Fitnat H. Yildiz

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.

Vibrio biofilms: so much the same yet so different

Vibrios are natural inhabitants of aquatic environments and form symbiotic or pathogenic relationships with eukaryotic hosts. Recent studies reveal that the ability of vibrios to form biofilms (i.e. matrix-enclosed, surface-associated communities) depends upon specific structural genes (flagella, pili and exopolysaccharide biosynthesis) and regulatory processes (two-component regulators, quorum sensing and c-di-GMP signaling). Here, we compare and contrast mechanisms and regulation of biofilm formation by Vibrio species, with a focus on Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio fischeri. Although many aspects are the same, others differ dramatically. Crucial questions that remain to be answered regarding the molecular underpinnings of Vibrio biofilm formation are also discussed.

Molecular analysis of rugosity in a Vibrio cholerae O1 El Tor phase variant.

Reversible phase variation between the rugose and smooth colony variants is predicted to be important for the survival of Vibrio cholerae in natural aquatic habitats. Microarray expression profiling studies of the  rugose  and  smooth  variants  of  the  same  strain led to the identification of 124 differentially regulated genes. Further expression profiling experiments showed how these genes are regulated by the VpsR and HapR transcription factors, which, respectively, positively and negatively regulate production of VPSEl Tor, a rugose‐associated extracellular polysaccharide. The study of mutants of rpoN and rpoS demonstrated the effects of these alternative sigma factors on phase variation‐specific gene expression. Bioinformatics analysis of these expression data shows that ‘rugosity’ and ‘smoothness’ are determined by a complex hierarchy of positive and negative regulators, which also affect the biofilm, surface hydrophobicity and motility phenotypes of the organism.

VpsR, a Member of the Response Regulators of the Two-Component Regulatory Systems, Is Required for Expression of vps Biosynthesis Genes and EPS(ETr)-Associated Phenotypes in Vibrio cholerae O1 El Tor.

The rugose colonial variant of Vibrio cholerae O1 El Tor produces an exopolysaccharide (EPS(ETr)) that enables the organism to form a biofilm and to resist oxidative stress and the bactericidal action of chlorine. Transposon mutagenesis of the rugose variant led to the identification of vpsR, which codes for a homologue of the NtrC subclass of response regulators. Targeted disruption of vpsR in the rugose colony genetic background yielded a nonreverting smooth-colony morphotype that produced no detectable EPS(ETr) and did not form an architecturally mature biofilm. Analysis of two genes, vpsA and vpsL, within the vps cluster of EPS(ETr) biosynthesis genes revealed that their expression is induced above basal levels in the rugose variant, compared to the smooth colonial variant, and requires vpsR. These results show that VpsR functions as a positive regulator of vpsA and vpsL and thus acts to positively regulate EPS(ETr) production and biofilm formation.

Molecular Architecture and Assembly Principles of Vibrio cholerae Biofilms

Biofilms Up Close Many bacterial infections involve biofilm formation. Cells within a biofilm are significantly more resistant to immune clearance and antibiotics compared to unattached, planktonic cells. Berk et al. (p. 236) applied superresolution optical methods to image living bacteria with nanometer-scale precision as they form a biofilm. Vibrio cholerae biofilms were observed to have three distinct levels of spatial organization: cells, clusters of cells, and collections of clusters. Each cell cluster was wrapped in a flexible, elastic envelope. Several V. cholerae matrix proteins played complementary architectural roles during biofilm development. RbmA provided cell-cell adhesion, Bap1 allowed the developing biofilm to adhere to surfaces, and heterogeneous mixtures of VPS, RbmC, and Bap1 formed the dynamic, flexible, and ordered envelopes that encase the cell clusters. When imaged in real time, biofilm development can be seen to rely on spatial and temporal intercellular contacts. In their natural environment, microbes organize into communities held together by an extracellular matrix composed of polysaccharides and proteins. We developed an in vivo labeling strategy to allow the extracellular matrix of developing biofilms to be visualized with conventional and superresolution light microscopy. Vibrio cholerae biofilms displayed three distinct levels of spatial organization: cells, clusters of cells, and collections of clusters. Multiresolution imaging of living V. cholerae biofilms revealed the complementary architectural roles of the four essential matrix constituents: RbmA provided cell-cell adhesion; Bap1 allowed the developing biofilm to adhere to surfaces; and heterogeneous mixtures of Vibrio polysaccharide, RbmC, and Bap1 formed dynamic, flexible, and ordered envelopes that encased the cell clusters.

