RflM mediates target specificity of the RcsCDB phosphorelay system for transcriptional repression of flagellar synthesis in Salmonella enterica

The bacterial flagellum enables directed movement of Salmonella enterica towards favorable conditions in liquid environments. Regulation of flagellar synthesis is tightly controlled by various environmental signals at transcriptional and post-transcriptional levels. The flagellar master regulator FlhD₄C₂ resides on top of the flagellar transcriptional hierarchy and is under autogenous control by FlhD₄C₂-dependent activation of the repressor rflM. The inhibitory activity of RflM depends on the presence of RcsB, the response regulator of the RcsCDB phosphorelay system. In this study, we elucidated the molecular mechanism of RflM-dependent repression of flhDC. We show that RcsB and RflM form a heterodimer that coordinately represses flhDC transcription independent of RcsB phosphorylation. RcsB-RflM complex binds to a RcsB box downstream the P1 transcriptional start site of the flhDC promoter with increased affinity compared to RcsB in the absence of RflM. We propose that RflM stabilizes binding of unphosphorylated RcsB to the flhDC promoter in absence of environmental cues. Thus, RflM is a novel auxiliary regulatory protein that mediates target specificity of RcsB for flhDC repression. The cooperative action of the RcsB-RflM repressor complex allows Salmonella to fine-tune initiation of flagellar gene expression and adds another level to the complex regulation of flagellar synthesis.