Establishing differential gene expression in sporulating Bacillus subtilis: Phosphorylation of SpoIIAA (anti-anti-sigma(F)) alters its conformation and prevents formation of a SpoIIAA/SpoIIAB/ADP complex

Sigma-factor F (sigma(F)) is a key transcription factor that initiates prespore development in Bacillus subtilis, Its activity is controlled by an anti-sigma factor, SpollAB, which is also a protein kinase that phosphorylates the anti-anti-sigma factor SpollAA. We have examined our earlier prediction that SpollAA must undergo a major change in its properties when phosphorylated, Upon gel filtration in the presence of ADP, SpollAA-P was eluted from a Superdex column much later than SpollAB, whereas SpollAA was co-eluted with SpollAB, indicating the formation of a protein/protein complex. The complex contained ADP, and had two monomers of SpollAA to each SpollAB dimer, Its dissociation constant was 13 mu M. Gel permeation on high-performance liquid chromatography (HPLC) suggested an apparent molecular mass for SpollAA-P which was much higher (23.5 kDa) than that of SpollAA (15.8 kDa), but Ferguson plots showed that SpollAA-P was not a phosphorylated dimer of SpollAA, Our tentative conclusion, that SpollAA and SpollAA-P differ markedly in conformation, was confirmed by the results of partial digestion with chymotrypsin.