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.

Sac1, a putative regulator that is critical for survival of Chlamydomonas reinhardtii during sulfur deprivation

The sac1 mutant of Chlamydomonas reinhardtii is aberrant in most of the normal responses to sulfur limitation; it cannot synthesize arylsulfatase, does not take up sulfate as rapidly as wild‐type cells, and does not synthesize periplasmic proteins that normally accumulate during sulfur‐limited growth. Here, we show that the sac1 mutant dies much more rapidly than wild‐type cells during sulfur deprivation; this emphasizes the vital role of the acclimation process. The loss of viability of the sac1 mutant during sulfur deprivation is only observed in the light and is mostly inhibited by DCMU. During sulfur‐stress, wild‐type cells, but not the sac1 mutant, downregulate photosynthesis. Thus, death of the sac1 mutant during sulfur deprivation is probably a consequence of its inability to downregulate photosynthesis. Furthermore, since SAC1 is necessary for the downregulation of photosynthesis, the process must be highly controlled and not simply the result of a general decrease in protein synthesis due to sulfur limitation. Genomic and cDNA copies of the SAC1 gene have been cloned. The deduced amino acid sequence of Sac1 is similar to an Escherichia coli gene that may involved in the response of E.coli to nutrient deprivation.

You've come a long way: c-di-GMP signaling.

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.

VpsT Is a Transcriptional Regulator Required for Expression of vps Biosynthesis Genes and the Development of Rugose Colonial Morphology in Vibrio cholerae O1 El Tor

Vibrio cholerae switches between smooth and rugose colonial variants. The rugose variant produces more vibrio polysaccharides (VPS(El Tor)) and forms well-developed biofilms. Both phenotypes depend on expression of vps biosynthesis genes. We identified a positive transcriptional regulator of vps gene expression, VpsT, which is homologous to response regulators of two-component regulatory systems. Disruption of vpsT in the rugose variant yields smooth colonies, prevents formation of mature biofilms, and decreases vps gene expression. The interaction between VpsT and VpsR, a previously identified positive regulator of vps genes, was also investigated.

Mutants of Chlamydomonas with Aberrant Responses to Sulfur Deprivation

In the absence of sulfur, Chlamydomonas reinhardtii, a unicellular green alga, increases its rate of sulfate import and synthesizes several periplasmic proteins, including an arylsulfatase (Ars). These changes appear to help cells acclimate to a sulfur-deficient environment. The elevated rate of sulfate import results from an increase in the capacity and affinity of the transport system for sulfate. The synthesis of Ars, a periplasmic enzyme that cleaves sulfate from aromatic compounds, enables cells to use these molecules as a source of sulfur when free sulfate is not available. To characterize the ways in which C. reinhardtii perceives changes in the sulfur status of the environment and regulates its responses to these changes, we mutagenized cells and isolated strains exhibiting aberrant accumulation of Ars activity. These mutants were characterized for Ars activity, ars mRNA accumulation, periplasmic protein accumulation, and sulfate transport activity when grown in both sulfur-sufficient and sulfur-deficient conditions. All of the mutants exhibited pleiotropic effects with respect to several of these responses. Strains harboring double mutant combinations were constructed and characterized for Ars activity and ars mRNA accumulation. From the mutant phenotypes, we inferred that both positive and negative regulatory elements were involved in the acclimation process. Both the epistatic relationships among the mutations and the effects of the lesions on the responses of C. reinhardtii to sulfur limitation distinguished these mutants from similar mutants in Neurospora crassa.