Roles of RseB, σE,and DegP in Virulence and Phase Variation of Colony Morphotype of Vibrio vulnificus

Vibrio vulnificus is an estuarine bacterium capable of causing serious and often fatal wound infections and primary septicemia. We used alkaline phosphatase insertion mutagenesis to identify genes necessary for the virulence of this pathogen. One mutant had an in-frame fusion of ′phoA to the gene encoding RseB, a periplasmic negative regulator of the alternative sigma factor σ^E. σ^E controls an extensive regulon involved in responding to cell envelope stresses. Colonies of the rseB mutant were less opaque than wild-type colonies and underwent phase variation between translucent and opaque morphologies. rseB mutants were attenuated for virulence in subcutaneously inoculated iron-dextran-treated mice. To obtain insight into the role of rseB and the extracytoplasmic stress response in V. vulnificus, mutants with defined mutations in rseB and two important members of the extracytoplasmic stress regulon, rpoE and degP, were constructed for analysis of virulence, colony morphology, and stress-associated phenotypes. Deletion of rseB caused reversible phase variation in the colony morphotype that was associated with extracellular polysaccharides. Translucent and transparent morphotype strains were attenuated for virulence. rpoE and degP deletion mutants were sensitive to membrane-perturbing agents and heat but were not significantly attenuated for V. vulnificus virulence in mice. These results reveal complex relationships between regulation of the extracytoplasmic stress response, exopolysaccharides, and the virulence of V. vulnificus.Vibrio vulnificus is a gram-negative estuarine bacterium responsible for severe opportunistic infections (for a review, see reference 17). Ingestion of raw contaminated seafood can lead to septicemia in susceptible patients, while contact with contaminated seawater or seafood can cause wound infection, which may progress to necrotizing fasciitis and sepsis. Mortality rates for sepsis and wound infection can be as high as 75% and 50%, respectively. Predisposing conditions for septicemia include liver disease, cirrhosis, hemochromatosis, diabetes, and immune compromise, while wound infection can occur in otherwise healthy persons.Several virulence factors have been identified for V. vulnificus, most notably the antiphagocytic capsule (55, 65), RtxA toxin (26, 28, 32), and iron acquisition systems (31, 64). For a review, see reference 17. However, much is yet to be discovered, particularly the mechanisms of extreme tissue damage and rapid growth in host tissues (17). To examine these traits, we focused on the factors that are localized to the bacterial cell surface or are secreted into the extracellular space, considering that most virulence factors are exported to interact with the host. Alkaline phosphatase (phoA) mutagenesis is a useful tool for identifying genes encoding exported products (33), based on the principle that alkaline phosphatase is active only when it is exported beyond the bacterial cytoplasm. Randomly generated phoA gene fusions, most often generated via TnphoA (33), must be in genes encoding exported proteins to have enzyme activity detected by plating on the chromogenic substrate 5-bromo-4-chloro-3-indolylphosphate (BCIP). In our studies, TnphoA did not work effectively in V. vulnificus for unknown reasons. We therefore created a mini-Tn5 transposon-based ′phoA delivery system, miniTn5phoA (8), that works well in V. vulnificus.Using this method, we identified a phoA mutant that had an in-frame fusion of ′phoA to the gene encoding RseB, a periplasmic negative regulator of sigma E (σ^E) activity. The rseB mutant exhibited several interesting phenotypes, including phase variation between translucent and opaque colony morphologies and attenuated virulence. σ^E is an alternative RNA polymerase sigma factor that controls an extensive regulon involved in responding to cell envelope stresses (48). This response, termed the extracytoplasmic stress response (ESR), is essential for maintaining the envelope integrity of gram-negative bacteria under certain stress conditions (for a review, see reference 48). Because rseB is involved in the ESR, we determined the role of the σ^E-mediated ESR in V. vulnificus. We also investigated the possible reasons for the translucent morphology of RseB variants by comparing these variants with an acapsular translucent mutant of V. vulnificus. This study uncovered a possible role for RseB in phase variation of extracellular polysaccharide (EPS) expression and is the first study to investigate the role of the ESR in the virulence of V. vulnificus.