As Bacillus subtilis proceeds 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 ςA and two mother cell ς factors, ςE and ςK, which successively replace ςA on RNAP. Although the relative

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... undance of ςA with 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 (T 0) to 2 to 8% by 3 h into the process (T 3). In a strain that failed to synthesize ςE, the first of the mother cell-specific ς factors, approximately 40% of the ςA remained associated with RNAP at T 3. The level of ςA-RNAP cosedimentation dropped to less than 10% in a strain which synthesized a ςE variant (ςECR119) that could bind to RNAP but was unable to direct ςE-dependent transcription. The E-ςE-to-E-ςK changeover was characterized by both the displacement of ςE from RNAP and the disappearance of ςE from the cell. Analyses of extracts from wild-type and mutant B. subtilis showed that the ςK protein is required for the displacement of ςE from RNAP and also confirmed that ςK is needed for the loss of the ςE 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.