Parallel regulatory circuits orchestrate biofilm formation in response to c-di-GMP levels and growth phase.

Biofilm formation is a highly regulated process that contributes to the environmental fitness of microorganisms, including pathogenic bacteria. The second messenger c-di-GMP is a critical regulator of biofilm formation whose cellular levels are tightly regulated by the abundance and activity of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs). These enzymes synthesize and degrade c-di-GMP, respectively. The Vibrio cholerae VpvABC system encodes a DGC and is critical for biofilm formation; however, much remains unknown about its regulation. Here we demonstrate that the vpvABC system is transcriptionally regulated by c-di-GMP and the master biofilm regulators VpsT and VpsR. However, we also identify the alternative sigma factor RpoS as a positive regulator of vpvABC. RpoS is involved in the regulation of many c-di-GMP metabolism genes and plays a role in biofilm architecture, likely mediated in part through vpvC. In mature biofilms, vpvA transcription was highest near the biofilm substratum and VpsT, VpsR, and RpoS were critical for vpvABC transcription. Overall, our genetic dissection reveals the vpvABC system is regulated by two parallel circuits: a c-di-GMP sensing-circuit acting through VpsT and VpsR and a stationary growth phase circuit via RpoS. These findings underscore the multilayered regulatory mechanisms that precisely govern biofilm formation by a pathogen.