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Genetic experiments have suggested that a^, the first compartment-specific transcription factor in sporulating B. subtilis, is regulated by an anti-o-factor SpoIIAB and an anti-anti-cr factor SpoIIAA. Previously, we reported biochemical results demonstrating that SpoIIAB is both a phosphokinase whose substrate is SpoIIAA and an inhibitor of cr^-directed transcription. We now show that in the presence of SpoIIAB and ATP or ADP, SpoIIAA can undergo two alternative reactions. When ATP is present, SpoIIAA is phosphorylated rapidly and completely to SpoIIAA-phosphate, and SpoIIAB is immediately released; but in the presence of ADP, SpoIIAA forms a long-lasting complex with SpoIIAB. ADP is an inhibitor of the phosphorylation by ATP. Furthermore, we have mutated SpoIIAA at residue Ser 58, the target for phosphorylation, to aspartate or alanine. SpoIIAAS58D, which apparently resembles SpoIIAA-phosphate, is unable to make a complex with SpoIIAB and is devoid of anti-anti-o-^ activity, whereas SpoIIAAS58A, which cannot be phosphorylated, makes complexes with SpoIIAB in the presence of ADP or ATP and has constitutive anti-anti-cr^ activity both in vivo and in vitro. It seems likely that the alternative reactions of SpoIIAA and SpoIIAB, involving ADP or ATP, regulate the anti-anti-a capacity of SpoIIAA and hence the activity of a^.

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During sporulation in Bacillus subtilis an asymmetric cell division gives rise to unequal progeny called the prepore and the mother cell. Gene expression in the prespore is initiated by cell-specific activation of the transcription factor cr F. Three proteins participate in the regulation of orF activity. The first, SpoIIAB, is an inhibitor of OrF, that is, an anti-or factor. SpoIIAB is also a protein kinase that catalyzes phosphorylation of the second regulatory protein SpoIIAA (the anti-anti-or factor), and thus inactivates it. A third protein, SpoIIE, was shown recently to be able to dephosphorylate SpoIIAA-P in vitro. Here we show that SpoIIE is a bifunctional protein with two critical roles in the establishment of cell fate. First, we confirm by the use of in vivo experiments that it regulates the release of orF activity by dephosphorylating SpoIIAA-P. Second, we show that SpoIIE is needed for normal formation of the asymmetric septum that separates the prespore from the mother cell. Combination of these two functions in a single polypeptide may serve to couple the release of the cell-specific transcription factors with the formation of the differentiating cells.

F, the first compartment-specific transcription factor in sporulating Bacillus subtilis, is negatively regulated by an anti-factor, SpoIIAB. SpoIIAB has an alternative binding partner, SpoIIAA. To see whether (as has been proposed) SpoIIAB's binding preference for SpoIIAA or 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, 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 was detectable only from t 1.5 , reached a maximum at 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 for the regulation of 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 F .Early in the sporulation of Bacillus subtilis, the cell divides asymmetrically to form two compartments that remain attached to each other, a small prespore and a larger mother cell. Although the two compartments have identical genomes, they show different patterns of gene expression. The prespore gradually becomes transformed into the mature spore, which is eventually released by lysis of the mother cell. Gene expression in the two compartments depends on sigma factors that show both spatial and temporal specificity: in the prespore, F is followed by G , and in the mother cell, E is followed by K (9, 16). The first sporulation-specific sigma factor, F