The kinase activity of the antisigma factor SpoIIAB is required for activation as well as inhibition of transcription factor sigmaF during sporulation in Bacillus subtilis.

The activity of the developmental transcription factor sigmaF in the spore-forming bacterium Bacillus subtilis is controlled by SpoIIAB, which sequesters sigmaF in an inactive complex. sigmaF is released from the SpoIIAB-sigmaF complex by the action of SpoIIAA, which triggers the dissociation of the complex. SpoIIAB is also a protein kinase that phosphorylates SpoIIAA on serine residue 58 (S58). This phosphorylation inactivates SpoIIAA and thus indirectly prevents the activation of sigmaF. Here, we report the identification of a patch of amino acid residues located in the vicinity of the adenosine nucleotide binding pocket of SpoIIAB that is required for the phosphorylation of SpoIIAA. A lysine substitution (E104K) at one of these residues (Glu104) markedly impaired the capacity of SpoIIAB to phosphorylate SpoIIAA in vitro as well as during sporulation. Kinetic analysis and evidence from the construction of alanine substitution mutants indicates that the side-chains of these amino acids could be contact sites for the SpoIIAA substrate during the phosphorylation reaction. Importantly, E104K and other kinase mutants blocked the activation of sigmaF during sporulation. This is paradoxical, because a mutant of SpoIIAA (S58A) that cannot be phosphorylated is known to cause higher than normal levels of sigmaF activity during sporulation. In resolution of this paradox, we present biochemical evidence indicating that SpoIIAA directly attacks the SpoIIAB-sigmaF complex and that SpoIIAA is phosphorylated as a result of this reaction. Consistent with this idea, mutations impairing kinase function of SpoIIAB were found to be epistatic to a mutation causing the S58A substitution in SpoIIAA; that is, cells producing mutant forms of both proteins were blocked in the activation of sigmaF. We conclude that phosphorylation of SpoIIAA plays a dual role in the sigmaF pathway, and that the kinase function of SpoIIAB is required for the activation as well as the inhibition of sigmaF during sporulation.