Physical Evidence for the Induced Release of the Bacillus subtilis Transcription Factor, σF, from its Inhibitory Complex

The release of the transcription factor σF from its inhibitory complex with SpoIIAB is a key regulatory step in the control of sporulation in Bacillus subtilis as it initiates a pattern of differential gene expression in the mother cell and prespore compartments. The σF·SpoIIAB complex is dissociated by the unphosphorylated form of the protein SpoIIAA, the alternative binding partner of SpoIIAB. Here, we employ fluorescence spectroscopy to examine the mechanism by which SpoIIAA acts on the σF·SpoIIAB complex. We constructed a mutant of σF, σF-W46L, which displayed a reproducible fluorescence response on binding to SpoIIAB. Using this mutant we were able to quantify the amount of σF bound to SpoIIAB in real time. The results provide physical evidence for the “induced release” mechanism of σF activation. We demonstrate that SpoIIAA interacts directly with the σF·SpoIIAB complex, greatly decreasing the affinity of SpoIIAB for σF and thus causing the release of the latter. We also demonstrate that σF is released before SpoIIAA is phosphorylated and that release occurs on a similar time scale to the binding of SpoIIAA to SpoIIAB.