The rugose colony variant of Vibrio cholerae O1, biotype El Tor, is shown to produce an exopolysaccharide, EPS ETr , that confers chlorine resistance and biofilm-forming capacity. EPS ETr production requires a chromosomal locus, vps, that contains sequences homologous to carbohydrate biosynthesis genes of other bacterial species. Mutations within this locus yield chlorine-sensitive, smooth colony variants that are biofilm deficient. The biofilm-forming properties of EPS ETr may enable the survival of V. cholerae O1 within environmental aquatic habitats between outbreaks of human disease.The epidemiology of cholera in the Bengal region of India and Bangladesh is marked by periodic, seasonal outbreaks followed by long intervals during which the disease occurs sporadically or not at all (1). Failure to regularly identify chronic carriers of Vibrio cholerae O1 or infected animal reservoirs, its capacity to colonize the gut of copepods (2), and detection of the organism attached to phytoplankton (3-5) and in water samples throughout the year (6) has led to the idea that V. cholerae O1 resides within natural aquatic habitats during interepidemic periods (7). Possibilities suggested by other investigators include its persistence in a viable, but nonculturable, state in water (8, 9) and its association as a commensal or symbiont of other members of the aquatic flora (7). These microenvironments have in common the survival of the organism under physiological constraints that likely differ markedly from conditions within the human gastrointestinal tract. A common feature of most environmental reservoirs is the low availability of nutrients, compared with the intestinal milieu. Additionally, environmental habitats are subject to seasonally determined changes of the microflora and to physicochemical fluctuations. Here we report the following: the rugose colonial variant of V. cholerae O1, biotype El Tor produces a unique extracellular polysaccharide, designated EPS ETr , that confers resistance to chlorine and promotes biofilm formation. Compositional and linkage analysis of this material shows it to be unrelated to previously described V. cholerae carbohydrates, and mutational experiments led to the identification of a chromosomal cluster of genes that is required for the production of this compound and for rugose-associated phenotypes. In view of the functional properties it confers, we now propose a role for EPS ETr in the survival of the organism within environmental aquatic habitats. MATERIALS AND METHODSBacterial Strains. Escherichia coli strains DH5␣ and S17-1 (10) were used for standard DNA manipulations and mating, respectively. The V. cholerae strains used were smooth and rugose variants of 92A1552 (wild type, El Tor, Inaba, and Rif r ) and mutants of these strains listed in Table 2.Microscopy. Scanning and transmission electron microscopy, ruthenium red staining, and immunogold electron microscopy were performed as described (11).Isolation of EPS. Smooth or rugose colonies were cultivated for 24 hr at 30°...

Microorganisms can switch from a planktonic, free-swimming life style to a sessile, colonial state, called a biofilm, conferring resistance to environmental stress. Conversion between the motile and biofilm life style 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, identifying VpsT as a master regulator for biofilm formation. Rather than being regulated by phosphorylation, VpsT undergoes a change in oligomerization upon c-di-GMP binding.

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). In this review, 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. While many aspects are the same, others differ dramatically. Critical questions that remain to be answered regarding the molecular underpinnings of Vibrio biofilm formation will also be discussed.

SummaryReversible 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 VPS El 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.

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 super-resolution 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 (VPS), RbmC, and Bap1 formed dynamic, flexible and ordered envelopes that encased the cell clusters.