Serotype conversion gene rfbT is directly regulated by histone-like nucleoid structuring protein (H-NS) in V. cholerae O1.

serogroup O1 ( O1) is closely associated with cholera epidemics and has two main immunologically distinguishable serotypes, Ogawa and Inaba. Isolates serotype as Ogawa if the O-antigen polysaccharide (O-PS) is methylated or as Inaba if the O-PS is not methylated. This methylation is mediated by a methyltransferase encoded by the gene, and the mutation and low expression of results in serotype switch from Ogawa to Inaba. Previously, we have shown that cAMP receptor protein (CRP) activates . In this study, we demonstrated that histone-like nucleoid structuring protein (H-NS) is directly involved in the transcriptional repression of . This finding is supported by the analyses of mRNA level, - reporter fusions, electrophoretic mobility shift assay (EMSA), and DNase I footprinting assay. The mRNA abundances were significantly increased by deleting rather than which also preferentially associates with AT-rich sequences. A . Analysis of - reporter fusions validated the transcriptional repression of Subsequent EMSA and DNase I footprinting assay confirmed the direct binding of H-NS to promoter and mapped the exact binding site which was further verified by site-directed mutagenesis and promoter functional analysis. Furthermore, we found that in deletion mutant, CRP is no longer required for transcriptionally activating , suggesting that CRP functions as a dedicated transcription factor to relieve H-NS repression at . Together, this study expanded our understanding of the genetic regulatory mechanism of serotype conversion by global regulators in O1.