The ClpX protein ofBacillus subtilisindirectly influences RNA polymerase holoenzyme composition and directly stimulates σH-dependent transcription

Abstract

In Bacillus subtilis, several processes associated with the onset of stationary phase, including the initiation of sporulation, require the activity of the minor σ^H form of RNA polymerase (RNAP). The induction of σ^H-dependent gene transcription requires the regulatory ATPase, ClpX. The ClpX-dependent post-exponential increase in σ^H activity is not dependent on the activator of sporulation gene expression, Spo0A. By determining the level of σ^H and σ^A in whole-cell extracts and RNAP preparations, evidence is presented that clpX does not influence the concentration of sigma subunits, but is required for the stationary phase reduction in σ^A-RNAP holoenzyme. This is probably an indirect consequence of ClpX activity, because the ClpX-dependent decrease in σ^A-RNAP concentration does not occur in a spo0A abrB mutant. The addition of ClpX to in vitro transcription reactions resulted in the stimulation of RNAP holoenzyme activity, but σ^H-RNAP was observed to be more sensitive to ClpX-dependent stimulation than σ^A-RNAP. No difference in transcriptional activity was observed in single-cycle in vitro transcription reactions, suggesting that ClpX acted at a step in transcription initiation after closed- and open-promoter complex formation. ClpX is proposed to function indirectly in the displacement of σ^A from core RNAP and to act directly in the stimulation of σ^H-dependent transcription in sporulating B. subtilis cells.