Contribution of partner switching and SpoIIAA cycling to regulation of F activity in sporulating Bacillus subtilis

sF, the first compartment-specific transcription factor in sporulating Bacillus subtilis, is negatively regulated by an anti-s factor, SpoIIAB. SpoIIAB has an alternative binding partner, SpoIIAA. To see whether (as has been proposed) SpoIIAB’s binding preference for SpoIIAA or sF depends on the nature of the adenine nucleo-tide present, we used surface plasmon resonance to measure the dissociation constants of the three complexes SpoIIAA-SpoIIAB-ADP, sF-SpoIIAB-ADP, and sF-SpoIIAB-ATP. The results suggested that SpoIIAB’s choice of binding partner is unlikely to depend on the ATP/ADP ratio in the cell. The intracellular concentrations of sF, SpoIIAB, SpoIIAA, and SpoIIAA-phosphate (SpoIIAA-P) were measured by quantitative immunoblotting between 0 and 3 h after the beginning of sporulation (t0 to t3). s F and SpoIIAB were barely detectable at t0, but their concentrations increased in parallel to reach maxima at about t1.5. SpoIIAA-P increased steadily to a maximum at t3, but nonphosphorylated SpoIIAA was detectable only from t1.5, reached a maximum at t2.5, and then declined. Kinetic studies of the phosphorylation of SpoIIAA catalyzed by SpoIIAB suggested that the reaction was limited by a very slow release of one of the products (SpoIIAA-P or ADP) from SpoIIAB, with a turnover of about once per 20 min. This remarkable kinetic property provides an unexpected mechanism for the regulation of sF. We propose that when SpoIIE (which dephosphorylates SpoIIAA-P) is active at the sam