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The sigB operon of Bacillus subtilis encodes σB and three additional open reading frames (orfV, orfW, and orfX). Having previously mapped several mutations that alter the induction pattern of a σB-dependent promoter (ctc) to regions of cloned B. subtilis DNA which contain these three open reading frames, we directly tested the regulatory potential of orfV, orfW, and orfX by creating null alleles of each of these genes and examining the effects of the mutations, either singly or in pairs, on transcription of ctc and the sigB operon. Using lacZ reporter gene fusions and Northern (RNA) blot analyses, we have determined that all three genes modulate the activation of the σB-dependent promoters at both the sigB operon and ctc. Our data are consistent with the three gene products participating in a single pathway of negative control. orfW and orfX single-mutant strains have high levels of sigB and ctc transcription. sigB and ctc transcription in an orfV strain is similar to that found in mutant strains which lack σB itself. The orfV mutation is dominant to orfX but recessive to orfW. These results suggest that OrfW is the primary inhibitor of σB-dependent transcription and that OrfV is capable of counteracting the negative control of OrfW but is prevented from doing this by the orJX gene product.