Evidence in support of a docking model for the release of the transcription factor σF from the antisigma factor SpoIIAB in Bacillus subtilis

Abstract

Cell-specific activation of the transcription factor σF during the process of sporulation in Bacillus subtilis is governed by an antisigma factor SpoIIAB and an anti-antisigma factor SpoIIAA. SpoIIAB, which exists as a dimer, binds to σF in a complex of stoichiometry σF·SpoIIAB2. Escape from the complex is mediated by SpoIIAA, which reacts with the complex to cause the release of free σF. Previous evidence indicated that Arg-20 in SpoIIAB is a contact site for both σF and SpoIIAA and that contact with σF is mediated by Arg-20 on only one of the two subunits in the σF·SpoIIAB2 complex. Here we report the construction of heterodimers of SpoIIAB in which one subunit is wild type and the other subunit is a mutant for Arg-20. We show that the dissociation constant for the binding of σF to the heterodimer was similar to that for the wild type, a finding consistent with the idea that σF contacts Arg-20 on only one of the two subunits. Although SpoIIAA was highly effective in causing the release of σF from the wild type homodimer, the anti-antisigma factor had little effect on the release of σF from the heterodimer. This finding is consistent with a model in which SpoIIAA docks on the σF·SpoIIAB2 complex, making contact with the subunit in which Arg-20 is not in contact with σF. SpoIIAB is both an anti-σF factor and a protein kinase that phosphorylates and thereby inactivates SpoIIAA. We show that SpoIIAA effectively displaces σF from a complex of σF with a mutant (SpoIIABR105A) that is impaired in the kinase function of SpoIIAB. This result shows that SpoIIAA-mediated displacement of σF from SpoIIAB does not require concomitant phosphorylation of SpoIIAA.