guanosine-triphosphate and bis(3--5-)-cyclic-diguanylic-acid

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

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 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

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

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

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

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

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

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

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

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

Group I self-splicing ribozymes commonly function as components of selfish mobile genetic elements. We identified an allosteric group I ribozyme, wherein self-splicing is regulated by a distinct riboswitch class that senses the bacterial second messenger c-di-GMP. The tandem RNA sensory system resides in the 5' untranslated region of the messenger RNA for a putative virulence gene in the pathogenic bacterium Clostridium difficile. c-di-GMP binding by the riboswitch induces folding changes at atypical splice site junctions to modulate alternative RNA processing. Our findings indicate that some self-splicing ribozymes are not selfish elements but are harnessed by cells as metabolite sensors and genetic regulators.

Topics: 5' Untranslated Regions; Aptamers, Nucleotide; Base Pairing; Base Sequence; Clostridioides difficile; Codon, Initiator; Cyclic GMP; Exons; Genes, Bacterial; Guanosine Triphosphate; Molecular Sequence Data; Nucleic Acid Conformation; Regulatory Sequences, Ribonucleic Acid; RNA Splicing; RNA, Bacterial; RNA, Catalytic; RNA, Messenger; Second Messenger Systems

Pseudomonas aeruginosa encodes many enzymes that are potentially associated with the synthesis or degradation of the widely conserved second messenger cyclic-di-GMP (c-di-GMP). In this study, we show that mutation of rbdA, which encodes a fusion protein consisting of PAS-PAC-GGDEF-EAL multidomains, results in decreased biofilm dispersal. RbdA contains a highly conserved GGDEF domain and EAL domain, which are involved in the synthesis and degradation of c-di-GMP, respectively. However, in vivo and in vitro analyses show that the full-length RbdA protein only displays phosphodiesterase activity, causing c-di-GMP degradation. Further analysis reveals that the GGDEF domain of RbdA plays a role in activating the phosphodiesterase activity of the EAL domain in the presence of GTP. Moreover, we show that deletion of the PAS domain or substitution of the key residues implicated in sensing low-oxygen stress abrogates the functionality of RbdA. Subsequent study showed that RbdA is involved in positive regulation of bacterial motility and production of rhamnolipids, which are associated with biofilm dispersal, and in negative regulation of production of exopolysaccharides, which are required for biofilm formation. These data indicate that the c-di-GMP-degrading regulatory protein RbdA promotes biofilm dispersal through its two-pronged effects on biofilm development, i.e., downregulating biofilm formation and upregulating production of the factors associated with biofilm dispersal.

Topics: Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Glycolipids; Guanosine Triphosphate; Hydrolysis; Hypoxia; Locomotion; Phosphoric Diester Hydrolases; Polysaccharides, Bacterial; Protein Structure, Tertiary; Pseudomonas aeruginosa

Cyclic diguanylic acid (c-di-GMP) is a global second messenger controlling motility and adhesion in bacterial cells. Synthesis and degradation of c-di-GMP is catalyzed by diguanylate cyclases (DGC) and c-di-GMP-specific phosphodiesterases (PDE), respectively. Whereas the DGC activity has recently been assigned to the widespread GGDEF domain, the enzymatic activity responsible for c-di-GMP cleavage has been associated with proteins containing an EAL domain. Here we show biochemically that CC3396, a GGDEF-EAL composite protein from Caulobacter crescentus is a soluble PDE. The PDE activity, which rapidly converts c-di-GMP into the linear dinucleotide pGpG, is confined to the C-terminal EAL domain of CC3396, depends on the presence of Mg2+ ions, and is strongly inhibited by Ca2+ ions. Remarkably, the associated GGDEF domain, which contains an altered active site motif (GEDEF), lacks detectable DGC activity. Instead, this domain is able to bind GTP and in response activates the PDE activity in the neighboring EAL domain. PDE activation is specific for GTP (K(D) 4 microM) and operates by lowering the K(m) for c-di-GMP of the EAL domain to a physiologically significant level (420 nM). Mutational analysis suggested that the substrate-binding site (A-site) of the GGDEF domain is involved in the GTP-dependent regulatory function, arguing that a catalytically inactive GGDEF domain has retained the ability to bind GTP and in response can activate the neighboring EAL domain. Based on this we propose that the c-di-GMP-specific PDE activity is confined to the EAL domain, that GGDEF domains can either catalyze the formation of c-di-GMP or can serve as regulatory domains, and that c-di-GMP-specific phosphodiesterase activity is coupled to the cellular GTP level in bacteria.

