The Rvv two-component regulatory system regulates biofilm formation and colonization in Vibrio cholerae

The facultative human pathogen, Vibrio cholerae, employs two-component signal transduction systems (TCS) to sense and respond to environmental signals encountered during its infection cycle. TCSs consist of a sensor histidine kinase (HK) and a response regulator (RR); the V. cholerae genome encodes 43 HKs and 49 RRs, of which 25 are predicted to be cognate pairs. Using deletion mutants of each HK gene, we analyzed the transcription of vpsL, a biofilm gene required for Vibrio polysaccharide and biofilm formation. We found that a V. cholerae TCS that had not been studied before, now termed Rvv, controls biofilm gene transcription. The Rvv TCS is part of a three-gene operon that is present in 30% of Vibrionales species. The rvv operon encodes RvvA, the HK; RvvB, the cognate RR; and RvvC, a protein of unknown function. Deletion of rvvA increased transcription of biofilm genes and altered biofilm formation, while deletion of rvvB or rvvC lead to no changes in biofilm gene transcription. The phenotypes observed in Delta rvvA depend on RvvB. Mutating RvvB to mimic constitutively active and inactive versions of the RR only impacted phenotypes in the Delta rvvA genetic background. Mutating the conserved residue required for kinase activity in RvvA did not affect phenotypes, whereas mutation of the conserved residue required for phosphatase activity mimicked the phenotype of the rvvA mutant. Furthermore, Delta rvvA displayed a significant colonization defect which was dependent on RvvB and RvvB phosphorylation state, but not on VPS production. We found that RvvA's phosphatase activity regulates biofilm gene transcription, biofilm formation, and colonization phenotypes. This is the first systematic analysis of the role of V. cholerae HKs in biofilm gene transcription and resulted in the identification of a new regulator of biofilm formation and virulence, advancing our understanding of the role TCSs play in regulating these critical cellular processes in V. cholerae. Author summaryTwo-component signal transduction systems are one of the most used regulatory networks by microorganisms to sense, respond, and adapt to their environments and increase their environmental fitness. We systematically analyzed V. cholerae HKs and identified Rvv, a new regulator of biofilm gene transcription and virulence. This study lays the groundwork for understanding the signal transduction pathways that regulate V. cholerae's environmental fitness. Additionally, it enhances our understanding of the role TCSs play in regulating these critical cellular processes in V. cholerae.