The assembly status of the V. cholerae flagellum regulates biofilm formation, suggesting that the bacterium senses a lack of movement to commit to a sessile lifestyle. Motility and biofilm formation are inversely regulated by the second messenger molecule cyclic dimeric guanosine monophosphate (c-di-GMP). Therefore, we sought to define the flagellum-associated c-di-GMP-mediated signaling pathways that regulate the transition from a motile to a sessile state. Here we report that elimination of the flagellum, via loss of the FlaA flagellin, results in a flagellum-dependent biofilm regulatory (FDBR) response, which elevates cellular c-di-GMP levels, increases biofilm gene expression, and enhances biofilm formation. The strength of the FDBR response is linked with status of the flagellar stator: it can be reversed by deletion of the T ring component MotX, and reduced by mutations altering either the Na+ binding ability of the stator or the Na+ motive force. Absence of the stator also results in reduction of mannose-sensitive hemagglutinin (MSHA) pilus levels on the cell surface, suggesting interconnectivity of signal transduction pathways involved in biofilm formation. Strains lacking flagellar rotor components similarly launched an FDBR response, however this was independent of the status of assembly of the flagellar stator. We found that the FDBR response requires at least three specific diguanylate cyclases that contribute to increased c-di-GMP levels, and propose that activation of biofilm formation during this response relies on c-di-GMP-dependent activation of positive regulators of biofilm production. Together our results dissect how flagellum assembly activates c-di-GMP signaling circuits, and how V. cholerae utilizes these signals to transition from a motile to a sessile state.

Production of an extracellular matrix is essential for biofilm formation, as this matrix both secures and protects the cells it encases. Mechanisms underlying production and assembly of matrices are poorly understood. Vibrio cholerae, relies heavily on biofilm formation for survival, infectivity, and transmission. Biofilm formation requires Vibrio polysaccharide (VPS), which is produced by vps gene-products, yet the function of these products remains unknown. Here, we demonstrate that the vps gene-products vpsO and vpsU encode respectively for a tyrosine kinase and a cognate tyrosine phosphatase. Collectively, VpsO and VpsU act as a tyrosine phosphoregulatory system to modulate VPS production. We present structures of VpsU and the kinase domain of VpsO, and we report observed autocatalytic tyrosine phosphorylation of the VpsO C-terminal tail. The position and amount of tyrosine phosphorylation in the VpsO C-terminal tail represses VPS production and biofilm formation through a mechanism involving the modulation of VpsO oligomerization. We found that tyrosine phosphorylation enhances stability of VpsO. Regulation of VpsO phosphorylation by the phosphatase VpsU is vital for maintaining native VPS levels. This study provides new insights into the mechanism and regulation of VPS production and establishes general principles of biofilm matrix production and its inhibition.