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

sigma(F), the first compartment-specific transcription factor in sporulating Bacillus subtilis, is negatively regulated by an anti-sigma factor, SpoIIAB. SpoIIAB has an alternative binding partner, SpoIIAA. To see whether (as has been proposed) SpoIIAB's binding preference for SpoIIAA or sigma(F) depends on the nature of the adenine nucleotide present, we used surface plasmon resonance to measure the dissociation constants of the three complexes SpoIIAA-SpoIIAB-ADP, sigma(F)-SpoIIAB-ADP, and sigma(F)-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 sigma(F), SpoIIAB, SpoIIAA, and SpoIIAA-phosphate (SpoIIAA-P) were measured by quantitative immunoblotting between 0 and 3 h after the beginning of sporulation (t(0) to t(3)), sigma(F) and SpoIIAB were barely detectable at t(0), but their concentrations increased in parallel to reach maxima at about t(1.5). SpoIIAA-P increased steadily to a maximum at t(3), but nonphosphorylated SpoIIAA mas detectable only from t(1.5), reached a maximum al t(2.5), 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 fur the regulation of sigma(F). We propose that when SpoIIE (which dephosphorylates SpoIIAA-P) is active at the same time as SpoIIAB, SpoIIAA cycles repeatedly between the phosphorylated and nonphosphorylated forms. This cycling sequesters SpoIIAB in a long-lived complex and prevents it from inhibiting sigma(F).