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

The anti-sigma factor FlgM of Salmonella typhimurium inhibits transcription of class 3 flagellar genes through a direct interaction with the flagellar-specific sigma factor, sigma28. FlgM is believed to prevent RNA polymerase (RNAP) holoenzyme formation by sequestering free sigma28. We have analyzed FlgM-mediated inhibition of sigma28 activity in vitro. FlgM is able to inhibit sigma28 activity even when sigma28 is first allowed to associate with core RNAP. Surface plasmon resonance (SPR) was used to evaluate the interaction between FlgM and both sigma28 and sigma28 holoenzyme (Esigma28). The Kd of the sigma28-FlgM complex is approximately 2 x 10(-10) M; missense mutations in FlgM that cause a defect in sigma28 inhibition in vivo increase the Kd of this interaction by 4- to 10-fold. SPR measurements of Esigma28 dissociation in the presence of FlgM indicate that FlgM destabilizes Esigma28, presumably via an interaction with the sigma subunit. Our data provide the first direct evidence of an interaction between FlgM and Esigma28. 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.