c-di-GMP signaling in Vibrio cholerae: The link between rugosity, biofilm formation and motility

Vibrio cholerae, the causative agent of diarrheal disease cholera, is a facultative human pathogen. The pathogen is an inhabitant of aquatic environments and its environmental survival is critical for occurrence of cholera epidemics. Growth in biofilms---surface attached microbial communities---facilitates environmental survival of the pathogen. V. cholerae produces two variants, termed smooth and rugose, which differ in their biofilm forming capacity, motility and virulence. Rugose variant produces high amounts of exopolysaccharide, forms more structured biofilms, and is more resistant to osmotic and oxidative stresses, acid and chlorine treatments, phage infections and protozoan grazing compared to smooth. In this study, I sought to elucidate the molecular mechanism of smooth-to-rugose phase variation, which is fundamental to our understanding of environmental survival strategies utilized by V. cholerae.;Using mutational and complementational analyses, I found that the smooth-to-rugose transition in our prototype rugose variant of V. cholerae is resulted from a single nucleotide change in a gene encoding a diguanylate cyclase. Diguanylate cyclases are enzymes capable of producing cyclic diguanosine monophosphate (c-di-GMP), which is a newly recognized second messenger in prokaryotes. I determined another diguanylate cyclase, CdgA, is responsible for the rugosity observed in Hap---the master regulator of quorum sensing-mutants. V. cholerae has 62 genes predicted to encode c-di-GMP proteins. To elucidate roles of other c-di-GMP signaling components, I screened a library of c-di-GMP signaling mutants for rugosity-associated phenotypes. I found two additional proteins, CdgG and CdgH, that controls rugosity, biofilm formation and motility. Through whole-genome expression profiling analysis, I determined that V. cholerae responds to changes in intracellular [c-di-GMP] by modulating transcription of genes involved in Vibrio polysaccharide (VPS) production and motility. Taken together, these studies provided insight into the molecular mechanism of c-di-GMP signaling systems and their role in biofilm formation in V. cholerae.