Temporal and selective association of multiple sigma factors with RNA polymerase during sporulation in Bacillus subtilis

Background: During sporulation in Bacillus subtilis, an asymmetric division produces two cells, a forespore and mother cell, with which follow different developmental paths. The highly ordered programme of temporal and spatial gene activation during sporulation is governed by the principal RNA polymerase holoenzyme (E sigma(A)) and alternative holoenzyme forms containing the developmental sigma factors sigma(H), sigma(F), sigma(E), sigma(G) and sigma(K), which appear successively during development. The control mechanism(s) of temporal and selective association of multiple sigma factors with core RNA polymerase is unclear. As a first step to addressing these issues, this report quantifies the amount of each subunit of RNA polymerase that is present in the sporangium during sporulation, and analyses in vitro the relative affinities of each sigma subunit for core RNA polymerase. Results: Using quantitative immunoblot analysis, the amounts of E sigma(A), E sigma(H), E sigma(E) and E sigma(K) in relation to the total amount of RNA polymerase at appropriate time-points were found to be 15%, 1%, 6% and 2%, respectively. Therefore, the core RNA polymerase is predicted to be in excess. The level of core RNA polymerase and sigma(A) remained constant during the transition from vegetative growth to sporulation, whereas the sporulation-specific sigma factors appeared successively, in the order sigma(H), sigma(E) and sigma(K). Competition experiments between sigma factors in an in vitro transcription system revealed the dominance of sigma(A) over sigma(H) and sigma(E) for open promoter complex formation. These results are inconsistent with the idea that late appearing sigma factors can displace earlier appearing sigmas from the core enzyme. Conclusions: As the core RNA polymerase is in excess, the results suggest that successive sigma factors can bind to core RNA polymerase without having to displace earlier appearing sigma factors. Thus, the programme of gene expression during sporulation might not require mechanisms for the substitution of one sigma factor by another on the core RNA polymerase.