Sinem Beyhan

Vibrio cholerae VpsT Regulates Matrix Production and Motility by Directly Sensing Cyclic di-GMP

Decoding a Second-Messengers Message Biofilms are aggregates of bacteria on a surface often associated with increased resistance to antibiotics and stress. In Vibrio cholerae, the bacterial species that causes cholera, biofilm formation is promoted by the bacterial second-messenger cyclic diguanylate (c-di-GMP) and involves the transcription regulator, VpsT. Krasteva et al. (p. 866) show that VpsT is itself a receptor for c-di-GMP and that binding of the small signaling molecule promotes VpsT dimerization, which is required for DNA recognition and transcriptional regulation. As well as activating components of the biofilm pathway, VpsT also down-regulates motility genes in a c-di-GMP–dependent manner. A bacterial signaling molecule induces the dimerization and activation of a biofilm-promoting transcription factor. Microorganisms can switch from a planktonic, free-swimming life-style to a sessile, colonial state, called a biofilm, which confers resistance to environmental stress. Conversion between the motile and biofilm life-styles has been attributed to increased levels of the prokaryotic second messenger cyclic di-guanosine monophosphate (c-di-GMP), yet the signaling mechanisms mediating such a global switch are poorly understood. Here we show that the transcriptional regulator VpsT from Vibrio cholerae directly senses c-di-GMP to inversely control extracellular matrix production and motility, which identifies VpsT as a master regulator for biofilm formation. Rather than being regulated by phosphorylation, VpsT undergoes a change in oligomerization on c-di-GMP binding.

Cyclic‐diGMP signal transduction systems in Vibrio cholerae: modulation of rugosity and biofilm formation

Cyclic di‐guanylic acid (c‐diGMP) is a second messenger that modulates the cell surface properties of several microorganisms. Concentrations of c‐diGMP in the cell are controlled by the opposing activities of diguanylate cyclases and phosphodiesterases, which are carried out by proteins harbouring GGDEF and EAL domains respectively. In this study, we report that the cellular levels of c‐diGMP are higher in the Vibrio cholerae rugose variant compared with the smooth variant. Modulation of cellular c‐diGMP levels by overexpressing proteins with GGDEF or EAL domains increased or decreased colony rugosity respectively. Several genes encoding proteins with either GGDEF or EAL domains are differentially expressed between the two V. cholerae variants. The generation and characterization of null mutants of these genes (cdgA–E, rocS and mbaA) revealed that rugose colony formation, exopolysaccharide production, motility and biofilm formation are controlled by their action. Furthermore, epistasis analysis suggested that cdgC, rocS and mbaA act in convergent pathways to regulate the phenotypic properties of the rugose and smooth variants, and are part of the VpsR, VpsT and HapR signal transduction pathway.

Regulation of Rugosity and Biofilm Formation in Vibrio cholerae: Comparison of VpsT and VpsR Regulons and Epistasis Analysis of vpsT, vpsR, and hapR

Vibrio cholerae undergoes phenotypic variation that generates two morphologically different variants, termed smooth and rugose. The transcriptional profiles of the two variants differ greatly, and many of the differentially regulated genes are controlled by a complex regulatory circuitry that includes the transcriptional regulators VpsR, VpsT, and HapR. In this study, we identified the VpsT regulon and compared the VpsT and VpsR regulons to elucidate the contribution of each positive regulator to the rugose variant transcriptional profile and associated phenotypes. We have found that although the VpsT and VpsR regulons are very similar, the magnitude of the gene regulation accomplished by each regulator is different. We also determined that cdgA, which encodes a GGDEF domain protein, is partially responsible for the altered vps gene expression between the vpsT and vpsR mutants. Analysis of epistatic relationships among hapR, vpsT, and vpsR with respect to a whole-genome expression profile, colony morphology, and biofilm formation revealed that vpsR is epistatic to hapR and vpsT. Expression of virulence genes was increased in a vpsR hapR double mutant relative to a hapR mutant, suggesting that VpsR negatively regulates virulence gene expression in the hapR mutant. These results show that a complex regulatory interplay among VpsT, VpsR, HapR, and GGDEF/EAL family proteins controls transcription of the genes required for Vibrio polysaccharide and virulence factor production in V. cholerae.

