A Negative Feedback Loop That Limits the Ectopic Activation of a Cell Type–Specific Sporulation Sigma Factor of Bacillus subtilis

Two highly similar RNA polymerase sigma subunits, sF and sG, govern the early and late phases of forespore-specific gene expression during spore differentiation in Bacillus subtilis. sF drives synthesis of sG but the latter only becomes active once engulfment of the forespore by the mother cell is completed, its levels rising quickly due to a positive feedback loop. The mechanisms that prevent premature or ectopic activation of sG while discriminating between sF and sG in the forespore are not fully comprehended. Here, we report that the substitution of an asparagine by a glutamic acid at position 45 of sG (N45E) strongly reduced binding by a previously characterized anti-sigma factor, CsfB (also known as Gin), in vitro, and increased the activity of sG in vivo. The N45E mutation caused the appearance of a sub-population of pre-divisional cells with strong activity of sG. CsfB is normally produced in the forespore, under sF control, but sigGN45E mutant cells also expressed csfB and did so in a sG-dependent manner, autonomously from sF. Thus, a negative feedback loop involving CsfB counteracts the positive feedback loop resulting from ectopic sG activity. N45 is invariant in the homologous position of sG orthologues, whereas its functional equivalent in sF proteins, E39, is highly conserved. While CsfB does not bind to wild-type sF, a E39N substitution in sF resulted in efficient binding of CsfB to sF. Moreover, under certain conditions, the E39N alteration strongly restrains the activity of sF in vivo, in a csfB-dependent manner, and the efficiency of sporulation. Therefore, a single amino residue, N45/E39, is sufficient for the ability of CsfB to discriminate between the two forespore