Replacement of vegetative σ(A) by sporulation-specific σ(F) as a component of the RNA polymerase holoenzyme in sporulating Bacillus subtilis

Abstract

Soon after asymmetric septation in sporulating Bacillus subtilis cells, σ(F) is liberated in the prespore from inhibition by SpoIIAB. To initiate transcription from its cognate promoters, σ(F) must compete with σ(A), the housekeeping sigma factor in the predivisional cell, for binding to core RNA polymerase (E). To estimate the relative affinity of E for σ(A) and σ(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 σ(F) was found to be about 25-fold lower than that for σ(A). We used quantitative Western blotting to estimate the concentrations of E, σ(A) and σ(F) in sporulating cells. The cellular concentrations of E and era were both about 7.5 μM, and neither changed significantly during the first 3 h of sporulation. The concentration of err 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 σ(F) was some twofold higher than that of σ(A). This difference in concentration cannot adequately account for the replacement of σ(A) holoenzyme by σ(F) holoenzyme in the prespore, and it seems that some further mechanism - perhaps the synthesis or activation of an anti-σ(A) factor - must be responsible for this replacement.