Reciprocal c-di-GMP signaling: Incomplete flagellum biogenesis triggers c-di-GMP signaling pathways that promote biofilm formation

Author summary A key regulator of Vibrio cholerae physiology is the nucleotide-based, second messenger cyclic dimeric guanosine monophosphate (c-di-GMP). We found that the status of flagellar biosynthesis at different stages of flagellar assembly modulates c-di-GMP signaling in V. cholerae and identified diguanylate cyclases involved in this regulatory process. The effect of motility status on the cellular c-di-GMP level is partly dependent on the flagellar stator and Na+ flux through the flagellum. Finally, we showed that c-di-GMP-dependent positive regulators of biofilm formation are critical for the signaling cascade that connects motility status to biofilm formation. Our results show that in addition to c-di-GMP promoting motile to biofilm lifestyle switch, "motility status" of V. cholerae modulates c-di-GMP signaling and biofilm formation. 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.