Indole acts as an Extracellular Cue Regulating Gene Expression in Vibrio cholerae

Indole has been proposed to act as an extracellular signal molecule influencing biofilm formation in a range of bacteria. For this study, the role of indole in Vibrio cholerae biofilm formation was examined. It was shown that indole activates genes involved in vibrio polysaccharide (VPS) production, which is essential for V. cholerae biofilm formation. In addition to activating these genes, it was determined using microarrays that indole influences the expression of many other genes, including those involved in motility, protozoan grazing resistance, iron utilization, and ion transport. A transposon mutagenesis screen revealed additional components of the indole-VPS regulatory circuitry. The indole signaling cascade includes the DksA protein along with known regulators of VPS production, VpsR and CdgA. A working model is presented in which global control of gene expression by indole is coordinated through sigma(54) and associated transcriptional regulators.

The Vibrio cholerae flagellar regulatory hierarchy controls expression of virulence factors.

Vibrio cholerae is a motile bacterium responsible for the disease cholera, and motility has been hypothesized to be inversely regulated with virulence. We examined the transcription profiles of V. cholerae strains containing mutations in flagellar regulatory genes (rpoN, flrA, flrC, and fliA) by utilizing whole-genome microarrays. Results revealed that flagellar transcription is organized into a four-tiered hierarchy. Additionally, genes with proven or putative roles in virulence (e.g., ctx, tcp, hemolysin, and type VI secretion genes) were upregulated in flagellar regulatory mutants, which was confirmed by quantitative reverse transcription-PCR. Flagellar regulatory mutants exhibit increased hemolysis of human erythrocytes, which was due to increased transcription of the thermolabile hemolysin (tlh). The flagellar regulatory system positively regulates transcription of a diguanylate cyclase, CdgD, which in turn regulates transcription of a novel hemagglutinin (frhA) that mediates adherence to chitin and epithelial cells and enhances biofilm formation and intestinal colonization in infant mice. Our results demonstrate that the flagellar regulatory system modulates the expression of nonflagellar genes, with induction of an adhesin that facilitates colonization within the intestine and repression of virulence factors maximally induced following colonization. These results suggest that the flagellar regulatory hierarchy facilitates correct spatiotemporal expression patterns for optimal V. cholerae colonization and disease progression.

Identification and Characterization of Cyclic Diguanylate Signaling Systems Controlling Rugosity in Vibrio cholerae

Vibrio cholerae, the causative agent of the disease cholera, can generate rugose variants that have an increased capacity to form biofilms. Rugosity and biofilm formation are critical for the environmental survival and transmission of the pathogen, and these processes are controlled by cyclic diguanylate (c-di-GMP) signaling systems. c-di-GMP is produced by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). Proteins that contain GGDEF domains act as DGCs, whereas proteins that contain EAL or HD-GYP domains act as PDEs. In the V. cholerae genome there are 62 genes that are predicted to encode proteins capable of modulating the cellular c-di-GMP concentration. We previously identified two DGCs, VpvC and CdgA, that can control the switch between smooth and rugose. To identify other c-di-GMP signaling proteins involved in rugosity, we generated in-frame deletion mutants of all genes predicted to encode proteins with GGDEF and EAL domains and then searched for mutants with altered rugosity. In this study, we identified two new genes, cdgG and cdgH, involved in rugosity control. We determined that CdgH acts as a DGC and positively regulates rugosity, whereas CdgG does not have DGC activity and negatively regulates rugosity. In addition, epistasis analysis with CdgG, CdgH, and other DGCs and PDEs controlling rugosity revealed that CdgG and CdgH act in parallel with previously identified c-di-GMP signaling proteins to control rugosity in V. cholerae. We also determined that PilZ domain-containing c-di-GMP binding proteins contribute minimally to rugosity, indicating that there are additional c-di-GMP binding proteins controlling rugosity in V. cholerae.

Smooth to rugose phase variation in Vibrio cholerae can be mediated by a single nucleotide change that targets c‐di‐GMP signalling pathway

Microorganisms use phase variation to increase population diversity to maximize evolutionary success. One such variation is the smooth to rugose phenotype change in Vibrio cholerae. We determined that the variation between smooth and rugose phenotypes can be controlled by a single nucleotide change in a gene (vpvC) predicted to encode a diguanylate cyclase. The vpvC allele found in the rugose genetic background is more active at producing c‐di‐GMP while that in smooth genetic background is less active. In support of this finding, disruption of vpvC in the rugose genetic variant decreases cellular c‐di‐GMP levels, diminishes rugose‐associated phenotypes and yields a smooth variant. Furthermore, the frequency of phase variation decreases dramatically when the vpvC locus is deleted in the smooth genetic background. Evidence is presented that the rugose variant is less susceptible to phage infection than the smooth variant. As phage infection is known to control populations of V. cholerae and thus outbreaks of cholera, phase variation may increase the evolutionary success of the pathogen.

