The PAS domain of the Polarly-Localized Histidine Kinase FlrB in Vibrio cholerae Controls Class III Flagellar Transcription and Contributes to Intestinal Colonization

FlrB is a histidine kinase and along with FlrC it represents the two-component system that regulates flagellar transcription within Vibrio cholerae. FlrB autophosphorylates the conserved histidine at amino acid position 135, then transfers the phosphate to the conserved aspartate residue at position 54 in FlrC. FlrC-P binds promoter regions and activates sigma54-dependent transcription of class III flagellar genes. By mutating the histidine at position 135 to an asparagine (H135N), FlrB is unable to undergo autophosphorylation, prohibiting phosphorylation of FlrC and transcription of FlrC-dependent genes. Mutating the leucine residue at position 36 in the PAS domain of FlrB to a phenylalanine (L36F) results in increased FlrC-dependent transcription. We hypothesize that V. cholerae with the FlrB L36F mutant form produces significantly higher amounts of FlrC-dependent transcripts. Conversely, FlrB H135N mutants will be significantly downregulated for the same genes. Mid-log cultures of V. cholerae O395 strains WT (KKV598), DflrB, FlrB H135N, FlrB L36F, DflaBCDE, DflaBCDE + FlrB L36F, DflaBCDE + FlrB H135N, and DflaBCDE + FlrB L36F H135N underwent Trizol-mediated RNA extraction and RNA-seq analysis. RNA extraction was performed on 3 biological replicates. FlrB is a histidine kinase and member of the two-component flagellar-regulated transcriptional system. By phosphorylating the histidine at amino acid position 135, FlrB is activated, permitting transfer of the phosphate from H135 to aspartate residue D54 on FlrC, which in turn binds with sigma-54 and RNAP to facilitate transcription of class III and class IV flagellar genes. By mutating the histidine at position 135 to an asparagine (H135N), FlrB is unable to undergo autophosphorylation, prohibiting phosphorylation of FlrC and transcription of FlrC-dependent genes. Mutating the leucine residue at position 36 in the PAS domain of FlrB to a phenylalanine (L36F) results in constitutive activation of FlrB and FlrC-dependent transcription. We hypothesize that V. cholerae with the FlrB L36F mutant form produce significantly higher amounts of FlrC-dependent transcripts. Conversely, FlrB H135N mutants will be significantly down-regulated for the same genes.