of flagellar, chemotaxis, and type III secretion system proteins is independent of this enzymatic activity. Based on these unexpected effects of PA2133, we propose to rename this gene product FcsR, for Flagellar, chemotaxis and type III secretion system Regulator.

Topics: Bacterial Proteins; Biofilms; Cell Membrane; Chemotaxis; Cyclic GMP; Enzyme Activation; Flagella; Gene Expression Regulation, Bacterial; Mutation; Phenotype; Phosphoric Diester Hydrolases; Proteome; Proteomics; Pseudomonas aeruginosa; Type III Secretion Systems

Necrotic enteritis (NE) caused by netB-positive strains of Clostridium perfringens is an important disease of intensively-reared broiler chickens. It is widely controlled by antibiotic use, but this practice that has come under increasing scrutiny and alternative approaches are required. As part of the search for alternative approaches over the last decade, advances have been made in understanding its pathogenesis but much remains to be understood and applied to the control of NE. The objective of this work was to assess the effect on virulence of mutation of the cyclic-di-GMP signaling genes present on the large pathogenicity locus (NELoc-1) in the tcp-encoding conjugative virulence plasmid, pNetB. For this purpose, the diguanylate cyclase (dgc) and phosphodiesterase (pde) genes were individually insertionally inactivated and the two mutants were subsequently complemented with their respective genes. Southern blotting showed that a single gene insertion was present. Mutation of either gene resulted in almost total attenuation of the mutants to cause NE in experimentally-infected broiler chickens, which was fully restored in each case by complementation of the respective mutated gene. Production of NetB-associated cytotoxicity for Leghorn male hepatoma (LMH) cells was unaffected in mutants. We conclude that the cyclic-di-GMP signaling system is important in controlling virulence in a NE C. perfringens strain and might be a target for control of the disease.

Topics: Animals; Chickens; Clostridium Infections; Clostridium perfringens; Cyclic GMP; Enteritis; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Intestines; Mutation; Poultry Diseases

Diguanylate cyclases that synthesize and phosphodiesterases that hydrolyze the second messenger cyclic-di-GMP (c-di-GMP) are at the center of bacterial signaling pathways that control behaviors relevant to all aspects of microbial physiology and pathogenesis (Romling et al., Microbiol Mol Biol Rev 77(1):1-52, 2013). Bioinformatics tools can easily predict the presence of the diguanylate cyclase GGDEF domain, or the EAL and HD-GYP domains associated with phosphodiesterase activity. However, experimental confirmation of enzymatic activity is still necessary, as many proteins contain degenerate domains that lack catalytic activity but nonetheless function as c-di-GMP receptors.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Bacteria; Cyclic GMP; Enzyme Activation; Escherichia coli Proteins; Gene Expression; Phosphorus Radioisotopes; Phosphorus-Oxygen Lyases; Recombinant Proteins

C-di-GMP has emerged as a prevalent bacterial messenger that controls a multitude of bacterial behaviors. Having access to milligram or gram quantities of c-di-GMP is essential for the biochemical and structural characterization of enzymes and effectors involved in c-di-GMP signaling. Although c-di-GMP can be synthesized using chemical methods, diguanylate cyclases (DGC)-based enzymatic synthesis is the most efficient method of preparing c-di-GMP today. Many DGCs are not suitable for c-di-GMP production because of poor protein stability and the presence of a c-di-GMP-binding inhibitory site (I-site) in most DGCs. We have identified and engineered a thermophilic DGC for efficient production of c-di-GMP for characterizing c-di-GMP signaling proteins and riboswitches. Importantly, residue replacement in the inhibitory I-site of the thermophilic DGC drastically relieved product inhibition to enable the production of hundreds of milligrams of c-di-GMP using 5-10 mg of this robust biocatalyst.

Topics: Chromatography, High Pressure Liquid; Cyclic GMP; Escherichia coli Proteins; Gene Expression; Phosphorus-Oxygen Lyases; Recombinant Proteins; Structure-Activity Relationship; Temperature; Thermotoga maritima

The modulation of c-di-GMP levels plays a vital role in the regulation of various processes in a wide array of bacterial species. Thus, investigation of c-di-GMP regulation requires reliable methods for the assessment of c-di-GMP levels and turnover. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis has become a commonly used approach to accomplish these goals. The following describes the extraction and HPLC-based detection and quantification of c-di-GMP from Pseudomonas aeruginosa samples, a procedure that is amenable to modifications for the analysis of c-di-GMP in other bacterial species.

Topics: Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Cyclic GMP; Pseudomonas aeruginosa; Reference Standards

Cyclic dinucleotides such as bis-(3',5')-cyclic dimeric guanosine monophosphate (3',3'-c-di-GMP) represent an important class of second messengers in bacteria and are involved in numerous (patho)physiological settings. Here, we describe a sensitive and specific quantification method for 3',3'-c-di-GMP by HPLC-coupled tandem mass spectrometry (LC-MS/MS). Additionally, linear 3',3'-c-di-GMP metabolites, i.e., 5'-phosphoguanylyl-3',5'-guanosine (pGpG) and 5'-guanosine monophosphate (5'-GMP), as well as cyclic guanosine monophosphate (3',5'-cGMP) and 3',3' c-di-GMP analogues (2',3'-c-di-GMP and 2',2'-c-di-GMP) can be simultaneously determined by this method.

Topics: Bacteria; Chromatography, Reverse-Phase; Cyclic GMP; Metabolomics; Second Messenger Systems; Tandem Mass Spectrometry

Topics: Chromatography, High Pressure Liquid; Cyclic GMP; Escherichia coli Proteins; Kinetics; Mass Spectrometry; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Second Messenger Systems; Spectrophotometry; Tandem Mass Spectrometry

Recent research has shown that the molecule c-di-GMP is an important second messenger regulating various functions in bacteria. In particular, the implication of c-di-GMP as a positive regulator of adhesion and biofilm formation has gained momentum as a highly relevant research topic, as detailed knowledge about the underlying regulatory mechanisms may enable the development of measures to control biofilms in both industrial and medical settings. Accordingly, it is in many cases of interest to measure the c-di-GMP level in bacteria under specific conditions or in specific mutant strains. We have developed a collection of fluorescence-based c-di-GMP biosensors capable of gauging the c-di-GMP level in Pseudomonas aeruginosa and closely related bacteria. Here, we describe protocols for the use of these biosensors in gauging and visualizing cellular c-di-GMP levels of P. aeruginosa both in in vitro setups such as continuous-culture flow-cell biofilms, and in in vivo settings such as a murine corneal infection model.

Topics: Animals; Biofilms; Biosensing Techniques; Cyclic GMP; Disease Models, Animal; Female; Fluorescence; Keratitis; Mice; Molecular Imaging; Pseudomonas aeruginosa; Pseudomonas Infections

3',5'-cyclic diguanosine monophosphate (cyclic di-GMP) is a bacterial secondary messenger molecule that regulates many important cellular activities and behaviors, such as motility and biofilm formation. While mass spectrometry protocols for quantitative analyses of intracellular cyclic di-GMP concentrations have been developed, they are time intensive, expensive, low-throughput, and incapable of directly monitoring dynamic changes in vivo. In this protocol, we provide a Pseudomonas aeruginosa-specific detailed methodology to assay the intracellular levels of cyclic di-GMP using biological reporters.

Topics: Cyclic GMP; Gene Expression; Genes, Reporter; Luminescent Measurements; Microscopy, Fluorescence; Pseudomonas aeruginosa; Second Messenger Systems; Transcription, Genetic

By adopting elaborate three-dimensional morphologies that vary according to their extracellular matrix composition, macrocolony biofilms offer a unique opportunity to interrogate about the roles of specific matrix components in shaping biofilm architecture. Here, we describe two methods optimized for Escherichia coli that profit from morphology and the high level of structural organization of macrocolonies to gain insight into the production and assembly of amyloid curli and cellulose-the two major biofilm matrix elements of E. coli-in biofilms. The first method, the macrocolony morphology assay, is based on the ability of curli and cellulose-either alone or in combination-to generate specific morphological and Congo Red-staining patterns in E. coli macrocolonies, which can then be used as a direct visual readout for the production of these matrix components. The second method involves thin sectioning of macrocolonies, which along with in situ staining of amyloid curli and cellulose and microscopic imaging allows gaining fine details of the spatial arrangement of both matrix elements inside macrocolonies. Beyond their current use with E. coli and related curli and cellulose-producing Enterobacteriaceae, both the methods offer the potential to be adapted to other bacterial species.

Topics: Bacteria; Bacterial Physiological Phenomena; Biofilms; Cellulose; Cyclic GMP; Extracellular Matrix; Fluorescent Dyes; Microscopy, Fluorescence

Congo red is a diazo textile dye that has been used to visualize the production of amyloid fibers for nearly a century. Microbiological applications were later developed, especially in identifying strains that produce amyloid appendages called curli and overexpressing polysaccharides in the biofilm matrix. The second messenger cyclic diguanylate (c-di-GMP) regulates the production of biofilm matrix polysaccharides, and therefore Congo red staining of samples can be utilized as an indirect measurement of elevated c-di-GMP production in bacteria. Congo red allows the identification of strains producing high c-di-GMP in an inexpensive, quantitative, and high-throughput manner.

Topics: Bacteria; Bacterial Physiological Phenomena; Biofilms; Colorimetry; Congo Red; Cyclic GMP; Extracellular Matrix; Staining and Labeling

Second-generation RNA-based fluorescent biosensors have been developed that enable flow cytometry experiments to monitor the population dynamics of c-di-GMP signaling in live bacteria. These experiments are high-throughput, provide information at the single-cell level, and can be performed on cells grown in complex media and/or under anaerobic conditions. Here, we describe flow cytometry methods for three applications: (1) high-throughput screening for diguanylate cyclase activity, (2) analyzing c-di-GMP levels under anaerobic conditions, and (3) monitoring cell population dynamics of c-di-GMP levels upon environmental changes. These methods showcase RNA-based fluorescent biosensors as versatile tools for studying c-di-GMP signaling in bacteria.

Topics: Anaerobiosis; Biosensing Techniques; Cyclic GMP; Enzyme Activation; Escherichia coli; Escherichia coli Proteins; Flow Cytometry; Phosphorus-Oxygen Lyases; Single-Cell Analysis

The nucleotide-based second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulates multiple processes in bacteria including cellular motility. The rod-shaped Myxococcus xanthus cells move in the direction of their long axis using two distinct motility systems: type IV pili (T4P)-dependent motility and gliding motility. Manipulation of the c-di-GMP level by expression of either an active, heterologous diguanylate cyclase or an active, heterologous phosphodiesterase causes defects in T4P-dependent motility without affecting gliding motility. As both an increased and a decreased level of c-di-GMP affect T4P-dependent motility, M. xanthus represents a good model system to assess enzyme activity of diguanylate cyclases and phosphodiesterases using T4P-dependent motility as a readout. Here, we describe the assay, which allows correlating diguanylate cyclase and phosphodiesterase activity with T4P-dependent motility in M. xanthus.

Topics: Cyclic GMP; Escherichia coli Proteins; Fimbriae, Bacterial; Myxococcus xanthus; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases

Signaling pathways involving second messenger c-di-GMP regulate various aspects of bacterial physiology and behavior. We describe the use of a red light-activated diguanylate cyclase (c-di-GMP synthase) and a blue light-activated c-di-GMP phosphodiesterase (hydrolase) for manipulating intracellular c-di-GMP levels in bacterial cells. We illustrate the application of these enzymes in regulating several c-di-GMP-dependent phenotypes, i.e., motility and biofilm phenotypes in E. coli and chemotactic behavior in the alphaproteobacterium Azospirillum brasilense. We expect these light-activated enzymes to be also useful in regulating c-di-GMP-dependent processes occurring at the fast timescale, for spatial control of bacterial populations, as well as for analyzing c-di-GMP-dependent phenomena at the single-cell level.

Topics: Bacteria; Bacterial Physiological Phenomena; Bacterial Proteins; Biofilms; Biomass; Chemotaxis; Cyclic GMP; Enzyme Activators; Escherichia coli; Escherichia coli Proteins; Light; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Plasmids; Transformation, Bacterial

Diguanylate cyclases are enzymes that use two GTP molecules to produce one molecule cyclic dimeric guanosine monophosphate (c-di-GMP). This cyclic dinucleotide is an ubiquitous prokaryotic second messenger that controls a variety of cell functions. Several proteins have been described which contain a photoreceptor domain fused to a diguanylate cyclase. The cyanobacterial light sensor Cph2 is responsible for the blue-light induced synthesis of c-di-GMP in Synechocystis sp. PCC 6803. Here, we provide a detailed protocol for an in vitro enzymatic assay with a purified photoreceptor protein using light as the crucial reaction parameter for c-di-GMP synthesis. The assay is accomplished under continuous illumination with light of different quality with inactivation of the enzyme by heat denaturation. Analytics are performed using HPLC-UV.

Topics: Bacterial Proteins; Chromatography, High Pressure Liquid; Cyanobacteria; Cyclic GMP; Enzyme Activation; Enzyme Assays; Light; Reference Standards; Spectrophotometry, Ultraviolet

The determination of the genome sequences of pathogenic bacteria has facilitated functional analyses that aim to understand the molecular basis of virulence. In particular, genome sequence information of the pathogen Xanthomonas campestris pathovar campestris has allowed researchers to identify and functionally analyze the role of intracellular signaling involving cyclic di-GMP in black rot disease of crucifers. Here, we describe leaf clipping and spraying methods for testing the virulence of wild type and derived mutants of X. campestris in Chinese radish. These methods address different facets of the disease cycle, which requires the ability to survive epiphytically before entry into the plant and growth and systemic spread within the xylem.

Topics: Cyclic GMP; Phenotype; Plant Diseases; Raphanus; Second Messenger Systems; Signal Transduction; Virulence; Xanthomonas campestris

Bacteria produce toxins to enhance their competitiveness in the colonization of an environment as well as during an infection. The delivery of toxins into target cells is mediated by several types of secretion systems, among them our focus is Type III and Type VI Secretion Systems (T3SS and T6SS, respectively). A thorough methodology is provided detailing how to identify if cyclic di-GMP signaling plays a role in the P. aeruginosa toxin delivery mediated by T3SS or T6SS. This includes in vitro preparation of the samples for Western blot analysis aiming at detecting possible c-di-GMP-dependent T3SS/T6SS switch, as well as in vivo analysis using the model organism Galleria mellonella to demonstrate the ecological and pathogenic consequence of the switch between these two secretion systems.

Topics: Animals; Bacterial Toxins; Cyclic GMP; Moths; Mutation; Pseudomonas aeruginosa; Type III Secretion Systems; Type VI Secretion Systems

c-di-GMP is widely recognized as an important ubiquitous signaling molecule in bacteria. c-di-GMP phosphodiesterases (PDEs) regulate the intracellular concentration of c-di-GMP and some could be potential drug targets. Here, we describe a class of dinucleotide probes suitable for monitoring the enzymatic activities of c-di-GMP PDEs in real time. Such probes contain fluorescent nucleobases and can be readily cleaved by PDEs, resulting in a change in fluorescence. Fluorescent cyclic and linear dinucleotide probes could be used in diverse applications, such as confirming the activity of an expressed PDE or oligoribonuclease (Orns) or identifying inhibitors of PDEs or Orns using high-throughput screening formats.

Topics: 2-Aminopurine; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 6; Enzyme Activation; Enzyme Assays; Exoribonucleases; Fluorescent Dyes; Phosphoric Diester Hydrolases

The second messenger, cyclic diguanylate (c-di-GMP), regulates a variety of bacterial cellular and social behaviors. A key determinant of c-di-GMP levels in cells is its degradation by c-di-GMP-specific phosphodiesterases (PDEs). Here, we describe an assay to determine c-di-GMP degradation rates in vitro using 2'-O-(N'-methylanthraniloyl)-cyclic diguanylate (MANT-c-di-GMP). Additionally, a protocol for the production and purification of recombinant Pseudomonas aeruginosa RocR, a c-di-GMP-specific PDE that may serve as a control in MANT-c-di-GMP assays, is provided. The use of the fluorescent MANT-c-di-GMP analogue can deliver fundamental information about PDE function, and is suitable for identifying and investigating c-di-GMP-specific PDE activators and inhibitors.

Topics: Bacterial Proteins; Cyclic GMP; Enzyme Activation; Enzyme Assays; ortho-Aminobenzoates; Phosphoric Diester Hydrolases; Recombinant Proteins

Cyclic-di-GMP phosphodiesterases (PDEs) catalyze the hydrolysis of the bacterial second messenger c-di-GMP. This protocol describes a sensitive radioactive assay for PDE activity in which substrate and product can be quickly and easily separated by thin-layer chromatography.

Topics: Chromatography, Thin Layer; Cyclic GMP; Enzyme Activation; Enzyme Assays; Phosphoric Diester Hydrolases; Phosphorus Radioisotopes

The Congo Red (CR) assay is a standard biofilm test assessing the colony morphology of bacteria growing on agar plates supplemented with the diazo dye Congo Red. Biofilm forming Salmonella enterica serovar Typhimurium and Escherichia coli produce a red, dry, and rough (rdar) morphotype on CR-plates. The phenotype is characterized by staining of the extracellular matrix components curli (brown color) and cellulose (pink color) by CR. This method allows semiquantitative determination of the expression level of the individual matrix components and dissection of the regulatory networks controlling their production in response to c-di-GMP levels. Here, we describe the CR-assay and its variations and discuss the effect of deletion or overexpression of c-di-GMP turnover proteins on colony morphology.

Topics: Biofilms; Congo Red; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phenotype; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Salmonella typhimurium; Sequence Deletion; Serogroup

Modulation of signal transduction via binding of the secondary messenger molecule cyclic di-GMP to effector proteins is a near universal regulatory schema in bacteria. In particular, direct binding of c-di-GMP to transcriptional regulators has been shown to alter gene expression of a variety of processes. Here, we illustrate a pull-down-based DNA:protein binding reaction to determine the relative importance of c-di-GMP in the binding affinity of a target protein to specific DNA sequences. Specifically, the pull-down-based assay enables DNA binding to be analyzed with differing concentrations of c-di-GMP in the absence/presence of specific and nonspecific competitors.

Topics: Cyclic GMP; DNA; Gene Expression Regulation; Immunoblotting; Protein Binding; Transcription Factors; Transcription, Genetic

The transition of bacteria from a planktonic lifestyle to a collaborative, sessile biofilm lifestyle is a regulated process orchestrated by the intracellular second-messenger c-di-GMP (bis-(3'-5')-cyclic dimeric guanosine monophosphate). To modulate this transition, c-di-GMP acts at the transcriptional, posttranscriptional, and posttranslational levels. In this chapter, we describe a method to study of how a transcriptional regulator modulates gene expression in response to c-di-GMP binding. DNase I footprinting is a valuable tool for use in analyzing how regulatory proteins bind to DNA, the location of their binding sites or how c-di-GMP affects their binding to DNA. This chapter describes a protocol for nonradiochemical DNase I footprinting experiments using a capillary electrophoresis method based on the interaction of the Pseudomonas aeruginosa FleQ protein with the promoter regions of biofilm-related genes.

Topics: Binding Sites; Cyclic GMP; Deoxyribonuclease I; DNA Footprinting; DNA-Binding Proteins; DNA, Bacterial; Electrophoresis, Capillary; Fluorescent Dyes; Promoter Regions, Genetic; Staining and Labeling; Transcription Factors

Cyclic di-GMP is an important regulatory messenger molecule that often directly interacts with proteins to alter function. It is therefore important to find potential c-di-GMP binding proteins and verify a direct interaction between them. Here, we describe a pull-down assay using biotinylated-c-di-GMP to capture a specific protein of interest followed by immunoblot analysis to determine relative protein abundance. This method also allows for addition of both specific and nonspecific competitors to determine specificity of c-di-GMP-protein binding. We also discuss using densitometry analysis on resulting immunoblots to calculate the dissociation constant (K

Topics: Biotinylation; Cyclic GMP; Densitometry; DNA-Binding Proteins; Immunoblotting; Kinesics; Protein Binding

Capture compound technology coupled to mass spectrometry (CCMS) allows to biochemically identify ligand receptors. Using a c-di-GMP-specific Capture Compound, we adapted this method for the identification and characterization of c-di-GMP binding proteins in any bacterial species. Because in silico analysis often fails to predict novel c-di-GMP effectors, this universal method aims at better defining the cellular c-di-GMP network in a wide range of bacteria. CCMS was successfully applied in several bacterial species (Nesper et al., J Proteom 75:4874-4878, 2012; Steiner et al., EMBO J 32:354-368, 2013; Tschowri et al., Cell 158:1136-1147, 2014; Trampari et al., J Biol Chem 290:24470-24483, 2015; Rotem et al., J Bacteriol 198:127-137, 2015). To outline the detailed protocol and to illustrate its power, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control, as an example. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network.

Topics: Bacterial Proteins; Carrier Proteins; Chromatography, Liquid; Cross-Linking Reagents; Cyclic GMP; Databases, Genetic; Mass Spectrometry; Protein Binding; Pseudomonas aeruginosa; Tandem Mass Spectrometry

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is an important signaling molecule for community behavior control, cell morphogenesis, and virulence in bacteria. In addition to protein effectors, this second messenger binds RNA molecules that act as riboswitches to control target gene expression. In this chapter, we describe a method for experimental validation of the functionality of c-di-GMP-responsive riboswitches and the analysis of c-di-GMP control of target gene expression by qRT-PCR and Northern blot. This procedure can be used for the studies of in silico-predicted riboswitch candidates, as well as a targeted experimental approach for exploring the data from next-generation sequencing. The examples on the analysis of type I and type II c-di-GMP-responsive riboswitches in Clostridium difficile are provided to illustrate the application of the method.

Topics: Blotting, Northern; Clostridioides difficile; Cyclic GMP; Nucleic Acid Conformation; Riboswitch; RNA, Bacterial; Second Messenger Systems; Transcription Termination, Genetic; Transcription, Genetic; Workflow

Isothermal titration calorimetry (ITC) is a commonly used biophysical technique that enables the quantitative characterization of intermolecular interactions in solution. Based on enthalpy changes (ΔH) upon titration of the binding partner (e.g., a small-molecule ligand such as c-di-GMP) to the molecule of interest (e.g., a receptor protein), the resulting binding isotherms provide information on the equilibrium association/dissociation constants (K

Topics: Bacterial Proteins; Calorimetry; Chromatography, Gel; Cyclic GMP; DNA-Binding Proteins; Kinetics; Ligands; Models, Molecular; Molecular Conformation; Protein Binding; Structure-Activity Relationship

One of the most important signals involved in controlling biofilm formation is represented by the intracellular second messenger 3',5'-cyclic diguanylic acid (c-di-GMP). Since the pathways involved in c-di-GMP biosynthesis and breakdown are found only in bacteria, targeting c-di-GMP metabolism represents an attractive strategy for the development of biofilm-disrupting drugs. Here, we present the workflow required to perform a structure-based design of inhibitors of diguanylate cyclases, the enzymes responsible for c-di-GMP biosynthesis. Downstream of the virtual screening process, detailed in the first part of the chapter, we report the step-by-step protocols required to test the positive hits in vitro and to validate their selectivity, thus minimizing possible off-target effects.

Topics: Bacterial Proteins; Binding Sites; Catalytic Domain; Cell Line, Tumor; Chromatography, Liquid; Computer Simulation; Cyclic GMP; Drug Discovery; Enzyme Inhibitors; Escherichia coli Proteins; Humans; Models, Molecular; Molecular Conformation; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Binding; Quantitative Structure-Activity Relationship; Reproducibility of Results; Spectrum Analysis

Bacteria in the biofilm mode of growth cause numerous problematic infections due to their resistance to antimicrobials and the immune system. Because conventional antimicrobial compounds cannot efficiently eradicate biofilm infections, we urgently need new efficient anti-biofilm drugs. The secondary messenger c-di-GMP is a positive regulator of biofilm formation in many clinically relevant bacteria, and it is assumed that drugs that lower the intracellular level of c-di-GMP will force biofilm bacteria into a more treatable planktonic lifestyle. We describe a protocol for high-throughput screening of chemical libraries for compounds that lower the c-di-GMP level in bacteria, and potentially can serve as lead compounds in the development of novel biofilm dismantling drugs.

Topics: Anti-Bacterial Agents; Bacteria; Biofilms; Cyclic GMP; Drug Discovery; High-Throughput Screening Assays; Second Messenger Systems; Small Molecule Libraries; Workflow

Pseudomonas aeruginosa infections are often difficult or impossible to treat, mainly due to its ability to form antibiotic-resistant biofilms. Since c-di-GMP signaling strongly influences P. aeruginosa biofilm development and sensitivity to antibiotics, it is considered a promising target for the development of anti-biofilm drugs and it is under intensive investigation. However, studying c-di-GMP signaling in P. aeruginosa is challenging, mainly due to (1) the multiplicity of enzymes involved in c-di-GMP metabolism, (2) the difficulty to extract and measure c-di-GMP intracellular levels by chemical methods, and (3) the lack of genetic tools specifically dedicated to this purpose.Here, a bioluminescence-based reporter system convenient for studying cellular processes or compounds expected to cause an increase or a decrease in intracellular c-di-GMP levels produced by P. aeruginosa cultures is described. Bioluminescence is particularly appropriate in P. aeruginosa research, due to the high intensity of the signal and total lack of background noise. In addition, the use of genetic cassettes allowing the fine control of P. aeruginosa c-di-GMP intracellular levels via arabinose induction is described.Overall, the genetic tools described here could facilitate investigations tackling the c-di-GMP signaling process on different fields, from cellular physiology to drug-discovery research.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression; Gene Expression Regulation, Bacterial; Genes, Reporter; Genetic Testing; Luminescent Measurements; Plasmids; Promoter Regions, Genetic; Pseudomonas aeruginosa; Signal Transduction

Production of cellulose, a stress response-mediated process in enterobacteria, is modulated in Escherichia coli by the activity of the two pyrimidine nucleotide biosynthetic pathways, namely, the de novo biosynthetic pathway and the salvage pathway, which relies on the environmental availability of pyrimidine nitrogenous bases. We had previously reported that prevalence of the salvage over the de novo pathway triggers cellulose production via synthesis of the second messenger c-di-GMP by the DgcQ (YedQ) diguanylate cyclase. In this work, we show that DgcQ enzymatic activity is enhanced by UTP, whilst being inhibited by N-carbamoyl-aspartate, an intermediate of the de novo pathway. Thus, direct allosteric control by these ligands allows full DgcQ activity exclusively in cells actively synthesizing pyrimidine nucleotides via the salvage pathway. Inhibition of DgcQ activity by N-carbamoyl-aspartate appears to be favoured by protein-protein interaction between DgcQ and PyrB, a subunit of aspartate transcarbamylase, which synthesizes N-carbamoyl-aspartate. Our results suggest that availability of pyrimidine bases might be sensed, somehow paradoxically, as an environmental stress by E. coli. We hypothesize that this link might have evolved since stress events, leading to extensive DNA/RNA degradation or lysis of neighbouring cells, can result in increased pyrimidine concentrations and activation of the salvage pathway.

Topics: Aspartate Carbamoyltransferase; Aspartic Acid; Biosynthetic Pathways; Cellulose; Cyclic GMP; DNA; Escherichia coli; Escherichia coli Proteins; Phosphorus-Oxygen Lyases; RNA; Uridine Triphosphate

Agar plate-based biofilm of enterobacteria like Escherichia coli is characterized by expression of the extracellular matrix components amyloid curli and cellulose exopolysaccharide, which can be visually enhanced upon addition of the dye Congo Red, resulting in a red, dry, and rough (rdar) colony morphology. Expression of the rdar morphotype depends on the transcriptional regulator CsgD and occurs predominantly at ambient temperature in model strains. In contrast, commensal and pathogenic isolates frequently express the csgD-dependent rdar morphotype semi-constitutively, also at human host body temperature. To unravel the molecular basis of temperature-independent rdar morphotype expression, biofilm components and c-di-GMP turnover proteins of seven commensal and uropathogenic E. coli isolates were analyzed. A diversity within the c-di-GMP signaling network was uncovered which suggests alteration of activity of the trigger phosphodiesterase YciR to contribute to (up)regulation of csgD expression and consequently semi-constitutive rdar morphotype development.

Topics: Amino Acid Substitution; Biofilms; Cyclic GMP; Enzyme Activation; Escherichia coli Proteins; Gene Expression Profiling; Genome, Bacterial; Humans; Mutation; Phenotype; Phylogeny; Uropathogenic Escherichia coli

The

Topics: Animals; ATP-Binding Cassette Transporters; Cyclic GMP; Escherichia coli Infections; Escherichia coli Proteins; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Humans; Mice; Multigene Family; Mutation; Operon; Phosphates; Phosphorus-Oxygen Lyases; Recombinases; Regulon; Transcription Factors; Urinary Bladder; Urinary Tract Infections; Uropathogenic Escherichia coli; Virulence

Biofilm is prevalent in various ecological niches, in which microbial cells interconnect with each other through extracellular polymeric substances including polysaccharides, extracellular DNA, and proteins. When living in biofilms, the microbial cells employ small signalling chemicals as their "language" to communicate mutually, and exhibit remarkable differences in physiology compared to those living in planktonic state. It has been proven that the development of biofilm is subject to the regulation of c-di-GMP, an important second messenger found in prokaryotes. Given its important roles of biofilms in microbial infection, industry application, plant-microbe interactions and environmental pollustion, biofilm is one of frontier research areas in microbiology. This special issue of "Biofilm and c-di-GMP" systematically reviews the current progresses in the multiple research frontiers, including biotechnology, infectious diseases, environmental microbiology and plant pathology, with special focus on the methods and techniques in biofilm research. We hope that the issue will boost the interest of students and young scientists in this exciting area of microbiology.. 生物被膜 (Biofilm) 是微生物生存的主要形式。它是一种或多种微生物通过胞外多糖、胞外DNA、蛋白质等组成的基质聚集在一起形成的细胞多聚体。生物被膜中的微生物细胞之间存在密切的信号通讯，并且表现出与浮游生活时完全不同的生理代谢特征，其发育过程受到环二鸟苷单磷酸 (c-di-GMP) 等细胞第二信使的控制。由于生物被膜在基础生命科学和医疗、工业、农业、环境治理等应用科学中的重要性，相关研究是微生物学领域的前沿之一。本期专刊从生物工程、研究技术、感染与免疫、环境生物学和植物病理学等角度，较系统地对生物被膜的形成机制、调控分子机理、理化特性和应用技术等进行了综述或研究，展示了我国在本领域的研究水平，同时也对本领域未来发展趋势进行了有益的讨论。.

Topics: Biofilms; Cyclic GMP; Second Messenger Systems

The biofilm life cycle is characterized by the transition of planktonic cells exhibiting high susceptibly to antimicrobial agents to a biofilm mode of growth characterized by high tolerance to antimicrobials, followed by dispersion of cells from the biofilm back into the environment. Dispersed cells, however, are not identical to planktonic cells but have been characterized as having a unique transitionary phenotype relative to biofilm and planktonic cells, with dispersed cells attaching in a manner similar to exponential-phase cells, but demonstrating gene expression patterns that are distinct from both exponential and stationary-phase planktonic cells. This raised the question whether dispersed cells are as susceptible as planktonic cells and whether the dispersion inducer or the antibiotic class affects the drug susceptibility of dispersed cells. Dispersed cells obtained in response to dispersion cues glutamate and nitric oxide (NO) were thus exposed to tobramycin and colistin. Although NO-induced dispersed cells were as susceptible to colistin and tobramycin as exponential-phase planktonic cells, glutamate-induced dispersed cells were susceptible to tobramycin but resistant to colistin. The difference in colistin susceptibility was independent of cellular c-di-GMP levels, with modulation of c-di-GMP failing to induce dispersion. Instead, drug susceptibility was inversely correlated with LPS modification system and the biofilm-specific transcriptional regulator BrlR. The susceptibility phenotype of glutamate-induced dispersed cells to colistin was found to be reversible, with dispersed cells being rendered as susceptible to colistin within 2 h postdispersion, though additional time was required for dispersed cells to display expression of genes indicative of exponential growth.

Topics: Anti-Bacterial Agents; Bacterial Adhesion; Biofilms; Colistin; Cyclic GMP; Drug Resistance, Multiple, Bacterial; Gene Expression Regulation, Bacterial; Glutamic Acid; Humans; Microbial Sensitivity Tests; Pseudomonas aeruginosa; Tobramycin

Overuse of antibiotics is contributing to an emerging antimicrobial resistance crisis. To better understand how bacteria adapt tolerance and resist antibiotic treatment, Pseudomonas aeruginosa isolates obtained from infection sites sampled from companion animals were collected and evaluated for phenotypic differences. Selected pairs of clonal isolates were obtained from individual infection samples and were assessed for antibiotic susceptibility, cyclic di-GMP levels, biofilm production, motility and genetic-relatedness. A total of 18 samples from equine, feline and canine origin were characterized. A sample from canine otitis media produced a phenotypically heterogeneous pair of P. aeruginosa isolates, 42121A and 42121B, which during growth on culture medium respectively exhibited hyper dye-binding small colony morphology and wild-type phenotypes. Antibiotic susceptibility to gentamicin and ciprofloxacin also differed between this pair of clonal isolates. Sequence analysis of gyrA, a gene known to be involved in ciprofloxacin resistance, indicated that 42121A and 42121B both contained mutations that confer ciprofloxacin resistance, but this did not explain the differences in ciprofloxacin resistance that were observed. Cyclic di-GMP levels also varied between this pair of isolates and were shown to contribute to the observed colony morphology variation and ability to form a biofilm. Our results demonstrate the role of cyclic di-GMP in generating the observed morphological phenotypes that are known to contribute to biofilm-mediated antibiotic tolerance. The generation of phenotypic diversity may go unnoticed during standard diagnostic evaluation, which potentially impacts the therapeutic strategy chosen to treat the corresponding infection and may contribute to the spread of antibiotic resistance.

Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Cats; Ciprofloxacin; Cyclic GMP; DNA Gyrase; Dogs; Drug Resistance, Bacterial; Escherichia coli Proteins; Gene Expression; Genome, Bacterial; Gentamicins; Horses; Microbial Sensitivity Tests; Mutation; Phenotype; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa

Vibrio cholerae of serogroups O1 and O139, the causative agent of Asiatic cholera, continues to be a major global health threat. This pathogen utilizes substratum-specific pili to attach to distinct surfaces in the aquatic environment and the human small intestine and detaches when conditions become unfavorable. Both attachment and detachment are critical to bacterial environmental survival, pathogenesis and disease transmission. However, the factors that promote detachment are less understood. In this study, we examine the role of flagellar motility and hemagglutinin/protease (HapA) in vibrio detachment from a non-degradable abiotic surface and from the suckling mouse intestine. Flagellar motility facilitated V. cholerae detachment from abiotic surfaces. HapA had no effect on the stability of biofilms formed on abiotic surfaces despite representing >50% of the proteolytic activity present in the extracellular matrix. We developed a balanced lethal plasmid system to increase the bacterial cyclic diguanylate (c-di-GMP) pool late in infection, a condition that represses motility and HapA expression. Increasing the c-di-GMP pool enhanced V. cholerae colonization of the suckling mouse intestine. The c-di-GMP effect was fully abolished in hapA isogenic mutants. These results suggest that motility facilitates detachment in a substratum-independent manner. Instead, HapA appears to function as a substratum-specific detachment factor.

Topics: Animals; Bacterial Adhesion; Biofilms; Cholera; Cyclic GMP; Fimbriae, Bacterial; Flagella; Gene Expression Regulation, Bacterial; Intestinal Mucosa; Intestine, Small; Metalloendopeptidases; Mice; Movement; Polystyrenes; Vibrio cholerae

The polyamine norspermidine is one of the major polyamines synthesized by Vibrionales and has also been found in various aquatic organisms. Norspermidine is among the environmental signals that positively regulate Vibrio cholerae biofilm formation. The NspS/MbaA signaling complex detects extracellular norspermidine and mediates the response to this polyamine. Norspermidine binding to the NspS periplasmic binding protein is thought to inhibit the phosphodiesterase activity of MbaA, increasing levels of the biofilm-promoting second messenger cyclic diguanylate monophosphate, thus enhancing biofilm formation. V. cholerae can also synthesize norspermidine using the enzyme NspC as well as import it from the environment. Deletion of the nspC gene was shown to reduce accumulation of bacteria in biofilms, leading to the conclusion that intracellular norspermidine is also a positive regulator of biofilm formation. Because V. cholerae uses norspermidine to synthesize the siderophore vibriobactin it is possible that intracellular norspermidine is required to obtain sufficient amounts of iron, which is also necessary for robust biofilm formation. The objective of this study was to assess the relative contributions of intracellular and extracellular norspermidine to the regulation of biofilm formation in V. cholerae. We show the biofilm defect of norspermidine synthesis mutants does not result from an inability to produce vibriobactin as vibriobactin synthesis mutants do not have diminished biofilm forming abilities. Furthermore, our work shows that extracellular, but not intracellular norspermidine, is mainly responsible for promoting biofilm formation. We establish that the NspS/MbaA signaling complex is the dominant mediator of biofilm formation in response to extracellular norspermidine, rather than norspermidine synthesized by NspC or imported into the cell.

Topics: Biofilms; Catechols; Cyclic GMP; Iron; Oxazoles; Periplasmic Binding Proteins; Phosphoric Diester Hydrolases; Signal Transduction; Spermidine; Vibrio cholerae

The cyclic dinucleotides (CDNs) c-di-GMP, c-di-AMP, and c-AMP-GMP are widely utilized as second messengers in bacteria, where they signal lifestyle changes such as motility and biofilm formation, cell wall and membrane homeostasis, virulence, and exo-electrogenesis. For all known bacterial CDNs, specific riboswitches have been identified that alter gene expression in response to the second messengers. In addition, bacterial CDNs trigger potent immune responses, making them attractive as adjuvants in immune therapies. Besides the three naturally occurring CDNs, seven further CDNs containing canonical 3'-5'-linkages are possible by combining the four natural ribonucleotides. Herein, we have synthesized all ten possible combinations of 3'-5'-linked CDNs. The binding affinity of novel CDNs and GEMM riboswitch variants was assessed utilizing a spinach aptamer fluorescence assay and in-line probing assays. The immune-stimulatory effect of CDNs was evaluated by induction of type I interferons (IFNs), and a novel CDN c-AMP-CMP was identified as a new immune-stimulatory agent.

Topics: Cyclic GMP; Dinucleoside Phosphates; Geobacter; Molecular Conformation

Phytochromes and cyanobacteriochromes (CBCRs) use double-bond photoisomerization of their linear tetrapyrrole (bilin) chromophores within cGMP-specific phosphodiesterases/adenylyl cyclases/FhlA (GAF) domain-containing photosensory modules to regulate activity of C-terminal output domains. CBCRs exhibit photocycles that are much more diverse than those of phytochromes and are often found in large modular proteins such as Tlr0924 (SesA), one of three blue light regulators of cell aggregation in the cyanobacterium Thermosynechococcus elongatus. Tlr0924 contains a single bilin-binding GAF domain adjacent to a C-terminal diguanylate cyclase (GGDEF) domain whose catalytic activity requires formation of a dimeric transition state presumably supported by a multidomain extension at its N-terminus. To probe the structural basis of light-mediated signal propagation from the photosensory input domain to a signaling output domain for a representative CBCR, these studies explore the properties of a bidomain GAF-GGDEF construct of Tlr0924 (Tlr0924Δ) that retains light-regulated diguanylate cyclase activity. Surprisingly, circular dichroism spectroscopy and size exclusion chromatography data do not support formation of stable dimers in either the blue-absorbing

Topics: Adenylyl Cyclases; Bacterial Proteins; Bile Pigments; Cyanobacteria; Cyclic GMP; Escherichia coli Proteins; Light; Phosphorus-Oxygen Lyases; Protein Domains; Protein Multimerization

When bacteria encounter surfaces, they respond with surface colonization and virulence induction. The mechanisms of bacterial mechanosensation and downstream signaling remain poorly understood. Here, we describe a tactile sensing cascade in

Topics: Adhesins, Bacterial; Caulobacter crescentus; Cyclic GMP; Escherichia coli Proteins; Fimbriae, Bacterial; Flagella; Glycosyltransferases; Mechanotransduction, Cellular; Phosphorus-Oxygen Lyases; Polysaccharides, Bacterial; Rotation; Second Messenger Systems; Surface Properties

Cyclic-di-GMP (c-di-GMP) is an intracellular secondary messenger which controls the biofilm life cycle in many bacterial species. High intracellular c-di-GMP content enhances biofilm formation via the reduction of motility and production of biofilm matrix, while low c-di-GMP content in biofilm cells leads to increased motility and biofilm dispersal. While the effect of high c-di-GMP levels on bacterial lifestyles is well studied, the physiology of cells at low c-di-GMP levels remains unclear. Here, we showed that

Topics: Animals; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Glycolipids; Mice; Operon; Pseudomonas aeruginosa; Pyocyanine; Quorum Sensing; RAW 264.7 Cells; Transcription Factors; Transcriptome; Virulence

The flagellar motor is a sophisticated rotary machine facilitating locomotion and signal transduction. Owing to its important role in bacterial behavior, its assembly and activity are tightly regulated. For example, chemotaxis relies on a sensory pathway coupling chemical information to rotational bias of the motor through phosphorylation of the motor switch protein CheY. Using a chemical proteomics approach, we identified a novel family of CheY-like (Cle) proteins in

Topics: Bacterial Adhesion; Bacterial Proteins; Caulobacter crescentus; Chemotaxis; Cyclic GMP; Flagella; Gene Expression Regulation, Bacterial; Protein Binding; Proteome

Quorum sensing (QS) signals are used by bacteria to regulate biological functions in response to cell population densities. Cyclic diguanosine monophosphate (c-di-GMP) regulates cell functions in response to diverse environmental chemical and physical signals that bacteria perceive. In

Topics: Animals; Bacterial Load; Bacterial Proteins; Biofilms; Burkholderia cenocepacia; Burkholderia Infections; Cyclic GMP; Fatty Acids, Monounsaturated; Gene Expression Regulation, Bacterial; Mice; Mutation; Phenotype; Quorum Sensing; Signal Transduction; Virulence

The attachment strength of biofilm microbes is responsible for the adherence of the cells to surfaces and thus is a critical parameter in biofilm processes. In tubular microreactors, aqueous-air segmented flow ensures an optimal oxygen supply and prevents excessive biofilm growth. However, organisms growing in these systems depend on an adaptation phase of several days, before mature and strong biofilms can develop. This is due to strong interfacial forces. In this study, a hyperadherent mutant of Pseudomonas taiwanensis VLB120ΔCeGFP possessing an engineered cyclic diguanylate metabolism, was applied to a continuous biofilm process for the production of (S)-styrene oxide. Cells of the mutant P.  taiwanensis VLB120ΔCeGFP Δ04710, showing the same specific activity as the wild type, adhered substantially stronger to the substratum. Adaptation to the high interfacial forces was not necessary in these cases. Thereby, 40% higher final product concentrations were achieved and the maximal volumetric productivity of the parent strain was significantly surpassed by P. taiwanensis VLB120ΔCeGFP Δ04710. Applying mutants with strong adhesion in biofilm-based catalysis opens the door to biological process control in future applications of catalytic biofilms using other industrially relevant strains.

Topics: Bacterial Adhesion; Bioreactors; Cyclic GMP; Epoxy Compounds; Metabolic Engineering; Metabolic Networks and Pathways; Pseudomonas

The intracellular signal molecule cyclic di-GMP (c-di-GMP) is an important element in regulation of biofilm formation by bacteria. In Pseudomonas aeruginosa, FleQ functions as a c-di-GMP-dependent transcriptional regulator of expression of flagellar genes and the exopolysaccharide (EPS) Pel, a component of the biofilm extracellular matrix. In the plant-beneficial bacterium Pseudomonas putida KT2440, a mutation in fleQ reduces biofilm formation and colonization of plant surfaces. Using isothermal titration calorimetry and electrophoretic mobility shift assays, we show in this work that FleQ of P. putida interacts with c-di-GMP and directly binds the promoter regions of flagellar and EPS genes. Data obtained by analytical gel filtration and ultracentrifugation indicate that FleQ is in multiple oligomeric states in solution (dimers, tetramers and hexamers), which do not show altered equilibrium in the presence of c-di-GMP. DNA binding is independent of c-diGMP, although it is favored by the second messenger in the case of the promoter of the operon responsible for synthesis of the species-specific EPS Pea. Analysis of expression using transcriptional fusions showed an influence of FleQ upon two of the four EPS operons under regular growth conditions. Finally, a consensus sequence for promoter recognition by FleQ in P. putida is also proposed.

Topics: Adenosine Triphosphatases; Artificial Gene Fusion; Bacterial Proteins; Binding Sites; Biofilms; Calorimetry; Chromatography, Gel; Consensus Sequence; Cyclic GMP; DNA-Binding Proteins; DNA, Bacterial; Electrophoretic Mobility Shift Assay; Flagella; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Polysaccharides, Bacterial; Promoter Regions, Genetic; Protein Binding; Protein Multimerization; Pseudomonas putida; Ultracentrifugation

Flagella-mediated motility is an important capability of many bacteria to survive in nutrient-depleted and harsh environments. Decreasing the intracellular cyclic di-GMP (c-di-GMP) level by overexpression of phosphodiesterase BifA promotes flagellar-mediated motility and induces planktonic lifestyle in Pseudomonas. The mechanism that regulates expression of bifA gene was poorly studied. Here we showed that expression of BifA was partly controlled by flagellar sigma factor FliA (σ

Topics: Bacterial Proteins; Cyclic GMP; Flagella; Gene Expression Regulation, Bacterial; Phosphoric Diester Hydrolases; Promoter Regions, Genetic; Pseudomonas putida; Sigma Factor; Transcription Initiation Site

Azotobacter vinelandii is a soil bacterium that produces the polysaccharide alginate. In this work, we identified a miniTn5 mutant, named GG9, which showed increased alginate production of higher molecular mass, and increased expression of the alginate biosynthetic genes algD and alg8 when compared to its parental strain. The miniTn5 was inserted within ORF Avin07920 encoding a hypothetical protein. Avin07910, located immediately downstream and predicted to form an operon with Avin07920, encodes an inner membrane multi-domain signaling protein here named mucG. Insertional inactivation of mucG resulted in a phenotype of increased alginate production of higher molecular mass similar to that of mutant GG9. The MucG protein contains a periplasmic and putative HAMP and PAS domains, which are linked to GGDEF and EAL domains. The last two domains are potentially involved in the synthesis and degradation, respectively, of bis-(3'-5')-cyclic dimeric GMP (c-di-GMP), a secondary messenger that has been reported to be essential for alginate production. Therefore, we hypothesized that the negative effect of MucG on the production of this polymer could be explained by the putative phosphodiesterase activity of the EAL domain. Indeed, we found that alanine replacement mutagenesis of the MucG EAL motif or deletion of the entire EAL domain resulted in increased alginate production of higher molecular mass similar to the GG9 and mucG mutants. To our knowledge, this is the first reported protein that simultaneous affects the production of alginate and its molecular mass.

Topics: Alginates; Azotobacter vinelandii; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Glucuronic Acid; Hexuronic Acids; Operon

The structure of bacterial biofilms is predominantly established through the secretion of extracellular polymeric substances (EPS). They show that Bacillus subtilis contains an operon (ydaJ-N) whose induction leads to increased Congo Red staining of biofilms and strongly altered biofilm architecture, suggesting that it mediates the production of an unknown exopolysaccharide. Supporting this idea, overproduction of YdaJKLMN leads to cell clumping during exponential growth in liquid culture, and also causes colony morphology alterations in wild type cells, as well as in a mutant background lacking the major exopolysaccharide of B. subtilis. The first gene product of the operon, YdaJ, appears to modify the overproduction effects, but is not essential for cell clumping or altered colony morphology, while the presence of the c-di-GMP receptor YdaK is required, suggesting an involvement of second messenger c-di-GMP. YdaM, YdaN and YdaK colocalize to clusters predominantly at the cell poles and are statically positioned at this subcellular site, similar to other exopolysaccharide machinery components in other bacteria. Their analysis reveals that B. subtilis contains a static subcellular assembly of an EPS machinery that affects cell aggregation and biofilm formation.

Topics: Bacillus subtilis; Bacterial Adhesion; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Polyenes; Polysaccharides, Bacterial

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a dynamic intracellular signaling molecule that plays a central role in the biofilm life cycle. Current methodologies for the quantification of c-di-GMP are typically based on chemical extraction, representing end point measurements. Chemical methodologies also fail to take into consideration the physiological heterogeneity of the biofilm and thus represent an average c-di-GMP concentration across the entire biofilm. To address these problems, a ratiometric, image-based quantification method has been developed based on expression of the green fluorescence protein (GFP) under the control of the c-di-GMP-responsive cdrA promoter (Rybtke, M. T., Borlee, B. R., Murakami, K., Irie, Y., Hentzer, M., Nielsen, T. E., Givskov, M., Parsek, M. R., and Tolker-Nielsen, T. (2012) Appl. Environ. Microbiol. 78, 5060-5069). The methodology uses the cyan fluorescent protein (CFP) as a biomass indicator and the GFP as a c-di-GMP reporter. Thus, the CFP/GFP ratio gives the effective c-di-GMP per biomass. A binary mask was applied to alleviate background fluorescence, and fluorescence was calibrated against known c-di-GMP concentrations. Using flow cells for biofilm formation, c-di-GMP showed a non-uniform distribution across the biofilm, with concentrated hot spots of c-di-GMP. Additionally, c-di-GMP was found to be localized at the outer boundary of mature colonies in contrast to a uniform distribution in early stage, small colonies. These data demonstrate the application of a method for the in situ, real time quantification of c-di-GMP and show that the amount of this biofilm-regulating second messenger was dynamic with time and colony size, reflecting the extent of biofilm heterogeneity in real time.

Topics: Biofilms; Cyclic GMP; Green Fluorescent Proteins; Pseudomonas aeruginosa

Manipulation of biofilm formation in Shewanella is beneficial for application to industrial and environmental biotechnology. BpfA is an adhesin largely responsible for biofilm formation in many Shewanella species. However, the mechanism underlying BpfA production and the resulting biofilm remains vaguely understood. We previously described the finding that BpfA expression is enhanced by DosD, an oxygen-stimulated diguanylate cyclase, under aerobic growth. In the present work, we identify FlrA as a critical transcription regulator of the bpfA operon in Shewanella putrefaciens CN32 by transposon mutagenesis. FlrA acted as a repressor of the operon promoter by binding to two boxes overlapping the -10 and -35 sites recognized by σ. Motility and biofilm are mutually exclusive lifestyles, shifts between which are under the strict regulation of bacteria attempting to adapt to the fluctuation of diverse environmental conditions. The FlrA protein in many bacteria is known to control motility as a master regulator of flagellum synthesis. This work elucidates its effect on biofilm formation by controlling the expression of the adhesin BpfA in S. putrefaciens CN32 in response to c-di-GMP. Therefore, FlrA plays a dual role in controlling motility and biofilm formation in S. putrefaciens CN32. The cooccurrence of flrA, bpfA, and the FlrA box in the promoter region of the bpfA operon in diverse Shewanella strains suggests that bpfA is a common mechanism that controls biofilm formation in this bacterial species.

Topics: Adhesins, Bacterial; Bacterial Proteins; Base Sequence; Biofilms; Cyclic GMP; Flagella; Gene Expression Regulation, Bacterial; Promoter Regions, Genetic; Repressor Proteins; Shewanella putrefaciens; Transcription, Genetic

Coordination of cell movement with cell differentiation is a major feat of embryonic development. The Dictyostelium stalk always forms at the organizing tip, by a mechanism that is not understood. We previously reported that cyclic diguanylate (c-di-GMP), synthesized by diguanylate cyclase A (DgcA), induces stalk formation. Here we used transcriptional profiling of dgca- structures to identify target genes for c-di-GMP, and used these genes to investigate the c-di-GMP signal transduction pathway. We found that knockdown of cAMP-dependent protein kinase (PKA) activity in prestalk cells reduced stalk gene induction by c-di-GMP, whereas PKA activation bypassed the c-di-GMP requirement for stalk gene expression. c-di-GMP caused a persistent increase in cAMP, which still occurred in mutants lacking the adenylate cyclases ACG or ACR, or the cAMP phosphodiesterase RegA. However, both inhibition of adenylate cyclase A (ACA) with SQ22536 and incubation of a temperature-sensitive ACA mutant at the restrictive temperature prevented c-di-GMP-induced cAMP synthesis as well as c-di-GMP-induced stalk gene transcription. ACA produces the cAMP pulses that coordinate Dictyostelium morphogenetic cell movement and is highly expressed at the organizing tip. The stalk-less dgca- mutant regained its stalk by expression of a light-activated adenylate cyclase from the ACA promoter and exposure to light, indicating that cAMP is also the intermediate for c-di-GMP in vivo. Our data show that the more widely expressed DgcA activates tip-expressed ACA, which then acts on PKA to induce stalk genes. These results explain why stalk formation in Dictyostelia always initiates at the site of the morphogenetic organizer.

Topics: Adenylyl Cyclases; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Dictyostelium; Escherichia coli Proteins; Gene Expression Regulation, Developmental; Genes, Protozoan; Mutation; Phosphorus-Oxygen Lyases; Protozoan Proteins; Signal Transduction; Spores, Protozoan

The objective of this study was to investigate the influence of extracellular polymeric substance (EPS) on the coupling effects between ammonia-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing (anammox) bacteria for the completely autotrophic nitrogen removal over nitrite (CANON) biofilm formation in a moving bed biofilm reactor (MBBR). Analysis of the quantity of EPS and cyclic diguanylate (c-di-GMP) confirmed that the contents of polysaccharides and c-di-GMP were correlated in the AOB sludge, anammox sludge, and CANON biofilm. The anammox sludge secreted more EPS (especially polysaccharides) than AOB with a markedly higher c-di-GMP content, which could be used by the bacteria to regulate the synthesis of exopolysaccharides that are ultimately used as a fixation matrix, for the adhesion of biomass. Indeed, increased intracellular c-di-GMP concentrations in the anammox sludge enhanced the regulation of polysaccharides to promote the adhesion of AOB and formation of the CANON biofilm. Overall, the results of this study provide new comprehensive information regarding the coupling effects of AOB and anammox bacteria for the nitrogen removal process.

Topics: Ammonia; Ammonium Compounds; Anaerobiosis; Bacteria; Biofilms; Bioreactors; Cyclic GMP; Denitrification; Gene Expression Regulation, Bacterial; Nitrites; Nitrogen; Oxidation-Reduction; Polysaccharides, Bacterial; Second Messenger Systems; Sewage

Topics: Antifungal Agents; Bacterial Proteins; Cyclic GMP; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Gene Library; Lysobacter; Multigene Family; Mutation; Signal Transduction; Soil Microbiology

A novel, biofilm-based AND logic gate was constructed in Shewanella oneidensis through a near-infrared (NIR) light responsive c-di-GMP module. The logic gate was demonstrated in microbial fuel cells with isopropyl β-d-thiogalactoside (IPTG) and NIR light as the inputs and electrical signals as the output.

Topics: Biofilms; Cyclic GMP; Infrared Rays; Logic; Shewanella

Although bacterial cellulose synthase (bcs) operons are widespread within the Proteobacteria phylum, subunits required for the partial-acetylation of the polymer appear to be restricted to a few γ-group soil, plant-associated and phytopathogenic pseudomonads, including Pseudomonas fluorescens SBW25 and several Pseudomonas syringae pathovars. However, a bcs operon with acetylation subunits has also been annotated in the unrelated β-group respiratory pathogen, Bordetella avium 197N. Our comparison of subunit protein sequences and GC content analyses confirms the close similarity between the B. avium 197N and pseudomonad operons and suggests that, in both cases, the cellulose synthase and acetylation subunits were acquired as a single unit. Using static liquid microcosms, we can confirm that B. avium 197N expresses low levels of cellulose in air-liquid interface biofilms and that biofilm strength and attachment levels could be increased by elevating c-di-GMP levels like the pseudomonads, but cellulose was not required for biofilm formation itself. The finding that B. avium 197N is capable of producing cellulose from a highly-conserved, but relatively uncommon bcs operon raises the question of what functional role this modified polymer plays during the infection of the upper respiratory tract or survival between hosts, and what environmental signals control its production.

Topics: Animals; Bacterial Adhesion; Biofilms; Bird Diseases; Birds; Bordetella avium; Bordetella Infections; Cellulose; Cyclic GMP; Gene Expression Regulation, Bacterial; Glucosyltransferases; Humans; Operon; Opportunistic Infections; Pseudomonas fluorescens; Respiratory Tract Infections

The secondary messenger cyclic di-GMP promotes biofilm formation by up regulating the expression of csgD, encoding the major regulator of rdar biofilm formation in Salmonella typhimurium. The GGDEF/EAL domain proteins regulate the c-di-GMP turnover. There are twenty- two GGDEF/EAL domain proteins in the genome of S. typhimurium. In this study, we dissect the role of individual GGDEF/EAL proteins for csgD expression and rdar biofilm development.. Among twelve GGDEF domains, two proteins upregulate and among fifteen EAL domains, four proteins down regulate csgD expression. We identified two additional GGDEF proteins required to promote optimal csgD expression. With the exception of the EAL domain of STM1703, solely, diguanylate cyclase and phosphodiesterase activities are required to regulate csgD mediated rdar biofilm formation. Identification of corresponding phosphodiesterases and diguanylate cyclases interacting in the csgD regulatory network indicates various levels of regulation by c-di-GMP. The phosphodiesterase STM1703 represses transcription of csgD via a distinct promoter upstream region.. The enzymatic activity and the protein scaffold of GGDEF/EAL domain proteins regulate csgD expression. Thereby, c-di-GMP adjusts csgD expression at multiple levels presumably using a multitude of input signals.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Deletion; Gene Expression Regulation, Bacterial; Mutation; Phenotype; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Domains; Salmonella typhimurium; Signal Transduction; Up-Regulation

For

Topics: Bacterial Secretion Systems; Cyclic GMP; Escherichia coli Proteins; Fimbriae, Bacterial; Gene Deletion; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Movement; Oxygen; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Polysaccharides, Bacterial; Pseudomonas aeruginosa

The bacterial second messenger cyclic di-3',5'-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825

Topics: Bacterial Proteins; Biocatalysis; Catalytic Domain; Cations, Divalent; Cloning, Molecular; Crystallography, X-Ray; Cyclic GMP; Escherichia coli; Gene Expression; Genetic Vectors; Hydrolysis; Kinetics; Magnesium; Manganese; Models, Molecular; Phosphoric Diester Hydrolases; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Multimerization; Pseudomonas aeruginosa; Recombinant Proteins; Substrate Specificity

The exopolysaccharide alginate, produced by the opportunistic human pathogen

Topics: Alginates; Bacterial Proteins; Catalytic Domain; Cyclic GMP; Cytosol; DNA Mutational Analysis; Enzyme Activators; Glucuronic Acid; Hexuronic Acids; Membrane Proteins; Models, Molecular; Mutagenesis, Site-Directed; Protein Binding; Protein Conformation; Protein Multimerization; Pseudomonas aeruginosa

The second messenger cyclic di-guanylate (c-di-GMP) plays an important role in controlling the switch between planktonic and biofilm lifestyles. The synthesis of c-di-GMP is catalyzed by di-guanylate cyclases (DGCs) and the enzymes are characterized by the presence of a conserved GGDEF domain. In the sequenced staphylococcal genomes, gdpS is the only gene encoding a GGDEF domain-containing protein. Previous studies have shown that gdpS contributes to staphylococcal biofilm formation, but its effect remains under debate. In the present study, we deleted gdpS in Staphylococcus epidermidis strain RP62A. Disruption of gdpS in this strain impaired biofilm formation under both static and dynamic flow conditions, suggesting that gdpS act as a positive regulator of biofilm development in this high-biofilm-forming isolate. The predicted translational start site of gdpS in S. epidermidis differs between the Refseq database and the Genbank database. By using site-directed mutagenesis and Western blot analysis, we determined GdpS is translated from the start codon annotated in the Refseq database. In addition, mutation in the GGDEF domain did not affect the ability of gdpS to complement the biofilm defect of the gdpS mutant. Heterologous di-guanylate cyclases expressed in trans failed to complement the gdpS mutant. These results confirmed that gdpS modulates staphylococcal biofilm independently of c-di-GMP signaling pathway. Furthermore, mutations of the start codon did not abolish the capacity of gdpS to enhance biofilm formation. Taken together, these findings indicated that the S. epidermidis gdpS regulates biofilm formation independently of its protein-coding function.

Topics: Bacterial Proteins; Base Sequence; Biofilms; Blotting, Western; Cyclic GMP; DNA, Bacterial; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Mutagenesis, Site-Directed; Staphylococcus epidermidis; Transcription Factors; Transcription, Genetic

The opportunistic pathogen Pseudomonas aeruginosa forms antimicrobial resistant biofilms through sequential steps requiring several two-component regulatory systems. The sensor-regulator hybrid SagS plays a central role in biofilm development by enabling the switch from the planktonic to the biofilm mode of growth, and by facilitating the transition of biofilm cells to a highly tolerant state. However, the mechanism by which SagS accomplishes both functions is unknown. SagS harbours a periplasmic sensory HmsP, and phosphorelay HisKA and Rec domains. SagS domain was used as constructs and site-directed mutagenesis to elucidate how SagS performs its dual functions. It was demonstrated that HisKA-Rec and the phospho-signalling between SagS and BfiS contribute to the switch to the biofilm mode of growth, but not to the tolerant state. Instead, expression of SagS domain constructs harbouring HmsP rendered ΔsagS biofilm cells as recalcitrant to antimicrobial agents as wild-type biofilms, likely by restoring BrlR production and cellular c-di-GMP levels to wild-type levels. Restoration of biofilm tolerance by HmsP was independent of biofilm biomass accumulation, RsmA, RsmYZ, HptB and BfiSR-downstream targets. Our findings thus suggest that SagS likely makes use of a "divide-and-conquer" mechanism to regulate its dual switch function, by activating two distinct regulatory networks via its individual domains.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Cyclic GMP; Drug Resistance, Multiple, Bacterial; Gene Expression Regulation, Bacterial; Histidine Kinase; Mutagenesis, Site-Directed; Protein Domains; Pseudomonas aeruginosa; Signal Transduction

Pseudomonas aeruginosa is a Gram-negative bacterial pathogen associated with acute and chronic infections. The universal cyclic-di-GMP second messenger is instrumental in the switch from a motile lifestyle to resilient biofilm as in the cystic fibrosis lung. The SadC diguanylate cyclase is associated with this patho-adaptive transition. Here, we identify an unrecognized SadC partner, WarA, which we show is a methyltransferase in complex with a putative kinase, WarB. We established that WarA binds to cyclic-di-GMP, which potentiates its methyltransferase activity. Together, WarA and WarB have structural similarities with the bifunctional Escherichia coli lipopolysaccharide (LPS) O antigen regulator WbdD. Strikingly, WarA influences P. aeruginosa O antigen modal distribution and interacts with the LPS biogenesis machinery. LPS is known to modulate the immune response in the host, and by using a zebrafish infection model, we implicate WarA in the ability of P. aeruginosa to evade detection by the host.

Topics: Animals; Cyclic GMP; Disease Models, Animal; Immune Evasion; Lipopolysaccharides; Methyltransferases; Protein Binding; Pseudomonas aeruginosa; Pseudomonas Infections; Zebrafish

Riboswitches are RNAs that form complex, folded structures that selectively bind small molecules or ions. As with certain groups of protein enzymes and receptors, some riboswitch classes have evolved to change their ligand specificity. We developed a procedure to systematically analyze known riboswitch classes to find additional variants that have altered their ligand specificity. This approach uses multiple-sequence alignments, atomic-resolution structural information, and riboswitch gene associations. Among the discoveries are unique variants of the guanine riboswitch class that most tightly bind the nucleoside 2'-deoxyguanosine. In addition, we identified variants of the glycine riboswitch class that no longer recognize this amino acid, additional members of a rare flavin mononucleotide (FMN) variant class, and also variants of c-di-GMP-I and -II riboswitches that might recognize different bacterial signaling molecules. These findings further reveal the diverse molecular sensing capabilities of RNA, which highlights the potential for discovering a large number of additional natural riboswitch classes.

Topics: Base Sequence; Binding Sites; Computational Biology; Cyclic GMP; Deoxyguanosine; Glycine; Guanine; Ligands; Models, Molecular; Nucleic Acid Conformation; Riboswitch; RNA

In order to resist harmful environmental conditions, many bacteria form multicellular aggregates called biofilms. In these biofilms, they protect themselves in a self-produced matrix consisting of extracellular polysaccharides, proteins and DNA. In many bacteria, biofilm formation is stimulated in the presence of the second messenger cyclic di-GMP. In this issue of Environmental Microbiology Reports, Bedrunka and Graumann have studied matrix production by the proteins encoded in the Bacillus subtilis ydaJKLMN operon. For the first time, they were able to provide a link between c-di-GMP signalling and matrix production in this bacterium. The work demonstrates that the c-di-GMP receptor protein YdaK forms a membrane-bound complex with the YdaM and YdaN proteins, and that this interaction with YdaK is required for polysaccharide production by YdaL, YdaM and YdaN.

Topics: Bacillus subtilis; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Polysaccharides, Bacterial

We previously identified a second-messenger-regulated signaling system in the environmental bacterium Pseudomonas fluorescens which controls biofilm formation in response to levels of environmental inorganic phosphate. This system contains the transmembrane cyclic di-GMP (c-di-GMP) receptor LapD and the periplasmic protease LapG. LapD regulates LapG and controls the ability of this protease to process a large cell surface adhesin protein, LapA. While LapDG orthologs can be identified in diverse bacteria, predictions of LapG substrates are sparse. Notably, the opportunistic pathogen Pseudomonas aeruginosa harbors LapDG orthologs, but neither the substrate of LapG nor any associated secretion machinery has been identified to date. Here, we identified P. aeruginosa CdrA, a protein known to mediate cell-cell aggregation and biofilm maturation, as a substrate of LapG. We also demonstrated LapDG to be a minimal system sufficient to control CdrA localization in response to changes in the intracellular concentration of c-di-GMP. Our work establishes this biofilm signaling node as a regulator of a type Vb secretion system substrate in a clinically important pathogen.. Here, the biological relevance of a conserved yet orphan signaling system in the opportunistic pathogen Pseudomonas aeruginosa is revealed. In particular, we identified the adhesin CdrA, the cargo of a two-partner secretion system, as a substrate of a periplasmic protease whose activity is controlled by intracellular c-di-GMP levels and a corresponding transmembrane receptor via an inside-out signaling mechanism. The data indicate a posttranslational control mechanism of CdrA via c-di-GMP, in addition to its established transcriptional regulation via the same second messenger.

Topics: Amino Acid Sequence; Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Models, Molecular; Molecular Sequence Data; Periplasm; Phylogeny; Protein Conformation; Pseudomonas aeruginosa; Type V Secretion Systems

The nucleotide-based second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is involved in regulating a plethora of processes in bacteria that are typically associated with lifestyle changes. Myxococcus xanthus undergoes major lifestyle changes in response to nutrient availability, with the formation of spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. Here, we investigated the function of c-di-GMP in M. xanthus and show that this bacterium synthesizes c-di-GMP during growth. Manipulation of the c-di-GMP level by expression of either an active, heterologous diguanylate cyclase or an active, heterologous phosphodiesterase correlated with defects in type IV pilus (T4P)-dependent motility, whereas gliding motility was unaffected. An increased level of c-di-GMP correlated with reduced transcription of the pilA gene (which encodes the major pilin of T4P), reduced the assembly of T4P, and altered cell agglutination, whereas a decreased c-di-GMP level correlated with altered cell agglutination. The systematic inactivation of the 24 genes in M. xanthus encoding proteins containing GGDEF, EAL, or HD-GYP domains, which are associated with c-di-GMP synthesis, degradation, or binding, identified three genes encoding proteins important for T4P-dependent motility, whereas all mutants had normal gliding motility. Purified DmxA had diguanylate cyclase activity, whereas the hybrid histidine protein kinases TmoK and SgmT, each of which contains a GGDEF domain, did not have diguanylate cyclase activity. These results demonstrate that c-di-GMP is important for T4P-dependent motility in M. xanthus.. We provide the first direct evidence that M. xanthus synthesizes c-di-GMP and demonstrate that c-di-GMP is important for T4P-dependent motility, whereas we did not obtain evidence that c-di-GMP regulates gliding motility. The data presented uncovered a novel mechanism for regulation of T4P-dependent motility, in which increased levels of c-di-GMP inhibit transcription of the pilA gene (which encodes the major pilin of T4P), ultimately resulting in the reduced assembly of T4P. Moreover, we identified an enzymatically active diguanylate cyclase that is important for T4P-dependent motility.

Topics: Bacterial Proteins; Bacterial Secretion Systems; Cyclic GMP; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Movement; Myxococcus xanthus; Protein Structure, Tertiary

In recent years, Escherichia coli has served as one of a few model bacterial species for studying cyclic di-GMP (c-di-GMP) signaling. The widely used E. coli K-12 laboratory strains possess 29 genes encoding proteins with GGDEF and/or EAL domains, which include 12 diguanylate cyclases (DGC), 13 c-di-GMP-specific phosphodiesterases (PDE), and 4 "degenerate" enzymatically inactive proteins. In addition, six new GGDEF and EAL (GGDEF/EAL) domain-encoding genes, which encode two DGCs and four PDEs, have recently been found in genomic analyses of commensal and pathogenic E. coli strains. As a group of researchers who have been studying the molecular mechanisms and the genomic basis of c-di-GMP signaling in E. coli, we now propose a general and systematic dgc and pde nomenclature for the enzymatically active GGDEF/EAL domain-encoding genes of this model species. This nomenclature is intuitive and easy to memorize, and it can also be applied to additional genes and proteins that might be discovered in various strains of E. coli in future studies.

Topics: Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Signal Transduction; Terminology as Topic

The second messenger cyclic dimeric (3'→5') GMP (cyclic di-GMP or c-di-GMP) has been implicated in the transition between motile and sessile lifestyles in bacteria. In this study, we demonstrate that biofilm formation, cellular aggregation or flocculation, and cellular buoyancy are under the control of c-di-GMP in Synechocystis sp. PCC 6803 (Synechocystis) and Fremyella diplosiphon. Synechocystis is a unicellular cyanobacterium and displays lower levels of c-di-GMP; F. diplosiphon is filamentous and displays higher intracellular c-di-GMP levels. We transformed Synechocystis and F. diplosiphon with a plasmid for constitutive expression of genes encoding diguanylate cylase (DGC) and phosphodiesterase (PDE) proteins from Vibrio cholerae or Escherichia coli, respectively. These engineered strains allowed us to modulate intracellular c-di-GMP levels. Biofilm formation and cellular deposition were induced in the DGC-expressing Synechocystis strain which exhibited high intracellular levels of c-di-GMP; whereas strains expressing PDE in Synechocystis and F. diplosiphon to drive low intracellular levels of c-di-GMP exhibited enhanced cellular buoyancy. In addition, the PDE-expressing F. diplosiphon strain showed elevated chlorophyll levels. These results imply roles for coordinating c-di-GMP homeostasis in regulating native cyanobacterial phenotypes. Engineering exogenous DGC or PDE proteins to regulate intracellular c-di-GMP levels represents an effective tool for uncovering cryptic phenotypes or modulating phenotypes in cyanobacteria for practical applications in biotechnology applicable in photobioreactors and in green biotechnologies, such as energy-efficient harvesting of cellular biomass or the treatment of metal-containing wastewaters.

Topics: Biofilms; Cyanobacteria; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Metabolic Engineering; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Vibrio cholerae

Bis-(3'-5') cyclic dimeric GMP (c-di-GMP) controls the lifestyle transition between the sessile and motile states in many Gram-negative bacteria, including the opportunistic human pathogen Pseudomonas aeruginosa. Under laboratory conditions, high concentrations of c-di-GMP decrease motility and promote biofilm formation, while low concentrations of c-di-GMP promote motility and decease biofilm formation. Here we sought to determine the contribution of c-di-GMP signaling to biofilm formation during P. aeruginosa-mediated catheter-associated urinary tract infection (CAUTI). Using a murine CAUTI model, a decrease in CFU was detected in the bladders and kidneys of mice infected with strains overexpressing the phosphodiesterases (PDEs) encoded by PA3947 and PA2133 compared to those infected with wild-type P. aeruginosa. Conversely, overexpression of the diguanylate cyclases (DGCs) encoded by PA3702 and PA1107 increased the number of bacteria in bladder and significantly increased dissemination of bacteria to the kidneys compared to wild-type infection. To determine which of the DGCs and PDEs contribute to c-di-GMP signaling during infection, a panel of PA14 in-frame deletion mutants lacking DGCs and PDEs were tested in the CAUTI model. Results from these infections revealed five mutants, three containing GGDEF domains (ΔPA14_26970, ΔPA14_72420, and ΔsiaD) and two containing dual GGDEF-EAL domains (ΔmorA and ΔPA14_07500), with decreased colonization of the bladder and dissemination to the kidneys. These results indicate that c-di-GMP signaling influences P. aeruginosa-mediated biofilms during CAUTI.. Biofilm-based infections are an important cause of nosocomial infections, since they resist the immune response and traditional antibiotic treatment. Cyclic di-GMP (c-di-GMP) is a second messenger that promotes biofilm formation in many Gram-negative pathogens, including Pseudomonas aeruginosa. Here we determined the contribution of c-di-GMP signaling to catheter-associated urinary tract infection (CAUTI), an animal model of biofilm-based infection. P. aeruginosa with elevated levels of c-di-GMP during the initial infection produces an increased bacterial burden in the bladder and kidneys. Conversely, low concentrations of c-di-GMP decreased the bacterial burden in the bladder and kidneys. We screened a library of mutants with mutations in genes regulating c-di-GMP signaling and found several mutants that altered colonization of the urinary tract. This study implicates c-di-GMP signaling during CAUTI.

Topics: Animals; Catheter-Related Infections; Cyclic GMP; Female; Gene Deletion; Gene Expression Regulation, Bacterial; Humans; Mice; Pseudomonas aeruginosa; Pseudomonas Infections; Signal Transduction

The plant innate immune system employs plasma membrane-localized receptors that specifically perceive pathogen/microbe-associated molecular patterns (PAMPs/MAMPs). This induces a defence response called pattern-triggered immunity (PTI) to fend off pathogen attack. Commensal bacteria are also exposed to potential immune recognition and must employ strategies to evade and/or suppress PTI to successfully colonize the plant. During plant infection, the flagellum has an ambiguous role, acting as both a virulence factor and also as a potent immunogen as a result of the recognition of its main building block, flagellin, by the plant pattern recognition receptors (PRRs), including FLAGELLIN SENSING2 (FLS2). Therefore, strict control of flagella synthesis is especially important for plant-associated bacteria. Here, we show that cyclic-di-GMP [bis-(3'-5')-cyclic di-guanosine monophosphate], a central regulator of bacterial lifestyle, is involved in the evasion of PTI. Elevated cyclic-di-GMP levels in the pathogen Pseudomonas syringae pv. tomato (Pto) DC3000, the opportunist P. aeruginosa PAO1 and the commensal P. protegens Pf-5 inhibit flagellin synthesis and help the bacteria to evade FLS2-mediated signalling in Nicotiana benthamiana and Arabidopsis thaliana. Despite this, high cellular cyclic-di-GMP concentrations were shown to drastically reduce the virulence of Pto DC3000 during plant infection. We propose that this is a result of reduced flagellar motility and/or additional pleiotropic effects of cyclic-di-GMP signalling on bacterial behaviour.

