Vibrio cholerae O1 El Tor: Identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation

Abstract

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°...

Rugosity in Grimontia hollisae

“…Both rugose and smooth cells grown at 30°C produce significantly more biofilm material than their counterpart rugose and smooth cells grown at 37°C. The observation that rugose cells produce more biofilm than do smooth cells is similar to what has been shown for rugose-expressing organisms (26,36).…”

“…The observations by microscopy of living rugose cells in this study showed that over time the cellular behavior of cells was to align themselves in chains, which then eventually formed laterally arranged aggregates or large multicellular cell masses. This description is markedly similar to what Yildiz and Schoolnik reported for V. cholerae O1 El Tor strains (36). However, one noted deviation in the results from these two studies is in the time frame that the aggregate formations occurred between these two Vibrio species.…”

“…Regulation of rugosity seems to be multifactorial and highly complex (5,17,26,36,37). Although there are some similarities, great differences are observed among organisms capable of expressing rugosity (3,36,37). Even though Hickman et al (11) originally described mixed opaque and nonopaque colonies for V. hollisae, now named Grimontia hollisae, we feel that the present study is the first complete description of the rugose phenotype expressed by this organism.…”

“…It is thought that the adeptness of Vibrio cells at changing back and forth from either phenotypic variant would favor the variant derivative that is more fit for survival under the prevailing environment conditions, such as those encountered in a human host or on an environmental surface. Regulation of rugosity seems to be multifactorial and highly complex (5,17,26,36,37). Although there are some similarities, great differences are observed among organisms capable of expressing rugosity (3,36,37).…”

Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus

“…V. cholerae strains (O139 as well as O1) also produce another form of extracellular polysaccharide, designated VPS. In electron micrographs, VPS appears amorphous, filling the spaces between cells Yildiz and Schoolnik, 1999). Strains overproducing VPS form rugose colonies, exhibit enhanced biofilm formation, and are more resistant to environmental insult Wai et al, 1998;1999;Yildiz and Schoolnik, 1999;Hammer and Bassler, 2003;Zhu and Mekalanos, 2003).…”