Bacillus subtilis mutations that alter the pathway of phosphorylation of the anti‐anti‐σF factor SpoIIAA lead to a Spo− phenotype

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Abstract
Sigma‐F, the first sporulation‐specific transcription factor of Bacillus subtilis, is regulated by an anti‐sigma factor SpoIIAB, which can also act as a protein kinase that phosphorylates the anti‐anti‐sigma factor SpoIIAA. The time course of phosphorylation reaction is biphasic, a fact that has been interpreted in terms of a mechanism for sequestering SpoIIAB away from σF and thus allowing activation of σF when needed. Site‐directed mutagenesis of SpoIIAA has allowed us to isolate two mutants that cannot activate σF and which are therefore Spo−. The two mutant SpoIIAA proteins, SpoIIAAL61A and SpoIIAAL90A, are phosphorylated with linear kinetics; in addition they are less able to form the stable non‐covalent complex that wild‐type SpoIIAA makes with SpoIIAB in the presence of ADP. The phosphorylated form of SpoIIAAL90A was hydrolysed by the specific phosphatase SpoIIE at the same rate as wild‐type SpoIIAA‐P, but the rate of hydrolysis of SpoIIAAL61A‐P was much slower. The secondary structure and the global fold of the mutant proteins were unchanged from the wild type. The results are interpreted in terms of a model for the wild type in which SpoIIAB, after phosphorylating SpoIIAA, is released in a form that is tightly bound to ADP and which then makes a ternary complex with an unreacted SpoIIAA. We propose that it is the inability to make this ternary complex that deprives the mutant cells of a means of keeping SpoIIAB from inhibiting σF.

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