Topics: Arabidopsis; Cyclic GMP; Flagellin; Gene Expression Regulation, Bacterial; Immune Evasion; Nicotiana; Plant Diseases; Plant Immunity; Pseudomonas syringae; Reactive Oxygen Species; Virulence

The ubiquitous bacterial second messenger cyclic di-GMP (c-di-GMP) has recently become prominent as a trigger for biofilm formation in many bacteria. It is generated by diguanylate cyclases (DGCs; with GGDEF domains) and degraded by specific phosphodiesterases (PDEs; containing either EAL or HD-GYP domains). Most bacterial species contain multiples of these proteins with some having specific functions that are based on direct molecular interactions in addition to their enzymatic activities. Escherichia coli K-12 laboratory strains feature 29 genes encoding GGDEF and/or EAL domains, resulting in a set of 12 DGCs, 13 PDEs, and four enzymatically inactive "degenerate" proteins that act by direct macromolecular interactions. We present here a comparative analysis of GGDEF/EAL domain-encoding genes in 61 genomes of pathogenic, commensal, and probiotic E. coli strains (including enteric pathogens such as enteroaggregative, enterohemorrhagic, enteropathogenic, enterotoxigenic, and adherent and invasive Escherichia coli and the 2011 German outbreak O104:H4 strain, as well as extraintestinal pathogenic E. coli, such as uropathogenic and meningitis-associated E. coli). We describe additional genes for two membrane-associated DGCs (DgcX and DgcY) and four PDEs (the membrane-associated PdeT, as well as the EAL domain-only proteins PdeW, PdeX, and PdeY), thus showing the pangenome of E. coli to contain at least 35 GGDEF/EAL domain proteins. A core set of only eight proteins is absolutely conserved in all 61 strains: DgcC (YaiC), DgcI (YliF), PdeB (YlaB), PdeH (YhjH), PdeK (YhjK), PdeN (Rtn), and the degenerate proteins CsrD and CdgI (YeaI). In all other GGDEF/EAL domain genes, diverse point and frameshift mutations, as well as small or large deletions, were discovered in various strains.. Our analysis reveals interesting trends in pathogenic Escherichia coli that could reflect different host cell adherence mechanisms. These may either benefit from or be counteracted by the c-di-GMP-stimulated production of amyloid curli fibers and cellulose. Thus, EAEC, which adhere in a "stacked brick" biofilm mode, have a potential for high c-di-GMP accumulation due to DgcX, a strongly expressed additional DGC. In contrast, EHEC and UPEC, which use alternative adherence mechanisms, tend to have extra PDEs, suggesting that low cellular c-di-GMP levels are crucial for these strains under specific conditions. Overall, our study also indicates that GGDEF/EAL domain proteins evolve rapidly and thereby contribute to adaptation to host-specific and environmental niches of various types of E. coli.

Topics: Amino Acid Sequence; Conserved Sequence; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genome, Bacterial; Protein Structure, Tertiary; Signal Transduction

Over the course of the last 3 decades the role of the second messenger cyclic di-GMP (c-di-GMP) as a master regulator of bacterial physiology was determined. Although the control over c-di-GMP levels via synthesis and breakdown and the allosteric regulation of c-di-GMP over receptor proteins (effectors) and riboswitches have been extensively studied, relatively few effectors have been identified and most are of unknown functions. The obligate predatory bacterium Bdellovibrio bacteriovorus has a peculiar dimorphic life cycle, in which a phenotypic transition from a free-living attack phase (AP) to a sessile, intracellular predatory growth phase (GP) is tightly regulated by specific c-di-GMP diguanylate cyclases. B. bacteriovorus also bears one of the largest complement of defined effectors, almost none of known functions, suggesting that additional proteins may be involved in c-di-GMP signaling. In order to uncover novel c-di-GMP effectors, a c-di-GMP capture-compound mass-spectroscopy experiment was performed on wild-type AP and host-independent (HI) mutant cultures, the latter serving as a proxy for wild-type GP cells. Eighty-four proteins were identified as candidate c-di-GMP binders. Of these proteins, 65 did not include any recognized c-di-GMP binding site, and 3 carried known unorthodox binding sites. Putative functions could be assigned to 59 proteins. These proteins are included in metabolic pathways, regulatory circuits, cell transport, and motility, thereby creating a potentially large c-di-GMP network. False candidate effectors may include members of protein complexes, as well as proteins binding nucleotides or other cofactors that were, respectively, carried over or unspecifically interacted with the capture compound during the pulldown. Of the 84 candidates, 62 were found to specifically bind the c-di-GMP capture compound in AP or in HI cultures, suggesting c-di-GMP control over the whole-cell cycle of the bacterium. High affinity and specificity to c-di-GMP binding were confirmed using microscale thermophoresis with a hypothetical protein bearing a PilZ domain, an acyl coenzyme A dehydrogenase, and a two-component system response regulator, indicating that additional c-di-GMP binding candidates may be bona fide novel effectors.. In this study, 84 putative c-di-GMP binding proteins were identified in B. bacteriovorus, an obligate predatory bacterium whose lifestyle and reproduction are dependent on c-di-GMP signaling, using a c-di-GMP capture compound precipitation approach. This predicted complement covers metabolic, energy, transport, motility and regulatory pathways, and most of it is phase specific, i.e., 62 candidates bind the capture compound at defined modes of B. bacteriovorus lifestyle. Three of the putative binders further demonstrated specificity and high affinity to c-di-GMP via microscale thermophoresis, lending support for the presence of additional bona fide c-di-GMP effectors among the pulled-down protein repertoire.

Topics: Bacterial Proteins; Bdellovibrio; Cyclic GMP; Gene Expression Regulation, Bacterial; Gene Regulatory Networks; Protein Binding; Signal Transduction

High levels of the universal bacterial second messenger cyclic di-GMP (c-di-GMP) promote the establishment of surface-attached growth in many bacteria. Not only can c-di-GMP bind to nucleic acids and directly control gene expression, but it also binds to a diverse array of proteins of specialized functions and orchestrates their activity. Since its development in the early 1990s, the synthetic peptide array technique has become a powerful tool for high-throughput approaches and was successfully applied to investigate the binding specificity of protein-ligand interactions. In this study, we used peptide arrays to uncover the c-di-GMP binding site of a Pseudomonas aeruginosa protein (PA3740) that was isolated in a chemical proteomics approach. PA3740 was shown to bind c-di-GMP with a high affinity, and peptide arrays uncovered LKKALKKQTNLR to be a putative c-di-GMP binding motif. Most interestingly, different from the previously identified c-di-GMP binding motif of the PilZ domain (RXXXR) or the I site of diguanylate cyclases (RXXD), two leucine residues and a glutamine residue and not the charged amino acids provided the key residues of the binding sequence. Those three amino acids are highly conserved across PA3740 homologs, and their singular exchange to alanine reduced c-di-GMP binding within the full-length protein.. In many bacterial pathogens the universal bacterial second messenger c-di-GMP governs the switch from the planktonic, motile mode of growth to the sessile, biofilm mode of growth. Bacteria adapt their intracellular c-di-GMP levels to a variety of environmental challenges. Several classes of c-di-GMP binding proteins have been structurally characterized, and diverse c-di-GMP binding domains have been identified. Nevertheless, for several c-di-GMP receptors, the binding motif remains to be determined. Here we show that the use of a synthetic peptide array allowed the identification of a c-di-GMP binding motif of a putative c-di-GMP receptor protein in the opportunistic pathogen P. aeruginosa. The application of synthetic peptide arrays will facilitate the search for additional c-di-GMP receptor proteins and aid in the characterization of c-di-GMP binding motifs.

Topics: Amino Acid Motifs; Amino Acid Sequence; Consensus Sequence; Cyclic GMP; Molecular Sequence Data; Molecular Structure; Movement; Protein Array Analysis; Protein Binding; Protein Structure, Tertiary; Pseudomonas aeruginosa

Five mutants of Pseudomonas taiwanensis VLB120ΔCeGFP showed significant autoaggregation when growing on defined carbohydrates or gluconate, while they grew as suspended cells on complex medium and on organic acids like citrate and succinate. Surprisingly, the respective mutations affected very different genes, although all five strains exhibited the same behaviour of aggregate formation. To elucidate the mechanism of the aggregative behaviour, the microbial adhesion to hydrocarbons (MATH) assay and contact angle measurements were performed that pointed to an increased cell surface hydrophobicity. Moreover, investigations of the outer layer of the cell membrane revealed a reduced amount of O-specific polysaccharides in the lipopolysaccharide of the mutant cells. To determine the regulation of the aggregation, reverse transcription quantitative real-time PCR was performed and, irrespective of the mutation, the transcription of a gene encoding a putative phosphodiesterase, which is degrading the global second messenger cyclic diguanylate, was decreased or even deactivated in all mutants. In summary, it appears that the trophic autoaggregation was regulated via cyclic diguanylate and a link between the cellular cyclic diguanylate concentration and the lipopolysaccharide composition of P. taiwanensis VLB120ΔCeGFP is suggested.

Topics: Bacterial Adhesion; Carbohydrate Metabolism; Culture Media; Cyclic GMP; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Gluconates; Hydrophobic and Hydrophilic Interactions; Pseudomonas; Real-Time Polymerase Chain Reaction; Surface Properties

To develop new genetic tools for studying 3',5'-cyclic diguanylic acid (c-di-GMP) signalling in Pseudomonas aeruginosa.. Plasmid pPcdrA::lux, carrying a transcriptional fusion between the c-di-GMP responsive promoter PcdrA and the luxCDABE reporter genes, has been generated and validated in purpose-built P. aeruginosa strains in which c-di-GMP levels can be increased or reduced upon arabinose-dependent induction of c-di-GMP synthetizing or degrading enzymes.. The reporter systems described so far were able to detect a decrease in the c-di-GMP levels only in engineered strains overproducing c-di-GMP. Conversely, pPcdrA::lux could be used for studying any process or chemical compound expected to cause both an increase or a decrease with respect to the c-di-GMP levels produced by wild type P. aeruginosa. Another relevant aspect of this study has been the development of novel and improved genetic devices for the fine arabinose-dependent control of c-di-GMP levels in P. aeruginosa.. The genetic tools developed and validated in this study could facilitate investigations tackling the c-di-GMP signalling process on different fields, from cellular physiology to drug-discovery research.

Topics: Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Genes, Reporter; Genetic Techniques; Plasmids; Promoter Regions, Genetic; Pseudomonas aeruginosa; Signal Transduction

The importance of cyclic di-GMP (c-di-GMP) and its control of biofilm matrix assembly and production has been a focal point of researchers in recent history. In this issue, Cooley et al. (Cooley RB, Smith TJ, Leung W, Tierney V, Borlee BR, O'Toole GA, Sondermann H, J Bacteriol 198:66-77, http://dx.doi.org/10.1128/JB.00369-15) demonstrate that two c-di-GMP controlled features of Pseudomonas aeruginosa, the periplasmic protease LapG and the surface adhesin CdrA, are linked. CdrA is shown to be a substrate of LapG, with LapG activity controlled by intracellular c-di-GMP levels. This commentary discusses the significance of this finding.

Topics: Bacteria; Bacterial Adhesion; Bacterial Physiological Phenomena; Biofilms; Cyclic GMP; Pseudomonas aeruginosa

Intracellular levels of the bacterial second messenger cyclic di-GMP (c-di-GMP) are controlled by antagonistic activities of diguanylate cyclases and phosphodiesterases. The phosphodiesterase PdeH was identified as a key regulator of motility in Escherichia coli, while deletions of any of the other 12 genes encoding potential phosphodiesterases did not interfere with motility. To analyze the roles of E. coli phosphodiesterases, we demonstrated that most of these proteins are expressed under laboratory conditions. We next isolated suppressor mutations in six phosphodiesterase genes, which reinstate motility in the absence of PdeH by reducing cellular levels of c-di-GMP. Expression of all mutant alleles also led to a reduction of biofilm formation. Thus, all of these proteins are bona fide phosphodiesterases that are capable of interfering with different c-di-GMP-responsive output systems by affecting the global c-di-GMP pool. This argues that E. coli possesses several phosphodiesterases that are inactive under laboratory conditions because they lack appropriate input signals. Finally, one of these phosphodiesterases, PdeL, was studied in more detail. We demonstrated that this protein acts as a transcription factor to control its own expression. Motile suppressor alleles led to a strong increase of PdeL activity and elevated pdeL transcription, suggesting that enzymatic activity and transcriptional control are coupled. In agreement with this, we showed that overall cellular levels of c-di-GMP control pdeL transcription and that this control depends on PdeL itself. We thus propose that PdeL acts both as an enzyme and as a c-di-GMP sensor to couple transcriptional activity to the c-di-GMP status of the cell.. Most bacteria possess multiple diguanylate cyclases and phosphodiesterases. Genetic studies have proposed that these enzymes show signaling specificity by contributing to distinct cellular processes without much cross talk. Thus, spatial separation of individual c-di-GMP signaling units was postulated. However, since most cyclases and phosphodiesterases harbor N-terminal signal input domains, it is equally possible that most of these enzymes lack their activating signals under laboratory conditions, thereby simulating signaling specificity on a genetic level. We demonstrate that a subset of E. coli phosphodiesterases can be activated genetically to affect the global c-di-GMP pool and thus influence different c-di-GMP-dependent processes. Although this does not exclude spatial confinement of individual phosphodiesterases, this study emphasizes the importance of environmental signals for activation of phosphodiesterases.

Topics: Amino Acid Sequence; Catalytic Domain; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Movement; Phosphoric Diester Hydrolases; Video Recording

In Pseudomonas syringae pv. tomato DC3000, the second messenger c-di-GMP has been previously shown to stimulate pellicle formation and cellulose biosynthesis. A screen for genes involved in cellulose production under high c-di-GMP intracellular levels led to the identification of insertions in two genes, wssB and wssE, belonging to the Pto DC3000 cellulose biosynthesis operon wssABCDEFGHI. Interestingly, beside cellulose-deficient mutants, colonies with a rougher appearance than the wild type also arouse among the transposants. Those mutants carry insertions in amrZ, a gene encoding a transcriptional regulator in different Pseudomonas. Here, we provide evidence that AmrZ is involved in the regulation of bacterial cellulose production at transcriptional level by binding to the promoter region of the wssABCDEFGHI operon and repressing cellulose biosynthesis genes. Mutation of amrZ promotes wrinkly colony morphology, increased cellulose production and loss of motility in Pto DC3000. AmrZ regulon includes putative c-di-GMP metabolising proteins, like AdcA and MorA, which may also impact those phenotypes. Furthermore, an amrZ but not a cellulose-deficient mutant turned out to be impaired in pathogenesis, indicating that AmrZ is a key regulator of Pto DC3000 virulence probably by controlling bacterial processes other than cellulose production.

Topics: Bacterial Proteins; Cellulose; Cyclic GMP; DNA-Binding Proteins; Gene Expression Regulation, Bacterial; Glucosyltransferases; Mutagenesis, Insertional; Mutagenesis, Site-Directed; Operon; Plant Diseases; Plant Leaves; Pseudomonas syringae; Regulon; Solanum lycopersicum

Sinorhizobium meliloti undergoes major lifestyle changes between planktonic states, biofilm formation, and symbiosis with leguminous plant hosts. In many bacteria, the second messenger 3',5'-cyclic di-GMP (c-di-GMP, or cdG) promotes a sessile lifestyle by regulating a plethora of processes involved in biofilm formation, including motility and biosynthesis of exopolysaccharides (EPS). Here, we systematically investigated the role of cdG in S. meliloti Rm2011 encoding 22 proteins putatively associated with cdG synthesis, degradation, or binding. Single mutations in 21 of these genes did not cause evident changes in biofilm formation, motility, or EPS biosynthesis. In contrast, manipulation of cdG levels by overproducing endogenous or heterologous diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) affected these processes and accumulation of N-Acyl-homoserine lactones in the culture supernatant. Specifically, individual overexpression of the S. meliloti genes pleD, SMb20523, SMb20447, SMc01464, and SMc03178 encoding putative DGCs and of SMb21517 encoding a single-domain PDE protein had an impact and resulted in increased levels of cdG. Compared to the wild type, an S. meliloti strain that did not produce detectable levels of cdG (cdG(0)) was more sensitive to acid stress. However, it was symbiotically potent, unaffected in motility, and only slightly reduced in biofilm formation. The SMc01790-SMc01796 locus, homologous to the Agrobacterium tumefaciens uppABCDEF cluster governing biosynthesis of a unipolarly localized polysaccharide, was found to be required for cdG-stimulated biofilm formation, while the single-domain PilZ protein McrA was identified as a cdG receptor protein involved in regulation of motility.. We present the first systematic genome-wide investigation of the role of 3',5'-cyclic di-GMP (c-di-GMP, or cdG) in regulation of motility, biosynthesis of exopolysaccharides, biofilm formation, quorum sensing, and symbiosis in a symbiotic alpha-rhizobial species. Phenotypes of an S. meliloti strain unable to produce cdG (cdG(0)) demonstrated that this second messenger is not essential for root nodule symbiosis but may contribute to acid tolerance. Our data further suggest that enhanced levels of cdG promote sessility of S. meliloti and uncovered a single-domain PilZ protein as regulator of motility.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Mutation; Polysaccharides, Bacterial; Sinorhizobium meliloti

The intracellular infections of Mycobacterium tuberculosis, which is the causative agent of tuberculosis, are regulated by many cyclic dinucleotide signaling. Rv2837c from M. tuberculosis is a soluble, stand-alone DHH-DHHA1 domain phosphodiesterase that down-regulates c-di-AMP through catalytic degradation and plays an important role in M. tuberculosis infections. Here, we report the crystal structure of Rv2837c (2.0 Å), and its complex with hydrolysis intermediate 5'-pApA (2.35 Å). Our structures indicate that both DHH and DHHA1 domains are essential for c-di-AMP degradation. Further structural analysis shows that Rv2837c does not distinguish adenine from guanine, which explains why Rv2837c hydrolyzes all linear dinucleotides with almost the same efficiency. We observed that Rv2837c degraded other c-di-NMPs at a lower rate than it did on c-di-AMP. Nevertheless, our data also showed that Rv2837c significantly decreases concentrations of both c-di-AMP and c-di-GMP in vivo. Our results suggest that beside its major role in c-di-AMP degradation Rv2837c could also regulate c-di-GMP signaling pathways in bacterial cell.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Amino Acid Sequence; Bacterial Proteins; Biocatalysis; Catalytic Domain; Conserved Sequence; Cyclic AMP; Cyclic GMP; Dinucleoside Phosphates; Exoribonucleases; Models, Molecular; Molecular Sequence Data; Mutation; Mycobacterium tuberculosis; Peptide Fragments; Protein Conformation; Recombinant Fusion Proteins; Recombinant Proteins; Sequence Alignment; Substrate Specificity

In bacteria, proteins containing GGDEF domains are involved in production of the second messenger c-di-GMP. Here we report that the cdgA gene encoding diguanylate cyclase A (CdgA) is involved in biofilm formation and exopolysaccharide (EPS) production in Azospirillum brasilense Sp7. Biofilm quantification using crystal violet staining revealed that inactivation of cdgA decreased biofilm formation. In addition, confocal laser scanning microscopy analysis of green-fluorescent protein-labeled bacteria showed that, during static growth, the biofilms had differential levels of development: bacteria harboring a cdgA mutation exhibited biofilms with considerably reduced thickness compared with those of the wild-type Sp7 strain. Moreover, DNA-specific staining and treatment with DNase I, and epifluorescence studies demonstrated that extracellular DNA and EPS are components of the biofilm matrix in Azospirillum. After expression and purification of the CdgA protein, diguanylate cyclase activity was detected. The enzymatic activity of CdgA-producing cyclic c-di-GMP was determined using GTP as a substrate and flavin adenine dinucleotide (FAD(+)) and Mg(2)(+) as cofactors. Together, our results revealed that A. brasilense possesses a functional c-di-GMP biosynthesis pathway.

Topics: Azospirillum brasilense; Bacteriological Techniques; Biofilms; Coenzymes; Cyclic GMP; Escherichia coli Proteins; Flavin-Adenine Dinucleotide; Guanosine Triphosphate; Magnesium; Microscopy, Confocal; Phosphorus-Oxygen Lyases; Polysaccharides, Bacterial; Staining and Labeling

Yersinia pestis, the cause of plague, forms a biofilm in the proventriculus of its flea vector to enhance transmission. Biofilm formation in Y. pestis is regulated by the intracellular levels of cyclic diguanylate (c-di-GMP). In this study, we investigated the role of the 3' untranslated region (3'UTR) in hmsT mRNA, a transcript that encodes a diguanylate cyclase that stimulates biofilm formation in Y. pestis by synthesizing the second messenger c-di-GMP. Deletion of the 3'UTR increased the half-life of hmsT mRNA, thereby upregulating c-di-GMP levels and biofilm formation. Our findings indicate that multiple regulatory sequences might be present in the hmsT 3'UTR that function together to mediate mRNA turnover. We also found that polynucleotide phosphorylase is partially responsible for hmsT 3'UTR-mediated mRNA decay. In addition, the hmsT 3'UTR strongly repressed gene expression at 37°C and 26°C, but affected gene expression only slightly at 21°C. Our findings suggest that the 3'UTR might be involved in precise and rapid regulation of hmsT expression, allowing Y. pestis to fine-tune c-di-GMP synthesis and consequently regulate biofilm production to adapt to the changing host environment.

Topics: 3' Untranslated Regions; Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Phosphorus-Oxygen Lyases; Yersinia pestis

Bacterial biofilm formation during chronic infections confers increased fitness, antibiotic tolerance, and cytotoxicity. In many pathogens, the transition from a planktonic lifestyle to collaborative, sessile biofilms represents a regulated process orchestrated by the intracellular second-messenger c-di-GMP. A main effector for c-di-GMP signaling in the opportunistic pathogen Pseudomonas aeruginosa is the transcription regulator FleQ. FleQ is a bacterial enhancer-binding protein (bEBP) with a central AAA+ ATPase σ(54)-interaction domain, flanked by a C-terminal helix-turn-helix DNA-binding motif and a divergent N-terminal receiver domain. Together with a second ATPase, FleN, FleQ regulates the expression of flagellar and exopolysaccharide biosynthesis genes in response to cellular c-di-GMP. Here we report structural and functional data that reveal an unexpected mode of c-di-GMP recognition that is associated with major conformational rearrangements in FleQ. Crystal structures of FleQ's AAA+ ATPase domain in its apo-state or bound to ADP or ATP-γ-S show conformations reminiscent of the activated ring-shaped assemblies of other bEBPs. As revealed by the structure of c-di-GMP-complexed FleQ, the second messenger interacts with the AAA+ ATPase domain at a site distinct from the ATP binding pocket. c-di-GMP interaction leads to active site obstruction, hexameric ring destabilization, and discrete quaternary structure transitions. Solution and cell-based studies confirm coupling of the ATPase active site and c-di-GMP binding, as well as the functional significance of crystallographic interprotomer interfaces. Taken together, our data offer unprecedented insight into conserved regulatory mechanisms of gene expression under direct c-di-GMP control via FleQ and FleQ-like bEBPs.

Topics: Amino Acid Motifs; Amino Acid Sequence; Bacterial Proteins; Base Sequence; Binding Sites; Biofilms; Calorimetry; Conserved Sequence; Cross-Linking Reagents; Crystallography, X-Ray; Cyclic GMP; DNA, Bacterial; Gene Expression Regulation, Bacterial; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutant Proteins; Promoter Regions, Genetic; Protein Multimerization; Protein Stability; Protein Structure, Quaternary; Protein Structure, Tertiary; Pseudomonas aeruginosa; Sequence Alignment; Solutions; Temperature; Trans-Activators; Transcription, Genetic

Several bacterial pathogens produce diffusible signal factor (DSF)-type quorum sensing (QS) signals to control biofilm formation and virulence. Previous work showed that in Burkholderia cenocepacia the RpfFBc/RpfR system is involved in sensing and responding to DSF signals and that this signal/sensor gene pair is highly conserved in several bacterial species including Cronobacter spp. Here we show that C. turicensis LMG 23827(T) possesses a functional RpfF/R system that is involved in the regulation of various phenotypes, including colony morphology, biofilm formation and swarming motility. In vivo experiments using the zebrafish embryo model revealed a role of this regulatory system in virulence of this opportunistic pathogen. We provide evidence that the RpfF/R system modulates the intracellular c-di-GMP level of the organism, an effect that may underpin the alteration in phenotype and thus the regulated phenotypes may be a consequence thereof. This first report on an RpfF/R-type QS system of an organism outside the genus Burkholderia revealed that both the underlying molecular mechanisms as well as the regulated functions show a high degree of conservation.

Topics: Animals; Bacterial Proteins; Biofilms; Biosensing Techniques; Cronobacter; Cyclic GMP; Enterobacteriaceae Infections; Peptide Hydrolases; Phenotype; Quorum Sensing; Zebrafish

A cyclic dinucleotide riboswitch has been fused with a G-quadruplex motif to produce a conditional riboswitch-peroxidase-mimicking sensor that oxidizes both colorimetric and fluorogenic substrates in the presence of c-di-GMP. We find that signal-to-noise ratio could be improved by using a two-, not three-, floor split G-quadruplex for this conditional peroxidase-mimicking riboswitch.

Topics: Base Sequence; Cyclic GMP; Dimerization; Fluorescence; G-Quadruplexes; Molecular Sequence Data; Nucleic Acid Heteroduplexes; Oxidation-Reduction; Peroxidase; Riboswitch; Tyramine

Pseudomonas aeruginosa produces increased levels of alginate in response to oxygen-deprived conditions. The regulatory pathway(s) that links oxygen limitation to increased synthesis of alginate has remained elusive. In the present study, using immunofluorescence microscopy, we show that anaerobiosis-induced alginate production by planktonic PAO1 requires the diguanylate cyclase (DGC) SadC, previously identified as a regulator of surface-associated lifestyles. Furthermore, we found that the gene products of PA4330 and PA4331, located in a predicted operon with sadC, have a major impact on alginate production: deletion of PA4330 (odaA, for oxygen-dependent alginate synthesis activator) caused an alginate production defect under anaerobic conditions, whereas a PA4331 (odaI, for oxygen-dependent alginate synthesis inhibitor) deletion mutant produced alginate also in the presence of oxygen, which would normally inhibit alginate synthesis. Based on their sequence, OdaA and OdaI have predicted hydratase and dioxygenase reductase activities, respectively. Enzymatic assays using purified protein showed that unlike OdaA, which did not significantly affect DGC activity of SadC, OdaI inhibited c-di-GMP production by SadC. Our data indicate that SadC, OdaA and OdaI are components of a novel response pathway of P. aeruginosa that regulates alginate synthesis in an oxygen-dependent manner.

Topics: Alginates; Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Glucuronic Acid; Hexuronic Acids; Operon; Oxygen; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa

There is a compelling need for more effective vaccine adjuvants to augment induction of Ag-specific adaptive immune responses. Recent reports suggested the bacterial second messenger bis-(3'-5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) acts as an innate immune system modulator. We recently incorporated a Vibrio cholerae diguanylate cyclase into an adenovirus vaccine, fostering production of c-di-GMP as well as proinflammatory responses in mice. In this study, we recombined a more potent diguanylate cyclase gene, VCA0848, into a nonreplicating adenovirus serotype 5 (AdVCA0848) that produces elevated amounts of c-di-GMP when expressed in mammalian cells in vivo. This novel platform further improved induction of type I IFN-β and activation of innate and adaptive immune cells early after administration into mice as compared with control vectors. Coadministration of the extracellular protein OVA and the AdVCA0848 adjuvant significantly improved OVA-specific T cell responses as detected by IFN-γ and IL-2 ELISPOT, while also improving OVA-specific humoral B cell adaptive responses. In addition, we found that coadministration of AdVCA0848 with another adenovirus serotype 5 vector expressing the HIV-1-derived Gag Ag or the Clostridium difficile-derived toxin B resulted in significant inhibitory effects on the induction of Gag and toxin B-specific adaptive immune responses. As a proof of principle, these data confirm that in vivo synthesis of c-di-GMP stimulates strong innate immune responses that correlate with enhanced adaptive immune responses to concomitantly administered extracellular Ag, which can be used as an adjuvant to heighten effective immune responses for protein-based vaccine platforms against microbial infections and cancers.

Topics: Adaptive Immunity; Adenoviridae; Adjuvants, Immunologic; Animals; Antigens; Blotting, Western; Cyclic GMP; Enzyme-Linked Immunosorbent Assay; Enzyme-Linked Immunospot Assay; Flow Cytometry; Genetic Vectors; Immunotherapy; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Transduction, Genetic

Post-transcriptional control of protein abundance is a highly important, underexplored regulatory process by which organisms respond to their environments. Here we describe an important and previously unidentified regulatory pathway involving the ribosomal modification protein RimK, its regulator proteins RimA and RimB, and the widespread bacterial second messenger cyclic-di-GMP (cdG). Disruption of rimK affects motility and surface attachment in pathogenic and commensal Pseudomonas species, with rimK deletion significantly compromising rhizosphere colonisation by the commensal soil bacterium P. fluorescens, and plant infection by the pathogens P. syringae and P. aeruginosa. RimK functions as an ATP-dependent glutamyl ligase, adding glutamate residues to the C-terminus of ribosomal protein RpsF and inducing specific effects on both ribosome protein complement and function. Deletion of rimK in P. fluorescens leads to markedly reduced levels of multiple ribosomal proteins, and also of the key translational regulator Hfq. In turn, reduced Hfq levels induce specific downstream proteomic changes, with significant increases in multiple ABC transporters, stress response proteins and non-ribosomal peptide synthetases seen for both ΔrimK and Δhfq mutants. The activity of RimK is itself controlled by interactions with RimA, RimB and cdG. We propose that control of RimK activity represents a novel regulatory mechanism that dynamically influences interactions between bacteria and their hosts; translating environmental pressures into dynamic ribosomal changes, and consequently to an adaptive remodeling of the bacterial proteome.

Topics: Adaptation, Physiological; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Humans; Models, Biological; Movement; Mutation; Plant Roots; Protein Binding; Proteome; Pseudomonas; Pseudomonas Infections; Regulon; Rhizosphere; Ribosomes; Second Messenger Systems; Triticum; Up-Regulation; Virulence

In many bacterial species, including the aquatic bacterium and human pathogen Vibrio cholerae, the second messenger cyclic diguanylate (c-di-GMP) modulates processes such as biofilm formation, motility, and virulence factor production. By interacting with various effectors, c-di-GMP regulates gene expression or protein function. One type of c-di-GMP receptor is the class I riboswitch, representatives of which have been shown to bind c-di-GMP in vitro. Herein, we examined the in vitro and in vivo function of the putative class I riboswitch in Vibrio cholerae, Vc1, which lies upstream of the gene encoding GbpA, a colonization factor that contributes to attachment of V. cholerae to environmental and host surfaces containing N-acetylglucosamine moieties. We provide evidence that Vc1 RNA interacts directly with c-di-GMP in vitro, and that nucleotides conserved among this class of riboswitch are important for binding. Yet the mutation of these conserved residues individually in the V. cholerae chromosome inconsistently affects the expression of gbpA and production of the GbpA protein. By isolating the regulatory function of Vc1, we show that the Vc1 element positively regulates downstream gene expression in response to c-di-GMP. Together these data suggest that the Vc1 element responds to c-di-GMP in vivo. Positive regulation of gbpA expression by c-di-GMP via Vc1 may influence the ability of V. cholerae to associate with chitin in the aquatic environment and the host intestinal environment.

Topics: Amino Acid Sequence; Binding Sites; Cyclic GMP; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Mutation; Response Elements; Riboswitch; RNA, Bacterial; Vibrio cholerae

The division of labour is a central feature of the most sophisticated biological systems, including genomes, multicellular organisms and societies, which took millions of years to evolve. Here we show that a well-organized and robust division of labour can evolve in a matter of days. Mutants emerge within bacterial colonies and work with the parent strain to gain new territory. The two strains self-organize in space: one provides a wetting polymer at the colony edge, whereas the other sits behind and pushes them both along. The emergence of the interaction is repeatable, bidirectional and only requires a single mutation to alter production of the intracellular messenger, cyclic-di-GMP. Our work demonstrates the power of the division of labour to rapidly solve biological problems without the need for long-term evolution or derived sociality. We predict that the division of labour will evolve frequently in microbial populations, where rapid genetic diversification is common.

Topics: Bacteria; Bacterial Proteins; Biological Evolution; Cyclic GMP; Frameshift Mutation; Microbial Interactions; Pseudomonas fluorescens

c-di-GMP riboswitches are structured RNAs located in the 5'-untranslated regions (5'-UTRs) of mRNAs that regulate expression of downstream genes in response to changing concentrations of the second messenger c-di-GMP. We discovered three complete c-di-GMP riboswitches (Bc3, Bc4 and Bc5 RNA) with similar structures, which are arranged in tandem to constitute a triple-tandem (Bc3-5 RNA) riboswitch in the 5'-UTR of the cspABCDE mRNA in Bacillus thuringiensis subsp. chinensis CT-43. Our results showed that this natural triple-tandem riboswitch controlled the expression of the reporter gene more stringently and digitally than the double-tandem or single riboswitch. A sandwich-like dual-fluorescence reporter was further constructed by fusing the Bc3-5 RNA gene between the two fluorescence protein genes amcyan and turborfp. This reporter strain was found to exhibit detectable fluorescence color changes under bright field in response to intracellular c-di-GMP level altered by induced expression of diguanylate cyclase (DGC) PleD. Using this system, two putative membrane-bound DGCs from B. thuringiensis and Xanthomonas oryzae were verified to be functional by replacing pleD with the corresponding DGC genes. This report represented the first native triple-tandem riboswitch that was applied to serve as a riboswitch-based dual-fluorescence reporter for the efficient and convenient verification of putative DGC activity in vivo.

Topics: Base Sequence; Computational Biology; Cyclic GMP; Escherichia coli; Gene Expression Regulation; Gene Expression Regulation, Bacterial; Genes, Reporter; Nucleic Acid Conformation; Riboswitch; RNA, Messenger

The alarmone (p)ppGpp regulates transcription, translation, replication, virulence, lipid synthesis, antibiotic sensitivity, biofilm formation, and other functions in bacteria. Signaling nucleotide cyclic di-GMP (c-di-GMP) regulates biofilm formation, motility, virulence, the cell cycle, and other functions. In Mycobacterium smegmatis, both (p)ppGpp and c-di-GMP are synthesized and degraded by bifunctional proteins Rel(Msm) and DcpA, encoded by rel(Msm) and dcpA genes, respectively. We have previously shown that the Δrel(Msm) and ΔdcpA knockout strains are antibiotic resistant and defective in biofilm formation, show altered cell surface properties, and have reduced levels of glycopeptidolipids and polar lipids in their cell wall (K. R. Gupta, S. Kasetty, and D. Chatterji, Appl Environ Microbiol 81:2571-2578, 2015,http://dx.doi.org/10.1128/AEM.03999-14). In this work, we have explored the phenotypes that are affected by both (p)ppGpp and c-di-GMP in mycobacteria. We have shown that both (p)ppGpp and c-di-GMP are needed to maintain the proper growth rate under stress conditions such as carbon deprivation and cold shock. Scanning electron microscopy showed that low levels of these second messengers result in elongated cells, while high levels reduce the cell length and embed the cells in a biofilm-like matrix. Fluorescence microscopy revealed that the elongated Δrel(Msm) and ΔdcpA cells are multinucleate, while transmission electron microscopy showed that the elongated cells are multiseptate. Gene expression analysis also showed that genes belonging to functional categories such as virulence, detoxification, lipid metabolism, and cell-wall-related processes were differentially expressed. Our results suggests that both (p)ppGpp and c-di-GMP affect some common phenotypes in M. smegmatis, thus raising a possibility of cross talk between these two second messengers in mycobacteria.. Our work has expanded the horizon of (p)ppGpp and c-di-GMP signaling in Gram-positive bacteria. We have come across a novel observation that M. smegmatis needs (p)ppGpp and c-di-GMP for cold tolerance. We had previously shown that the Δrel(Msm) and ΔdcpA strains are defective in biofilm formation. In this work, the overproduction of (p)ppGpp and c-di-GMP encased M. smegmatis in a biofilm-like matrix, which shows that both (p)ppGpp and c-di-GMP are needed for biofilm formation. The regulation of cell length and cell division by (p)ppGpp was known in mycobacteria, but our work shows that c-di-GMP also affects the cell size and cell division in mycobacteria. This is perhaps the first report of c-di-GMP regulating cell division in mycobacteria.

Topics: Biofilms; Cell Division; Cold Temperature; Cyclic GMP; Gene Expression; Gene Expression Regulation, Bacterial; Guanosine Pentaphosphate; Mycobacterium smegmatis; Phenotype; Second Messenger Systems; Signal Transduction; Stress, Physiological

Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger that is synthesized by diguanylate cyclase (DGC) with the GGDEF-domain, regulates diverse virulence phenotypes in pathogenic bacteria. Although 11 genes encoding GGDEF-domain proteins have been shown in the genome of Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99(A) , the causal pathogen of bacterial blight of rice, however, little is known about their roles in the c-di-GMP regulation of virulence in the pathogen. GdpX1, one of the GGDEF-domain proteins in Xoo was investigated in this study to reveal its regulatory function of bacterial virulence expression through genetic analysis.. GdpX1 was functionally characterized in virulence expression through deletion and overexpression analysis. Bioinformatics analysis revealed the GGDEF-domain in GdpX1 was well conserved, indicating it is a putative DGC. Deletion of gdpX1 resulted in significant increases in virulence, exopolysaccharide (EPS) production and flagellar motility. In contrast, overexpression of gdpX1 dramatically reduced these virulence phenotypes. qRT-PCR analysis showed genes related to the type III secretion system (T3SS), EPS synthesis, and flagellar motility, were up-regulated in ∆gdpX1 and down-regulated in the gdpX1-overexpressed strains. In addition, overexpression of gdpX1 promoted biofilm formation and xylanase activity.. GdpX1 is the first GGDEF-domain protein functionally characterized in Xoo, which functions as a negative regulator of bacterial virulence via suppression of virulence-related gene transcription.. Identification and functional characterization of GdpX1 provided additional insights into molecular mechanisms of c-di-GMP regulation of bacterial virulence expression.

Topics: Amino Acid Sequence; Bacterial Proteins; Computational Biology; Cyclic GMP; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Oryza; Plant Diseases; Polysaccharides, Bacterial; Protein Domains; Sequence Alignment; Virulence; Xanthomonas

Cyclic diguanylate (c-di-GMP) is a second messenger of bacteria and its detection is an important issue in basic and applied microbiology. As c-di-GMP riboswitch ligand-binding domains (aptamer domains) capture c-di-GMP with high affinity and selectivity, they are promising platforms for the development of RNA-based c-di-GMP sensors. We analyzed two previously reported c-di-GMP sensor RNAs derived from the Vc2 riboswitch. We also designed and tested their variants, some of which showed improved properties as RNA-based c-di-GMP sensors.

Topics: Aptamers, Nucleotide; Base Sequence; Biosensing Techniques; Cyclic GMP; Fluorescence; Ligands; Riboswitch; RNA; Second Messenger Systems; Spinacia oleracea

The ecological and evolutionary forces that promote and maintain diversity in biofilms are not well understood. To quantify these forces, three Pseudomonas aeruginosa populations were experimentally evolved from strain PA14 in a daily cycle of attachment, assembly, and dispersal for 600 generations. Each biofilm population evolved diverse colony morphologies and mutator genotypes defective in DNA mismatch repair. This diversity enhanced population fitness and biofilm output, owing partly to rare, early colonizing mutants that enhanced attachment of others. Evolved mutants exhibited various levels of the intracellular signal cyclic-di-GMP, which associated with their timing of adherence. Manipulating cyclic-di-GMP levels within individual mutants revealed a network of interactions in the population that depended on various attachment strategies related to this signal. Diversification in biofilms may therefore arise and be reinforced by initial colonists that enable community assembly.. How biofilm diversity assembles, evolves, and contributes to community function is largely unknown. This presents a major challenge for understanding evolution during chronic infections and during the growth of all surface-associated microbes. We used experimental evolution to probe these dynamics and found that diversity, partly related to altered cyclic-di-GMP levels, arose and persisted due to the emergence of ecological interdependencies related to attachment patterns. Clonal isolates failed to capture population attributes, which points to the need to account for diversity in infections. More broadly, this study offers an experimental framework for linking phenotypic variation to distinct ecological strategies in biofilms and for studying eco-evolutionary interactions.

Topics: Bacterial Proteins; Bacteriological Techniques; Biofilms; Cyclic GMP; Directed Molecular Evolution; Ecosystem; Gene Expression Regulation, Bacterial; Mutation; Pseudomonas aeruginosa; Signal Transduction

Polyphosphate (polyP) degradation in Escherichia coli stationary phase triggers biofilm formation via the LuxS quorum sensing system. In media containing excess of phosphate (Pi), high polyP levels are maintained in the stationary phase with the consequent inhibition of biofilm formation. The transcriptional-response regulator PhoB, which is activated under Pi limitation, is involved in the inhibition of biofilm formation in several bacterial species. In the current study, we report, for the first time, we believe that E. coli PhoB can be activated in non-limiting Pi conditions, leading to inhibition of biofilm formation. In fact, PhoB was activated when high polyP levels were maintained in the stationary phase, whereas it remained inactive when the polymer was degraded or absent. PhoB activation was mediated by acetyl phosphate with the consequent repression of biofilm formation owing to the downregulation of c-di-GMP synthesis and the inhibition of autoinducer-2 production. These results allowed us to propose a model showing that PhoB is a component in the signal cascade regulating biofilm formation triggered by fluctuations of polyP levels in E. coli cells during stationary phase.

Topics: Bacterial Proteins; Biofilms; Carbon-Sulfur Lyases; Cyclic GMP; Enzyme Activation; Escherichia coli; Gene Expression Regulation, Bacterial; Homoserine; Lactones; Organophosphates; Polyphosphates; Quorum Sensing; Signal Transduction

The Vc2 riboswitch possesses an aptamer domain belonging to the class-I c-di-GMP riboswitch family. This domain has been analysed and the molecular mechanism by which it recognizes the c-di-GMP ligand has been elucidated. On the other hand, the regulatory mechanism of the full-length Vc2 riboswitch to control its downstream open reading frame (ORF) remains largely unknown. In this study, we performed in vivo reporter assays and in vitro biochemical analyses of the full-length riboswitch and its aptamer domain. We evaluated the results of in vivo and in vitro analyses to elucidate the regulatory mechanism of the Vc2 riboswitch. The present results suggest that recognition of c-di-GMP ligand by the Vc2 riboswitch aptamer domain downregulates expression of its downstream ORF primarily at the translational level.

Topics: Cyclic GMP; Nucleic Acid Conformation; Open Reading Frames; Riboswitch

The second messenger c-di-GMP is implicated in regulation of various aspects of the lifestyles and virulence of Gram-negative bacteria. Cyclic di-GMP is formed by diguanylate cyclases with a GGDEF domain and degraded by phosphodiesterases with either an EAL or HD-GYP domain. Proteins with tandem GGDEF-EAL domains occur in many bacteria, where they may be involved in c-di-GMP turnover or act as enzymatically-inactive c-di-GMP effectors. Here, we report a systematic study of the regulatory action of the eleven GGDEF-EAL proteins in Xanthomonas oryzae pv. oryzicola, an important rice pathogen causing bacterial leaf streak. Mutational analysis revealed that XOC_2335 and XOC_2393 positively regulate bacterial swimming motility, while XOC_2102, XOC_2393 and XOC_4190 negatively control sliding motility. The ΔXOC_2335/XOC_2393 mutant that had a higher intracellular c-di-GMP level than the wild type and the ΔXOC_4190 mutant exhibited reduced virulence to rice after pressure inoculation. In vitro purified XOC_4190 and XOC_2102 have little or no diguanylate cyclase or phosphodiesterase activity, which is consistent with unaltered c-di-GMP concentration in ΔXOC_4190. Nevertheless, both proteins can bind to c-di-GMP with high affinity, indicating a potential role as c-di-GMP effectors. Overall our findings advance understanding of c-di-GMP signaling and its links to virulence in an important rice pathogen.

Topics: Bacterial Proteins; Cyclic GMP; DNA Mutational Analysis; Gene Expression Regulation, Bacterial; Oryza; Protein Structure, Tertiary; Signal Transduction; Virulence; Xanthomonas

Biofilm-associated infections are hard to treat because of their high antibiotic resistance and the presence of a very persistent subpopulation of bacteria. The second messenger molecule cyclic di-guanosine monophosphate (c-di-GMP) plays a very important role in this biofilm physiology. Here, we evaluated the role of YddV, an enzyme with a c-di-GMP synthesis function, in the formation and maturation of Escherichia coli biofilms. Our results suggest that YddV stimulates biofilm growth via its role in the production of c-di-GMP and this likely by influencing the production of matrix (e.g. poly-N-acetylglucosamine (PGA)). However, lowering the YddV expression did not alter the biofilm formation since there was no significant difference between the biofilm phenotypes of WT E. coli and YddV-knockout bacteria. Additionally, YddV expression had no significant influence on the amount of persister cells within the biofilm population, questioning the use of YddV as therapeutic target.

Topics: Biofilms; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Phosphorus-Oxygen Lyases

YfiBNR is a recently identified bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens. It is a key regulator of biofilm formation, which is correlated with prolonged persistence of infection and antibiotic drug resistance. In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. However, the detailed regulatory mechanism remains elusive. Here, we report the crystal structures of YfiB alone and of an active mutant YfiB(L43P) complexed with YfiR with 2:2 stoichiometry. Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiB(L43P) adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiB(L43P) shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiB(L43P)-induced biofilm formation. Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system.

Topics: Amino Acid Sequence; Bacterial Proteins; Binding Sites; Biofilms; Crystallography, X-Ray; Cyclic GMP; Dimerization; Molecular Dynamics Simulation; Molecular Sequence Data; Mutagenesis; Protein Structure, Quaternary; Pseudomonas aeruginosa; Sequence Alignment; Tryptophan; Vitamin B 6

The second messenger cyclic diguanylate (c-di-GMP) is an important regulator of motility in many bacterial species. In Pseudomonas aeruginosa, elevated levels of c-di-GMP promote biofilm formation and repress flagellum-driven swarming motility. The rotation of P. aeruginosa's polar flagellum is controlled by two distinct stator complexes, MotAB, which cannot support swarming motility, and MotCD, which promotes swarming motility. Here we show that when c-di-GMP levels are elevated, swarming motility is repressed by the PilZ domain-containing protein FlgZ and by Pel polysaccharide production. We demonstrate that FlgZ interacts specifically with the motility-promoting stator protein MotC in a c-di-GMP-dependent manner and that a functional green fluorescent protein (GFP)-FlgZ fusion protein shows significantly reduced polar localization in a strain lacking the MotCD stator. Our results establish FlgZ as a c-di-GMP receptor affecting swarming motility by P. aeruginosa and support a model wherein c-di-GMP-bound FlgZ impedes motility via its interaction with the MotCD stator.. The regulation of surface-associated motility plays an important role in bacterial surface colonization and biofilm formation. c-di-GMP signaling is a widespread means of controlling bacterial motility, and yet the mechanism whereby this signal controls surface-associated motility in P. aeruginosa remains poorly understood. Here we identify a PilZ domain-containing c-di-GMP effector protein that contributes to c-di-GMP-mediated repression of swarming motility by P. aeruginosa We provide evidence that this effector, FlgZ, impacts swarming motility via its interactions with flagellar stator protein MotC. Thus, we propose a new mechanism for c-di-GMP-mediated regulation of motility for a bacterium with two flagellar stator sets, increasing our understanding of surface-associated behaviors, a key prerequisite to identifying ways to control the formation of biofilm communities.

Topics: Amino Acid Sequence; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Protein Binding; Protein Domains; Pseudomonas aeruginosa; Sequence Alignment

Biofilm formation can be considered a bacterial virulence mechanism. In a range of Gram-negatives, increased levels of the second messenger cyclic diguanylate (c-di-GMP) promotes biofilm formation and reduces motility. Other bacterial processes known to be regulated by c-di-GMP include cell division, differentiation and virulence. Among Gram-positive bacteria, where the function of c-di-GMP signalling is less well characterized, c-di-GMP was reported to regulate swarming motility in Bacillus subtilis while having very limited or no effect on biofilm formation. In contrast, we show that in the Bacillus cereus group c-di-GMP signalling is linked to biofilm formation, and to several other phenotypes important to the lifestyle of these bacteria. The Bacillus thuringiensis 407 genome encodes eleven predicted proteins containing domains (GGDEF/EAL) related to c-di-GMP synthesis or breakdown, ten of which are conserved through the majority of clades of the B. cereus group, including Bacillus anthracis. Several of the genes were shown to affect biofilm formation, motility, enterotoxin synthesis and/or sporulation. Among these, cdgF appeared to encode a master diguanylate cyclase essential for biofilm formation in an oxygenated environment. Only two cdg genes (cdgA, cdgJ) had orthologs in B. subtilis, highlighting differences in c-di-GMP signalling between B. subtilis and B. cereus group bacteria.

Topics: Bacillus cereus; Bacillus subtilis; Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Deletion; Phosphorus-Oxygen Lyases; Second Messenger Systems

Type VI secretion systems (T6SSs) are nanomachines used for interbacterial killing and intoxication of eukaryotes. Although Vibrio cholerae is a model organism for structural studies on T6SSs, the underlying regulatory network is less understood. A recent study showed that the T6SS is part of the natural competence regulon in V. cholerae and is activated by the regulator TfoX. Here, we identify the TfoX homolog TfoY as a second activator of the T6SS. Importantly, despite inducing the same T6SS core machinery, the overall regulons differ significantly for TfoX and TfoY. We show that TfoY does not contribute to competence induction. Instead, TfoY drives the production of T6SS-dependent and T6SS-independent toxins, together with an increased motility phenotype. Hence, we conclude that V. cholerae uses its sole T6SS in response to diverse cues and for distinctive outcomes: either to kill for the prey's DNA, leading to horizontal gene transfer, or as part of a defensive escape reaction.

Topics: Bacterial Proteins; Bacterial Secretion Systems; Cyclic GMP; Gene Expression Regulation, Bacterial; Models, Biological; Sequence Homology, Amino Acid; Vibrio cholerae

Biofilm formation on biotic or abiotic surfaces has unwanted consequences in medical, clinical, and industrial settings. Treatments with antibiotics or biocides are often ineffective in eradicating biofilms. Promising alternatives to conventional agents are biofilm-inhibiting compounds regulating biofilm development without toxicity to growth. Here, we screened a biofilm inhibitor, raffinose, derived from ginger. Raffinose, a galactotrisaccharide, showed efficient biofilm inhibition of Pseudomonas aeruginosa without impairing its growth. Raffinose also affected various phenotypes such as colony morphology, matrix formation, and swarming motility. Binding of raffinose to a carbohydrate-binding protein called LecA was the cause of biofilm inhibition and altered phenotypes. Furthermore, raffinose reduced the concentration of the second messenger, cyclic diguanylate (c-di-GMP), by increased activity of a c-di-GMP specific phosphodiesterase. The ability of raffinose to inhibit P. aeruginosa biofilm formation and its molecular mechanism opens new possibilities for pharmacological and industrial applications.

Topics: Adhesins, Bacterial; Anti-Bacterial Agents; Biofilms; Cyclic GMP; Pseudomonas aeruginosa; Raffinose; Zingiber officinale

The transcription factor FliA, also called sigma 28, is a major regulator of bacterial flagellar biosynthesis genes. Growing evidence suggest that in addition to motility, FliA is involved in controlling numerous bacterial behaviors, even though the underlying regulatory mechanism remains unclear. By using a transcriptional fusion to gfp that responds to cyclic (c)-di-GMP, this study revealed a higher c-di-GMP concentration in the fliA deletion mutant of Pseudomonas aeruginosa than in its wild-type strain PAO1. A comparative analysis of transcriptome profiles of P. aeruginosa PAO1 and its fliA deletion mutant revealed an altered expression of several c-di-GMP-modulating enzyme-encoding genes in the fliA deletion mutant. Moreover, the downregulation of PA4367 (bifA), a Glu-Ala-Leu motif-containing phosphodiesterase, in the fliA deletion mutant was confirmed using the β-glucuronidase reporter gene assay. FliA also altered pyocyanin and pyorubin production by modulating the c-di-GMP concentration. Complementing the fliA mutant strain with bifA restored the motility defect and pigment overproduction of the fliA mutant. Our results indicate that in addition to regulating flagellar gene transcription, FliA can modulate the c-di-GMP concentration to regulate the swarming motility and phenazine pigment production in P. aeruginosa.

Topics: Bacterial Proteins; Base Sequence; Computational Biology; Cyclic GMP; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genes, Reporter; Mutation; Phenazines; Pigments, Biological; Promoter Regions, Genetic; Pseudomonas aeruginosa; Sigma Factor

Topics: Acetobacter; Caulobacter; Cyclic GMP; Fimbriae, Bacterial; Flagella; Locomotion; Phosphoric Diester Hydrolases

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice blight disease as well as a serious phytopathogen worldwide. It is also one of the model organisms for studying bacteria-plant interactions. Current progress in bacterial signal transduction pathways has identified cyclic di-GMP as a major second messenger molecule in controlling Xanthomonas pathogenicity. However, it still remains largely unclear how c-di-GMP regulates the secretion of bacterial virulence factors in Xoo. In this study, we focused on the important roles played by DgcA (XOO3988), one of our previously identified diguanylate cyclases in Xoo, through further investigating the phenotypes of several dgcA-related mutants, namely, the dgcA-knockout mutant ΔdgcA, the dgcA overexpression strain OdgcA, the dgcA complemented strain CdgcA and the wild-type strain. The results showed that dgcA negatively affected virulence, EPS production, bacterial autoaggregation and motility, but positively triggered biofilm formation via modulating the intracellular c-di-GMP levels. RNA-seq data further identified 349 differentially expressed genes controlled by DgcA, providing a foundation for a more solid understanding of the signal transduction pathways in Xoo. Collectively, the present study highlights DgcA as a major regulator of Xoo virulence, and can serve as a potential target for preventing rice blight diseases.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Mutation; Oryza; Phosphorus-Oxygen Lyases; Plant Diseases; Polysaccharides, Bacterial; Sequence Analysis, RNA; Virulence Factors; Xanthomonas

Bioaugmentation has been frequently proposed in wastewater and soil treatment to remove toxic aromatic compounds. The performance of bioaugmentation is affected by a number of biological and environmental factors, including the interaction between the target pollutant and the augmented bacterial cells. In this study, using Comamonas testosteroni and 3-chloroaniline (3-CA) as the model organism and target pollutant, we explored the influence of toxic aromatic pollutants on the biofilm lifestyle of bacteria capable of degrading aromatic compounds toward a better understanding of cell-pollutant interaction in bioaugmentation. Our results showed that the exposure to 3-CA greatly reduced the retention of C. testosteroni cells in packed-bed bioreactors (from 22% to 15% after three pore volumes), which could be attributed to the altered bacterial motility and cell surface hydrophobicity. To further understand the molecular mechanisms, we employed an integrated genomic and transcriptomic analysis to examine the influence of 3-CA on the expression of genes important to the biofilm lifestyle of C. testosteroni We found that exposure to 3-CA reduced the intracellular c-di-GMP level by downregulating the expression of genes encoding c-di-GMP synthases and induced massive cell dispersal from the biofilms. Our findings provide novel environmental implications on bioaugmentation, particularly in biofilm reactors, for the treatment of wastewater containing recalcitrant industrial pollutants.. Bioaugmentation is a bioremediation approach that often has been described in the literature but has almost never been successfully applied in practice. Many biological and environmental factors influence the overall performance of bioaugmentation. Among these, the interaction between the target pollutant and the augmented bacterial cells is one of the most important factors. In this study, we revealed the influence of toxic aromatic pollutants on the biofilm lifestyle of bacteria capable of degrading aromatic compounds toward a better understanding of cell-pollutant interaction in bioaugmentation. Our findings provide novel environmental implications on bioaugmentation for the treatment of wastewater containing recalcitrant industrial pollutants; in particular, the exposure to toxic pollutants may reduce the retention of augmented organisms in biofilm reactors by reducing the c-di-GMP level, and approaches to elevating or maintaining a high c-di-GMP level may be promising to establish and maintain sustainable bioaugmentation activity.

Topics: Aniline Compounds; Biofilms; Comamonas testosteroni; Cyclic GMP; Gene Expression Profiling; Water Pollutants

Generally, the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulates the switch between motile and sessile lifestyles in bacteria. Here, we show that c-di-GMP is an essential regulator of multicellular development in the social bacterium Myxococcus xanthus. In response to starvation, M. xanthus initiates a developmental program that culminates in formation of spore-filled fruiting bodies. We show that c-di-GMP accumulates at elevated levels during development and that this increase is essential for completion of development whereas excess c-di-GMP does not interfere with development. MXAN3735 (renamed DmxB) is identified as a diguanylate cyclase that only functions during development and is responsible for this increased c-di-GMP accumulation. DmxB synthesis is induced in response to starvation, thereby restricting DmxB activity to development. DmxB is essential for development and functions downstream of the Dif chemosensory system to stimulate exopolysaccharide accumulation by inducing transcription of a subset of the genes encoding proteins involved in exopolysaccharide synthesis. The developmental defects in the dmxB mutant are non-cell autonomous and rescued by co-development with a strain proficient in exopolysaccharide synthesis, suggesting reduced exopolysaccharide accumulation as the causative defect in this mutant. The NtrC-like transcriptional regulator EpsI/Nla24, which is required for exopolysaccharide accumulation, is identified as a c-di-GMP receptor, and thus a putative target for DmxB generated c-di-GMP. Because DmxB can be-at least partially-functionally replaced by a heterologous diguanylate cyclase, these results altogether suggest a model in which a minimum threshold level of c-di-GMP is essential for the successful completion of multicellular development in M. xanthus.

Topics: Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Myxococcus xanthus; Phosphorus-Oxygen Lyases; Spores, Bacterial; Starvation

The bacterial messenger cyclic di-GMP (c-di-GMP) binds to a diverse range of effectors to exert its biological effect. Despite the fact that free-standing PilZ proteins are by far the most prevalent c-di-GMP effectors known to date, their physiological function and mechanism of action remain largely unknown. Here we report that the free-standing PilZ protein PA2799 from the opportunistic pathogen Pseudomonas aeruginosa interacts directly with the hybrid histidine kinase SagS. We show that PA2799 (named as HapZ: histidine kinase associated PilZ) binds directly to the phosphoreceiver (REC) domain of SagS, and that the SagS-HapZ interaction is further enhanced at elevated c-di-GMP concentration. We demonstrate that binding of HapZ to SagS inhibits the phosphotransfer between SagS and the downstream protein HptB in a c-di-GMP-dependent manner. In accordance with the role of SagS as a motile-sessile switch and biofilm growth factor, we show that HapZ impacts surface attachment and biofilm formation most likely by regulating the expression of a large number of genes. The observations suggest a previously unknown mechanism whereby c-di-GMP mediates two-component signaling through a PilZ adaptor protein.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Histidine Kinase; Pseudomonas aeruginosa; Second Messenger Systems

Biofilm formation is important for infection by many pathogens. Bordetella bronchiseptica causes respiratory tract infections in mammals and forms biofilm structures in nasal epithelium of infected mice. We previously demonstrated that cyclic di-GMP is involved in biofilm formation in B. bronchiseptica. In the present work, based on their previously reported function in Pseudomonas fluorescens, we identified three genes in the B. bronchiseptica genome likely involved in c-di-GMP-dependent biofilm formation: brtA, lapD and lapG. Genetic analysis confirmed a role for BrtA, LapD and LapG in biofilm formation using microtiter plate assays, as well as scanning electron and fluorescent microscopy to analyze the phenotypes of mutants lacking these proteins. In vitro and in vivo studies showed that the protease LapG of B. bronchiseptica cleaves the N-terminal domain of BrtA, as well as the LapA protein of P. fluorescens, indicating functional conservation between these species. Furthermore, while BrtA and LapG appear to have little or no impact on colonization in a mouse model of infection, a B. bronchiseptica strain lacking the LapG protease has a significantly higher rate of inducing a severe disease outcome compared to the wild type. These findings support a role for c-di-GMP acting through BrtA/LapD/LapG to modulate biofilm formation, as well as impact pathogenesis, by B. bronchiseptica.

Topics: Animals; Bacterial Proteins; Biofilms; Blotting, Western; Bordetella bronchiseptica; Bordetella Infections; Cyclic GMP; Female; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Green Fluorescent Proteins; Host-Pathogen Interactions; Mice, Inbred BALB C; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Mutation; Pseudomonas fluorescens; Respiratory Tract Infections; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

Cyclic di-GMP is a ubiquitous second messenger that regulates diverse cellular processes in bacteria by binding to various protein or riboswitch effectors. In Bacillus thuringiensis BMB171, a c-di-GMP riboswitch termed Bc2 RNA resides in the 5'-untranslated region (5'-UTR) of an mRNA that encodes a collagen adhesion protein (Cap). The expression of cap was strongly repressed in parent strain BMB171 because of the presence of Bc2 RNA but was significantly promoted in the Bc2 RNA markerless deletion mutant. Bc2 RNA acts as a genetic "on" switch, which forms an anti-terminator structure to promote cap read-through transcription upon c-di-GMP binding. As a result, cap transcription was de-repressed under high c-di-GMP levels. Therefore, Bc2 RNA regulates cap expression using a repression/de-repression model. Bc2 RNA-regulated Cap was also found to be tightly associated with motility, aggregation, exopolysaccharide secretion, biofilm formation, and virulence of B. thuringiensis BMB171 against its host insect Helicoverpa armigera.

Topics: 5' Untranslated Regions; Animals; Bacillus thuringiensis; Bacterial Adhesion; beta-Galactosidase; Biofilms; Cell Adhesion; Collagen; Computational Biology; Cyclic GMP; Gene Deletion; Gene Expression Regulation, Bacterial; Insecta; Microscopy, Electron, Scanning; Mutation; Nucleic Acid Conformation; Open Reading Frames; Protein Binding; Riboswitch; RNA; Virulence

Bacteria occupy a diverse set of environmental niches with differing oxygen availability. Anaerobic environments such as mammalian digestive tracts and industrial reactors harbor an abundance of both obligate and facultative anaerobes, many of which play significant roles in human health and biomanufacturing. Studying bacterial function under partial or fully anaerobic conditions, however, is challenging given the paucity of suitable live-cell imaging tools. Here, we introduce a series of RNA-based fluorescent biosensors that respond selectively to cyclic di-GMP, an intracellular bacterial second messenger that controls cellular motility and biofilm formation. We demonstrate the utility of these biosensors in vivo under both aerobic and anaerobic conditions, and we show that biosensor expression does not interfere with the native motility phenotype. Together, our results attest to the effectiveness and versatility of RNA-based fluorescent biosensors, priming further development and application of these and other analogous sensors to study host-microbial and microbial-microbial interactions through small molecule signals.

Topics: Anaerobiosis; Aptamers, Nucleotide; Biosensing Techniques; Cyclic GMP; Escherichia coli; Flow Cytometry; Green Fluorescent Proteins; Movement; Phenotype; Phylogeny; RNA; Spectrometry, Fluorescence

C-di-GMP (3',5' -Cyclic diguanylic acid) is an important second messenger in bacteria that influences virulence, motility, biofilm formation, and cell division. The level of c-di-GMP in cells is controlled by diguanyl cyclases (DGCs) and phosphodiesterases (PDEs). Here, we report the biochemical functions and crystal structure of the potential diguanylase Dcsbis (PA2771, a diguanylate cyclase with a self-blocked I-site) from Pseudomonas aeruginosa PAO1. The full-length Dcsbis protein contains an N-terminal GAF domain and a C-terminal GGDEF domain. We showed that Dcsbis tightly coordinates cell motility without markedly affecting biofilm formation and is a diguanylate cyclase with a catalytic activity much higher than those of many other DGCs. Unexpectedly, we found that a peptide loop (protecting loop) extending from the GAF domain occupies the conserved inhibition site, thereby largely relieving the product-inhibition effect. A large hydrophobic pocket was observed in the GAF domain, thus suggesting that an unknown upstream signaling molecule may bind to the GAF domain, moving the protecting loop from the I-site and thereby turning off the enzymatic activity.

Topics: Bacterial Proteins; Biofilms; Catalytic Domain; Crystallography, X-Ray; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Hydrogen Bonding; Models, Molecular; Phosphorus-Oxygen Lyases; Protein Conformation; Protein Domains; Pseudomonas aeruginosa

The second messenger, bis-(3',5')-cyclic dimeric guanosine monophosphate (cyclic di-GMP), is involved in the control of multiple bacterial phenotypes, including those that impact host-pathogen interactions. Bioinformatics analyses predicted that Mycobacterium leprae, an obligate intracellular bacterium and the causative agent of leprosy, encodes three active diguanylate cyclases. In contrast, the related pathogen Mycobacterium tuberculosis encodes only a single diguanylate cyclase. One of the M. leprae unique diguanylate cyclases (ML1419c) was previously shown to be produced early during the course of leprosy. Thus, functional analysis of ML1419c was performed. The gene encoding ML1419c was cloned and expressed in Pseudomonas aeruginosa PAO1 to allow for assessment of cyclic di-GMP production and cyclic di-GMP-mediated phenotypes. Phenotypic studies revealed that ml1419c expression altered colony morphology, motility and biofilm formation of P. aeruginosa PAO1 in a manner consistent with increased cyclic di-GMP production. Direct measurement of cyclic di-GMP levels by liquid chromatography-mass spectrometry confirmed that ml1419c expression increased cyclic di-GMP production in P. aeruginosa PAO1 cultures in comparison to the vector control. The observed phenotypes and increased levels of cyclic di-GMP detected in P. aeruginosa expressing ml1419c could be abrogated by mutation of the active site in ML1419c. These studies demonstrated that ML1419c of M. leprae functions as diguanylate cyclase to synthesize cyclic di-GMP. Thus, this protein was renamed DgcA (Diguanylate cyclase A). These results also demonstrated the ability to use P. aeruginosa as a heterologous host for characterizing the function of proteins involved in the cyclic di-GMP pathway of a pathogen refractory to in vitro growth, M. leprae.

Topics: Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Mutation; Mycobacterium leprae; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa

We report for the first time a hydrolysis mechanism of the cyclic dimeric guanosine monophosphate (c-di-GMP) by the EAL domain phosphodiesterases as revealed by molecular simulations. A model system for the enzyme-substrate complex was prepared on the base of the crystal structure of the EAL domain from the BlrP1 protein complexed with c-di-GMP. The nucleophilic hydroxide generated from the bridging water molecule appeared in a favorable position for attack on the phosphorus atom of c-di-GMP. The most difficult task was to find a pathway for a proton transfer to the O3' atom of c-di-GMP to promote the O3'P bond cleavage. We show that the hydrogen bond network extended over the chain of water molecules in the enzyme active site and the Glu359 and Asp303 side chains provides the relevant proton wires. The suggested mechanism is consistent with the structural, mutagenesis, and kinetic experimental studies on the EAL domain phosphodiesterases. Proteins 2016; 84:1670-1680. © 2016 Wiley Periodicals, Inc.

Topics: Amino Acid Substitution; Aspartic Acid; Catalytic Domain; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression; Glutamic Acid; Hydrogen Bonding; Hydrolysis; Kinetics; Molecular Dynamics Simulation; Phosphoric Diester Hydrolases; Phosphorus; Protein Structure, Secondary; Protons; Quantum Theory; Structure-Activity Relationship; Thermodynamics; Water

The ability of Yersinia pestis to form a biofilm is an important characteristic in flea transmission of this pathogen. Y. pestis laterally acquired two plasmids (pPCP1and pMT1) and the ability to form biofilms when it evolved from Yersinia pseudotuberculosis. Small regulatory RNAs (sRNAs) are thought to play a crucial role in the processes of biofilm formation and pathogenesis.. A pPCP1-derived sRNA HmsA (also known as sR084) was found to contribute to the enhanced biofilm formation phenotype of Y. pestis. The concentration of c-di-GMP was significantly reduced upon deletion of the hmsA gene in Y. pestis. The abundance of mRNA transcripts determining exopolysaccharide production, crucial for biofilm formation, was measured by primer extension, RT-PCR and lacZ transcriptional fusion assays in the wild-type and hmsA mutant strains. HmsA positively regulated biofilm synthesis-associated genes (hmsHFRS, hmsT and hmsCDE), but had no regulatory effect on the biofilm degradation-associated gene hmsP. Interestingly, the recently identified biofilm activator sRNA, HmsB, was rapidly degraded in the hmsA deletion mutant. Two genes (rovM and rovA) functioning as biofilm regulators were also found to be regulated by HmsA, whose regulatory effects were consistent with the HmsA-mediated biofilm phenotype.. HmsA potentially functions as an activator of biofilm formation in Y. pestis, implying that sRNAs encoded on the laterally acquired plasmids might be involved in the chromosome-based regulatory networks implicated in Y. pestis-specific physiological processes.

Topics: Bacterial Proteins; beta-Galactosidase; Biofilms; Cyclic GMP; Phenotype; Plasmids; Real-Time Polymerase Chain Reaction; RNA, Bacterial; Sequence Analysis, DNA; Transcription Factors; Yersinia pestis

The waterborne pathogen Legionella pneumophila grows as a biofilm, freely or inside amoebae. Cyclic-di-GMP (c-di-GMP), a bacterial second messenger frequently implicated in biofilm formation, is synthesized and degraded by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), respectively. To characterize the c-di-GMP-metabolizing enzymes involved in L. pneumophila biofilm regulation, the consequences on biofilm formation and the c-di-GMP concentration of each corresponding gene inactivation were assessed in the Lens strain. The results showed that one DGC and two PDEs enhance different aspects of biofilm formation, while two proteins with dual activity (DGC/PDE) inhibit biofilm growth. Surprisingly, only two mutants exhibited a change in global c-di-GMP concentration. This study highlights that specific c-di-GMP pathways control L. pneumophila biofilm formation, most likely via temporary and/or local modulation of c-di-GMP concentration. Furthermore, Lpl1054 DGC is required to enable the formation a dense biofilm in response to nitric oxide, a signal for biofilm dispersion in many other species.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Legionella pneumophila; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Signal Transduction

Current antibiotic treatments are insufficient in eradicating bacterial biofilms, which represent the primary cause of chronic bacterial infections. Thus, there is an urgent need for new strategies to eradicate biofilm infections. The second messenger c-di-GMP is a positive regulator of biofilm formation in many clinically relevant bacteria. It is hypothesized that drugs lowering the intracellular level of c-di-GMP will force biofilm bacteria into a more treatable planktonic lifestyle. To identify compounds capable of lowering c-di-GMP levels in Pseudomonas aeruginosa, we screened 5000 compounds for their potential c-di-GMP-lowering effect using a recently developed c-di-GMP biosensor strain. Our screen identified the anti-cancerous drug doxorubicin as a potent c-di-GMP inhibitor. In addition, the drug decreased the transcription of many biofilm-related genes. However, despite its effect on the c-di-GMP content in P. aeruginosa, doxorubicin was unable to inhibit biofilm formation or disperse established biofilms. On the contrary, the drug was found to promote P. aeruginosa biofilm formation, possibly through release of extracellular DNA from a subpopulation of killed bacteria. Our findings emphasize that lowering of the c-di-GMP content in bacteria might not be sufficient to mediate biofilm inhibition or dispersal.

Topics: Antineoplastic Agents; Bacterial Proteins; Biofilms; Cyclic GMP; Doxorubicin; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa

Salmonellosis is one of the most important bacterial zoonotic diseases transmitted through the consumption of contaminated food, with chicken and pig related products being key reservoirs of infection. Although numerous studies on animal vaccination have been performed in order to reduce Salmonella prevalence, there is still a need for an ideal vaccine. Here, with the aim of constructing a novel live attenuated Salmonella vaccine candidate, we firstly analyzed the impact of the absence of cyclic-di-GMP (c-di-GMP) in Salmonella virulence. C-di-GMP is an intracellular second messenger that controls a wide range of bacterial processes, including biofilm formation and synthesis of virulence factors, and also modulates the host innate immune response. Our results showed that a Salmonella multiple mutant in the twelve genes encoding diguanylate cyclase proteins that, as a consequence, cannot synthesize c-di-GMP, presents a moderate attenuation in a systemic murine infection model. An additional mutation of the rpoS gene resulted in a synergic attenuating effect that led to a highly attenuated strain, referred to as ΔXIII, immunogenic enough to protect mice against a lethal oral challenge of a S. Typhimurium virulent strain. ΔXIII immunogenicity relied on activation of both antibody and cell mediated immune responses characterized by the production of opsonizing antibodies and the induction of significant levels of IFN-γ, TNF-α, IL-2, IL-17 and IL-10. ΔXIII was unable to form a biofilm and did not survive under desiccation conditions, indicating that it could be easily eliminated from the environment. Moreover, ΔXIII shows DIVA features that allow differentiation of infected and vaccinated animals. Altogether, these results show ΔXIII as a safe and effective live DIVA vaccine.

Topics: Administration, Oral; Animals; Bacterial Proteins; Cyclic GMP; Female; Interferon-gamma; Interleukin-10; Interleukin-17; Interleukin-2; Mice; Mice, Inbred BALB C; Salmonella Infections, Animal; Salmonella typhimurium; Salmonella Vaccines; Sigma Factor; Tumor Necrosis Factor-alpha; Vaccines, Attenuated

Mycobacterium tuberculosis (Mtb) has been a threat to humans since ancient times, and it is the main causative agent of tuberculosis (TB). Until today, the only licensed vaccine against Mtb is the live attenuated M. bovis Bacillus Calmette-Guérin (BCG), which has variable levels of protection against the pulmonary form of infection. The quest for a new vaccine is a priority given the rise of multidrug-resistant Mtb around the world, as well as the tremendous burden imposed by latent TB. The objective of this study was to evaluate the immunogenicity and capacity of protection of a modified BCG strain (BCGΔBCG1419c) lacking the c-di-GMP phosphodiesterase gene BCG1419c, in diverse mice models. In a previous report, we have shown that BCGΔBCG1419c was capable of increasing biofilm production and after intravenous infection of immunocompetent mice; this strain persisted longer in lungs than parental BCG Pasteur. This led us to hypothesize that BCGΔBCG1419c might therefore possess some advantage as vaccine candidate. Our results in this report indicate that compared to conventional BCG, vaccination with BCGΔBCG1419c induced a better activation of specific T-lymphocytes population, was equally effective in preventing weight loss despite being used at lower dose, reduced tissue damage (pneumonic scores), increased local IFNγ(+) T cells, and diminished bacterial burden in lungs of BALB/c mice infected intratracheally with high dose Mtb H37Rv to induce progressive TB. Moreover, vaccination with BCGΔBCG1419c improved resistance to reactivation after immunosuppression induced by corticosterone in a murine model of chronic infection similar to latent TB. Furthermore, despite showing increased persistence in immunocompetent mice, BCGΔBCG1419c was as attenuated as parental BCG in nude mice. To our knowledge, this is the first demonstration that a modified BCG vaccine candidate with increased pellicle/biofilm production has the capacity to protect against Mtb challenge in chronic and reactivation models of infection.

