A negative feedback loop that limits the ectopic activation of a cell type-specific sporulation sigma factor of bacillus subtilis

Abstract

Two highly similar RNA polymerase sigma subunits, σ F and σ G, govern the early and late phases of forespore-specific gene expression during spore differentiation in Bacillus subtilis. σ F drives synthesis of σ 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 σ G while discriminating between σ F and σ 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 σ G (N45E) strongly reduced binding by a previously characterized anti-sigma factor, CsfB (also known as Gin), in vitro, and increased the activity of σ G in vivo. The N45E mutation caused the appearance of a sub-population of pre-divisional cells with strong activity of σ G. CsfB is normally produced in the forespore, under σ F control, but sigGN45E mutant cells also expressed csfB and did so in a σ G-dependent manner, autonomously from σ F. Thus, a negative feedback loop involving CsfB counteracts the positive feedback loop resulting from ectopic σ G activity. N45 is invariant in the homologous position of σ G orthologues, whereas its functional equivalent in σ F proteins, E39, is highly conserved. While CsfB does not bind to wild-type σ F, a E39N substitution in σ F resulted in efficient binding of CsfB to σ F. Moreover, under certain conditions, the E39N alteration strongly restrains the activity of σ 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. © 2011 Serrano et al.