Replacement of vegetative σA by sporulation-specific σF as a component of the RNA polymerase holoenzyme in sporulating Bacillus subtilis

Soon after asymmetric septation in sporulating Bacillus subtilis cells, sigma(F) is liberated in the prespore from inhibition by SpoIIAB. To initiate transcription from its cognate promoters, sigma(F) must compete with sigma(A), the housekeeping sigma factor in the predivisional cell, for binding to core RNA polymerase (E). To estimate the relative affinity of E for sigma(A) and sigma(F), we made separate mixtures of E with each of the two sigma factors, allowed reconstitution of the holoenzyme, and measured the concentration of free E remaining in each mixture. The affinity of E for sigma(F) was found to be about 25-fold lower than that for sigma(A). We used quantitative Western blotting to estimate the concentrations of E, sigma(A), and sigma(F) in sporulating cells. The cellular concentrations of E and sigma(A) were both about 7.5 mu M, and neither changed significantly during the first 3 h of sporulation. The concentration of sigma(F) was extremely low at the beginning of sporulation, but it rose rapidly to a peak after about 2 h. At its peak, the concentration of sigma(F) was some twofold higher than that of sigma(A). This difference in concentration cannot adequately account for the replacement of sigma(A) holoenzyme by sigma(F) holoenzyme in the prespore, and it seems that some further mechanism-perhaps the synthesis or activation of an anti-sigma(A) factor-must be responsible for this replacement.