wo highly similar RNA polymerase sigma subunits, sigma(F) and sigma(G), govern the early and late phases of forespore-specific gene expression during spore differentiation in Bacillus subtilis. sigma(F) drives synthesis of sigma(G) 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 sigma(G) while discriminating between sigma(F) and sigma(G) in the forespore are not fully comprehended. Here, we report that the substitution of an asparagine by a glutamic acid at position 45 of sigma(G) (N45E) strongly reduced binding by a previously characterized anti-sigma factor, CsfB (also known as Gin), in vitro, and increased the activity of sigma(G) in vivo. The N45E mutation caused the appearance of a sub-population of pre-divisional cells with strong activity of sigma(G). CsfB is normally produced in the forespore, under sigma(F) control, but sigGN45E mutant cells also expressed csfB and did so in a sigma(G) -dependent manner, autonomously from sigma(F). Thus, a negative feedback loop involving CsfB counteracts the positive feedback loop resulting from ectopic sigma(G) activity. N45 is invariant in the homologous position of sigma(G) orthologues, whereas its functional equivalent in sigma(F) proteins, E39, is highly conserved. While CsfB does not bind to wildtype sigma(F), a E39N substitution in sigma(F) resulted in efficient binding of CsfB to sigma(F). Moreover, under certain conditions, the E39N alteration strongly restrains the activity of sigma(F) 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-specific sigma factors in B. subtilis.