Characterization of the RpoN Regulon Reveals Differential Regulation of T6SS and New Flagellar Operons in Vibrio cholerae

The alternative sigma factor RpoN is an essential colonization factor of Vibrio cholerae and controls important cellular functions including motility and type VI secretion (T6SS). The RpoN regulon has yet to be clearly defined in T6SS-active V. cholerae isolates, which use T6SS to target both bacterial competitors and eukaryotic cells. We hypothesize that T6SS-dependent secreted effectors are co-regulated by RpoN. To systemically identify RpoN-controlled genes, we used chromatin immunoprecipitation coupled with sequencing (ChIP-Seq) and transcriptome analysis (RNA-Seq) to determine RpoN-binding sites and RpoNcontrolled gene expression. There were 68 RpoNbinding sites and 82 operons positively controlled by RpoN, among which 37 operons had ChIPidentified binding sites. A consensus RpoN-binding motif was identified with a highly conserved thymine ( 14) and an AT-rich region in the middle between the hallmark RpoN-recognized motif GG( 24)/ GC( 12). There were seven new RpoN-dependent promoters in the flagellar regions. We identified a small RNA, flaX, downstream of the major flagellin gene flaA. Mutation of flaX substantially reduced motility. In contrast to previous results, we report that RpoN positively regulates the expression of hcp operons and vgrG3 that encode T6SS secreted proteins but has no effect on the expression of the main T6SS cluster encoding sheath and other struc-