Sigma factor displacement from RNA polymerase during Bacillus subtilis sporulation.

Abstract

As Bacillus subtilis proceeds through sporulation, the principal vegetative cell sigma subunit (sigma(A)) persists in the cell but is replaced in the extractable RNA polymerase (RNAP) by sporulation-specific sigma 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 sigma(A) and two mother cell sigma factors, sigma(E) and sigma(K), which successively replace sigma(A) on RNAP. Although the relative abundance of sigma(A) with respect to RNAP remained virtually unchanged during sporulation, the percentage of the detectable sigma(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 sigma(E), the first of the mother cell-specific sigma factors, approximately 40% of the sigma(A) remained associated with RNAP at T(3). The level of sigma(A)-RNAP cosedimentation dropped to less than 10% in a strain which synthesized a sigma(E) variant (sigma(ECR119)) that could bind to RNAP but was unable to direct sigma(E)-dependent transcription. The E-sigma(E)-to-E-sigma(K) changeover was characterized by both the displacement of sigma(E) from RNAP and the disappearance of sigma(E) from the cell. Analyses of extracts from wild-type and mutant B. subtilis showed that the sigma(K) protein is required for the displacement of sigma(E) from RNAP and also confirmed that sigma(K) is needed for the loss of the sigma(E) protein. The results indicate that the successive appearance of mother cell sigma factors, but not necessarily their activities, is an important element in the displacement of preexisting sigma factors from RNAP. It suggests that competition for RNAP by consecutive sporulation sigma factors may be an important feature of the holoenzyme changeovers that occur during sporulation.