Identification of genetic and environmental factors that control exopolysaccharide expression and phase variation in the human pathogen Vibrio vulnificus

Vibrio vulnificus is a gram-negative bacterium found in estuaries and coastal waters and is associated with human disease caused by the ingestion of raw shellfish. Pathogenesis is directly related to the presence of capsular polysaccharide (CPS). Encapsulated virulent strains exhibit an opaque colony phenotype (OpS), while unencapsulated attenuated strains appear translucent (TrS). A third colony type, rugose (R), is caused by expression of rugose extracellular polysaccharide (rEPS) and forms robust biofilms. V. vulnificus undergoes spontaneous phase variation associated with altered levels of CPS and rEPS, and the work presented here identifies genetic and environmental parameters that control this process. A cluster of nine genes (brpABCDFHIJK) was found to be up-regulated in R isolates when compared to OpS or TrS isolates. We assessed the role of the brp gene cluster in CPS and rEPS production by creating non-polar mutants and characterizing their colony morphotypes, rEPS production, biofilm formation, and motility phenotypes. We demonstrate that the brp genes are required for rEPS production and robust biofilm formation, and that the decreased motility of R isolates is subject to regulation by the second messenger cyclic-di-GMP. Approximately 130-135bp upstream of brpA, we identify a promoter, which is activated at significantly higher levels in R variants than in TrS or OpS variants. In addition to characterizing the genetic components of rEPS phase variation, we show that environmental factors, such as media composition and temperature, influence the rate of polysaccharide phase variation. Specifically, calcium (Ca2+) significantly increases the rate of CPS and rEPS phase variation in V. vulnificus. Multiple phenotypic responses to increased [Ca2+] were observed among strains, which suggests the existence of underlying cognate genetic or epigenetic differences. Certain TrS isolates contained deletions at the group I CPS operon, inferring increased [Ca2+] up-regulates existing phase variation mechanisms. Expanding on a previous observation, increased [Ca2+] also enhanced biofilm formation for all phase variants. Our results show that [Ca2+] increases polysaccharide phase variation and contributes to biofilm formation, thereby likely playing a dual role in the persistence of V. vulnificus in the environment