Topics: Animals; Bacterial Load; BCG Vaccine; Cyclic GMP; Female; Latent Tuberculosis; Lung; Mice; Mice, Inbred BALB C; Mice, Nude; Mycobacterium tuberculosis; T-Lymphocytes; Tuberculosis, Pulmonary; Virulence

The pathogenesis of Klebsiella pneumonia is linked to the bacteria's ability to form biofilms. Mannose-resistant Klebsiella-like (Mrk) hemagglutinins are critical for K pneumonia biofilm development, and the expression of the genes encoding these proteins is activated by a 3',5'-cyclic diguanylic acid (c-di-GMP)-regulated transcription factor, MrkH. To gain insight into MrkH function, we performed structural and biochemical analyses. Data revealed MrkH to be a monomer with a two-domain architecture consisting of a PilZ C-domain connected to an N domain that unexpectedly also harbors a PilZ-like fold. Comparison of apo- and c-di-GMP-bound MrkH structures reveals a large 138° interdomain rotation that is induced by binding an intercalated c-di-GMP dimer. c-di-GMP interacts with PilZ C-domain motifs 1 and 2 (RxxxR and D/NxSxxG) and a newly described c-di-GMP-binding motif in the MrkH N domain. Strikingly, these c-di-GMP-binding motifs also stabilize an open state conformation in apo MrkH via contacts from the PilZ motif 1 to residues in the C-domain motif 2 and the c-di-GMP-binding N-domain motif. Use of the same regions in apo structure stabilization and c-di-GMP interaction allows distinction between the states. Indeed, domain reorientation by c-di-GMP complexation with MrkH, which leads to a highly compacted structure, suggests a mechanism by which the protein is activated to bind DNA. To our knowledge, MrkH represents the first instance of specific DNA binding mediated by PilZ domains. The MrkH structures also pave the way for the rational design of inhibitors that target K pneumonia biofilm formation.

Topics: Amino Acid Motifs; Bacterial Proteins; Binding Sites; Biofilms; Cloning, Molecular; Crystallography, X-Ray; Cyclic GMP; DNA, Bacterial; Gene Expression; Gene Expression Regulation, Bacterial; Klebsiella pneumoniae; Models, Molecular; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Folding; Protein Interaction Domains and Motifs; Recombinant Proteins; Transcription Factors

C-di-GMP is a bacterial second messenger regulating various cellular functions. Many bacteria contain c-di-GMP-metabolizing enzymes but lack known c-di-GMP receptors. Recently, two MshE-type ATPases associated with bacterial type II secretion system and type IV pilus formation were shown to specifically bind c-di-GMP. Here we report crystal structure of the MshE N-terminal domain (MshEN1-145) from Vibrio cholerae in complex with c-di-GMP at a 1.37 Å resolution. This structure reveals a unique c-di-GMP-binding mode, featuring a tandem array of two highly conserved binding motifs, each comprising a 24-residue sequence RLGxx(L/V/I)(L/V/I)xxG(L/V/I)(L/V/I)xxxxLxxxLxxQ that binds half of the c-di-GMP molecule, primarily through hydrophobic interactions. Mutating these highly conserved residues markedly reduces c-di-GMP binding and biofilm formation by V. cholerae. This c-di-GMP-binding motif is present in diverse bacterial proteins exhibiting binding affinities ranging from 0.5 μM to as low as 14 nM. The MshEN domain contains the longest nucleotide-binding motif reported to date.

Topics: Adenosine Triphosphatases; Amino Acid Motifs; Bacterial Proteins; Biofilms; Crystallography, X-Ray; Cyclic GMP; Mutation; Protein Binding; Protein Domains; Type II Secretion Systems; Vibrio cholerae

As an important bacterial second messenger, bis-(3',5')-cyclic diguanylate (cyclic di-GMP or c-di-GMP) has been implicated in numerous biological activities, including biofilm formation, motility, survival and virulence. These processes are manipulated by the binding of c-di-GMP to its receptors. XC_3703 from the plant pathogen Xanthomonas campestris pv. campestris, which belongs to the YajQ family of proteins, has recently been identified as a potential c-di-GMP receptor. XC_3703, together with XC_2801, functions as a transcription factor activating virulence-related genes, which can be reversed by the binding of c-di-GMP to XC_3703. However, the structural basis of how c-di-GMP regulates XC_3703 remains elusive. In this study, the structure of XC_3703 was determined to 2.1 Å resolution using the molecular-replacement method. The structure of XC_3703 consists of two domains adopting the same topology, which is similar to that of the RNA-recognition motif (RRM). Arg65, which is conserved among the c-di-GMP-binding subfamily of the YajQ family of proteins, together with Phe80 in domain II, forms a putative c-di-GMP binding site.

Topics: Amino Acid Sequence; Arginine; Bacterial Proteins; Binding Sites; Cloning, Molecular; Crystallography, X-Ray; Cyclic GMP; Escherichia coli; Gene Expression; Models, Molecular; Phenylalanine; Plasmids; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Recombinant Proteins; RNA-Binding Proteins; Second Messenger Systems; Sequence Alignment; Structural Homology, Protein; Xanthomonas campestris

Klebsiella pneumoniae is the predominant pathogen isolated from liver abscesses of diabetic patients in Asian countries. However, the effects of elevated blood glucose levels on the virulence of this pathogen remain largely unknown. Type 3 fimbriae, encoded by the mrkABCDF genes, are important virulence factors in K. pneumoniae pathogenesis. In this study, the effects of exogenous glucose and the intracellular cyclic AMP (cAMP) signaling pathway on type 3 fimbriae expression regulation were investigated. The production of MrkA, the major subunit of type 3 fimbriae, was increased in glucose-rich medium, whereas cAMP supplementation reversed the effect. MrkA production was markedly increased by cyaA or crp deletion, but slightly decreased by cpdA deletion. In addition, the mRNA levels of mrkABCDF genes and the activity of PmrkA were increased in Δcrp strain, as well as the mRNA levels of mrkHIJ genes that encode cyclic di-GMP (c-di-GMP)-related regulatory proteins that influence type 3 fimbriae expression. Moreover, the activities of PmrkHI and PmrkJ were decreased in ΔlacZΔcrp strain. These results indicate that CRP-cAMP down-regulates mrkABCDF and mrkHIJ at the transcriptional level. Further deletion of mrkH or mrkI in Δcrp strain diminished the production of MrkA, indicating that MrkH and MrkI are required for the CRP regulation of type 3 fimbriae expression. Furthermore, the high activity of PmrkHI in the ΔlacZΔcrp strain was diminished in ΔlacZΔcrpΔmrkHI, but increased in the ΔlacZΔcrpΔmrkJ strain. Deletion of crp increased the intracellular c-di-GMP concentration and reduced the phosphodiesterase activity. Moreover, we found that the mRNA levels of multiple genes related to c-di-GMP metabolism were altered in Δcrp strain. These indicate that CRP regulates type 3 fimbriae expression indirectly via the c-di-GMP signaling pathway. In conclusion, we found evidence of a coordinated regulation of type 3 fimbriae expression by the CRP-cAMP and c-di-GMP signaling pathways in K. pneumoniae.

Topics: Bacterial Proteins; C-Reactive Protein; Cyclic AMP; Cyclic GMP; Fimbriae, Bacterial; Glucose; Klebsiella pneumoniae

Most bacteria alternate between a free living planktonic lifestyle and the formation of structured surface-associated communities named biofilms. The transition between these two lifestyles requires a precise and timely regulation of the factors involved in each of the stages that has been likened to a developmental process. Here we characterize the involvement of the transcriptional regulator FleQ and the second messenger cyclic diguanylate in the coordinate regulation of multiple functions related to motility and surface colonization in Pseudomonas putida. Disruption of fleQ caused strong defects in flagellar motility, biofilm formation and surface attachment, and the ability of this mutation to suppress multiple biofilm-related phenotypes associated to cyclic diguanylate overproduction suggests that FleQ mediates cyclic diguanylate signaling critical to biofilm growth. We have constructed a library containing 94 promoters potentially involved in motility and biofilm development fused to gfp and lacZ, screened this library for FleQ and cyclic diguanylate regulation, and assessed the involvement of alternative σ factors σN and FliA in the transcription of FleQ-regulated promoters. Our results suggest a dual mode of action for FleQ. Low cyclic diguanylate levels favor FleQ interaction with σN-dependent promoters to activate the flagellar cascade, encompassing the flagellar cluster and additional genes involved in cyclic diguanylate metabolism, signal transduction and gene regulation. On the other hand, characterization of the FleQ-regulated σN- and FliA-independent PlapA and PbcsD promoters revealed two disparate regulatory mechanisms leading to a similar outcome: the synthesis of biofilm matrix components in response to increased cyclic diguanylate levels.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Promoter Regions, Genetic; Pseudomonas aeruginosa; Sigma Factor; Trans-Activators

This study revealed that the gross indicators commonly adopted for monitoring the performance of aerobic granular sludge processes are not capable of probing the microstructural deterioration of granule interior upon organic loading swaps. These granules subjected to loading swaps retained their global characteristics: appearances, sizes and settling velocities, chemical oxygen demand (COD) and ammonia-nitrogen removal capacities. However, the granule interior strength, as determined by ultrasound method, was largely weakened upon COD switch-off and was not recovered in the subsequent COD re-supply stage. In response to COD switch-off, the 5.6kDa polysaccharides component of granule extracellular polymeric substances (EPS) was diminished. Correspondingly, two bacterial species, Thauera and Sphingomonas sp., were faded away together with the significant decline in contents of intracellular cyclic dimeric GMP (c-di-GMP). The microstructural integrity of granules was seriously deteriorated upon COD switch-off, which was not detectable by the commonly adopted gross indicators.

Topics: Aerobiosis; Biological Oxygen Demand Analysis; Bioreactors; Cyclic GMP; Homoserine; Lactones; Microbial Consortia; Polysaccharides; Sewage; Waste Disposal, Fluid

Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host-Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a ΔbpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the ΔbpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase ΔcgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, ΔbpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection.

Topics: Adaptation, Physiological; Animals; Biofilms; Brucella; Brucellosis; Cells, Cultured; Cyclic GMP; Genetic Fitness; Macrophages; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mutation; Type IV Secretion Systems; Virulence

Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger that regulates diverse cellular processes, is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). GcbA is a well conserved DGC among Pseudomonas species, and has been reported to influence biofilm formation and flagellar motility in Pseudomonas fluorescens and Pseudomonas aeruginosa. Here we confirm the function of GcbA in Pseudomonas putida and reveal that expression of GcbA is regulated by FleQ in response to c-di-GMP. GcbA deletion impaired initial biofilm formation and enhanced swimming motility, but showed no influence on biofilm maturation in Pseudomonas putida. Deletion of the c-di-GMP effector FleQ led to a significant decrease in transcription of gcbA. Moreover, reducing c-di-GMP levels promoted gcbA transcription in a FleQ dependent way, while enhancing c-di-GMP levels abolished the promotion. In in vitro experiments we found that FleQ bound to gcbA promoter DNA and the binding was inhibited by c-di-GMP. Besides, FleN, an anti-activator of FleQ, and the sigma factor RpoN also participated in transcription of gcbA. Our finding expands the complexity of FleQ-dependent regulation and reveals a self-regulation function of c-di-GMP by regulating GcbA expression via FleQ.

Topics: Cyclic GMP; DNA, Bacterial; Escherichia coli Proteins; Gene Deletion; Gene Expression Regulation; Phosphorus-Oxygen Lyases; Promoter Regions, Genetic; Protein Binding; Pseudomonas putida; Trans-Activators

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that controls diverse functions in bacteria, including transitions from planktonic to biofilm lifestyles, virulence, motility, and cell cycle. Here we describe TolR, a hybrid two-component system (HTCS), from the β-proteobacterium Azoarcus sp. strain CIB that degrades c-di-GMP in response to aromatic hydrocarbons, including toluene. This response protects cells from toluene toxicity during anaerobic growth. Whereas wild-type cells tolerated a sudden exposure to a toxic concentration of toluene, a tolR mutant strain or a strain overexpressing a diguanylate cyclase gene lost viability upon toluene shock. TolR comprises an N-terminal aromatic hydrocarbon-sensing Per-Arnt-Sim (PAS) domain, followed by an autokinase domain, a response regulator domain, and a C-terminal c-di-GMP phosphodiesterase (PDE) domain. Autophosphorylation of TolR in response to toluene exposure initiated an intramolecular phosphotransfer to the response regulator domain that resulted in c-di-GMP degradation. The TolR protein was engineered as a functional sensor histidine kinase (TolR

Topics: Azoarcus; Bacterial Proteins; Cyclic GMP; Membrane Proteins; Toluene

The bacterial messenger cyclic diguanylate monophosphate (c-di-GMP) binds to various effectors, the most common of which are single-domain PilZ proteins. These c-di-GMP effectors control various cellular functions and multicellular behaviors at the transcriptional or posttranslational level. We found that MapZ (methyltransferase-associated PilZ; formerly known as PA4608), a single-domain PilZ protein from the opportunistic pathogen Pseudomonas aeruginosa, directly interacted with the methyltransferase CheR1 and that this interaction was enhanced by c-di-GMP. In vitro assays indicated that, in the presence of c-di-GMP, MapZ inhibited CheR1 from methylating the chemoreceptor PctA, which would be expected to increase its affinity for chemoattractants and promote chemotaxis. MapZ localized to the poles of P. aeruginosa cells, where the flagellar motor and other chemotactic proteins, including PctA and CheR1, are also located. P. aeruginosa cells exhibit a random walk behavior by frequently switching the direction of flagellar rotation in a uniform solution. We showed that binding of c-di-GMP to MapZ decreased the frequency of flagellar motor switching and that MapZ was essential for generating the heterogeneous motility typical of P. aeruginosa cell populations and for efficient surface attachment during biofilm formation. Collectively, the studies revealed that c-di-GMP affects flagellar motor output by regulating the methylation of chemoreceptors through a single-domain PilZ adaptor protein.

Topics: Adaptor Proteins, Signal Transducing; Bacterial Proteins; Chemotaxis; Cyclic GMP; Flagella; Methyltransferases; Pseudomonas aeruginosa

The c-di-GMP network of Borrelia burgdorferi, a causative agent of Lyme disease, consists of Rrp1, a diguanylate cyclase/response regulator; Hpk1, a histidine kinase; PdeA and PdeB, c-di-GMP phosphodiesterases; and PlzA, a PilZ domain c-di-GMP receptor. Borrelia hermsii, a causative agent of tick-borne relapsing fever, possesses a putative c-di-GMP regulatory network that is uncharacterized. While B. burgdorferi requires c-di-GMP to survive within ticks, the associated effector mechanisms are poorly defined. Using site-directed mutagenesis, size exclusion chromatography, isothermal titration calorimetry and fluorescence resonance energy transfer, we investigate the interaction of c-di-GMP with the Borrelia PilZ domain-containing Plz proteins: B. burgdorferi PlzA and B. hermsii PlzC. The Plz proteins were determined to be monomeric in their apo and holo forms and to bind c-di-GMP with high affinity with a 1:1 stoichiometry. C-di-GMP binding induced structural rearrangements in PlzA and PlzC. C-di-GMP binding proved to be dependent on positive charge at R

Topics: Amino Acid Substitution; Bacterial Proteins; Borrelia burgdorferi; Cyclic GMP; Humans; Lyme Disease; Mutation; Protein Binding; Protein Conformation; Protein Multimerization; Relapsing Fever; Spirochaetaceae

The frequent outbreaks of rice foot rot disease caused by Dickeya zeae have become a significant concern in rice planting regions and countries, but the regulatory mechanisms that govern the virulence of this important pathogen remain vague. Given that the second messenger cyclic di-GMP (c-di-GMP) is associated with modulation of various virulence-related traits in various microorganisms, here we set to investigate the role of the genes encoding c-di-GMP metabolism in the regulation of the bacterial physiology and virulence by construction all in-frame deletion mutants targeting the annotated c-di-GMP turnover genes in D. zeae strain EC1. Phenotype analyses identified individual mutants showing altered production of exoenzymes and phytotoxins, biofilm formation and bacterial motilities. The results provide useful clues and a valuable toolkit for further characterization and dissection of the regulatory complex that modulates the pathogenesis and persistence of this important bacterial pathogen.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Enterobacteriaceae; Flagella; Genetic Association Studies; Macrolides; Mutation; Oryza; Plant Diseases; Polyamines; Protein Interaction Domains and Motifs; Quantitative Trait, Heritable; Virulence; Virulence Factors

The host immune system offers a hostile environment with antimicrobials and reactive oxygen species (ROS) that are detrimental to bacterial pathogens, forcing them to adapt and evolve for survival. However, the contribution of oxidative stress to pathogen evolution remains elusive. Using an experimental evolution strategy, we show that exposure of the opportunistic pathogen Pseudomonas aeruginosa to sub-lethal hydrogen peroxide (H

Topics: Bacterial Proteins; Biofilms; Biological Evolution; Cyclic GMP; Gene Expression Regulation, Bacterial; Glutathione; Hydrogen Peroxide; Pseudomonas aeruginosa; Reactive Oxygen Species

Bacteria sense their environment to alter phenotypes, including biofilm formation, to survive changing conditions. Heme proteins play important roles in sensing the bacterial gaseous environment and controlling the switch between motile and sessile (biofilm) states. Globin coupled sensors (GCS), a family of heme proteins consisting of a globin domain linked by a central domain to an output domain, are often found with diguanylate cyclase output domains that synthesize c-di-GMP, a major regulator of biofilm formation. Characterization of diguanylate cyclase-containing GCS proteins from Bordetella pertussis and Pectobacterium carotovorum demonstrated that cyclase activity is controlled by ligand binding to the heme within the globin domain. Both O

Topics: Amino Acid Sequence; Bacterial Proteins; Binding Sites; Biocatalysis; Biofilms; Bordetella pertussis; Cyclic GMP; Escherichia coli Proteins; Globins; Heme; Hemeproteins; Kinetics; Models, Molecular; Mutation; Oxygen; Pectobacterium carotovorum; Phosphorus-Oxygen Lyases; Protein Binding; Protein Domains; Protein Multimerization; Sequence Homology, Amino Acid

The second messenger c-di-GMP (or cyclic diguanylate) regulates biofilm formation, a physiological adaptation process in bacteria, via a widely conserved signaling node comprising a prototypical transmembrane receptor for c-di-GMP, LapD, and a cognate periplasmic protease, LapG. Previously, we reported a structure-function study of a soluble LapD•LapG complex, which established conformational changes in the receptor that lead to c-di-GMP-dependent protease recruitment (Chatterjee et al., 2014). This work also revealed a basal affinity of c-di-GMP-unbound receptor for LapG, the relevance of which remained enigmatic. Here, we elucidate the structural basis of coincidence detection that relies on both c-di-GMP and LapG binding to LapD for receptor activation. The data indicate that high-affinity for LapG relies on the formation of a receptor dimer-of-dimers, rather than a simple conformational change within dimeric LapD. The proposed mechanism provides a rationale of how external proteins can regulate receptor function and may also apply to c-di-GMP-metabolizing enzymes that are akin to LapD.

Topics: Bacterial Proteins; Cyclic GMP; Models, Biological; Protein Conformation; Protein Multimerization; Pseudomonas fluorescens; Scattering, Small Angle; Second Messenger Systems; Signal Transduction

Flagella-mediated swimming and swarming motility in Salmonella enterica serovar Typhimurium is intercalated with the cyclic di-guanylate monophosphate (c-di-GMP) signalling network. In this study, we identified the GGDEF domain proteins STM2672, STM4551 and STM1987 as key di-guanylate cyclases involved in regulation of motility in a ΔyhjH phosphodiesterase gene deletion mutant with elevated c-di-GMP levels inhibiting motility. Surprisingly, these di-guanylate cyclases distinctively inhibited motility through the c-di-GMP receptors YcgR and the cellulose synthase BcsA, whereby STM2672 corresponded to YcgR, STM1987 to BcsA and STM4551 to both receptors. Although downregulation of motility is believed to prepare the bacterial cells for surface adhesion and biofilm formation, the major biofilm regulator CsgD of S. sv. Typhimurium was not involved in the regulation of swimming or swarming motility. Together with previously identified c-di-GMP networks regulating flagella-related phenotypes, flagella biosynthesis is a major target of c-di-GMP signalling in S. sv. Typhimurium.

Topics: Bacterial Adhesion; Biofilms; Carrier Proteins; Cyclic GMP; Flagella; Gene Deletion; Gene Expression Regulation, Bacterial; Glucosyltransferases; Phosphoric Diester Hydrolases; Protein Structure, Tertiary; Salmonella typhimurium; Trans-Activators

Persisters are bacteria that are highly tolerant to antibiotics due to their dormant state and are of clinical significance owing to their role in infections. Given that the population of persisters increases in biofilms and that cyclic diguanylate (c-di-GMP) is an intracellular signal that increases biofilm formation, we sought to determine whether c-di-GMP has a role in bacterial persistence. By examining the effect of 30 genes from Escherichia coli, including diguanylate cyclases that synthesize c-di-GMP and phosphodiesterases that breakdown c-di-GMP, we determined that DosP (direct oxygen sensing phosphodiesterase) increases persistence by over a thousand fold. Using both transcriptomic and proteomic approaches, we determined that DosP increases persistence by decreasing tryptophanase activity and thus indole. Corroborating this effect, addition of indole reduced persistence. Despite the role of DosP as a c-di-GMP phosphodiesterase, the decrease in tryptophanase activity was found to be a result of cyclic adenosine monophosphate (cAMP) phosphodiesterase activity. Corroborating this result, the reduction of cAMP via CpdA, a cAMP-specific phosphodiesterase, increased persistence and reduced indole levels similarly to DosP. Therefore, phosphodiesterase DosP increases persistence by reducing the interkingdom signal indole via reduction of the global regulator cAMP.

Topics: Cyclic AMP; Cyclic GMP; Drug Resistance, Bacterial; Escherichia coli; Escherichia coli Proteins; Indoles; Phosphoric Diester Hydrolases

The bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous second messenger that determines bacterial lifestyle between the planktonic and biofilm modes of life. Although the role of c-di-GMP signaling in biofilm development and dispersal has been extensively studied, how c-di-GMP signaling influences environmental bioprocess activities such as biodegradation remains unexplored. To elucidate the impacts of elevating c-di-GMP level on environmental bioprocesses, we constructed a Comamonas testosteroni strain constitutively expressing a c-di-GMP synthase YedQ from Escherichia coli and examined its capability in biofilm formation and biodegradation of 3-chloroaniline (3-CA). The high c-di-GMP strain exhibited an increased binding to Congo red dye, a decreased motility, and an enhanced biofilm formation capability. In planktonic cultures, the strain with an elevated c-di-GMP concentration and the wild type could degrade 3-CA comparably well. However, under batch growth conditions with a high surface to volume ratio, an elevated c-di-GMP concentration in C. testosteroni significantly increased the contribution of biofilms in 3-CA biodegradation. In continuous submerged biofilm reactors, C. testosteroni with an elevated c-di-GMP level exhibited an enhanced 3-CA biodegradation and a decreased cell detachment rate. Taken together, this study provides a novel strategy to enhance biofilm-based biodegradation of toxic xenobiotic compounds through manipulating bacterial c-di-GMP signaling.

Topics: Aniline Compounds; Biofilms; Bioreactors; Biotransformation; Comamonas testosteroni; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression; Phosphorus-Oxygen Lyases; Recombinant Proteins; Second Messenger Systems

Biofilm dispersion is a highly regulated process that allows biofilm bacteria to respond to changing environmental conditions and to disseminate to new locations. The dispersion of biofilms formed by the opportunistic pathogen Pseudomonas aeruginosa is known to require a number of cyclic di-GMP (c-di-GMP)-degrading phosphodiesterases (PDEs) and the chemosensory protein BdlA, with BdlA playing a pivotal role in regulating PDE activity and enabling dispersion in response to a wide array of cues. BdlA is activated during biofilm growth via posttranslational modifications and nonprocessive cleavage in a manner that is dependent on elevated c-di-GMP levels. Here, we provide evidence that the diguanylate cyclase (DGC) GcbA contributes to the regulation of BdlA cleavage shortly after initial cellular attachment to surfaces and, thus, plays an essential role in allowing biofilm cells to disperse in response to increasing concentrations of a variety of substances, including carbohydrates, heavy metals, and nitric oxide. DGC activity of GcbA was required for its function, as a catalytically inactive variant could not rescue impaired BdlA processing or the dispersion-deficient phenotype of gcbA mutant biofilms to wild-type levels. While modulating BdlA cleavage during biofilm growth, GcbA itself was found to be subject to c-di-GMP-dependent and growth-mode-specific regulation. GcbA production was suppressed in mature wild-type biofilms and could be induced by reducing c-di-GMP levels via overexpression of genes encoding PDEs. Taken together, the present findings demonstrate that the regulatory functions of c-di-GMP-synthesizing DGCs expand beyond surface attachment and biofilm formation and illustrate a novel role for DGCs in the regulation of the reverse sessile-motile transition of dispersion.

Topics: Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Phosphorus-Oxygen Lyases; Protein Processing, Post-Translational; Pseudomonas aeruginosa

The second messenger cyclic diguanylate (c-di-GMP) plays a critical role in the regulation of motility. In Pseudomonas aeruginosa PA14, c-di-GMP inversely controls biofilm formation and surface swarming motility, with high levels of this dinucleotide signal stimulating biofilm formation and repressing swarming. P. aeruginosa encodes two stator complexes, MotAB and MotCD, that participate in the function of its single polar flagellum. Here we show that the repression of swarming motility requires a functional MotAB stator complex. Mutating the motAB genes restores swarming motility to a strain with artificially elevated levels of c-di-GMP as well as stimulates swarming in the wild-type strain, while overexpression of MotA from a plasmid represses swarming motility. Using point mutations in MotA and the FliG rotor protein of the motor supports the conclusion that MotA-FliG interactions are critical for c-di-GMP-mediated swarming inhibition. Finally, we show that high c-di-GMP levels affect the localization of a green fluorescent protein (GFP)-MotD fusion, indicating a mechanism whereby this second messenger has an impact on MotCD function. We propose that when c-di-GMP level is high, the MotAB stator can displace MotCD from the motor, thereby affecting motor function. Our data suggest a newly identified means of c-di-GMP-mediated control of surface motility, perhaps conserved among Pseudomonas, Xanthomonas, and other organisms that encode two stator systems.

Topics: Bacterial Proteins; Cyclic GMP; Gene Deletion; Gene Expression; Gene Expression Regulation, Bacterial; Locomotion; Point Mutation; Pseudomonas aeruginosa

There are numerous ways by which cyclic dimeric GMP (c-di-GMP) inhibits motility. Kuchma et al. (S. L. Kuchma, N. J. Delalez, L. M. Filkins, E. A. Snavely, J. P. Armitage, and G. A. O'Toole, J. Bacteriol. 197:420-430, 2015, http://dx.doi.org/10.1128/JB.02130-14) offer a new, previously unseen way of swarming motility inhibition in Pseudomonas aeruginosa PA14. This bacterium possesses a single flagellum with one rotor and two sets of stators, only one of which can provide torque for swarming. The researchers discovered that elevated levels of c-di-GMP inhibit swarming by skewing stator selection in favor of the nonfunctional, "bad" stators.

Topics: Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Locomotion; Pseudomonas aeruginosa

Aerobic granulation is a novel biotechnology for wastewater treatment. This study refined existing aerobic granulation mechanisms as a sequencing process including formation of calcium precipitate under alkaline pH to form inorganic cores, followed by bacterial attachment and growth on these cores to form the exopolysaccharide matrix. Mature granules comprised an inner core and a matrix layer and a rim layer with enriched microbial strains. The inorganic core was a mix of different crystals of calcium and phosphates. Functional strains including Sphingomonas sp., Paracoccus sp. Sinorhizobium americanum strain and Flavobacterium sp. attached onto the cores. These functional strains promote c-di-GMP production and the expression by Psl and Alg genes for exopolysaccharide production to enhance formation of mature granules.

Topics: Aerobiosis; Bacteria, Aerobic; Biological Oxygen Demand Analysis; Biotechnology; Calcium; Chemical Precipitation; Cyclic GMP; DNA Primers; Hydrogen-Ion Concentration; Microscopy, Electron, Scanning; Models, Biological; Phylogeny; Polysaccharides, Bacterial; Real-Time Polymerase Chain Reaction; Wastewater; Water Purification

XAC0610, from Xanthomonas citri subsp. citri, is a large multi-domain protein containing one GAF (cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA) domain, four PAS (Per-Arnt-Sim) domains and one GGDEF domain. This protein has a demonstrable in vivo and in vitro diguanylate cyclase (DGC) activity that leads to the production of cyclic di-GMP (c-di-GMP), a ubiquitous bacterial signaling molecule. Analysis of a XacΔ0610 knockout strain revealed that XAC0610 plays a role in the regulation of Xac motility and resistance to H2O2. Site-directed mutagenesis of a conserved DGC lysine residue (Lys759 in XAC0610) resulted in a severe reduction in XAC0610 DGC activity. Furthermore, experimental and in silico analyses suggest that XAC0610 is not subject to allosteric product inhibition, a common regulatory mechanism for DGC activity control. Instead, steady-state kinetics of XAC0610 DGC activity revealed a positive cooperative effect of the GTP substrate with a dissociation constant for the binding of the first GTP molecule (K1) approximately 5× greater than the dissociation constant for the binding of the second GTP molecule (K2). We present a general kinetics scheme that should be used when analyzing DGC kinetics data and propose that cooperative GTP binding could be a common, though up to now overlooked, feature of these enzymes that may in some cases offer a physiologically relevant mechanism for regulation of DGC activity in vivo.

Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; Circular Dichroism; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Hydrogen Peroxide; Hydrogen-Ion Concentration; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphorus-Oxygen Lyases; Plasmids; Protein Binding; Sequence Alignment; Substrate Specificity; Xanthomonas

Cell death in the model organism Dictyostelium, as studied in monolayers in vitro, can be induced by the polyketide DIF-1 or by the cyclical dinucleotide c-di-GMP. c-di-GMP, a universal bacterial second messenger, can trigger innate immunity in bacterially infected animal cells and is involved in developmental cell death in Dictyostelium. We show here that c-di-GMP was not sufficient to induce cell death in Dictyostelium cell monolayers. Unexpectedly, it also required the DIF-1 polyketide. The latter could be exogenous, as revealed by a telling synergy between c-di-GMP and DIF-1. The required DIF-1 polyketide could also be endogenous, as shown by the inability of c-di-GMP to induce cell death in Dictyostelium HMX44A cells and DH1 cells upon pharmacological or genetic inhibition of DIF-1 biosynthesis. In these cases, c-di-GMP-induced cell death was rescued by complementation with exogenous DIF-1. Taken together, these results demonstrated that c-di-GMP could trigger cell death in Dictyostelium only in the presence of the DIF-1 polyketide or its metabolites. This identified another element of control to this cell death and perhaps also to c-di-GMP effects in other situations and organisms.

Topics: Cell Death; Cyclic GMP; Dictyostelium; Polyketides; Second Messenger Systems; Signal Transduction; Species Specificity

Recognition of pathogen-derived nucleic acids by immune cells is critical for the activation of protective innate immune responses. Bacterial cyclic dinucleotides (CDNs) are small nucleic acids that are directly recognized by the cytosolic DNA sensor STING (stimulator of IFN genes), initiating a response characterized by proinflammatory cytokine and type I IFN production. Strategies to improve the immune stimulatory activities of CDNs can further their potential for clinical development. Here, we demonstrate that a simple complex of cylic-di-GMP with a cell-penetrating peptide enhances both cellular delivery and biological activity of the cyclic-di-GMP in murine splenocytes. Furthermore, our findings establish that activation of the TLR-dependent and TLR-independent DNA recognition pathways through combined use of CpG oligonucleotide (ODN) and CDN results in synergistic activity, augmenting cytokine production (IFN-α/β, IL-6, TNF-α, IP-10), costimulatory molecule upregulation (MHC class II, CD86), and antigen-specific humoral and cellular immunity. Results presented herein indicate that 3'3'-cGAMP, a recently identified bacterial CDN, is a superior stimulator of IFN genes ligand than cyclic-di-GMP in human PBMCs. Collectively, these findings suggest that the immune-stimulatory properties of CDNs can be augmented through peptide complexation or synergistic use with CpG oligonucleotide and may be of interest for the development of CDN-based immunotherapeutic agents.

Topics: Adjuvants, Immunologic; Animals; Cell-Penetrating Peptides; CpG Islands; Cyclic GMP; Cytokines; Humans; Immunity, Innate; Interferon Type I; Membrane Proteins; Mice; Mice, Inbred C57BL; Nucleotides, Cyclic; Oligodeoxyribonucleotides; Peptides; Spleen; Tumor Cells, Cultured

The second messenger molecule cyclic diguanylate is essential for Yersinia pestis biofilm formation that is important for blockage-dependent plague transmission from fleas to mammals. Two diguanylate cyclases (DGCs) HmsT and Y3730 (HmsD) are responsible for biofilm formation in vitro and biofilm-dependent blockage in the oriental rat flea Xenopsylla cheopis respectively. Here, we have identified a tripartite signalling system encoded by the y3729-y3731 operon that is responsible for regulation of biofilm formation in different environments. We present genetic evidence that a putative inner membrane-anchored protein with a large periplasmic domain Y3729 (HmsC) inhibits HmsD DGC activity in vitro while an outer membrane Pal-like putative lipoprotein Y3731 (HmsE) counteracts HmsC to activate HmsD in the gut of X. cheopis. We propose that HmsE is a critical element in the transduction of environmental signal(s) required for HmsD-dependent biofilm formation.

Topics: Animals; Base Sequence; Biofilms; Cyclic GMP; DNA, Bacterial; Escherichia coli Proteins; Phosphorus-Oxygen Lyases; Plague; Rats; Sequence Analysis, DNA; Signal Transduction; Xenopsylla; Yersinia pestis

Anthranilate and indole are alternative degradation products of tryptophan, depending on the bacterial species. While indole enhances the biofilm formation of Pseudomonas aeruginosa, we found that anthranilate, the tryptophan degradation product of P. aeruginosa, had an opposite effect on P. aeruginosa biofilm formation, in which anthranilate deteriorated the mushroom structure of biofilm. The anthranilate effect on biofilm formation was differentially exerted depending on the developmental stage and the presence of shear force. Anthranilate slightly accelerated the initial attachment of P. aeruginosa at the early stage of biofilm development and appeared to build more biofilm without shear force. But anthranilate weakened the biofilm structure in the late stage, deteriorating the mushroom structure of biofilms with shear force to make a flat biofilm. To investigate the interplay of anthranilate with indole in biofilm formation, biofilms were cotreated with anthranilate and indole, and the results showed that anthranilate antagonized the biofilm-enhancing effect of indole. Anthranilate was able to deteriorate the preformed biofilm. The effect of anthranilate and indole on biofilm formation was quorum sensing independent. AntR, a regulator of anthranilate-degrading metabolism was synergistically activated by cotreatment with anthranilate and indole, suggesting that indole might enhance biofilm formation by facilitating the degradation of anthranilate. Anthranilate slightly but significantly affected the cyclic diguaniylate (c-di-GMP) level and transcription of major extracellular polysaccharide (Psl, Pel, and alginate) operons. These results suggest that anthranilate may be a promising antibiofilm agent and antagonize the effect of indole on P. aeruginosa biofilm formation.

Topics: Anti-Bacterial Agents; Biofilms; Cyclic GMP; Indoles; ortho-Aminobenzoates; Polysaccharides, Bacterial; Pseudomonas aeruginosa; Transcription, Genetic

Biofilms are surface-attached multicellular communities. Using single-cell tracking microscopy, we showed that a pilY1 mutant of Pseudomonas aeruginosa is defective in early biofilm formation. We leveraged the observation that PilY1 protein levels increase on a surface to perform a genetic screen to identify mutants altered in surface-grown expression of this protein. Based on our genetic studies, we found that soon after initiating surface growth, cyclic AMP (cAMP) levels increase, dependent on PilJ, a chemoreceptor-like protein of the Pil-Chp complex, and the type IV pilus (TFP). cAMP and its receptor protein Vfr, together with the FimS-AlgR two-component system (TCS), upregulate the expression of PilY1 upon surface growth. FimS and PilJ interact, suggesting a mechanism by which Pil-Chp can regulate FimS function. The subsequent secretion of PilY1 is dependent on the TFP assembly system; thus, PilY1 is not deployed until the pilus is assembled, allowing an ordered signaling cascade. Cell surface-associated PilY1 in turn signals through the TFP alignment complex PilMNOP and the diguanylate cyclase SadC to activate downstream cyclic di-GMP (c-di-GMP) production, thereby repressing swarming motility. Overall, our data support a model whereby P. aeruginosa senses the surface through the Pil-Chp chemotaxis-like complex, TFP, and PilY1 to regulate cAMP and c-di-GMP production, thereby employing a hierarchical regulatory cascade of second messengers to coordinate its program of surface behaviors.. Biofilms are surface-attached multicellular communities. Here, we show that a stepwise regulatory circuit, involving ordered signaling via two different second messengers, is required for Pseudomonas aeruginosa to control early events in cell-surface interactions. We propose that our studies have uncovered a multilayered "surface-sensing" system that allows P. aeruginosa to effectively coordinate its surface-associated behaviors. Understanding how cells transition into the biofilm state on a surface may provide new approaches to prevent formation of these communities.

Topics: Bacterial Proteins; Biofilms; Cyclic AMP; Cyclic GMP; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa; Second Messenger Systems

The bacterial second messengers (p)ppGpp and bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulate important functions, such as transcription, virulence, biofilm formation, and quorum sensing. In mycobacteria, they regulate long-term survival during starvation, pathogenicity, and dormancy. Recently, a Pseudomonas aeruginosa strain lacking (p)ppGpp was shown to be sensitive to multiple classes of antibiotics and defective in biofilm formation. We were interested to find out whether Mycobacterium smegmatis strains lacking the gene for either (p)ppGpp synthesis (ΔrelMsm) or c-di-GMP synthesis (ΔdcpA) would display similar phenotypes. We used phenotype microarray technology to compare the growth of the wild-type and the knockout strains in the presence of several antibiotics. Surprisingly, the ΔrelMsm and ΔdcpA strains showed enhanced survival in the presence of many antibiotics, but they were defective in biofilm formation. These strains also displayed altered surface properties, like impaired sliding motility, rough colony morphology, and increased aggregation in liquid cultures. Biofilm formation and surface properties are associated with the presence of glycopeptidolipids (GPLs) in the cell walls of M. smegmatis. Thin-layer chromatography analysis of various cell wall fractions revealed that the levels of GPLs and polar lipids were reduced in the knockout strains. As a result, the cell walls of the knockout strains were significantly more hydrophobic than those of the wild type and the complemented strains. We hypothesize that reduced levels of GPLs and polar lipids may contribute to the antibiotic resistance shown by the knockout strains. Altogether, our data suggest that (p)ppGpp and c-di-GMP may be involved in the metabolism of glycopeptidolipids and polar lipids in M. smegmatis.

Topics: Anti-Bacterial Agents; Bacterial Adhesion; Biofilms; Chromatography, Thin Layer; Cyclic GMP; Gene Deletion; Glycolipids; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Ligases; Locomotion; Microarray Analysis; Microbial Viability; Mycobacterium smegmatis; Phenotype; Phospholipids; Surface Properties

An artificial increase of cyclic diguanylate (c-di-GMP) levels in Sinorhizobium meliloti 8530, a bacterium that does not carry known cellulose synthesis genes, leads to overproduction of a substance that binds the dyes Congo red and calcofluor. Sugar composition and methylation analyses and NMR studies identified this compound as a linear mixed-linkage (1 → 3)(1 → 4)-β-D-glucan (ML β-glucan), not previously described in bacteria but resembling ML β-glucans found in plants and lichens. This unique polymer is hydrolyzed by the specific endoglucanase lichenase, but, unlike lichenan and barley glucan, it generates a disaccharidic → 4)-β-D-Glcp-(1 → 3)-β-D-Glcp-(1 → repeating unit. A two-gene operon bgsBA required for production of this ML β-glucan is conserved among several genera within the order Rhizobiales, where bgsA encodes a glycosyl transferase with domain resemblance and phylogenetic relationship to curdlan synthases and to bacterial cellulose synthases. ML β-glucan synthesis is subjected to both transcriptional and posttranslational regulation. bgsBA transcription is dependent on the exopolysaccharide/quorum sensing ExpR/SinI regulatory system, and posttranslational regulation seems to involve allosteric activation of the ML β-glucan synthase BgsA by c-di-GMP binding to its C-terminal domain. To our knowledge, this is the first report on a linear mixed-linkage (1 → 3)(1 → 4)-β-glucan produced by a bacterium. The S. meliloti ML β-glucan participates in bacterial aggregation and biofilm formation and is required for efficient attachment to the roots of a host plant, resembling the biological role of cellulose in other bacteria.

Topics: Carbohydrate Sequence; Chromatography, Thin Layer; Cyclic GMP; Medicago sativa; Molecular Sequence Data; Operon; Phylogeny; Plant Roots; Polymerase Chain Reaction; Proteoglycans; Receptors, Transforming Growth Factor beta; Sinorhizobium meliloti; Transcription, Genetic

Elevated levels of the second messenger c-di-GMP activate biosynthesis of an unknown exopolysaccharide (EPS) in the food-borne pathogen Listeria monocytogenes. This EPS strongly protects cells against disinfectants and desiccation, indicating its potential significance for listerial persistence in the environment and for food safety. We analyzed the potential phylogenetic origin of this EPS, determined its complete structure, characterized genes involved in its biosynthesis and hydrolysis and identified diguanylate cyclases activating its synthesis. Phylogenetic analysis of EPS biosynthesis proteins suggests that they have evolved within monoderms. Scanning electron microscopy revealed that L. monocytogenes EPS is cell surface-bound. Secreted carbohydrates represent exclusively cell-wall debris. Based on carbohydrate composition, linkage and NMR analysis, the structure of the purified EPS is identified as a β-1,4-linked N-acetylmannosamine chain decorated with terminal α-1,6-linked galactose. All genes of the pssA-E operon are required for EPS production and so is a separately located pssZ gene. We show that PssZ has an EPS-specific glycosylhydrolase activity. Exogenously added PssZ prevents EPS-mediated cell aggregation and disperses preformed aggregates, whereas an E72Q mutant in the presumed catalytic residue is much less active. The diguanylate cyclases DgcA and DgcB, whose genes are located next to pssZ, are primarily responsible for c-di-GMP-dependent EPS production.

Topics: Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Glycosyltransferases; Hexosamines; Listeria monocytogenes; Microscopy, Electron, Scanning; Operon; Phosphorus-Oxygen Lyases; Phylogeny; Polysaccharides, Bacterial

The nucleotide signalling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays an essential role in regulating microbial virulence and biofilm formation. C-di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. One intrinsic feature of c-di-GMP signalling is the abundance of DGCs and PDEs encoded by many bacterial species. It is unclear whether the different DGCs or PDEs coordinately establish the c-di-GMP regulation or function independently of each other. Here, we provide evidence that multiple DGCs are involved in regulation of c-di-GMP on synthesis of the major iron siderophore pyoverdine in Pseudomonas aeruginosa. Constitutive expression of the WspG or YedQ DGC in P. aeruginosa is able to induce its pyoverdine synthesis. Induction of pyoverdine synthesis by high intracellular c-di-GMP depends on the synthesis of exopolysaccharides and another two DGCs, SiaD and SadC. SiaD was found to boost the c-di-GMP synthesis together with constitutively expressing YedQ. The exopolysaccharides and the SiaD DGC were found to modulate the expression of the RsmY/RsmZ ncRNAs. Induction of the RsmY/RsmZ ncRNAs might enhance the pyoverdine synthesis through SadC. Our study sheds light on a novel multiple DGC-coordinated c-di-GMP regulatory mechanism of bacteria.

Topics: Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Oligopeptides; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa

Biofilm formation is crucial to the environmental survival and transmission of Vibrio cholerae, the facultative human pathogen responsible for the disease cholera. During its infectious cycle, V. cholerae experiences fluctuations in temperature within the aquatic environment and during the transition between human host and aquatic reservoirs. In this study, we report that biofilm formation is induced at low temperatures through increased levels of the signalling molecule, cyclic diguanylate (c-di-GMP). Strains harbouring in frame deletions of all V. cholerae genes that are predicted to encode diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) were screened for their involvement in low-temperature-induced biofilm formation and Vibrio polysaccharide gene expression. Of the 52 mutants tested, deletions of six DGCs and three PDEs were found to affect these phenotypes at low temperatures. Unlike wild type, a strain lacking all six DGCs did not exhibit a low-temperature-dependent increase in c-di-GMP, indicating that these DGCs are required for temperature modulation of c-di-GMP levels. We also show that temperature modulates c-di-GMP levels in a similar fashion in the Gram-negative pathogen Pseudomonas aeruginosa but not in the Gram-positive pathogen Listeria monocytogenes. This study uncovers the role of temperature in environmental regulation of biofilm formation and c-di-GMP signalling.

Topics: Biofilms; Cholera; Cyclic GMP; Escherichia coli Proteins; Humans; Listeria monocytogenes; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa; Sequence Deletion; Signal Transduction; Temperature; Vibrio cholerae

An understanding of biofilm formation is relevant to the design of biological strategies to improve the efficiency of the bioleaching process and to prevent environmental damages caused by acid mine/rock drainage. For this reason, our laboratory is focused on the characterization of the molecular mechanisms involved in biofilm formation in different biomining bacteria. In many bacteria, the intracellular levels of c-di-GMP molecules regulate the transition from the motile planktonic state to sessile community-based behaviors, such as biofilm development, through different kinds of effectors. Thus, we recently started a study of the c-di-GMP pathway in several biomining bacteria including Acidithiobacillus caldus. C-di-GMP molecules are synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). We previously reported the existence of intermediates involved in c-di-GMP pathway from different Acidithiobacillus species. Here, we report our work related to At. caldus ATCC 51756. We identified several putative-ORFs encoding DGC and PDE and effector proteins. By using total RNA extracted from At. caldus cells and RT-PCR, we demonstrated that these genes are expressed. We also demonstrated the presence of c-di-GMP by mass spectrometry and showed that genes for several of the DGC enzymes were functional by heterologous genetic complementation in Salmonella enterica serovar Typhimurium mutants. Moreover, we developed a DGC defective mutant strain (Δc1319) that strongly indicated that the c-di-GMP pathway regulates the swarming motility and adherence to sulfur surfaces by At. caldus. Together, our results revealed that At. caldus possesses a functional c-di-GMP pathway which could be significant for ores colonization during the bioleaching process.

Topics: Acidithiobacillus; Bacterial Adhesion; Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Genetic Complementation Test; Metabolic Networks and Pathways; Mutation; Phosphorus-Oxygen Lyases; Protein Interaction Domains and Motifs; Signal Transduction

Highly pathogenic avian influenza H5N1 infection remains a public health threat and vaccination is the best measure of limiting the impact of a potential pandemic. Mucosal vaccines have the advantage of eliciting immune responses at the site of viral entry, thereby preventing infection as well as further viral transmission. In this study, we assessed the protective efficacy of hemagglutinin (HA) from the A/Indonesia/05/05 (H5N1) strain of influenza virus that was produced by transient expression in plants. The plant-derived vaccine, in combination with the mucosal adjuvant (3',5')-cyclic dimeric guanylic acid (c-di-GMP) was used for intranasal immunization of mice and ferrets, before challenge with a lethal dose of the A/Indonesia/05/05 (H5N1) virus. Mice vaccinated with 15 μg or 5 μg of adjuvanted HA survived the viral challenge, while all control mice died within 10 d of challenge. Vaccinated animals elicited serum hemagglutination inhibition, IgG and IgA antibody titers. In the ferret challenge study, all animals vaccinated with the adjuvanted plant vaccine survived the lethal viral challenge, while 50% of the control animals died. In both the mouse and ferret models, the vaccinated animals were better protected from weight loss and body temperature changes associated with H5N1 infection compared with the non-vaccinated controls. Furthermore, the systemic spread of the virus was lower in the vaccinated animals compared with the controls. Results presented here suggest that the plant-produced HA-based influenza vaccine adjuvanted with c-di-GMP is a promising vaccine/adjuvant combination for the development of new mucosal influenza vaccines.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Cyclic GMP; Disease Models, Animal; Female; Ferrets; Hemagglutinin Glycoproteins, Influenza Virus; Influenza A Virus, H5N1 Subtype; Influenza Vaccines; Male; Mice, Inbred BALB C; Orthomyxoviridae Infections; Plants, Genetically Modified; Survival Analysis; Treatment Outcome; Vaccines, Synthetic

Understanding the numerous functions of RNAs depends critically on the knowledge of their three-dimensional (3D) structure. In contrast to the protein field, a much smaller number of RNA 3D structures have been assessed using X-ray crystallography, NMR spectroscopy, and cryomicroscopy. This has led to a great demand to obtain the RNA 3D structures using prediction methods. The 3D structure prediction, especially of large RNAs, still remains a significant challenge and there is still a great demand for high-resolution structure prediction methods. In this chapter, we describe RNAComposer, a method and server for the automated prediction of RNA 3D structures based on the knowledge of secondary structure. Its applications are supported by other automated servers: RNA FRABASE and RNApdbee, developed to search and analyze secondary and 3D structures. Another method, RNAlyzer, offers new way to analyze and visualize quality of RNA 3D models. Scope and limitations of RNAComposer in application for an automated prediction of riboswitches' 3D structure will be presented and discussed. Analysis of the cyclic di-GMP-II riboswitch from Clostridium acetobutylicum (PDB ID 3Q3Z) as an example allows for 3D structure prediction of related riboswitches from Clostridium difficile 4, Bacillus halodurans 1, and Thermus aquaticus Y5.1 of yet unknown structures.

Topics: Base Sequence; Clostridioides difficile; Clostridium acetobutylicum; Cyclic GMP; Internet; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Riboswitch; RNA; RNA, Bacterial; Software

Klebsiella pneumoniae is an important cause of nosocomial infections, primarily through the formation of surface-associated biofilms to promote microbial colonization on host tissues. Expression of type 3 fimbriae by K. pneumoniae facilitates surface adherence, a process strongly activated by the cyclic di-GMP (c-di-GMP)-dependent transcriptional activator MrkH. In this study, we demonstrated the critical importance of MrkH in facilitating K. pneumoniae attachment on a variety of medically relevant materials and demonstrated the mechanism by which bacteria activate expression of type 3 fimbriae to colonize these materials. Sequence analysis revealed a putative MrkH recognition DNA sequence ("MrkH box"; TATCAA) located in the regulatory region of the mrkHI operon. Mutational analysis, electrophoretic mobility shift assay, and quantitative PCR experiments demonstrated that MrkH binds to the cognate DNA sequence to autoregulate mrkHI expression in a c-di-GMP-dependent manner. A half-turn deletion, but not a full-turn deletion, between the MrkH box and the -35 promoter element rendered MrkH ineffective in activating mrkHI expression, implying that a direct interaction between MrkH and RNA polymerase exists. In vivo analyses showed that residues L260, R265, N268, C269, E273, and I275 in the C-terminal domain of the RNA polymerase α subunit are involved in the positive control of mrkHI expression by MrkH and revealed the regions of MrkH required for DNA binding and transcriptional activation. Taken together, the data suggest a model whereby c-di-GMP-dependent MrkH recruits RNA polymerase to the mrkHI promoter to autoactivate mrkH expression. Increased MrkH production subsequently drives mrkABCDF expression when activated by c-di-GMP, leading to biosynthesis of type 3 fimbriae and biofilm formation.. Bacterial biofilms can cause persistent infections that are refractory to antimicrobial treatments. This study investigated how a commonly encountered hospital-acquired pathogen, Klebsiella pneumoniae, controls the expression of MrkH, the principal regulator of type 3 fimbriae and biofilm formation. We discovered a regulatory circuit whereby MrkH acts as a c-di-GMP-dependent transcriptional activator of both the gene cluster of type 3 fimbriae and the mrkHI operon. In this positive-feedback loop, whereby MrkH activates its own production, K. pneumoniae has evolved a mechanism to ensure rapid MrkH production, expression of type 3 fimbriae, and subsequent biofilm formation under favorable conditions. Deciphering the molecular mechanisms of biofilm formation by bacterial pathogens is important for the development of innovative treatment strategies for biofilm infections.

Topics: Bacterial Adhesion; Binding Sites; Biofilms; Cyclic GMP; DNA Mutational Analysis; DNA-Directed RNA Polymerases; DNA, Bacterial; Electrophoretic Mobility Shift Assay; Fimbriae, Bacterial; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Homeostasis; Klebsiella pneumoniae; Operon; Promoter Regions, Genetic; Protein Binding; Real-Time Polymerase Chain Reaction; Sequence Analysis, DNA; Transcription Factors

The second messenger cyclic di-GMP (c-di-GMP) controls the transition between different lifestyles in bacterial pathogens. Here, we report the identification of DgcP (diguanylate cyclase conserved in Pseudomonads), whose activity in the olive tree pathogen Pseudomonas savastanoi pv. savastanoi is dependent on the integrity of its GGDEF domain. Furthermore, deletion of the dgcP gene revealed that DgcP negatively regulates motility and positively controls biofilm formation in both the olive tree pathogen P. savastanoi pv. savastanoi and the human opportunistic pathogen Pseudomonas aeruginosa. Overexpression of the dgcP gene in P. aeruginosa PAK led to increased exopolysaccharide production and upregulation of the type VI secretion system; in turn, it repressed the type III secretion system, which is a hallmark of chronic infections and persistence for P. aeruginosa. Deletion of the dgcP gene in P. savastanoi pv. savastanoi NCPPB 3335 and P. aeruginosa PAK reduced their virulence in olive plants and in a mouse acute lung injury model respectively. Our results show that diguanylate cyclase DgcP is a conserved Pseudomonas protein with a role in virulence, and confirm the existence of common c-di-GMP signalling pathways that are capable of regulating plant and human Pseudomonas spp. infections.

Topics: Acute Lung Injury; Animals; Biofilms; Cyclic GMP; Disease Models, Animal; Escherichia coli Proteins; Humans; Mice; Olea; Phosphorus-Oxygen Lyases; Plant Diseases; Protein Structure, Tertiary; Pseudomonas aeruginosa; Pseudomonas Infections; Sequence Deletion; Signal Transduction; Type III Secretion Systems; Type VI Secretion Systems; Virulence

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases including Pierce's disease of grapevines. X. fastidiosa is thought to induce disease by colonizing and clogging xylem vessels through the formation of cell aggregates and bacterial biofilms. Here we examine the role in X. fastidiosa virulence of an uncharacterized gene, PD1671, annotated as a two-component response regulator with potential GGDEF and EAL domains. GGDEF domains are found in c-di-GMP diguanylate cyclases while EAL domains are found in phosphodiesterases, and these domains are for c-di-GMP production and turnover, respectively. Functional analysis of the PD1671 gene revealed that it affected multiple X. fastidiosa virulence-related phenotypes. A Tn5 PD1671 mutant had a hypervirulent phenotype in grapevines presumably due to enhanced expression of gum genes leading to increased exopolysaccharide levels that resulted in elevated biofilm formation. Interestingly, the PD1671 mutant also had decreased motility in vitro but did not show a reduced distribution in grapevines following inoculation. Given these responses, the putative PD1671 protein may be a negative regulator of X. fastidiosa virulence.

Topics: Amino Acid Sequence; Bacterial Proteins; Biofilms; Cyclic GMP; Extracellular Space; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Mutation; Plant Diseases; Polymers; Protein Interaction Domains and Motifs; Sequence Alignment; Virulence; Xylella

Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.

Topics: Alginates; Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Cyclic GMP; Glucuronic Acid; Hexuronic Acids; Humans; Membrane Proteins; Mutation; Protein Multimerization; Protein Structure, Quaternary; Pseudomonas aeruginosa; Virulence Factors

Pathogenic bacteria often need to survive in the host and the environment, and it is not well understood how cells transition between these equally challenging situations. For the human and animal pathogen Salmonella enterica serovar Typhimurium, biofilm formation is correlated with persistence outside a host, but the connection to virulence is unknown. In this study, we analyzed multicellular-aggregate and planktonic-cell subpopulations that coexist when S. Typhimurium is grown under biofilm-inducing conditions. These cell types arise due to bistable expression of CsgD, the central biofilm regulator. Despite being exposed to the same stresses, the two cell subpopulations had 1,856 genes that were differentially expressed, as determined by transcriptome sequencing (RNA-seq). Aggregated cells displayed the characteristic gene expression of biofilms, whereas planktonic cells had enhanced expression of numerous virulence genes. Increased type three secretion synthesis in planktonic cells correlated with enhanced invasion of a human intestinal cell line and significantly increased virulence in mice compared to the aggregates. However, when the same groups of cells were exposed to desiccation, the aggregates survived better, and the competitive advantage of planktonic cells was lost. We hypothesize that CsgD-based differentiation is a form of bet hedging, with single cells primed for host cell invasion and aggregated cells adapted for persistence in the environment. This allows S. Typhimurium to spread the risks of transmission and ensures a smooth transition between the host and the environment.

Topics: Animals; Bacterial Proteins; Caco-2 Cells; Cyclic GMP; Gene Expression Regulation, Bacterial; Humans; Mice; Protein Transport; Salmonella typhimurium; Trans-Activators; Transcription, Genetic; Virulence

The CckA-ChpT-CtrA phosphorelay is central to the regulation of the cell cycle in Caulobacter crescentus. The three proteins are conserved in Alphaproteobacteria, but little is known about their roles in most members of this class. Here, we characterized the system in Sphingomonas melonis. We found that the transcription factor CtrA is the master regulator of flagella synthesis genes, the hierarchical transcriptional organization of which is herein described. CtrA also regulates genes involved in exopolysaccharide synthesis and cyclic-di-GMP signaling, and is important for biofilm formation. In addition, the ctrA mutant exhibits an aberrant morphology, suggesting a role for CtrA in cell division. An analysis of the regulation of CtrA indicates that the phosphorelay composed of CckA and ChpT is conserved and that the absence of the bifunctional kinase/phosphatase CckA apparently results in overactivation of CtrA through ChpT. Suppressors of this phenotype identified the hybrid histidine kinase CcsA. Phosphorelays initiated by CckA or CcsA were reconstituted in vitro, suggesting that in S. melonis, CtrA phosphorylation is controlled by a branched pathway upstream of ChpT. This study thus suggests that signals can directly converge at the level of ChpT phosphorylation through multiple hybrid kinases to coordinate a number of important physiological processes.

Topics: Bacterial Proteins; Biofilms; Cell Cycle; Cell Division; Cyclic GMP; DNA-Binding Proteins; Flagella; Gene Expression Regulation, Bacterial; Histidine Kinase; Mutation; Phosphorylation; Protein Kinases; Signal Transduction; Sphingomonas; Transcription Factors

Biofilm formation in flea gut is important for flea-borne transmission of Yersinia pestis. There are enhancing factors (HmsHFRS, HmsCDE, and HmsT) and inhibiting one (HmsP) for Yersinia pestis biofilm formation. The RcsAB regulatory complex acts as a repressor of Yesinia biofilm formation, and adaptive pseudogenization of rcsA promotes Y. pestis to evolve the ability of biofilm formation in fleas. In this study, we constructed a set of isogenic strains of Y. pestis biovar Microtus, namely WT (RscB+ and RcsA-), c-rcsA (RscB+ and RcsA+), ΔrcsB (RscB- and RcsA-), and ΔrcsB/c-rcsA (RscB- and RcsA+). The phenotypic assays confirmed that RcsB alone (but not RcsA alone) had an inhibiting effect on biofilm/c-di-GMP production whereas assistance of RcsA to RcsB greatly enhanced this inhibiting effect. Further gene regulation experiments showed that RcsB in assistance of RcsA tightly bound to corresponding promoter-proximal regions to achieve transcriptional repression of hmsCDE, hmsT and hmsHFRS and, meanwhile, RcsAB positively regulated hmsP most likely in an indirect manner. Data presented here disclose that pseudogenization of rcsA leads to dramatic remodeling of RcsAB-dependent hms gene expression between Y. pestis and its progenitor Y. pseudotuberculosis, enabling potent production of Y. pestis biofilms in fleas.

Topics: Bacterial Proteins; Base Sequence; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Molecular Sequence Data; Mutation; Operon; Phenotype; Promoter Regions, Genetic; Protein Binding; Yersinia

Upon systemic bacterial infection, hematopoietic stem and progenitor cells (HSPCs) migrate to the periphery in order to supply a sufficient number of immune cells. Although pathogen-associated molecular patterns reportedly mediate HSPC activation, how HSPCs detect pathogen invasion in vivo remains elusive. Bacteria use the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) for a variety of activities. Here, we report that c-di-GMP comprehensively regulated both HSPCs and their niche cells through an innate immune sensor, STING, thereby inducing entry into the cell cycle and mobilization of HSPCs while decreasing the number and repopulation capacity of long-term hematopoietic stem cells. Furthermore, we show that type I interferon acted as a downstream target of c-di-GMP to inhibit HSPC expansion in the spleen, while transforming growth factor-β was required for c-di-GMP-dependent splenic HSPC expansion. Our results define machinery underlying the dynamic regulation of HSPCs and their niches during bacterial infection through c-di-GMP/STING signaling.

Topics: Animals; Bacteria; Cyclic GMP; Gene Expression Regulation; Hematopoietic Stem Cells; Immunity, Innate; Interferon Regulatory Factor-3; Membrane Proteins; Mice; Signal Transduction; Stem Cell Niche; Transforming Growth Factor beta

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail. We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms(1). Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

Topics: Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Mass Spectrometry; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa; Signal Transduction

We previously showed the isolation of biofilmpersistent Pseudomonas putida mutants that fail to undergo biofilm dispersal upon entry in stationary phase. Two such mutants were found to bear insertions in PP0914, encoding a GGDEF/EAL domain protein with high similarity to Pseudomon asaeruginosa BifA. Here we show the phenotypic characterization of a ΔbifA mutant in P. putida KT2442.This mutant displayed increased biofilm and pellicle formation, cell aggregation in liquid medium and decreased starvation-induced biofilm dispersal relative to the wild type. Unlike its P. aeruginosa counterpart, P. putida BifA did not affect swarming motility. The hyperadherent phenotype of the ΔbifA mutant correlates with a general increase in cyclic diguanylate (c-di-GMP) levels, Congo Red-binding exopolyaccharide production and transcription of the adhesin-encoding lapA gene. Integrity of the EAL motif and a modified GGDEF motif (altered to GGDQF)were crucial for BifA activity, and c-di-GMP depletion by overexpression of a heterologous c-di-GMP phosphodiesterase in the ΔbifA mutant restored wild-type biofilm dispersal and lapA expression.Our results indicate that BifA is a phosphodiesterase involved in the regulation of the c-di-GMP pool and required for the generation of the low c-di-GMP signal that triggers starvation-induced biofilm dispersal.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Phosphoric Diester Hydrolases; Protein Structure, Tertiary; Pseudomonas putida

Most bacteria can form multicellular communities called biofilms on biotic and abiotic surfaces. This multicellular response to surface contact correlates with an increased resistance to various adverse environmental conditions, including those encountered during infections of the human host and exposure to antimicrobial compounds. Biofilm formation occurs when freely swimming (planktonic) cells encounter a surface, which stimulates the chemosensory-like, surface-sensing system Wsp and leads to generation of the intracellular second messenger 3',5'-cyclic-di-guanosine monophosphate (c-di-GMP). We identified adaptive mutations in a clinical small colony variant (SCV) of Pseudomonas aeruginosa and correlated their presence with self-aggregating growth behavior and an enhanced capacity to form biofilms. We present evidence that a point mutation in the 5' untranslated region of the accBC gene cluster, which encodes components of an enzyme responsible for fatty acid biosynthesis, was responsible for a stabilized mRNA structure that resulted in reduced translational efficiency and an increase in the proportion of short-chain fatty acids in the plasma membrane. We propose a model in which these changes in P. aeruginosa serve as a signal for the Wsp system to constitutively produce increased amounts of c-di-GMP and thus play a role in the regulation of adhesion-stimulated bacterial responses.

Topics: 5' Untranslated Regions; Acetyl-CoA Carboxylase; Bacterial Proteins; Base Sequence; Biofilms; Cell Membrane; Cyclic GMP; Cytosol; Fatty Acids; Multigene Family; Mutation; Nucleic Acid Conformation; Phenotype; Protein Biosynthesis; Pseudomonas aeruginosa; RNA, Messenger; Sequence Homology, Nucleic Acid; Signal Transduction; Sodium Chloride

Bacterial invasion plays a critical role in the establishment of Pseudomonas aeruginosa infection and is aided by two major virulence factors--surface appendages and secreted proteases. The second messenger cyclic diguanylate (c-di-GMP) is known to affect bacterial attachment to surfaces, biofilm formation and related virulence phenomena. Here we report that MorA, a global regulator with GGDEF and EAL domains that was previously reported to affect virulence factors, negatively regulates protease secretion via the type II secretion system (T2SS) in P. aeruginosa PAO1. Infection assays with mutant strains carrying gene deletion and domain mutants show that host cell invasion is dependent on the active domain function of MorA. Further investigations suggest that the MorA-mediated c-di-GMP signaling affects protease secretion largely at a post-translational level. We thus report c-di-GMP second messenger system as a novel regulator of T2SS function in P. aeruginosa. Given that T2SS is a central and constitutive pump, and the secreted proteases are involved in interactions with the microbial surroundings, our data broadens the significance of c-di-GMP signaling in P. aeruginosa pathogenesis and ecological fitness.

Topics: Bacterial Proteins; Bacterial Secretion Systems; Cell Shape; Cyclic GMP; Endopeptidases; Extracellular Space; Humans; Pancreatic Elastase; Pseudomonas aeruginosa; Signal Transduction; Virulence

The opportunistic human pathogen Pseudomonas aeruginosa expresses numerous acute virulence factors in the initial phase of infection, and during long-term colonization it undergoes adaptations that optimize survival in the human host. Adaptive changes that often occur during chronic infection give rise to rugose small colony variants (RSCVs), which are hyper-biofilm-forming mutants that commonly possess mutations that increase production of the biofilm-promoting secondary messenger cyclic di-GMP (c-di-GMP). We show that RSCVs display a decreased production of acute virulence factors as a direct result of elevated c-di-GMP content. Overproduction of c-di-GMP causes a decrease in the transcription of virulence factor genes that are regulated by the global virulence regulator Vfr. The low level of Vfr-dependent transcription is caused by a low level of its coactivator, cyclic AMP (cAMP), which is decreased in response to a high level of c-di-GMP. Mutations that cause reversion of the RSCV phenotype concomitantly reactivate Vfr-cAMP signaling. Attempts to uncover the mechanism underlying the observed c-di-GMP-mediated lowering of cAMP content provided evidence that it is not caused by inhibition of adenylate cyclase production or activity and that it is not caused by activation of cAMP phosphodiesterase activity. In addition to the studies of the RSCVs, we present evidence that the deeper layers of wild-type P. aeruginosa biofilms have high c-di-GMP levels and low cAMP levels.. Our work suggests that cross talk between c-di-GMP and cAMP signaling pathways results in downregulation of acute virulence factors in P. aeruginosa biofilm infections. Knowledge about this cross-regulation adds to our understanding of virulence traits and immune evasion by P. aeruginosa in chronic infections and may provide new approaches to eradicate biofilm infections.

Topics: Bacterial Proteins; Cyclic AMP; Cyclic AMP Receptor Protein; Cyclic GMP; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa; Signal Transduction

Electroactive biofilms play essential roles in determining the power output of microbial fuel cells (MFCs). To engineer the electroactive biofilm formation of Shewanella oneidensis MR-1, a model exoelectrogen, we herein heterologously overexpressed a c-di-GMP biosynthesis gene ydeH in S. oneidensis MR-1, constructing a mutant strain in which the expression of ydeH is under the control of IPTG-inducible promoter, and a strain in which ydeH is under the control of a constitutive promoter. Such engineered Shewanella strains had significantly enhanced biofilm formation and bioelectricity generation. The MFCs inoculated with these engineered strains accomplished a maximum power density of 167.6 ± 3.6 mW/m(2) , which was ∼ 2.8 times of that achieved by the wild-type MR-1 (61.0 ± 1.9 mW/m(2) ). In addition, the engineered strains in the bioelectrochemical system at poised potential of 0.2 V vs. saturated calomel electrode (SCE) generated a stable current density of 1100 mA/m(2) , ∼ 3.4 times of that by wild-type MR-1 (320 mA/m(2) ).

Topics: Bioelectric Energy Sources; Biofilms; Cyclic GMP; Electricity; Gene Expression; Isopropyl Thiogalactoside; Phosphorus-Oxygen Lyases; Shewanella; Transcriptional Activation

The PilZ domain proteins have been demonstrated to be one of the major types of receptors mediating cyclic di-GMP (c-di-GMP) signaling pathways in several pathogenic bacteria. However, little is known about the function of PilZ domain proteins in c-di-GMP regulation of virulence in the bacterial blight pathogen of rice Xanthomonas oryzae pv. oryzae. Here, the roles of PilZ domain proteins PXO_00049 and PXO_02374 in c-di-GMP binding, regulation of virulence and motility, and subcellular localization were characterized in comparison with PXO_02715, identified previously as an interactor with the c-di-GMP receptor Filp to regulate virulence. The c-di-GMP binding motifs in the PilZ domains were conserved in PXO_00049 and PXO_02374 but were less well conserved in PXO_02715. PXO_00049 and PXO_02374 but not PXO_02715 proteins bound to c-di-GMP with high affinity in vitro, and the R(141) and R(10) residues in the PilZ domains of PXO_00049 and PXO_02374, respectively, were crucial for c-di-GMP binding. Gene deletion of PXO_00049 and PXO_02374 resulted in significant increases in virulence and hrp gene transcription, indicating their negative regulation of virulence via type III secretion system expression. All mutants showed significant changes in sliding motility but not exopolysaccharide production and biofilm formation. In trans expression of the full-length open reading frame (ORF) of each gene in the relevant mutants led to restoration of the phenotype to wild-type levels. Moreover, PXO_00049 and PXO_02374 displayed mainly multisite subcellular localizations, whereas PXO_02715 showed nonpolar distributions in the X. oryzae pv. oryzae cells. Therefore, this study demonstrated the different functions of the PilZ domain proteins in mediation of c-di-GMP regulation of virulence and motility in X. oryzae pv. oryzae.

Topics: Amino Acid Motifs; Bacterial Proteins; Binding Sites; Cyclic GMP; Gene Deletion; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Locomotion; Protein Binding; Virulence; Xanthomonas

Alginates exhibit unique material properties suitable for medical and industrial applications. However, if produced by Pseudomonas aeruginosa, it is an important virulence factor in infection of cystic fibrosis patients. The alginate biosynthesis machinery is activated by c-di-GMP imparted by the inner membrane protein, MucR. Here, it was shown that MucR impairs alginate production in response to nitrate in P. aeruginosa. Subsequent site-specific mutagenesis of MucR revealed that the second MHYT sensor motif (MHYT II, amino acids 121-124) of MucR sensor domain was involved in nitrate sensing. We also showed that both c-di-GMP synthesizing and degrading active sites of MucR were important for alginate production. Although nitrate and deletion of MucR impaired alginate promoter activity and global c-di-GMP levels, alginate yields were not directly correlated with alginate promoter activity or c-di-GMP levels, suggesting that nitrate and MucR modulate alginate production at a post-translational level through a localized pool of c-di-GMP. Nitrate increased pel promoter activity in the mucR mutant while in the same mutant the psl promoter activity was independent of nitrate. Nitrate and deletion of mucR did not impact on swarming motility but impaired attachment to solid surfaces. Nitrate and deletion of mucR promoted the formation of biofilms with increased thickness, cell density, and survival. Overall, this study provided insight into the functional role of MucR with respect to nitrate-mediated regulation of alginate biosynthesis.

Topics: Alginates; Cyclic GMP; Gene Deletion; Gene Expression Regulation, Bacterial; Glucuronic Acid; Hexuronic Acids; Membrane Proteins; Mutagenesis, Site-Directed; Nitrates; Pseudomonas aeruginosa

For the investigation and comparison of microbial biofilms, a variety of analytical methods have been established, all focusing on different growth stages and application areas of biofilms. In this study, a novel quantitative assay for analysing biofilm maturation under the influence of continuous flow conditions was developed using the interesting biocatalyst Pseudomonas taiwanensis VLB120. In contrast to other tubular-based assay systems, this novel assay format delivers three readouts using a single setup in a total assay time of 40 h. It combines morphotype analysis of biofilm colonies with the direct quantification of biofilm biomass and pellicle formation on an air/liquid interphase. Applying the Tube-Assay, the impact of the second messenger cyclic diguanylate on biofilm formation of P. taiwanensis VLB120 was investigated. To this end, 41 deletions of genes encoding for protein homologues to diguanylate cyclase and phosphodiesterase were generated in the genome of P. taiwanensis VLB120. Subsequently, the biofilm formation of the resulting mutants was analysed using the Tube-Assay. In more than 60 % of the mutants, a significantly altered biofilm formation as compared to the parent strain was detected. Furthermore, the potential of the proposed Tube-Assay was validated by investigating the biofilms of several other bacterial species.

Topics: Bacteriological Techniques; Biofilms; Cyclic GMP; Pseudomonas

Cyclic dinucleotides (CDNs) are agonists of stimulator of IFN genes (STING) and have potential as vaccine adjuvants. However, cyclic di-GMP (cdGMP) injected s.c. shows minimal uptake into lymphatics/draining lymph nodes (dLNs) and instead is rapidly distributed to the bloodstream, leading to systemic inflammation. Here, we encapsulated cdGMP within PEGylated lipid nanoparticles (NP-cdGMP) to redirect this adjuvant to dLNs. Compared with unformulated CDNs, encapsulation blocked systemic dissemination and markedly enhanced dLN accumulation in murine models. Delivery of NP-cdGMP increased CD8+ T cell responses primed by peptide vaccines and enhanced therapeutic antitumor immunity. A combination of a poorly immunogenic liposomal HIV gp41 peptide antigen and NP-cdGMP robustly induced type I IFN in dLNs, induced a greater expansion of vaccine-specific CD4+ T cells, and greatly increased germinal center B cell differentiation in dLNs compared with a combination of liposomal HIV gp41 and soluble CDN. Further, NP-cdGMP promoted durable antibody titers that were substantially higher than those promoted by the well-studied TLR agonist monophosphoryl lipid A and comparable to a much larger dose of unformulated cdGMP, without the systemic toxicity of the latter. These results demonstrate that nanoparticulate delivery safely targets CDNs to the dLNs and enhances the efficacy of this adjuvant. Moreover, this approach can be broadly applied to other small-molecule immunomodulators of interest for vaccines and immunotherapy.

Topics: Adjuvants, Immunologic; AIDS Vaccines; Animals; Cyclic GMP; Drug Delivery Systems; HIV Envelope Protein gp41; HIV-1; Lipid A; Lymph Nodes; Membrane Proteins; Mice; Mice, Inbred BALB C; Nanoparticles; Polyethylene Glycols

Nitric oxide (NO) plays an important signaling role in all domains of life. Many bacteria contain a heme-nitric oxide/oxygen binding (H-NOX) protein that selectively binds NO. These H-NOX proteins often act as sensors that regulate histidine kinase (HK) activity, forming part of a bacterial two-component signaling system that also involves one or more response regulators. In several organisms, NO binding to the H-NOX protein governs bacterial biofilm formation; however, the source of NO exposure for these bacteria is unknown. In mammals, NO is generated by the enzyme nitric oxide synthase (NOS) and signals through binding the H-NOX domain of soluble guanylate cyclase. Recently, several bacterial NOS proteins have also been reported, but the corresponding bacteria do not also encode an H-NOX protein. Here, we report the first characterization of a bacterium that encodes both a NOS and H-NOX, thus resembling the mammalian system capable of both synthesizing and sensing NO. We characterized the NO signaling pathway of the marine alphaproteobacterium Silicibacter sp. strain TrichCH4B, determining that the NOS is activated by an algal symbiont, Trichodesmium erythraeum. NO signaling through a histidine kinase-response regulator two-component signaling pathway results in increased concentrations of cyclic diguanosine monophosphate, a key bacterial second messenger molecule that controls cellular adhesion and biofilm formation. Silicibacter sp. TrichCH4B biofilm formation, activated by T. erythraeum, may be an important mechanism for symbiosis between the two organisms, revealing that NO plays a previously unknown key role in bacterial communication and symbiosis.. Bacterial nitric oxide (NO) signaling via heme-nitric oxide/oxygen binding (H-NOX) proteins regulates biofilm formation, playing an important role in protecting bacteria from oxidative stress and other environmental stresses. Biofilms are also an important part of symbiosis, allowing the organism to remain in a nutrient-rich environment. In this study, we show that in Silicibacter sp. strain TrichCH4B, NO mediates symbiosis with the alga Trichodesmium erythraeum, a major marine diazotroph. In addition, Silicibacter sp. TrichCH4B is the first characterized bacteria to harbor both the NOS and H-NOX proteins, making it uniquely capable of both synthesizing and sensing NO, analogous to mammalian NO signaling. Our study expands current understanding of the role of NO in bacterial signaling, providing a novel role for NO in bacterial communication and symbiosis.

