Molecular analysis of Vibrio vulnificus phase variants and characterization of a new biofilm-proficient rugose phenotype

Vibrio vulnificus is a marine bacterium capable of causing severe and fatal illness in susceptible people. The primary virulence factor is a capsular polysaccharide (CPS) that allows the bacteria to evade the host immune system. Two phenotypic forms were previously known to exist, opaque (virulent) and translucent (avirulent), and had been shown to undergo spontaneous phase variation, each switching to the other phenotype. Provided here is the first report of a third phenotype, rugose. The rugose variant is dry and wrinkled compared to the smooth colony morphology of the opaque and translucent forms. Both opaque and translucent strains produce rugose variants that are similar in some ways, but show important differences between the two. Rugose V. vulnificus produce copious amounts of rugose exopolysaccharide (EPS) that allows them to form well-defined biofilms in attachment to glass, as a pellicle on the surface of broth, and as a 3-dimensional colony of distinct architecture. The rugose forms survived better in normal human serum than did their opaque and translucent parents, while only the translucent parent and its rugose variant survived exposure to chlorine. It is proposed that opaque-derived rugose variants also produce CPS, in addition to EPS, and that it is the CPS that actually confers susceptibility to chlorine. Five genes associated with CPS and/or EPS production were expressed by all phase variants at all points of the life cycle. Also reported here is a group of genes, the rugose gene cluster, differentially expressed by the three phenotypes. Opaque and translucent forms expressed genes in this cluster at low levels or not at all, while the rugose variants expressed all of these genes at higher levels throughout exponential growth. As biofilms have been shown to protect bacteria from adverse environmental conditions and the rugose variants produce robust biofilms, this form may contribute to the survival of this bacteria in its natural environment. Future study of these genes should provide insight into the expression and regulation of the rugose phenotype and the resulting biofilms, as well as survival and virulence of V. vulnificus