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

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

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

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

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

Biofilms are communities of surface-attached, matrix-embedded microbial cells that can resist antimicrobial chemotherapy and contribute to persistent infections. Using an Escherichia coli biofilm model we found that exposure of bacteria to subinhibitory concentrations of ribosome-targeting antibiotics leads to strong biofilm induction. We present evidence that this effect is elicited by the ribosome in response to translational stress. Biofilm induction involves upregulation of the polysaccharide adhesin poly-beta-1,6-N-acetyl-glucosamine (poly-GlcNAc) and two components of the poly-GlcNAc biosynthesis machinery, PgaA and PgaD. Poly-GlcNAc control depends on the bacterial signalling molecules guanosine-bis 3', 5'(diphosphate) (ppGpp) and bis-(3'-5')-cyclic di-GMP (c-di-GMP). Treatment with translation inhibitors causes a ppGpp hydrolase (SpoT)-mediated reduction of ppGpp levels, resulting in specific derepression of PgaA. Maximal induction of PgaD and poly-GlcNAc synthesis requires the production of c-di-GMP by the dedicated diguanylate cyclase YdeH. Our results identify a novel regulatory mechanism that relies on ppGpp signalling to relay information about ribosomal performance to the Pga machinery, thereby inducing adhesin production and biofilm formation. Based on the important synergistic roles of ppGpp and c-di-GMP in this process, we suggest that interference with bacterial second messenger signalling might represent an effective means for biofilm control during chronic infections.

Topics: Adhesins, Bacterial; Anti-Bacterial Agents; beta-Glucans; Biofilms; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Guanosine Tetraphosphate; Phosphorus-Oxygen Lyases; Protein Biosynthesis; Pyrophosphatases; Ribosomes; RNA Processing, Post-Transcriptional; Second Messenger Systems