Topics: Bacterial Adhesion; Biofilms; Cyanobacteria; Cyclic GMP; Gene Expression Regulation, Bacterial; Histidine Kinase; Microbial Interactions; Nitric Oxide; Protein Kinases; Rhodobacteraceae; Signal Transduction; Symbiosis; Transcription Factors

Fundamental to all living organisms is the capacity to coordinate cell division and cell differentiation to generate appropriate numbers of specialized cells. Whereas eukaryotes use cyclins and cyclin-dependent kinases to balance division with cell fate decisions, equivalent regulatory systems have not been described in bacteria. Moreover, the mechanisms used by bacteria to tune division in line with developmental programs are poorly understood. Here we show that Caulobacter crescentus, a bacterium with an asymmetric division cycle, uses oscillating levels of the second messenger cyclic diguanylate (c-di-GMP) to drive its cell cycle. We demonstrate that c-di-GMP directly binds to the essential cell cycle kinase CckA to inhibit kinase activity and stimulate phosphatase activity. An upshift of c-di-GMP during the G1-S transition switches CckA from the kinase to the phosphatase mode, thereby allowing replication initiation and cell cycle progression. Finally, we show that during division, c-di-GMP imposes spatial control on CckA to install the replication asymmetry of future daughter cells. These studies reveal c-di-GMP to be a cyclin-like molecule in bacteria that coordinates chromosome replication with cell morphogenesis in Caulobacter. The observation that c-di-GMP-mediated control is conserved in the plant pathogen Agrobacterium tumefaciens suggests a general mechanism through which this global regulator of bacterial virulence and persistence coordinates behaviour and cell proliferation.

Topics: Agrobacterium tumefaciens; Bacterial Proteins; Catalytic Domain; Caulobacter crescentus; Cell Cycle; Cell Division; Chromosomes; Conserved Sequence; Cyclic GMP; Cyclins; DNA Replication; Models, Molecular; Phosphoric Monoester Hydrolases; Phosphotransferases; Protein Binding; Protein Structure, Tertiary

The substrate specificity of recombinant full-length diguanylate cyclase (DGC) of Thermotoga maritima with mutant allosteric site was investigated. It has been originally shown that the enzyme could use GTP closest analogues - 2'-deoxyguanosine-5'-triphosphate (dGTP) and 9-β-D-arabinofuranosyl-guanine-5'-triphosphate (araGTP) as the substrates. The first demonstrations of an enzymatic synthesis of bis-(3'-5')-cyclic dimeric deoxyguanosine monophosphate (c-di-dGMP) and the previously unknown bis-(3'-5')-cyclic dimeric araguanosine monophosphate (c-di-araGMP) using DGC of T. maritima in the form of inclusion bodies have been provided.

Topics: Arabinonucleotides; Bacterial Proteins; Cyclic GMP; Deoxyguanine Nucleotides; Escherichia coli Proteins; Guanosine Triphosphate; Phosphorus-Oxygen Lyases; Thermotoga maritima

The development of fluorescent biosensors is motivated by the desire to monitor cellular metabolite levels in real time. Most genetically encodable fluorescent biosensors are based on receptor proteins fused to fluorescent protein domains. More recently, small molecule-binding riboswitches have been adapted for use as fluorescent biosensors through fusion to the in vitro selected Spinach aptamer, which binds a pro-fluorescent, cell-permeable small molecule mimic of the GFP chromophore, DFHBI. Here we describe methods to prepare and analyze riboswitch-Spinach tRNA fusions for ligand-dependent activation of fluorescence in vivo. Example procedures describe the use of the Vc2-Spinach tRNA biosensor to monitor perturbations in cellular levels of cyclic di-GMP using either fluorescence microscopy or flow cytometry. The relative ease of cloning and imaging of these biosensors, as well as their modular nature, should make this method appealing to other researchers interested in utilizing riboswitch-based biosensors for metabolite sensing.

Topics: Biosensing Techniques; Cell Tracking; Cyclic GMP; Flow Cytometry; Genetic Vectors; Microscopy, Fluorescence; Riboswitch; RNA, Transfer; Spinacia oleracea

The molecular mechanisms of alginate polymerization/modification/secretion by a proposed envelope-spanning multiprotein complex are unknown. Here, bacterial two-hybrid assays and pulldown experiments showed that the catalytic subunit Alg8 directly interacts with the proposed copolymerase Alg44 while embedded in the cytoplasmic membrane. Alg44 additionally interacts with the lipoprotein AlgK bridging the periplasmic space. Site-specific mutagenesis of Alg44 showed that protein-protein interactions and stability were independent of conserved amino acid residues R17 and R21, which are involved in c-di-GMP binding, the N-terminal PilZ domain, and the C-terminal 26 amino acids. Site-specific mutagenesis was employed to investigate the c-di-GMP-mediated activation of alginate polymerization by the PilZAlg44 domain and Alg8. Activation was found to be different from the proposed activation mechanism for cellulose synthesis. The interactive role of Alg8, Alg44, AlgG (epimerase), and AlgX (acetyltransferase) on alginate polymerization and modification was studied by using site-specific deletion mutants, inactive variants, and overproduction of subunits. The compositions, molecular masses, and material properties of resulting novel alginates were analyzed. The molecular mass was reduced by epimerization, while it was increased by acetylation. Interestingly, when overproduced, Alg44, AlgG, and the nonepimerizing variant AlgG(D324A) increased the degree of acetylation, while epimerization was enhanced by AlgX and its nonacetylating variant AlgX(S269A). Biofilm architecture analysis showed that acetyl groups promoted cell aggregation while nonacetylated polymannuronate alginate promoted stigmergy. Overall, this study sheds new light on the arrangement of the multiprotein complex involved in alginate production. Furthermore, the activation mechanism and the interplay between polymerization and modification of alginate were elucidated.. This study provides new insights into the molecular mechanisms of the synthesis of the unique polysaccharide, alginate, which not only is an important virulence factor of the opportunistic human pathogen Pseudomonas aeruginosa but also has, due to its material properties, many applications in medicine and industry. Unraveling the assembly and composition of the alginate-synthesizing and envelope-spanning multiprotein complex will be of tremendous significance for the scientific community. We identified a protein-protein interaction network inside the multiprotein complex and studied its relevance with respect to alginate polymerization/modification as well as the c-di-GMP-mediated activation mechanism. A relationship between alginate polymerization and modification was shown. Due to the role of alginate in pathogenesis as well as its unique material properties harnessed in numerous applications, results obtained in this study will aid the design and development of inhibitory drugs as well as the commercial bacterial production of tailor-made alginates.

Topics: Acetylation; Acetyltransferases; Alginates; Bacterial Proteins; Biofilms; Cellulose; Cyclic GMP; Humans; Membrane Proteins; Multiprotein Complexes; Mutagenesis, Site-Directed; Mutation; Polymerization; Pseudomonas aeruginosa; Racemases and Epimerases; Two-Hybrid System Techniques; Virulence Factors

Expression of Vibrio cholerae genes required for the biosynthesis of exopolysacchide (vps) and protein (rbm) components of the biofilm matrix is enhanced by cyclic diguanylate (c-di-GMP). In a previous study, we reported that the histone-like nucleoid structuring (H-NS) protein represses the transcription of vpsA, vpsL and vpsT. Here we demonstrate that the regulator VpsT can disrupt repressive H-NS nucleoprotein complexes at the vpsA and vpsL promoters in the presence of c-di-GMP, while H-NS could disrupt the VpsT-promoter complexes in the absence of c-di-GMP. Chromatin immunoprecipitation-Seq showed a remarkable trend for H-NS to cluster at loci involved in biofilm development such as the rbmABCDEF genes. We show that the antagonistic relationship between VpsT and H-NS regulates the expression of the rbmABCDEF cluster. Epistasis analysis demonstrated that VpsT functions as an antirepressor at the rbmA/F, vpsU and vpsA/L promoters. Deletion of vpsT increased H-NS occupancy at these promoters while increasing the c-di-GMP pool had the opposite effect and included the vpsT promoter. The negative effect of c-di-GMP on H-NS occupancy at the vpsT promoter required the regulator VpsR. These results demonstrate that c-di-GMP activates the transcription of genes required for the biosynthesis of the biofilm matrix by triggering a coordinated VpsR- and VpsT-dependent H-NS antirepression cascade.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; DNA-Binding Proteins; Extracellular Matrix; Gene Expression Regulation, Bacterial; Protein Binding; Protein Biosynthesis; Second Messenger Systems; Vibrio cholerae

Therapeutic vaccines to induce anti-tumor CD8 T cells have been used in clinical trials for advanced melanoma patients, but the clinical response rate and overall survival time have not improved much. We believe that these dismal outcomes are caused by inadequate number of antigen-specific CD8 T cells generated by most vaccines. In contrast, huge CD8 T cell responses readily occur during acute viral infections. High levels of type-I interferon (IFN-I) are produced during these infections, and this cytokine not only exhibits anti-viral activity but also promotes CD8 T cell responses. The studies described here were performed to determine whether promoting the production of IFN-I could enhance the potency of a peptide vaccine. We report that cyclic diguanylate monophosphate (c-di-GMP), which activates the stimulator of interferon genes, potentiated the immunogenicity and anti-tumor effects of a peptide vaccine against mouse B16 melanoma. The synergistic effects of c-di-GMP required co-administration of costimulatory anti-CD40 antibody, the adjuvant poly-IC, and were mediated in part by IFN-I. These findings demonstrate that peptides representing CD8 T cell epitopes can be effective inducers of large CD8 T cell responses in vaccination strategies that mimic acute viral infections.

Topics: Animals; Antibodies, Monoclonal; Cancer Vaccines; CD40 Antigens; CD8-Positive T-Lymphocytes; Cyclic GMP; Epitopes, T-Lymphocyte; Humans; Immunotherapy; Interferon Type I; Lymphocyte Activation; Melanoma, Experimental; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Poly I-C; Receptors, Interferon; Skin Neoplasms; Vaccines, Subunit

Borrelia burgdorferi, the Lyme disease spirochete, couples environmental sensing and gene regulation primarily via the Hk1/Rrp1 two-component system (TCS) and Rrp2/RpoN/RpoS pathways. Beginning with acquisition, we reevaluated the contribution of these pathways to spirochete survival and gene regulation throughout the enzootic cycle. Live imaging of B. burgdorferi caught in the act of being acquired revealed that the absence of RpoS and the consequent derepression of tick-phase genes impart a Stay signal required for midgut colonization. In addition to the behavioral changes brought on by the RpoS-off state, acquisition requires activation of cyclic di-GMP (c-di-GMP) synthesis by the Hk1/Rrp1 TCS; B. burgdorferi lacking either component is destroyed during the blood meal. Prior studies attributed this dramatic phenotype to a metabolic lesion stemming from reduced glycerol uptake and utilization. In a head-to-head comparison, however, the B. burgdorferi Δglp mutant had a markedly greater capacity to survive tick feeding than B. burgdorferi Δhk1 or Δrrp1 mutants, establishing unequivocally that glycerol metabolism is only one component of the protection afforded by c-di-GMP. Data presented herein suggest that the protective response mediated by c-di-GMP is multifactorial, involving chemotactic responses, utilization of alternate substrates for energy generation and intermediary metabolism, and remodeling of the cell envelope as a means of defending spirochetes against threats engendered during the blood meal. Expression profiling of c-di-GMP-regulated genes through the enzootic cycle supports our contention that the Hk1/Rrp1 TCS functions primarily, if not exclusively, in ticks. These data also raise the possibility that c-di-GMP enhances the expression of a subset of RpoS-dependent genes during nymphal transmission.

Topics: Animals; Bacterial Proteins; Borrelia burgdorferi; Cyclic GMP; Female; Gene Expression Regulation, Bacterial; Humans; Lyme Disease; Mice; Mice, Inbred C3H; Microbial Viability; Rats; Rats, Sprague-Dawley; Ticks

Stress response plays an important role on microbial adaptation under hostile environmental conditions. It is generally unclear how the signaling transduction pathway mediates a stress response in planktonic and biofilm modes of microbial communities simultaneously. Here, we showed that metalloid tellurite (TeO3(2-)) exposure induced the intracellular content of the secondary messenger cyclic di-GMP (c-di-GMP) of Pseudomonas aeruginosa. Two diguanylate cyclases (DGCs), SadC and SiaD, were responsible for the increased intracellular content of c-di-GMP. Enhanced c-di-GMP levels by TeO3(2-) further increased P. aeruginosa biofilm formation and resistance to TeO3(2-). P. aeruginosa ΔsadCΔsiaD and PAO1/p(lac)-yhjH mutants with low intracellular c-di-GMP content were more sensitive to TeO3(2-) exposure and had low relative fitness compared to the wild-type PAO1 planktonic and biofilm cultures exposed to TeO3(2-). Our study provided evidence that c-di-GMP level can play an important role in mediating stress response in microbial communities during both planktonic and biofilm modes of growth.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Drug Resistance, Bacterial; Escherichia coli Proteins; Microscopy, Electron, Scanning; Phosphorus-Oxygen Lyases; Proteome; Pseudomonas aeruginosa; Reactive Oxygen Species; Signal Transduction; Spectrometry, X-Ray Emission; Tellurium

In this issue of Science Signaling, Mills et al. show that the amino acid ʟ-arginine increases the concentration of the second messenger c-di-GMP in Salmonella enterica serovar Typhimurium through a specific diguanylate cyclase, leading to increased production of the exopolysaccharide cellulose, which is an extracellular matrix component of environmental and host-associated biofilms.

Topics: Arginine; Cyclic GMP; Periplasm; Salmonella typhimurium; Signal Transduction

Cyclic-di-GMP (c-di-GMP) is a bacterial second messenger that transduces internal and external signals and regulates bacterial motility and biofilm formation. Some organisms encode more than 100 c-di-GMP-modulating enzymes, but only for a few has a signal been defined that modulates their activity. We developed and applied a high-throughput, real-time flow cytometry method that uses a fluorescence resonance energy transfer (FRET)-based biosensor of free c-di-GMP to screen for signals that modulate its concentration within Salmonella Typhimurium. We identified multiple compounds, including glucose, N-acetyl-d-glucosamine, salicylic acid, and ʟ-arginine, that modulated the FRET signal and therefore the free c-di-GMP concentration. By screening a library of mutants, we identified proteins required for the c-di-GMP response to each compound. Furthermore, low micromolar concentrations of ʟ-arginine induced a rapid translation-independent increase in c-di-GMP concentrations and c-di-GMP-dependent cellulose synthesis, responses that required the regulatory periplasmic domain of the diguanylate cyclase STM1987. ʟ-Arginine signaling also required the periplasmic putative ʟ-arginine-binding protein ArtI, implying that ʟ-arginine sensing occurred in the periplasm. Among the 20 commonly used amino acids, S. Typhimurium specifically responded to ʟ-arginine with an increase in c-di-GMP, suggesting that ʟ-arginine may serve as a signal during S. Typhimurium infection. Our results demonstrate that a second-messenger biosensor can be used to identify environmental signals and define pathways that alter microbial behavior.

Topics: Amino Acids; Arginine; Bacterial Proteins; Carrier Proteins; Cellulose; Cyclic GMP; Escherichia coli Proteins; Flow Cytometry; Fluorescence Resonance Energy Transfer; Mutation; Periplasm; Phosphorus-Oxygen Lyases; Salmonella typhimurium; Second Messenger Systems; Signal Transduction

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that have diverse spectral properties and domain compositions. Although large numbers of CBCR genes exist in cyanobacterial genomes, no studies have assessed whether multiple CBCRs work together. We recently showed that the diguanylate cyclase (DGC) activity of the CBCR SesA from Thermosynechococcus elongatus is activated by blue-light irradiation and that, when irradiated, SesA, via its product cyclic dimeric GMP (c-di-GMP), induces aggregation of Thermosynechococcus vulcanus cells at a temperature that is suboptimum for single-cell viability. For this report, we first characterize the photobiochemical properties of two additional CBCRs, SesB and SesC. Blue/teal light-responsive SesB has only c-di-GMP phosphodiesterase (PDE) activity, which is up-regulated by teal light and GTP. Blue/green light-responsive SesC has DGC and PDE activities. Its DGC activity is enhanced by blue light, whereas its PDE activity is enhanced by green light. A ΔsesB mutant cannot suppress cell aggregation under teal-green light. A ΔsesC mutant shows a less sensitive cell-aggregation response to ambient light. ΔsesA/ΔsesB/ΔsesC shows partial cell aggregation, which is accompanied by the loss of color dependency, implying that a nonphotoresponsive DGC(s) producing c-di-GMP can also induce the aggregation. The results suggest that SesB enhances the light color dependency of cell aggregation by degrading c-di-GMP, is particularly effective under teal light, and, therefore, seems to counteract the induction of cell aggregation by SesA. In addition, SesC seems to improve signaling specificity as an auxiliary backup to SesA/SesB activities. The coordinated action of these three CBCRs highlights why so many different CBCRs exist.

Topics: Color; Cyclic GMP; Light; Photoreceptors, Microbial; Point Mutation; Signal Transduction; Synechococcus

Cyclic di-GMP (c-di-GMP) is a second messenger that is important in regulating bacterial physiology and behavior, including motility and virulence. Many questions remain about the role and regulation of this signaling molecule, but current methods of detection are limited by either modest sensitivity or requirements for extensive sample purification. We have taken advantage of a natural, high affinity receptor of c-di-GMP, the Vc2 riboswitch aptamer, to develop a sensitive and rapid electrophoretic mobility shift assay (EMSA) for c-di-GMP quantitation that required minimal engineering of the RNA.

Topics: Aptamers, Nucleotide; Base Sequence; Biosensing Techniques; Cyclic GMP; Electrophoretic Mobility Shift Assay; Escherichia coli; Mutation; Nucleic Acid Conformation; Riboswitch; Second Messenger Systems; Signal Transduction

The motile-to-sessile transition is an important lifestyle switch in diverse bacteria and is often regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). In general, high c-di-GMP concentrations promote attachment to surfaces, whereas cells with low levels of signal remain motile. In the plant pathogen Agrobacterium tumefaciens, c-di-GMP controls attachment and biofilm formation via regulation of a unipolar polysaccharide (UPP) adhesin. The levels of c-di-GMP in A. tumefaciens are controlled in part by the dual-function diguanylate cyclase-phosphodiesterase (DGC-PDE) protein DcpA. In this study, we report that DcpA possesses both c-di-GMP synthesizing and degrading activities in heterologous and native genetic backgrounds, a binary capability that is unusual among GGDEF-EAL domain-containing proteins. DcpA activity is modulated by a pteridine reductase called PruA, with DcpA acting as a PDE in the presence of PruA and a DGC in its absence. PruA enzymatic activity is required for the control of DcpA and through this control, attachment and biofilm formation. Intracellular pterin analysis demonstrates that PruA is responsible for the production of a novel pterin species. In addition, the control of DcpA activity also requires PruR, a protein encoded directly upstream of DcpA with a predicted molybdopterin-binding domain. PruR is hypothesized to be a potential signaling intermediate between PruA and DcpA through an as-yet-unidentified mechanism. This study provides the first prokaryotic example of a pterin-mediated signaling pathway and a new model for the regulation of dual-function DGC-PDE proteins.. Pathogenic bacteria often attach to surfaces and form multicellular communities called biofilms. Biofilms are inherently resilient and can be difficult to treat, resisting common antimicrobials. Understanding how bacterial cells transition to the biofilm lifestyle is essential in developing new therapeutic strategies. We have characterized a novel signaling pathway that plays a dominant role in the regulation of biofilm formation in the model pathogen Agrobacterium tumefaciens. This control pathway involves small metabolites called pterins, well studied in eukaryotes, but this is the first example of pterin-dependent signaling in bacteria. The described pathway controls levels of an important intracellular second messenger (cyclic diguanylate monophosphate) that regulates key bacterial processes such as biofilm formation, motility, and virulence. Pterins control the balance of activity for an enzyme that both synthesizes and degrades the second messenger. These findings reveal a complex, multistep pathway that modulates this enzyme, possibly identifying new targets for antibacterial intervention.

Topics: Agrobacterium tumefaciens; Bacterial Adhesion; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Models, Biological; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Pterins; Signal Transduction

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a global secondary bacterial messenger that controls the formation of drug-resistant multicellular biofilms. Lowering the intracellular c-di-GMP content can disperse biofilms, and it is proposed as a biofilm eradication strategy. However, freshly dispersed biofilm cells exhibit a physiology distinct from biofilm and planktonic cells, and they might have a clinically relevant role in infections. Here we present in vitro and in vivo protocols for the generation and characterization of dispersed cells from Pseudomonas aeruginosa biofilms by reducing the intracellular c-di-GMP content through modulation of phosphodiesterases (PDEs). Unlike conventional protocols that demonstrate biofilm dispersal by biomass quantification, our protocols enable physiological characterization of the dispersed cells. Biomarkers of dispersed cells are identified and quantified, serving as potential targets for treating the dispersed cells. The in vitro protocol can be completed within 4 d, whereas the in vivo protocol requires 7 d.

Topics: Animals; Bacteriological Techniques; Biofilms; Cyclic GMP; Female; Mice, Inbred BALB C; Pseudomonas aeruginosa

A number of investigations of Escherichia coli have suggested that the DNA-binding protein H-NS, in addition to its well-known functions in chromosome organization and gene regulation, interacts directly with the flagellar motor to modulate its function. Here, in a study initially aimed at characterizing the H-NS/motor interaction further, we identify problems and limitations in the previous work that substantially weaken the case for a direct H-NS/motor interaction. Null hns mutants are immotile, largely owing to the downregulation of the flagellar master regulators FlhD and FlhC. We, and others, previously reported that an hns mutant remains poorly motile even when FlhDC are expressed constitutively. In the present work, we use better-engineered strains to show that the motility defect in a Δhns, FlhDC-constitutive strain is milder than that reported previously and does not point to a direct action of H-NS at the motor. H-NS regulates numerous genes and might influence motility via a number of regulatory molecules besides FlhDC. To examine the sources of the motility defect that persists in an FlhDC-constitutive Δhns mutant, we measured transcript levels and overexpression effects of a number of genes in candidate regulatory pathways. The results indicate that H-NS influences motility via multiple regulatory linkages that include, minimally, the messenger molecule cyclic di-GMP, the biofilm regulatory protein CsgD, and the sigma factors σ(S) and σ(F). The results are in accordance with the more standard view of H-NS as a regulator of gene expression rather than a direct modulator of flagellar motor performance.. Data from a number of previous studies have been taken to indicate that the nucleoid-organizing protein H-NS influences motility not only by its well-known DNA-based mechanisms but also by binding directly to the flagellar motor to alter function. In this study, H-NS is shown to influence motility through diverse regulatory pathways, but a direct interaction with the motor is not supported. Previous indications of a direct action at the motor appear to be related to the use of nonnull strains and, in some cases, a failure to effectively bypass the requirement for H-NS in the expression of the flagellar regulon. These findings call for a substantially revised interpretation of the literature concerning H-NS and flagellar motility and highlight the importance of H-NS in diverse regulatory processes involved in the motile-sessile transition.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; DNA-Binding Proteins; Escherichia coli; Escherichia coli Proteins; Flagella; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Membrane Proteins; Mutation; Phosphorus-Oxygen Lyases; Sigma Factor

Bacteria have developed a repertoire of signalling mechanisms that enable adaptive responses to fluctuating environmental conditions. The formation of biofilm, for example, allows persisting in times of external stresses, e.g. induced by antibiotics or a lack of nutrients. Adhesive curli fibers, the major extracellular matrix components in Escherichia coli biofilms, exhibit heterogeneous expression in isogenic cells exposed to identical external conditions. The dynamical mechanisms underlying this heterogeneity remain poorly understood. In this work, we elucidate the potential role of post-translational bistability as a source for this heterogeneity.. We introduce a structured modelling workflow combining logical network topology analysis with time-continuous deterministic and stochastic modelling. The aim is to evaluate the topological structure of the underlying signalling network and to identify and analyse model parameterisations that satisfy observations from a set of genetic knockout experiments. Our work supports the hypothesis that the phenotypic heterogeneity of curli expression in biofilm cells is induced by bistable regulation at the post-translational level. Stochastic modelling suggests diverse noise-induced switching behaviours between the stable states, depending on the expression levels of the c-di-GMP-producing (diguanylate cyclases, DGCs) and -degrading (phosphodiesterases, PDEs) enzymes and reveals the quantitative difference in stable c-di-GMP levels between distinct phenotypes. The most dominant type of behaviour is characterised by a fast switching from curli-off to curli-on with a slow switching in the reverse direction and the second most dominant type is a long-term differentiation into curli-on or curli-off cells. This behaviour may implicate an intrinsic feature of the system allowing for a fast adaptive response (curli-on) versus a slow transition to the curli-off state, in line with experimental observations.. The combination of logical and continuous modelling enables a thorough analysis of different determinants of bistable regulation, i.e. network topology and biochemical kinetics, and allows for an incorporation of experimental data from heterogeneous sources. Our approach yields a mechanistic explanation for the phenotypic heterogeneity of curli fiber expression. Furthermore, the presented work provides a detailed insight into the interactions between the multiple DGC- and PDE-type enzymes and the role of c-di-GMP in dynamical regulation of cellular decisions.

Topics: Biofilms; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Extracellular Matrix; Gene Expression Regulation, Bacterial; Gene Knockout Techniques; Models, Genetic; Phenotype; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Biosynthesis; Signal Transduction; Stochastic Processes

AlgR is a key transcriptional regulator required for the expression of multiple virulence factors, including type IV pili and alginate in Pseudomonas aeruginosa. However, the regulon and molecular regulatory mechanism of AlgR have yet to be fully elucidated. Here, among 157 loci that were identified by a ChIP-seq assay, we characterized a gene, mucR, which encodes an enzyme that synthesizes the intracellular second messenger cyclic diguanylate (c-di-GMP). A ΔalgR strain produced lesser biofilm than did the wild-type strain, which is consistent with a phenotype controlled by c-di-GMP. AlgR positively regulates mucR via direct binding to its promoter. A ΔalgRΔmucR double mutant produced lesser biofilm than did the single ΔalgR mutant, demonstrating that c-di-GMP is a positive regulator of biofilm formation. AlgR controls the levels of c-di-GMP synthesis via direct regulation of mucR. In addition, the cognate sensor of AlgR, FimS/AlgZ, also plays an important role in P. aeruginosa virulence. Taken together, this study provides new insights into the AlgR regulon and reveals the involvement of c-di-GMP in the mechanism underlying AlgR regulation.

Topics: Bacterial Proteins; Binding Sites; Biofilms; Chromatin Immunoprecipitation; Cyclic GMP; Escherichia coli Proteins; Gene Deletion; Gene Expression Regulation, Bacterial; Genome, Bacterial; High-Throughput Nucleotide Sequencing; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa; Pyocyanine; Sequence Analysis, DNA; Trans-Activators; Virulence

The widespread second messenger molecule cyclic di-GMP (cdG) regulates the transition from motile and virulent lifestyles to sessile, biofilm-forming ones in a wide range of bacteria. Many pathogenic and commensal bacterial-host interactions are known to be controlled by cdG signaling. Although the biochemistry of cyclic dinucleotide metabolism is well understood, much remains to be discovered about the downstream signaling pathways that induce bacterial responses upon cdG binding. As part of our ongoing research into the role of cdG signaling in plant-associated Pseudomonas species, we carried out an affinity capture screen for cdG binding proteins in the model organism Pseudomonas fluorescens SBW25. The flagella export AAA+ ATPase FliI was identified as a result of this screen and subsequently shown to bind specifically to the cdG molecule, with a KD in the low micromolar range. The interaction between FliI and cdG appears to be very widespread. In addition to FliI homologs from diverse bacterial species, high affinity binding was also observed for the type III secretion system homolog HrcN and the type VI ATPase ClpB2. The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro. Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits. Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins.

Topics: Adenosine Triphosphatases; Allosteric Site; Amino Acid Sequence; Bacteria; Bacterial Proteins; Binding Sites; Cyclic GMP; Flagella; Gene Expression Regulation; Gene Expression Regulation, Bacterial; L-Lactate Dehydrogenase; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Nucleotides; Protein Binding; Protein Conformation; Protein Transport; Proton-Translocating ATPases; Pseudomonas aeruginosa; Pyruvate Kinase; Sequence Homology, Amino Acid; Signal Transduction; Surface Plasmon Resonance

Malignant melanomas escape immunosurveillance via the loss/down-regulation of MHC-I expression. Natural killer (NK) cells have the potential to function as essential effector cells for eliminating melanomas. Cyclic di-GMP (c-di-GMP), a ligand of the stimulator of interferon genes (STING) signal pathway, can be thought of as a new class of adjuvant against cancer. However, it is yet to be tested, because technologies for delivering c-di-GMP to the cytosol are required. Herein, we report that c-di-GMP efficiently activates NK cells and induces antitumor effects against malignant melanomas when loaded in YSK05 lipid containing liposomes, by assisting in the efficient delivery of c-di-GMP to the cytosol. The intravenous administration of c-di-GMP encapsulated within YSK05-liposomes (c-di-GMP/YSK05-Lip) into mice efficiently induced the production of type I interferon (IFN) as well as the activation of NK cells, resulting in a significant antitumor effect in a lung metastasis mouse model using B16-F10. This antitumor effect was dominated by NK cells. The infiltration of NK cells was observed in the lungs with B16-F10 melanomas. These findings indicate that the c-di-GMP/YSK05-Lip induces MHC-I non-restricted antitumor immunity mediated by NK cells. Consequently, c-di-GMP/YSK05-Lip represents a potentially new adjuvant system for use in immunotherapy against malignant melanomas.

Topics: Animals; Chemotherapy, Adjuvant; Cyclic GMP; Cytokines; Cytosol; Drug Delivery Systems; Immunotherapy; Injections, Intravenous; Killer Cells, Natural; Ligands; Lipids; Liposomes; Lung; Macrophage Activation; Melanoma, Experimental; Membrane Proteins; Mice; Neoplasm Metastasis; Piperidines

In Gram-positive pathogens, surface proteins may be covalently anchored to the bacterial peptidoglycan by sortase, a cysteine transpeptidase enzyme. In contrast to other Gram-positive bacteria, only one single sortase enzyme, SrtB, is conserved between strains of Clostridium difficile. Sortase-mediated peptidase activity has been reported in vitro, and seven potential substrates have been identified. Here, we demonstrate the functionality of sortase in C. difficile. We identify two sortase-anchored proteins, the putative adhesins CD2831 and CD3246, and determine the cell wall anchor structure of CD2831. The C-terminal PPKTG sorting motif of CD2831 is cleaved between the threonine and glycine residues, and the carboxyl group of threonine is amide-linked to the side chain amino group of diaminopimelic acid within the peptidoglycan peptide stem. We show that CD2831 protein levels are elevated in the presence of high intracellular cyclic diGMP (c-diGMP) concentrations, in agreement with the control of CD2831 expression by a c-diGMP-dependent type II riboswitch. Low c-diGMP levels induce the release of CD2831 and presumably CD3246 from the surface of cells. This regulation is mediated by proteolytic cleavage of CD2831 and CD3246 by the zinc metalloprotease ZmpI, whose expression is controlled by a type I c-diGMP riboswitch. These data reveal a novel regulatory mechanism for expression of two sortase substrates by the secondary messenger c-diGMP, on which surface anchoring is dependent.

Topics: Adhesins, Bacterial; Amino Acid Motifs; Aminoacyltransferases; Bacterial Proteins; Cell Membrane; Cell Wall; Clostridioides difficile; Cyclic GMP; Cysteine Endopeptidases; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Metalloproteases; Microscopy, Fluorescence; Mutation; Oligonucleotides; Peptide Hydrolases; Peptidoglycan; Plasmids; Protein Binding; Protein Structure, Tertiary; Tandem Mass Spectrometry; Virulence Factors

The nucleotide second messengers cAMP and c-di-GMP allow many bacteria, including the human intestinal pathogen Vibrio cholerae, to respond to environmental stimuli with appropriate physiological adaptations. In response to limitation of specific carbohydrates, cAMP and its receptor CRP control the transcription of genes important for nutrient acquisition and utilization; c-di-GMP controls the transition between motile and sessile lifestyles often, but not exclusively, through transcriptional mechanisms. In this study, we investigated the convergence of cAMP and c-di-GMP signaling pathways in regulating the expression of gbpA. GbpA is a colonization factor that participates in the attachment of V. cholerae to N-acetylglucosamine-containing surfaces in its native aquatic environment and the host intestinal tract.. We show that c-di-GMP inhibits gbpA activation in a fashion independent of the known transcription factors that directly sense c-di-GMP. Interestingly, inhibition of gbpA activation by c-di-GMP only occurs during growth on non-PTS dependent nutrient sources. Consistent with this result, we show that CRP binds to the gbpA promoter in a cAMP-dependent manner in vitro and drives transcription of gbpA in vivo. The interplay between cAMP and c-di-GMP does not broadly impact the CRP-cAMP regulon, but occurs more specifically at the gbpA promoter.. These findings suggest that c-di-GMP directly interferes with the interaction of CRP-cAMP and the gbpA promoter via an unidentified regulator. The use of two distinct second messenger signaling mechanisms to regulate gbpA transcription may allow V. cholerae to finely modulate GbpA production, and therefore colonization of aquatic and host surfaces, in response to discrete environmental stimuli.

Topics: Adhesins, Bacterial; Cyclic AMP; Cyclic GMP; Gene Expression Regulation, Bacterial; Humans; Second Messenger Systems; Vibrio cholerae

The second messenger cyclic diguanylate (c-di-GMP) controls diverse cellular processes among bacteria. Diguanylate cyclases synthesize c-di-GMP, whereas it is degraded by c-di-GMP-specific phosphodiesterases (PDEs). Nearly 80% of these PDEs are predicted to depend on the catalytic function of glutamate-alanine-leucine (EAL) domains, which hydrolyze a single phosphodiester group in c-di-GMP to produce 5'-phosphoguanylyl-(3',5')-guanosine (pGpG). However, to degrade pGpG and prevent its accumulation, bacterial cells require an additional nuclease, the identity of which remains unknown. Here we identify oligoribonuclease (Orn)-a 3'→5' exonuclease highly conserved among Actinobacteria, Beta-, Delta- and Gammaproteobacteria-as the primary enzyme responsible for pGpG degradation in Pseudomonas aeruginosa cells. We found that a P. aeruginosa Δorn mutant had high intracellular c-di-GMP levels, causing this strain to overexpress extracellular polymers and overproduce biofilm. Although recombinant Orn degraded small RNAs in vitro, this enzyme had a proclivity for degrading RNA oligomers comprised of two to five nucleotides (nanoRNAs), including pGpG. Corresponding with this activity, Δorn cells possessed highly elevated pGpG levels. We found that pGpG reduced the rate of c-di-GMP degradation in cell lysates and inhibited the activity of EAL-dependent PDEs (PA2133, PvrR, and purified recombinant RocR) from P. aeruginosa. This pGpG-dependent inhibition was alleviated by the addition of Orn. These data suggest that elevated levels of pGpG exert product inhibition on EAL-dependent PDEs, thereby increasing intracellular c-di-GMP in Δorn cells. Thus, we propose that Orn provides homeostatic control of intracellular pGpG under native physiological conditions and that this activity is fundamental to c-di-GMP signal transduction.

Topics: Bacterial Proteins; Blotting, Western; Cyclic GMP; Deoxyguanine Nucleotides; Escherichia coli Proteins; Exoribonucleases; Gene Expression Regulation, Bacterial; Homeostasis; Mutation; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

The bacterial second messenger cyclic di-GMP (c-di-GMP) controls biofilm formation and other phenotypes relevant to pathogenesis. Cyclic-di-GMP is synthesized by diguanylate cyclases (DGCs). Phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG), which is then hydrolyzed to two GMP molecules by yet unidentified enzymes termed PDE-Bs. We show that pGpG inhibits a PDE-A from Pseudomonas aeruginosa. In a dual DGC and PDE-A reaction, excess pGpG extends the half-life of c-di-GMP, indicating that removal of pGpG is critical for c-di-GMP homeostasis. Thus, we sought to identify the PDE-B enzyme(s) responsible for pGpG degradation. A differential radial capillary action of ligand assay-based screen for pGpG binding proteins identified oligoribonuclease (Orn), an exoribonuclease that hydrolyzes two- to five-nucleotide-long RNAs. Purified Orn rapidly converts pGpG into GMP. To determine whether Orn is the primary enzyme responsible for degrading pGpG, we assayed cell lysates of WT and ∆orn strains of P. aeruginosa PA14 for pGpG stability. The lysates from ∆orn showed 25-fold decrease in pGpG hydrolysis. Complementation with WT, but not active site mutants, restored hydrolysis. Accumulation of pGpG in the ∆orn strain could inhibit PDE-As, increasing c-di-GMP concentration. In support, we observed increased transcription from the c-di-GMP-regulated pel promoter. Additionally, the c-di-GMP-governed auto-aggregation and biofilm phenotypes were elevated in the ∆orn strain in a pel-dependent manner. Finally, we directly detect elevated pGpG and c-di-GMP in the ∆orn strain. Thus, we identified that Orn serves as the primary PDE-B enzyme that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway.

Topics: Amino Acid Sequence; Bacterial Proteins; Biofilms; Chromatography, Liquid; Cyclic GMP; Deoxyguanine Nucleotides; Escherichia coli Proteins; Exoribonucleases; Guanosine Monophosphate; Homeostasis; Hydrolysis; Molecular Sequence Data; Mutation; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Binding; Pseudomonas aeruginosa; Sequence Homology, Amino Acid; Tandem Mass Spectrometry

Two-component signal transduction systems are the primary mechanisms by which bacteria perceive and respond to changes in their environment. The Hk1/Rrp1 two-component system (TCS) in Borrelia burgdorferi consists of a hybrid histidine kinase and a response regulator with diguanylate cyclase activity, respectively. Phosphorylated Rrp1 catalyzes the synthesis of c-di-GMP, a second messenger associated with bacterial life-style control networks. Spirochetes lacking either Hk1 or Rrp1 are virulent in mice but destroyed within feeding ticks. Activation of Hk1 by exogenous stimuli represents the seminal event for c-di-GMP signaling. We reasoned that structural characterization of Hk1's sensor would provide insights into the mechanism underlying signal transduction and aid in the identification of activating ligands. The Hk1 sensor is composed of three ligand-binding domains (D1-3), each with homology to periplasmic solute-binding proteins (PBPs) typically associated with ABC transporters. Herein, we determined the structure for D1, the most N-terminal PBP domain. As expected, D1 displays a bilobed Venus Fly Trap-fold. Similar to the prototypical sensor PBPs HK29S from Geobacter sulfurreducens and VFT2 from Bordetella pertussis, apo-D1 adopts a closed conformation. Using complementary approaches, including SAXS, we established that D1 forms a dimer in solution. The D1 structure enabled us to model the D2 and D3 domains. Differences in the ligand-binding pockets suggest that each PBP recognizes a different ligand. The ability of Hk1 to recognize multiple stimuli provides spirochetes with a means of distinguishing between the acquisition and transmission blood meals and generate a graded output response that is reflective of the perceived environmental threats.

Topics: Amino Acid Sequence; Borrelia burgdorferi; Catalytic Domain; Conserved Sequence; Crystallography, X-Ray; Cyclic GMP; Histidine Kinase; Models, Molecular; Molecular Sequence Data; Periplasm; Periplasmic Proteins; Protein Kinases; Protein Structure, Quaternary; Protein Structure, Secondary

Comamonas is one of the most abundant microorganisms in biofilm communities driving wastewater treatment. Little has been known about the role of this group of organisms and their biofilm mode of life. In this study, using Comamonas testosteroni as a model organism, we demonstrated the involvement of Comamonas biofilms in denitrification under bulk aerobic conditions and elucidated the influence of nitrate respiration on its biofilm lifestyle. Our results showed that C. testosteroni could use nitrate as the sole electron acceptor for anaerobic growth. Under bulk aerobic condition, biofilms of C. testosteroni were capable of reducing nitrate, and intriguingly, nitrate reduction significantly enhanced viability of the biofilm-cells and reduced cell detachment from the biofilms. Nitrate respiration was further shown to play an essential role in maintaining high cell viability in the biofilms. RNA-seq analysis, quantitative polymerase chain reaction, and liquid chromatography-mass spectrometry revealed a higher level of bis(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) in cells respiring on nitrate than those grown aerobically (1.3 × 10(-4) fmol/cell vs 7.9 × 10(-6) fmol/cell; P < 0.01). C-di-GMP is one universal signaling molecule that regulates the biofilm mode of life, and a higher c-di-GMP concentration reduces cell detachment from biofilms. Taking these factors together, this study reveals that nitrate reduction occurs in mature biofilms of C. testosteroni under bulk aerobic conditions, and the respiratory reduction of nitrate is beneficial to the biofilm lifestyle by providing more metabolic energy to maintain high viability and a higher level of c-di-GMP to reduce cell detachment.

Topics: Aerobiosis; Biofilms; Comamonas testosteroni; Cyclic GMP; Denitrification; Gene Expression Regulation, Bacterial; Nitrates; Polymerase Chain Reaction; Sequence Analysis, RNA; Wastewater

FleQ is an AAA+ ATPase enhancer-binding protein that regulates both flagella and biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa. FleQ belongs to the NtrC subfamily of response regulators, but lacks the corresponding aspartic acid for phosphorylation in the REC domain (FleQ(R), also named FleQ domain). Here, we show that the atypical REC domain of FleQ is essential for the function of FleQ. Crystal structure of FleQ(R) at 2.3Å reveals that the structure of FleQ(R) is significantly different from the REC domain of NtrC1 which regulates gene expression in a phosphorylation dependent manner. FleQ(R) forms a novel active dimer (transverse dimer), and mediates the dimerization of full-length FleQ in an unusual manner. Point mutations that affect the dimerization of FleQ lead to loss of function of the protein. Moreover, a c-di-GMP binding site deviating from the previous reported one is identified through structure analysis and point mutations.

Topics: Amino Acid Sequence; Amino Acid Substitution; Bacterial Proteins; Binding Sites; Biofilms; Crystallography, X-Ray; Cyclic GMP; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Molecular Sequence Data; Protein Interaction Domains and Motifs; Protein Multimerization; Protein Structure, Secondary; Pseudomonas aeruginosa; Trans-Activators

Propionate presents as one of the major volatile fatty acids in municipal wastewaters, which are not readily degraded as acetate by microorganisms. This study cultivated aerobic granules from column reactors with acetate, acetate/propionate mix (3:1 and 1:3) and propionate as carbon sources and noted that propionate-rich feed would delay granulation, but could generate granules with high structural strength. Propionate feed enriched strains fractionated into the hydrophobic phase, Sphaerotilus sp., Sphingomonadaceae and Thauera sp., in granules and altered hydrophobicity of Thauera sp. and Zoogloea sp. The enriched strains could secret high quantities of cyclic-di-diguanylate to increase production of extracellular polymeric substances (EPS). The hydrophobic cell surface and increased EPS quantity led to integrated propionate-fed granules. Feed with high propionate concentration is proposed as promising way to cultivate strong aerobic granules for practical use.

Topics: Acetates; Aerobiosis; Bioreactors; Cyclic GMP; Hydrophobic and Hydrophilic Interactions; Microbial Consortia; Propionates; Thauera; Waste Disposal, Fluid; Wastewater

Cyclic diguanylate monophosphate (c-di-GMP) is a well-conserved second messenger in bacteria. During infection, the innate immune system can also sense c-di-GMP; however, whether bacterial pathogens utilize c-di-GMP as a weapon to fight against host defense for survival and possible mechanisms underlying this process remain poorly understood. Siderocalin (LCN2) is a key antibacterial component of the innate immune system and sequesters bacterial siderophores to prevent acquisition of iron. Here we show that c-di-GMP can directly target the human LCN2 protein to inhibit its antibacterial activity. We demonstrate that c-di-GMP specifically binds to LCN2. In addition, c-di-GMP can compete with bacterial ferric siderophores to bind LCN2. Furthermore, c-di-GMP can significantly reduce LCN2-mediated inhibition on the in vitro growth of Escherichia coli. Thus, LCN2 acts as a c-di-GMP receptor. Our findings provide insight into the mechanism by which bacteria utilize c-di-GMP to interfere with the innate immune system for survival.

Topics: Acute-Phase Proteins; Anti-Bacterial Agents; Carrier Proteins; Cyclic GMP; Databases, Factual; Escherichia coli; Gene Expression Regulation; Humans; Lipocalin-2; Lipocalins; Models, Chemical; Models, Molecular; Molecular Structure; Mycobacterium tuberculosis; Protein Binding; Protein Conformation; Proto-Oncogene Proteins; Recombinant Proteins

The cyclic diguanylate (c-di-GMP) is currently considered an ubiquitous second messenger in bacteria that influences a wide range of cellular processes. One of the methodological approaches to unravel c-di-GMP regulatory networks involves raising the c-di-GMP intracellular levels, e.g. by expressing a diguanylate cyclase (DGC), to provoke phenotypic changes.. We have constructed mini-Tn7 delivery vectors for the integration and stable expression of the pleD* gene encoding a highly active DGC, which can be used to artificially increase the intracellular levels of c-di-GMP in Gram negative bacteria. The functionality of these new vectors has been validated in several plant-interacting α- and γ-proteobacteria. Similarly to vector plasmid-borne pleD*, the genome-borne mini-Tn7pleD* constructs provide significant increases in intracellular c-di-GMP, provoking expected phenotypic changes such as enhanced polysaccharide production, biofilm formation and reduced motility. However, the mini-Tn7pleD* constructs resulted far more stable in the absence of antibiotics than the plasmid-based pleD* constructs. Furthermore, we have also implemented an inducible system to modulate pleD* expression and intracellular c-di-GMP rises "on demand".. mini-Tn7pleD* constructs are very stable and are maintained during bacterial free-living growth as well as during interaction with eukaryotic hosts, in the absence of selective pressure. This high stability ensures experimental homogeneity in time and space with regard to enhancing c-di-GMP intracellular levels in bacteria of interest.

Topics: Cyclic GMP; DNA Transposable Elements; Escherichia coli Proteins; Gene Expression; Genetics, Microbial; Genomic Instability; Gram-Negative Bacteria; Molecular Biology; Phosphorus-Oxygen Lyases; Recombination, Genetic

Dickeya dadantii is a globally dispersed phytopathogen which causes diseases on a wide range of host plants. This pathogen utilizes the type III secretion system (T3SS) to suppress host defense responses, and secretes pectate lyase (Pel) to degrade the plant cell wall. Although the regulatory small RNA (sRNA) RsmB, cyclic diguanylate monophosphate (c-di-GMP) and flagellar regulator have been reported to affect the regulation of these two virulence factors or multiple cell behaviours such as motility and biofilm formation, the linkage between these regulatory components that coordinate the cell behaviours remain unclear. Here, we revealed a sophisticated regulatory network that connects the sRNA, c-di-GMP signalling and flagellar master regulator FlhDC. We propose multi-tiered regulatory mechanisms that link the FlhDC to the T3SS through three distinct pathways including the FlhDC-FliA-YcgR3937 pathway; the FlhDC-EcpC-RpoN-HrpL pathway; and the FlhDC-rsmB-RsmA-HrpL pathway. Among these, EcpC is the most dominant factor for FlhDC to positively regulate T3SS expression.

Topics: Amino Acid Sequence; Bacterial Proteins; Cyclic GMP; Enterobacteriaceae; Fimbriae Proteins; Flagella; Flagellin; Gene Expression Regulation, Bacterial; Plant Diseases; Polysaccharide-Lyases; Regulatory Sequences, Ribonucleic Acid; Signal Transduction; Transcription Factors; Type III Secretion Systems; Vegetables; Virulence; Virulence Factors

In many bacteria, including Vibrio cholerae, cyclic dimeric guanosine monophosphate (c-di-GMP) controls the motile to biofilm life style switch. Yet, little is known about how this occurs. In this study, we report that changes in c-di-GMP concentration impact the biosynthesis of the MshA pili, resulting in altered motility and biofilm phenotypes in V. cholerae. Previously, we reported that cdgJ encodes a c-di-GMP phosphodiesterase and a ΔcdgJ mutant has reduced motility and enhanced biofilm formation. Here we show that loss of the genes required for the mannose-sensitive hemagglutinin (MshA) pilus biogenesis restores motility in the ΔcdgJ mutant. Mutations of the predicted ATPase proteins mshE or pilT, responsible for polymerizing and depolymerizing MshA pili, impair near surface motility behavior and initial surface attachment dynamics. A ΔcdgJ mutant has enhanced surface attachment, while the ΔcdgJmshA mutant phenocopies the high motility and low attachment phenotypes observed in a ΔmshA strain. Elevated concentrations of c-di-GMP enhance surface MshA pilus production. MshE, but not PilT binds c-di-GMP directly, establishing a mechanism for c-di-GMP signaling input in MshA pilus production. Collectively, our results suggest that the dynamic nature of the MshA pilus established by the assembly and disassembly of pilin subunits is essential for transition from the motile to sessile lifestyle and that c-di-GMP affects MshA pilus assembly and function through direct interactions with the MshE ATPase.

Topics: Biofilms; Cyclic GMP; Epistasis, Genetic; Fimbriae Proteins; Fimbriae, Bacterial; Mannose-Binding Lectin; Movement; Vibrio cholerae

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates a variety of complex processes through a diverse set of c-di-GMP receptor proteins. We have utilized a systematic approach to identify c-di-GMP receptors from the pathogen Vibrio cholerae using the Differential Radial Capillary Action of Ligand Assay (DRaCALA). The DRaCALA screen identified a majority of known c-di-GMP binding proteins in V. cholerae and revealed a novel c-di-GMP binding protein, MshE (VC0405), an ATPase associated with the mannose sensitive hemagglutinin (MSHA) type IV pilus. The known c-di-GMP binding proteins identified by DRaCALA include diguanylate cyclases, phosphodiesterases, PilZ domain proteins and transcription factors VpsT and VpsR, indicating that the DRaCALA-based screen of open reading frame libraries is a feasible approach to uncover novel receptors of small molecule ligands. Since MshE lacks the canonical c-di-GMP-binding motifs, a truncation analysis was utilized to locate the c-di-GMP binding activity to the N-terminal T2SSE_N domain. Alignment of MshE homologs revealed candidate conserved residues responsible for c-di-GMP binding. Site-directed mutagenesis of these candidate residues revealed that the Arg9 residue is required for c-di-GMP binding. The ability of c-di-GMP binding to MshE to regulate MSHA dependent processes was evaluated. The R9A allele, in contrast to the wild type MshE, was unable to complement the ΔmshE mutant for the production of extracellular MshA to the cell surface, reduction in flagella swimming motility, attachment to surfaces and formation of biofilms. Testing homologs of MshE for binding to c-di-GMP identified the type II secretion ATPase of Pseudomonas aeruginosa (PA14_29490) as a c-di-GMP receptor, indicating that type II secretion and type IV pili are both regulated by c-di-GMP.

Topics: Adenosine Triphosphatases; Cyclic GMP; Fimbriae Proteins; Fimbriae, Bacterial; Mannose-Binding Lectin; Open Reading Frames; Type II Secretion Systems; Vibrio cholerae

Cyclic di-GMP is a bacterial second messenger that is involved in switching between motile and sessile lifestyles. Given the medical importance of biofilm formation, there has been increasing interest in understanding the synthesis and degradation of cyclic di-GMPs and their regulation in various bacterial pathogens. Environmental cues are detected by sensing domains coupled to GGDEF and EAL or HD-GYP domains that have diguanylate cyclase and phosphodiesterase activities, respectively, producing and degrading cyclic di-GMP. The Escherichia coli protein DosC (also known as YddV) consists of an oxygen-sensing domain belonging to the class of globin sensors that is coupled to a C-terminal GGDEF domain via a previously uncharacterized middle domain. DosC is one of the most strongly expressed GGDEF proteins in E. coli, but to date structural information on this and related proteins is scarce. Here, the high-resolution structural characterization of the oxygen-sensing globin domain, the middle domain and the catalytic GGDEF domain in apo and substrate-bound forms is described. The structural changes between the iron(III) and iron(II) forms of the sensor globin domain suggest a mechanism for oxygen-dependent regulation. The structural information on the individual domains is combined into a model of the dimeric DosC holoprotein. These findings have direct implications for the oxygen-dependent regulation of the activity of the cyclase domain.

Topics: Crystallography, X-Ray; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Models, Molecular; Oxygen; Phosphorus-Oxygen Lyases; Protein Structure, Tertiary

The structure and biological properties of RNAs are a function of changing cellular conditions, but comprehensive, simultaneous investigation of the effect of multiple interacting environmental variables is not easily achieved. We have developed an efficient, high-throughput method to characterize RNA structure and thermodynamic stability as a function of multiplexed solution conditions using Förster resonance energy transfer (FRET). In a single FRET experiment using conventional quantitative PCR instrumentation, 19,400 conditions of MgCl2, ligand and temperature are analysed to generate detailed empirical conformational and stability landscapes of the cyclic diguanylate (c-di-GMP) riboswitch. The method allows rapid comparison of RNA structure modulation by cognate and non-cognate ligands. Landscape analysis reveals that kanamycin B stabilizes a non-native, idiosyncratic conformation of the riboswitch that inhibits c-di-GMP binding. This demonstrates that allosteric control of folding, rather than direct competition with cognate effectors, is a viable approach for pharmacologically targeting riboswitches and other structured RNA molecules.

Topics: Cyclic GMP; Fluorescence Resonance Energy Transfer; High-Throughput Screening Assays; Ligands; Nucleic Acid Conformation; Riboswitch; RNA Stability; RNA, Bacterial; Vibrio cholerae

Cyclic diguanylate (c-di-GMP) is a bacterial second messenger that controls multiple cellular processes. c-di-GMP networks have up to dozens of diguanylate cyclases (DGCs) that synthesize c-di-GMP along with many c-di-GMP-responsive target proteins that can bind and respond to this signal. For such networks to have order, a mechanism(s) likely exists that allow DGCs to specifically signal their targets, and it has been suggested that physical interactions might provide such specificity. Our results show a DGC from Pseudomonas fluorescens physically interacting with its target protein at a conserved interface, and this interface can be predictive of DGC-target protein interactions. Furthermore, we demonstrate that physical interaction is necessary for the DGC to maximally signal its target. If such "local signaling" is a theme for even a fraction of the DGCs used by bacteria, it becomes possible to posit a model whereby physical interaction allows a DGC to directly signal its target protein, which in turn may help curtail undesired cross talk with other members of the network.. An important question in microbiology is how bacteria make decisions using a signaling network made up of proteins that make, break, and bind the second messenger c-di-GMP, which is responsible for controlling many cellular behaviors. Previous work has shown that a given DGC enzyme will signal for specific cellular outputs, despite making the same diffusible molecule as its sibling DGCs in the unpartitioned space of the bacterial cell. Understanding how one DGC differentiates its output from the dozens of other such enzymes in the cell is synonymous with understanding a large component of the bacterial decision-making machinery. We present evidence for a helix on a DGC used to physically associate with its target protein, which is necessary to achieve maximal signaling.

Topics: Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Protein Binding; Pseudomonas fluorescens; Signal Transduction

The dispersal phase that completes the biofilm lifecycle is of particular interest for its potential to remove recalcitrant, antimicrobial tolerant biofilm infections. Here we found that temperature is a cue for biofilm dispersal and a rise by 5 °C or more can induce the detachment of Pseudomonas aeruginosa biofilms. Temperature upshifts were found to decrease biofilm biomass and increase the number of viable freely suspended cells. The dispersal response appeared to involve the secondary messenger cyclic di-GMP, which is central to a genetic network governing motile to sessile transitions in bacteria. Furthermore, we used poly((oligo(ethylene glycol) methyl ether acrylate)-block-poly(monoacryloxy ethyl phosphate)-stabilized iron oxide nanoparticles (POEGA-b-PMAEP@IONPs) to induce local hyperthermia in established biofilms upon exposure to a magnetic field. POEGA-b-PMAEP@IONPs were non-toxic to bacteria and when heated induced the detachment of biofilm cells. Finally, combined treatments of POEGA-b-PMAEP@IONPs and the antibiotic gentamicin reduced by 2-log the number of colony-forming units in both biofilm and planktonic phases after 20 min, which represent a 3.2- and 4.1-fold increase in the efficacy against planktonic and biofilm cells, respectively, compared to gentamicin alone. The use of iron oxide nanoparticles to disperse biofilms may find broad applications across a range of clinical and industrial settings.

Topics: Anti-Bacterial Agents; Biofilms; Cyclic GMP; Dynamic Light Scattering; Ferric Compounds; Hyperthermia, Induced; Magnetic Fields; Metal Nanoparticles; Microscopy, Electron, Transmission; Polymers; Pseudomonas aeruginosa; Thermogravimetry

Pseudomonas aeruginosa is a versatile bacterial pathogen capable of occupying diverse ecological niches. To cope with iron limitation, P. aeruginosa secretes two siderophores, pyoverdine and pyochelin, whose ability to deliver iron to the cell is crucial for biofilm formation and pathogenicity. In this study, we describe a link between iron uptake and the Gac/Rsm system, a conserved signal transducing pathway of P. aeruginosa that controls the production of extracellular products and virulence factors, as well as the switch from planktonic to biofilm lifestyle. We have observed that pyoverdine and pyochelin production in P. aeruginosa is strongly dependent on the activation state of the Gac/Rsm pathway, which controls siderophore regulatory and biosynthetic genes at the transcriptional level, in a manner that does not involve regulation of ferric uptake regulator (Fur) expression. Gac/Rsm-mediated regulation of iron uptake genes appears to be conserved in different P. aeruginosa strains. Further experiments led to propose that the Gac/Rsm system regulates siderophore production through modulation of the intracellular levels of the second messenger c-di-GMP, indicating that the c-di-GMP and the Gac/Rsm regulatory networks essential for biofilm formation can also coordinately control iron uptake in P. aeruginosa.

Topics: Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Iron; Oligopeptides; Phenols; Pseudomonas aeruginosa; Repressor Proteins; RNA-Binding Proteins; Siderophores; Sigma Factor; Thiazoles; Virulence Factors

In many species of the marine Roseobacter clade, periods of attached life, in association with phytoplankton or particles, are interspersed with planktonic phases. The purpose of this study was to determine whether shifts between motile and sessile life in the globally abundant Roseobacter clade species Ruegeria mobilis are associated with intracellular concentrations of the signal compound cyclic dimeric guanosinmonophosphate (c-di-GMP), which in bacteria regulates transitions between motile and sessile life stages. Genes for diguanylate cyclases and phosphodiesterases, which are involved in c-di-GMP signalling, were found in the genome of R. mobilis strain F1926. Ion pair chromatography-tandem mass spectrometry revealed 20-fold higher c-di-GMP concentrations per cell in biofilm-containing cultures than in planktonic cells. An introduced diguanylate cyclase gene increased c-di-GMP and enhanced biofilm formation and production of the potent antibiotic tropodithietic acid (TDA). An introduced phosphodiesterase gene decreased c-di-GMP and reduced biofilm formation and TDA production. tdaC, a key gene for TDA biosynthesis, was expressed only in attached or biofilm-forming cells, and expression was induced immediately after initial attachment. In conclusion, c-di-GMP signalling controls biofilm formation and biofilm-associated traits in R. mobilis and, as suggested by presence of GGDEF and EAL domain protein genes, also in other Roseobacter clade species.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Molecular Sequence Data; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Structure, Tertiary; Rhodobacteraceae; Tropolone

A host type I IFN response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP (c-di-GMP) by STING (stimulator of IFN genes). Because the STING, an adaptor protein, links the cytosolic detection of DNA by the cytosolic DNA sensors such as the IFN-inducible human IFI16 and murine p202 proteins to the TBK1/IRF3 axis, we investigated whether c-di-GMP-induced signaling could regulate expression of IFI16 and p202 proteins. Here, we report that activation of c-di-GMP-induced signaling in human and murine cells increased steady-state levels of IFI16 and p202 proteins. The increase was c-di-GMP concentration- and time-dependent. Unexpectedly, treatment of cells with type I IFN decreased levels of the adaptor protein STING. Therefore, we investigated whether the IFI16 or p202 protein could regulate the expression of STING and activation of the TBK1/IRF3 axis. We found that constitutive knockdown of IFI16 or p202 expression in cells increased steady-state levels of STING. Additionally, the knockdown of IFI16 resulted in activation of the TBK1/IRF3 axis. Accordingly, increased levels of the IFI16 or p202 protein in cells decreased STING levels. Together, our observations identify a novel negative feedback loop between c-di-GMP-induced levels of IFI16 and p202 cytosolic DNA sensors and the adaptor protein STING.

Topics: Animals; Cell Line; Cyclic GMP; Cytosol; DNA; Feedback, Physiological; Gene Knockdown Techniques; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Inbred C57BL; Nuclear Proteins; Phosphoproteins; Signal Transduction; Tumor Suppressor p53-Binding Protein 1

The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.

Topics: Anti-Bacterial Agents; Azoles; Biofilms; Capillary Action; Cyclic GMP; Escherichia coli Proteins; High-Throughput Screening Assays; Isoindoles; Ligands; Models, Molecular; Organoselenium Compounds; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa; Pseudomonas Infections

Bis-(3',5') cyclic di-guanylate (c-di-GMP) is a key bacterial second messenger that is implicated in the regulation of many crucial processes that include biofilm formation, motility and virulence. Cellular levels of c-di-GMP are controlled through synthesis by GGDEF domain diguanylate cyclases and degradation by two classes of phosphodiesterase with EAL or HD-GYP domains. Here, we have determined the structure of an enzymatically active HD-GYP domain protein from Persephonella marina (PmGH) alone, in complex with substrate (c-di-GMP) and final reaction product (GMP). The structures reveal a novel trinuclear iron binding site, which is implicated in catalysis and identify residues involved in recognition of c-di-GMP. This structure completes the picture of all domains involved in c-di-GMP metabolism and reveals that the HD-GYP family splits into two distinct subgroups containing bi- and trinuclear metal centres.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Amino Acid Motifs; Amino Acid Sequence; Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Cyclic GMP; Evolution, Molecular; Gram-Negative Bacteria; Iron; Mutation; Protein Conformation; Protein Structure, Tertiary; Sequence Alignment

Yersinia pestis, the cause of plague, forms a biofilm in the foregut of its flea vector to enhance transmission. Biofilm formation in Y. pestis is controlled by the intracellular levels of the second messenger molecule cyclic diguanylate (c-di-GMP). HmsT and Y3730, the two diguanylate cyclases (DGC) in Y. pestis, are responsible for the synthesis of c-di-GMP. Y3730, which we name here as HmsD, has little effect on in vitro biofilms, but has a major effect on biofilm formation in the flea. The mechanism by which HmsD plays differential roles in vivo and in vitro is not understood. In this study, we show that hmsD is part of a three-gene operon (y3729-31), which we designate as hmsCDE. Deletion of hmsC resulted in increased, hmsD-dependent biofilm formation, while deletion or overexpression of hmsE did not affect biofilm formation. Localization experiments suggest that HmsC resides in the periplasmic space. In addition, we provide evidence that HmsC might interact directly with the periplasmic domain of HmsD and cause the proteolysis of HmsD. We propose that HmsC senses the environmental signals, which in turn regulates HmsD, and controls the c-di-GMP synthesis and biofilm formation in Y. pestis.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Periplasmic Proteins; Phosphorus-Oxygen Lyases; Yersinia pestis

The major sessility-motility lifestyle change and additional fundamental aspects of bacterial physiology, behaviour and morphology are regulated by the secondary messenger cyclic di-GMP (c-di-GMP). Although the c-di-GMP metabolizing enzymes and many receptors have been readily characterized upon discovery, the HD-GYP domain c-di-GMP phosphodiesterase family remained underinvestigated. In this issue of Molecular Microbiology, Bellini et al. provide an important step towards functional and structural characterization of the previously neglected HD-GYP domain family by resolving the crystal structure of PmGH, a catalytically active family member from the thermophilic bacterium Persephonella marina. The crystal structure revealed a novel tri-nuclear catalytic iron centre involved in c-di-GMP binding and catalysis and provides the structural basis to subsequently characterize in detail the catalytic mechanism of hydrolysis of c-di-GMP to GMP by HD-GYP domains.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Bacterial Proteins; Catalytic Domain; Cyclic GMP; Gram-Negative Bacteria; Iron

Bacteria have evolved dedicated signaling mechanisms that enable the integration of a range of environmental stimuli and the accordant modulation of metabolic pathways. One central signaling molecule in bacteria is the second messenger cyclic dimeric GMP (c-di-GMP). Complex regulatory mechanisms for modulating c-di-GMP concentrations have evolved, in line with its importance for maintaining bacterial fitness under changing environmental conditions. One interesting example in this context is the blue-light-regulated phosphodiesterase 1 (BlrP1) of Klebsiella pneumoniae. This covalently linked system of a sensor of blue light using FAD (BLUF) and an EAL phosphodiesterase domain orchestrates the light-dependent down-regulation of c-di-GMP levels. To reveal details of light-induced structural changes involved in EAL activity regulation, we extended previous crystallographic studies with hydrogen-deuterium exchange experiments and small-angle X-ray scattering analysis of different functional BlrP1 states. The combination of hydrogen-deuterium exchange and small-angle X-ray scattering allows the integration of local and global structural changes and provides an improved understanding of light signaling via an allosteric communication pathway between the BLUF and EAL domains. This model is supported by results from a mutational analysis of the EAL dimerization region and the analysis of metal-coordination effects of the EAL active site on the dark-state recovery kinetics of the BLUF domain. In combination with structural information from other EAL domains, the observed bidirectional communication points to a general mechanism of EAL activity regulation and suggests that a similar allosteric coupling is maintained in catalytically inactive EAL domains that retain a regulatory function.

Topics: Allosteric Regulation; Amino Acid Sequence; Bacterial Proteins; Binding Sites; Calcium; Catalytic Domain; Conserved Sequence; Cyclic GMP; Deuterium Exchange Measurement; Klebsiella pneumoniae; Models, Molecular; Molecular Sequence Data; Mutation; Phosphoric Diester Hydrolases; Protein Conformation; Protein Multimerization; Protein Structure, Tertiary; Scattering, Small Angle; Signal Transduction; X-Ray Diffraction

The bacterial second messenger (3'-5')-cyclic-di-guanosine-monophosphate (CDG) is a promising mucosal adjuvant candidate that activates balanced Th1/Th2/Th17 responses. We showed previously that CDG activates stimulator of IFN genes (STING)-dependent IFN-I production in vitro. However, it is unknown whether STING or IFN-I is required for the CDG adjuvant activity in vivo. In this study, we show that STING(-/-) mice (Tmem173()) do not produce Ag-specific Abs or Th1/Th2/Th17 cytokines during CDG/Ag immunization. Intranasal administration of CDG did not induce TNF-α, IL-1β, IL-6, IL-12, or MCP-1 production in STING(-/-) mice. Surprisingly, we found that the cytokine and Ab responses were unaltered in CDG/Ag-immunized IFNAR(-/-) mice. Instead, we found that CDG activates STING-dependent, IFN-I-independent TNF-α production in vivo and in vitro. Furthermore, using a TNFR1(-/-) mouse, we demonstrate that TNF-α signaling is critical for CDG-induced Ag-specific Ab and Th1/Th2 cytokine production. This is distinct from STING-mediated DNA adjuvant activity, which requires IFN-I, but not TNF-α, production. Finally, we found that CDG activates STING-dependent, but IRF3 stimulation-independent, NF-κB signaling. Our results established an essential role for STING-mediated TNF-α production in the mucosal adjuvant activity of CDG in vivo and revealed a novel IFN-I stimulation-independent STING-NF-κB-TNF-α pathway.

Topics: Adjuvants, Immunologic; Animals; Chemokines; Cyclic GMP; Cytokines; Dendritic Cells; Female; Immunoglobulin G; Interferon Regulatory Factor-3; Interferon Type I; Membrane Proteins; Mice; Mucous Membrane; NF-kappa B; Ovalbumin; Signal Transduction; Tumor Necrosis Factor-alpha

The first syntheses of neutral thiourea, urea, and carbodiimide analogs, along with two guanidinium analogs, of the bacterial signaling molecule cyclic diguanosine monophosphate (c-di-GMP) are reported. The key intermediate, obtained in nine steps, is a 3'-amino-5'-azido-3',5'-dideoxy derivative. The 5'-azide serves as a masked amine from which the amine is obtained by Staudinger reduction, while the 3'-amine is converted to an isothiocyanate that, while stable to chromatography, and Staudinger conditions, nevertheless reacts well with the 5'-amine.

Topics: Carbodiimides; Cyclic GMP; Guanidine; Nucleic Acid Conformation; Urea

This study monitored the granulation process of an aggregating functional consortium X9 that was consisted of Pseudomonas putida X-1, Acinetobacter sp. X-2, Alcaligenes sp. X-3 and Comamonas testosteroni X-4 in shaken reactors. The growth curve of X9 was fit using logistic model as follows y=1.49/(1+21.3*exp(-0.33x)), the maximum specific cell growth rate for X9 was 0.33 h(-1). Initially X9 consumed polysaccharides (PS) and secreted proteins (PN) to trigger granulation. Then X9 grew in biomass and formed numerous micro-granules, driven by increasing hydrophobicity of cell membranes and of accumulated extracellular polymeric substances (EPS). In later stage the intracellular cyclic diguanylate (c-di-GMP) was at high levels for inhibiting bacteria swarming motility, thereby promotion formation of large aerobic granules. The findings reported herein advise the way to accelerate granule formation and to stabilize operation in aerobic granular reactors.

Topics: Aerobiosis; Bacteria; Bacterial Proteins; Biopolymers; Cyclic GMP; Flocculation; Hydrophobic and Hydrophilic Interactions; Microbial Consortia; Movement; Phylogeny; Polysaccharides; Sewage

Cholera is a waterborne diarrheal disease caused by Vibrio cholerae strains of serogroups O1 and O139. Expression of the general stress response regulator RpoS and formation of biofilm communities enhance the capacity of V. cholerae to persist in aquatic environments. The transition of V. cholerae between free-swimming (planktonic) and biofilm life-styles is regulated by the second messenger cyclic di-GMP (c-di-GMP). We previously reported that increasing the c-di-GMP pool by overexpression of a diguanylate cyclase diminished RpoS expression. Here we show that c-di-GMP repression of RpoS expression is eliminated by deletion of the genes vpsR and vpsT, encoding positive regulators of biofilm development. To determine the mechanism of this regulation, we constructed a strain expressing a vpsT-FLAG allele from native transcription and translation signals. Increasing the c-di-GMP pool induced vpsT-FLAG expression. The interaction between VpsT-FLAG and the rpoS promoter was demonstrated by chromatin immunoprecipitation. Furthermore, purified VpsT interacted with the rpoS promoter in a c-di-GMP-dependent manner. Primer extension analysis identified two rpoS transcription initiation sites located 43 bp (P1) and 63 bp (P2) upstream of the rpoS start codon. DNase I footprinting showed that the VpsT binding site at the rpoS promoter overlaps the primary P1 transcriptional start site. Deletion of vpsT significantly enhanced rpoS expression in V. cholerae biofilms that do not make HapR. This result suggests that VpsT and c-di-GMP contribute to the transcriptional silencing of rpoS in biofilms prior to cells entering the quorum-sensing mode.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Sigma Factor; Stress, Physiological; Transcription, Genetic; Vibrio cholerae

Legionella pneumophila is an intracellular pathogen which replicates within protozoan cells and can accidently infect alveolar macrophages, causing an acute pneumonia in humans. The second messenger cyclic di-GMP (c-di-GMP) has been shown to play key roles in the regulation of various bacterial processes, including virulence. While investigating the function of the 22 potential c-di-GMP-metabolizing enzymes of the L. pneumophila Lens strain, we found three that directly contribute to its ability to infect both protozoan and mammalian cells. These three enzymes display diguanylate cyclase (Lpl0780), phosphodiesterase (Lpl1118), and bifunctional diguanylate cyclase/phosphodiesterase (Lpl0922) activities, which are all required for the survival and intracellular replication of L. pneumophila. Mutants with deletions of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for the escape of the Legionella-containing vacuole (LCV) from the host degradative endocytic pathway and result in lower survival. In addition, Lpl1118 is required for efficient endoplasmic reticulum recruitment to the LCV. Trafficking and biogenesis of the LCV are dependent upon the orchestrated actions of several type 4 secretion system Dot/Icm effectors proteins, which exhibit differentially altered translocation in the three mutants. While translocation of some effectors remained unchanged, others appeared over- and undertranslocated. A general translocation offset of the large repertoire of Dot/Icm effectors may be responsible for the observed defects in the trafficking and biogenesis of the LCV. Our results suggest that L. pneumophila uses cyclic di-GMP signaling to fine-tune effector delivery and ensure effective evasion of the host degradative pathways and establishment of a replicative vacuole.

Topics: Bacterial Proteins; Cell Line, Tumor; Cyclic GMP; Endocytosis; Endoplasmic Reticulum; Escherichia coli Proteins; Humans; Legionella pneumophila; Legionnaires' Disease; Macrophages; Phagocytes; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Transport; Signal Transduction; U937 Cells; Virulence

Mediator of IFN regulatory transcription factor 3 activation (MITA) is an important adaptor protein to mediate the induction of type I IFNs. In this study, we identified an alternatively spliced isoform of MITA lacking exon 7, termed MITA-related protein (MRP). MRP shares the N-terminal portion aa 1-253 with MITA but possesses a unique 30-aa sequence at the carboxyl terminal part, therefore lacking the conserved domains including TANK-binding kinase 1 (TBK1) and cyclic diguanylate binding domain. MRP is expressed in multiple tissues and distinct cell lines. Overexpression of MRP inhibited MITA-mediated activation of IFN-β promoter by sendai virus infection and cyclic diguanylate treatment but enhanced that in HSV-1 infection. Interestingly, MRP expression was reduced after Sendai virus infection but was upregulated after HSV-1 infection. Overexpression of MRP inhibited MITA-mediated induction of IFN-β via TBK1-IFN regulatory transcription factor 3 by disrupting the MITA-TBK1 interaction. However, NF-κB pathway was still activated by MRP, as MRP retained the ability to interact with inducible inhibitor of NF-κB (iκB) kinase. Thus, MRP acts as a dominant negative regulator of MITA-mediated induction of IFN production.

