Sigma Factor Displacement from RNA Polymerase during Bacillus subtilis Sporulation

As Bacillus subtilis proceeds through sporulation, the principal vegetative cell ς subunit (ς(A)) persists in the cell but is replaced in the extractable RNA polymerase (RNAP) by sporulation-specific ς factors. To explore how this holoenzyme changeover might occur, velocity centrifugation techniques were used in conjunction with Western blot analyses to monitor the associations of RNAP with ς(A) and two mother cell ς factors, ς(E) and ς(K), which successively replace ς(A) on RNAP. Although the relative abundance of ς(A) with respect to RNAP remained virtually unchanged during sporulation, the percentage of the detectable ς(A) which cosedimented with RNAP fell from approximately 50% at the onset of sporulation (T(0)) to 2 to 8% by 3 h into the process (T(3)). In a strain that failed to synthesize ς(E), the first of the mother cell-specific ς factors, approximately 40% of the ς(A) remained associated with RNAP at T(3). The level of ς(A)-RNAP cosedimentation dropped to less than 10% in a strain which synthesized a ς(E) variant (ς(ECR119)) that could bind to RNAP but was unable to direct ς(E)-dependent transcription. The E-ς(E)-to-E-ς(K) changeover was characterized by both the displacement of ς(E) from RNAP and the disappearance of ς(E) from the cell. Analyses of extracts from wild-type and mutant B. subtilis showed that the ς(K) protein is required for the displacement of ς(E) from RNAP and also confirmed that ς(K) is needed for the loss of the ς(E) protein. The results indicate that the successive appearance of mother cell ς factors, but not necessarily their activities, is an important element in the displacement of preexisting ς factors from RNAP. It suggests that competition for RNAP by consecutive sporulation ς factors may be an important feature of the holoenzyme changeovers that occur during sporulation.