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The sigB operon of Bacilus subtilis encodes (σB plus three additional proteins (RsbV, RsbW, and RsbX) that regulate σB activity. Using an anti-σB monoclonal antibody to monitor the levels of (σB protein, PSPAC to control the expression of the sigB operon, and a ctc-lacZ reporter system to monitor σB activity, we observed that the rsbV and rsbW products control (σB activity at the ctc promoter independently of their effects on (σB levels. In contrast, RsbX was found to have no effect on expression of ctc when the sigB operon was controlled by PSPAc. The data are consistent with RsbV and RsbW being regulators of (σB activity and RsbX acting primarily as a negative regulator of sigB operon expression. Evidence that stationary-phase induction of the σB-dependent ctc promoter is accomplished by a reduction in RsbW-dependent inhibition of σB activity is also presented. In addition, Western blot (immunoblot) analyses of sigB operon expression demonstrated that (σB accumulation is coupled to the synthesis of its primary inhibitor (RsbW). This finding is consistent with RsbW and (σB being present within the cell in equivalent amounts, a circumstance that would permit RsbW to directly influence (σB activity by a direct protein-protein interaction.