Topics: Alternative Splicing; Cell Line; Cyclic GMP; DNA, Complementary; Down-Regulation; Exons; Gene Expression Regulation; Herpesvirus 1, Human; Humans; Immunity, Innate; Interferon Regulatory Factor-3; Interferon-beta; Membrane Proteins; Molecular Sequence Data; NF-kappa B; Protein Interaction Mapping; Protein Isoforms; Protein Serine-Threonine Kinases; Sendai virus; Signal Transduction

C-di-GMP has emerged as a ubiquitous second messenger, which regulates the transition between sessile and motile lifestyles and virulence factor expression in many pathogenic bacteria using both RNA riboswitches and protein effectors. We recently showed that two additional class I c-di-GMP riboswitch aptamers (Ct-E88 and Cb-17B) bind c-di-GMP with nanomolar affinity, and that Ct-E88 RNA binds 2'-F-c-di-GMP 422 times less tightly than class I Vc2 RNA. Based on sequence comparison, it was concluded that the global folds of Ct-E88 and Vc2 RNAs were similar and that differences in ligand binding were probably due to differences in binding site architectures. Herein, we utilized EMSA, aptamer sensing spinach modules, SAXS and 1D NMR titration to study the conformational transitions of Ct-E88. We conclude that whereas the global folds of the bound states of Vc2 and Ct-E88 RNAs are similar, the unbound states are different and this could explain differences in ligand affinities between these class I c-di-GMP riboswitches.

Topics: Aptamers, Nucleotide; Base Sequence; Clostridium tetani; Cyclic GMP; Models, Molecular; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Nucleic Acid Conformation; Riboswitch; RNA Folding; RNA, Bacterial; Sequence Alignment

The universal second messenger cyclic di-GMP (cdG) is involved in the regulation of a diverse range of cellular processes in bacteria. The intracellular concentration of the dinucleotide is determined by the opposing actions of diguanylate cyclases and cdG-specific phosphodiesterases (PDEs). Whereas most PDEs have accessory domains that are involved in the regulation of their activity, the regulatory mechanism of this class of enzymes has remained unclear. Here, we use biophysical and functional analyses to show that the isolated EAL domain of a PDE from Escherichia coli (YahA) is in a fast thermodynamic monomer-dimer equilibrium, and that the domain is active only in its dimeric state. Furthermore, our data indicate thermodynamic coupling between substrate binding and EAL dimerization with the dimerization affinity being increased about 100-fold upon substrate binding. Crystal structures of the YahA-EAL domain determined under various conditions (apo, Mg(2+), cdG·Ca(2+) complex) confirm structural coupling between the dimer interface and the catalytic center. The built-in regulatory properties of the EAL domain probably facilitate its modular, functional combination with the diverse repertoire of accessory domains.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Amino Acid Sequence; Catalysis; Catalytic Domain; Crystallography, X-Ray; Cyclic GMP; Escherichia coli Proteins; Hydrolysis; Molecular Sequence Data; Protein Multimerization; Protein Structure, Tertiary; Second Messenger Systems

LapA and LapF are large extracellular proteins that play a relevant role in biofilm formation by Pseudomonas putida. Current evidence favors a sequential model in which LapA is first required for the initial adhesion of individual bacteria to a surface, while LapF participates in later stages of biofilm development. In agreement with this model, lapF transcription was previously shown to take place at late times of growth and to respond to the stationary-phase sigma factor RpoS. We have now analyzed the transcription pattern of lapA and other regulatory elements that influence expression of both genes. The lapA promoter shows a transient peak of activation early during growth, with a second increase in stationary phase that is independent of RpoS. The same pattern is observed in biofilms although expression is not uniform in the population. Both lapA and lapF are under the control of the two-component regulatory system GacS/GacA, and their transcription also responds to the intracellular levels of the second messenger cyclic diguanylate (c-di-GMP), although in surprisingly reverse ways. Whereas expression from the lapA promoter increases with high levels of c-di-GMP, the opposite is true for lapF. The transcriptional regulator FleQ is required for the modulation of lapA expression by c-di-GMP but has a minor influence on lapF. This work represents a further step in our understanding of the regulatory interactions controlling biofilm formation in P. putida.

Topics: Adhesins, Bacterial; Bacterial Proteins; Cyclic GMP; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Pseudomonas putida; Transcription Factors

Diguanylate cyclases (DGCs) regulate biofilm formation and motility in bacteria by synthesizing the second messenger cyclic di-GMP (c-di-GMP) in response to environmental stimuli. DGC enzymatic activity is believed to be dependent on the presence of a GG(D/E)EF active site motif, however approximately 25% of known DGCs contain a degenerate active site. The Vibrio cholerae protein VCA0965 contains an AGDEF active site and is presumed to be an inactive DGC.. Ectopic expression of VCA0965 in V. cholerae causes a 3-fold reduction in flagellar-based motility. Additionally, an RXXD allosteric inhibition mutant of VCA0965 strongly inhibited motility and stimulated biofilm formation. This activity was lost when the active site of VCA0965 was mutated to AGDAF, suggesting that VCA0965 synthesizes c-di-GMP. In support of this, ectopic expression of VCA0965 and VCA0965 containing a mutation in its RXXD motif significantly increased the intracellular c-di-GMP levels in V. cholerae and Escherichia coli. Furthermore, we found that purified VCA0965 was able to synthesize c-di-GMP in vitro. Systematic mutation of the first amino acid in the AGDEF motif of VCA0965 revealed that glycine, methionine, and histidine also produced an active DGC capable of inhibiting motility and increasing the intracellular concentration of c-di-GMP in V. cholerae.. Based on these results, we conclude that VCA0965 is capable of c-di-GMP synthesis and that the first amino acid of the GG(D/E)EF motif is more tolerant of substitutions than currently appreciated.

Topics: Catalytic Domain; Cloning, Molecular; Cyclic GMP; DNA Mutational Analysis; Escherichia coli; Escherichia coli Proteins; Gene Expression; Mutagenesis, Site-Directed; Phosphorus-Oxygen Lyases; Vibrio cholerae

Diguanylate cyclase and phosphodiesterase enzymatic activities control c-di-GMP levels modulating planktonic versus sessile lifestyle behavior in bacteria. The PilZ domain is described as a sensor of c-di-GMP intracellular levels and the proteins containing a PilZ domain represent the best studied class of c-di-GMP receptors forming part of the c-di-GMP signaling cascade. In P. fluorescens F113 we have found two diguanylate cyclases (WspR, SadC) and one phosphodiesterase (BifA) implicated in regulation of swimming motility and biofilm formation. Here we identify a flgZ gene located in a flagellar operon encoding a protein that contains a PilZ domain. Moreover, we show that FlgZ subcellular localization depends on the c-di-GMP intracellular levels. The overexpression analysis of flgZ in P. fluorescens F113 and P. putida KT2440 backgrounds reveal a participation of FlgZ in Pseudomonas swimming motility regulation. Besides, the epistasis of flgZ over wspR and bifA clearly shows that c-di-GMP intracellular levels produced by the enzymatic activity of the diguanylate cyclase WspR and the phosphodiesterase BifA regulates biofilm formation through FlgZ.

Topics: Bacterial Proteins; Biofilms; Conserved Sequence; Cyclic GMP; Flagella; Genes, Bacterial; Movement; Protein Structure, Tertiary; Pseudomonas; Subcellular Fractions

The polyamines norspermidine and spermidine are among the environmental signals that regulate Vibrio cholerae biofilm formation. The effects of these polyamines are mediated by NspS, a member of the bacterial periplasmic solute binding protein superfamily. Almost all members of this superfamily characterized to date are components of ATP-binding cassette-type transporters involved in nutrient uptake. Consequently, in the current annotation of the V. cholerae genome, NspS has been assigned a function in transport. The objective of this study was to further characterize NspS and investigate its potential role in transport. Our results support a role for NspS in signal transduction in response to norspermidine and spermidine, but not their transport. In addition, we provide evidence that these polyamine signals are processed by c-di-GMP signalling networks in the cell. Furthermore, we present comparative genomics analyses which reveal the presence of NspS-like proteins in a variety of bacteria, suggesting that periplasmic ligand binding proteins may be widely utilized for sensory transduction.

Topics: ATP-Binding Cassette Transporters; Biofilms; Cyclic GMP; Signal Transduction; Spermidine; Vibrio cholerae O139

Degenerate GGDEF and EAL domain proteins represent major types of cyclic diguanylic acid (c-di-GMP) receptors in pathogenic bacteria. Here, we characterized a FimX-like protein (Filp) which possesses both GGDEF and EAL domains in Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice. Both in silico analysis and enzyme assays indicated that the GGDEF and EAL domains of Filp were degenerate and enzymatically inactive. However, Filp bound to c-di-GMP efficiently within the EAL domain, where Q(477), E(653), and F(654) residues were crucial for the binding. Deletion of the filp gene in X. oryzae pv. oryzae resulted in attenuated virulence in rice and reduced type III secretion system (T3SS) gene expression. Complementation analysis with different truncated proteins indicated that REC, PAS, and EAL domains but not the GGDEF domain were required for the full activity of Filp in vivo. In addition, a PilZ-domain protein (PXO_02715) was identified as a Filp interactor by yeast two-hybrid and glutathione-S-transferase pull-down assays. Deletion of the PXO_02715 gene demonstrated changes in bacterial virulence and T3SS gene expression similar to Δfilp. Moreover, both mutants were impaired in their ability to induce hypersensitive response in nonhost plants. Thus, we concluded that Filp was a novel c-di-GMP receptor of X. oryzae pv. oryzae, and its function to regulate bacterial virulence expression might be via the interaction with PXO_02715.

Topics: Amino Acid Sequence; Bacterial Proteins; Biofilms; Cyclic GMP; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Mutation; Nicotiana; Oryza; Plant Diseases; Plant Leaves; Polysaccharides, Bacterial; Protein Binding; Protein Structure, Tertiary; Sequence Alignment; Sequence Homology, Amino Acid; Signal Transduction; Two-Hybrid System Techniques; Virulence; Xanthomonas

Recently, a number of study have shown the ligand-dependent allosteric ribozymes can be harnessed as biosensors, high-throughput screening, and agents for the control of gene expression in vivo, called artificial riboswitches. In this chapter, we describe how in vitro selection can be used to create an allosteric ribozyme that senses bacterial second messenger cyclic-di-GMP (c-di-GMP). A hammerhead ribozyme was joined to a natural c-di-GMP class I riboswitch aptamer via communication modules. Both c-di-GMP-activating and -inhibiting ribozyme can be obtained by this approach.

Topics: Allosteric Regulation; Aptamers, Nucleotide; Base Sequence; Cyclic GMP; Genetic Engineering; Polymerase Chain Reaction; Riboswitch; RNA, Catalytic

Stimulator of interferon genes (STING, also named MITA, MYPS, or ERIS) is an intracellular DNA sensor that induces type I interferon through its interaction with TANK-binding kinase 1 (TBK1). Here we found that the nucleotide-binding, leucine-rich-repeat-containing protein, NLRC3, reduced STING-dependent innate immune activation in response to cytosolic DNA, cyclic di-GMP (c-di-GMP), and DNA viruses. NLRC3 associated with both STING and TBK1 and impeded STING-TBK1 interaction and downstream type I interferon production. By using purified recombinant proteins, we found NLRC3 to interact directly with STING. Furthermore, NLRC3 prevented proper trafficking of STING to perinuclear and punctated region, known to be important for its activation. In animals, herpes simplex virus 1 (HSV-1)-infected Nlrc3(-/-) mice exhibited enhanced innate immunity and reduced morbidity and viral load. This demonstrates the intersection of two key pathways of innate immune regulation, NLR and STING, to fine tune host response to intracellular DNA, DNA virus, and c-di-GMP.

Topics: Animals; Cell Line; Cyclic GMP; Cytokines; DNA; Herpes Simplex; Herpesvirus 1, Human; Humans; Immunity, Innate; Intercellular Signaling Peptides and Proteins; Interferon Type I; Membrane Proteins; Mice; Mice, Knockout; Protein Binding; Protein Serine-Threonine Kinases; Protein Transport; Signal Transduction

The transcriptional regulator BrlR is a member of the MerR family of multidrug transport activators that contributes to the high-level drug tolerance of Pseudomonas aeruginosa biofilms. While MerR regulators are known to activate both the expression of multidrug efflux pump genes and their own transcription upon inducer binding, little is known about BrlR activation. We demonstrate using promoter reporter strains, in vivo and in vitro DNA-binding assays combined with 5'RACE, that BrlR binds to its own promoter, likely via a MerR-like palindromic sequence. Unlike known MerR multidrug transport activators, BrlR and brlR expression are not activated by multidrug transporter substrates. Instead, BrlR-DNA binding was enhanced by the secondary messenger c-di-GMP. In addition to enhanced BrlR-DNA binding, c-di-GMP levels contributed to PbrlR promoter activity in initial attached cells with elevated c-di-GMP levels correlating with increased expression of brlR. While not harbouring amino acid motifs resembling previously defined c-di-GMP-binding domains, BrlR was found to bind c-di-GMP in vitro at a ratio of one c-di-GMP per two BrlR. Cross-linking assays confirmed dimer formation to be enhanced in the presence of elevated c-di-GMP levels. Our findings demonstrate BrlR to be an unusual MerR-family member in that BrlR function and expression require the secondary messenger c-di-GMP.

Topics: Cyclic GMP; DNA, Bacterial; Gene Expression Regulation, Bacterial; Genes, Reporter; Promoter Regions, Genetic; Protein Binding; Protein Multimerization; Pseudomonas aeruginosa; Transcription Factors

Despite a recent burst of research, knowledge on c-di-GMP signaling pathways remains largely fragmentary and molecular mechanisms of regulation and even c-di-GMP targets are yet unknown for most bacteria. Besides genomics or bioinformatics, accompanying alternative approaches are necessary to reveal c-di-GMP regulation in bacteria with complex lifestyles. We have approached this study by artificially altering the c-di-GMP economy of diverse pathogenic and mutualistic plant-interacting bacteria and examining the effects on the interaction with their respective host plants. Phytopathogenic Pseudomonas and symbiotic Rhizobium strains with enhanced levels of intracellular c-di-GMP displayed common free-living responses: reduction of motility, increased production of extracellular polysaccharides and enhanced biofilm formation. Regarding the interaction with the host plants, P. savastanoi pv. savastanoi cells containing high c-di-GMP levels formed larger knots on olive plants which, however, displayed reduced necrosis. In contrast, development of disease symptoms in P. syringae-tomato or P. syringae-bean interactions did not seem significantly affected by high c-di-GMP. On the other hand, increasing c-di-GMP levels in symbiotic R. etli and R. leguminosarum strains favoured the early stages of the interaction since enhanced adhesion to plant roots, but decreased symbiotic efficiency as plant growth and nitrogen contents were reduced. Our results remark the importance of c-di-GMP economy for plant-interacting bacteria and show the usefulness of our approach to reveal particular stages during plant-bacteria associations which are sensitive to changes in c-di-GMP levels.

Topics: Alginates; Bacterial Proteins; Benzenesulfonates; Biofilms; Cellulose; Cyclic GMP; Fluorescent Dyes; Gene Expression Regulation, Bacterial; Mutation; Olea; Phaseolus; Phenotype; Plant Roots; Plants; Pseudomonas; Rhizobium; Solanum lycopersicum; Species Specificity; Symbiosis

Biofilms are surface-attached communities of bacteria enclosed in a polysaccharide matrix. Bacteria in a biofilm are extremely resistant to antibiotics. Several recent reports have linked the signaling molecule nitric oxide (NO) with biofilm dispersal. We have previously reported that an H-NOX (heme-nitric oxide/oxygen binding) protein in the biofilm-dwelling bacterium Shewanella woodyi mediates NO-induced biofilm dispersal. In S. woodyi, H-NOX (SwH-NOX) is cocistronic with a gene encoding a dual-functioning diguanylate cyclase/phosphodiesterase enzyme, designated here as HaCE (H-NOX-associated cyclic-di-GMP processing enzyme). Enzymes such as these are responsible for regulating the intracellular concentrations of cyclic-di-GMP, a secondary signaling molecule essential to biofilm formation in bacteria. We have demonstrated that NO-bound SwH-NOX regulates both enzymatic activities of SwHaCE, resulting in decreased cellular cyclic-di-GMP levels and disruption of biofilm formation. Thus, H-NOX/HaCE represents a potential drug target for regulating biofilm formation. In this work, the SwH-NOX surface residues critical for the formation of a protein complex with SwHaCE are identified using nuclear magnetic resonance, fluorescence quenching, and cosedimentation. Enzyme assays confirm this protein-protein interface and its importance for H-NOX/HaCE function.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Heme; Models, Molecular; Molecular Structure; Mutagenesis, Site-Directed; Mutant Proteins; Nitric Oxide; Oxygen; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Shewanella

Biofilms are highly structured, surface-associated communities. A hallmark of biofilms is their extraordinary resistance to antimicrobial agents that is activated during early biofilm development of Pseudomonas aeruginosa and requires the regulatory hybrid SagS and BrlR, a member of the MerR family of multidrug efflux pump activators. However, little is known about the mechanism by which SagS contributes to BrlR activation or drug resistance. Here, we demonstrate that ΔsagS biofilm cells harbour the secondary messenger c-di-GMP at reduced levels similar to those observed in wild-type cells grown planktonically rather than as biofilms. Restoring c-di-GMP levels to wild-type biofilm-like levels restored brlR expression, DNA binding by BrlR, and recalcitrance to killing by antimicrobial agents of ΔsagS biofilm cells. We likewise found that increasing c-di-GMP levels present in planktonic cells to biofilm-like levels (≥ 55 pmol mg(-1) ) resulted in planktonic cells being significantly more resistant to antimicrobial agents, with increased resistance correlating with increased brlR, mexA, and mexE expression and BrlR production. In contrast, reducing cellular c-di-GMP levels of biofilm cells to ≤ 40 pmol mg(-1) correlated with increased susceptibility and reduced brlR expression. Our findings suggest that a signalling pathway involving a specific c-di-GMP pool regulated by SagS contributes to the resistance of P. aeruginosa biofilms.

Topics: Anti-Infective Agents; Bacterial Proteins; Cyclic GMP; Drug Resistance, Bacterial; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Microbial Viability; Pseudomonas aeruginosa; Second Messenger Systems

We constructed a library of in-frame deletion mutants targeting each gene in Pseudomonas aeruginosa PA14 predicted to participate in cyclic di-GMP (c-di-GMP) metabolism (biosynthesis or degradation) to provide a toolkit to assist investigators studying c-di-GMP-mediated regulation by this microbe. We present phenotypic assessments of each mutant, including biofilm formation, exopolysaccharide (EPS) production, swimming motility, swarming motility, and twitch motility, as a means to initially characterize these mutants and to demonstrate the potential utility of this library.

Topics: Biofilms; Biotransformation; Cyclic GMP; Gene Deletion; Gene Library; Locomotion; Metabolic Networks and Pathways; Polysaccharides, Bacterial; Pseudomonas aeruginosa

The ability of many pathogenic bacteria to grow as a biofilm results in lower susceptibility to antibiotic treatments and to the host immune response, thus leading to the development of chronic infections. The understanding that biofilms can play an important role in bacterial virulence has prompted the search for inhibitors of biofilm development and of biofilm-related cellular processes. In this report, we present two examples of target-based microbiological screenings for antimicrobials endowed with anti-biofilm activity, aimed respectively at the inhibition of the signal molecule cyclic di-GMP and of quorum sensing.

Topics: Anti-Infective Agents; Biofilms; Biosynthetic Pathways; Cyclic GMP; Drug Evaluation, Preclinical; Microbial Sensitivity Tests; Quorum Sensing

Bdellovibrio bacteriovorus invade Gram-negative bacteria in a predatory process requiring Type IV pili (T4P) at a single invasive pole, and also glide on surfaces to locate prey. Ras-like G-protein MglA, working with MglB and RomR in the deltaproteobacterium Myxococcus xanthus, regulates adventurous gliding and T4P-mediated social motility at both M. xanthus cell poles. Our bioinformatic analyses suggested that the GTPase activating protein (GAP)-encoding gene mglB was lost in Bdellovibrio, but critical residues for MglA(Bd) GTP-binding are conserved. Deletion of mglA(Bd) abolished prey-invasion, but not gliding, and reduced T4P formation. MglA(Bd) interacted with a previously uncharacterised tetratricopeptide repeat (TPR) domain protein Bd2492, which we show localises at the single invasive pole and is required for predation. Bd2492 and RomR also interacted with cyclic-di-GMP-binding receptor CdgA, required for rapid prey-invasion. Bd2492, RomR(Bd) and CdgA localize to the invasive pole and may facilitate MglA-docking. Bd2492 was encoded from an operon encoding a TamAB-like secretion system. The TamA protein and RomR were found, by gene deletion tests, to be essential for viability in both predatory and non-predatory modes. Control proteins, which regulate bipolar T4P-mediated social motility in swarming groups of deltaproteobacteria, have adapted in evolution to regulate the anti-social process of unipolar prey-invasion in the "lone-hunter" Bdellovibrio. Thus GTP-binding proteins and cyclic-di-GMP inputs combine at a regulatory hub, turning on prey-invasion and allowing invasion and killing of bacterial pathogens and consequent predatory growth of Bdellovibrio.

Topics: Bacterial Proteins; Bdellovibrio; Cell Movement; Cyclic GMP; GTP Phosphohydrolases; GTPase-Activating Proteins; Myxococcus xanthus; Operon; ras Proteins

Cyclic dinucleotides are of importance in the field of microbiology and immunology. They function as second messengers and are thought to participate in the signal transduction of cytosolic DNA immune responses. One such dinucleotide, cyclic di-GMP (c-di-GMP), stimulates the immune system. It is thought that c-di-GMP is recognized by ATP dependent RNA helicase (DDX41) in the cytosol, forms a complex with the Stimulator of interferon genes protein (STING), triggers a signal via the tank binding kinase 1-interferon regulatory factor 3 (TBK1-IRF3) pathway and induces the production of type I interferons. Therefore c-di-GMP can be thought of as a new class of adjuvant. However, because c-di-GMP contains two phosphate groups, this prevents its use as an adjuvant because it cannot pass through the cell membrane, even though the target molecule of c-di-GMP is located in the cytoplasm. Our group has been developing a series of liposomal drug delivery systems and recently investigated YSK05 which is a synthetic, pH sensitive lipid that has a high fusogenicity. We utilized this lipid as a carrier to transport c-di-GMP into the cytosol to then use c-di-GMP as an adjuvant. Based on screening experiments, YSK05/POPE/cholesterol=40/25/35 was found to induce IFN-β in Raw264.7 cells. The induction of IFN-β from c-di-GMP liposomes was inhibited by adding BX795, a TBK1 inhibitor, indicating that the production of IFN-β caused the activation of the STING-TBK1 pathway. C-di-GMP liposomes also showed significantly higher levels of expression of CD80, CD86 and MHC class I. The c-di-GMP/YSK05 liposome facilitated antigen specific cytotoxic T cell activity and the inhibition of tumor growth in a mouse model. These findings indicate that c-di-GMP/YSK05 liposomes could be used, not only to transfer c-di-GMP to the cytosol and induce an innate immune system but also as a platform for investigating the mechanism of immune sensing with cyclic dinucleotides in vitro and in vivo.

Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; Cell Line; Cell Line, Tumor; Cyclic GMP; Female; Immunotherapy; Interferon-beta; Lipids; Liposomes; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Piperidines; T-Lymphocytes, Cytotoxic; Tumor Burden

Rhizobia are soil bacteria that can fix nitrogen in symbiosis with leguminous plants or exist free living in the rhizosphere. Crucial to their complex lifestyle is the ability to sense and respond to diverse environmental stimuli, requiring elaborate signaling pathways. In the majority of bacteria, the nucleotide-based second messenger cyclic diguanosine monophosphate (c-di-GMP) is involved in signal transduction. Surprisingly, little is known about the importance of c-di-GMP signaling in rhizobia. We have analyzed the genome sequences of six well-studied type species (Bradyrhizobium japonicum, Mesorhizobium loti, Rhizobium etli, Rhizobium leguminosarum, Sinorhizobium fredii, and Sinorhizobium meliloti) for proteins possibly involved in c-di-GMP signaling based on the presence of four domains: GGDEF (diguanylate cyclase), EAL and HD-GYP (phosphodiesterase), and PilZ (c-di-GMP sensor). We find that rhizobia possess a high number of these proteins. Conservation analysis suggests that c-di-GMP signaling proteins modulate species-specific pathways rather than ancient rhizobia-specific processes. Two hybrid GGDEF-EAL proteins were selected for functional analysis, R. etli RHE_PD00105 (CdgA) and RHE_PD00137 (CdgB). Expression of cdgA and cdgB is repressed by the alarmone (p)ppGpp. cdgB is significantly expressed on plant roots and free living. Mutation of cdgA, cdgB, or both does not affect plant root colonization, nitrogen fixation capacity, biofilm formation, motility, and exopolysaccharide production. However, heterologous expression of the individual GGDEF and EAL domains of each protein in Escherichia coli strongly suggests that CdgA and CdgB are bifunctional proteins, possessing both diguanylate cyclase and phosphodiesterase activities. Taken together, our results provide a platform for future studies of c-di-GMP signaling in rhizobia.

Topics: Bacterial Proteins; Biofilms; Bradyrhizobium; Computational Biology; Cyclic GMP; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Mesorhizobium; Nitrogen Fixation; Plant Roots; Polysaccharides; Rhizobiaceae; Sequence Analysis, DNA; Soil Microbiology

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous bacterial signalling molecule produced by diguanylate cyclases of the GGDEF-domain family. Elevated c-di-GMP levels or increased GGDEF protein expression is frequently associated with the onset of sessility and biofilm formation in numerous bacterial species. Conversely, phosphodiesterase-dependent diminution of c-di-GMP levels by EAL- and HD-GYP-domain proteins is often accompanied by increased motility and virulence. In this study, we individually overexpressed 23 predicted GGDEF, EAL or HD-GYP-domain proteins encoded by the phytopathogen Pectobacterium atrosepticum strain SCRI1043. MS-based detection of c-di-GMP and 5'-phosphoguanylyl-(3'-5')-guanosine in these strains revealed that overexpression of most genes promoted modest 1-10-fold changes in cellular levels of c-di-GMP, with the exception of the GGDEF-domain proteins ECA0659 and ECA3374, which induced 1290- and 7660-fold increases, respectively. Overexpression of most EAL domain proteins increased motility, while overexpression of most GGDEF domain proteins reduced motility and increased poly-β-1,6-N-acetyl-glucosamine-dependent flocculation. In contrast to domain-based predictions, overexpression of the EAL protein ECA3549 or the HD-GYP protein ECA3548 increased c-di-GMP concentrations and reduced motility. Most overexpression constructs altered the levels of secreted cellulases, pectinases and proteases, confirming c-di-GMP regulation of virulence in Pe. atrosepticum. However, there was no apparent correlation between virulence-factor induction and the domain class expressed or cellular c-di-GMP levels, suggesting that regulation was in response to specific effectors within the network, rather than total c-di-GMP concentration. Finally, we demonstrated that the cellular localization patterns vary considerably for GGDEF/EAL/HD-GYP proteins, indicating it is a likely factor restricting specific interactions within the c-di-GMP network.

Topics: Bacterial Proteins; Computational Biology; Cyclic GMP; Gene Expression; Gene Expression Regulation, Bacterial; Pectobacterium; Phenotype; Plant Diseases; Plant Tubers; Recombinant Fusion Proteins; Signal Transduction; Solanum tuberosum; Virulence

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder β-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system.

Topics: Animals; Binding Sites; Blotting, Western; Crystallography, X-Ray; Cyclic AMP; Cyclic GMP; Dinucleoside Phosphates; HEK293 Cells; High-Throughput Screening Assays; Humans; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; Ion Channel Gating; Ligands; Mice; Mice, Inbred C57BL; Molecular Docking Simulation; Molecular Structure; Muscle Proteins; Myocytes, Cardiac; Patch-Clamp Techniques; Potassium Channels; Sinoatrial Node; Small Molecule Libraries; Transfection

Vibrio cholerae is a Gram-negative bacterium that persists in aquatic reservoirs and causes the diarrheal disease cholera upon entry into a human host. V. cholerae employs the second messenger molecule 3',5'-cyclic diguanylic acid (c-di-GMP) to transition between these two distinct lifestyles. c-di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and hydrolyzed by phosphodiesterase (PDE) enzymes. Bacteria typically encode many different DGCs and PDEs within their genomes. Presumably, each enzyme senses and responds to cognate environmental cues by alteration of enzymatic activity. c-di-GMP represses the expression of virulence factors in V. cholerae, and it is predicted that the intracellular concentration of c-di-GMP is low during infection. Contrary to this model, we found that bile acids, a prevalent constituent of the human proximal small intestine, increase intracellular c-di-GMP in V. cholerae. We identified four c-di-GMP turnover enzymes that contribute to increased intracellular c-di-GMP in the presence of bile acids, and deletion of these enzymes eliminates the bile induction of c-di-GMP and biofilm formation. Furthermore, this bile-mediated increase in c-di-GMP is quenched by bicarbonate, the intestinal pH buffer secreted by intestinal epithelial cells. Our results lead us to propose that V. cholerae senses distinct microenvironments within the small intestine using bile and bicarbonate as chemical cues and responds by modulating the intracellular concentration of c-di-GMP.

Topics: Bicarbonates; Bile Acids and Salts; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Vibrio cholerae

The secondary messenger molecule, 3',5'-cyclic diguanosine monophosphate (c-di-GMP), controls various cellular processes in bacteria. Direct measurement of intracellular concentration of c-di-GMP is fast becoming an important tool for studying prokaryotic biology. Here, we describe a comprehensive extraction protocol from live bacteria and quantitative analysis using LC/MS/MS.

Topics: Chromatography, Liquid; Cyclic GMP; Pseudomonas aeruginosa; Reference Standards; Second Messenger Systems; Tandem Mass Spectrometry

Deciphering the molecular basis of HCN channel regulation by cGMP leads to the serendipitous discovery of cyclic dinucleotides as potent inhibitors of I(f) current in the heart.

Topics: Animals; Cyclic AMP; Cyclic GMP; Dinucleoside Phosphates; Humans; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; Ion Channel Gating; Muscle Proteins; Potassium Channels

The bacterial plant pathogen Pseudomonas syringae causes disease in a wide range of plants. The associated decrease in crop yields results in economic losses and threatens global food security. Competition exists between the plant immune system and the pathogen, the basic principles of which can be applied to animal infection pathways. P. syringae uses a type III secretion system (T3SS) to deliver virulence factors into the plant that promote survival of the bacterium. The P. syringae T3SS is a product of the hypersensitive response and pathogenicity (hrp) and hypersensitive response and conserved (hrc) gene cluster, which is strictly controlled by the codependent enhancer-binding proteins HrpR and HrpS. Through a combination of bacterial gene regulation and phenotypic studies, plant infection assays, and plant hormone quantifications, we now report that Chp8 (i) is embedded in the Hrp regulon and expressed in response to plant signals and HrpRS, (ii) is a functional diguanylate cyclase, (iii) decreases the expression of the major pathogen-associated molecular pattern (PAMP) flagellin and increases extracellular polysaccharides (EPS), and (iv) impacts the salicylic acid/jasmonic acid hormonal immune response and disease progression. We propose that Chp8 expression dampens PAMP-triggered immunity during early plant infection.. The global demand for food is projected to rise by 50% by 2030 and, as such, represents one of the major challenges of the 21st century, requiring improved crop management. Diseases caused by plant pathogens decrease crop yields, result in significant economic losses, and threaten global food security. Gaining mechanistic insights into the events at the plant-pathogen interface and employing this knowledge to make crops more resilient is one important strategy for improving crop management. Plant-pathogen interactions are characterized by the sophisticated interplay between plant immunity elicited upon pathogen recognition and immune evasion by the pathogen. Here, we identify Chp8 as a contributor to the major effort of the plant pathogen Pseudomonas syringae pv. tomato DC3000 to evade immune responses of the plant.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Enzyme Activation; Escherichia coli Proteins; Extracellular Space; Flagellin; Gene Expression Regulation, Bacterial; Immune Evasion; Phenotype; Phosphorus-Oxygen Lyases; Plant Diseases; Polysaccharides; Pseudomonas syringae; Solanum lycopersicum

Pseudomonas aeruginosa biofilm formation was increased by addition of sucrose to Luria-Bertani medium, whereas addition of NaCl to a final similar osmolarity and use of maltose instead of sucrose, were ineffective. In a previous study, we showed that the extracytoplasmic sigma factor SigX is activated in the presence of sucrose. The sucrose-mediated pellicle increase was abolished in a sigX mutant strain. Sucrose addition led to an increase in pel expression and cyclic-diguanylate (c-di-GMP) pool level production. Interestingly, these two phenotypes were strongly decreased in a sigX mutant. Since pel is not known as a SigX-target, we suspect SigX to be involved in the c-di-GMP production. We found that expression of the diguanylate cyclase PA4843 gene was increased in the presence of sucrose at least partly through SigX activity. Our study shows that sucrose itself rather than osmolarity favours the biofilm mode of P. aeruginosa through the activation of SigX.

Topics: Biofilms; Culture Media; Cyclic GMP; Escherichia coli Proteins; Gene Deletion; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa; Sigma Factor; Sodium Chloride; Sucrose

Cyclic di-GMP is a conserved signaling molecule regulating the transitions between motile and sessile modes of growth in a variety of bacterial species. Recent evidence suggests that Pseudomonas species harbor separate intracellular pools of c-di-GMP to control different phenotypic outputs associated with motility, attachment, and biofilm formation, with multiple diguanylate cyclases (DGCs) playing distinct roles in these processes, yet little is known about the potential conservation of functional DGCs across Pseudomonas species. In the present study, we demonstrate that the P. aeruginosa homolog of the P. fluorescens DGC GcbA involved in promoting biofilm formation via regulation of swimming motility likewise synthesizes c-di-GMP to regulate surface attachment via modulation of motility, however, without affecting subsequent biofilm formation. P. aeruginosa GcbA was found to regulate flagellum-driven motility by suppressing flagellar reversal rates in a manner independent of viscosity, surface hardness, and polysaccharide production. P. fluorescens GcbA was found to be functional in P. aeruginosa and was capable of restoring phenotypes associated with inactivation of gcbA in P. aeruginosa to wild-type levels. Motility and attachment of a gcbA mutant strain could be restored to wild-type levels via overexpression of the small regulatory RNA RsmZ. Furthermore, epistasis analysis revealed that while both contribute to the regulation of initial surface attachment and flagellum-driven motility, GcbA and the phosphodiesterase DipA act within different signaling networks to regulate these processes. Our findings expand the complexity of c-di-GMP signaling in the regulation of the motile-sessile switch by providing yet another potential link to the Gac/Rsm network and suggesting that distinct c-di-GMP-modulating signaling pathways can regulate a single phenotypic output.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Flagella; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Movement; Phenotype; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Polysaccharides, Bacterial; Polystyrenes; Promoter Regions, Genetic; Pseudomonas aeruginosa; Sequence Deletion; Signal Transduction

Cancer vaccination may be our best and most benign option for preventing or treating metastatic cancer. However, breakthroughs are hampered by immune suppression in the tumor microenvironment. In this study, we analyzed whether cyclic diguanylate (c-di-GMP), a ligand for stimulator of interferon genes (STING), could overcome immune suppression and improve vaccination against metastatic breast cancer. Mice with metastatic breast cancer (4T1 model) were therapeutically immunized with an attenuated Listeria monocytogenes (LM)-based vaccine, expressing tumor-associated antigen Mage-b (LM-Mb), followed by multiple low doses of c-di-GMP (0.2 μmol/L). This treatment resulted in a striking and near elimination of all metastases. Experiments revealed that c-di-GMP targets myeloid-derived suppressor cells (MDSC) and tumor cells. Low doses of c-di-GMP significantly increased the production of IL12 by MDSCs, in correlation with improved T-cell responses to Mage-b, whereas a high dose of c-di-GMP (range, 0.3-3 mmol/L) activated caspase-3 in the 4T1 tumor cells and killed the tumor cells directly. On the basis of these results, we tested one administration of high-dose c-di-GMP (3 mmol/L) followed by repeated administrations of low-dose c-di-GMP (0.2 μmol/L) in the 4T1 model, and found equal efficacy compared with the combination of LM-Mb and c-di-GMP. This finding correlated with a mechanism of improved CD8 T-cell responses to tumor-associated antigens (TAA) Mage-b and Survivin, most likely through cross-presentation of these TAAs from c-di-GMP-killed 4T1 tumor cells, and through c-di-GMP-activated TAA-specific T cells. Our results demonstrate that activation of STING-dependent pathways by c-di-GMP is highly attractive for cancer immunotherapy.

Topics: Animals; Antigens, Neoplasm; Breast Neoplasms; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cross-Priming; Cyclic GMP; Female; HEK293 Cells; Humans; Immunotherapy; Listeria monocytogenes; Mammary Neoplasms, Experimental; Membrane Proteins; Mice; Mice, Inbred BALB C; Tumor Microenvironment; Vaccines, Attenuated

In contrast to numerous enzymes involved in c-di-GMP synthesis and degradation in enterobacteria, only a handful of c-di-GMP receptors/effectors have been identified. In search of new c-di-GMP receptors, we screened the Escherichia coli ASKA overexpression gene library using the Differential Radial Capillary Action of Ligand Assay (DRaCALA) with fluorescently and radioisotope-labelled c-di-GMP. We uncovered three new candidate c-di-GMP receptors in E. coli and characterized one of them, BcsE. The bcsE gene is encoded in cellulose synthase operons in representatives of Gammaproteobacteria and Betaproteobacteria. The purified BcsE proteins from E. coli, Salmonella enterica and Klebsiella pneumoniae bind c-di-GMP via the domain of unknown function, DUF2819, which is hereby designated GIL, GGDEF I-site like domain. The RxGD motif of the GIL domain is required for c-di-GMP binding, similar to the c-di-GMP-binding I-site of the diguanylate cyclase GGDEF domain. Thus, GIL is the second protein domain, after PilZ, dedicated to c-di-GMP-binding. We show that in S. enterica, BcsE is not essential for cellulose synthesis but is required for maximal cellulose production, and that c-di-GMP binding is critical for BcsE function. It appears that cellulose production in enterobacteria is controlled by a two-tiered c-di-GMP-dependent system involving BcsE and the PilZ domain containing glycosyltransferase BcsA.

Topics: Bacterial Proteins; Cellulose; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Glucosyltransferases; Glycosyltransferases; Klebsiella pneumoniae; Mutagenesis, Site-Directed; Operon; Phosphorus-Oxygen Lyases; Protein Binding; Protein Interaction Domains and Motifs; Salmonella typhimurium; Signal Transduction

A large number of genes coding for enzymes predicted to synthesize and degrade 3'-5'-cyclic diguanylic acid (c-di-GMP) is found in most bacterial genomes and this dinucleotide emerged as an intracellular signal-controlling bacterial behaviour. An association between high levels of c-di-GMP and antibiotic resistance may be expected because c-di-GMP regulates biofilm formation and this mode of growth leads to enhanced antibiotic resistance. However, a clear understanding of this correlation has not been established. We found that increased levels of c-di-GMP in Pseudomonas aeruginosa improve fitness in the presence of imipenem, even when grown as planktonic cells. P. aeruginosa post-transcriptionally regulates the amounts of five porins in response to c-di-GMP, including OprD, responsible for imipenem uptake. Cells with low c-di-GMP levels are consequently more sensitive to this antibiotic. Main efflux pumps or β-lactamase genes did not show altered mRNA levels in P. aeruginosa strains with modified different c-di-GMP concentrations. Together, our findings show that c-di-GMP levels modulate fitness of planktonic cultures in the presence of imipenem.

Topics: Anti-Bacterial Agents; beta-Lactamases; Biofilms; Cyclic GMP; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genetic Fitness; Imipenem; Molecular Sequence Annotation; Plankton; Porins; Pseudomonas aeruginosa

Topics: Bacteria; Bacterial Physiological Phenomena; Cyclic GMP; History, 20th Century; History, 21st Century; Microbiology; Signal Transduction; Virulence; Virulence Factors

We have reported previously that the long-term survival of Mycobacterium smegmatis is facilitated by a dual-active enzyme MSDGC-1 (renamed DcpA), which controls the cellular turnover of cyclic diguanosine monophosphate (c-di-GMP). Most mycobacterial species possess at least a single copy of a DcpA orthologue that is highly conserved in terms of sequence similarity and domain architecture. Here, we show that DcpA exists in monomeric and dimeric forms. The dimerization of DcpA is due to non-covalent interactions between two protomers that are arranged in a parallel orientation. The dimer shows both synthesis and hydrolysis activities, whereas the monomer shows only hydrolysis activity. In addition, we have shown that DcpA is associated with the cytoplasmic membrane and exhibits heterogeneous cellular localization with a predominance at the cell poles. Finally, we have also shown that DcpA is involved in the change in cell length and colony morphology of M. smegmatis. Taken together, our study provides additional evidence about the role of the bifunctional protein involved in c-di-GMP signalling in M. smegmatis.

Topics: Cell Membrane; Cyclic GMP; Escherichia coli Proteins; Hydrolysis; Mycobacterium smegmatis; Phosphorus-Oxygen Lyases; Protein Binding; Protein Multimerization

The two-component system (TCS), consisting of a response regulator (RR) and a cognate histidine kinase (HK), responds to extra-/intercellular cues and triggers adaptive changes. The RR, RavR, has been reported to act as a positive virulence regulator and a c-di-GMP hydrolase in Xanthomonas campestris pv. campestris (Xcc). Here, we identified the cognate HK, RavA, that regulate RavR phosphorylation levels and bacterial pathogenesis. Deletion of ravA, a putative HK gene flanking ravR, dramatically attenuated Xcc virulence. Phenotypes of the double mutant ΔravR/ΔravA were similar to those of ΔravR, suggesting that RavR is a downstream component of RavA signaling. RavA interacts with RavR and positively influences the phosphorylated RavR levels. In vitro analysis suggests that RavR is a bifunctional enzyme involved in c-di-GMP synthesis and degradation. Importantly, mutation and enzyme activity assays indicate that the phosphorylation level affects RavR c-di-GMP turnover activity. These results show that RavA acts as the RavR cognate HK, which fine-tunes RavR functions and enables bacteria to adapt quickly to intracellular changes.

Topics: Cyclic GMP; Gene Deletion; Histidine Kinase; Protein Kinases; Transcription Factors; Virulence; Xanthomonas campestris

Bacteria assume distinct lifestyles during the planktonic and biofilm modes of growth. Increased levels of the intracellular messenger c-di-GMP determine the transition from planktonic to biofilm growth, while a reduction causes biofilm dispersal. It is generally assumed that cells dispersed from biofilms immediately go into the planktonic growth phase. Here we use single-nucleotide resolution transcriptomic analysis to show that the physiology of dispersed cells from Pseudomonas aeruginosa biofilms is highly different from those of planktonic and biofilm cells. In dispersed cells, the expression of the small regulatory RNAs RsmY and RsmZ is downregulated, whereas secretion genes are induced. Dispersed cells are highly virulent against macrophages and Caenorhabditis elegans compared with planktonic cells. In addition, they are highly sensitive towards iron stress, and the combination of a biofilm-dispersing agent, an iron chelator and tobramycin efficiently reduces the survival of the dispersed cells.

Topics: Animals; Biofilms; Caenorhabditis elegans; Cells, Cultured; Cyclic GMP; Gene Expression Regulation, Bacterial; Iron Chelating Agents; Macrophages; Mice; Mutation; Oligopeptides; Pseudomonas aeruginosa; RNA, Bacterial; Transcriptome

Cyanobacteria have unique photoreceptors, cyanobacteriochromes, that show diverse spectral properties to sense near-UV/visible lights. Certain cyanobacteriochromes have been shown to regulate cellular phototaxis or chromatic acclimation of photosynthetic pigments. Some cyanobacteriochromes have output domains involved in bacterial signaling using a second messenger cyclic dimeric GMP (c-di-GMP), but its role in cyanobacteria remains elusive. Here, we characterize the recombinant Tlr0924 from a thermophilic cyanobacterium Thermosynechococcus elongatus, which was expressed in a cyanobacterial system. The protein reversibly photoconverts between blue- and green-absorbing forms, which is consistent with the protein prepared from Escherichia coli, and has diguanylate cyclase activity, which is enhanced 38-fold by blue light compared with green light. Therefore, Tlr0924 is a blue light-activated diguanylate cyclase. The protein's relatively low affinity (10.5 mM) for Mg(2+), which is essential for diguanylate cyclase activity, suggests that Mg(2+) might also regulate c-di-GMP signaling. Finally, we show that blue light irradiation under low temperature is responsible for Thermosynechococcus vulcanus cell aggregation, which is abolished when tlr0924 is disrupted, suggesting that Tlr0924 mediates blue light-induced cell aggregation by producing c-di-GMP. Given our results, we propose the name "sesA (sessility-A)" for tlr0924. This is the first report for cyanobacteriochrome-dependent regulation of a sessile/planktonic lifestyle in cyanobacteria via c-di-GMP.

Topics: Bacterial Proteins; Binding, Competitive; Cyanobacteria; Cyclic GMP; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Escherichia coli; Escherichia coli Proteins; Light; Magnesium; Models, Biological; Mutation; Phosphorus-Oxygen Lyases; Photoreceptors, Microbial; Protein Binding; Recombinant Proteins; Signal Transduction; Spectrophotometry; Temperature

Microbial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often encode dozens of redundant enzymes that synthesize and hydrolyze c-di-GMP, diguanylate cyclases (DGCs), and c-di-GMP phosphodiesterases (PDEs); thus, determining the contribution of any one particular enzyme is challenging. In an effort to develop a facile system to study c-di-GMP metabolic enzymes, we have engineered a suite of Bacillus subtilis strains to assess the effect of individual heterologously expressed proteins on c-di-GMP levels. As a proof of principle, we characterized all 37 known genes encoding predicted DGCs and PDEs in Clostridium difficile using parallel readouts of swarming motility and fluorescence from green fluorescent protein (GFP) expressed under the control of a c-di-GMP-controlled riboswitch. We found that 27 of the 37 putative C. difficile 630 c-di-GMP metabolic enzymes had either active cyclase or phosphodiesterase activity, with agreement between our motility phenotypes and fluorescence-based c-di-GMP reporter. Finally, we show that there appears to be a threshold level of c-di-GMP needed to inhibit motility in Bacillus subtilis.

Topics: Bacillus subtilis; Bacterial Proteins; Biofilms; Clostridioides difficile; Cyclic GMP; Escherichia coli Proteins; Fluorescence; Gene Expression; Genes, Reporter; Genetic Engineering; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Riboswitch; Signal Transduction; Transgenes; Virulence

We characterized key components and major targets of the c-di-GMP signaling pathways in the foodborne pathogen Listeria monocytogenes, identified a new c-di-GMP-inducible exopolysaccharide responsible for motility inhibition, cell aggregation, and enhanced tolerance to disinfectants and desiccation, and provided first insights into the role of c-di-GMP signaling in listerial virulence. Genome-wide genetic and biochemical analyses of c-di-GMP signaling pathways revealed that L. monocytogenes has three GGDEF domain proteins, DgcA (Lmo1911), DgcB (Lmo1912) and DgcC (Lmo2174), that possess diguanylate cyclase activity, and three EAL domain proteins, PdeB (Lmo0131), PdeC (Lmo1914) and PdeD (Lmo0111), that possess c-di-GMP phosphodiesterase activity. Deletion of all phosphodiesterase genes (ΔpdeB/C/D) or expression of a heterologous diguanylate cyclase stimulated production of a previously unknown exopolysaccharide. The synthesis of this exopolysaccharide was attributed to the pssA-E (lmo0527-0531) gene cluster. The last gene of the cluster encodes the fourth listerial GGDEF domain protein, PssE, that functions as an I-site c-di-GMP receptor essential for exopolysaccharide synthesis. The c-di-GMP-inducible exopolysaccharide causes cell aggregation in minimal medium and impairs bacterial migration in semi-solid agar, however, it does not promote biofilm formation on abiotic surfaces. The exopolysaccharide also greatly enhances bacterial tolerance to commonly used disinfectants as well as desiccation, which may contribute to survival of L. monocytogenes on contaminated food products and in food-processing facilities. The exopolysaccharide and another, as yet unknown c-di-GMP-dependent target, drastically decrease listerial invasiveness in enterocytes in vitro, and lower pathogen load in the liver and gallbladder of mice infected via an oral route, which suggests that elevated c-di-GMP levels play an overall negative role in listerial virulence.

Topics: Animals; Bacterial Proteins; Chromatography, High Pressure Liquid; Cyclic GMP; Disease Models, Animal; Escherichia coli Proteins; Female; Gene Expression Regulation, Bacterial; Listeria monocytogenes; Listeriosis; Mice; Mice, Inbred BALB C; Phosphorus-Oxygen Lyases; Signal Transduction; Virulence

A large number of genes coding for enzymes predicted to synthesize and degrade 3'-5-cyclic diguanylic acid (c-di-GMP) is found in most bacterial genomes and this dinucleotide emerged as an intracellular signal-controlling bacterial behaviour. An association between high levels of c-di-GMP and antibiotic resistance may be expected because c-di-GMP regulates biofilm formation and this mode of growth leads to enhanced antibiotic resistance. However, a clear understanding of this correlation has not been established. We found that increased levels of c-di-GMP in Pseudomonas aeruginosa improve fitness in the presence of imipenem, even when grown as planktonic cells. P. aeruginosa post-transcriptionally regulates the amounts of five porins in response to c-di-GMP, including OprD, responsible for imipenem uptake. Cells with low c-di-GMP levels are consequently more sensitive to this antibiotic. Main efflux pumps or β-lactamase genes did not show altered mRNA levels in P. aeruginosa strains with modified different c-di-GMP concentrations. Together, our findings show that c-di-GMP levels modulate fitness of planktonic cultures in the presence of imipenem.

Topics: Adaptation, Biological; Anti-Bacterial Agents; Bacterial Proteins; Cyclic GMP; Imipenem; Porins; Pseudomonas aeruginosa

When Caulobacter crescentus enters S-phase the replication initiation inhibitor CtrA dynamically positions to the old cell pole to be degraded by the polar ClpXP protease. Polar delivery of CtrA requires PopA and the diguanylate cyclase PleD that positions to the same pole. Here we present evidence that PopA originated through gene duplication from its paralogue response regulator PleD and subsequent co-option as c-di-GMP effector protein. While the C-terminal catalytic domain (GGDEF) of PleD is activated by phosphorylation of the N-terminal receiver domain, functional adaptation has reversed signal transduction in PopA with the GGDEF domain adopting input function and the receiver domain serving as regulatory output. We show that the N-terminal receiver domain of PopA specifically interacts with RcdA, a component required for CtrA degradation. In contrast, the GGDEF domain serves to target PopA to the cell pole in response to c-di-GMP binding. In agreement with the divergent activation and targeting mechanisms, distinct markers sequester PleD and PopA to the old cell pole upon S-phase entry. Together these data indicate that PopA adopted a novel role as topology specificity factor to help recruit components of the CtrA degradation pathway to the protease specific old cell pole of C. crescentus.

Topics: Bacterial Proteins; Caulobacter crescentus; Cell Cycle Checkpoints; Cyclic GMP; Models, Molecular; Phosphorylation; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; Protein Processing, Post-Translational

The cyclic dinucleotide c-di-GMP is a signaling molecule with diverse functions in cellular physiology. Here, we report that c-di-GMP can assemble into a tetramer that mediates the effective dimerization of a transcription factor, BldD, which controls the progression of multicellular differentiation in sporulating actinomycete bacteria. BldD represses expression of sporulation genes during vegetative growth in a manner that depends on c-di-GMP-mediated dimerization. Structural and biochemical analyses show that tetrameric c-di-GMP links two subunits of BldD through their C-terminal domains, which are otherwise separated by ~10 Å and thus cannot effect dimerization directly. Binding of the c-di-GMP tetramer by BldD is selective and requires a bipartite RXD-X8-RXXD signature. The findings indicate a unique mechanism of protein dimerization and the ability of nucleotide signaling molecules to assume alternative oligomeric states to effect different functions.

Topics: Amino Acid Sequence; Bacterial Proteins; Crystallography, X-Ray; Cyclic GMP; Dimerization; Models, Molecular; Molecular Sequence Data; Sequence Alignment; Spores, Bacterial; Streptomyces; Transcription Factors

Bacterial biofilm formation is regulated by enzymes, such as diguanylate cyclases, that respond to environmental signals and alter c-di-GMP levels. Diguanylate cyclase activity of two globin coupled sensors is shown to be regulated by gaseous ligands, with cyclase activity and O2 dissociation affected by protein oligomeric state.

Topics: Bacterial Proteins; Biofilms; Bordetella pertussis; Catalytic Domain; Cyclic GMP; Enzyme Activation; Oxygen; Pectobacterium; Phosphorus-Oxygen Lyases; Protein Multimerization

The bacterial soft rot pathogen Dickeya dadantii utilizes the type III secretion system (T3SS) to suppress host defense responses, and secretes pectate lyase (Pel) to disintegrate the plant cell wall. A transposon mutagenesis fluorescence-activated cell sorting screen was used to identify mutants with altered promoter activities of the T3SS pilus gene hrpA. Several insertion mutations, resulting in changes in hrpA expression, were mapped to a new locus, opgGH, which encodes the gene cluster responsible for osmoregulated periplasmic glucan (OPG) synthesis proteins. Our data showed that OPG was involved in T3SS and Pel regulation by altering the expression of the regulatory small RNA RsmB. Through genome searching, the mechanism of two novel regulatory components, the RcsCD-RcsB phosphorelay and CsrD on OPG and the rsmB gene, was further investigated. The Rcs phosphorelay and OPG inversely regulated rsmB at transcriptional and post-transcriptional levels, respectively. CsrD exhibited dual functionality in T3SS and Pel regulation by manipulating levels of RsmB RNA and cyclic diguanylate monophosphate (c-di-GMP). CsrD positively regulated the promoter activity of the rsmB gene but negatively controlled RsmB RNA at the post-transcriptional level via OpgGH. In addition, CsrD contains both GGDEF and EAL domains but acted as a c-di-GMP phosphodiesterase. When the expression of the csrD gene was induced, CsrD regulated T3SS expression and Pel production through controlling intracellular c-di-GMP levels.

Topics: Bacterial Proteins; Bacterial Secretion Systems; Cell Wall; Cyclic GMP; Enterobacteriaceae; Gene Expression Regulation, Bacterial; Models, Biological; Mutagenesis, Insertional; Mutagenesis, Site-Directed; Phenotype; Plant Diseases; Plants; Polysaccharide-Lyases; Promoter Regions, Genetic; Protein Structure, Tertiary; Sequence Analysis, DNA; Transcriptional Activation; Virulence; Virulence Factors

Stable surface adhesion of cells is one of the early pivotal steps in bacterial biofilm formation, a prevalent adaptation strategy in response to changing environments. In Pseudomonas fluorescens, this process is regulated by the Lap system and the second messenger cyclic-di-GMP. High cytoplasmic levels of cyclic-di-GMP activate the transmembrane receptor LapD that in turn recruits the periplasmic protease LapG, preventing it from cleaving a cell surface-bound adhesin, thereby promoting cell adhesion. In this study, we elucidate the molecular basis of LapG regulation by LapD and reveal a remarkably sensitive switching mechanism that is controlled by LapD's HAMP domain. LapD appears to act as a coincidence detector, whereby a weak interaction of LapG with LapD transmits a transient outside-in signal that is reinforced only when cyclic-di-GMP levels increase. Given the conservation of key elements of this receptor system in many bacterial species, the results are broadly relevant for cyclic-di-GMP- and HAMP domain-regulated transmembrane signaling.

Topics: Allosteric Regulation; Bacterial Proteins; Biofilms; Crystallography, X-Ray; Cyclic GMP; Electrophoresis, Polyacrylamide Gel; Models, Molecular; Mutation; Peptide Hydrolases; Periplasm; Periplasmic Proteins; Protein Binding; Protein Structure, Tertiary; Proteolysis; Pseudomonas fluorescens; Signal Transduction

The cell-division cycle of Caulobacter crescentus depends on periodic activation and deactivation of the essential response regulator CtrA. Although CtrA is critical for transcription during some parts of the cell cycle, its activity must be eliminated before chromosome replication because CtrA also blocks the initiation of DNA replication. CtrA activity is down-regulated both by dephosphorylation and by proteolysis, mediated by the ubiquitous ATP-dependent protease ClpXP. Here we demonstrate that proteins needed for rapid CtrA proteolysis in vivo form a phosphorylation-dependent and cyclic diguanylate (cdG)-dependent adaptor complex that accelerates CtrA degradation in vitro by ClpXP. The adaptor complex includes CpdR, a single-domain response regulator; PopA, a cdG-binding protein; and RcdA, a protein whose activity cannot be predicted. When CpdR is unphosphorylated and when PopA is bound to cdG, they work together with RcdA in an all-or-none manner to reduce the Km of CtrA proteolysis 10-fold. We further identified a set of amino acids in the receiver domain of CtrA that modulate its adaptor-mediated degradation in vitro and in vivo. Complex formation between PopA and CtrA depends on these amino acids, which reside on alpha-helix 1 of the CtrA receiver domain, and on cdG binding by PopA. These results reveal that each accessory factor plays an essential biochemical role in the regulated proteolysis of CtrA and demonstrate, to our knowledge, the first example of a multiprotein, cdG-dependent proteolytic adaptor.

Topics: Amino Acid Sequence; Bacterial Proteins; Caulobacter crescentus; Cell Cycle; Cyclic GMP; DNA-Binding Proteins; Endopeptidase Clp; Kinetics; Models, Molecular; Molecular Sequence Data; Phosphorylation; Protein Structure, Tertiary; Proteolysis; Second Messenger Systems; Sequence Homology, Amino Acid; Transcription Factors

To study the regulation mechanism of biofilm formation c-di-GMP synthesisby AphA in Vibrio parahaemolyticus, by using phenotypic and molecular biochemical experiments.. Colony morphology and crystal violet staining assays were used to analyze the phenotypic changes between the aphA null mutant (delta aphA) and the wide-type (WT) parent strain. The intracellular levels of c-di-GMP in the delta aphA and WT strains were determined by a chromatography-coupled tandem mass spectrometry (HPLC-MS/MS) method. Total RNAs were extracted from delta aphA and WT. Quantitative RT-PCR was applied to calculate the transcriptional variation of scrABC and scrG between delta aphA and WT. The promoter-proximal regions of scrABC and scrG were cloned into the pHRP309 vector containing a promoterless lacZ gene, respectively. Then, each of the two recombinant LacZ reporter plasmids was transformed into delta aphA and WT, respectively, to measure the promoter activity (the beta-Galactosidase activity) of the target genes in AaphA and WT by using the beta-Galactosidase Enzyme Assay System. The over-expressed His-AphA was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Then, the electrophoretic mobility shift assay (EMSA) was applied to analyze the DNA-binding activity of His-AphA to scrABC and scrG promoter regions in vitro. [Results] The phenotypic experiments disclosed that AphA was an activator of c-di-GMP synthesis and biofilm formation in Vibrio parahaemolyticus. The quantitative RT-PCR and LacZ fusion results showed that the transcription of scrABC and scrG was under negative control of AphA. However, the purified His-AphA could not bind to the upstream DNA regions of scrABC and scrG, as determined by EMSA.. The fact that AphA represses the transcription of scrABC and scrG will at least partially account for the positive regulation of e-di-GMP synthesis and biofilm formation by AphA in Vibrio parahaemolyticus.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Trans-Activators; Vibrio parahaemolyticus

Alginate-like exopolysaccharides (ALE) likely contribute markedly to strength of aerobic granules. This study cultivated aerobic granules from propionate wastewaters using strategies with different organic loading rates (OLRs) (4.4-17.4 kg/m(3)-d). When the OLR increased suddenly, the constituent cells (Pseudomonas, Clostridium, Thauera and Arthrobacter) were stimulated to secret extracellular cyclic diguanylate (c-di-GMP) and produced excess ALE, which formed a large quantity of sticky materials that served as the precursor of aerobic granules. Formation of excess ALE was the prerequisite for accelerated granulation. Conversely, this study observed no enrichment of poly guluronic acid blocks in ALE during granulation.

Topics: Alginates; Bacteria; Bacterial Adhesion; Base Sequence; Chromatography, High Pressure Liquid; Cyclic GMP; Denaturing Gradient Gel Electrophoresis; DNA Primers; Microscopy, Electron, Scanning; Molecular Sequence Data; Polymerase Chain Reaction; Polysaccharides, Bacterial; Propionates; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Waste Disposal, Fluid; Wastewater; Water Purification

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen and a threat for immunocompromised and cystic fibrosis patients. It is responsible for acute and chronic infections and can switch between these lifestyles upon taking an informed decision involving complex regulatory networks. The RetS/LadS/Gac/Rsm network and the cyclic-di-GMP (c-di-GMP) signaling pathways are both central to this phenomenon redirecting the P. aeruginosa population toward a biofilm mode of growth, which is associated with chronic infections. While these two pathways were traditionally studied independently from each other, we recently showed that cellular levels of c-di-GMP are increased in the hyperbiofilm retS mutant. Here, we have formally established the link between the two networks by showing that the SadC diguanylate cyclase is central to the Gac/Rsm-associated phenotypes, notably, biofilm formation. Importantly, SadC is involved in the signaling that converges onto the RsmA translational repressor either via RetS/LadS or via HptB/HsbR. Although the level of expression of the sadC gene does not seem to be impacted by the regulatory cascade, the production of the SadC protein is tightly repressed by RsmA. This adds to the growing complexity of the signaling network associated with c-di-GMP in P. aeruginosa. While this organism possesses more than 40 c-di-GMP-related enzymes, it remains unclear how signaling specificity is maintained within the c-di-GMP network. The finding that SadC but no other diguanylate cyclase is related to the formation of biofilm governed by the Gac/Rsm pathway further contributes to understanding of this insulation mechanism.

Topics: Biofilms; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa; Repressor Proteins; Signal Transduction

The bacterial second messenger cyclic di-GMP (c-di-GMP) stimulates inflammation by initiating innate immune cell recruitment and triggering the release of proinflammatory cytokines and chemokines. These properties make c-di-GMP a promising candidate for use as a vaccine adjuvant, and numerous studies have demonstrated that administration of purified c-di-GMP with different antigens increases protection against infection in animal models. Here, we have developed a novel approach to produce c-di-GMP inside host cells as an adjuvant to exploit a host-pathogen interaction and initiate an innate immune response. We have demonstrated that c-di-GMP can be synthesized in vivo by transducing a diguanylate cyclase (DGC) gene into mammalian cells using an adenovirus serotype 5 (Ad5) vector. Expression of DGC led to the production of c-di-GMP in vitro and in vivo, and this was able to alter proinflammatory gene expression in murine tissues and increase the secretion of numerous cytokines and chemokines when administered to animals. Furthermore, coexpression of DGC modestly increased T-cell responses to a Clostridium difficile antigen expressed from an adenovirus vaccine, although no significant differences in antibody titers were observed. This adenovirus c-di-GMP delivery system offers a novel method to administer c-di-GMP as an adjuvant to stimulate innate immunity during vaccination.

Topics: Adenoviridae; Adjuvants, Immunologic; Animals; Antigens, Bacterial; Bacterial Vaccines; Clostridioides difficile; Cyclic GMP; Escherichia coli Proteins; Immunity, Innate; Male; Mice, Inbred BALB C; Phosphorus-Oxygen Lyases; Recombinant Proteins; Transduction, Genetic

Dispersion enables the transition from the biofilm to the planktonic growth state in response to various cues. While several Pseudomonas aeruginosa proteins, including BdlA and the c-di-GMP phosphodiesterases DipA, RbdA, and NbdA, have been shown to be required for dispersion to occur, little is known about dispersion cue sensing and the signalling translating these cues into the modulation c-di-GMP levels to enable dispersion. Using glutamate-induced dispersion as a model, we report that dispersion-inducing nutrient cues are sensed via an outside-in signalling mechanism by the diguanylate cyclase NicD belonging to a family of seven transmembrane (7TM) receptors. NicD directly interacts with BdlA and the phosphodiesterase DipA, with NicD, BdlA, and DipA being part of the same pathway required for dispersion. Glutamate sensing by NicD results in NicD dephosphorylation and increased cyclase activity. Active NicD contributes to the non-processive proteolysis and activation of BdlA via phosphorylation and temporarily elevated c-di-GMP levels. BdlA, in turn, activates DipA, resulting in the overall reduction of c-di-GMP levels. Our results provide a basis for understanding the signalling mechanism based on NicD to induce biofilm dispersion that may be applicable to various biofilm-forming species and may have implications for the control of biofilm-related infections.

Topics: Biofilms; Cyclic GMP; Escherichia coli Proteins; Food; Glutamic Acid; Phosphorus-Oxygen Lyases; Phosphorylation; Protein Binding; Protein Processing, Post-Translational; Pseudomonas aeruginosa; Signal Transduction

Topics: Bacterial Proteins; Cyclic GMP; Streptomyces; Transcription Factors

Although type I IFNs play critical roles in antiviral and antitumor activity, it remains to be elucidated how type I IFNs are produced in sterile conditions of the tumor microenvironment and directly affect tumor-infiltrating immune cells. Mouse de novo gliomas show increased expression of type I IFN messages, and in mice, CD11b(+) brain-infiltrating leukocytes (BIL) are the main source of type I IFNs that are induced partially in a STING (stimulator of IFN genes)-dependent manner. Consequently, glioma-bearing Sting(Gt) (/Gt) mice showed shorter survival and lower expression levels of Ifns compared with wild-type mice. Furthermore, BILs of Sting(Gt) (/Gt) mice showed increased CD11b(+) Gr-1(+) immature myeloid suppressor and CD25(+) Foxp3(+) regulatory T cells (Treg) and decreased IFNγ-producing CD8(+) T cells. CD4(+) and CD8(+) T cells that received direct type I IFN signals showed lesser degrees of regulatory activity and increased levels of antitumor activity, respectively. Finally, intratumoral administration of a STING agonist (cyclic diguanylate monophosphate; c-di-GMP) improved the survival of glioma-bearing mice associated with enhanced type I IFN signaling, Cxcl10 and Ccl5, and T-cell migration into the brain. In combination with subcutaneous OVA peptide vaccination, c-di-GMP increased OVA-specific cytotoxicity of BILs and prolonged their survival. These data demonstrate significant contributions of STING to antitumor immunity via enhancement of type I IFN signaling in the tumor microenvironment and suggest a potential use of STING agonists for the development of effective immunotherapy, such as the combination with antigen-specific vaccinations.

Topics: Animals; Cancer Vaccines; CD11b Antigen; CD11c Antigen; Cell Line, Tumor; Cyclic GMP; Disease Models, Animal; Gene Expression Profiling; Glioma; Interferon Type I; Membrane Proteins; Mice; Mice, Knockout; Signal Transduction; T-Lymphocyte Subsets; Tumor Microenvironment

Bis-(3',5') cyclic di-guanylate (cyclic di-GMP) is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc). This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K(d)∼2 µM). Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence.

Topics: Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Humans; Mutation; Promoter Regions, Genetic; RNA-Binding Proteins; Second Messenger Systems; Transcription Factors; Virulence; Xanthomonas campestris

Innate immunity represents the first line of defense against invading pathogens in the respiratory tract. Innate immune cells such as monocytes, macrophages, dendritic cells, NK cells, and granulocytes contain specific pathogen-recognition molecules which induce the production of cytokines and subsequently activate the adaptive immune response. c-di-GMP is a ubiquitous second messenger that stimulates innate immunity and regulates biofilm formation, motility and virulence in a diverse range of bacterial species with potent immunomodulatory properties. In the present study, c-di-GMP was used to enhance the innate immune response against pertussis, a respiratory infection mainly caused by Bordetella pertussis. Intranasal treatment with c-di-GMP resulted in the induction of robust innate immune responses to infection with B. pertussis characterized by enhanced recruitment of neutrophils, macrophages, natural killer cells and dendritic cells. The immune responses were associated with an earlier and more vigorous expression of Th1-type cytokines, as well as an increase in the induction of nitric oxide in the lungs of treated animals, resulting in significant reduction of bacterial numbers in the lungs of infected mice. These results demonstrate that c-di-GMP is a potent innate immune stimulatory molecule that can be used to enhance protection against bacterial respiratory infections. In addition, our data suggest that priming of the innate immune system by c-di-GMP could further skew the immune response towards a Th1 type phenotype during subsequent infection. Thus, our data suggest that c-di-GMP might be useful as an adjuvant for the next generation of acellular pertussis vaccine to mount a more protective Th1 phenotype immune response, and also in other systems where a Th1 type immune response is required.

Topics: Animals; Bordetella pertussis; Cyclic GMP; Dendritic Cells; Disease Models, Animal; Female; Immune System; Immunity, Innate; Killer Cells, Natural; Macrophages; Mice; Neutrophils; Respiratory System; Whooping Cough

In chronic infections, pathogens are often in the presence of other microbial species. For example, Pseudomonas aeruginosa is a common and detrimental lung pathogen in individuals with cystic fibrosis (CF) and co-infections with Candida albicans are common. Here, we show that P. aeruginosa biofilm formation and phenazine production were strongly influenced by ethanol produced by the fungus C. albicans. Ethanol stimulated phenotypes that are indicative of increased levels of cyclic-di-GMP (c-di-GMP), and levels of c-di-GMP were 2-fold higher in the presence of ethanol. Through a genetic screen, we found that the diguanylate cyclase WspR was required for ethanol stimulation of c-di-GMP. Multiple lines of evidence indicate that ethanol stimulates WspR signaling through its cognate sensor WspA, and promotes WspR-dependent activation of Pel exopolysaccharide production, which contributes to biofilm maturation. We also found that ethanol stimulation of WspR promoted P. aeruginosa colonization of CF airway epithelial cells. P. aeruginosa production of phenazines occurs both in the CF lung and in culture, and phenazines enhance ethanol production by C. albicans. Using a C. albicans adh1/adh1 mutant with decreased ethanol production, we found that fungal ethanol strongly altered the spectrum of P. aeruginosa phenazines in favor of those that are most effective against fungi. Thus, a feedback cycle comprised of ethanol and phenazines drives this polymicrobial interaction, and these relationships may provide insight into why co-infection with both P. aeruginosa and C. albicans has been associated with worse outcomes in cystic fibrosis.

Topics: Biofilms; Candida albicans; Candidiasis; Cyclic GMP; Epithelial Cells; Ethanol; Humans; Phenazines; Pseudomonas aeruginosa

The second messenger cyclic diguanylate (c-di-GMP) plays a central role in bacterial adaptation to extracellular stimuli, controlling processes such as motility, biofilm development, cell development and, in some pathogens, virulence. The intracellular level of c-di-GMP is controlled by the complementary activities of diguanylate cyclases containing a GGDEF domain and two classes of c-di-GMP phosphodiesterases containing an EAL or HD-GYP hydrolytic domain. Compared to the GGDEF and EAL domains, the functions of HD-GYP domain family proteins are poorly characterized. The human diarrheal pathogen Vibrio cholerae encodes nine putative HD-GYP domain proteins. To determine the contributions of HD-GYP domain proteins to c-di-GMP signaling in V. cholerae, we systematically analyzed the enzymatic functionality of each protein and their involvement in processes known to be regulated by c-di-GMP: motility, biofilm development and virulence.. Complementary in vitro and in vivo experiments showed that four HD-GYP domain proteins are active c-di-GMP phosphodiesterases: VC1295, VC1348, VCA0210 and VCA0681. Mutation of individual HD-GYP domain genes, as well as combinatorial mutations of multiple HD-GYP domain genes, had no effect on motility or biofilm formation of V. cholerae under the conditions tested. Furthermore, no single HD-GYP domain gene affected intestinal colonization by V. cholerae in an infant mouse model. However, inactivation of multiple HD-GYP domain genes, including the four encoding functional phosphodiesterases, significantly attenuated colonization.. These results indicate that the HD-GYP family of c-di-GMP phosphodiesterases impacts signaling by this second messenger during infection. Altogether, this work greatly furthers the understanding of this important family of c-di-GMP metabolic enzymes and demonstrates a role for HD-GYP domain proteins in the virulence of V. cholerae.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Biofilms; Cholera; Cyclic GMP; Disease Models, Animal; Locomotion; Mice; Mutation; Signal Transduction; Vibrio cholerae; Virulence

In 2011, nearly 4,000 people in Germany were infected by Shiga toxin (Stx)-producing Escherichia coli O104:H4 with > 22% of patients developing haemolytic uraemic syndrome (HUS). Genome sequencing showed the outbreak strain to be related to enteroaggregative E. coli (EAEC), suggesting its high virulence results from EAEC-typical strong adherence and biofilm formation combined to Stx production. Here, we report that the outbreak strain contains a novel diguanylate cyclase (DgcX)--producing the biofilm-promoting second messenger c-di-GMP--that shows higher expression than any other known E. coli diguanylate cyclase. Unlike closely related E. coli, the outbreak strain expresses the c-di-GMP-controlled biofilm regulator CsgD and amyloid curli fibres at 37°C, but is cellulose-negative. Moreover, it constantly generates derivatives with further increased and deregulated production of CsgD and curli. Since curli fibres are strongly proinflammatory, with cellulose counteracting this effect, high c-di-GMP and curli production by the outbreak O104:H4 strain may enhance not only adherence but may also contribute to inflammation, thereby facilitating entry of Stx into the bloodstream and to the kidneys where Stx causes HUS.

Topics: Biofilms; Cyclic GMP; Disease Outbreaks; Escherichia coli Infections; Escherichia coli Proteins; Female; Germany; Hemolytic-Uremic Syndrome; Humans; Middle Aged; Phosphorus-Oxygen Lyases; Shiga Toxin; Shiga-Toxigenic Escherichia coli

Synthetic devices for traceless remote control of gene expression may provide new treatment opportunities in future gene- and cell-based therapies. Here we report the design of a synthetic mind-controlled gene switch that enables human brain activities and mental states to wirelessly programme the transgene expression in human cells. An electroencephalography (EEG)-based brain-computer interface (BCI) processing mental state-specific brain waves programs an inductively linked wireless-powered optogenetic implant containing designer cells engineered for near-infrared (NIR) light-adjustable expression of the human glycoprotein SEAP (secreted alkaline phosphatase). The synthetic optogenetic signalling pathway interfacing the BCI with target gene expression consists of an engineered NIR light-activated bacterial diguanylate cyclase (DGCL) producing the orthogonal second messenger cyclic diguanosine monophosphate (c-di-GMP), which triggers the stimulator of interferon genes (STING)-dependent induction of synthetic interferon-β promoters. Humans generating different mental states (biofeedback control, concentration, meditation) can differentially control SEAP production of the designer cells in culture and of subcutaneous wireless-powered optogenetic implants in mice.

Topics: Alkaline Phosphatase; Animals; Brain-Computer Interfaces; Cyclic GMP; Electroencephalography; Female; Gene Expression; Humans; Implants, Experimental; Mice; Optogenetics; Signal Transduction; Transcription, Genetic; Transgenes; Wireless Technology

Diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively synthesise and hydrolyse the secondary messenger cyclic dimeric GMP (c-di-GMP), and both activities are often found in a single protein. Intracellular c-di-GMP levels in turn regulate bacterial motility, virulence and biofilm formation. We report the first structure of a tandem DGC-PDE fragment, in which the catalytic domains are shown to be active. Two phosphodiesterase states are distinguished by active site formation. The structures, in the presence or absence of c-di-GMP, suggest that dimerisation and binding pocket formation are linked, with dimerisation being required for catalytic activity. An understanding of PDE activation is important, as biofilm dispersal via c-di-GMP hydrolysis has therapeutic effects on chronic infections.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Amino Acid Sequence; Biocatalysis; Catalytic Domain; Cyclic GMP; Enzyme Activation; Escherichia coli Proteins; Models, Molecular; Molecular Sequence Data; Phosphorus-Oxygen Lyases; Protein Multimerization; Protein Structure, Quaternary; Pseudomonas aeruginosa

The second messenger cyclic di-GMP (c-di-GMP) regulates numerous phenotypes in response to environmental stimuli to enable bacteria to transition between different lifestyles. Here we discuss our recent findings that the human pathogen Vibrio cholerae recognizes 2 host-specific signals, bile and bicarbonate, to regulate intracellular c-di-GMP. We have demonstrated that bile acids increase intracellular c-di-GMP to promote biofilm formation. We have also shown that this bile-mediated increase of intracellular c-di-GMP is negated by bicarbonate, and that this interaction is dependent on pH, suggesting that V. cholerae uses these 2 environmental cues to sense and adapt to its relative location in the small intestine. Increased intracellular c-di-GMP by bile is attributed to increased c-di-GMP synthesis by 3 diguanylate cyclases (DGCs) and decreased expression of one phosphodiesterase (PDE) in the presence of bile. The molecular mechanisms by which bile controls the activity of the 3 DGCs and the regulators of bile-mediated transcriptional repression of the PDE are not yet known. Moreover, the impact of varying concentrations of bile and bicarbonate at different locations within the small intestine and the response of V. cholerae to these cues remains unclear. The native microbiome and pharmaceuticals, such as omeprazole, can impact bile and pH within the small intestine, suggesting these are potential unappreciated factors that may alter V. cholerae pathogenesis.

