SummarySigma-F, the first sporulation-specific transcription factor of Bacillus subtilis, is regulated by an antisigma 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 s F and thus allowing activation of s F when needed.Site-directed mutagenesis of SpoIIAA has allowed us to isolate two mutants that cannot activate s F and which are therefore Spo 2 . 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 s F .

Phosphorylation of SpoIIAA catalyzed by SpoIIAB helps to regulate the first sporulation-specific factor, F , of Bacillus subtilis. The steady-state rate of phosphorylation is known to be exceptionally slow and to be limited by the return of the protein kinase, SpoIIAB, to a catalytically active state. Previous work from this laboratory has suggested that, after catalyzing the phosphorylation, SpoIIAB is in a form (SpoIIAB*) that does not readily release ADP. We now show that the rate of release of ADP from the SpoIIAB*-ADP complex was much diminished by the presence of unreacted SpoIIAA, suggesting that SpoIIAA can form a long-lived ternary complex with SpoIIAB*-ADP in which the SpoIIAB* form is stabilized. In kinetic studies of the phosphorylation of SpoIIAA, the ternary complex SpoIIAA-SpoIIAB*-ADP could be distinguished from the short-lived complex SpoIIAA-SpoIIAB-ADP, which can be readily produced in the absence of an enzymatic reaction.