Topics: Allosteric Regulation; Amino Acid Sequence; Bacterial Proteins; Binding Sites; Caulobacter crescentus; Cell Fractionation; Cyclic GMP; DNA Mutational Analysis; Enzyme Activation; Guanosine Triphosphate; Molecular Sequence Data; Phosphoric Diester Hydrolases; Protein Binding; Protein Structure, Tertiary; Substrate Specificity

Pole development is coordinated with the Caulobacter crescentus cell cycle by two-component signaling proteins. We show that an unusual response regulator, PleD, is required for polar differentiation and is sequestered to the cell pole only when it is activated by phosphorylation. Dynamic localization of PleD to the cell pole provides a mechanism to temporally and spatially control the signaling output of PleD during development. Targeting of PleD to the cell pole is coupled to the activation of a C-terminal guanylate cyclase domain, which catalyzes the synthesis of cyclic di-guanosine monophosphate. We propose that the local action of this novel-type guanylate cyclase might constitute a general regulatory principle in bacterial growth and development.

Topics: Bacterial Proteins; Caulobacter crescentus; Cell Cycle; Cyclic GMP; Guanosine Triphosphate; Guanylate Cyclase; Histidine Kinase; Phosphorylation; Protein Kinases

1. The effect of the novel, naturally occurring nucleotide 3'-5' cyclic diguanylic acid (c-di-GMP) on the lymphoblastoid Molt 4 cell line was studied. When exposed to this guanine nucleotide. Molt 4 cells exhibited a marked increase in [3H]thymidine incorporation, up to 200-fold at 50 microM c-di-GMP. Correspondingly, the DNA content of the treated cells was 9-fold higher than untreated cells. Stimulation of [3H]thymidine incorporation into the cells was time- and concentration-dependent. This effect was specific and was not observed with GMP or cyclic GMP, nor with the unhydrolysable GTP analogues, guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta gamma-imido]-triphosphate. C-di-GMP entrance into the cells was experimentally verified and occurred without using any means of cell permeabilization. SDS/PAGE analysis of cells exposed to [32P]c-di-GMP, followed by autoradiography, revealed the labelling of three low-molecular-mass proteins at 18-27 kDa. The labelling is highly specific to c-di-GMP and its extent was not affected by other guanine nucleotides. 2. One of the c-di-GMP-binding proteins was found to be the p21ras protein, by immunoprecipitation with the anti-Ras monoclonal antibody Y13-259. The effects described appear to be unique for c-di-GMP and, taken together, raise the possibility that an irreversible binding of this guanine nucleotide to the growth-promoting p21ras protein results in a fixed active conformation of this protein affecting DNA synthesis. Strikingly, although at 48 h of growth markedly high DNA levels were found in Molt 4 cells treated with c-di-GMP, this guanine nucleotide had no effect on cell replication during this period. Thus Molt 4 cells exposed to c-di-GMP enter the S phase uncoordinated with their overall replication rate.

Topics: Cell Cycle; Cell Division; Cell Line; Cell Membrane Permeability; Cyclic GMP; DNA; Guanosine Triphosphate; Humans; Lymphocytes; Proteins; Proto-Oncogene Proteins p21(ras); S Phase; Sensitivity and Specificity; Thymidine; Tritium

The occurrence of the novel regulatory nucleotide bis(3',5')-cyclic diguanylic acid (c-di-GMP) and its relation to cellulose biogenesis in the plant pathogen Agrobacterium tumefaciens was studied. c-di-GMP was detected in acid extracts of 32P-labeled cells grown in various media, and an enzyme responsible for its formation from GTP was found to be present in cell-free preparations. Cellulose synthesis in vivo was quantitatively assessed with [14C]glucose as a tracer. The organism produced cellulose during growth in the absence of plant cells, and this capacity was retained in resting cells. Synthesis of a cellulosic product from UDP-glucose in vitro with membrane preparations was markedly stimulated by c-di-GMP and its precursor GTP and was further enhanced by Ca2+. The calcium effect was attributed to inhibition of a c-di-GMP-degrading enzyme shown to be present in the cellulose synthase-containing membranes.

Topics: Arabidopsis Proteins; Cellulose; Cyclic GMP; Glucosyltransferases; Guanosine Triphosphate; Kinetics; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Rhizobium