Differences in Gene Expression between the Classical and El Tor Biotypes of Vibrio cholerae O1

ABSTRACT Differences in whole-genome expression patterns between the classical and El Tor biotypes of Vibrio cholerae O1 were determined under conditions that induce virulence gene expression in the classical biotype. A total of 524 genes (13.5% of the genome) were found to be differentially expressed in the two biotypes. The expression of genes encoding proteins required for biofilm formation, chemotaxis, and transport of amino acids, peptides, and iron was higher in the El Tor biotype. These gene expression differences may contribute to the enhanced survival capacity of the El Tor biotype in environmental reservoirs. The expression of genes encoding virulence factors was higher in the classical than in the El Tor biotype. In addition, the vieSAB genes, which were originally identified as regulators of ctxA transcription, were expressed at a fivefold higher level in the classical biotype. We determined the VieA regulon in both biotypes by transcriptome comparison of wild-type and vieA deletion mutant strains. VieA predominantly regulates gene expression in the classical biotype; 401 genes (10.3% of the genome), including those encoding proteins required for virulence, exopolysaccharide biosynthesis, and flagellum production as well as those regulated by σE, are differentially expressed in the classical vieA deletion mutant. In contrast, only five genes were regulated by VieA in the El Tor biotype. A large fraction (20.8%) of the genes that are differentially expressed in the classical versus the El Tor biotype are controlled by VieA in the classical biotype. Thus, VieA is a major regulator of genes in the classical biotype under virulence gene-inducing conditions.

Cell Envelope Perturbation Induces Oxidative Stress and Changes in Iron Homeostasis in Vibrio cholerae

The Vibrio cholerae type II secretion (T2S) machinery is a multiprotein complex that spans the cell envelope. When the T2S system is inactivated, cholera toxin and other exoproteins accumulate in the periplasmic compartment. Additionally, loss of secretion via the T2S system leads to a reduced growth rate, compromised outer membrane integrity, and induction of the extracytoplasmic stress factor RpoE (A. E. Sikora, S. R. Lybarger, and M. Sandkvist, J. Bacteriol. 189:8484-8495, 2007). In this study, gene expression profiling reveals that inactivation of the T2S system alters the expression of genes encoding cell envelope components and proteins involved in central metabolism, chemotaxis, motility, oxidative stress, and iron storage and acquisition. Consistent with the gene expression data, molecular and biochemical analyses indicate that the T2S mutants suffer from internal oxidative stress and increased levels of intracellular ferrous iron. By using a tolA mutant of V. cholerae that shares a similar compromised membrane phenotype but maintains a functional T2S machinery, we show that the formation of radical oxygen species, induction of oxidative stress, and changes in iron physiology are likely general responses to cell envelope damage and are not unique to T2S mutants. Finally, we demonstrate that disruption of the V. cholerae cell envelope by chemical treatment with polymyxin B similarly results in induction of the RpoE-mediated stress response, increased sensitivity to oxidants, and a change in iron metabolism. We propose that many types of extracytoplasmic stresses, caused either by genetic alterations of outer membrane constituents or by chemical or physical damage to the cell envelope, induce common signaling pathways that ultimately lead to internal oxidative stress and misregulation of iron homeostasis.

The Vibrio cholerae virulence regulatory cascade controls glucose uptake through activation of TarA, a small regulatory RNA

Vibrio cholerae causes the severe diarrhoeal disease cholera. A cascade of regulators controls expression of virulence determinants in V. cholerae at both transcriptional and post‐transcriptional levels. ToxT is the direct transcription activator of the major virulence genes in V. cholerae. Here we describe TarA, a highly conserved, small regulatory RNA, whose transcription is activated by ToxT from toxboxes present upstream of the ToxT‐activated gene tcpI. TarA regulates ptsG, encoding a major glucose transporter in V. cholerae. Cells overexpressing TarA exhibit decreased steady‐state levels of ptsG mRNA and grow poorly in glucose‐minimal media. A mutant lacking the ubiquitous regulatory protein Hfq expresses diminished TarA levels, indicating that TarA likely interacts with Hfq to regulate gene expression. RNAhybrid analysis of TarA and the putative ptsG mRNA leader suggests potential productive base‐pairing between these two RNA molecules. A V. cholerae mutant lacking TarA is compromised for infant mouse colonization in competition with wild type, suggesting a role in the in vivo fitness of V. cholerae. Although somewhat functionally analogous to SgrS of Escherichia coli, TarA does not encode a regulatory peptide, and its expression is activated by the virulence gene pathway in V. cholerae and not by glycolytic intermediates.