SummaryExtracellular polysaccharides, such as lipopolysaccharide and loosely associated exopolysaccharides, are essential for Vibrio vulnificus to form biofilms. The role of another major component of the V. vulnificus extracellular matrix, capsular polysaccharide (CPS), which contributes to colony opacity, has been characterized in biofilm formation. A CPS-deficient mutant, whose wbpP gene encoding UDP-GlcNAc C4-epimerase was knocked out, formed significantly more biofilm than wild type, due to increased hydrophobicity of the cell surface, adherence to abiotic surfaces and cell aggregation. To elucidate the direct effect of CPS on biofilm structure, extracted CPS and a CPS-degrading enzyme, α-N-acetylgalactosaminidase, were added in biofilm assays, resulting in reduction and increment of biofilm sizes respectively. Therefore, it is suggested that CPS play a critical role in determining biofilm size by restricting continual growth of mature biofilms. Since CPS is required after maturation, CPS biosynthesis should be controlled in a cell density-dependent manner, e.g. by quorumsensing (QS) regulation. Analysing transcription of the CPS gene cluster revealed that it was activated by SmcR, a QS master regulator, via binding to the upstream region of the cluster. Therefore, CPS was produced when biofilm cell density reached high enough to turn on QS regulation and limited biofilms to appropriate sizes.

NtrC-mediated production of exopolysaccharides (EPS), essential components for Vibrio vulnificus biofilms, is highly increased in the presence of dicarboxylic or tricarboxylic acids. Gel-shift assays showed that regulation of the EPS-gene cluster I (EPS-I cluster) by NtrC was direct via binding of phosphorylated NtrC (p-NtrC) to the regulatory region of the EPS-I cluster. In contrast, p-NtrC did not bind to the EPS-II and EPS-III clusters, suggesting that NtrC regulation was not direct and another transcription factor belonging to an NtrC-regulon might play a role in activating their transcription. A candidate transcription factor, DctD, of which expression was induced by NtrC, activated the expression of the EPS-II and EPS-III clusters via direct binding to their upstream regions. Under growth conditions with either dicarboxylic or tricarboxylic acids, the expression of NtrC was induced and the transcription of dctD was activated. Furthermore, DctD exhibited higher transcriptional activity under the conditions with dicarboxylic acids than with tricarboxylic acids. Therefore, this study demonstrates that under dicarboxylate-rich conditions, both the abundance and activity of DctD were markedly induced, which activates the expression of two EPS clusters to maximize biosynthesis of EPS facilitating biofilm maturation in V. vulnificus.

The interaction between fermentation-respiration switch (FrsA) protein and glucose-specific enzyme IIA(Glc) increases glucose fermentation under oxygen-limited conditions. We show that FrsA converts pyruvate to acetaldehyde and carbon dioxide in a cofactor-independent manner and that its pyruvate decarboxylation activity is enhanced by the dephosphorylated form of IIA(Glc) (d-IIA(Glc)). Crystal structures of FrsA and its complex with d-IIA(Glc) revealed residues required for catalysis as well as the structural basis for the activation by d-IIA(Glc).

The gram-negative bacterium Vibrio cholerae is the causative agent of the diarrhoeal disease cholera and is responsible for seven recorded pandemics. Several factors are postulated to have led to the decline of 6th pandemic classical strains and the rise of El Tor biotype V. cholerae, establishing the current 7th pandemic. We investigated the ability of classical V. cholerae of the 2nd and 6th pandemics to engage their type six secretion system (T6SS) in microbial competition against non-pandemic and 7th pandemic strains. We report that classical V. cholerae underwent sequential mutations in T6SS genetic determinants that initially exposed 2nd pandemic strains to microbial attack by non-pandemic strains and subsequently caused 6th pandemic strains to become vulnerable to El Tor biotype V. cholerae intraspecific competition. The chronology of these T6SS-debilitating mutations agrees with the decline of 6th pandemic classical strains and the emergence of 7th pandemic El Tor V. cholerae.

Transcription regulation by bacterial two-component systems is achieved by a response regulator upon its transition to the transcriptionally active form via kinase activity of its cognate sensor under specific conditions. A well-known response regulator, DctD, is converted to its phosphorylated form when DctB senses ambient dicarboxylic acids.