Sigma Factor Displacement from RNA Polymerase during Bacillus subtilis Sporulation

Abstract

ABSTRACTAsBacillus subtilisproceeds through sporulation, the principal vegetative cell ς subunit (ςA) 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 ςAand two mother cell ς factors, ςEand ςK, which successively replace ςAon RNAP. Although the relative abundance of ςAwith respect to RNAP remained virtually unchanged during sporulation, the percentage of the detectable ςAwhich cosedimented with RNAP fell from approximately 50% at the onset of sporulation (T0) to 2 to 8% by 3 h into the process (T3). In a strain that failed to synthesize ςE, the first of the mother cell-specific ς factors, approximately 40% of the ςAremained associated with RNAP atT3. The level of ςA-RNAP cosedimentation dropped to less than 10% in a strain which synthesized a ςEvariant (ςECR119) that could bind to RNAP but was unable to direct ςE-dependent transcription. The E-ςE-to-E-ςKchangeover was characterized by both the displacement of ςEfrom RNAP and the disappearance of ςEfrom the cell. Analyses of extracts from wild-type and mutantB. subtilisshowed that the ςKprotein is required for the displacement of ςEfrom RNAP and also confirmed that ςKis needed for the loss of the ςEprotein. 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.