The flagellar anti-ς factor FlgM actively dissociates Salmonella typhimurium ς28 RNA polymerase holoenzyme

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Abstract
The anti-ς factor FlgM of Salmonella typhimurium inhibits transcription of class 3 flagellar genes through a direct interaction with the flagellar-specific ς factor, ς28. FlgM is believed to prevent RNA polymerase (RNAP) holoenzyme formation by sequestering free ς28. We have analyzed FlgM-mediated inhibition of ς28 activity in vitro. FlgM is able to inhibit ς28 activity even when ς28 is first allowed to associate with core RNAP. Surface plasmon resonance (SPR) was used to evaluate the interaction between FlgM and both ς28 and ς28 holoenzyme (Eς28). TheKd of the ς28–FlgM complex is ∼2 × 10−10 m; missense mutations in FlgM that cause a defect in ς28 inhibition in vivo increase theKd of this interaction by 4- to 10-fold. SPR measurements of Eς28 dissociation in the presence of FlgM indicate that FlgM destabilizes Eς28, presumably via an interaction with the ς subunit. Our data provide the first direct evidence of an interaction between FlgM and Eς28. We propose that this secondary activity of FlgM, which we term holoenzyme destabilization, enhances the sensitivity of the cell to changes in FlgM levels during flagellar biogenesis.