Topics: Animals; Bicarbonates; Bile Acids and Salts; Cyclic GMP; Escherichia coli Proteins; Humans; Intestine, Small; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Vibrio cholerae

Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is an intracellular second messenger that controls the lifestyles of many bacteria. A high intracellular level of c-di-GMP induces a biofilm lifestyle, whereas a low intracellular level of c-di-GMP stimulates dispersal of biofilms and promotes a planktonic lifestyle. Here, we used the expression of different reporters to show that planktonic cells, biofilm cells, and cells dispersed from biofilms (DCells) had distinct intracellular c-di-GMP levels. Proteomics analysis showed that the low intracellular c-di-GMP level of DCells induced the expression of proteins required for the virulence and development of antimicrobial peptide resistance in Pseudomonas aeruginosa. In accordance with this, P. aeruginosa cells with low c-di-GMP levels were found to be more resistant to colistin than P. aeruginosa cells with high c-di-GMP levels. This finding contradicts the current dogma stating that dispersed cells are inevitably more susceptible to antibiotics than their sessile counterparts.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Colistin; Cyclic GMP; Drug Resistance, Bacterial; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genes, Reporter; Green Fluorescent Proteins; Plankton; Proteomics; Pseudomonas aeruginosa; Second Messenger Systems

The bacterial tyrosine-kinase (BY-kinase) family comprises the major group of bacterial enzymes endowed with tyrosine kinase activity. We previously showed that the BceF protein from Burkholderia cepacia IST408 belongs to this BY-kinase family and is involved in the biosynthesis of the exopolysaccharide cepacian. However, little is known about the extent of regulation of this protein kinase activity. In order to examine this regulation, we performed a comparative transcriptome profile between the bceF mutant and wild-type B. cepacia IST408. The analyses led to identification of 630 genes whose expression was significantly changed. Genes with decreased expression in the bceF mutant were related to stress response, motility, cell adhesion, and carbon and energy metabolism. Genes with increased expression were related to intracellular signaling and lipid metabolism. Mutation of bceF led to reduced survival under heat shock and UV light exposure, reduced swimming motility, and alteration in biofilm architecture when grown in vitro. Consistent with some of these phenotypes, the bceF mutant demonstrated elevated levels of cyclic-di-GMP. Furthermore, BceF contributed to the virulence of B. cepacia for larvae of the Greater wax moth, Galleria mellonella. Taken together, BceF appears to play a considerable role in many cellular processes, including biofilm formation and virulence. As homologues of BceF occur in a number of pathogenic and plant-associated Burkholderia strains, the modulation of bacterial behavior through tyrosine kinase activity is most likely a widely occurring phenomenon.

Topics: Animals; Bacterial Proteins; Biofilms; Burkholderia cepacia; Cyclic GMP; DNA, Bacterial; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Moths; Mutagenesis, Insertional; Oligonucleotide Array Sequence Analysis; Protein-Tyrosine Kinases; Stress, Physiological; Transcriptome; Virulence

The second messenger cyclic di-GMP (c-di-GMP) is a nearly ubiquitous intracellular signal molecule known to regulate various cellular processes, including biofilm formation, motility, and virulence. The intracellular concentration of c-di-GMP is inversely governed by diguanylate cyclase (DGC) enzymes and phosphodiesterase (PDE) enzymes, which synthesize and degrade c-di-GMP, respectively. The role of c-di-GMP in the plant pathogen and causal agent of fire blight disease Erwinia amylovora has not been studied previously. Here we demonstrate that three of the five predicted DGC genes in E. amylovora (edc genes, for Erwinia diguanylate cyclase), edcA, edcC, and edcE, are active diguanylate cyclases. We show that c-di-GMP positively regulates the secretion of the main exopolysaccharide in E. amylovora, amylovoran, leading to increased biofilm formation, and negatively regulates flagellar swimming motility. Although amylovoran secretion and biofilm formation are important for the colonization of plant xylem tissues and the development of systemic infections, deletion of the two biofilm-promoting DGCs increased tissue necrosis in an immature-pear infection assay and an apple shoot infection model, suggesting that c-di-GMP negatively regulates virulence. In addition, c-di-GMP inhibited the expression of hrpA, a gene encoding the major structural component of the type III secretion pilus. Our results are the first to describe a role for c-di-GMP in E. amylovora and suggest that downregulation of motility and type III secretion by c-di-GMP during infection plays a key role in the coordination of pathogenesis.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Erwinia amylovora; Escherichia coli Proteins; Mutagenesis, Insertional; Phosphorus-Oxygen Lyases; Polysaccharides, Bacterial; Virulence

Cyclic dinucleotides are an important class of signaling molecules that regulate a wide variety of pathogenic responses in bacteria, but tools for monitoring their regulation in vivo are lacking. We have designed RNA-based fluorescent biosensors for cyclic di-GMP and cyclic AMP-GMP by fusing the Spinach aptamer to variants of a natural GEMM-I riboswitch. In live cell imaging experiments, these biosensors demonstrate fluorescence turn-on in response to cyclic dinucleotides, and they were used to confirm in vivo production of cyclic AMP-GMP by the enzyme DncV.

Topics: Amino Acid Sequence; Aptamers, Nucleotide; Biosensing Techniques; Cyclic AMP; Cyclic GMP; Fluorescence; Molecular Imaging; RNA; Spinacia oleracea

Signal transduction pathways mediated by cyclic-bis(3'→5')-dimeric GMP (c-di-GMP) control many important and complex behaviors in bacteria. C-di-GMP is synthesized through the action of GGDEF domains that possess diguanylate cyclase activity and is degraded by EAL or HD-GYP domains with phosphodiesterase activity. There is mounting evidence that some important c-di-GMP-mediated pathways require protein-protein interactions between members of the GGDEF, EAL, HD-GYP and PilZ protein domain families. For example, interactions have been observed between PilZ and the EAL domain from FimX of Xanthomonas citri (Xac). FimX and PilZ are involved in the regulation of type IV pilus biogenesis via interactions of the latter with the hexameric PilB ATPase associated with the bacterial inner membrane. Here, we present the crystal structure of the ternary complex made up of PilZ, the FimX EAL domain (FimXEAL) and c-di-GMP. PilZ interacts principally with the lobe region and the N-terminal linker helix of the FimXEAL. These interactions involve a hydrophobic surface made up of amino acids conserved in a non-canonical family of PilZ domains that lack intrinsic c-di-GMP binding ability and strand complementation that joins β-sheets from both proteins. Interestingly, the c-di-GMP binds to isolated FimXEAL and to the PilZ-FimXEAL complex in a novel conformation encountered in c-di-GMP-protein complexes in which one of the two glycosidic bonds is in a rare syn conformation while the other adopts the more common anti conformation. The structure points to a means by which c-di-GMP and PilZ binding could be coupled to FimX and PilB conformational states.

Topics: Bacterial Proteins; Crystallography, X-Ray; Cyclic GMP; Fimbriae, Bacterial; Models, Biological; Models, Molecular; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; Protein Multimerization; Xanthomonas

Commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(propargyl)guanosine is converted to its 3'-O-levulinyl ester in a yield of 91%. The reaction of commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl]guanosine with N(2)-isobutyryl-2'-O-propargyl-3'-O-(levulinyl)guanosine provides, after P(III) oxidation, 3'-/5'-deprotection, and purification, the 2'-O-propargylated guanylyl(3'-5')guanosine 2-cyanoethyl phosphate triester in a yield of 88%. Phosphitylation of this dinucleoside phosphate triester with 2-cyanoethyl tetraisopropylphosphordiamidite and 1H-tetrazole, followed by an in situ intramolecular cyclization, gives the propargylated cyclic dinucleoside phosphate triester, which is isolated in a yield of 40% after P(III) oxidation and purification. Complete removal of the nucleobases, phosphates, and 2'-O-tert-butyldimethylsilyl protecting groups leads to the desired propargylated c-di-GMP diester. Cycloaddition of a biotinylated azide with the propargylated c-di-GMP diester under click conditions provides the biotinylated c-di-GMP conjugate in an isolated yield of 62%. Replacement of the 6-oxo function of N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-levulinyl-2'-O-(propargyl)guanosine with a 2-cyanoethylthio group is effected by treatment with 2,4,6-triisopropybenzenesulfonyl chloride and triethylamine to give a 6-(2,4,6-triisopropylbenzenesulfonic acid) ester intermediate. Reaction of this key intermediate with 3-mercaptoproprionitrile and triethylamine, followed by 5'-dedimethoxytritylation, affords the 6-(2-cyanoethylthio)guanosine derivative in a yield of 70%. The 5'-hydroxy function of this derivative is reacted with commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl]guanosine. The reaction product is then converted to the mono-6-thioated c-di- GMP biotinylated conjugate under conditions highly similar to those described above for the preparation of the biotinylated c-di-GMP conjugate, and isolated in similar yields.

Topics: Azides; Biotinylation; Click Chemistry; Cyclic GMP; Dinucleoside Phosphates; Guanosine; Guanosine Monophosphate; Tetrazoles

Elevated intracellular levels of the bacterial second messenger c-di-GMP are known to suppress motility and promote sessility. Bacterial chemotaxis guides motile cells in gradients of attractants and repellents over broad concentration ranges, thus allowing bacteria to quickly adapt to changes in their surroundings. Here, we describe a chemotaxis receptor that enhances, as opposed to suppresses, motility in response to temporary increases in intracellular c-di-GMP. Azospirillum brasilense's preferred metabolism is adapted to microaerophily, and these motile cells quickly navigate to zones of low oxygen concentration by aerotaxis. We observed that changes in oxygen concentration result in rapid changes in intracellular c-di-GMP levels. The aerotaxis and chemotaxis receptor, Tlp1, binds c-di-GMP via its C-terminal PilZ domain and promotes persistent motility by increasing swimming velocity and decreasing swimming reversal frequency, which helps A. brasilense reach low-oxygen zones. If c-di-GMP levels remain high for extended periods, A. brasilense forms nonmotile clumps or biofilms on abiotic surfaces. These results suggest that association of increased c-di-GMP levels with sessility is correct on a long-term scale, while in the short-term c-di-GMP may actually promote, as opposed to suppress, motility. Our data suggest that sensing c-di-GMP by Tlp1 functions similar to methylation-based adaptation. Numerous chemotaxis receptors contain C-terminal PilZ domains or other sensory domains, suggesting that intracellular c-di-GMP as well as additional stimuli can be used to modulate adaptation of bacterial chemotaxis receptors.. To adapt and compete under changing conditions, bacteria must not only detect and respond to various environmental cues but also be able to remain sensitive to further changes in the environmental conditions. In bacterial chemotaxis, chemosensory sensitivity is typically brought about by changes in the methylation status of chemotaxis receptors capable of modulating the ability of motile cells to navigate in gradients of various physicochemical cues. Here, we show that the ubiquitous second messenger c-di-GMP functions to modulate chemosensory sensitivity of a bacterial chemotaxis receptor in the alphaproteobacterium Azospirillum brasilense. Binding of c-di-GMP to the chemotaxis receptor promotes motility under conditions of elevated intracellular c-di-GMP levels. Our results revealed that the role of c-di-GMP as a sessile signal is overly simplistic. We also show that adaptation by sensing an intracellular metabolic cue, via PilZ or other domains, is likely widespread among bacterial chemotaxis receptors.

Topics: Azospirillum brasilense; Biofilms; Chemotaxis; Cyclic GMP; Locomotion; Oxygen; Second Messenger Systems; Signal Transduction

Recently, it has been reported that the montmorillonite-catalyzed oligomerization of activated nucleotides exhibits remarkable enantioselectivity. In the current paper we investigate the structures and intrinsic energies of homochiral and heterochiral cyclic dinucleotides by means of accurate quantum chemical calculations in gas-phase and in bulk water. The steric effect of the clay is represented with geometrical constraints. Our computations reveal that the heterochiral dimer geometries are systematically less stable than their homochiral counterparts due to steric clashes inside the sugar-phosphate ring geometry. Thus we suggest that the homochiral selectivity observed in the cyclic dinucleotide formation in confined spaces may arise from the energetic destabilization of the heterochiral ring geometries as compared to their homochiral analogues. In the present paper we provide the first model of the 3D structure of d,l cyclic dinucleotides, which until now has eluded experimental observation.

Topics: Cyclic GMP; Molecular Structure; Quantum Theory; Stereoisomerism

The mammalian ER protein STING (stimulator of interferon genes; also known as MITA, ERIS, MPYS or TMEM173) is an adaptor protein that links the detection of cytosolic dsDNA to the activation of TANK-binding kinase 1 (TBK1) and its downstream transcription factor interferon regulatory factor 3 (IFN3). Recently, STING itself has been found to be the direct receptor of bacterial c-di-GMP, and crystal structures of several human STING C-terminal domain (STING-CTD) dimers in the apo form or in complex with c-di-GMP have been published. Here, a novel set of structures of mouse STING-CTD (mSTING(137-344)) in apo and complex forms determined from crystals obtained under different crystallization conditions are reported. These novel closed-form structures exhibited considerable differences from previously reported open-form human STING-CTD structures. The novel mSTING structures feature extensive interactions between the two monomers, a unique asymmetric c-di-GMP molecule with one guanine base in an unusual syn conformation that is well accommodated in the dimeric interface with many direct specific interactions and two unexpected equivalent secondary peripheral c-di-GMP binding sites. Replacement of the amino acids crucial for specific c-di-GMP binding in mSTING significantly changes the ITC titration profiles and reduces the IFN-β reporter luciferase activity. Taken together, these results reveal a more stable c-di-GMP binding mode of STING proteins that could serve as a template for rational drug design to stimulate interferon production by mammalian cells.

Topics: Adaptive Immunity; Animals; Crystallography, X-Ray; Cyclic GMP; Immunity, Innate; Membrane Proteins; Mice

Escherichia coli O157:H7 is an important foodborne pathogen that causes serious illness in humans at low infectious doses. The main source of infections is beef or greens contaminated with E. coli O157:H7 shed by cattle. Here we investigated the role of c-di-GMP-dependent signal transduction in cattle gut colonization of E. coli O157:H7. To manipulate intracellular c-di-GMP levels, we introduced into E. coli O157:H7 a c-di-GMP specific phosphodiesterase (PDE). Liquid chromatography tandem mass spectrometry analysis confirmed that in E. coli O157:H7, over-expression of PDE decreased c-di-GMP level. Consistent with the altered c-di-GMP level, PDE overexpression resulted in decreased biofilm formation in E. coli O157:H7. Furthermore, this diminished c-di-GMP levels reduced adhesion of E. coli O157:H7 to both cultured HT-29 cells and cattle colon explants. Consistently, mRNA levels of genes involved in adhesion were down-regulated including genes encoding E. coli common pili, long polar fimbriae 1, hemorrhagic coli pilus, as well as intimin and tir. We further observed decreased curli fimbriae synthesis in the strain with decreased c-di-GMP levels, which was supported by the reduction in the transcription of curli large subunit gene csgA and the curli expression regulator gene csgD. Genes for enterocyte effacement encoded regulator (Ler) and type III secretion system effectors, EspA and EspB, were also down-regulated. Collectively, data indicated that c-di-GMP signaling positively regulates E. coli O157:H7 intestinal epithelial cell and tissue colonization and expression of associated adhesion factors.

Topics: Animals; Cattle; Cyclic GMP; Enterocytes; Epithelial Cells; Escherichia coli Infections; Escherichia coli O157; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; HT29 Cells; Humans; Intestines; Signal Transduction; Time Factors

C-di-GMP is a second messenger in bacteria and partly regulates bacterial physiology by binding to class I and II riboswitches. Four class I c-di-GMP riboswitch aptamer candidates, Ct-E88, Cb-17B, Cb-E43 and Cd-630 RNAs, selected from a GEMM RNA sequence motif in the Rfam database, were expressed and experimentally verified to bind to c-di-GMP. The two newly characterized c-di-GMP riboswitches, Ct-E88 and Cb-E43, bound c-di-GMP with nanomolar Kd whereas the affinities of Cb-17B and Cd-630 for c-di-GMP were at least a 100-fold weaker. Interestingly, whereas the three riboswitches (Vc2, Et-E88 and Cb-E43) bound c-di-GMP with similar Kd values, 2'-modified analogs of c-di-GMP differentially bound to these three class I aptamers. For example, 2'-F-c-di-GMP bound Vc2 with a Kd value of 102 nM whereas the Kd value of 2'-F-c-di-GMP-Ct-E88 is 43 μM (422× higher than that for Vc2 RNA), revealing that there are differences in the binding sites of functional class I c-di-GMP riboswitches.

Topics: Aptamers, Nucleotide; Base Sequence; Binding Sites; Clostridium botulinum; Clostridium tetani; Cyclic GMP; Nucleic Acid Conformation; Riboswitch; RNA, Bacterial; Sequence Alignment; Structure-Activity Relationship

The extracellular matrix of bacterial biofilms has at least two key functions: to serve as a structural scaffold for the multicellular community, and to play a protective role against external stress. In this work, we report a compensatory effect whereby Pseudomonas putida reacts to the lack of either of the two main surface proteins involved in biofilm formation, LapA and LapF, by increasing expression and production of a species-specific EPS. Elevated levels of the second messenger molecule cyclic di-GMP alter the balance of extracellular matrix components, and the phenotypes of lapA and lapF mutants under these conditions are indicative of direct interactions taking place between large secreted proteins and exopolysaccharides. Our data suggest the existence of a mechanism by which bacteria would sense alterations in the composition of the extracellular matrix, leading to changes in expression of the different elements.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Extracellular Matrix Proteins; Gene Expression Regulation, Bacterial; Pseudomonas putida

In Gram-negative bacteria, production of the signal molecule c-di-GMP by diguanylate cyclases (DGCs) is a key trigger for biofilm formation, which, in turn, is often required for the development of chronic bacterial infections. Thus, DGCs represent interesting targets for new chemotherapeutic drugs with anti-biofilm activity. We searched for inhibitors of the WspR protein, a Pseudomonas aeruginosa DGC involved in biofilm formation and production of virulence factors, using a set of microbiological assays developed in an Escherichia coli strain expressing the wspR gene. We found that azathioprine, an immunosuppressive drug used in the treatment of Crohn's disease, was able to inhibit WspR-dependent c-di-GMP biosynthesis in bacterial cells. However, in vitro enzymatic assays ruled out direct inhibition of WspR DGC activity either by azathioprine or by its metabolic derivative 2-amino-6-mercapto-purine riboside. Azathioprine is an inhibitor of 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase, an enzyme involved in purine biosynthesis, which suggests that inhibition of c-di-GMP biosynthesis by azathioprine may be due to perturbation of intracellular nucleotide pools. Consistent with this hypothesis, WspR activity is abolished in an E. coli purH mutant strain, unable to produce AICAR transformylase. Despite its effect on WspR, azathioprine failed to prevent biofilm formation by P. aeruginosa; however, it affected production of extracellular structures in E. coli clinical isolates, suggesting efficient inhibition of c-di-GMP biosynthesis in this bacterium. Our results indicate that azathioprine can prevent biofilm formation in E. coli through inhibition of c-di-GMP biosynthesis and suggest that such inhibition might contribute to its anti-inflammatory activity in Crohn's disease.

Topics: Anti-Bacterial Agents; Azathioprine; Biofilms; Cyclic GMP; Escherichia coli; Nucleotides; Pseudomonas aeruginosa

Extensive research on antiviral small molecules starting in the early 1970s has led to the identification of 10-carboxymethyl-9-acridanone (CMA) as a potent type I interferon (IFN) inducer. Up to date, the mode of action of this antiviral molecule has remained elusive. Here we demonstrate that CMA mediates a cell-intrinsic type I IFN response, depending on the ER-resident protein STING. CMA directly binds to STING and triggers a strong antiviral response through the TBK1/IRF3 route. Interestingly, while CMA displays extraordinary activity in phosphorylating IRF3 in the murine system, CMA fails to activate human cells that are otherwise responsive to STING ligands. This failure to activate human STING can be ascribed to its inability to bind to the C-terminal ligand-binding domain of human STING. Crystallographic studies show that two CMA molecules bind to the central Cyclic diguanylate (c-diGMP)-binding pocket of the STING dimer and fold the lid region in a fashion similar, but partially distinct, to c-diGMP. Altogether, these results provide novel insight into ligand-sensing properties of STING and, furthermore, unravel unexpected species-specific differences of this innate sensor.

Topics: Acridines; Animals; Binding Sites; Crystallography, X-Ray; Cyclic GMP; Humans; Interferon Inducers; Interferon Regulatory Factor-3; Interferon Type I; Macrophages; Membrane Proteins; Mice; Protein Conformation; Protein Multimerization; Protein Structure, Tertiary; Recombinant Proteins

WspR is a hybrid response regulator-diguanylate cyclase that is phosphorylated by the Wsp signal transduction complex in response to growth of Pseudomonas aeruginosa on surfaces. Active WspR produces cyclic di-GMP (c-di-GMP), which in turn stimulates biofilm formation. In previous work, we found that when activated by phosphorylation, yellow fluorescent protein (YFP)-tagged WspR forms clusters that are visible in individual cells by fluorescence microscopy. Unphosphorylated WspR is diffuse in cells and not visible. Thus, cluster formation is an assay for WspR signal transduction. To understand how and why WspR forms subcellular clusters, we analyzed cluster formation and the enzymatic activities of six single amino acid variants of WspR. In general, increased cluster formation correlated with increased in vivo and in vitro diguanylate cyclase activities of the variants. In addition, WspR specific activity was strongly concentration dependent in vitro, and the effect of the protein concentration on diguanylate cyclase activity was magnified when WspR was treated with the phosphor analog beryllium fluoride. Cluster formation appears to be an intrinsic property of phosphorylated WspR (WspR-P). These results support a model in which the formation of WspR-P subcellular clusters in vivo in response to a surface stimulus is important for potentiating the diguanylate cyclase activity of WspR. Subcellular cluster formation appears to be an additional means by which the activity of a response regulator protein can be regulated.. Bacterial sensor proteins often phosphorylate cognate response regulator proteins when stimulated by an environmental signal. Phosphorylated response regulators then mediate an appropriate adaptive cellular response. About 6% of response regulator proteins have an enzymatic domain that is involved in producing or degrading cyclic di-GMP (c-di-GMP), a molecule that stimulates bacterial biofilm formation. In this work, we examined the in vivo and in vitro behavior of the response regulator-diguanylate cyclase WspR. When phosphorylated in response to a signal associated with surface growth, WspR has a tendency to form oligomers that are visible in cells as subcellular clusters. Our results show that the formation of phosphorylated WspR (WspR-P) subcellular clusters is important for potentiating the diguanylate cyclase activity of WspR-P, making it more active in c-di-GMP production. We conclude that oligomer formation visualized as subcellular clusters is an additional mechanism by which the activities of response regulator-diguanylate cyclases can be regulated.

Topics: Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Models, Biological; Models, Molecular; Phosphorus-Oxygen Lyases; Phosphorylation; Protein Conformation; Protein Multimerization; Protein Processing, Post-Translational; Pseudomonas aeruginosa; Signal Transduction; Transcription Factors

Cyclic-di-GMP (c-di-GMP) is a central regulator of bacterial behavior. Various studies have implicated c-di-GMP in biofilm formation and virulence factor production in multitudes of bacteria. Hence it is expected that the disruption of c-di-GMP signaling could provide an effective means to disrupt biofilm and/or virulence factor formation in several bacteria of clinical relevance. C-di-GMP achieves the regulation of bacterial phenotype via binding to several effector molecules including transcription factors, enzymes and riboswitches. Crystal structure analyses of c-di-GMP effector molecules, in complex with the ligand, reveal that various classes of c-di-GMP receptors recognize this dinucleotide using different sets of recognition elements. Therefore, it is plausible that different analogues of c-di-GMP could be used to selectively modulate a specific class of c-di-GMP binding receptors, and hence modulate the bacterial phenotype. Thus far only a detailed study of the differential binding of c-di-GMP analogues to riboswitches, but not proteins, has been reported. In this report, we prepared various 2'-modified analogues of c-di-GMP and studied both polymorphisms of these analogues using DOSY NMR and the binding to several effector proteins, such as PilZ-containing proteins, diguanylate cyclases (DGC) containing I-sites, and phoshphodiesterases (PDE). 2'-Modification of c-di-GMP did not adversely affect the propensity to form higher aggregates, such as octameric forms, in the presence of potassium salts. Interestingly, we find that the selective binding to different classes of c-di-GMP binding proteins could be achieved with the 2'-modified analogues and that 2'-F analogue of c-di-GMP binds to the I-site of DGCs better (four times) than the native dinucleotide, c-di-GMP, whereas c-di-GMP binds to PDEs better (10 times) than 2'-F-c-di-GMP. 2'-F-c-di-GMP potently inhibits c-di-GMP synthesis by DGCs and hence raises the potential that cell permeable analogues of 2'-F-c-di-GMP could be used to disrupt c-di-GMP signaling in bacteria.

Topics: Allosteric Regulation; Bacteria; Cyclic GMP; Enzyme Inhibitors; Escherichia coli Proteins; Fluorine; Magnetic Resonance Spectroscopy; Molecular Structure; Phosphorus-Oxygen Lyases

Opportunistic pathogenic bacteria can engage in biofilm-based infections that evade immune responses and develop into chronic conditions. Because conventional antimicrobials cannot efficiently eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections. It has recently been established that the secondary messenger cyclic diguanosine monophosphate (c-di-GMP) functions as a positive regulator of biofilm formation in several different bacteria. In the present study we investigated whether manipulation of the c-di-GMP level in bacteria potentially can be used for biofilm control in vivo. We constructed a Pseudomonas aeruginosa strain in which a reduction in the c-di-GMP level can be achieved via induction of the Escherichia coli YhjH c-di-GMP phosphodiesterase. Initial experiments showed that induction of yhjH expression led to dispersal of the majority of the bacteria in in vitro-grown P. aeruginosa biofilms. Subsequently, we demonstrated that P. aeruginosa biofilms growing on silicone implants, located in the peritoneal cavity of mice, dispersed after induction of the YhjH protein. Bacteria accumulated temporarily in the spleen after induction of biofilm dispersal, but the mice tolerated the dispersed bacteria well. The present work provides proof of the concept that modulation of the c-di-GMP level in bacteria is a viable strategy for biofilm control.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Biofilms; Cyclic GMP; Disease Models, Animal; Escherichia coli Proteins; Female; Mice; Mice, Inbred BALB C; Pseudomonas aeruginosa; Pseudomonas Infections

The direct oxygen sensor DosP is a multidomain protein that contains a gas-sensing haem domain and an EAL effector domain. EAL domains are omnipresent signal transduction domains in bacteria. Many EAL domains are active phosphodiesterases and are involved in breakdown of the ubiquitous bacterial second messenger cyclic di-GMP. Despite a great deal of information on the functional and structural aspects of active and inactive EAL domains, little is known about the structural basis of their regulation by their associated sensory domains. Here, two crystal structures of the Escherichia coli DosP EAL domain derived from cubic and monoclinic crystal forms that were obtained under tartrate and PEG conditions, respectively, are described. Both of the structures display the typical TIM (triosephosphate isomerase) barrel fold with one antiparallel β-strand. However, unlike other EAL structures, access to the active site in DosP EAL is sterically restricted by the presence of a short helical stretch (Ser637-Ala-Leu-His640) in loop L3 between strand β3 and helix α3. This element, together with an unordered fragment, replaces the short α-helix (named α5 in Tbd1265 EAL) that is found in other EAL-domain structures. Since DosP EAL is an active c-di-GMP phosphodiesterase, the observed inactive conformation is suggested to be of functional relevance for the regulation mechanism of DosP.

Topics: Catalytic Domain; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Models, Molecular; Phosphoric Diester Hydrolases; X-Ray Diffraction

Klebsiella pneumoniae CG43, a heavy encapsulated liver abscess isolate, mainly expresses type 3 fimbriae. Type 1 fimbriae expression was only apparent in CG43S3ΔmrkA (the type 3 fimbriae-deficient strain). The expression of type 1 fimbriae in CG43S3ΔmrkA was reduced by deleting the fimK gene, but was unaffected by removing the 3' end of fimK encoding the C-terminal EIL domain (EILfimK). Quantitative RT-PCR and promoter activity analysis showed that the putative DNA-binding region at the N terminus, but not the C-terminal EIL domain, of FimK positively affects transcription of the type 1 fimbrial major subunit, fimA. An electrophoretic mobility shift assay demonstrated that the recombinant FimK could specifically bind to fimS, which is located upstream of fimA and contains a vegetative promoter for the fim operon, also reflecting possible transcriptional regulation. EILfimK was shown to encode a functional phosphodiesterase (PDE) via enhancing motility in Escherichia coli JM109 and in vitro using PDE activity assays. Moreover, EILfimK exhibited higher PDE activity than FimK, implying that the N-terminal DNA-binding domain may negatively affect the PDE activity of FimK. FimA expression was detected in CG43S3 expressing EILfimK or AILfimK, suggesting that FimA expression is not directly influenced by the c-di-GMP level. In summary, FimK influences type 1 fimbriation by binding to fimS at the N-terminal domain, and thereafter, the altered protein structure may activate C-terminal PDE activity to reduce the intracellular c-di-GMP level.

Topics: Bacterial Proteins; Cyclic GMP; Fimbriae Proteins; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Humans; Klebsiella pneumoniae; Operon; Phosphoric Diester Hydrolases

C-di-GMP-which is produced by diguanylate cyclases (DGC) and degraded by specific phosphodiesterases (PDEs)-is a ubiquitous second messenger in bacterial biofilm formation. In Escherichia coli, several DGCs (YegE, YdaM) and PDEs (YhjH, YciR) and the MerR-like transcription factor MlrA regulate the transcription of csgD, which encodes a biofilm regulator essential for producing amyloid curli fibres of the biofilm matrix. Here, we demonstrate that this system operates as a signalling cascade, in which c-di-GMP controlled by the DGC/PDE pair YegE/YhjH (module I) regulates the activity of the YdaM/YciR pair (module II). Via multiple direct interactions, the two module II proteins form a signalling complex with MlrA. YciR acts as a connector between modules I and II and functions as a trigger enzyme: its direct inhibition of the DGC YdaM is relieved when it binds and degrades c-di-GMP generated by module I. As a consequence, YdaM then generates c-di-GMP and-by direct and specific interaction-activates MlrA to stimulate csgD transcription. Trigger enzymes may represent a general principle in local c-di-GMP signalling.

Topics: Biofilms; Cyclic GMP; Escherichia coli K12; Escherichia coli Proteins; Models, Biological; Multiprotein Complexes; Phosphorus-Oxygen Lyases; Protein Interaction Domains and Motifs; Second Messenger Systems; Signal Transduction; Trans-Activators; Transcription, Genetic

In Escherichia coli and Salmonella enterica, bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a ubiquitous bacterial second-messenger molecule that participates in many cellular processes, can regulate flagellar motor speed and reduce cell swimming velocity by binding to the PilZ-containing protein YcgR. Here, the crystallization and preliminary X-ray crystallographic analysis of YcgR with c-di-GMP are reported. The crystals diffracted to 2.3 Å resolution and belonged to space group R3:H, with unit-cell parameters a = b = 93.96, c = 109.61 Å. The asymmetric unit appeared to contain one subunit with a Matthews coefficient of 3.21 Å(3) Da(-1). The results reported here provide a sound basis for solving the crystal structure of YcgR with c-di-GMP and revealing its structure-function relationship based on the three-dimensional structure.

Topics: Crystallization; Crystallography, X-Ray; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Flagella; Structure-Activity Relationship

Although oxygen has been reported to regulate biofilm formation by several Shewanella species, the exact regulatory mechanism mostly remains unclear. Here, we identify a direct oxygen-sensing diguanylate cyclase (DosD) and reveal its regulatory role in biofilm formation by Shewanella putrefaciens CN32 under aerobic conditions. In vitro and in vivo analyses revealed that the activity of DosD culminates to synthesis of cyclic diguanylate (c-di-GMP) in the presence of oxygen. DosD regulates the transcription of bpfA operon which encodes seven proteins including a large repetitive adhesin BpfA and its cognate type I secretion system (TISS). Regulation of DosD in aerobic biofilms is heavily dependent on an adhesin BpfA and the TISS. This study offers an insight into the molecular mechanism of oxygen-stimulated biofilm formation by S. putrefaciens CN32.

Topics: Adhesins, Bacterial; Bacterial Proteins; Biofilms; Biosensing Techniques; Blotting, Western; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Operon; Oxygen; Phosphorus-Oxygen Lyases; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Shewanella putrefaciens

Complex 3'-5'-cyclic diguanylic acid (c-di-GMP) responsive regulatory networks that are modulated by the action of multiple diguanylate cyclases (DGC; GGDEF domain proteins) and phosphodiesterases (PDE; EAL domain proteins) have evolved in many bacteria. YfgF proteins possess a membrane-anchoring domain (MASE1), a catalytically inactive GGDEF domain and a catalytically active EAL domain. Here, sustained expression of the Salmonella enterica spp. Enterica ser. Enteritidis YfgF protein is shown to mediate inhibition of the formation of the aspartate chemotactic ring on motility agar under aerobic conditions. This phenomenon was c-di-GMP-independent because it occurred in a Salmonella strain that lacked the ability to synthesize c-di-GMP and also when PDE activity was abolished by site-directed mutagenesis of the EAL domain. YfgF-mediated inhibition of aspartate chemotactic ring formation was impaired in the altered redox environment generated by exogenous p-benzoquinone. This ability of YfgF to inhibit the response to aspartate required a motif, (213)Lys-Lys-Glu(215), in the predicted cytoplasmic loop between trans-membrane regions 5 and 6 of the MASE1 domain. Thus, for the first time the function of a MASE1 domain as a redox-responsive regulator of bacterial responses to aspartate has been shown.

Topics: Amino Acid Motifs; Aspartic Acid; Bacterial Proteins; Benzoquinones; Chemotaxis; Cyclic GMP; Mutagenesis, Site-Directed; Phosphoric Diester Hydrolases; Protein Structure, Tertiary; Salmonella enterica

Topics: Cyclic GMP; Escherichia coli K12; Escherichia coli Proteins

During urinary tract infections (UTIs), uropathogenic Escherichia coli must maintain a delicate balance between sessility and motility to achieve successful infection of both the bladder and kidneys. Previous studies showed that cyclic dimeric GMP (c-di-GMP) levels aid in the control of the transition between motile and nonmotile states in E. coli. The yfiRNB locus in E. coli CFT073 contains genes for YfiN, a diguanylate cyclase, and its activity regulators, YfiR and YfiB. Deletion of yfiR yielded a mutant that was attenuated in both the bladder and the kidneys when tested in competition with the wild-type strain in the murine model of UTI. A double yfiRN mutant was not attenuated in the mouse model, suggesting that unregulated YfiN activity and likely increased cytoplasmic c-di-GMP levels cause a survival defect. Curli fimbriae and cellulose production were increased in the yfiR mutant. Expression of yhjH, a gene encoding a proven phosphodiesterase, in CFT073 ΔyfiR suppressed the overproduction of curli fimbriae and cellulose and further verified that deletion of yfiR results in c-di-GMP accumulation. Additional deletion of csgD and bcsA, genes necessary for curli fimbriae and cellulose production, respectively, returned colonization levels of the yfiR deletion mutant to wild-type levels. Peroxide sensitivity assays and iron acquisition assays displayed no significant differences between the yfiR mutant and the wild-type strain. These results indicate that dysregulation of c-di-GMP production results in pleiotropic effects that disable E. coli in the urinary tract and implicate the c-di-GMP regulatory system as an important factor in the persistence of uropathogenic E. coli in vivo.

Topics: Animals; Bacterial Proteins; Cellulose; Cyclic GMP; Cytoplasm; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Female; Fimbriae, Bacterial; Gene Deletion; Hydrogen Peroxide; Iron; Mice; Phosphorus-Oxygen Lyases; Urinary Tract; Urinary Tract Infections; Urine; Uropathogenic Escherichia coli

Vibrio cholerae senses its environment, including the surrounding bacterial community, using both the second messenger cyclic di-GMP (c-di-GMP) and quorum sensing (QS) to regulate biofilm formation and other bacterial behaviors. Cyclic di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. V. cholerae encodes a complex network of 61 enzymes predicted to mediate changes to the levels of c-di-GMP in response to extracellular signals, and the transcription of many of these enzymes is influenced by QS. Because of the complexity of the c-di-GMP signaling system in V. cholerae, it is difficult to determine if modulation of intracellular c-di-GMP in response to different stimuli is driven primarily by changes in c-di-GMP synthesis or hydrolysis. Here, we describe a novel method, named the ex vivo lysate c-di-GMP assay (TELCA), that systematically measures total DGC and PDE cellular activity. We show that V. cholerae grown in different environments exhibits significantly different intracellular levels of c-di-GMP, and we used TELCA to determine that these differences correspond to changes in both c-di-GMP synthesis and hydrolysis. Furthermore, we show that the increased concentration of c-di-GMP at low cell density is primarily due to increased DGC activity due to the DGC CdgA. Our findings highlight the idea that modulation of both total DGC and PDE activity alters the intracellular concentration of c-di-GMP, and we present a new method that is widely applicable to the systematic analysis of complex c-di-GMP signaling networks.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Signal Transduction; Vibrio cholerae

Shewanella oneidensis MR-1, a gammaproteobacterium with respiratory versatility, forms biofilms on mineral surfaces through a process controlled by the cyclic dinucleotide messenger c-di-GMP. Cellular concentrations of c-di-GMP are maintained by proteins containing GGDEF and EAL domains, which encode diguanylate cyclases for c-di-GMP synthesis and phosphodiesterases for c-di-GMP hydrolysis, respectively. The S. oneidensis MR-1 genome encodes several GGDEF and EAL domain proteins (50 and 31, respectively), with a significant fraction (∼10) predicted to be multidomain (e.g., GGDEF-EAL) enzymes containing an additional Per-Arnt-Sim (PAS) sensor domain. However, the biochemical activities and physiological functions of these multidomain enzymes remain largely unknown. Here, we present genetic and biochemical analyses of a predicted PAS-GGDEF-EAL domain-containing protein, SO0437, here named PdeB. A pdeB deletion mutant exhibited decreased swimming motility and increased biofilm formation under rich growth medium conditions, which was consistent with an increase in intracellular c-di-GMP. A mutation inactivating the EAL domain also produced similar swimming and biofilm phenotypes, indicating that the increase in c-di-GMP was likely due to a loss in phosphodiesterase activity. Therefore, we also examined the enzymatic activity of purified PdeB and found that the protein exhibited phosphodiesterase activity via the EAL domain. No diguanylate cyclase activity was observed. In addition to the motility and biofilm phenotypes, transcriptional profiling by DNA microarray analysis of biofilms of pdeB (in-frame deletion and EAL) mutant cells revealed that expression of genes involved in sulfate uptake and assimilation were repressed. Addition of sulfate to the growth medium resulted in significantly less motile pdeB mutants. Together, these results indicate a link between c-di-GMP metabolism, S. oneidensis MR-1 biofilm development, and sulfate uptake/assimilation.

Topics: Biofilms; Culture Media; Cyclic GMP; Gene Deletion; Gene Expression Profiling; Locomotion; Microarray Analysis; Mutant Proteins; Phosphoric Diester Hydrolases; Protein Structure, Tertiary; Shewanella; Sulfates

As a ubiquitous second messenger, cyclic dimeric GMP (c-di-GMP) has been studied in numerous bacteria. The oral spirochete Treponema denticola, a periodontal pathogen associated with human periodontitis, has a complex c-di-GMP signaling network. However, its function remains unexplored. In this report, a PilZ-like c-di-GMP binding protein (TDE0214) was studied to investigate the role of c-di-GMP in the spirochete. TDE0214 harbors a PilZ domain with two signature motifs: RXXXR and DXSXXG. Biochemical studies showed that TDE0214 binds c-di-GMP in a specific manner, with a dissociation constant (Kd) value of 1.73 μM, which is in the low range compared to those of other reported c-di-GMP binding proteins. To reveal the role of c-di-GMP in T. denticola, a TDE0214 deletion mutant (TdΔ214) was constructed and analyzed in detail. First, swim plate and single-cell tracking analyses showed that TdΔ214 had abnormal swimming behaviors: the mutant was less motile and reversed more frequently than the wild type. Second, we found that biofilm formation of TdΔ214 was substantially repressed (∼6.0-fold reduction). Finally, in vivo studies using a mouse skin abscess model revealed that the invasiveness and ability to induce skin abscesses and host humoral immune responses were significantly attenuated in TdΔ214, indicative of the impact that TDE0214 has on the virulence of T. denticola. Collectively, the results reported here indicate that TDE0214 plays important roles in motility, biofilm formation, and virulence of the spirochete. This report also paves a way to further unveil the roles of the c-di-GMP signaling network in the biology and pathogenicity of T. denticola.

Topics: Abscess; Animals; Biofilms; Carrier Proteins; Cyclic GMP; Disease Models, Animal; Gene Knockout Techniques; Kinetics; Locomotion; Mice; Protein Binding; Protein Structure, Tertiary; Skin Diseases, Bacterial; Treponema denticola; Virulence; Virulence Factors

Many bacteria colonize surfaces and transition to a sessile mode of growth. The plant pathogen Agrobacterium tumefaciens produces a unipolar polysaccharide (UPP) adhesin at single cell poles that contact surfaces. Here we report that elevated levels of the intracellular signal cyclic diguanosine monophosphate (c-di-GMP) lead to surface-contact-independent UPP production and a red colony phenotype due to production of UPP and the exopolysaccharide cellulose, when A. tumefaciens is incubated with the polysaccharide stain Congo Red. Transposon mutations with elevated Congo Red staining identified presumptive UPP-negative regulators, mutants for which were hyperadherent, producing UPP irrespective of surface contact. Multiple independent mutations were obtained in visN and visR, activators of flagellar motility in A. tumefaciens, now found to inhibit UPP and cellulose production. Expression analysis in a visR mutant and isolation of suppressor mutations, identified three diguanylate cyclases inhibited by VisR. Null mutations for two of these genes decrease attachment and UPP production, but do not alter cellular c-di-GMP levels. However, analysis of catalytic site mutants revealed their GGDEF motifs are required to increase UPP production and surface attachment. Mutations in a specific presumptive c-di-GMP phosphodiesterase also elevate UPP production and attachment, consistent with c-di-GMP activation of surface-dependent adhesin deployment.

Topics: Adhesins, Bacterial; Agrobacterium tumefaciens; Bacterial Adhesion; Congo Red; Cyclic GMP; DNA Transposable Elements; Gene Deletion; Gene Expression Regulation, Bacterial; Genes, Bacterial; Locomotion; Mutagenesis, Insertional; Polysaccharides, Bacterial; Staining and Labeling

The ESF-EMBO Conference on 'Bacterial Networks' (BacNet13) was held in March 2013, in Pultusk, Poland. It brought together 164 molecular microbiologists, bacterial systems biologists and synthetic biologists to discuss the architecture, function and dynamics of regulatory networks in bacteria.

Topics: Antibiosis; Bacteria; Cyclic GMP; Gene Regulatory Networks; Microbial Consortia; Quorum Sensing; Signal Transduction

Lack of the Pseudomonas putida PP2258 protein or its overexpression results in defective motility on solid media. The PP2258 protein is tripartite, possessing a PAS domain linked to two domains associated with turnover of c-di-GMP - a cyclic nucleotide that controls the switch between motile and sessile lifestyles. The second messenger c-di-GMP is produced by diguanylate cyclases and degraded by phosphodiesterases containing GGDEF and EAL or HD-GYP domains respectively. It is common for enzymes involved in c-di-GMP signalling to contain two domains with potentially opposing c-di-GMP turnover activities; however, usually one is degenerate and has been adopted to serve regulatory functions. Only a few proteins have previously been found to have dual enzymatic activities - being capable of both synthesizing and hydrolysing c-di-GMP. Here, using truncated and mutant derivatives of PP2258, we show that despite a lack of complete consensus in either the GGDEF or EAL motifs, the two c-di-GMP turnover domains can function independently of each other, and that the diguanylate cyclase activity is regulated by an inhibitory I-site within its GGDEF domain. Thus, motility-associated PP2258 can be added to the short list of bifunctional c-di-GMP signalling proteins.

Topics: Amino Acid Motifs; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Locomotion; Models, Molecular; Mutant Proteins; Protein Conformation; Pseudomonas putida; Sequence Deletion; Signal Transduction

The intracellular level of the ubiquitous bacterial secondary messenger, cyclic di-(3',5')-guanosine monophosphate (c-di-GMP), represents a balance between its biosynthesis and degradation, the latter via specific phosphodiesterases (PDEs). One class of c-di-GMP PDEs contains a characteristic HD-GYP domain. Here we report that an HD-GYP PDE from Vibrio cholerae contains a non-heme diiron-carboxylate active site, and that only the reduced form is active. An engineered D-to-A substitution in the HD dyad caused loss of c-di-GMP PDE activity and of two iron atoms. This report constitutes the first demonstration that a non-heme diiron-carboxylate active site can catalyze the c-di-GMP PDE reaction and that this activity can be redox regulated in the HD-GYP class.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Bacterial Proteins; Catalytic Domain; Cyclic GMP; Heme; Iron; Models, Molecular; Protein Structure, Tertiary; Vibrio cholerae

Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is an intracellular second messenger that regulates adaptation processes, including biofilm formation, motility, and virulence in Gram-negative bacteria. In this study, we have characterized the core components of a c-di-GMP signaling pathway in the model Gram-positive bacterium Bacillus subtilis. Specifically, we have directly identified and characterized three active diguanylate cyclases, DgcP, DgcK, and DgcW (formerly YtrP, YhcK, and YkoW, respectively), one active c-di-GMP phosphodiesterase, PdeH (formerly YuxH), and a cyclic-diguanylate (c-di-GMP) receptor, DgrA (formerly YpfA). Furthermore, elevation of c-di-GMP levels in B. subtilis led to inhibition of swarming motility, whereas biofilm formation was unaffected. Our work establishes paradigms for Gram-positive c-di-GMP signaling, and we have shown that the concise signaling system identified in B. subtilis serves as a powerful heterologous host for the study of c-di-GMP enzymes from bacteria predicted to possess larger, more-complex signaling systems.

Topics: Amino Acid Sequence; Bacillus subtilis; Bacterial Proteins; Cloning, Molecular; Cyclic GMP; Gene Deletion; Gene Expression Regulation, Bacterial; Molecular Structure; Protein Binding; Signal Transduction

C-di-GMP [Bis-(3'-5')-cyclic-dimeric-guanosine monophosphate], a second messenger is involved in intracellular communication in the bacterial species. As a result several multi-cellular behaviors in both Gram-positive and Gram-negative bacteria are directly linked to the intracellular level of c-di-GMP. The cellular concentration of c-di-GMP is maintained by two opposing activities, diguanylate cyclase (DGC) and phosphodiesterase (PDE-A). In Mycobacterium smegmatis, a single bifunctional protein MSDGC-1 is responsible for the cellular concentration of c-di-GMP. A better understanding of the regulation of c-di-GMP at the genetic level is necessary to control the function of above two activities. In this work, we have characterized the promoter element present in msdgc-1 along with the +1 transcription start site and identified the sigma factors that regulate the transcription of msdgc-1. Interestingly, msdgc-1 utilizes SigA during the initial phase of growth, whereas near the stationary phase SigB containing RNA polymerase takes over the expression of msdgc-1. We report here that the promoter activity of msdgc-1 increases during starvation or depletion of carbon source like glucose or glycerol. When msdgc-1 is deleted, the numbers of viable cells are ~10 times higher in the stationary phase in comparison to that of the wild type. We propose here that msdgc-1 is involved in the regulation of cell population density.

Topics: Bacterial Proteins; Base Sequence; beta-Galactosidase; Chromosome Mapping; Culture Media; Cyclic GMP; Enzyme Induction; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Gene Knockout Techniques; Genes, Reporter; Glucose; Glycerol; Molecular Sequence Annotation; Molecular Sequence Data; Mutagenesis, Site-Directed; Mycobacterium smegmatis; Phosphorus-Oxygen Lyases; Point Mutation; Promoter Regions, Genetic; Protein Binding; Sigma Factor; Stress, Physiological; Transcription Initiation Site

This study shows that the expression of yjcC, an in vivo expression (IVE) gene, and the stress response regulatory genes soxR, soxS, and rpoS are paraquat inducible in Klebsiella pneumoniae CG43. The deletion of rpoS or soxRS decreased yjcC expression, implying an RpoS- or SoxRS-dependent control. After paraquat or H2O2 treatment, the deletion of yjcC reduced bacterial survival. These effects could be complemented by introducing the ΔyjcC mutant with the YjcC-expression plasmid pJR1. The recombinant protein containing only the YjcC-EAL domain exhibited phosphodiesterase (PDE) activity; overexpression of yjcC has lower levels of cyclic di-GMP. The yjcC deletion mutant also exhibited increased reactive oxygen species (ROS) formation, oxidation damage, and oxidative stress scavenging activity. In addition, the yjcC deletion reduced capsular polysaccharide production in the bacteria, but increased the LD50 in mice, biofilm formation, and type 3 fimbriae major pilin MrkA production. Finally, a comparative transcriptome analysis showed 34 upregulated and 29 downregulated genes with the increased production of YjcC. The activated gene products include glutaredoxin I, thioredoxin, heat shock proteins, chaperone, and MrkHI, and proteins for energy metabolism (transporters, cell surface structure, and transcriptional regulation). In conclusion, the results of this study suggest that YjcC positively regulates the oxidative stress response and mouse virulence but negatively affects the biofilm formation and type 3 fimbriae expression by altering the c-di-GMP levels after receiving oxidative stress signaling inputs.

Topics: Animals; Bacterial Capsules; Bacterial Proteins; Base Sequence; Biofilms; Cyclic GMP; Down-Regulation; Female; Fimbriae, Bacterial; Gene Deletion; Gene Expression Regulation, Bacterial; Klebsiella Infections; Klebsiella pneumoniae; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Oxidative Stress; Paraquat; Phosphoric Diester Hydrolases; Polysaccharides, Bacterial; Transcriptome; Up-Regulation; Virulence

Microorganisms use a variety of metabolites to respond to external stimuli, including second messengers that amplify primary signals and elicit biochemical changes in a cell. Levels of the second messenger cyclic dimeric GMP (c-di-GMP) are regulated by a variety of environmental stimuli and play a critical role in regulating cellular processes such as biofilm formation and cellular motility. Cyclic di-GMP signaling systems have been largely characterized in pathogenic bacteria; however, proteins that can impact the synthesis or degradation of c-di-GMP are prominent in cyanobacterial species and yet remain largely underexplored. In cyanobacteria, many putative c-di-GMP synthesis or degradation domains are found in genes that also harbor light-responsive signal input domains, suggesting that light is an important signal for altering c-di-GMP homeostasis. Indeed, c-di-GMP-associated domains are often the second most common output domain in photoreceptors-outnumbered only by a histidine kinase output domain. Cyanobacteria differ from other bacteria regarding the number and types of photoreceptor domains associated with c-di-GMP domains. Due to the widespread distribution of c-di-GMP domains in cyanobacteria, we investigated the evolutionary origin of a subset of genes. Phylogenetic analyses showed that c-di-GMP signaling systems were present early in cyanobacteria and c-di-GMP genes were both vertically and horizontally inherited during their evolution. Finally, we compared intracellular levels of c-di-GMP in two cyanobacterial species under different light qualities, confirming that light is an important factor for regulating this second messenger in vivo.. This study shows that many proteins containing cyclic dimeric GMP (c-di-GMP)-regulatory domains in cyanobacteria are associated with photoreceptor domains. Although the functional roles of c-di-GMP domain-containing proteins in cyanobacteria are only beginning to emerge, the abundance of these multidomain proteins in cyanobacteria that occupy diverse habitats ranging from freshwater to marine to soil environments suggests an important role for the regulation of c-di-GMP in these organisms. Indeed, we showed that light distinctly regulates c-di-GMP levels in Fremyella diplosiphon and Synechocystis sp. strain PCC6803. Our findings are consistent with the occurrence of c-di-GMP domains based on evolutionary origin and as an adaptation to specific habitat characteristics. Phylogenetic analyses of these domains clearly separate two distinctive clades, one composed of domains belonging predominantly to cyanobacteria and the other belonging to a mix of cyanobacteria and other bacteria. We further demonstrate that in cyanobacteria the acquisition of c-di-GMP signaling domains occurred both vertically and horizontally.

Topics: Bacterial Proteins; Cyanobacteria; Cyclic GMP; Evolution, Molecular; Gene Expression Regulation; Light; Phylogeny; Signal Transduction

Loss of structural stability of aerobic granular process is the challenge for its field applications to treat wastewaters. The second messenger, cyclic diguanylate (c-di-GMP), is widely used by bacteria to regulate the synthesis of exopolysaccharide. This study for the first time confirmed the correlation between concentration of intracellular c-di-GMP and the granular stability under sequencing batch reactor (MBR) mode. In the presence of manganese ions (Mn(2+)), the concentrations of intracellular c-di-GMP and of extracellular polysaccharides and proteins in granules were declined. Clone library study revealed that the polysaccharide producers. Acinetobacter sp., Thauera sp., Bdellovibrio sp. and Paracoccus sp. were lost after Mn(2+) addition. The findings reported herein confirmed that the c-di-GMP is a key chemical factor epistatic to quorum sensing to determine granular stability. Stimulation of synthesis of intracellular c-di-GMP presents a potential way to enhance long-term stability of aerobic granules.

Topics: Acinetobacter; Aerobiosis; Bacterial Proteins; Bdellovibrio; Bioreactors; Cyclic GMP; Escherichia coli; Gene Expression Regulation, Bacterial; Industrial Waste; Ions; Manganese; Paracoccus; Polysaccharides; Proteins; Quorum Sensing; RNA, Ribosomal, 16S; Second Messenger Systems; Sewage; Thauera; Time Factors; Waste Disposal, Fluid; Wastewater

Topics: Bacillus subtilis; Cyclic GMP; Gene Expression Regulation, Bacterial

Cytosolic DNA sensing via the stimulator of IFN genes (STING) adaptor incites autoimmunity by inducing type I IFN (IFN-αβ). In this study, we show that DNA is also sensed via STING to suppress immunity by inducing IDO. STING gene ablation abolished IFN-αβ and IDO induction by dendritic cells (DCs) after DNA nanoparticle (DNP) treatment. Marginal zone macrophages, some DCs, and myeloid cells ingested DNPs, but CD11b(+) DCs were the only cells to express IFN-β, whereas CD11b(+) non-DCs were major IL-1β producers. STING ablation also abolished DNP-induced regulatory responses by DCs and regulatory T cells, and hallmark regulatory responses to apoptotic cells were also abrogated. Moreover, systemic cyclic diguanylate monophosphate treatment to activate STING induced selective IFN-β expression by CD11b(+) DCs and suppressed Th1 responses to immunization. Thus, previously unrecognized functional diversity among physiologic innate immune cells regarding DNA sensing via STING is pivotal in driving immune responses to DNA.

Topics: Animals; CD11b Antigen; Cyclic GMP; Dendritic Cells; DNA; Epitopes, T-Lymphocyte; Immunity, Innate; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interferon-alpha; Interferon-beta; Male; Membrane Proteins; Mice; Mice, Knockout; Myeloid Cells; Nanoparticles; Th1 Cells

The cyclic dinucleotides 3'-5'diadenylate (c-diAMP) and 3'-5' diguanylate (c-diGMP) are important bacterial second messengers that have recently been shown to stimulate the secretion of type I Interferons (IFN-Is) through the c-diGMP-binding protein MPYS/STING. Here, we show that physiologically relevant levels of cyclic dinucleotides also stimulate a robust secretion of IL-1β through the NLRP3 inflammasome. Intriguingly, this response is independent of MPYS/STING. Consistent with most NLRP3 inflammasome activators, the response to c-diGMP is dependent on the mobilization of potassium and calcium ions. However, in contrast to other NLRP3 inflammasome activators, this response is not associated with significant changes in mitochondrial potential or the generation of mitochondrial reactive oxygen species. Thus, cyclic dinucleotides activate the NLRP3 inflammasome through a unique pathway that could have evolved to detect pervasive bacterial pathogen-associated molecular patterns associated with intracellular infections.

Topics: Animals; Calcium; Carrier Proteins; Cell Line, Tumor; Cyclic GMP; Dinucleoside Phosphates; Humans; Inflammasomes; Interleukin-1beta; Macrophages; Membrane Potential, Mitochondrial; Membrane Proteins; Mice; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Potassium; Reactive Oxygen Species

The Gram-positive obligate anaerobe Clostridium difficile causes potentially fatal intestinal diseases. How this organism regulates virulence gene expression is poorly understood. In many bacterial species, the second messenger cyclic di-GMP (c-di-GMP) negatively regulates flagellar motility and, in some cases, virulence. c-di-GMP was previously shown to repress motility of C. difficile. Recent evidence indicates that flagellar gene expression is tightly linked with expression of the genes encoding the two C. difficile toxins TcdA and TcdB, which are key virulence factors for this pathogen. Here, the effect of c-di-GMP on expression of the toxin genes tcdA and tcdB was determined, and the mechanism connecting flagellar and toxin gene expressions was examined. In C. difficile, increasing c-di-GMP levels reduced the expression levels of tcdA and tcdB, as well as that of tcdR, which encodes an alternative sigma factor that activates tcdA and tcdB expression. We hypothesized that the C. difficile orthologue of the flagellar alternative sigma factor SigD (FliA; σ(28)) mediates regulation of toxin gene expression in response to c-di-GMP. Indeed, ectopic expression of sigD in C. difficile resulted in increased expression levels of tcdR, tcdA, and tcdB. Furthermore, sigD expression enhanced toxin production and increased the cytopathic effect of C. difficile on cultured fibroblasts. Finally, evidence is provided that SigD directly activates tcdR expression and that SigD cannot activate tcdA or tcdB expression independent of TcdR. Taken together, these data suggest that SigD positively regulates toxin genes in C. difficile and that c-di-GMP can inhibit both motility and toxin production via SigD, making this signaling molecule a key virulence gene regulator in C. difficile.

Topics: Bacterial Proteins; Bacterial Toxins; Clostridioides difficile; Cyclic GMP; Enterotoxins; Gene Expression Regulation, Bacterial; Sigma Factor

Many bacteria mediate important life-style decisions by varying levels of the second messenger c-di-GMP. Behavioral transitions result from the coordination of complex cellular processes such as motility, surface adherence or the production of virulence factors and toxins. While the regulatory mechanisms responsible for these processes have been elucidated in some cases, the global pleiotropic effects of c-di-GMP are poorly understood, primarily because c-di-GMP networks are inherently complex in most bacteria. Moreover, the quantitative relationships between cellular c-di-GMP levels and c-di-GMP dependent phenotypes are largely unknown. Here, we dissect the c-di-GMP network of Caulobacter crescentus to establish a global and quantitative view of c-di-GMP dependent processes in this organism. A genetic approach that gradually reduced the number of diguanylate cyclases identified novel c-di-GMP dependent cellular processes and unraveled c-di-GMP as an essential component of C. crescentus cell polarity and its bimodal life cycle. By varying cellular c-di-GMP concentrations, we determined dose response curves for individual c-di-GMP-dependent processes. Relating these values to c-di-GMP levels modeled for single cells progressing through the cell cycle sets a quantitative frame for the successive activation of c-di-GMP dependent processes during the C. crescentus life cycle. By reconstructing a simplified c-di-GMP network in a strain devoid of c-di-GMP we defined the minimal requirements for the oscillation of c-di-GMP levels during the C. crescentus cell cycle. Finally, we show that although all c-di-GMP dependent cellular processes were qualitatively restored by artificially adjusting c-di-GMP levels with a heterologous diguanylate cyclase, much higher levels of the second messenger are required under these conditions as compared to the contribution of homologous c-di-GMP metabolizing enzymes. These experiments suggest that a common c-di-GMP pool cannot fully explain spatiotemporal regulation by c-di-GMP in C. crescentus and that individual enzymes preferentially regulate specific phenotypes during the cell cycle.

Topics: Caulobacter; Cell Cycle; Cell Division; Cell Lineage; Cell Movement; Cyclic GMP; Escherichia coli Proteins; Phosphorus-Oxygen Lyases; Second Messenger Systems

Eukaryotic morphogenesis is seeded with the establishment and subsequent amplification of polarity cues at key times during the cell cycle, often using (cyclic) nucleotide signals. We discovered that flagellum de- and repolarization in the model prokaryote Caulobacter crescentus is precisely orchestrated through at least three spatiotemporal mechanisms integrated at TipF. We show that TipF is a cell cycle-regulated receptor for the second messenger--bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP)--that perceives and transduces this signal through the degenerate c-di-GMP phosphodiesterase (EAL) domain to nucleate polar flagellum biogenesis. Once c-di-GMP levels rise at the G1 → S transition, TipF is activated, stabilized, and polarized, enabling the recruitment of downstream effectors, including flagellar switch proteins and the PflI positioning factor, at a preselected pole harboring the TipN landmark. These c-di-GMP-dependent events are coordinated with the onset of tipF transcription in early S phase and together enable the correct establishment and robust amplification of TipF-dependent polarization early in the cell cycle. Importantly, these mechanisms also govern the timely removal of TipF at cell division coincident with the drop in c-di-GMP levels, thereby resetting the flagellar polarization state in the next cell cycle after a preprogrammed period during which motility must be suspended.

Topics: Amino Acid Sequence; Bacterial Proteins; Caulobacter crescentus; Cell Cycle; Cell Polarity; Cyclic GMP; Enzyme Activation; Flagella; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Protein Binding; Protein Transport; Sequence Alignment; Signal Transduction

In biofilms, the bacterial community optimizes the strategies to sense the environment and to communicate from cell to cell. A key player in the development of a bacterial biofilm is the second messenger c-di-GMP, whose intracellular levels are modulated by the opposite activity of diguanylate cyclases and phosphodiesterases. Given the huge impact of bacterial biofilms on human health, understanding the molecular details of c-di-GMP metabolism represents a critical step in the development of novel therapeutic approaches against biofilms. In this study, we present a detailed biochemical characterization of two c-di-GMP phosphodiesterases of the HD-GYP subtype from the human pathogen Pseudomonas aeruginosa, namely PA4781 and PA4108. Upstream of the catalytic HD-GYP domain, PA4781 contains a REC domain typical of two-component systems, while PA4108 contains an uncharacterized domain of unknown function. Our findings shed light on the activity and catalytic mechanism of these phosphodiesterases. We show that both enzymes hydrolyse c-di-GMP in a two-step reaction via the linear intermediate pGpG and that they produce GMP in vitro at a surprisingly low rate. In addition, our data indicate that the non-phosphorylated REC domain of PA4781 prevents accessibility of c-di-GMP to the active site. Both PA4108 and phosphorylated PA4781 are also capable to use pGpG as an alternative substrate and to hydrolyse it into GMP; the affinity of PA4781 for pGpG is one order of magnitude higher than that for c-di-GMP. These results suggest that these enzymes may not work (primarily) as genuine phosphodiesterases. Moreover, the unexpected affinity of PA4781 for pGpG may indicate that pGpG could also act as a signal molecule in its own right, thus further widening the c-di-GMP-related signalling scenario.

Topics: Bacterial Proteins; Cyclic GMP; Phosphoric Diester Hydrolases; Pseudomonas aeruginosa

Dormancy of Mycobacterium tuberculosis is likely to be a major cause of extended chemotherapeutic regimens and wide prevalence of tuberculosis. The molecular mechanisms underlying M. tuberculosis dormancy are not well understood. In this study, single-copy genes responsible for synthesis (dgc) and degradation (pde) of the ubiquitous bacterial second messenger, cyclic di-GMP (c-di-GMP), were deleted in the virulent M. tuberculosis strain H37Rv to generate dgc(mut) and Δpde, respectively. Under aerobic growth conditions, the two mutants and wild-type cells showed similar phenotypes. However, dgc(mut) and Δpde exhibited increased and reduced dormancy, respectively, in both anaerobiosis-triggered and vitamin C-triggered in vitro dormancy models, as determined by survival and growth recovery from dormancy. The transcriptomes of aerobic cultures of dgc(mut) and wild-type H37Rv exhibited no difference, whereas those of anaerobic cultures showed a significant difference with 61 genes that are not a part of the dosR regulon. Furthermore, Δpde but not dgc(mut) showed decreased infectivity with human THP-1 cells. Δpde also showed attenuated pathogenicity in a C57BL/6 mouse infection model. These findings are explained by c-di-GMP-mediated signaling negatively regulating M. tuberculosis dormancy and pathogenicity.

Topics: Anaerobiosis; Animals; Cyclic GMP; Disease Models, Animal; Gene Deletion; Mice; Mice, Inbred C57BL; Microbial Viability; Mycobacterium tuberculosis; Oxidation-Reduction; Oxygen Consumption; Signal Transduction; Stress, Physiological; Tuberculosis, Pulmonary; Virulence

Bacterial biofilm formation can cause serious problems in clinical and industrial settings, which drives the development or screening of biofilm inhibitors. Some biofilm inhibitors have been screened from natural products or modified from natural compounds. Ginger has been used as a medicinal herb to treat infectious diseases for thousands of years, which leads to the hypothesis that it may contain chemicals inhibiting biofilm formation. To test this hypothesis, we evaluated ginger's ability to inhibit Pseudomonas aeruginosa PA14 biofilm formation. A static biofilm assay demonstrated that biofilm development was reduced by 39-56% when ginger extract was added to the culture. In addition, various phenotypes were altered after ginger addition of PA14. Ginger extract decreased production of extracellular polymeric substances. This finding was confirmed by chemical analysis and confocal laser scanning microscopy. Furthermore, ginger extract formed noticeably less rugose colonies on agar plates containing Congo red and facilitated swarming motility on soft agar plates. The inhibition of biofilm formation and the altered phenotypes appear to be linked to a reduced level of a second messenger, bis-(3'-5')-cyclic dimeric guanosine monophosphate. Importantly, ginger extract inhibited biofilm formation in both Gram-positive and Gram-negative bacteria. Also, surface biofilm cells formed with ginger extract detached more easily with surfactant than did those without ginger extract. Taken together, these findings provide a foundation for the possible discovery of a broad spectrum biofilm inhibitor.

Topics: Biofilms; Congo Red; Cyclic GMP; Microscopy, Confocal; Plant Extracts; Pseudomonas aeruginosa; Zingiber officinale

Cyclic di-GMP (c-di-GMP) controls the transition between sessility and motility in many bacterial species. This regulation is achieved by a variety of mechanisms including alteration of transcription initiation and inhibition of flagellar function. How c-di-GMP inhibits the motility of Vibrio cholerae has not been determined. FlrA, a homologue of the c-di-GMP binding Pseudomonas aeruginosa motility regulator FleQ, is the master regulator of the V. cholerae flagellar biosynthesis regulon. Here we show that binding of c-di-GMP to FlrA abrogates binding of FlrA to the promoter of the flrBC operon, deactivating expression of the flagellar biosynthesis regulon. FlrA does not regulate expression of extracellular Vibrio polysaccharide (VPS) synthesis genes. Mutation of the FlrA amino acids R135 and R176 to histidine abrogates binding of c-di-GMP to FlrA, rendering FlrA active in the presence of high levels of c-di-GMP. Surprisingly, c-di-GMP still inhibited the motility of V. cholerae only expressing the c-di-GMP blind FlrA(R176H) mutant. We determined that this flagellar transcription-independent inhibition is due to activation of VPS production by c-di-GMP. Therefore, c-di-GMP prevents motility of V. cholerae by two distinct but functionally redundant mechanisms.

Topics: Arginine; Bacterial Proteins; Binding Sites; Cyclic GMP; Flagella; Gene Expression Regulation, Bacterial; Histidine; Models, Molecular; Movement; Mutation; Operon; Polysaccharides, Bacterial; Protein Conformation; Protein Structure, Tertiary; Transcription, Genetic; Vibrio cholerae

The transcription factor FleQ is a bacterial AAA+ ATPase enhancer-binding protein that is the master activator of flagella gene expression in the opportunistic bacterial pathogen Pseudomonas aeruginosa. Homologs of FleQ are present in all Pseudomonas species and in many polarly flagellated gamma proteobacteria. Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that controls the transition between planktonic and biofilm modes of growth in bacteria in response to diverse environmental signals. C-di-GMP binds to FleQ to dampen its activity, causing down-regulation of flagella gene expression. This action is potentiated in the simultaneous presence of another protein, FleN. We explored the effect of c-di-GMP and FleN on the ATPase activity of FleQ and found that a relatively low concentration of c-di-GMP competitively inhibited FleQ ATPase activity, suggesting that c-di-GMP competes with ATP for binding to the Walker A motif of FleQ. Confirming this, a FleQ Walker A motif mutant failed to bind c-di-GMP. FleN, whose gene is regulated by FleQ, also inhibited FleQ ATPase activity, and FleQ ATPase activity was much more inhibited by c-di-GMP in the presence of FleN than in its absence. These results indicate that FleN and c-di-GMP cooperate to inhibit FleQ activity and, by extension, flagella synthesis in P. aeruginosa. The Walker A motif of FleQ is perfectly conserved, opening up the possibility that other AAA+ ATPases may respond to c-di-GMP.

Topics: Amino Acid Motifs; Bacterial Proteins; Chromatography, Gel; Cyclic GMP; Electrophoresis, Polyacrylamide Gel; Flagella; Gene Expression Regulation, Bacterial; Mutagenesis; Pseudomonas aeruginosa; Trans-Activators

The STING (stimulator of interferon genes) protein can bind cyclic dinucleotides to activate the production of type I interferons and inflammatory cytokines. The cyclic dinucleotides can be bacterial second messengers c-di-GMP and c-di-AMP, 3'5'-3'5' cyclic GMP-AMP (3'3' cGAMP) produced by Vibrio cholerae and metazoan second messenger 2'5'-3'5' Cyclic GMP-AMP (2'3' cGAMP). Analysis of single nucleotide polymorphism (SNP) data from the 1000 Genome Project revealed that R71H-G230A-R293Q (HAQ) occurs in 20.4%, R232H in 13.7%, G230A-R293Q (AQ) in 5.2%, and R293Q in 1.5% of human population. In the absence of exogenous ligands, the R232H, R293Q and AQ SNPs had only modest effect on the stimulation of IFN-β and NF-κB promoter activities in HEK293T cells, while HAQ had significantly lower intrinsic activity. The decrease was primarily due to the R71H substitution. The SNPs also affected the response to the cyclic dinucleotides. In the presence of c-di-GMP, the R232H variant partially decreased the ability to activate IFN-βsignaling, while it was defective for the response to c-di-AMP and 3'3' cGAMP. The R293Q dramatically decreased the stimulatory response to all bacterial ligands. Surprisingly, the AQ and HAQ variants maintained partial abilities to activate the IFN-β signaling in the presence of ligands due primarily to the G230A substitution. Biochemical analysis revealed that the recombinant G230A protein could affect the conformation of the C-terminal domain of STING and the binding to c-di-GMP. Comparison of G230A structure with that of WT revealed that the conformation of the lid region that clamps onto the c-di-GMP was significantly altered. These results suggest that hSTING variation can affect innate immune signaling and that the common HAQ haplotype expresses a STING protein with reduced intrinsic signaling activity but retained the ability to response to bacterial cyclic dinucleotides.

Topics: Amino Acid Sequence; Blotting, Western; Calorimetry, Differential Scanning; Cyclic GMP; HEK293 Cells; Humans; Immunity, Innate; Interferon Regulatory Factor-3; Interferon Type I; Luciferases; Membrane Proteins; Molecular Sequence Data; NF-kappa B; Phosphorylation; Phylogeny; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Protein Conformation; Sequence Homology, Amino Acid; Signal Transduction

Pseudomonas aeruginosa is responsible for a plethora of biofilm mediated chronic infections among which cystic fibrosis pneumonia is the most frightening. The long-term survival strategy of P. aeruginosa in the patients lungs is based on a fine balance of virulence vs dormant states and on genetic adaptation, in order to select persistent phenotypes as the small colony variants (SCVs), which strongly correlate with antibiotic resistance and poor lung function. Recent studies have coupled SCV with increased levels of the signaling molecule cyclic di-GMP, and demonstrated the central role of the diguanylate cyclase YfiN, part of the tripartite signaling module YifBNR, in c-di-GMP dependent SCV regulation. YfiN, also called TpbB, is a multi-domain membrane enzyme connecting periplasmic stimuli to cytosolic c-di-GMP production by an allosteric inside-out signaling mechanism that, due to the lack of structural data, is still largely hypothetical. We have solved the crystal structure of the catalytic domain (GGDEF), and measured the enzymatic activity of the cytosolic portion in real-time by means of a newly developed method. Based on these results we demonstrate that, unlike other diguanylate cyclase, YfiN does not undergo product feedback inhibition, and that the presence of the HAMP domain is required for dimerization and catalysis. Coupling our structural and kinetic data with an in silico study we are now able to propose a model for the allosteric regulation of YfiN.

Topics: Allosteric Regulation; Amino Acid Sequence; Bacterial Proteins; Binding Sites; Catalytic Domain; Conserved Sequence; Cyclic GMP; Kinetics; Models, Molecular; Molecular Sequence Data; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; Protein Multimerization; Pseudomonas aeruginosa; Sequence Alignment

Individual cell heterogeneity is commonly observed within populations, although its molecular basis is largely unknown. Previously, using FRET-based microscopy, we observed heterogeneity in cellular c-di-GMP levels. In this study, we show that c-di-GMP heterogeneity in Pseudomonas aeruginosa is promoted by a specific phosphodiesterase partitioned after cell division. We found that subcellular localization and reduction of c-di-GMP levels by this phosphodiesterase is dependent on the histidine kinase component of the chemotaxis machinery, CheA, and its phosphorylation state. Therefore, individual cell heterogeneity in c-di-GMP concentrations is regulated by the activity and the asymmetrical inheritance of the chemotaxis organelle after cell division. c-di-GMP heterogeneity results in a diversity of motility behaviors. The generation of diverse intracellular concentrations of c-di-GMP by asymmetric partitioning is likely important to the success and survival of bacterial populations within the environment by allowing a variety of motility behaviors. DOI: http://dx.doi.org/10.7554/eLife.01402.001.

Topics: Chemotaxis; Cyclic GMP; Flagella; Phosphorylation; Pseudomonas aeruginosa

To understand the regulatory mechanism by cyclic diguanylate (c-di-GMP) receptor and transcriptional regulator Clpxoo of expression of endoglucanase gene (engAxoo) in Xanthomonas oryzae pv. oryzae, the bacterial leaf blight pathogen of rice.. A plasmid to expressclpxoo gene was constructed and transformed into Escherichia coli for expression by isopropylthio-beta-D-galactoside (IPTG) induction. The recombinant protein was purified by Ni-NTA resin. The binding affinity between purified Clpxoo protein and the promoter of endoglucanase gene (engAxoo-p) was determined by electrophoretic mobility shift assay using fluoresce in (FAM)-labeled probes. The role of c-di-GMP on the binding was also examined.. Under the optimized conditions, Clpxoo was expressed and purified successfully. Mobility shift of engAxoo-p in the presence of Clpxoo was observed, indicating that specific binding occurred between them. Moreover, addition of c-di-GMP molecules in the above reaction system abolished such binding.. Once interacting with the signal molecule c-di-GMP, Clpxoo conformational structure may change substantially, which results in inhibition of binding to engAxoo-p; The optimized methods for Clpxoo protein purification and electrophoretic mobility shift assay (EMSA) can be used for subsequent identification of Clp regulon in a larger scale.

