Regulation of the flagellar specific sigma factor, sigma28, of Salmonella typhimurium by the anti-sigma factor FlgM

Abstract

Expression of the flagellar genes of Salmonella typhimurium occurs in an ordered hierarchy that is coupled to the morphogenesis of the flagellar organelle. Transcription of the late (Class 3) flagellar genes is dependent upon a positive transcriptional regulatory protein, the flagellar specific sigma factor, sigma28, encoded by the fliA gene. sigma28 activity is inhibited by an anti-sigma factor, FlgM, encoded by the flgM gene. Inhibition of sigma 28 is accomplished through a direct interaction between sigma 28 and FlgM that interferes with the association of sigma 28 with core RNA polymerase (RNAP) to form a transcriptionally active holoenzyme complex (Esigma28). The mechanism of FlgM-mediated inhibition of Esigma28 formation became the subject of my thesis research.A genetic analysis of fliA mutants defective for negative regulation by FlgM identified potential FlgM binding domains in conserved sigma factor regions 2.1, 3.1 and 4.1/4.2 of sigma28. It was proposed that FlgM could interfere directly with holoenzyme formation by masking potential core RNAP binding determinants in these regions. Alternatively, FlgM could inhibit the core binding activity of sigma28 allosterically.Previous biochemical analyses of the sigma28/FlgM interaction had suggested that FlgM might be capable of binding to Esigma28 in addition to free sigma28. A surface plasmon resonance (SPR) based assay suggested that FlgM was able to interact transiently with Esigma28 to increase the rate of holoenzyme dissociation. FlgM mutants defective for sigma28 binding (FlgM* mutants) were also defective for this activity, termed "holoenzyme destabilization."SPR measurements of the affinities of the FlgM/sigma28* complexes revealed that most of the sigma28* mutants were defective for FlgM binding. These defects were reflected by the decreased sensitivity of these mutants to FlgM in in vitro transcription assays.We propose that FlgM inhibits sigma28 activity on two levels during flagellar biogenesis. Our model predicts that FlgM sequesters free sigma28 from core RNAP by binding to regions 2.1, 3.1 and 4.1/4.2. In addition, FlgM may also interact with sigma28 bound to core RNAP, at the subset of FlgM binding determinants that are accessible in the Esigma28 complex, to effect destabilization of that complex.