THE FLAGELLAR ANTI-SIGMA FACTOR FLGM ACTIVELY DISSOCIATES SALMONELLA-TYPHIMURIUM SIGMA-28 RNA-POLYMERASE HOLOENZYME

The anti-a factor FlgM of Salmonella typhimurium inhibits transcriptio n of class 3 flagellar genes through a direct interaction with the fla gellar-specific a factor, sigma(28). FlgM is believed to prevent RNA p olymerase (RNAP) holoenzyme formation by sequestering free sigma(28). We have analyzed FlgM-mediated inhibition of sigma(28) activity in vit ro. FlgM is able to inhibit sigma(28) activity even when sigma(28) is first allowed to associate with core RNAP. Surface plasmon resonance ( SPR) was used to evaluate the interaction between FlgM and both sigma( 28) and sigma(28) holoenzyme (E sigma(28)). The K-d of the sigma(28)-F lgM complex is similar to 2 x 10(-10) M; missense mutations in FlgM th at cause a defect in sigma(28) inhibition in vivo increase the K-d of this interaction by 4- to 10-fold. SPR measurements of E sigma(28) dis sociation in the presence of FlgM indicate that FlgM destabilizes E si gma(28), presumably via an interaction with the sigma subunit. Our dat a provide the first direct evidence of an interaction between FlgM and E sigma(28). We propose that this secondary activity of FlgM, which w e term holoenzyme destabilization, enhances the sensitivity of the cel l to changes in FlgM levels during flagellar biogenesis.