Topics: Cellulase; Cloning, Molecular; Cyclic AMP Receptor Protein; Cyclic GMP; Electrophoretic Mobility Shift Assay; Promoter Regions, Genetic; Recombinant Proteins; Xanthomonas

Vibrio alginolyticus, a Gram-negative marine bacterium, has brought about severe economic damage to the mariculture industry by causing vibriosis in various fish species. We are intrigued in the regulation of the pathogenesis in this bacterium. Here, we reported a complex regulatory connection among the newly defined type VI secretion system (T6SS), quorum sensing (QS), and 3',5'-cyclic diguanylic acid (c-di-GMP) signal through the phosphatase PppA encoded in the T6SS gene cluster of V. alginolyticus. Whole-genome transcriptome analysis revealed various regulatory targets of PppA including the T6SS substrate hemolysin coregulated protein (Hcp), quorum sensing regulator LuxR, exotoxin alkaline serine protease (Asp), flagellar proteins, as well as proteins involved in polysaccharide biosynthesis and transport. Western blot analysis showed PppA served as a negative regulator of the expression and secretion of Hcp1. Mutation of pppA resulted in an increased level of the intracellular second messenger c-di-GMP and a decreased expression of the QS regulator LuxR as well as exotoxin Asp. Complementation of intact pppA gene in ΔpppA mutant restored the production of c-di-GMP, LuxR, and Asp to the wild-type level. Phenotypic studies suggested that PppA takes part in the modulation of biofilm formation, motility, and cell aggregation. These results demonstrated new roles of PppA in controlling virulence factors and pleiotropic phenotypes and contributed to our understanding of the regulation of pathogenesis in V. alginolyticus.

Topics: Animals; Bacterial Proteins; Bacterial Secretion Systems; Cyclic GMP; Endopeptidases; Fishes; Gene Expression Regulation, Bacterial; Hemolysin Proteins; Phosphoprotein Phosphatases; Quorum Sensing; Serine Endopeptidases; Vibrio alginolyticus; Vibrio Infections; Virulence Factors

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen that resists the acidic gastric environment, colonizes the gut epithelium, and causes hemorrhagic colitis and hemolytic-uremic syndrome, especially in children. The genomic island OI-47 of E. coli O157:H7 contains a gene, z1528, encoding an EAL-domain protein potentially involved in c-di-GMP hydrolysis that is absent in non-pathogenic E. coli. This gene, designated vmpA, is co-transcribed with ycdT, which is present in non pathogenic E. coli and encodes a diguanylate cyclase involved in c-di-GMP synthesis. To test for vmpA function, we constructed a vmpA knockout mutant. We also overexpressed vmpA, purified the VmpA protein and assayed for its activity in vitro. We found that VmpA possesses c-di-GMP phosphodiesterase activity and that the vmpA mutation results in increased biofilm formation, and reduced swimming motility, which is consistent with the function determined in vitro. Unexpectedly, suppressor mutations arise frequently in the vmpA background suggesting that VmpA plays an important regulatory role in E. coli O157:H7. These findings represent an example of remarkable flexibility in the organization of c-di-GMP signaling pathways in closely related species.

Topics: Amino Acid Sequence; Biofilms; Cell Movement; Cyclic GMP; Escherichia coli O157; Genomic Islands; Molecular Sequence Data; Mutagenesis, Site-Directed; Nucleic Acid Hybridization; Phosphoric Diester Hydrolases; Reverse Transcriptase Polymerase Chain Reaction; RNA; Sequence Alignment

Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of morbidity and mortality due to diarrheal illness in developing countries. There is currently no effective vaccine against these important pathogens. Because genes modulated by pathogen-host interactions potentially encode putative vaccine targets, we investigated changes in gene expression and surface morphology of ETEC upon interaction with intestinal epithelial cells in vitro. Pan-genome microarrays, quantitative reverse transcriptase PCR (qRT-PCR), and transcriptional reporter fusions of selected promoters were used to study changes in ETEC transcriptomes. Flow cytometry, immunofluorescence microscopy, and scanning electron microscopy were used to investigate alterations in surface antigen expression and morphology following pathogen-host interactions. Following host cell contact, genes for motility, adhesion, toxin production, immunodominant peptides, and key regulatory molecules, including cyclic AMP (cAMP) receptor protein (CRP) and c-di-GMP, were substantially modulated. These changes were accompanied by visible changes in both ETEC architecture and the expression of surface antigens, including a novel highly conserved adhesin molecule, EaeH. The studies reported here suggest that pathogen-host interactions are finely orchestrated by ETEC and are characterized by coordinated responses involving the sequential deployment of multiple virulence molecules. Elucidation of the molecular details of these interactions could highlight novel strategies for development of vaccines for these important pathogens.

Topics: Adhesins, Bacterial; Antigens, Surface; Caco-2 Cells; Cell Line, Tumor; Cyclic GMP; DNA-Binding Proteins; Enterotoxigenic Escherichia coli; Epithelial Cells; Escherichia coli Infections; Escherichia coli Proteins; Gene Expression; Host-Pathogen Interactions; Humans; Intestinal Mucosa; Intestines; Promoter Regions, Genetic; Receptors, Cyclic AMP; Transcription, Genetic; Transcriptome; Virulence

Cyclic di-GMP (c-di-GMP) is a secondary messenger that controls a variety of cellular processes, including the switch between a biofilm and a planktonic bacterial lifestyle. This nucleotide binds to cellular effectors in order to exert its regulatory functions. In Salmonella, two proteins, BcsA and YcgR, both of them containing a c-di-GMP binding PilZ domain, are the only known c-di-GMP receptors. BcsA, upon c-di-GMP binding, synthesizes cellulose, the main exopolysaccharide of the biofilm matrix. YcgR is dedicated to c-di-GMP-dependent inhibition of motility through its interaction with flagellar motor proteins. However, previous evidences indicate that in the absence of YcgR, there is still an additional element that mediates motility impairment under high c-di-GMP levels. Here we have uncovered that cellulose per se is the factor that further promotes inhibition of bacterial motility once high c-di-GMP contents drive the activation of a sessile lifestyle. Inactivation of different genes of the bcsABZC operon, mutation of the conserved residues in the RxxxR motif of the BcsA PilZ domain, or degradation of the cellulose produced by BcsA rescued the motility defect of ΔycgR strains in which high c-di-GMP levels were reached through the overexpression of diguanylate cyclases. High c-di-GMP levels provoked cellulose accumulation around cells that impeded flagellar rotation, probably by means of steric hindrance, without affecting flagellum gene expression, exportation, or assembly. Our results highlight the relevance of cellulose in Salmonella lifestyle switching as an architectural element that is both essential for biofilm development and required, in collaboration with YcgR, for complete motility inhibition.

Topics: Alcohol Oxidoreductases; Bacterial Proteins; Cellulose; Cyclic GMP; Flagella; Gene Expression Regulation, Bacterial; Movement; Polysaccharides, Bacterial; Rotation; Salmonella enteritidis; Salmonella typhimurium; Signal Transduction

Highly pathogenic avian influenza A/H5N1 virus remains a potential pandemic threat, and it is essential to continue vaccine development against this subtype. A local mucosal immune response in the upper respiratory tract may stop influenza transmission. It is therefore important to develop effective intranasal pandemic influenza vaccines that induce mucosal immunity at the site of viral entry.. We evaluated the humoral and cellular immune responses of two promising mucosal adjuvants (Chitosan and c-di-GMP) for intranasal influenza H5N1 vaccine in a murine model. Furthermore, we evaluated the concept of co-adjuvanting an experimental adjuvant (c-di-GMP) with chitosan.. BALB/c mice were intranasally immunised with two doses of subunit NIBRG-14 (H5N1) vaccine (7·5, 1·5 or 0·3 μg haemagglutinin (HA) adjuvanted with chitosan (CSN), c-di-GMP or both adjuvants.. All adjuvant formulations improved the serum and local antibody responses, with the highest responses observed in the 7·5 μg HA CSN and c-di-GMP-adjuvanted groups. The c-di-GMP provided dose sparing with protective single radial haemolysis (SRH), and haemagglutination inhibition (HI) antibody responses found in the 0·3 μg HA group. CSN elicited a Th2 response, whereas c-di-GMP induced higher frequencies of virus-specific CD4+T cells producing one or more Th1 cytokines (IFN-γ+, IL-2+, TNF-α+). A combination of the two adjuvants demonstrated effectiveness at 7·5 μg HA and triggered a more balanced Th cytokine profile.. These data show that combining adjuvants can modulate the Th response and in combination with ongoing studies of adjuvanted intranasal vaccines will dictate the way forward for optimal mucosal influenza vaccines.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animal Experimentation; Animals; Antibodies, Viral; CD4-Positive T-Lymphocytes; Chitosan; Cyclic GMP; Cytokines; Female; Hemagglutination Inhibition Tests; Influenza A Virus, H5N1 Subtype; Influenza Vaccines; Mice; Mice, Inbred BALB C; Vaccination

In many bacterial pathogens, the second messenger c-di-GMP stimulates the production of an exopolysaccharide (EPS) matrix to shield bacteria from assaults of the immune system. How c-di-GMP induces EPS biogenesis is largely unknown. Here, we show that c-di-GMP allosterically activates the synthesis of poly-β-1,6-N-acetylglucosamine (poly-GlcNAc), a major extracellular matrix component of Escherichia coli biofilms. C-di-GMP binds directly to both PgaC and PgaD, the two inner membrane components of the poly-GlcNAc synthesis machinery to stimulate their glycosyltransferase activity. We demonstrate that the PgaCD machinery is a novel type c-di-GMP receptor, where ligand binding to two proteins stabilizes their interaction and promotes enzyme activity. This is the first example of a c-di-GMP-mediated process that relies on protein-protein interaction. At low c-di-GMP concentrations, PgaD fails to interact with PgaC and is rapidly degraded. Thus, when cells experience a c-di-GMP trough, PgaD turnover facilitates the irreversible inactivation of the Pga machinery, thereby temporarily uncoupling it from c-di-GMP signalling. These data uncover a mechanism of c-di-GMP-mediated EPS control and provide a frame for c-di-GMP signalling specificity in pathogenic bacteria.

Topics: Allosteric Regulation; beta-Glucans; Biofilms; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Extracellular Matrix Proteins; Glycosyltransferases; Immunoblotting; Immunoprecipitation; Models, Molecular; Polysaccharides, Bacterial; Second Messenger Systems

Topics: Bacterial Proteins; Cellulose; Cyclic GMP; Salmonella enteritidis; Salmonella typhimurium

Cellulose, the most abundant biological macromolecule, is an extracellular, linear polymer of glucose molecules. It represents an essential component of plant cell walls but is also found in algae and bacteria. In bacteria, cellulose production frequently correlates with the formation of biofilms, a sessile, multicellular growth form. Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the membrane-integrated catalytic BcsA subunit and the membrane-anchored, periplasmic BcsB protein. Here we present the crystal structure of a complex of BcsA and BcsB from Rhodobacter sphaeroides containing a translocating polysaccharide. The structure of the BcsA-BcsB translocation intermediate reveals the architecture of the cellulose synthase, demonstrates how BcsA forms a cellulose-conducting channel, and suggests a model for the coupling of cellulose synthesis and translocation in which the nascent polysaccharide is extended by one glucose molecule at a time.

Topics: Amino Acid Sequence; Bacterial Proteins; Biocatalysis; Biological Transport; Catalytic Domain; Cell Membrane; Cellulose; Crystallography, X-Ray; Cyclic GMP; Enzyme Activation; Models, Molecular; Multiprotein Complexes; Polysaccharides; Protein Structure, Tertiary; Rhodobacter

The bacterial second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) controls important biological processes such as biofilm formation, virulence response, and motility. This second messenger is sensed by macromolecular targets inside the cell, both protein and RNA, which induce specific phenotypic responses critical for bacterial survival. One class of enzymes responsible for regulating the intracellular concentration of c-di-GMP, and therefore the physiological behavior of the cell, consists of the EAL domain phosphodiesterases, which degrade the second messenger to its linear form, pGpG. Here, we investigate how base and backbone modifications of c-di-GMP affect the rate of cyclic dinucleotide degradation by an EAL domain protein (CC3396 from Caulobacter crescentus). The doubly substituted thiophosphate analogue is highly resistant to hydrolysis by this metabolizing enzyme but can still bind c-di-GMP riboswitch targets. We used these findings to develop a novel ribosyl phosphate-modified derivative of c-di-GMP containing 2'-deoxy and methylphosphonate substitutions that is charge neutral and demonstrate that this analogue is also resistant to EAL domain-catalyzed degradation. This suggests a general strategy for designing c-di-GMP derivatives with increased enzymatic stability that also possess desirable properties for development as chemical probes of c-di-GMP signaling.

Topics: Bacterial Proteins; Caulobacter crescentus; Cyclic GMP; Hydrolysis; Models, Molecular; Phosphates; Phosphoric Diester Hydrolases; Protein Structure, Tertiary; Ribose; Riboswitch; Second Messenger Systems; Substrate Specificity

How diversity evolves and persists in biofilms is essential for understanding much of microbial life, including the uncertain dynamics of chronic infections. We developed a biofilm model enabling long-term selection for daily adherence to and dispersal from a plastic bead in a test tube. Focusing on a pathogen of the cystic fibrosis lung, Burkholderia cenocepacia, we sequenced clones and metagenomes to unravel the mutations and evolutionary forces responsible for adaptation and diversification of a single biofilm community during 1,050 generations of selection. The mutational patterns revealed recurrent evolution of biofilm specialists from generalist types and multiple adaptive alleles at relatively few loci. Fitness assays also demonstrated strong interference competition among contending mutants that preserved genetic diversity. Metagenomes from five other independently evolved biofilm lineages revealed extraordinary mutational parallelism that outlined common routes of adaptation, a subset of which was found, surprisingly, in a planktonic population. These mutations in turn were surprisingly well represented among mutations that evolved in cystic fibrosis isolates of both Burkholderia and Pseudomonas. These convergent pathways included altered metabolism of cyclic diguanosine monophosphate, polysaccharide production, tricarboxylic acid cycle enzymes, global transcription, and iron scavenging. Evolution in chronic infections therefore may be driven by mutations in relatively few pathways also favored during laboratory selection, creating hope that experimental evolution may illuminate the ecology and selective dynamics of chronic infections and improve treatment strategies.

Topics: Bacterial Adhesion; Base Sequence; Biofilms; Burkholderia cenocepacia; Burkholderia Infections; Chronic Disease; Cyclic GMP; Cystic Fibrosis; Directed Molecular Evolution; DNA, Bacterial; Ecosystem; Genome, Bacterial; Humans; Lung Diseases; Mannose; Metagenome; Mutation; Opportunistic Infections; Phylogeny; Selection, Genetic

In Burkholderia cenocepacia, the second messenger cyclic diguanosine monophosphate (c-di-GMP) has previously been shown to positively regulate biofilm formation and the expression of cellulose and type-I fimbriae genes through binding to the transcriptional regulator Bcam1349. Here, we provide evidence that cellulose and type-I fimbriae are not involved in B. cenocepacia biofilm formation in flow chambers, and we identify a novel Bcam1349/c-di-GMP-regulated exopolysaccharide gene cluster which is essential for B. cenocepacia biofilm formation. Overproduction of Bcam1349 in trans promotes wrinkly colony morphology, pellicle, and biofilm formation in B. cenocepacia. A screen for transposon mutants unable to respond to the overproduction of Bcam1349 led to the identification of a 12-gene cluster, Bcam1330-Bcam1341, the products of which appear to be involved in the production of a putative biofilm matrix exopolysaccharide and to be essential for flow-chamber biofilm formation. We demonstrate that Bcam1349 binds to the promoter region of genes in the Bcam1330-Bcam1341 cluster and that this binding is enhanced by the presence of c-di-GMP. Furthermore, we demonstrate that overproduction of both c-di-GMP and Bcam1349 leads to increased transcription of these genes, indicating that c-di-GMP and Bcam1349 functions together in regulating exopolysaccharide production from the Bcam1330-Bcam1341 gene cluster. Our results suggest that the product encoded by the Bcam1330-Bcam1341 gene cluster is a major exopolysaccharide that provides structural stability to the biofilms formed by B. cenocepacia, and that its production is regulated by c-di-GMP through binding to and promotion of the activity of the transcriptional regulator Bcam1349.

Topics: Biofilms; Biosynthetic Pathways; Burkholderia cenocepacia; Cyclic GMP; DNA Transposable Elements; DNA, Bacterial; Gene Expression; Gene Expression Regulation, Bacterial; Multigene Family; Mutagenesis, Insertional; Polysaccharides, Bacterial; Promoter Regions, Genetic; Protein Binding; Transcription Factors

Cyclic di-GMP (c-di-GMP) has emerged as a prominent intracellular messenger that coordinates biofilm formation and pathogenicity in many bacterial species. Developing genetically encoded biosensors for c-di-GMP will help us understand how bacterial cells respond to environmental changes via the modulation of cellular c-di-GMP levels. Here we report the design of two genetically encoded c-di-GMP fluorescent biosensors with complementary dynamic ranges. By using the biosensors, we found that several compounds known to promote biofilm dispersal trigger a decline in c-di-GMP levels in Escherichia coli cells. In contrast, cellular c-di-GMP levels were elevated when the bacterial cells were treated with subinhibitory concentrations of biofilm-promoting antibiotics. The biosensors also revealed that E. coli cells engulfed by macrophages exhibit lower c-di-GMP levels, most likely as a response to the enormous pressures of survival during phagocytosis.

Topics: Biosensing Techniques; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Fluorescent Dyes; Molecular Structure

In some Proteobacteria and Firmicutes such as Pseudomonas aeruginosa, Vibrio cholerae, Xanthomonas campestris, and Clostridium difficile, cyclic dimeric guanosine monophosphate (c-di-GMP) is known to regulate cellular processes, including motility, biofilm formation, and virulence, as a second messenger. Cellulose production in Acetobacter xylinum, a model organism of cellulose biosynthesis, also depends on by cellular c-di-GMP level. In cellulose-synthesizing bacteria, a terminal complex localized in the cell membrane synthesizes cellulose and regulates the production of cellulose sensed by c-di-GMP. Although previous studies indicated that the PilZ domain conserved in cellulose synthase subunit A (CeSA) was part of a receptor for c-di-GMP, the recognition mechanism by PilZ domain of CeSA remains unclear. In the present study, we studied the interaction between c-di-GMP and the PilZ domain of CeSA from a structural viewpoint. First, we solved the crystal structure of the PilZ domain of CeSA from A. xylinum (AxCeSA-PilZ) at 2.1Å resolution. Then, comparison of the sequence and structure of AxCeSA-PilZ to those of known structures of PilZ, such as VCA0042, PP4397, and PA4608, indicated the involvement of Lys573 and Arg643 of AxCeSA-PilZ in the recognition of c-di-GMP besides the RxxxR motif. Finally, the binding characteristics of c-di-GMP to AxCeSA-PilZ and mutants were determined with isothermal titration calorimetry, indicating that the residues corresponding to Lys573 and Arg643 in AxCeSA-PilZ generally contribute to the binding of c-di-GMP to PilZ.

Topics: Alanine; Amino Acid Substitution; Crystallography, X-Ray; Cyclic GMP; Gluconacetobacter xylinus; Glucosyltransferases; Models, Chemical; Protein Binding; Protein Structure, Tertiary

Topics: Allosteric Regulation; Biofilms; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Extracellular Matrix Proteins; Polysaccharides, Bacterial; Second Messenger Systems

Bacterial signaling networks control a wide variety of cellular processes including growth, metabolism, and pathogenesis. Bis-(3'-5')-cyclic dimeric guanosine monophosphate (cdiGMP) is a secondary signaling nucleotide that controls cellulose synthesis, biofilm formation, motility and virulence in a wide range of gram-negative bacterial species. CdiGMP is a dynamic molecule that forms different tertiary structures in vitro, including a trans-monomer, cis-monomer, cis-dimer and G-octaplex (G8). Although the monomer and dimer have been shown to be physiologically relevant in modulating protein activity and transcription, the biological effects of the cdiGMP G8 has not yet been described. Here, we have developed a TLC-based assay to detect radiolabeled cdiGMP G8 formation. Utilizing the radiolabeled cdiGMP G8, we have also shown a novel inhibitory interaction between the cdiGMP G8 and HIV-1 reverse transcriptase and that the cdiGMP G8 does not interact with proteins from Pseudomonas aeruginosa known to bind monomeric and dimeric cdiGMP. These results suggest that the radiolabeled cdiGMP G8 can be used to measure interactions between the cdiGMP G8 and cellular proteins, providing an avenue through which the biological significance of this molecule could be investigated.

Topics: Bacterial Proteins; Chromatography, Thin Layer; Cyclic GMP; HIV Reverse Transcriptase; HIV-1; Phosphorus Radioisotopes; Polymerization; Protein Binding; Protein Structure, Tertiary; Pseudomonas aeruginosa; Signal Transduction; Solutions; Staining and Labeling; Stereoisomerism

Accurate RNA structure modeling is an important, incompletely solved, challenge. Single-nucleotide resolution SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) yields an experimental measurement of local nucleotide flexibility that can be incorporated as pseudo-free energy change constraints to direct secondary structure predictions. Prior work from our laboratory has emphasized both the overall accuracy of this approach and the need for nuanced interpretation of modeled structures. Recent studies by Das and colleagues [Kladwang, W., et al. (2011) Biochemistry 50, 8049; Nat. Chem. 3, 954], focused on analyzing six small RNAs, yielded poorer RNA secondary structure predictions than expected on the basis of prior benchmarking efforts. To understand the features that led to these divergent results, we re-examined four RNAs yielding the poorest results in this recent work: tRNA(Phe), the adenine and cyclic-di-GMP riboswitches, and 5S rRNA. Most of the errors reported by Das and colleagues reflected nonstandard experiment and data processing choices, and selective scoring rules. For two RNAs, tRNA(Phe) and the adenine riboswitch, secondary structure predictions are nearly perfect if no experimental information is included but were rendered inaccurate by the SHAPE data of Das and colleagues. When best practices were used, single-sequence SHAPE-directed secondary structure modeling recovered ~93% of individual base pairs and >90% of helices in the four RNAs, essentially indistinguishable from the results of the mutate-and-map approach with the exception of a single helix in the 5S rRNA. The field of experimentally directed RNA secondary structure prediction is entering a phase focused on the most difficult prediction challenges. We outline five constructive principles for guiding this field forward.

Topics: Acylation; Bacterial Proteins; Base Sequence; Cyclic GMP; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Ribonuclease P; Riboswitch; RNA-Directed DNA Polymerase; RNA, Bacterial; RNA, Ribosomal, 5S; RNA, Transfer, Phe; Staining and Labeling; Thermodynamics

Bacteria react to adverse environmental stimuli by clustering into organized communities called biofilms. A remarkably sophisticated control system based on the dinucleotide 3'-5' cyclic diguanylic acid (c-di-GMP) is involved in deciding whether to form or abandon biofilms. The ability of c-di-GMP to form self-intercalated dimers is also thought to play a role in this complex regulation. A great advantage in the quest of elucidating the catalytic properties of the enzymes involved in c-di-GMP turnover (diguanylate cyclases and phosphodiesterases) would come from the availability of an experimental approach for in vitro quantification of c-di-GMP in real-time. Here, we show that c-di-GMP can be detected and quantified by circular dichroism (CD) spectroscopy in the low micromolar range. The method is based on the selective ability of manganese ions to induce formation of the intercalated dimer of the c-di-GMP dinucleotide in solution, which displays an intense sigmoidal CD spectrum in the near-ultraviolet region. This characteristic spectrum originates from the stacking interaction of the four mutually intercalated guanines, as it is absent in the other cyclic dinucleotide 3'-5' cyclic adenilic acid (c-di-AMP). Thus, near-ultraviolet CD can be used to effectively quantify in real-time the activity of diguanylate cyclases and phosphodiesterases in solution.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Circular Dichroism; Cyclic GMP; Guanylate Cyclase; Manganese; Models, Molecular

The biotinylated c-di-GMP and c-di-AMP conjugates 10a/b were synthesized by a straightforward set of procedures from standard, commercially available phosphoramidites. Their availability should allow isolation and characterization of new protein and RNA receptors for these key bacterial signaling molecules.

Topics: Biotinylation; Chemical Precipitation; Click Chemistry; Crystallization; Cyclic GMP; Dinucleoside Phosphates; Organophosphorus Compounds

Macrophages respond to infection with Legionella pneumophila by the induction of inflammatory mediators, including type I Interferons (IFN-Is). To explore whether the bacterial second messenger cyclic 3'-5' diguanylate (c-diGMP) activates some of these mediators, macrophages were infected with L. pneumophila strains in which the levels of bacterial c-diGMP had been altered. Intriguingly, there was a positive correlation between c-diGMP levels and IFN-I expression. Subsequent studies with synthetic derivatives of c-diGMP, and newly described cyclic 3'-5' diadenylate (c-diAMP), determined that these molecules activate overlapping inflammatory responses in human and murine macrophages. Moreover, UV crosslinking studies determined that both dinucleotides physically associate with a shared set of host proteins. Fractionation of macrophage extracts on a biotin-c-diGMP affinity matrix led to the identification of a set of candidate host binding proteins. These studies suggest that mammalian macrophages can sense and mount a specific inflammatory response to bacterial dinucleotides.

Topics: Animals; Bacterial Proteins; Cell Line; Cyclic GMP; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Host-Pathogen Interactions; Humans; Interferon Type I; Interferon-beta; Legionella pneumophila; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Signal Transduction

The RNA binding protein CsrA (RsmA) represses biofilm formation in several proteobacterial species. In Escherichia coli, it represses the production of the polysaccharide adhesin poly-β-1,6-N-acetyl-D-glucosamine (PGA) by binding to the pgaABCD mRNA leader, inhibiting pgaA translation, and destabilizing this transcript. In addition, CsrA represses genes responsible for the synthesis of cyclic di-GMP, an activator of PGA production. Here we determined that CsrA also represses NhaR, a LysR-type transcriptional regulator which responds to elevated [Na(+)] and alkaline pH and activates the transcription of the pgaABCD operon. Gel shift studies revealed that CsrA binds at two sites in the 5' untranslated segment of nhaR, one of which overlaps the Shine-Dalgarno sequence. An epitope-tagged NhaR protein, expressed from the nhaR chromosomal locus, and an nhaR posttranscriptional reporter fusion (PlacUV5-nhaR'-'lacZ) both showed robust repression by CsrA. Northern blotting revealed a complex transcription pattern for the nhaAR locus. Nevertheless, CsrA did not repress nhaR mRNA levels. Toeprinting assays showed that CsrA competes effectively with the ribosome for binding to the translation initiation region of nhaR. Together, these findings indicate that CsrA blocks nhaR translation. Epistasis studies with a pgaA-lacZ transcriptional fusion confirmed a model in which CsrA indirectly represses pgaABCD transcription via NhaR. We conclude that CsrA regulates the horizontally acquired pgaABCD operon and PGA biosynthesis at multiple levels. Furthermore, nhaR repression exemplifies an expanding role for CsrA as a global regulator of stress response systems.

Topics: beta-Glucans; Biofilms; Cyclic GMP; DNA-Binding Proteins; Down-Regulation; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Hydrogen-Ion Concentration; Porins; Protein Biosynthesis; Repressor Proteins; RNA-Binding Proteins; RNA, Messenger; Sodium; Transcription Factors

Riboswitches are a class of structural RNAs that regulate transcription and translation through specific recognition of small molecules. Riboswitches are attractive not only as drug targets for novel antibiotics but also as modular tools for controlling gene expression. Sequence comparison of a class of riboswitches that sense cyclic di-GMP (type-I c-di-GMP riboswitches) revealed that this type of riboswitch frequently shows a GAAA loop/receptor interaction between P1 and P3 elements. In the crystal structures of a type-I c-di-GMP riboswitch from Vibrio cholerae (the Vc2 riboswitch), the GNRA loop/receptor interaction assembled P2 and P3 stems to organize a ligand-binding pocket. In this study, the functional importance of the GAAA loop-receptor interaction in the Vc2 riboswitch was examined. A series of variant Vc2 riboswitches with mutations in the GAAA loop/receptor interaction were assayed for their switching abilities. In mutants with mutations in the P2 GAAA loop, expression of the reporter gene was reduced to approximately 40% - 60% of that in the wild-type. However, mutants in which the P3 receptor motif was substituted with base pairs were as active as the wild-type. These results suggested that the GAAA loop/receptor interaction does not simply establish the RNA 3D structure but docking of P2 GAAA loop reduces the flexibility of the GAAA receptor motif in the P3 element. This mechanism was supported by a variant riboswitch bearing a theophylline aptamer module in P3 the structural rigidity of which could be modulated by the small molecule theophylline.

Topics: Aptamers, Nucleotide; Base Pairing; Base Sequence; Cyclic GMP; Genes, Reporter; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Riboswitch; RNA, Bacterial; Vibrio cholerae

  The primary goal of this study was to characterize the existence of a functional c-di-GMP pathway in the bioleaching bacterium Acidithiobacillus ferrooxidans..   A bioinformatic search revealed that the genome sequence of At. ferrooxidans ATCC 23270 codes for several proteins involved in the c-di-GMP pathway, including diguanylate cyclases (DGC), phosphodiesterases and PilZ effector proteins. Overexpression in Escherichia coli demonstrated that four At. ferrooxidans genes code for proteins containing GGDEF/EAL domains with functional DGC activity. MS/MS analysis allowed the identification of c-di-GMP in nucleotide preparations obtained from At. ferrooxidans cells. In addition, c-di-GMP levels in cells grown on the surface of solid energetic substrates such as sulfur prills or pyrite were higher than those measured in ferrous iron planktonic cells..   At. ferrooxidans possesses a functional c-di-GMP pathway that could play a key role in At. ferrooxidans biofilm formation during bioleaching processes..   This is the first global study about the c-di-GMP pathway in an acidophilic bacterium of great interest for the biomining industry. It opens a new way to explore the regulation of biofilm formation by biomining micro-organisms during the bioleaching process.

Topics: Acidithiobacillus; Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli; Intracellular Space; Minerals; Recombinant Proteins; Signal Transduction; Tandem Mass Spectrometry

Cyclic diguanosine-monophosphate (c-di-GMP) is a bacterial signaling molecule that triggers a switch from motile to sessile bacterial lifestyles. This mechanism is of considerable pharmaceutical interest, since it is related to bacterial virulence, biofilm formation, and persistence of infection. Previously, c-di-GMP has been reported to display a rich polymorphism of various oligomeric forms at millimolar concentrations, which differ in base stacking and G-quartet interactions. Here, we have analyzed the equilibrium and exchange kinetics between these various forms by NMR spectroscopy. We find that the association of the monomer into a dimeric form is in fast exchange (

Topics: Bacteria; Cyclic GMP; Hydrogen-Ion Concentration; Kinetics; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Structure; Sodium Chloride; Spectrophotometry, Ultraviolet

In many bacteria, high levels of the ubiquitous second messenger c-di-GMP have been demonstrated to suppress motility and to promote the establishment of surface-adherent biofilm communities. While molecular mechanisms underlying the synthesis and degradation of c-di-GMP have been comprehensively characterized, little is known about how c-di-GMP mediates its regulatory effects. In this study, we have established a chemical proteomics approach to identify c-di-GMP interacting proteins in the opportunistic pathogen Pseudomonas aeruginosa. A functionalized c-di-GMP analog, 2'-aminohexylcarbamoyl-c-di-GMP (2'-AHC-c-di-GMP), was chemically synthesized and following its immobilization used to perform affinity pull down experiments. Enriched proteins were subsequently identified by high-resolution mass spectrometry. 2'-AHC-c-di-GMP was also employed in surface plasmon resonance studies to evaluate and quantify the interaction of c-di-GMP with its potential target molecules in vitro. The biochemical tools presented here may serve the identification of novel classes of c-di-GMP effectors and thus contribute to a better characterization and understanding of the complex c-di-GMP signaling network.

Topics: Bacterial Proteins; Carrier Proteins; Cyclic GMP; Proteomics; Pseudomonas aeruginosa; Signal Transduction; Surface Plasmon Resonance

C-di-GMP has emerged as a signalling molecule that regulates a variety of processes in several bacteria; therefore there is interest in the development of biotinylated analogs for the identification of binding partners. No detailed study has been done to evaluate if biotinylated analogs of c-di-GMP are capable of binding to c-di-GMP receptors. Herein, we evaluate the binding of commercially available 2'-biotinylated c-di-GMP and phosphorothioate 2'-biotinylated c-di-GMP, prepared via a facile solid-phase synthesis, to several c-di-GMP receptors. Docking, using Autodock vina software, as well as experimental studies of these analogs, with c-di-GMP class I and II riboswitches and binding proteins, reveal that some, but not all, c-di-GMP receptors can tolerate the 2'-modification of c-di-GMP with biotin.

Topics: Bacterial Proteins; Biotin; Biotinylation; Carrier Proteins; Cyclic GMP; Ligands; Models, Molecular; Nucleic Acid Conformation; Riboswitch; RNA, Bacterial; Solid-Phase Synthesis Techniques; Vibrio cholerae

Topics: Bacterial Adhesion; Cyclic GMP; Gene Expression Regulation, Bacterial; Transcription, Genetic; Vibrio parahaemolyticus

The Vibrio parahaemolyticus Scr system modulates decisions pertinent to surface colonization by affecting the cellular level of cyclic dimeric GMP (c-di-GMP). In this work, we explore the scope and mechanism of this regulation. Transcriptome comparison of ΔscrABC and wild-type strains revealed expression differences with respect to ∼100 genes. Elevated c-di-GMP repressed genes in the surface-sensing regulon, including those encoding the lateral flagellar and type III secretion systems and N-acetylglucosamine-binding protein GpbA while inducing genes encoding other cell surface molecules and capsular polysaccharide. The transcription of a few regulatory genes was also affected, and the role of one was characterized. Mutations in cpsQ suppressed the sticky phenotype of scr mutants. cpsQ encodes one of four V. parahaemolyticus homologs in the CsgD/VpsT family, members of which have been implicated in c-di-GMP signaling. Here, we demonstrate that CpsQ is a c-di-GMP-binding protein. By using a combination of mutant and reporter analyses, CpsQ was found to be the direct, positive regulator of cpsA transcription. This c-di-GMP-responsive regulatory circuit could be reconstituted in Escherichia coli, where a low level of this nucleotide diminished the stability of CpsQ. The molecular interplay of additional known cps regulators was defined by establishing that CpsS, another CsgD family member, repressed cpsR, and the transcription factor CpsR activated cpsQ. Thus, we are developing a connectivity map of the Scr decision-making network with respect to its wiring and output strategies for colonizing surfaces and interaction with hosts; in doing so, we have isolated and reproduced a c-di-GMP-sensitive regulatory module in the circuit.

Topics: Bacterial Adhesion; Bacterial Proteins; Cyclic GMP; Escherichia coli; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Protein Binding; Transcription, Genetic; Vibrio parahaemolyticus

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) allows detection of protein interactions with low-molecular weight ligands based on separation of the protein-ligand complex by differential capillary action. Here, we present an application of DRaCALA to the study of nucleic acid-protein interactions using the Escherichia coli cyclic AMP receptor protein (CRP). CRP bound in DRaCALA specifically to (32)P-labeled oligonucleotides containing the consensus CRP binding site, but not to oligonucleotides with point mutations known to abrogate binding. Affinity and kinetic studies using DRaCALA yielded a dissociation constant and dissociation rate similar to previously reported values. Because DRaCALA is not subject to ligand size restrictions, whole plasmids with a single CRP-binding site were used as probes, yielding similar results. DNA can also function as an easily labeled carrier molecule for a conjugated ligand. Sequestration of biotinylated nucleic acids by streptavidin allowed nucleic acids to take the place of the protein as the immobile binding partner. Therefore, any molecular interactions involving nucleic acids can be tested. We demonstrate this principle utilizing a bacterial riboswitch that binds cyclic-di-guanosine monophosphate. DRaCALA is a flexible and complementary approach to other biochemical methods for rapid and accurate measurements of affinity and kinetics at near-equilibrium conditions.

Topics: Binding Sites; Cyclic GMP; DNA; DNA Probes; DNA-Binding Proteins; Escherichia coli Proteins; Kinetics; Ligands; Molecular Probe Techniques; Nucleic Acids; Oligonucleotide Probes; Plasmids; Receptors, Cyclic AMP; Riboswitch

C-di-GMP has emerged as an important bacterial signaling molecule that is involved in biofilm formation. Small molecules that can form biologically inactive complexes with c-di-GMP have the potential to be used as anti-biofilm agents. Herein, we report that water-soluble diamidinium/iminium aromatics (such as berenil), which are traditionally considered as minor groove binders of nucleic acids, are capable of aggregating c-di-GMP into G-quadruplexes via π-stacking interactions.

Topics: Amidines; Cyclic GMP; G-Quadruplexes; Imines; Molecular Structure; Solubility; Stereoisomerism; Water

Herein, we demonstrate that the bacterial signaling molecule, c-di-GMP, can enhance the peroxidation of hemin when proflavine is present. The c-di-GMP-proflavine-hemin nucleotidezyme can oxidize the colorless compound 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS, to the colored radical cation ABTS˙(+) and hence provides simple colorimetric detection of c-di-GMP at low micromolar concentrations.

Topics: Benzothiazoles; Colorimetry; Cyclic GMP; Hemin; Oxidation-Reduction; Peroxidase; Proflavine; Sulfonic Acids

Many pathogens regulate or modify their immune-stimulating ligands to avoid detection by their infected hosts. Listeria monocytogenes, a facultative intracellular bacterial pathogen, interacts with multiple components of mammalian innate immunity during its infection cycle. During replication within the cytosol of infected cells, L. monocytogenes utilizes two multidrug efflux pumps, MdrM and MdrT, to secrete the small nucleic acid second messenger cyclic-di-AMP (c-di-AMP). Host recognition of c-di-AMP triggers the production of type I interferons, including beta interferon (IFN-β), which, surprisingly, promote L. monocytogenes virulence. In this study, we have examined the capacity of multiple laboratory and clinical isolates of L. monocytogenes to stimulate host production of IFN-β. We have identified the L. monocytogenes strain LO28 as able to hyperinduce IFN-β production in infected cells ∼30-fold more than the common laboratory clone L. monocytogenes strain 10403S. Genomic analyses determined that LO28 contains a naturally occurring loss-of-function allele of the transcriptional regulator BrtA and correspondingly derepresses expression of MdrT. Surprisingly, while derepression of MdrT resulted in hyperstimulation of IFN-β, it results in significant attenuation in multiple mouse models of infection. While type I interferons may promote L. monocytogenes virulence, this study demonstrates that unregulated expression of the c-di-AMP-secreting efflux pump MdrT significantly restricts virulence in vivo by an unknown mechanism.

Topics: Animals; Bacterial Proteins; Base Sequence; Cells, Cultured; Cyclic GMP; Genes, MDR; Interferon-beta; Listeria monocytogenes; Macrophages; Membrane Transport Proteins; Mice; Mice, Inbred C57BL; Sequence Analysis, DNA

The ability to synthesize exopolysaccharides (EPS) is widespread among microorganisms, and microbial EPS play important roles in biofilm formation, pathogen persistence, and applications in the food and medical industries. Although it is well established that EPS synthesis is invariably in response to environmental cues, it remains largely unknown how various environmental signals trigger activation of the biochemical synthesis machinery.. We report here the transcriptome profiling of Agrobacterium sp. ATCC 31749, a microorganism that produces large amounts of a glucose polymer known as curdlan under nitrogen starvation. Transcriptome analysis revealed a nearly 100-fold upregulation of the curdlan synthesis operon upon transition to nitrogen starvation, thus establishing the prominent role that transcriptional regulation plays in the EPS synthesis. In addition to known mechanisms of EPS regulation such as activation by c-di-GMP, we identify novel mechanisms of regulation in ATCC 31749, including RpoN-independent NtrC regulation and intracellular pH regulation by acidocalcisomes. Furthermore, we show evidence that curdlan synthesis is also regulated by conserved cell stress responses, including polyphosphate accumulation and the stringent response. In fact, the stringent response signal, pppGpp, appears to be indispensible for transcriptional activation of curdlan biosynthesis.. This study identifies several mechanisms regulating the synthesis of curdlan, an EPS with numerous applications. These mechanisms are potential metabolic engineering targets for improving the industrial production of curdlan from Agrobacterium sp. ATCC 31749. Furthermore, many of the genes identified in this study are highly conserved across microbial genomes, and we propose that the molecular elements identified in this study may serve as universal regulators of microbial EPS synthesis.

Topics: Agrobacterium; Bacterial Proteins; beta-Glucans; Cyclic GMP; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genes, Bacterial; Guanosine Pentaphosphate; Metabolic Engineering; Nitrogen; Polyphosphates; Up-Regulation

Bdellovibrio bacteriovorus is a Delta-proteobacterium that oscillates between free-living growth and predation on Gram-negative bacteria including important pathogens of man, animals and plants. After entering the prey periplasm, killing the prey and replicating inside the prey bdelloplast, several motile B. bacteriovorus progeny cells emerge. The B. bacteriovorus HD100 genome encodes numerous proteins predicted to be involved in signalling via the secondary messenger cyclic di-GMP (c-di-GMP), which is known to affect bacterial lifestyle choices. We investigated the role of c-di-GMP signalling in B. bacteriovorus, focussing on the five GGDEF domain proteins that are predicted to function as diguanylyl cyclases initiating c-di-GMP signalling cascades. Inactivation of individual GGDEF domain genes resulted in remarkably distinct phenotypes. Deletion of dgcB (Bd0742) resulted in a predation impaired, obligately axenic mutant, while deletion of dgcC (Bd1434) resulted in the opposite, obligately predatory mutant. Deletion of dgcA (Bd0367) abolished gliding motility, producing bacteria capable of predatory invasion but unable to leave the exhausted prey. Complementation was achieved with wild type dgc genes, but not with GGAAF versions. Deletion of cdgA (Bd3125) substantially slowed predation; this was restored by wild type complementation. Deletion of dgcD (Bd3766) had no observable phenotype. In vitro assays showed that DgcA, DgcB, and DgcC were diguanylyl cyclases. CdgA lacks enzymatic activity but functions as a c-di-GMP receptor apparently in the DgcB pathway. Activity of DgcD was not detected. Deletion of DgcA strongly decreased the extractable c-di-GMP content of axenic Bdellovibrio cells. We show that c-di-GMP signalling pathways are essential for both the free-living and predatory lifestyles of B. bacteriovorus and that obligately predatory dgcC- can be made lacking a propensity to survive without predation of bacterial pathogens and thus possibly useful in anti-pathogen applications. In contrast to many studies in other bacteria, Bdellovibrio shows specificity and lack of overlap in c-di-GMP signalling pathways.

Topics: Bacterial Proteins; Bdellovibrio; Cyclic GMP; Escherichia coli Proteins; Gene Deletion; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Signal Transduction

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) plays an important role in a variety of cellular functions, including biofilm formation, alterations in the cell surface, host colonization and regulation of bacterial flagellar motility, which enable bacteria to survive changing environmental conditions. The cellular level of c-di-GMP is regulated by a balance between opposing activities of diguanylate cyclases (DGCs) and cognate phosphodiesterases (PDE-As). Here, we report the presence and importance of a protein, MSDGC-1 (an orthologue of Rv1354c in Mycobacterium tuberculosis), involved in c-di-GMP turnover in Mycobacterium smegmatis. MSDGC-1 is a multidomain protein, having GAF, GGDEF and EAL domains arranged in tandem, and exhibits both c-di-GMP synthesis and degradation activities. Most other proteins containing GGDEF and EAL domains have been demonstrated to have either DGC or PDE-A activity. Unlike other bacteria, which harbour several copies of the protein involved in c-di-GMP turnover, M. smegmatis has a single genomic copy, deletion of which severely affects long-term survival under conditions of nutrient starvation. Overexpression of MSDGC-1 alters the colony morphology and growth profile of M. smegmatis. In order to gain insights into the regulation of the c-di-GMP level, we cloned individual domains and tested their activities. We observed a loss of activity in the separated domains, indicating the importance of full-length MSDGC-1 for controlling bifunctionality.

Topics: Amino Acid Sequence; Bacterial Proteins; Cyclic GMP; Microbial Viability; Molecular Sequence Data; Mycobacterium smegmatis; Protein Structure, Tertiary; Sequence Alignment

Although several reports have documented nitric oxide (NO) regulation of biofilm formation, the molecular basis of this phenomenon is unknown. In many bacteria, an H-NOX (heme-nitric oxide/oxygen-binding) gene is found near a diguanylate cyclase (DGC) gene. H-NOX domains are conserved hemoproteins that are known NO sensors. It is widely recognized that cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates the transition between motility and biofilm. Therefore, NO may influence biofilm formation through H-NOX regulation of DGC, thus providing a molecular-level explanation for NO regulation of biofilm formation. This work demonstrates that, indeed, NO-bound H-NOX negatively affects biofilm formation by directly regulating c-di-GMP turnover in Shewanella woodyi strain MS32. Exposure of wild-type S. woodyi to a nanomolar level of NO resulted in the formation of thinner biofilms, and less intracellular c-di-GMP, than in the absence of NO. Also, a mutant strain in the gene encoding SwH-NOX showed a decreased level of biofilm formation (and a decreased amount of intracellular c-di-GMP) with no change observed upon NO addition. Furthermore, using purified proteins, it was demonstrated that SwH-NOX and SwDGC are binding partners. SwDGC is a dual-functioning DGC; it has diguanylate cyclase and phosphodiesterase activities. These data indicate that NO-bound SwH-NOX enhances c-di-GMP degradation, but not synthesis, by SwDGC. These results support the biofilm growth data and indicate that S. woodyi senses nanomolar NO with an H-NOX domain and that SwH-NOX regulates SwDGC activity, resulting in a reduction in c-di-GMP concentration and a decreased level of biofilm growth in the presence of NO. These data provide a detailed molecular mechanism for NO regulation of c-di-GMP signaling and biofilm formation.

Topics: Bacterial Proteins; Base Sequence; Biofilms; Cyclic GMP; DNA, Bacterial; Escherichia coli Proteins; Genes, Bacterial; Hydrolysis; Kinetics; Models, Biological; Mutation; Nitric Oxide; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Shewanella; Signal Transduction

Cyclic diguanylate (c-di-GMP) is a bacterial second messenger important for physiologic adaptation and virulence. Class-I c-di-GMP riboswitches are phylogenetically widespread and thought to mediate pleiotropic genetic responses to the second messenger. Previous studies suggest that the RNA aptamer domain switches from an extended free state to a compact, c-di-GMP-bound conformation in which two helical stacks dock side-by-side. Single molecule fluorescence resonance energy transfer (smFRET) experiments now reveal that the free RNA exists in four distinct populations that differ in dynamics in the extended and docked conformations. In the presence of c-di-GMP and Mg(2+), a stably docked population (>30 min) becomes predominant. smFRET mutant analysis demonstrates that tertiary interactions distal to the c-di-GMP binding site strongly modulate the RNA population structure, even in the absence of c-di-GMP. These allosteric interactions accelerate ligand recognition by preorganizing the RNA, favoring rapid c-di-GMP binding.

Topics: Cyclic GMP; Fluorescence Resonance Energy Transfer; Ligands; Magnesium; Nucleic Acid Conformation; Riboswitch; RNA, Bacterial; Vibrio cholerae

In Myxococcus xanthus the extracellular matrix is essential for type IV pili-dependent motility and starvation-induced fruiting body formation. Proteins of two-component systems including the orphan DNA binding response regulator DigR are essential in regulating the composition of the extracellular matrix. We identify the orphan hybrid histidine kinase SgmT as the partner kinase of DigR. In addition to kinase and receiver domains, SgmT consists of an N-terminal GAF domain and a C-terminal GGDEF domain. The GAF domain is the primary sensor domain. The GGDEF domain binds the second messenger bis-(3'-5')-cyclic-dimeric-GMP (c-di-GMP) and functions as a c-di-GMP receptor to spatially sequester SgmT. We identify the DigR binding site in the promoter of the fibA gene, which encodes an abundant extracellular matrix metalloprotease. Whole-genome expression profiling experiments in combination with the identified DigR binding site allowed the identification of the DigR regulon and suggests that SgmT/DigR regulates the expression of genes for secreted proteins and enzymes involved in secondary metabolite synthesis. We suggest that SgmT/DigR regulates extracellular matrix composition and that SgmT activity is regulated by two sensor domains with ligand binding to the GAF domain resulting in SgmT activation and c-di-GMP binding to the GGDEF domain resulting in spatial sequestration of SgmT.

Topics: Bacterial Proteins; Carrier Proteins; Cyclic GMP; DNA-Binding Proteins; Extracellular Matrix; Gene Expression Regulation, Bacterial; Histidine Kinase; Intracellular Signaling Peptides and Proteins; Myxococcus xanthus; Oligonucleotide Array Sequence Analysis; Protein Kinases; Regulon

The bacterial signaling molecule, cyclic diguanosine monophosphate (c-di-GMP), plays a key role in controlling biofilm formation and pathogenic virulence, among many other functions. It has widespread consequences for human health, and current research is actively exploring its molecular mechanisms. The convenient one-flask, gram-scale synthesis of c-di-GMP described here will facilitate these efforts.

Topics: Bacteria; Chromatography, High Pressure Liquid; Crystallization; Cyclic GMP; Cyclization; Dichloroacetic Acid; Ethylamines; Pyridines; Solutions; Time Factors

c-di-GMP is a major secondary-messenger molecule in regulation of bacterial pathogenesis. Therefore, the c-di-GMP-mediated signal transduction network is of considerable interest. The PilZ domain was the first c-di-GMP receptor to be predicted and identified. However, every PilZ domain binds c-di-GMP with a different binding affinity. Intriguingly, a noncanonical PilZ domain has recently been found to serve as a mediator to link FimX(EAL) to the PilB or PilT ATPase to control the function of type IV pili (T4P). It is thus essential to determine the structure of the FimX(EAL)-PilZ complex in order to determine how the binding of c-di-GMP to the FimX(EAL) domain induces conformational change of the adjoining noncanonical PilZ domain, which may transmit information to PilB or PilT to control T4P function. Here, the preparation and preliminary X-ray diffraction studies of the XccFimX(EAL)-c-di-GMP and XccFimX(EAL)-c-di-GMP-XccPilZ complexes from Xcc (Xanthomonas campestris pv. campesteris) are reported. Detailed studies of these complexes may allow a more thorough understanding of how c-di-GMP transmits its effects through the degenerate EAL domain and the noncanonical PilZ domain.

Topics: Bacterial Proteins; Crystallization; Crystallography, X-Ray; Cyclic GMP; Protein Binding; Xanthomonas campestris

A series of allosteric ribozymes that respond to the bacterial second messenger cyclic diguanosyl-5'-monophosphate (c-di-GMP) have been created by using in vitro selection. An RNA library was generated by using random-sequence bridges to join a hammerhead self-cleaving ribozyme to an aptamer from a natural c-di-GMP riboswitch. Specific bridge sequences, called communication modules, emerged through two in vitro selection efforts that either activate or inhibit ribozyme self-cleavage upon ligand binding to the aptamer. Representative RNAs were found that exhibit EC(50) (half-maximal effective concentration) values for c-di-GMP as low as 90 nM and IC(50) (half-maximal inhibitory concentration) values as low as 180 nM. The allosteric RNAs display molecular recognition characteristics that mimic the high discriminatory ability of the natural aptamer. Some engineered RNAs operate with ribozyme rate constants approaching that of the parent hammerhead ribozyme. By use of these allosteric ribozymes, cytoplasmic concentrations of c-di-GMP in three mutant strains of Escherichia coli were quantitatively estimated from cell lysates. Our findings demonstrate that engineered c-di-GMP-sensing ribozymes can be used as convenient tools to monitor c-di-GMP levels from complex biological or chemical samples. Moreover, these ribozymes could be employed in high-throughput screens to identify compounds that trigger c-di-GMP riboswitch function.

Topics: Allosteric Regulation; Aptamers, Nucleotide; Cyclic GMP; Escherichia coli; Nucleic Acid Conformation; Protein Engineering; Riboswitch; RNA; RNA, Catalytic; Second Messenger Systems

Clostridium difficile-associated disease is increasing in incidence and is costly to treat. Our understanding of how this organism senses its entry into the host and adapts for growth in the large bowel is limited. The small-molecule second messenger cyclic diguanylate (c-di-GMP) has been extensively studied in gram-negative bacteria and has been shown to modulate motility, biofilm formation, and other processes in response to environmental signals, yet little is known about the functions of this signaling molecule in gram-positive bacteria or in C. difficile specifically. In the current study, we investigated the function of the second messenger c-di-GMP in C. difficile. To determine the role of c-di-GMP in C. difficile, we ectopically expressed genes encoding a diguanylate cyclase enzyme, which synthesizes c-di-GMP, or a phosphodiesterase enzyme, which degrades c-di-GMP. This strategy allowed us to artificially elevate or deplete intracellular c-di-GMP, respectively, and determine that c-di-GMP represses motility in C. difficile, consistent with previous studies in gram-negative bacteria, in which c-di-GMP has a negative effect on myriad modes of bacterial motility. Elevated c-di-GMP levels also induced clumping of C. difficile cells, which may signify that C. difficile is capable of forming biofilms in the host. In addition, we directly quantified, for the first time, c-di-GMP production in a gram-positive bacterium. This work demonstrates the effect of c-di-GMP on the motility of a gram-positive bacterium and on aggregation of C. difficile, which may be relevant to the function of this signaling molecule during infection.

Topics: Bacterial Adhesion; Bacterial Proteins; Clostridioides difficile; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Locomotion; Phosphorus-Oxygen Lyases

Nitric oxide (NO) signaling in vertebrates is well characterized and involves the heme-nitric oxide/oxygen-binding (H-NOX) domain of soluble guanylate cyclase as a selective NO sensor. In contrast, little is known about the biological role or signaling output of bacterial H-NOX proteins. Here, we describe a molecular pathway for H-NOX signaling in Shewanella oneidensis. NO stimulates biofilm formation by controlling the levels of the bacterial secondary messenger cyclic diguanosine monophosphate (c-di-GMP). Phosphotransfer profiling was used to map the connectivity of a multicomponent signaling network that involves integration from two histidine kinases and branching to three response regulators. A feed-forward loop between response regulators with phosphodiesterase domains and phosphorylation-mediated activation intricately regulated c-di-GMP levels. Phenotypic characterization established a link between NO signaling and biofilm formation. Cellular adhesion may provide a protection mechanism for bacteria against reactive and damaging NO. These results are broadly applicable to H-NOX-mediated NO signaling in bacteria.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Amino Acid Sequence; Bacterial Proteins; Biofilms; Cyclic GMP; Gammaproteobacteria; Guanylate Cyclase; Histidine Kinase; Models, Biological; Molecular Sequence Data; Nitric Oxide; Phosphorylation; Protein Kinases; Protein Structure, Tertiary; Receptors, Cytoplasmic and Nuclear; Second Messenger Systems; Sequence Homology, Amino Acid; Shewanella; Signal Transduction; Soluble Guanylyl Cyclase

STING is an essential signaling molecule for DNA and cyclic di-GMP (c-di-GMP)-mediated type I interferon (IFN) production via TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) pathway. It contains an N-terminal transmembrane region and a cytosolic C-terminal domain (CTD). Here, we describe crystal structures of STING CTD alone and complexed with c-di-GMP in a unique binding mode. The strictly conserved aa 153-173 region was shown to be cytosolic and participated in dimerization via hydrophobic interactions. The STING CTD functions as a dimer and the dimerization was independent of posttranslational modifications. Binding of c-di-GMP enhanced interaction of a shorter construct of STING CTD (residues 139-344) with TBK1. This suggests an extra TBK1 binding site, other than serine 358. This study provides a glimpse into the unique architecture of STING and sheds light on the mechanism of c-di-GMP-mediated TBK1 signaling.

Topics: Amino Acid Sequence; Conserved Sequence; Crystallography, X-Ray; Cyclic GMP; Dimerization; HEK293 Cells; Humans; Hydrophobic and Hydrophilic Interactions; Membrane Proteins; Models, Molecular; Molecular Sequence Data; Peptide Fragments; Protein Binding; Protein Conformation; Protein Interaction Mapping; Protein Serine-Threonine Kinases; Recombinant Fusion Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) modulates the transition between planktonic and biofilm life styles. In response to c-di-GMP, the enhancer binding protein FleQ from Pseudomonas aeruginosa derepresses the expression of Pel exopolysaccharide genes required for biofilm formation when a second protein, FleN is present. A model is that binding of c-di-GMP to FleQ induces its dissociation from the pelA promoter allowing RNA polymerase to access this site. To test this, we analyzed pelA DNA footprinting patterns with various combinations of FleQ, FleN and c-di-GMP, coupled to in vivo promoter activities. FleQ binds to two sites called box 1 and 2. FleN binds to FleQ bound at these sites causing the intervening DNA to bend. Binding of c-di-GMP to FleQ relieves the DNA distortion but FleQ remains bound to the two sites. Analysis of wild type and mutated versions of pelA-lacZ transcriptional fusions suggests that FleQ represses gene expression from box 2 and activates gene expression in response to c-di-GMP from box 1. The role of c-di-GMP is thus to convert FleQ from a repressor to an activator. The mechanism of action of FleQ is distinct from that of other bacterial transcription factors that both activate and repress gene expression from a single promoter.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Bacterial Proteins; Binding Sites; Cyclic GMP; DNA, Bacterial; Gene Expression Regulation, Bacterial; Operon; Promoter Regions, Genetic; Pseudomonas aeruginosa; Repressor Proteins; Trans-Activators

To investigate the effect of exogenous c-di-GMP in preventing dental caries formation in SD rats.. Twenty-day-old SD rats with dental caries induced by S. Mutans infection were randomly divided into 3 groups for treatment with dental application of exogenous c-di-GMP, NaF solution or 0.9% NaCl, and changes in the bacterial number and scores of dental caries following the treatments were recorded.. Compared with 0.9% NaCl treatment, exogenous c-di-GMP treatment significantly lowered the scores of dental caries on the occlusal surface and smooth surface (P<0.05) but produced no obvious effect on the number of bacterial plagues (P>0.05).. Exogenous c-di-GMP can be a novel agent for prevention and treatment of tooth decay.

Topics: Animals; Cyclic GMP; Dental Caries; Disease Models, Animal; Female; Rats; Streptococcus mutans

High cellular concentrations of bis-(3',5')-cyclic dimeric guanosine mono-phosphate (c-di-GMP) regulate a diverse range of phenotypes in bacteria including biofilm development. The opportunistic pathogen Pseudomonas aeruginosa produces the PEL polysaccharide to form a biofilm at the air-liquid interface of standing cultures. Among the proteins required for PEL polysaccharide production, PelD has been identified as a membrane-bound c-di-GMP-specific receptor. In this work, we present the x-ray crystal structure of a soluble cytoplasmic region of PelD in its apo and c-di-GMP complexed forms. The structure of PelD reveals an N-terminal GAF domain and a C-terminal degenerate GGDEF domain, the latter of which binds dimeric c-di-GMP at an RXXD motif that normally serves as an allosteric inhibition site for active diguanylate cyclases. Using isothermal titration calorimetry, we demonstrate that PelD binds c-di-GMP with low micromolar affinity and that mutation of residues involved in binding not only decreases the affinity of this interaction but also abrogates PEL-specific phenotypes in vivo. Bioinformatics analysis of the juxtamembrane region of PelD suggests that it contains an α-helical stalk region that connects the soluble region to the transmembrane domains and that similarly to other GAF domain containing proteins, this region likely forms a coiled-coil motif that mediates dimerization. PelD with Alg44 and BcsA of the alginate and cellulose secretion systems, respectively, collectively constitute a group of c-di-GMP receptors that appear to regulate exopolysaccharide assembly at the protein level through activation of their associated glycosyl transferases.

Topics: Amino Acid Motifs; Amino Acid Sequence; Bacterial Proteins; Binding Sites; Binding, Competitive; Biofilms; Blotting, Western; Crystallography, X-Ray; Cyclic GMP; Membrane Proteins; Models, Molecular; Molecular Sequence Data; Mutation; Polysaccharide-Lyases; Polysaccharides, Bacterial; Protein Structure, Secondary; Protein Structure, Tertiary; Pseudomonas aeruginosa

Cph2 from the cyanobacterium Synechocystis sp. PCC 6803 is a hybrid photoreceptor that comprises an N-terminal module for red/far-red light reception and a C-terminal module switching between a blue- and a green-receptive state. This unusual photoreceptor exerts complex, light quality-dependent control of the motility of Synechocystis sp. PCC 6803 cells by inhibiting phototaxis towards blue light. Cph2 perceives blue light by its third GAF domain that bears all characteristics of a cyanobacteriochrome (CBCR) including photoconversion between green- and blue-absorbing states as well as formation of a bilin species simultaneously tethered to two cysteines, C994 and C1022. Upon blue light illumination the CBCR domain activates the subsequent C-terminal GGDEF domain, which catalyses formation of the second messenger c-di-GMP. Accordingly, expression of the CBCR-GGDEF module in Δcph2 mutant cells restores the blue light-dependent inhibition of motility. Additional expression of the N-terminal Cph2 fragment harbouring a red/far-red interconverting phytochrome fused to a c-di-GMP degrading EAL domain restores the complex behaviour of the intact Cph2 photosensor. c-di-GMP was shown to regulate flagellar and pili-based motility in several bacteria. Here we provide the first evidence that this universal bacterial second messenger is directly involved in the light-dependent regulation of cyanobacterial phototaxis.

Topics: Bacterial Proteins; Cyclic GMP; Light; Locomotion; Phytochrome; Synechocystis

Riboswitches for the bacterial second messenger c-di-GMP control the expression of genes involved in numerous cellular processes such as virulence, competence, biofilm formation, and flagella synthesis. Therefore, the two known c-di-GMP riboswitch classes represent promising targets for developing novel modulators of bacterial physiology. Here, we examine the binding characteristics of circular and linear c-di-GMP analogues for representatives of both class I and II c-di-GMP riboswitches derived from the pathogenic bacterium Vibrio choleae (class I) and Clostridium difficile (class II). Some compounds exhibit values for apparent dissociation constant (K(D)) below 1 μM and associate with riboswitch RNAs during transcription with a speed that is sufficient to influence riboswitch function. These findings are consistent with the published structural models for these riboswitches and suggest that large modifications at various positions on the ligand can be made to create novel compounds that target c-di-GMP riboswitches. Moreover, we demonstrate the potential of an engineered allosteric ribozyme for the rapid screening of chemical libraries for compounds that bind c-di-GMP riboswitches.

Topics: Allosteric Site; Anti-Infective Agents; Bacterial Physiological Phenomena; Binding Sites; Clostridioides difficile; Computational Biology; Cyclic GMP; Kinetics; Ligands; Models, Chemical; Ribose; Second Messenger Systems; Transcription, Genetic; Vibrio cholerae

The second messenger, cyclic diguanylate (c-di-GMP), regulates diverse cellular processes in bacteria. C-di-GMP is produced by diguanylate cyclases (DGCs), degraded by phosphodiesterases (PDEs), and receptors couple c-di-GMP production to cellular responses. In many bacteria, including Vibrio cholerae, multiple DGCs and PDEs contribute to c-di-GMP signaling, and it is currently unclear whether the compartmentalization of c-di-GMP signaling components is required to mediate c-di-GMP signal transduction. In this study we show that the transcriptional regulator, VpsT, requires c-di-GMP binding for subcellular localization and activity. Only the additive deletion of five DGCs markedly decreases the localization of VpsT, while single deletions of each DGC do not impact VpsT localization. Moreover, mutations in residues required for c-di-GMP binding, c-di-GMP-stabilized dimerization and DNA binding of VpsT abrogate wild type localization and activity. VpsT does not co-localize or interact with DGCs suggesting that c-di-GMP from these DGCs diffuses to VpsT, supporting a model in which c-di-GMP acts at a distance. Furthermore, VpsT localization in a heterologous host, Escherichia coli, requires a catalytically active DGC and is enhanced by the presence of VpsT-target sequences. Our data show that c-di-GMP signaling can be executed through an additive cellular c-di-GMP level from multiple DGCs affecting the localization and activity of a c-di-GMP receptor and furthers our understanding of the mechanisms of second messenger signaling.

Topics: Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Phosphorus-Oxygen Lyases; Second Messenger Systems; Signal Transduction; Transcription Factors; Transcription, Genetic; Vibrio cholerae; Viral Proteins

Our earlier studies showed that AhyRI- (AI-1) and LuxS-based (AI-2) quorum sensing (QS) systems were positive and negative regulators of virulence, respectively, in a diarrheal isolate SSU of Aeromonas hydrophila. Recently, we demonstrated that deletion of the QseBC two-component signal transduction system (AI-3 QS in enterohemorrhagic Escherichia coli) also led to an attenuation of A. hydrophila in a septicemic mouse model of infection, and that interplay exists between AI-1, AI-2, and the second-messenger cyclic-di-guanosine monophosphate (c-di-GMP) in modulating bacterial virulence. To further explore a network connection between all of the three QS systems in A. hydrophila SSU and their cross talk with c-di-GMP, we overproduced a protein with a GGDEF domain, which increases c-di-GMP levels in bacteria, and studied phenotypes and transcriptional profiling of genes involved in biofilm formation and motility of the wild-type (WT) A. hydrophila and its ΔqseB mutant. Over-expression of the GGDEF domain-encoding gene (aha0701h) resulted in a significantly reduced motility of the WT A. hydrophila similar to that of the ΔqseB mutant. While enhanced protease production was noted in WT A. hydrophila that had increased c-di-GMP, no enzymatic activity was detected in the ΔqseB mutant overexpressing the aha0701h gene. Likewise, denser biofilm formation was noted for WT bacteria when c-di-GMP was overproduced compared to its respective control; however, overproduction of c-di-GMP in the ΔqseB mutant led to reduced biofilm formation, a finding similar to that noted for the parental A. hydrophila strain. These effects on bacterial motility and biofilm formation in the ΔqseB mutant or the mutant with increased c-di-GMP were correlated with altered levels of fleN and vpsT genes. While we noted transcript levels of qseB and qseC genes to be increased in the ahyRI mutant, down-regulation of the ahyR and ahyI genes was observed in the ΔqseB mutant, which correlated with decreased protease activity. Finally, an enhanced virulence of WT A. hydrophila with increased c-di-GMP was noted in a mouse model when compared to findings in the parental strain with vector alone. Overall, we conclude that cross talk between AI-1 and QseBC systems exists in A. hydrophila SSU, and c-di-GMP modulation on QseBC system is dependent on the expression of the AI-1 system.

Topics: Aeromonas hydrophila; Animals; Bacterial Proteins; Biofilms; Cyclic GMP; Female; Gene Expression Regulation, Bacterial; Gram-Negative Bacterial Infections; Humans; Mice; Quorum Sensing; Virulence

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that utilizes polar type IV pili (T4P) for twitching motility and adhesion in the environment and during infection. Pilus assembly requires FimX, a GGDEF/EAL domain protein that binds and hydrolyzes cyclic di-GMP (c-di-GMP). Bacteria lacking FimX are deficient in twitching motility and microcolony formation. We carried out an extragenic suppressor screen in PA103ΔfimX bacteria to identify additional regulators of pilus assembly. Multiple suppressor mutations were mapped to PA0171, PA1121 (yfiR), and PA3703 (wspF), three genes previously associated with small-colony-variant phenotypes. Multiple independent techniques confirmed that suppressors assembled functional surface pili, though at both polar and nonpolar sites. Whole-cell c-di-GMP levels were elevated in suppressor strains, in agreement with previous studies that had shown that the disrupted genes encoded negative regulators of diguanylate cyclases. Overexpression of the regulated diguanylate cyclases was sufficient to suppress the ΔfimX pilus assembly defect, as was overexpression of an unrelated diguanylate cyclase from Caulobacter crescentus. Furthermore, under natural conditions of high c-di-GMP, PA103ΔfimX formed robust biofilms that showed T4P staining and were structurally distinct from those formed by nonpiliated bacteria. These results are the first demonstration that P. aeruginosa assembles a surface organelle, type IV pili, over a broad range of c-di-GMP concentrations. Assembly of pili at low c-di-GMP concentrations requires a polarly localized c-di-GMP binding protein and phosphodiesterase, FimX; this requirement for FimX is bypassed at high c-di-GMP concentrations. Thus, P. aeruginosa can assemble the same surface organelle in distinct ways for motility or adhesion under very different environmental conditions.

Topics: Bacterial Secretion Systems; Caulobacter crescentus; Cyclic GMP; Fimbriae, Bacterial; Gene Deletion; Hydrolysis; Pseudomonas aeruginosa; Suppression, Genetic

Detection of foreign materials is the first step of successful immune responses. Stimulator of interferon genes (STING) was shown to directly bind cyclic diguanylate monophosphate (c-di-GMP), a bacterial second messenger, and to elicit strong interferon responses. Here we elucidate the structural features of the cytosolic c-di-GMP binding domain (CBD) of STING and its complex with c-di-GMP. The CBD exhibits an α + β fold and is a dimer in the crystal and in solution. Surprisingly, one c-di-GMP molecule binds to the central crevice of a STING dimer, using a series of stacking and hydrogen bonding interactions. We show that STING is autoinhibited by an intramolecular interaction between the CBD and the C-terminal tail (CTT) and that c-di-GMP releases STING from this autoinhibition by displacing the CTT. The structures provide a remarkable example of pathogen-host interactions in which a unique microbial molecule directly engages the innate immune system.

Topics: Amino Acid Sequence; Binding Sites; Cyclic GMP; Dimerization; Humans; Hydrogen Bonding; Immunity, Innate; Membrane Proteins; Molecular Sequence Data; Protein Structure, Tertiary; Signal Transduction

Two-component systems (TCS) consisting of histidine kinases (HK) and response regulators (RR) play essential roles in bacteria to sense environmental signals and regulate cell functions. One type of RR is involved in metabolism of cyclic diguanylate (c-di-GMP), a ubiquitous bacterial second messenger. Although genomic studies predicted a large number of them existing in different bacteria, only a few have been studied. In this work, we characterized a novel TCS consisting of PdeK(PXO_01018)/PdeR(PXO_ 01019) from Xanthomonas oryzae pv. oryzae, which causes the bacterial leaf blight of rice. PdeR (containing GGDEF, EAL, and REC domains) was shown to have phosphodiesterase (PDE) activity in vitro by colorimetric assays and high-performance liquid chromatography analysis. The PDE activity of full-length PdeR needs to be triggered by HK PdeK. Deletion of pdeK or pdeR in X. oryzae pv. oryzae PXO99(A) had attenuated its virulence on rice. ΔpdeK and ΔpdeR secreted less exopolysaccharide than the wild type but there were no changes in terms of motility or extracellular cellulase activity, suggesting the activity of PdeK/PdeR might be specific.

Topics: Bacterial Proteins; Cloning, Molecular; Computational Biology; Cyclic GMP; Gene Expression Regulation, Bacterial; Histidine Kinase; Oryza; Phosphoric Diester Hydrolases; Protein Kinases; Two-Hybrid System Techniques; Virulence; Xanthomonas

Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger, has emerged as a key controller of several biological processes. Numbers of reports that deal with the mechanistic aspects of this second messenger have appeared in the literature. However, the lack of a reporter tag attached to the c-di-GMP at times limits the understanding of further details. In this study, we have chemically coupled N-methylisatoic anhydride (MANT) with c-di-GMP, giving rise to Mant-(c-di-GMP) or MANT-CDG. We have characterized the chemical and physical properties and spectral behavior of MANT-CDG. The fluorescence of MANT-CDG is sensitive to changes in the microenvironment, which helped us study its interaction with three different c-di-GMP binding proteins (a diguanylate cyclase, a phosphodiesterase, and a PilZ domain-containing protein). In addition, we have shown here that MANT-CDG can inhibit diguanylate cyclase activity; however, it is hydrolyzed by c-di-GMP specific phosphodiesterase. Taken together, our data suggest that MANT-CDG behaves like native c-di-GMP, and this study raises the possibility that MANT-CDG will be a valuable research tool for the in vitro characterization of c-di-GMP signaling factors.

Topics: Amino Acid Sequence; Bacterial Proteins; Chemistry Techniques, Synthetic; Cyclic GMP; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Hydrolysis; Kinetics; Molecular Sequence Data; Reproducibility of Results

STING (stimulator of interferon genes) is an innate immune sensor of cyclic dinucleotides that regulates the induction of type I interferons. STING's C-terminal domain forms a V-shaped dimer and binds a cyclic diguanylate monophosphate (c-di-GMP) at the dimer interface by both direct and solvent-mediated hydrogen bonds. Guanines of c-di-GMP stack against the phenolic rings of a conserved tyrosine, and mutations at the c-di-GMP binding surface reduce nucleotide binding and affect signaling.

Topics: Animals; Cell Line; Cyclic GMP; Humans; Hydrogen Bonding; Membrane Proteins; Mice; Models, Molecular; Protein Conformation

STING (stimulator of interferon genes) is an essential signaling adaptor that mediates cytokine production in response to microbial invasion by directly sensing bacterial secondary messengers such as the cyclic dinucleotide bis-(3'-5')-cyclic dimeric GMP (c-di-GMP). STING's structure and its binding mechanism to cyclic dinucleotides were unknown. We report here the crystal structures of the STING cytoplasmic domain and its complex with c-di-GMP, thus providing the structural basis for understanding STING function.

Topics: Amino Acid Sequence; Crystallography, X-Ray; Cyclic GMP; Humans; Membrane Proteins; Models, Molecular; Molecular Sequence Data; Protein Binding; Protein Conformation; Sequence Homology, Amino Acid

STING functions as both an adaptor protein signaling cytoplasmic double-stranded DNA and a direct immunosensor of cyclic diguanylate monophosphate (c-di-GMP). The crystal structures of the C-terminal domain of human STING (STING(CTD)) and its complex with c-di-GMP reveal how STING recognizes c-di-GMP. In response to c-di-GMP binding, two surface loops, which serve as a gate and latch of the cleft formed by the dimeric STING(CTD), undergo rearrangements to interact with the ligand.

Topics: Cyclic GMP; Humans; Membrane Proteins; Models, Molecular; Protein Conformation

Cyclic dimeric GMP (c-di-GMP) regulates numerous processes in Gram-negative bacteria, yet little is known about its role in Gram-positive bacteria. Here we characterize two c-di-GMP phosphodiesterases from the filamentous high-GC Gram-positive actinobacterium Streptomyces coelicolor, involved in controlling colony morphology and development. A transposon mutation in one of the two phosphodiesterase genes, SCO0928, hereby designated rmdA (regulator of morphology and development A), resulted in decreased levels of spore-specific gray pigment and a delay in spore formation. The RmdA protein contains GGDEF-EAL domains arranged in tandem and possesses c-di-GMP phosphodiesterase activity, as is evident from in vitro enzymatic assays using the purified protein. RmdA contains a PAS9 domain and is a hemoprotein. Inactivation of another GGDEF-EAL-encoding gene, SCO5495, designated rmdB, resulted in a phenotype identical to that of the rmdA mutant. Purified soluble fragment of RmdB devoid of transmembrane domains also possesses c-di-GMP phosphodiesterase activity. The rmdA rmdB double mutant has a bald phenotype and is impaired in aerial mycelium formation. This suggests that RmdA and RmdB functions are additive and at least partially overlapping. The rmdA and rmdB mutations likely result in increased local pools of intracellular c-di-GMP, because intracellular c-di-GMP levels in the single mutants did not differ significantly from those of the wild type, whereas in the double rmdA rmdB mutant, c-di-GMP levels were 3-fold higher than those in the wild type. This study highlights the importance of c-di-GMP-dependent signaling in actinomycete colony morphology and development and identifies two c-di-GMP phosphodiesterases controlling these processes.

