Bacillus subtilis sigma-B is an alternate sigma factor implicated in controlling stationary-phase gene expression. We characterized the genetic organization and regulation of the region containing the sigma-B structural gene (sigB) to learn which metabolic signals and protein factors govern sigma-B function. sigB lay in an operon with four open reading frames (orfs) in the order orfV-orfW-sigB-orfX, and lacZ gene fusions showed that all four frames were translated in vivo. Experiments with primer extension, Si nuclease mapping, and lacZ transcriptional fusions found that sigB operon transcription initiated early in stationary phase from a site 32 nucleotides upstream of orfV and terminated 34 nucleotides downstream of orfX. Fusion expression was abolished in a strain carrying an in-frame deletion in sigB, suggesting that sigma-B positively regulated its own synthesis, and deletions in the sigB promoter region showed that sequences identical to the sigma-Bdependent ctc promoter were essential for promoter activity. Fusion expression was greatly enhanced in a strain carrying an insertion mutation in orfX, suggesting that the 22-kilodalton (kDa) orfX product was a negative effector of sigma-B expression or activity. Notably, the genetic organization of the sigB operon was strikingly similar to that of the B. subtilis spollA operon, which has the gene order spoIIAA-spoIIAB-spoIIAC, with spollAC encoding the sporulation-essential sigma-F. The predicted sequence of the 12-kDa orfV product was 32% identical to that of the 13-kDa SpoIIAA protein, and the 18-kDa orfW product was 27% identical to the 16-kDa SpoILAB protein. On the basis of this clear evolutionary conservation, we speculate these protein pairs regulate their respective sigma factors by a similar molecular mechanism and that the spolIA and sigB operons might control divergent branches of stationary-phase gene expression.Alternate sigma factors associate with the catalytic core of procaryotic RNA polymerases to reprogram the pattern of gene expression in response to nutritional or environmental stress or, in some cases, in response to morphological change. Examples include regulation of the heat shock and nitrogen regulons of enteric bacteria (14,22,23,44,47), regulation of the chemotaxis and motility regulons of enteric bacteria and Bacillus subtilis (1, 16), and regulation of developmental gene expression in B. subtilis and Streptomyces coelicolor (5,20,21,27,46). However, it is not well understood how cellular and metabolic signals command the transcriptional apparatus to modulate gene expression.In the best-studied example, the NtrA sigma factor of enteric bacteria is controlled at the level of activity, not synthesis. The NtrA sigma determines promoter specificity for genes of diverse function, and it is a two-component regulatory system that controls activation of the closed initiation complex (22). For nitrogen-regulated promoters in Escherichia coli and Salmonella typhimurium, this twocomponent activation system responds to an elegant metabolic casca...

We began an analysis of rpoF, the gene encoding the cryptic, 37,000-dalton minor sigma factor (sigma-37) of Bacillus subtilis RNA polymerase. Using antibody raised against sigma-37 holoenzyme to Genetic analysis has begun to define the physiological roles of the multiple sigma factors which associate with Bacillus subtilis RNA polymerase core and which determine promoter recognition specificity in vitro (21). The major sigma factor (sigma-43; 43,000 daltons) is analogous to the Escherichia coli major sigma (8) and predominates during logarithmic growth (21). The minor form sigma-29 (13), which is the product of the spoIIG gene (32,37), and the sigma-like spoIIA gene product (6, 7) both control some aspect of stationary-phase physiology essential to sporulation.Less is known regarding the roles of the minor forms sigma-37 (14), sigma-32 (18), and sigma-28 (38). These are present during logarithmic growth and may have a vegetative function, but in the absence of authentic sigma-gene mutations their physiological roles remain unknown. We have therefore undertaken a genetic analysis to determine the role of sigma-37 during growth and sporulation and to determine which additional factors and metabolic signals govern its expression and activity. We report here the isolation, nucleotide sequence, and genetic mapping of the sigma-37 structural gene rpoF. During the course of these experiments, Binnie et al. (3) independently isolated the sigma-37 gene by using a different screening method. Our results agree with theirs in all major respects and provide additional data on rpoF map location and the possible genetic organization of the rpoF region.

The gene encoding the beta subunit of Bacillus subtilis RNA polymerase was isolated from a lambda gt11 expression library using an antibody probe. Gene identity was confirmed by the similarity of its predicted product to the Escherichia coli beta subunit and by mapping an alteration conferring rifampicin resistance within the conserved rif coding region. Including the rif region, four colinear blocks of sequence similarity were shared between the B. subtilis and E. coli beta subunits. In E. coli, these conserved blocks are separated by three regions that either were not conserved or were entirely absent from the B. subtilis protein. The B. subtilis beta gene was part of a cluster with the order rplL (encoding ribosomal protein L7/L12), orf23 (encoding a 22,513-dalton protein that is apparently essential for growth), rpoB (beta), and rpoC (beta'). This organization differs from the corresponding region in E. coli by the inclusion of orf23. Experiments using promoter probe vectors and site-directed mutagenesis located a major rpoB promoter overlapping the 3'-coding region of orf23, 250 nucleotides upstream from the beta initiation codon. Thus, the B. subtilis rpoB region differs from its E. coli counterpart in both genetic and transcriptional organization.

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