Environmental regulation of biofilm formation in Vibrio cholerae

In this dissertation, cellular processes and environmental signals that govern Vibrio cholerae biofilm formation is studied. The first section presents results from studies designed to understand the role of motility and chemotaxis in biofilm formation under dynamic conditions. The second section discusses the effect of changes in calcium levels to V. cholerae physiology including biofilm formation and virulence. The third section focuses on characterization of a calcium regulated two-component system CarRS, and its effects on biofilm formation, lipopolysaccharide modification, antimicrobial peptide resistance and in vivo fitness.;V. cholerae is a natural inhabitant of aquatic ecosystems and its complex lifecycle involves aquatic environments, ocean and freshwater, and human intestinal tract. V. cholerae's ability to form biofilms, surface-attached microbial communities embedded in a matrix, during aquatic and intestinal growth have long been thought critical for its environmental survival and transmission. To study biofilm formation in V. cholerae under the conditions simulating dynamic conditions in riverine and estuarine environments, we analyzed biofilm development dynamics using the "once-through" flow-cell reactor in wild-type V. cholerae and mutant strains lacking genes known to be critical and/or predicted to be important for biofilm formation. Our results showed that mannose-sensitive hemagglutinin (MSHA) is needed for initial surface contact and attachment, but it is not crucial at later stages as DeltamshA mutants could form mature biofilms. Flagellar motility appears to be important during formation of microcolonies but not as critical for initial attachment. Consistent with previous studies, we observed that mutants unable to produce Vibrio polysaccharide (VPS) could only form monolayers and cannot form three-dimensional biofilm structures. We also characterized the difference between the absence of a flagellum and the presence of a non-functional flagellum on initial attachment and biofilm formation. Strains with a non-functional flagellum, Delta pomAB and DeltamotX, attached less and formed thinner biofilms compared to a strain lacking flagellum, DeltaflaA and wild type. Finally, we assessed the role of chemotaxis in biofilm formation by using a strain lacking V. cholerae cheA homologs. Our results indicated that only cheA-2 has a role in biofilm formation in V. cholerae.;V. cholerae is a facultative human pathogen. The pathogen's capacity to cause outbreaks of cholera is linked to its survival and adaptability to changes in aquatic environments. One of the environmental conditions that can vary in V. cholerae's natural aquatic habitats is Ca +2. In the second part of this project, we investigated the response of V. cholerae to changes in extracellular Ca2+ levels. Whole-genome expression profiling revealed that Ca2+ reduced the expression of genes required for biofilm matrix production and iron acquisition, but increased the expression of virulence genes. LB supplemented with Ca2+ (LBCa2+) caused V. cholerae to form biofilms with reduced surface coverage and compactness, as compared to biofilms formed in LB. Furthermore, addition of Ca2+ to preformed biofilms lead to biofilm dissolution. Transcription of two genes encoding a two-component regulatory system pair, now termed calcium-regulated sensor (carS) and regulator (carR ), was decreased in cells grown in LBCa2+. Analysis of null and overexpression alleles of carS and carR revealed that expression of vps ( Vibrio polysaccharide) genes and biofilm formation are negatively regulated by the CarRS two-component regulatory system. Through epistasis analysis we determined that CarR acts in parallel with HapR, the negative regulator of vps gene expression. Taken together, our results suggest that Ca2+ causes V. cholerae to reprogram its transcriptome, cell surface properties and virulence potential.;In the final part, we characterized the CarRS two-component system and its function in V. cholerae. We identified CarRS regulons and showed that they positively regulate transcription of a three gene operon, termed crgA, crgB and crgC for CarR regulated genes. Crg proteins appear to be involved in LPS modification. crgA and crgC genes show high homology to late acyltransferases of Escherichia coli and V. cholerae, and our lipid modification analysis indicated a possible role for crg genes in lipid A modification. We also showed that crgC gene negatively regulates vps gene expression and biofilm formation, indicating that CarR might exert its biofilm phenotype through crgC, predicted to encode a lipid A modification enzyme. DeltacarR and Deltacrg mutants showed an increased sensitivity to antimicrobial peptide polymyxin-B. In bacteria lipopolysaccharide modification in response to environmental stimuli is an important part of adaptation and survival in diverse environments. Altogether, our results show that CarRS two-component system regulates biofilm formation and antimicrobial peptide resistance in response to changes in environmental calcium concentration.