Topics: Bacterial Proteins; Cyclic GMP; DNA Transposable Elements; Gene Expression Regulation, Bacterial; Mutation; Phosphoric Diester Hydrolases; Spores, Bacterial; Streptomyces coelicolor

The nucleotide messenger cyclic di-GMP (c-di-GMP) plays a central role in the regulation of motility, virulence, and biofilm formation in many pathogenic bacteria. EAL domain-containing phosphodiesterases are the major signaling proteins responsible for the degradation of c-di-GMP and maintenance of its cellular level. We determined the crystal structure of a single mutant (R286W) of the response regulator RocR from Pseudomonas aeruginosa to show that RocR exhibits a highly unusual tetrameric structure arranged around a single dyad, with the four subunits adopting two distinctly different conformations. Subunits A and B adopt a conformation with the REC domain located above the c-di-GMP binding pocket, whereas subunits C and D adopt an open conformation with the REC domain swung to the side of the EAL domain. Remarkably, the access to the substrate-binding pockets of the EAL domains of the open subunits C and D are blocked in trans by the REC domains of subunits A and B, indicating that only two of the four active sites are engaged in the degradation of c-di-GMP. In conjunction with biochemical and biophysical data, we propose that the structural changes within the REC domains triggered by the phosphorylation are transmitted to the EAL domain active sites through a pathway that traverses the dimerization interfaces composed of a conserved regulatory loop and the neighboring motifs. This exquisite mechanism reinforces the crucial role of the regulatory loop and suggests that similar regulatory mechanisms may be operational in many EAL domain proteins, considering the preservation of the dimerization interface and the spatial arrangement of the regulatory domains.

Topics: Crystallography, X-Ray; Cyclic GMP; Models, Molecular; Mutant Proteins; Mutation, Missense; Protein Multimerization; Protein Structure, Quaternary; Pseudomonas aeruginosa; Signal Transduction; Transcription Factors

Cyclic di-GMP (c-di-GMP) is a second messenger molecule that regulates the transition between sessile and motile lifestyles in bacteria. Bacteria often encode multiple diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes that produce and degrade c-di-GMP, respectively. Because of multiple inputs into the c-di-GMP-signaling network, it is unclear whether this system functions via high or low specificity. High-specificity signaling is characterized by individual DGCs or PDEs that are specifically associated with downstream c-di-GMP-mediated responses. In contrast, low-specificity signaling is characterized by DGCs or PDEs that modulate a general signal pool, which, in turn, controls a global c-di-GMP-mediated response. To determine whether c-di-GMP functions via high or low specificity in Vibrio cholerae, we correlated the in vivo c-di-GMP concentration generated by seven DGCs, each expressed at eight different levels, to the c-di-GMP-mediated induction of biofilm formation and transcription. There was no correlation between total intracellular c-di-GMP levels and biofilm formation or gene expression when considering all states. However, individual DGCs showed a significant correlation between c-di-GMP production and c-di-GMP-mediated responses. Moreover, the rate of phenotypic change versus c-di-GMP concentration was significantly different between DGCs, suggesting that bacteria can optimize phenotypic output to c-di-GMP levels via expression or activation of specific DGCs. Our results conclusively demonstrate that c-di-GMP does not function via a simple, low-specificity signaling pathway in V. cholerae.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Phosphorus-Oxygen Lyases; Second Messenger Systems; Vibrio cholerae

The second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays a vital role in the global regulation in bacteria. Here, we describe structural and biochemical characterization of a novel c-di-GMP effector PelD that is critical to the formation of pellicles by Pseudomonas aeruginosa. We present high-resolution structures of a cytosolic fragment of PelD in apo form and its complex with c-di-GMP. The structure contains a bi-domain architecture composed of a GAF domain (commonly found in cyclic nucleotide receptors) and a GGDEF domain (found in c-di-GMP synthesizing enzymes), with the latter binding to one molecule of c-di-GMP. The GGDEF domain has a degenerate active site but a conserved allosteric site (I-site), which we show binds c-di-GMP with a K(d) of 0.5 μm. We identified a series of residues that are crucial for c-di-GMP binding, and confirmed the roles of these residues through biochemical characterization of site-specific variants. The structures of PelD represent a novel class of c-di-GMP effector and expand the knowledge of scaffolds that mediate c-di-GMP recognition.

Topics: Bacterial Proteins; Carrier Proteins; Cell Wall; Crystallography, X-Ray; Cyclic GMP; Protein Structure, Tertiary; Pseudomonas aeruginosa

Cyclic di-GMP (c-di-GMP) is a second messenger that regulates diverse cellular processes in bacteria, including motility, biofilm formation, cell-cell signaling, and host colonization. Studies of c-di-GMP signaling have chiefly focused on Gram-negative bacteria. Here, we investigated c-di-GMP signaling in the Gram-positive bacterium Bacillus subtilis by constructing deletion mutations in genes predicted to be involved in the synthesis, breakdown, or response to the second messenger. We found that a putative c-di-GMP-degrading phosphodiesterase, YuxH, and a putative c-di-GMP receptor, YpfA, had strong influences on motility and that these effects depended on sequences similar to canonical EAL and RxxxR-D/NxSxxG motifs, respectively. Evidence indicates that YpfA inhibits motility by interacting with the flagellar motor protein MotA and that yuxH is under the negative control of the master regulator Spo0A∼P. Based on these findings, we propose that YpfA inhibits motility in response to rising levels of c-di-GMP during entry into stationary phase due to the downregulation of yuxH by Spo0A∼P. We also present evidence that YpfA has a mild influence on biofilm formation. In toto, our results demonstrate the existence of a functional c-di-GMP signaling system in B. subtilis that directly inhibits motility and directly or indirectly influences biofilm formation.

Topics: Bacillus subtilis; Biofilms; Cyclic GMP; Gene Deletion; Genes, Bacterial; Locomotion; Signal Transduction

Recent studies of signal transduction in bacteria have revealed a unique second messenger, bis-(3'-5')-cyclic dimeric GMP (c-di-GMP), which regulates transitions between motile states and sessile states, such as biofilms. C-di-GMP is synthesized from two GTP molecules by diguanylate cyclases (DGC). The catalytic activity of DGCs depends on a conserved GG(D/E)EF domain, usually part of a larger multi-domain protein organization. The domains other than the GG(D/E)EF domain often control DGC activation. This paper presents the 1.83 Å crystal structure of an isolated catalytically competent GG(D/E)EF domain from the A1U3W3_MARAV protein from Marinobacter aquaeolei. Co-crystallization with GTP resulted in enzymatic synthesis of c-di-GMP. Comparison with previously solved DGC structures shows a similar orientation of c-di-GMP bound to an allosteric regulatory site mediating feedback inhibition of the enzyme. Biosynthesis of c-di-GMP in the crystallization reaction establishes that the enzymatic activity of this DGC domain does not require interaction with regulatory domains.

Topics: Allosteric Regulation; Allosteric Site; Amino Acid Sequence; Bacterial Proteins; Conserved Sequence; Crystallography, X-Ray; Cyclic GMP; Enzyme Activation; Escherichia coli Proteins; Guanosine Triphosphate; Marinobacter; Molecular Sequence Data; Phosphorus-Oxygen Lyases; Protein Conformation; Protein Interaction Mapping; Protein Structure, Tertiary; Sequence Analysis, Protein

Bacterial biofilm formation is responsible for numerous chronic infections, causing a severe health burden. Many of these infections cannot be resolved, as bacteria in biofilms are resistant to the host's immune defenses and antibiotic therapy. New strategies to treat biofilm-based infections are critically needed. Cyclic di-GMP (c-di-GMP) is a widely conserved second-messenger signal essential for biofilm formation. As this signaling system is found only in bacteria, it is an attractive target for the development of new antibiofilm interventions. Here, we describe the results of a high-throughput screen to identify small-molecule inhibitors of diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP. We report seven small molecules that antagonize these enzymes and inhibit biofilm formation by Vibrio cholerae. Moreover, two of these compounds significantly reduce the total concentration of c-di-GMP in V. cholerae, one of which also inhibits biofilm formation by Pseudomonas aeruginosa in a continuous-flow system. These molecules represent the first compounds described that are able to inhibit DGC activity to prevent biofilm formation.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa; Vibrio cholerae

C-di-GMP regulates important processes involved in biofilm formation and virulence factors production in several bacteria. Herein we report a simple fluorescent strategy that allows for the detection of c-di-GMP (as low as 320 nM) using a Vc2 class I riboswitch domain as the sensing region and spinach as the fluorescent reporting module.

Topics: Aptamers, Nucleotide; Base Sequence; Biosensing Techniques; Cyclic GMP; Riboswitch; RNA, Plant; Spectrometry, Fluorescence

Cyclic di-(3′:5′)-guanosine monophosphate (c-di-GMP) is a major prokaryote signalling intermediate that is synthesized by diguanylate cyclases and triggers sessility and biofilm formation. We detected the first eukaryote diguanylate cyclases in all major groups of Dictyostelia. On food depletion, Dictyostelium discoideum amoebas collect into aggregates, which first transform into migrating slugs and then into sessile fruiting structures. These structures consist of a spherical spore mass that is supported by a column of stalk cells and a basal disk. A polyketide, DIF-1, which induces stalk-like cells in vitro, was isolated earlier. However, its role in vivo proved recently to be restricted to basal disk formation. Here we show that the Dictyostelium diguanylate cyclase, DgcA, produces c-di-GMP as the morphogen responsible for stalk cell differentiation. Dictyostelium discoideum DgcA synthesized c-di-GMP in a GTP-dependent manner and was expressed at the slug tip, which is the site of stalk cell differentiation. Disruption of the DgcA gene blocked the transition from slug migration to fructification and the expression of stalk genes. Fructification and stalk formation were restored by exposing DgcA-null slugs to wild-type secretion products or to c-di-GMP. Moreover, c-di-GMP, but not cyclic di-(3′:5′)-adenosine monophosphate, induced stalk gene expression in dilute cell monolayers. Apart from identifying the long-elusive stalk-inducing morphogen, our work also identifies a role for c-di-GMP in eukaryotes.

Topics: Biological Assay; Cell Differentiation; Cyclic GMP; Dictyostelium; Fruiting Bodies, Fungal; Guanosine Triphosphate; Molecular Sequence Data; Prokaryotic Cells; Protozoan Proteins; RNA, Messenger; Second Messenger Systems

Pseudomonas aeruginosa exhibits distinct surface-associated behaviors, including biofilm formation, flagellum-mediated swarming motility, and type IV pilus-driven twitching. Here, we report a role for the minor pilins, PilW and PilX, components of the type IV pilus assembly machinery, in the repression of swarming motility. Mutating either the pilW or pilX gene alleviates the inhibition of swarming motility observed for strains with elevated levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP) due to loss of BifA, a c-di-GMP-degrading phosphodiesterase. Blocking PilD peptidase-mediated processing of PilW and PilX renders the unprocessed proteins defective for pilus assembly but still functional in c-di-GMP-mediated swarming repression, indicating our ability to separate these functions. Strains with mutations in pilW or pilX also fail to exhibit the increase in c-di-GMP levels observed when wild-type (WT) or bifA mutant cells are grown on a surface. We also provide data showing that c-di-GMP levels are increased upon PilY1 overexpression in surface-grown cells and that this c-di-GMP increase does not occur in the absence of the SadC diguanylate cyclase. Increased levels of endogenous PilY1, PilX, and PilA are observed when cells are grown on a surface compared to liquid growth, linking surface growth and enhanced signaling via SadC. Our data support a model wherein PilW, PilX, and PilY1, in addition to their role(s) in type IV pilus biogenesis, function to repress swarming via modulation of intracellular c-di-GMP levels. By doing so, these pilus assembly proteins contribute to P. aeruginosa's ability to coordinately regulate biofilm formation with its two surface motility systems.

Topics: Amino Acid Sequence; Cyclic GMP; Fimbriae Proteins; Fimbriae, Bacterial; Gene Deletion; Gene Expression Regulation, Bacterial; Movement; Mutation; Phenotype; Pseudomonas aeruginosa

Yersinia pestis, the cause of the disease plague, forms biofilms to enhance flea-to-mammal transmission. Biofilm formation is dependent on exopolysaccharide synthesis and is controlled by the intracellular levels of the second messenger molecule cyclic diguanylate (c-di-GMP), but the mechanisms by which Y. pestis regulates c-di-GMP synthesis and turnover are not fully understood. Here we show that the small RNA chaperone Hfq contributes to the regulation of c-di-GMP levels and biofilm formation by modulating the abundance of both the c-di-GMP phosphodiesterase HmsP and the diguanylate cyclase HmsT. To do so, Hfq co-ordinately promotes hmsP mRNA accumulation while simultaneously decreasing the stability of the hmsT transcript. Hfq-dependent regulation of HmsP occurs at the transcriptional level while the regulation of HmsT is post-transcriptional and is localized to the 5' untranslated region/proximal coding sequence of the hmsT transcript. Decoupling HmsP from Hfq-based regulation is sufficient to overcome the effects of Δhfq on c-di-GMP and biofilm formation. We propose that Y. pestis utilizes Hfq to link c-di-GMP levels to environmental conditions and that the disregulation of c-di-GMP turnover in the absence of Hfq may contribute to the severe attenuation of Y. pestis lacking this RNA chaperone in animal models of plague.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Bacterial Proteins; Base Sequence; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Host Factor 1 Protein; Humans; Molecular Sequence Data; Phosphorus-Oxygen Lyases; Plague; Yersinia pestis

Many bacterial pathogens produce diffusible signal factor (DSF)-type quorum sensing (QS) signals in modulation of virulence and biofilm formation. Previous work on Xanthomonas campestris showed that the RpfC/RpfG two-component system is involved in sensing and responding to DSF signals, but little is known in other microorganisms. Here we show that in Burkholderia cenocepacia the DSF-family signal cis-2-dodecenoic acid (BDSF) negatively controls the intracellular cyclic dimeric guanosine monophosphate (c-di-GMP) level through a receptor protein RpfR, which contains Per/Arnt/Sim (PAS)-GGDEF-EAL domains. RpfR regulates the same phenotypes as BDSF including swarming motility, biofilm formation, and virulence. In addition, the BDSF(-) mutant phenotypes could be rescued by in trans expression of RpfR, or its EAL domain that functions as a c-di-GMP phosphodiesterase. BDSF is shown to bind to the PAS domain of RpfR with high affinity and stimulates its phosphodiesterase activity through induction of allosteric conformational changes. Our work presents a unique and widely conserved DSF-family signal receptor that directly links the signal perception to c-di-GMP turnover in regulation of bacterial physiology.

Topics: Bacterial Proteins; Burkholderia cenocepacia; Cell Communication; Cyclic GMP; Dimerization; Fatty Acids, Monounsaturated; Guanosine Monophosphate; Models, Genetic; Mutagenesis; Mutation; Phenotype; Protein Binding; Quorum Sensing; Receptors, Cell Surface; Signal Transduction; Virulence

Pseudomonas aeruginosa responds to growth on agar surfaces to produce cyclic-di-GMP, which stimulates biofilm formation. This is mediated by an alternative cellular function chemotaxis-like system called Wsp. The receptor protein WspA, is bioinformatically indistinguishable from methyl-accepting chemotaxis proteins. However, unlike standard chemoreceptors, WspA does not form stable clusters at cell poles. Rather, it forms dynamic clusters at both polar and lateral subcellular locations. To begin to study the mechanism of Wsp signal transduction in response to surfaces, we carried out a structure-function study of WspA and found that its C-terminus is important for its lateral subcellular localization and function. When this region was replaced with that of a chemoreceptor for amino acids, WspA became polarly localized. In addition, introduction of mutations in the C-terminal region of WspA that rendered this protein able to form more stable receptor-receptor interactions, also resulted in a WspA protein that was less capable of activating signal transduction. Receptor chimeras with a WspA C-terminus and N-terminal periplasmic domains from chemoreceptors that sense amino acids or malate responded to surfaces to produce c-di-GMP. Thus, the amino acid sequence of the WspA periplasmic region did not need to be conserved for the Wsp system to respond to surfaces.

Topics: Amino Acid Sequence; Bacterial Proteins; Cyclic GMP; Membrane Proteins; Molecular Sequence Data; Protein Transport; Pseudomonas aeruginosa; Sequence Alignment

Francisella tularensis is a gram-negative bacterium that is highly virulent in humans, causing the disease tularemia. F. novicida is closely related to F. tularensis and exhibits high virulence in mice, but it is avirulent in healthy humans. An F. novicida-specific gene cluster (FTN0451 to FTN0456) encodes two proteins with diguanylate cyclase (DGC) and phosphodiesterase (PDE) domains that modulate the synthesis and degradation of cyclic di-GMP (cdGMP). No DGC- or PDE-encoding protein genes are present in the F. tularensis genome. F. novicida strains lacking either the two DGC/PDE genes (cdgA and cdgB) or the entire gene cluster (strain KKF457) are defective for biofilm formation. In addition, expression of CdgB or a heterologous DGC in strain KKF457 stimulated F. novicida biofilms, even in a strain lacking the biofilm regulator QseB. Genetic evidence suggests that CdgA is predominantly a PDE, while CdgB is predominantly a DGC. The F. novicida qseB strain showed reduced cdgA and cdgB transcript levels, demonstrating an F. novicida biofilm signaling cascade that controls cdGMP levels. Interestingly, KKF457 with elevated cdGMP levels exhibited a decrease in intramacrophage replication and virulence in mice, as well as increased growth yields and biofilm formation in vitro. Microarray analyses revealed that cdGMP stimulated the transcription of a chitinase (ChiB) known to contribute to biofilm formation. Our results indicate that elevated cdGMP in F. novicida stimulates biofilm formation and inhibits virulence. We suggest that differences in human virulence between F. novicida and F. tularensis may be due in part to the absence of cdGMP signaling in F. tularensis.

Topics: Animals; Bacterial Proteins; Biofilms; Chitinases; Cyclic GMP; Female; Francisella; Gene Expression Regulation, Bacterial; Gram-Negative Bacterial Infections; Humans; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Sequence Analysis, DNA; Virulence

Cyclic di-GMP (c-di-GMP) is a novel secondary-messenger molecule that is involved in regulating a plethora of important bacterial activities through binding to an unprecedented array of effectors. Proteins with a canonical PilZ domain that bind c-di-GMP play crucial roles in regulating flagellum-based motility. In contrast, noncanonical type II PilZ domains that do not effectively bind c-di-GMP regulate twitching motility, which is dependent on type IV pili (T4P). Recent data indicate that T4P biogenesis is initiated via the interaction of a noncanonical type II PilZ protein with the GGDEF/EAL-domain protein FimX and the pilus motor protein PilB at high c-di-GMP concentrations. However, the molecular details of such interactions remain to be elucidated. In this manuscript, the first hetero-complex crystal structure between a type II PilZ protein and the EAL domain of the FimX protein (FimX(EAL)) from Xanthomonas campestris pv. campestris (Xcc) in the presence of c-di-GMP is reported. This work reveals two novel conformations of monomeric c-di-GMP in the XccFimX(EAL)-c-di-GMP and XccFimX(EAL)-c-di-GMP-XccPilZ complexes, as well as a unique interaction mode of a type II PilZ domain with FimX(EAL). These findings indicate that c-di-GMP is sufficiently flexible to adjust its conformation to match the corresponding recognition motifs of different cognate effectors. Together, these results represent a first step towards an understanding of how T4P biogenesis is controlled by c-di-GMP at the molecular level and also of the ability of c-di-GMP to bind to a wide variety of effectors.

Topics: Bacterial Proteins; Crystallography, X-Ray; Cyclic GMP; Protein Binding; Protein Structure, Tertiary; Pseudomonas aeruginosa; Sequence Homology, Amino Acid; Xanthomonas campestris

Dispersion enables biofilm bacteria to transit from the biofilm to the planktonic growth state and to spawn novel communities in new locales. Although the chemotaxis protein BdlA plays a role in the dispersion of Pseudomonas aeruginosa biofilms in response to environmental cues, little is known about regulation of BdlA activity or how BdlA modulates the dispersion response. Here, we demonstrate that BdlA in its native form is inactive and is activated upon nonprocessive proteolysis at a ClpP-protease-like cleavage site located between the Per Arnt Sim (PAS) sensory domains PASa and PASb. Activation of BdlA to enable biofilm dispersion requires phosphorylation at tyrosine-238 as a signal, elevated c-di-GMP levels, the chaperone ClpD, and the protease ClpP. The resulting truncated BdlA polypeptide chains directly interact and are required for P. aeruginosa biofilms to disperse. Our results provide a basis for understanding the mechanism of biofilm dispersion that may be applicable to a large number of biofilm-forming pathogenic species. Insights into the mechanism of BdlA function have implications for the control of biofilm-related infections.

Topics: Bacterial Proteins; Base Sequence; Binding Sites; Biofilms; Cyclic GMP; Endopeptidase Clp; Immunoblotting; Mutation; Phosphorylation; Protein Processing, Post-Translational; Proteolysis; Pseudomonas aeruginosa; Signal Transduction; Tyrosine

Bacterial polynucleotide phosphorylase (PNPase) is a 3'-5' processive exoribonuclease that participates in mRNA turnover and quality control of rRNA precursors in many bacterial species. It also associates with the RNase E scaffold and other components to form a multi-enzyme RNA degradasome machinery that performs a wider regulatory role in degradation, quality control and maturation of mRNA and noncoding RNA. Several crystal structures of bacterial PNPases, as well as some biological activity studies, have been published. However, how the enzymatic activity of PNPase is regulated is less well understood. Recently, Escherichia coli PNPase was found to be a direct c-di-GMP binding target, raising the possibility that c-di-GMP may participate in the regulation of RNA processing. Here, the successful cloning, purification and crystallization of S1-domain-truncated Xanthomonas campestris PNPase (XcPNPaseΔS1) in the presence of c-di-GMP are reported. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 132.76, b = 128.38, c = 133.01 Å, γ = 93.3°, and diffracted to a resolution of 2.00 Å.

Topics: Crystallization; Crystallography, X-Ray; Cyclic GMP; Polyribonucleotide Nucleotidyltransferase; Xanthomonas campestris

Intracellular concentration of cyclic diguanylate monophosphate (c-di-GMP), a second messenger molecule, is regulated in bacteria by diguanylate cyclases (DGCs) (synthesizing c-di-GMP) and phosphodiesterases (PDEs) (degrading c-di-GMP). c-di-GMP concentration ([c-di-GMP]) affects motility and sessility in a reciprocal fashion; high [c-di-GMP] typically inhibits motility and promotes sessility. A c-di-GMP sensor domain, PilZ, also regulates motility and sessility. Uropathogenic Escherichia coli regulates these processes during infection; motility is necessary for ascending the urinary tract, while sessility is essential for colonization of anatomical sites. Here, we constructed and screened 32 mutants containing deletions of genes encoding each PDE (n = 11), DGC (n = 13), PilZ (n = 2), and both PDE and DGC (n = 6) domains for defects in motility, biofilm formation, and adherence for the prototypical pyelonephritis isolate E. coli CFT073. Three of 32 mutations affected motility, all of which were in genes encoding enzymatically inactive PDEs. Four PDEs, eight DGCs, four PDE/DGCs, and one PilZ regulated biofilm formation in a medium-specific manner. Adherence to bladder epithelial cells was regulated by [c-di-GMP]. Four PDEs, one DGC, and three PDE/DGCs repress adherence and four DGCs and one PDE/DGC stimulate adherence. Thus, specific effectors of [c-di-GMP] and catalytically inactive DGCs and PDEs regulate adherence and motility in uropathogenic E. coli. IMPORTANCE Uropathogenic Escherichia coli (UPEC) contains several genes annotated as encoding enzymes that increase or decrease the abundance of the second messenger molecule, c-di-GMP. While this class of enzymes has been studied in an E. coli K-12 lab strain, these proteins have not been comprehensively examined in UPEC. UPEC utilizes both swimming motility and adherence to colonize and ascend the urinary tract; both of these processes are hypothesized to be regulated by the concentration of c-di-GMP. Here, for the first time, in a uropathogenic strain, E. coli CFT073, we have characterized mutants lacking each protein and demonstrated that the uropathogen has diverged from E. coli K-12 to utilize these enzymes to regulate adherence and motility by distinct mechanisms.

Topics: Bacterial Adhesion; Cells, Cultured; Cyclic GMP; Epithelial Cells; Escherichia coli Proteins; Gene Deletion; Humans; Locomotion; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Uropathogenic Escherichia coli

In a bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP)/transcription factor binding screen, we identified Mycobacterium smegmatis Ms6479 as the first c-di-GMP-responsive transcriptional factor in mycobacteria. Ms6479 could specifically bind with c-di-GMP and recognize the promoters of 37 lipid transport and metabolism genes. c-di-GMP could enhance the ability of Ms6479 to bind to its target DNA. Furthermore, our results establish Ms6479 as a global activator that positively regulates the expression of diverse target genes. Overexpression of Ms6479 in M. smegmatis significantly reduced the permeability of the cell wall to crystal violet and increased mycobacterial resistance to anti-tuberculosis antibiotics. Interestingly, Ms6479 lacks the previously reported c-di-GMP binding motifs. Our findings introduce Ms6479 (here designated LtmA for lipid transport and metabolism activator) as a new c-di-GMP-responsive regulator.

Topics: Bacterial Proteins; Biological Transport; Cyclic GMP; Drug Resistance, Bacterial; Gene Expression Regulation, Bacterial; Inverted Repeat Sequences; Lipid Metabolism; Mycobacterium smegmatis; Promoter Regions, Genetic; Transcription Factors

The induction of type I interferons by the bacterial secondary messengers cyclic di-GMP (c-di-GMP) or cyclic di-AMP (c-di-AMP) is dependent on a signaling axis that involves the adaptor STING, the kinase TBK1 and the transcription factor IRF3. Here we identified the heliase DDX41 as a pattern-recognition receptor (PRR) that sensed both c-di-GMP and c-di-AMP. DDX41 specifically and directly interacted with c-di-GMP. Knockdown of DDX41 via short hairpin RNA in mouse or human cells inhibited the induction of genes encoding molecules involved in the innate immune response and resulted in defective activation of STING, TBK1 and IRF3 in response to c-di-GMP or c-di-AMP. Our results suggest a mechanism whereby c-di-GMP and c-di-AMP are detected by DDX41, which forms a complex with STING to signal to TBK1-IRF3 and activate the interferon response.

Topics: Animals; Cell Line; Cyclic GMP; DEAD-box RNA Helicases; Dinucleoside Phosphates; Humans; Immunity, Innate; Interferon Regulatory Factor-3; Interferon Type I; Listeria monocytogenes; Macrophages; Membrane Proteins; Mice; Protein Serine-Threonine Kinases; Receptors, Pattern Recognition; RNA Interference; RNA, Small Interfering; Second Messenger Systems; Signal Transduction

Topics: Animals; Cyclic GMP; DEAD-box RNA Helicases; Dinucleoside Phosphates; Humans; Interferon Type I; Listeria monocytogenes; Receptors, Pattern Recognition

c-di-GMP is a bacterial second messenger that is enzymatically synthesized and degraded in response to environmental signals. Cellular processes are affected when c-di-GMP binds to receptors which include proteins that contain the PilZ domain. Although each c-di-GMP synthesis or degradation enzyme metabolizes the same molecule, many of these enzymes can be linked to specific downstream processes. Here we present evidence that c-di-GMP signalling specificity is achieved through differences in affinities of receptor macromolecules. We show that the PilZ domain proteins of Salmonella Typhimurium, YcgR and BcsA, demonstrate a 43-fold difference in their affinity for c-di-GMP. Modulation of the affinities of these proteins altered their activities in a predictable manner in vivo. Inactivation of yhjH, which encodes a predicted c-di-GMP degrading enzyme, increased the fraction of the cellular population that demonstrated c-di-GMP levels high enough to bind to the higher-affinity YcgR protein and inhibit motility, but not high enough to bind to the lower-affinity BcsA protein and stimulate cellulose production. Finally, PilZ domain proteins of Pseudomonas aeruginosa demonstrated a 145-fold difference in binding affinities, suggesting that regulation by binding affinity may be a conserved mechanism that allows organisms with many c-di-GMP binding macromolecules to rapidly integrate multiple environmental signals into one output.

Topics: Bacterial Proteins; Cellulose; Cyclic GMP; Gene Expression Regulation, Bacterial; Locomotion; Protein Binding; Salmonella typhimurium; Signal Transduction

Bacteria have a tendency to attach to surfaces and grow as structured communities called biofilms. Chronic biofilm infections are a problem because they tend to resist antibiotic treatment and are difficult to eradicate. Bacterial biofilms have an extracellular matrix that is usually composed of a mixture of polysaccharides, proteins, and nucleic acids. This matrix has long been assumed to play a passive structural and protective role for resident biofilm cells. Here we show that this view is an oversimplification and that the biofilm matrix can play an active role in stimulating its own synthesis. Working with the model biofilm bacterium Pseudomonas aeruginosa, we found that Psl, a major biofilm matrix polysaccharide for this species, acts as a signal to stimulate two diguanylate cyclases, SiaD and SadC, to produce the intracellular secondary messenger molecule c-di-GMP. Elevated intracellular concentrations of c-di-GMP then lead to the increased production of Psl and other components of the biofilm. This mechanism represents a unique positive feedback regulatory circuit, where the expression of an extracellular polysaccharide promotes biofilm growth in a manner analogous to autocrine signaling in eukaryotes.

Topics: Bacterial Proteins; Base Sequence; Biofilms; Cyclic GMP; DNA, Bacterial; Escherichia coli Proteins; Extracellular Matrix; Feedback, Physiological; Genes, Bacterial; Phosphorus-Oxygen Lyases; Polysaccharides, Bacterial; Pseudomonas aeruginosa; Second Messenger Systems; Signal Transduction

Recombinant full-length diguanylate cyclases (DGCs) of Thermotoga maritima with native and mutant allosteric sites were overexpressed in Escherichia coli cells and characterized. It has been shown that target enzymes are produced substantially in the form of active inclusion bodies. Introduction of the mutation in allosteric site resulted in 7-fold increase of the T. maritima DGC activity. Possibility of applying full-length DGC of T. maritima in the form of inclusion bodies for synthesis of c-di-GMP was originally demonstrated.

Topics: Allosteric Site; Biotechnology; Cyclic GMP; Electrophoresis, Polyacrylamide Gel; Escherichia coli Proteins; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Inclusion Bodies; Kinetics; Mutation; Phosphorus-Oxygen Lyases; Recombinant Proteins; Salts; Thermotoga maritima

Yersinia pestis synthesizes the attached biofilms in the flea proventriculus, which is important for the transmission of this pathogen by fleas. The hmsHFRS operons is responsible for the synthesis of exopolysaccharide (the major component of biofilm matrix), which is activated by the signaling molecule 3', 5'-cyclic diguanylic acid (c-di-GMP) synthesized by the only two diguanylate cyclases HmsT, and YPO0449 (located in a putative operonYPO0450-0448).. The phenotypic assays indicated that the transcriptional regulator Fur inhibited the Y. pestis biofilm production in vitro and on nematode. Two distinct Fur box-like sequences were predicted within the promoter-proximal region of hmsT, suggesting that hmsT might be a direct Fur target. The subsequent primer extension, LacZ fusion, electrophoretic mobility shift, and DNase I footprinting assays disclosed that Fur specifically bound to the hmsT promoter-proximal region for repressing the hmsT transcription. In contrast, Fur had no regulatory effect on hmsHFRS and YPO0450-0448 at the transcriptional level. The detection of intracellular c-di-GMP levels revealed that Fur inhibited the c-di-GMP production.. Y. pestis Fur inhibits the c-di-GMP production through directly repressing the transcription of hmsT, and thus it acts as a repressor of biofilm formation. Since the relevant genetic contents for fur, hmsT, hmsHFRS, and YPO0450-0448 are extremely conserved between Y. pestis and typical Y. pseudotuberculosis, the above regulatory mechanisms can be applied to Y. pseudotuberculosis.

Topics: Animals; Bacterial Proteins; Base Sequence; Biofilms; Biological Assay; Caenorhabditis elegans; Cyclic GMP; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Promoter Regions, Genetic; Repressor Proteins; Transcription, Genetic; Yersinia pestis

FimX is a large multidomain protein containing an EAL domain and involved in twitching motility in Pseudomonas aeruginosa. We present here two crystallographic structures of the EAL domain of FimX (residues 438-686): one of the apo form and the other of a complex with 5'-pGpG, the reaction product of the hydrolysis of c-di-GMP. In both crystal forms, the EAL domains form a dimer delimiting a large cavity encompassing the catalytic pockets. The ligand is trapped in this cavity by its sugar phosphate moiety. We confirmed by NMR that the guanine bases are not involved in the interaction in solution. We solved here the first structure of an EAL domain bound to the reaction product 5'-pGpG. Though isolated FimX EAL domain has a very low catalytic activity, which would not be significant compared to other catalytic EAL domains, the structure with the product of the reaction can provides some hints in the mechanism of hydrolysis of the c-di-GMP by EAL domains.

Topics: Amino Acid Sequence; Apoproteins; Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Cyclic GMP; Deoxyguanine Nucleotides; Hydrolysis; Ligands; Magnesium; Models, Molecular; Molecular Sequence Data; Nitrophenols; Phosphoric Diester Hydrolases; Protein Binding; Protein Multimerization; Protein Structure, Tertiary; Pseudomonas aeruginosa; Sequence Alignment; Solutions

Biofilm formation and dispersal in the black rot pathogen Xanthomonas campestris pathovar campestris (Xcc) is influenced by a number of factors. The extracellular mannanase ManA has been implicated in biofilm dispersal whereas biofilm formation requires a putative glycosyl transferase encoded by the xag gene cluster. Previously we demonstrated that the post-transcriptional regulator RsmA exerts a negative regulatory influence on biofilm formation in Xcc. Here we address the mechanisms by which RsmA exerts this action. We show that RsmA binds to the transcripts of three genes encoding GGDEF domain diguanylate cyclases to influence their expression. Accordingly, mutation of rsmA leads to an increase in cellular levels of cyclic di-GMP. This effect is associated with a down-regulation of transcription of manA, but an upregulation of xag gene transcription. Mutation of clp, which encodes a cyclic di-GMP-responsive transcriptional regulator of the CRP-FNR family, has similar divergent effects on the expression of manA and xag. Nevertheless Clp binding to manA and xag promoters is inhibited by cyclic di-GMP. The data support the contention that, in common with other CRP-FNR family members, Clp can act as both an activator and repressor of transcription of different genes to influence biofilm formation as a response to cyclic di-GMP.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Models, Biological; Mutation; Promoter Regions, Genetic; Protein Binding; Protein Interaction Domains and Motifs; RNA, Messenger; Transcription Factors; Transcription, Genetic; Xanthomonas campestris

Bacteria prefer to grow attached to themselves or an interface, and it is important for an array of applications to make biofilms disperse. Here we report simultaneously the discovery and protein engineering of BdcA (formerly YjgI) for biofilm dispersal using the universal signal 3,5-cyclic diguanylic acid (c-di-GMP). The bdcA deletion reduced biofilm dispersal, and production of BdcA increased biofilm dispersal to wild-type level. Since BdcA increases motility and extracellular DNA production while decreasing exopolysaccharide, cell length and aggregation, we reasoned that BdcA decreases the concentration of c-di-GMP, the intracellular messenger that controls cell motility through flagellar rotation and biofilm formation through synthesis of curli and cellulose. Consistently, c-di-GMP levels increase upon deleting bdcA, and purified BdcA binds c-di-GMP but does not act as a phosphodiesterase. Additionally, BdcR (formerly YjgJ) is a negative regulator of bdcA. To increase biofilm dispersal, we used protein engineering to evolve BdcA for greater c-di-GMP binding and found that the single amino acid change E50Q causes nearly complete removal of biofilms via dispersal without affecting initial biofilm formation.

Topics: Biofilms; Cyclic GMP; Escherichia coli K12; Escherichia coli Proteins; Phosphoric Diester Hydrolases; Protein Engineering

Type I interferons (IFNs) are central regulators of the innate and adaptive immune responses to viral and bacterial infections. Type I IFNs are induced upon cytosolic detection of microbial nucleic acids, including DNA, RNA, and the bacterial second messenger cyclic-di-GMP (c-di-GMP). In addition, a recent study demonstrated that the intracellular bacterial pathogen Listeria monocytogenes stimulates a type I IFN response due to cytosolic detection of bacterially secreted c-di-AMP. The transmembrane signaling adaptor Sting (Tmem173, Mita, Mpys, Eris) has recently been implicated in the induction of type I IFNs in response to cytosolic DNA and/or RNA. However, the role of Sting in response to purified cyclic dinucleotides or during in vivo L. monocytogenes infection has not been addressed. In order to identify genes important in the innate immune response, we have been conducting a forward genetic mutagenesis screen in C57BL/6 mice using the mutagen N-ethyl-N-nitrosourea (ENU). Here we describe a novel mutant mouse strain, Goldenticket (Gt), that fails to produce type I IFNs upon L. monocytogenes infection. By genetic mapping and complementation experiments, we found that Gt mice harbor a single nucleotide variant (T596A) of Sting that functions as a null allele and fails to produce detectable protein. Analysis of macrophages isolated from Gt mice revealed that Sting is absolutely required for the type I interferon response to both c-di-GMP and c-di-AMP. Additionally, Sting is required for the response to c-di-GMP and L. monocytogenes in vivo. Our results provide new functions for Sting in the innate interferon response to pathogens.

Topics: Alleles; Animals; Cell Line; Cyclic GMP; Dinucleoside Phosphates; Ethylnitrosourea; Female; Gene Expression Regulation; Genetic Complementation Test; Humans; Interferon Type I; Listeria monocytogenes; Listeriosis; Macrophages; Male; Membrane Proteins; Mice; Mice, Mutant Strains; Mutation; Polymorphism, Single Nucleotide

The second messenger 3'-5'-cyclic diguanylic acid (c-di-GMP) promotes biofilm formation, and c-di-GMP is synthesized by diguanylate cyclases (characterized by a GGDEF domain) and degraded by phosphodiesterases. Here, we evaluated the effect of the 12 E. coli GGDEF-only proteins on biofilm formation and motility. Deletions of the genes encoding the GGDEF proteins YeaI, YedQ, YfiN, YeaJ, and YneF increased swimming motility as expected for strains with reduced c-di-GMP. Alanine substitution in the EGEVF motif of YeaI abolished its impact on swimming motility. In addition, extracellular DNA (eDNA) was increased as expected for the deletions of yeaI (tenfold), yedQ (1.8-fold), and yfiN (3.2-fold). As a result of the significantly enhanced motility, but contrary to current models of decreased biofilm formation with decreased diguanylate cyclase activity, early biofilm formation increased dramatically for the deletions of yeaI (30-fold), yedQ (12-fold), and yfiN (18-fold). Our results indicate that YeaI, YedQ, and YfiN are active diguanylate cyclases that reduce motility, eDNA, and early biofilm formation and contrary to the current paradigm, the results indicate that c-di-GMP levels should be reduced, not increased, for initial biofilm formation so c-di-GMP levels must be regulated in a temporal fashion in biofilms.

Topics: Biofilms; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Deletion; Gene Expression Regulation, Bacterial; Genes, Bacterial; Mutagenesis, Site-Directed; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Structure, Tertiary; Second Messenger Systems

Cyclic di-GMP (c-di-GMP) is a signalling molecule that governs the transition between planktonic and biofilm states. Previously, we showed that the diguanylate cyclase HmsT and the putative c-di-GMP phosphodiesterase HmsP inversely regulate biofilm formation through control of HmsHFRS-dependent poly-β-1,6-N-acetylglucosamine synthesis. Here, we systematically examine the functionality of the genes encoding putative c-di-GMP metabolic enzymes in Yersinia pestis. We determine that, in addition to hmsT and hmsP, only the gene y3730 encodes a functional enzyme capable of synthesizing c-di-GMP. The seven remaining genes are pseudogenes or encode proteins that do not function catalytically or are not expressed. Furthermore, we show that HmsP has c-di-GMP-specific phosphodiesterase activity. We report that a mutant incapable of c-di-GMP synthesis is unaffected in virulence in plague mouse models. Conversely, an hmsP mutant, unable to degrade c-di-GMP, is defective in virulence by a subcutaneous route of infection due to poly-β-1,6-N-acetylglucosamine overproduction. This suggests that c-di-GMP signalling is not only dispensable but deleterious for Y. pestis virulence. Our results show that a key event in the evolution of Y. pestis from the ancestral Yersinia pseudotuberculosis was a significant reduction in the complexity of its c-di-GMP signalling network likely resulting from the different disease cycles of these human pathogens.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Bacterial Proteins; Biofilms; Cyclic GMP; Disease Models, Animal; Humans; Mice; Plague; Signal Transduction; Virulence; Virulence Factors; Yersinia pestis

Proteins that metabolize or bind the nucleotide second messenger cyclic diguanylate regulate a wide variety of important processes in bacteria. These processes include motility, biofilm formation, cell division, differentiation, and virulence. The role of cyclic diguanylate signaling in the lifestyle of Legionella pneumophila, the causative agent of Legionnaires' disease, has not previously been examined. The L. pneumophila genome encodes 22 predicted proteins containing domains related to cyclic diguanylate synthesis, hydrolysis, and recognition. We refer to these genes as cdgS (cyclic diguanylate signaling) genes. Strains of L. pneumophila containing deletions of all individual cdgS genes were created and did not exhibit any observable growth defect in growth medium or inside host cells. However, when overexpressed, several cdgS genes strongly decreased the ability of L. pneumophila to grow inside host cells. Expression of these cdgS genes did not affect the Dot/Icm type IVB secretion system, the major determinant of intracellular growth in L. pneumophila. L. pneumophila strains overexpressing these cdgS genes were less cytotoxic to THP-1 macrophages than wild-type L. pneumophila but retained the ability to resist grazing by amoebae. In many cases, the intracellular-growth inhibition caused by cdgS gene overexpression was independent of diguanylate cyclase or phosphodiesterase activities. Expression of the cdgS genes in a Salmonella enterica serovar Enteritidis strain that lacks all diguanylate cyclase activity indicated that several cdgS genes encode potential cyclases. These results indicate that components of the cyclic diguanylate signaling pathway play an important role in regulating the ability of L. pneumophila to grow in host cells.

Topics: Acanthamoeba castellanii; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Humans; Legionella pneumophila; Legionnaires' Disease; Macrophages; Signal Transduction

Recently, we demonstrated that the LuxS-based quorum sensing (QS) system (AI-2) negatively regulated the virulence of a diarrheal isolate SSU of Aeromonas hydrophila, while the ahyRI-based (AI-1) N-acyl-homoserine lactone system was a positive regulator of bacterial virulence. Thus, these QS systems had opposing effects on modulating biofilm formation and bacterial motility in vitro models and in vivo virulence in a speticemic mouse model of infection. In this study, we linked these two QS systems with the bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) in the regulation of virulence in A. hydrophila SSU. To accomplish this, we examined the effect of overproducing a protein with GGDEF domain, which increases c-di-GMP levels in bacteria, on the phenotype and transcriptional profiling of genes involved in biofilm formation and bacterial motility in wild-type (WT) versus its QS null mutants. We provided evidence that c-di-GMP overproduction dramatically enhanced biofilm formation and reduced motility of the WT A. hydrophila SSU, which was equitable with that of the ΔluxS mutant. On the contrary, the ∆ahyRI mutant exhibited only a marginal increase in the biofilm formation with no effect on motility when c-di-GMP was overproduced. Overall, our data indicated that c-di-GMP overproduction modulated transcriptional levels of genes involved in biofilm formation and motility phenotype in A. hydrophila SSU in a QS-dependent manner, involving both AI-1 and AI-2 systems.

Topics: Aeromonas hydrophila; Biofilms; Cyclic GMP; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Gram-Negative Bacterial Infections; Humans; Locomotion; Phenotype; Quorum Sensing; Transcription, Genetic; Virulence Factors

Cyclic diguanylic acid (CDG) is a ubiquitous messenger involved in bacterial signaling networks. Despite its central role in motility, biofilm formation, virulence, and flagellum development, fundamental properties such as its aggregation state are still poorly understood. Here the dynamics and stability of metal-free and Mg(2+)-bound CDG are characterized. Atomistic simulations establish that the CDG dimer is slightly favored (by -5 kcal mol(-1)) over its dissociated form (2 CDG), while the Mg(2+) ion coordinated in the X-ray structure readily dissociates from (CDG)(2) in solution and prefers water coordination. As a ligand in a protein, CDG binds both as a U-shaped and a quasilinear monomer. The current results indicate that the energy difference between these two conformations is only a few kilocalories per mole, which explains the facile adaptation to different protein environments. This, together with the slight preference of (CDG)(2) over 2 CDG suggests that (CDG)(2) binding to a protein does probably not occur via sequential binding of two individual monomers.

Topics: Cyclic GMP; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Dynamics Simulation; Solutions; Thermodynamics

The GGDEF domain protein MxdA, which is important for biofilm formation in Shewanella oneidensis MR-1, was hypothesized to possess diguanylate cyclase activity. Here, we demonstrate that while MxdA controls the cellular level of c-di-GMP in S. oneidensis, it modulates the c-di-GMP pool indirectly.

Topics: Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Phosphorus-Oxygen Lyases; Shewanella

PA4608 is a single PilZ domain protein from Pseudomonas aeruginosa that binds to cyclic dimeric guanosine monophosphate (c-di-GMP). Although the monomeric structure of unbound PA4608 has been studied in detail, the molecular details of c-di-GMP binding to this protein are still uncharacterized. Hence, we determined the solution structure of c-di-GMP bound PA4608. We found that PA4608 undergoes conformational changes to expose the c-di-GMP binding site by ejection of the C-terminal 3(10) helix. A dislocation of the C-terminal tail in the presence of c-di-GMP implies that this region acts as a lid that alternately covers and exposes the hydrophobic surface of the binding site. In addition, mutagenesis and NOE data for PA4608 revealed that conserved residues are in contact with the c-di-GMP molecule. The unique structural characteristics of PA4608, including its monomeric state and its ligand binding characteristics, yield insight into its function as a c-di-GMP receptor.

Topics: Bacterial Proteins; Binding Sites; Cyclic GMP; Molecular Dynamics Simulation; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; Protein Conformation

In Pseudomonas fluorescens Pf0-1 the availability of inorganic phosphate (Pi) is an environmental signal that controls biofilm formation through a cyclic dimeric GMP (c-di-GMP) signaling pathway. In low Pi conditions, a c-di-GMP phosphodiesterase (PDE) RapA is expressed, depleting cellular c-di-GMP and causing the loss of a critical outer-membrane adhesin LapA from the cell surface. This response involves an inner membrane protein LapD, which binds c-di-GMP in the cytoplasm and exerts a periplasmic output promoting LapA maintenance on the cell surface. Here we report how LapD differentially controls maintenance and release of LapA: c-di-GMP binding to LapD promotes interaction with and inhibition of the periplasmic protease LapG, which targets the N-terminus of LapA. We identify conserved amino acids in LapA required for cleavage by LapG. Mutating these residues in chromosomal lapA inhibits LapG activity in vivo, leading to retention of the adhesin on the cell surface. Mutations with defined effects on LapD's ability to control LapA localization in vivo show concomitant effects on c-di-GMP-dependent LapG inhibition in vitro. To establish the physiological importance of the LapD-LapG effector system, we track cell attachment and LapA protein localization during Pi starvation. Under this condition, the LapA adhesin is released from the surface of cells and biofilms detach from the substratum. This response requires c-di-GMP depletion by RapA, signaling through LapD, and proteolytic cleavage of LapA by LapG. These data, in combination with the companion study by Navarro et al. presenting a structural analysis of LapD's signaling mechanism, give a detailed description of a complete c-di-GMP control circuit--from environmental signal to molecular output. They describe a novel paradigm in bacterial signal transduction: regulation of a periplasmic enzyme by an inner membrane signaling protein that binds a cytoplasmic second messenger.

Topics: Adhesins, Bacterial; Amino Acid Sequence; Bacterial Adhesion; Binding Sites; Biofilms; Cell Membrane; Conserved Sequence; Cyclic GMP; Cysteine Proteases; Lectins; Molecular Sequence Data; Phenotype; Pseudomonas fluorescens; Sequence Alignment; Signal Transduction

The bacterial second messenger bis-(3'-5') cyclic dimeric guanosine monophosphate (c-di-GMP) has emerged as a central regulator for biofilm formation. Increased cellular c-di-GMP levels lead to stable cell attachment, which in Pseudomonas fluorescens requires the transmembrane receptor LapD. LapD exhibits a conserved and widely used modular architecture containing a HAMP domain and degenerate diguanylate cyclase and phosphodiesterase domains. c-di-GMP binding to the LapD degenerate phosphodiesterase domain is communicated via the HAMP relay to the periplasmic domain, triggering sequestration of the protease LapG, thus preventing cleavage of the surface adhesin LapA. Here, we elucidate the molecular mechanism of autoinhibition and activation of LapD based on structure-function analyses and crystal structures of the entire periplasmic domain and the intracellular signaling unit in two different states. In the absence of c-di-GMP, the intracellular module assumes an inactive conformation. Binding of c-di-GMP to the phosphodiesterase domain disrupts the inactive state, permitting the formation of a trans-subunit dimer interface between adjacent phosphodiesterase domains via interactions conserved in c-di-GMP-degrading enzymes. Efficient mechanical coupling of the conformational changes across the membrane is realized through an extensively domain-swapped, unique periplasmic fold. Our structural and functional analyses identified a conserved system for the regulation of periplasmic proteases in a wide variety of bacteria, including many free-living and pathogenic species.

Topics: Bacterial Adhesion; Bacterial Proteins; Binding Sites; Biofilms; Crystallography, X-Ray; Cyclic GMP; Dimerization; Peptide Hydrolases; Periplasm; Phosphoric Diester Hydrolases; Protein Interaction Mapping; Protein Structure, Tertiary; Pseudomonas fluorescens; Signal Transduction; Structure-Activity Relationship

The second messenger cyclic diguanylic acid (c-di-GMP) is implicated in key lifestyle decisions of bacteria, including biofilm formation and changes in motility and virulence. Some challenges in deciphering the physiological roles of c-di-GMP are the limited knowledge about the cellular targets of c-di-GMP, the signals that control its levels, and the proportion of free cellular c-di-GMP, if any. Here, we identify the target and the regulatory signal for a c-di-GMP-responsive Escherichia coli ribonucleoprotein complex. We show that a direct c-di-GMP target in E. coli is polynucleotide phosphorylase (PNPase), an important enzyme in RNA metabolism that serves as a 3' polyribonucleotide polymerase or a 3'-to-5' exoribonuclease. We further show that a complex of polynucleotide phosphorylase with the direct oxygen sensors DosC and DosP can perform oxygen-dependent RNA processing. We conclude that c-di-GMP can mediate signal-dependent RNA processing and that macromolecular complexes can compartmentalize c-di-GMP signaling.

Topics: Cyclic GMP; Endoribonucleases; Enzyme Activation; Escherichia coli; Escherichia coli Proteins; Heme; Macromolecular Substances; Oxygen; Phosphopyruvate Hydratase; Polyribonucleotide Nucleotidyltransferase; RNA; Second Messenger Systems

Recently, there has been an explosion of research activities in the cyclic dinucleotides field. Cyclic dinucleotides, such as c-di-GMP and c-di-AMP, have been shown to regulate bacterial virulence and biofilm formation. c-di-GMP can exist in different aggregate forms, and it has been demonstrated that the polymorphism of c-di-GMP is influenced by the nature of cation that is present in solution. In previous work, polymorphism of c-di-GMP could only be demonstrated at hundreds of micromolar concentrations of the dinucleotide, and it has been a matter of debate if polymorphism of c-di-GMP exists under in vivo conditions. In this Article, we demonstrate that c-di-GMP can form G-quadruplexes at low micromolar concentrations when aromatic molecules such as thiazole orange template the quadruplex formation. We then use this property of aromatic molecule-induced G-quadruplex formation of c-di-GMP to design a thiazole orange-based fluorescent detection of this important signaling molecule. We determine, using this thiazole orange assay on a crude bacterial cell lysate, that WspR D70E (a constitutively activated diguanylate cyclase) is functional in vivo when overexpressed in E. Coli . The intracellular concentration of c-di-GMP in an E. Coli cell that is overexpressed with WspR D70E is very high and can reach 2.92 mM.

Topics: Benzothiazoles; Biofilms; Cyclic GMP; Escherichia coli; Fluorescence; G-Quadruplexes; Molecular Conformation; Quinolines

The ubiquitous bacterial biofilm regulator, c-di-GMP can form G-quadruplexes at physiological conditions in the presence of some aromatic compounds, such as acriflavine and proflavine. The fluorescence of these compounds is quenched upon c-di-GMP binding and some of the formed c-di-GMP G-quadruplexes are stable even at 75 °C.

Topics: Acriflavine; Cyclic GMP; Fluorescence; G-Quadruplexes; Proflavine; Temperature

Clostridium difficile infections have become a major healthcare concern in the last decade during which the emergence of new strains has underscored this bacterium's capacity to cause persistent epidemics. c-di-GMP is a bacterial second messenger regulating diverse bacterial phenotypes, notably motility and biofilm formation, in proteobacteria such as Vibrio cholerae, Pseudomonas aeruginosa, and Salmonella. c-di-GMP is synthesized by diguanylate cyclases (DGCs) that contain a conserved GGDEF domain. It is degraded by phosphodiesterases (PDEs) that contain either an EAL or an HD-GYP conserved domain. Very little is known about the role of c-di-GMP in the regulation of phenotypes of Gram-positive or fastidious bacteria. Herein, we exposed the main components of c-di-GMP signalling in 20 genomes of C. difficile, revealed their prevalence, and predicted their enzymatic activity. Ectopic expression of 31 of these conserved genes was carried out in V. cholerae to evaluate their effect on motility and biofilm formation, two well-characterized phenotype alterations associated with intracellular c-di-GMP variation in this bacterium. Most of the predicted DGCs and PDEs were found to be active in the V. cholerae model. Expression of truncated versions of CD0522, a protein with two GGDEF domains and one EAL domain, suggests that it can act alternatively as a DGC or a PDE. The activity of one purified DGC (CD1420) and one purified PDE (CD0757) was confirmed by in vitro enzymatic assays. GTP was shown to be important for the PDE activity of CD0757. Our results indicate that, in contrast to most Gram-positive bacteria including its closest relatives, C. difficile encodes a large assortment of functional DGCs and PDEs, revealing that c-di-GMP signalling is an important and well-conserved signal transduction system in this human pathogen.

Topics: Bacterial Proteins; Biofilms; Cell Migration Assays; Clostridioides difficile; Clostridium Infections; Cyclic GMP; Escherichia coli Proteins; Humans; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Signal Transduction; Vibrio cholerae

Acinetobacter baumannii has emerged as a major cause of both community-associated and nosocomial infections worldwide. A. baumannii rapidly develops resistance to multiple antibiotics; as a result, infections by this pathogen have become increasingly difficult to treat. In this study, we evaluated the effect of 3',5'-cyclic diguanylic acid (c-di-GMP), a bacterial second messenger and immunomodulator, in the host defense against A. baumannii infection in a mouse model of intranasal infection. Our results showed that 50 μg of c-di-GMP administered 18 h prior to infection provided the best protection against intranasal infection with A. baumannii. Mechanistically, administration of c-di-GMP induced the production of chemokines KC, MCP-1, MIP-1α, MIP-2 and RANTES, and enhanced neutrophil recruitment in the lung. Moreover, depletion of neutrophils abolished the protective role of c-di-GMP. Taken together, our data suggest that c-di-GMP confers resistance against intranasal A. baumannii infection in mice through a neutrophil-dependent mechanism and that c-di-GMP should be further explored as an immunomodulator for the treatment of A. baumannii infection.

Topics: Acinetobacter baumannii; Acinetobacter Infections; Administration, Intranasal; Animals; Chemokine CCL2; Chemokine CCL3; Chemokine CCL5; Chemokine CXCL2; Chemokines; Cyclic GMP; Female; Lung; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Time Factors

The bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling pathway regulates biofilm formation, virulence, and other processes in many bacterial species and is critical for their survival. Two classes of c-di-GMP-binding riboswitches have been discovered that bind this second messenger with high affinity and regulate diverse downstream genes, underscoring the importance of RNA receptors in this pathway. We have solved the structure of a c-di-GMP-II riboswitch, which reveals that the ligand is bound as part of a triplex formed with a pseudoknot. The structure also shows that the guanine bases of c-di-GMP are recognized through noncanonical pairings and that the phosphodiester backbone is not contacted by the RNA. Recognition is quite different from that observed in the c-di-GMP-I riboswitch, demonstrating that at least two independent solutions for RNA second messenger binding have evolved. We exploited these differences to design a c-di-GMP analog that selectively binds the c-di-GMP-II aptamer over the c-di-GMP-I RNA. There are several bacterial species that contain both types of riboswitches, and this approach holds promise as an important tool for targeting one riboswitch, and thus one gene, over another in a selective fashion.

Topics: Aptamers, Nucleotide; Base Sequence; Binding Sites; Clostridium acetobutylicum; Cyclic GMP; Kinetics; Ligands; Models, Molecular; Nucleic Acid Conformation; Riboswitch; RNA, Bacterial; Second Messenger Systems

GGDEF and EAL/HD-GYP protein domains are responsible for the synthesis and hydrolysis of the bacterial secondary messenger cyclic diguanylate (c-di-GMP) through their diguanylate cyclase and phosphodiesterase activities, respectively. Forty-three genes in Pseudomonas putida KT2440 are putatively involved in the turnover of c-di-GMP. Of them only rup4959 (locus PP4959) encodes a GGDEF/EAL response regulator, which was identified in a genome wide analysis as preferentially induced while this bacterium colonizes roots and adjacent soil areas (the rhizosphere). By using fusions to reporter genes it was confirmed that the rup4959 promoter is active in the rhizosphere and inducible by corn plant root exudates and microaerobiosis. Transcription of rup4959 was strictly dependent on the alternative transcriptional factor σ(S) . The inactivation of the rup4959-4957 operon altered the expression of 22 genes in the rhizosphere and had a negative effect upon oligopeptide utilization and biofilm formation. In multicopy or when overexpressed, rup4959 enhanced adhesin LapA-dependent biofilm formation, the development of wrinkly colony morphology, and increased Calcofluor stainable exopolysaccharides (EPS). Under these conditions the inhibition of swarming motility was total and plant root tip colonization considerably less efficient, whereas swimming was partially diminished. This pleiotropic phenotype, which correlated with an increase in the global level of c-di-GMP, was not acquired with increased levels of Rup4959 catalytic mutant at GGDEF as a proof of this response regulator exhibiting diguanylate cyclase activity. A screen for mutants in putative targets of c-di-GMP led to the identification of a surface polysaccharide specific to KT2440, which is encoded by the genes cluster PP3133-PP3141, as essential for phenotypes associated with increased c-di-GMP. Cellulose and alginate were discarded as the overproduced EPS, and lipopolysaccharide (LPS) core and O-antigen were found to be essential for the development of wrinkly colony morphology.

Topics: Aerobiosis; Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Multigene Family; Mutation; Operon; Phenotype; Phosphorus-Oxygen Lyases; Plant Roots; Polysaccharides, Bacterial; Promoter Regions, Genetic; Protein Structure, Tertiary; Pseudomonas putida; Rhizosphere; Second Messenger Systems; Sigma Factor; Transcription, Genetic; Zea mays

To colonize the cystic fibrosis lung, Pseudomonas aeruginosa establishes sessile communities referred to as biofilms. Although the signaling molecule c-di-GMP governs the transition from motile to sessile growth, the environmental signal(s) required to modulate biofilm formation remain unclear. Using relevant in vivo concentrations of the 19 amino acids previously identified in cystic fibrosis sputum, we demonstrated that arginine, ornithine, isoleucine, leucine, valine, phenylalanine and tyrosine robustly promoted biofilm formation in vitro. Among the seven biofilm-promoting amino acids, only arginine also completely repressed the ability of P. aeruginosa to swarm over semi-solid surfaces, suggesting that arginine may be an environmental cue favoring a sessile lifestyle. Mutating two documented diguanylate cyclases required for biofilm formation (SadC and RoeA) reduced biofilm formation and restored swarming motility on arginine-containing medium. Growth on arginine increased the intracellular levels of c-di-GMP, and this increase was dependent on the SadC and RoeA diguanylate cyclases. Strains mutated in sadC, roeA or both also showed a reduction in biofilm formation when grown with the other biofilm-promoting amino acids. Taken together, these results suggest that amino acids can modulate biofilm formation and swarming motility, at least in part, by controlling the intracellular levels of c-di-GMP.

Topics: Amino Acids; Bacterial Proteins; Biofilms; Cyclic GMP; Escherichia coli Proteins; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa; Signal Transduction

Yersinia pestis forms a biofilm in the foregut of its flea vector that promotes transmission by flea bite. As in many bacteria, biofilm formation in Y. pestis is controlled by intracellular levels of the bacterial second messenger c-di-GMP. Two Y. pestis diguanylate cyclase (DGC) enzymes, encoded by hmsT and y3730, and one phosphodiesterase (PDE), encoded by hmsP, have been shown to control biofilm production in vitro via their opposing c-di-GMP synthesis and degradation activities, respectively. In this study, we provide further evidence that hmsT, hmsP, and y3730 are the only three genes involved in c-di-GMP metabolism in Y. pestis and evaluated the two DGCs for their comparative roles in biofilm formation in vitro and in the flea vector. As with HmsT, the DGC activity of Y3730 depended on a catalytic GGDEF domain, but the relative contribution of the two enzymes to the biofilm phenotype was influenced strongly by the environmental niche. Deletion of y3730 had a very minor effect on in vitro biofilm formation, but resulted in greatly reduced biofilm formation in the flea. In contrast, the predominant effect of hmsT was on in vitro biofilm formation. DGC activity was also required for the Hms-independent autoaggregation phenotype of Y. pestis, but was not required for virulence in a mouse model of bubonic plague. Our results confirm that only one PDE (HmsP) and two DGCs (HmsT and Y3730) control c-di-GMP levels in Y. pestis, indicate that hmsT and y3730 are regulated post-transcriptionally to differentially control biofilm formation in vitro and in the flea vector, and identify a second c-di-GMP-regulated phenotype in Y. pestis.

Topics: Animals; Bacterial Proteins; Biofilms; Cyclic GMP; Disease Models, Animal; Escherichia coli Proteins; Mice; Mutation; Phenotype; Phosphorus-Oxygen Lyases; Plague; Protein Structure, Tertiary; Siphonaptera; Virulence; Yersinia pestis

Vaccination is the best available measure of limiting the impact of the next influenza pandemic. Ideally, a candidate pandemic influenza vaccine should be easy to administer and should elicit strong mucosal and systemic immune responses. Production of influenza subunit antigen in transient plant expression systems is an alternative to overcome the bottleneck in vaccine supply during influenza pandemic. Furthermore, a needle-free intranasal influenza vaccine is an attractive approach, which may provide immunity at the portal of virus entry. The present study investigated the detailed humoral and cellular immune responses in mice vaccinated intranasally or intramuscularly with plant-derived influenza H5N1 (A/Anhui/1/05) antigen alone or formulated with bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) as adjuvant. The use of c-di-GMP as intramuscular adjuvant did not enhance the immune response to plant-derived influenza H5 antigen. However, intranasal c-di-GMP-adjuvanted vaccine induced strong mucosal and systemic humoral immune responses. Additionally, the intranasal vaccine elicited a balanced Th1/Th2 profile and, most importantly, high frequencies of multifunctional Th1 CD4(+) cells. Our results highlight that c-di-GMP is a promising mucosal adjuvant for pandemic influenza vaccine development.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Antibodies, Viral; Cyclic GMP; Female; Hemagglutinin Glycoproteins, Influenza Virus; Immunity, Mucosal; Influenza A Virus, H5N1 Subtype; Influenza Vaccines; Injections, Intramuscular; Mice; Mice, Inbred BALB C; Plants, Genetically Modified; Th1 Cells; Vaccines, Synthetic

The cyclic dinucleotide c-di-GMP is a master regulator of bacterial virulence and biofilm formation. The activations of c-di-GMP metabolism proteins, diguanylate cyclases (DGCs) and phosophodiesterases (PDEs), usually lead to diametrically opposite phenotypes in bacteria. Analogues of c-di-GMP, which can selectively modulate the activities of c-di-GMP processing proteins, will be useful chemical tools for studying and altering bacterial behavior. Herein we report that a conservative modification of one of the phosphate groups in c-di-GMP with a bridging sulfur in the phosphodiester linkage affords an analogue called endo-S-c-di-GMP. Computational, NMR (including DOSY), and CD experiments all reveal that, unlike c-di-GMP, endo-S-c-di-GMP does not readily form higher aggregates. The lower propensity of endo-S-c-di-GMP to form aggregates (as compared to that of c-di-GMP) is probably due to a higher activation barrier to convert from the "open" conformer (where the two guanines are on opposite faces) to the "closed" conformer (where the two guanines are on the same face). Consequently, endo-S-c-di-GMP has selectivity for proteins that bind monomeric but not dimeric c-di-GMP, which form from the "closed" conformer. For example, endo-S-c-di-GMP can inhibit the hydrolysis of c-di-GMP by RocR (a PDE enzyme that binds monomeric c-di-GMP) but did not bind to Alg44 (a PilZ protein) or regulate WspR (a DGC enzyme that has been shown to bind to dimeric c-di-GMP). This work demonstrates that selective binding to different classes of c-di-GMP binding proteins could be achieved by altering analogue conformer populations (conformational steering). We provide important design principles for the preparation of selective PDE inhibitors and reveal the role played by the c-di-GMP backbone in c-di-GMP polymorphism and binding to processing proteins.

Topics: Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Models, Molecular; Phosphates; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Binding; Protein Conformation; Pseudomonas aeruginosa

Biofilms contribute to Pseudomonas aeruginosa persistence in a variety of diseases, including cystic fibrosis, burn wounds, and chronic suppurative otitis media. However, few studies have directly addressed P. aeruginosa biofilms in vivo. We used a chinchilla model of otitis media, which has previously been used to study persistent Streptococcus pneumoniae and Haemophilus influenzae infections, to show that structures formed in vivo are biofilms of bacterial and host origin within a matrix that includes Psl, a P. aeruginosa biofilm polysaccharide. We evaluated three biofilm and/or virulence mediators of P. aeruginosa known to affect biofilm formation in vitro and pathogenesis in vivo--bis-(3',5')-cyclic dimeric GMP (c-di-GMP), flagella, and quorum sensing--in a chinchilla model. We show that c-di-GMP overproduction has a positive impact on bacterial persistence, while quorum sensing increases virulence. We found no difference in persistence attributed to flagella. We conclude from these studies that a chinchilla otitis media model provides a means to evaluate pathogenic mediators of P. aeruginosa and that in vitro phenotypes should be examined in multiple infection systems to fully understand their role in disease.

Topics: Animals; Biofilms; Chinchilla; Chronic Disease; Cyclic GMP; Disease Models, Animal; Gene Expression Regulation, Bacterial; Humans; Otitis Media; Pseudomonas aeruginosa; Pseudomonas Infections; Quorum Sensing; Rodent Diseases; Virulence

HD-GYP domain cyclic dimeric GMP (c-di-GMP) phosphodiesterases are implicated in motility and virulence in bacteria. Borrelia burgdorferi possesses a single set of c-di-GMP-metabolizing enzymes, including a putative HD-GYP domain protein, BB0374. Recently, we characterized the EAL domain phosphodiesterase PdeA. A mutation in pdeA resulted in cells that were defective in motility and virulence. Here we demonstrate that BB0374/PdeB specifically hydrolyzed c-di-GMP with a K(m) of 2.9 nM, confirming that it is a functional phosphodiesterase. Furthermore, by measuring phosphodiesterase enzyme activity in extracts from cells containing the pdeA pdeB double mutant, we demonstrate that no additional phosphodiesterases are present in B. burgdorferi. pdeB single mutant cells exhibit significantly increased flexing, indicating a role for c-di-GMP in motility. Constructing and analyzing a pilZ pdeB double mutant suggests that PilZ likely interacts with chemotaxis signaling. While virulence in needle-inoculated C3H/HeN mice did not appear to be altered significantly in pdeB mutant cells, these cells exhibited a reduced ability to survive in Ixodes scapularis ticks. Consequently, those ticks were unable to transmit the infection to naïve mice. All of these phenotypes were restored when the mutant was complemented. Identification of this role of pdeB increases our understanding of the c-di-GMP signaling network in motility regulation and the life cycle of B. burgdorferi.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Borrelia burgdorferi; Cyclic GMP; Disease Models, Animal; Female; Gene Deletion; Genetic Complementation Test; Ixodes; Kinetics; Locomotion; Lyme Disease; Mice; Mice, Inbred C3H; Rodent Diseases; Virulence

(3'-5')-Cyclic diguanylate (c-di-GMP) is a bacterial second messenger with immunomodulatory activities in mice suggesting potential applications as a vaccine adjuvant and as a therapeutic agent. Clinical studies in larger animals or humans will require larger doses that are difficult and expensive to generate by currently available chemical or enzymatic synthesis and purification methods. Here we report the production of c-di-GMP at the multi-gram scale from the economical precursors guanosine monophosphate (GMP) and adenosine triphosphate by a "one-pot" three enzyme cascade consisting of GMP kinase, nucleoside diphosphate kinase, and a mutated form of diguanylate cyclase engineered to lack product inhibition. The c-di-GMP was purified to apparent homogeneity by a combination of anion exchange chromatography and solvent precipitation and was characterized by reversed phase high performance liquid chormatography and mass spectrometry, nuclear magnetic resonance spectroscopy, and further compositional analyses. The immunomodulatory activity of the c-di-GMP preparation was confirmed by its potentiating effect on the lipopolysaccharide-induced interleukin 1β, tumor necrosis factor α, and interleukin 6 messenger RNA expression in J774A.1 mouse macrophages.

Topics: Adenosine Triphosphate; Animals; Biotechnology; Cell Line; Chromatography, Ion Exchange; Cloning, Molecular; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Guanosine Monophosphate; Guanylate Kinases; Immunologic Factors; Interleukin-1beta; Interleukin-6; Lipopolysaccharides; Macrophages; Magnetic Resonance Spectroscopy; Mice; Mutation; Nucleoside-Diphosphate Kinase; Phosphorus-Oxygen Lyases; Recombinant Proteins; Transformation, Bacterial; Tumor Necrosis Factor-alpha

We report the identification and characterization of a previously unidentified protein domain found in bacterial chemoreceptors and other bacterial signal transduction proteins. This domain contains a motif of three noncontiguous histidines and one cysteine, arranged as Hxx[WFYL]x(21-28)Cx[LFMVI]Gx[WFLVI]x(18-27)HxxxH(boldface type indicates residues that are nearly 100% conserved). This domain was first identified in the soluble Helicobacter pylori chemoreceptor TlpD. Using inductively coupled plasma mass spectrometry on heterologously and natively expressed TlpD, we determined that this domain binds zinc with a subfemtomolar dissociation constant. We thus named the domain CZB, for chemoreceptor zinc binding. Further analysis showed that many bacterial signaling proteins contain the CZB domain, most commonly proteins that participate in chemotaxis but also those that participate in c-di-GMP signaling and nitrate/nitrite sensing, among others. Proteins bearing the CZB domain are found in several bacterial phyla. The variety of signaling proteins using the CZB domain suggests that it plays a critical role in several signal transduction pathways.

Topics: Amino Acid Motifs; Bacterial Proteins; Chemotaxis; Cloning, Molecular; Cyclic GMP; Cysteine; Cytoplasm; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genes, Bacterial; Helicobacter pylori; Histidine; Mutation; Protein Binding; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Zinc

Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that modulates many biological processes. Although its role in bacterial pathogenesis during mammalian infection has been documented, the role of c-di-GMP in a pathogen's life cycle within a vector host is less understood. The enzootic cycle of the Lyme disease pathogen Borrelia burgdorferi involves both a mammalian host and an Ixodes tick vector. The B. burgdorferi genome encodes a single copy of the diguanylate cyclase gene (rrp1), which is responsible for c-di-GMP synthesis. To determine the role of c-di-GMP in the life cycle of B. burgdorferi, an Rrp1-deficient B. burgdorferi strain was generated. The rrp1 mutant remains infectious in the mammalian host but cannot survive in the tick vector. Microarray analyses revealed that expression of a four-gene operon involved in glycerol transport and metabolism, bb0240-bb0243, was significantly downregulated by abrogation of Rrp1. In vitro, the rrp1 mutant is impaired in growth in the media containing glycerol as the carbon source (BSK-glycerol). To determine the contribution of the glycerol metabolic pathway to the rrp1 mutant phenotype, a glp mutant, in which the entire bb0240-bb0243 operon is not expressed, was generated. Similar to the rrp1 mutant, the glp mutant has a growth defect in BSK-glycerol medium. In vivo, the glp mutant is also infectious in mice but has reduced survival in ticks. Constitutive expression of the bb0240-bb0243 operon in the rrp1 mutant fully rescues the growth defect in BSK-glycerol medium and partially restores survival of the rrp1 mutant in ticks. Thus, c-di-GMP appears to govern a catabolic switch in B. burgdorferi and plays a vital role in the tick part of the spirochetal enzootic cycle. This work provides the first evidence that c-di-GMP is essential for a pathogen's survival in its vector host.

Topics: Animals; Borrelia burgdorferi Group; Cyclic GMP; Disease Vectors; Escherichia coli Proteins; Gene Knockout Techniques; Glycerol; Ixodes; Lyme Disease; Mice; Mice, Inbred C3H; Microarray Analysis; Phosphorus-Oxygen Lyases; Polymerase Chain Reaction; Sequence Deletion

A significant part of bacterial two-component system response regulators contains effector domains predicted to be involved in metabolism of bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a second messenger that plays a key role in many physiological processes. The intracellular level of c-di-GMP is controlled by diguanylate cyclase and phosphodiesterases activities associated with GGDEF and EAL domains, respectively. The Legionella pneumophila Lens genome displays 22 GGDEF/EAL domain-encoding genes. One of them, lpl0329, encodes a protein containing a two-component system receiver domain and both GGDEF and EAL domains. Here, we demonstrated that the GGDEF and EAL domains of Lpl0329 are both functional and lead to simultaneous synthesis and hydrolysis of c-di-GMP. Moreover, these two opposite activities are finely regulated by Lpl0329 phosphorylation due to the atypical histidine kinase Lpl0330. Indeed, Lpl0330 was found to autophosphorylate on a histidine residue in an atypical H box, which is conserved in various bacteria species and thus defines a new histidine kinase subfamily. Lpl0330 also catalyzes the phosphotransferase to Lpl0329, which results in a diguanylate cyclase activity decrease whereas phosphodiesterase activity remains efficient. Altogether, these data present (i) a new histidine kinase subfamily based on the conservation of an original H box that we named HGN H box, and (ii) the first example of a bifunctional enzyme that modulates synthesis and turnover of c-di-GMP in response to phosphorylation of its receiver domain.

Topics: Bacterial Proteins; Cyclic GMP; Escherichia coli Proteins; Genes, Bacterial; Legionella pneumophila; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Phosphorylation; Protein Kinases