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The association of a sigma factor (sigma) with RNA polymerase in bacteria determines its specificity of promoter utilization. To identify amino acid residues in sigma E from Bacillus subtilis that determine the specificity of its interaction with the nucleotides at the -10 region of its cognate promoters, we tested whether base pair substitutions in the -10 region of a sigma B-dependent promoter could signal its utilization by sigma E-RNA polymerase. We found that a combination of base pair substitutions at positions -15 and -14 of the sigma B-dependent ctc promoter resulted in its utilization by sigma E-RNA polymerase in vivo. We also found that the combination of two amino acid substitutions at positions 119 and 120 in sigma E changed its specificity for promoter utilization, resulting in a sigma factor that directed transcription from the sigma B-dependent ctc promoter, but not from sigma E-dependent promoters. These results suggest that amino acid residues at positions 119 and 120 determine, at least in part, the specificity of interactions between sigma E and the nucleotides in the -10 region of its cognate promoters.

In order to determine which amino acyl residues in a secondary sigma factor govern its specificity of recognition at the -35 region of promoters, we examined the effects of amino acid substitutions in sigma E in Bacillus subtilis that made the sequence of its putative -35 recognition region more similar to another sigma factor in B. subtilis, sigma K. We found that a single amino acid substitution at position 217 of sigma E resulted in a sigma factor that could direct transcription from sigma K-dependent promoters. Furthermore, we tested whether this amino acid substitution in sigma E had changed the specificity of interactions of the sigma with -35 region sequences by examining the activity of the mutant sigma E on derivatives of sigma E-dependent promoters that contained single base-pair substitutions. We found that this substitution in sigma E specifically suppressed the effect of a single base-pair substitution at position -31 in a sigma E-dependent promoter spoIIID. The amino acyl residue at another position (219) on sigma E affected the specificity of interaction with position -33 in spoIIID promoter. The amino acyl residues at the two positions in sigma E, 217 and 219, that determine the specificity of interactions between the sigma and base-pairs in the -35 region of its cognate promoters (positions -33 and -31, respectively, in the spoIIID promoter) probably closely contact these base-pairs.

We have examined the role of the most highly conserved region of bacterial RNA polymerase sigma factors by analyzing the effect of amino acid substitutions and small deletions in sigma E from Bacillus subtilis. sigma E is required for the production of endospores in B. subtilis but not for vegetative growth. Strains expressing each of several mutant forms of sigE were found to be deficient in their ability to form endospores. Single amino acid substitutions at positions 68 and 94 resulted in sigma factors that bind with less affinity to the core subunits of RNA polymerase. The substitution at position 68 did not affect the stability of the protein in B. subtilis; therefore, this substitution probably did not have large effects on the overall structure of the sigma factor. The substitution at position 68 probably defines a position in sigma E that closely contacts a subunit of RNA polymerase, while the substitution at position 94 may define a position that is important for protein stability or for binding to core RNA polymerase.

Bacillus subtilis possesses at least five different forms of RNA polymerase holoenzyme which are distinguished by their sigma subunit and their promoter recognition specificity. Sigma-37 RNA polymerase, a minor form of RNA polymerase, recognizes a class of promoters, which includes promoters for genes transcribed early during endospore formation. We have used site-directed bisulfite mutagenesis to construct a series of single and multiple base substitutions in a promoter recognized by sigma-37 RNA polymerase. In vitro transcription analysis of this series of mutant promoters demonstrated that single base substitutions at positions -36, -16, -15 and -14 most dramatically reduced the efficiency of promoter utilization by sigma-37 RNA polymerase. These results support a model in which sigma-37 RNA polymerase recognizes its cognate promoters by interacting with a sequence of nucleotides near the -10 region and the -35 region of the promoter--a sequence not recognized by B. subtilis sigma-55 RNA polymerase or Escherichia coli RNA polymerase.

Bacillus subtilis possesses several forms of RNA polymerase, each differing in its sigma subunit and its specificity of promoter recognition. The sequential appearance of sigma subunits, which change the promoter recognition specificity of RNA polymerase, may have a key role in controlling the temporal pattern of gene expression required for endospore development in B. subtilis. Several genes that are expressed over relatively long periods of time during the developmental cycle are transcribed by more than one form of RNA polymerase, which initiate transcription from either tandem or overlapping promoter. The promoter region for the ctc gene is interesting because transcription is initiated at or near the same position by both sigma 37 RNA polymerase (E sigma 37), a minor form in growing cells, and sigma 29 RNA polymerase (E sigma 29), a form which appears approximately 2 h after the initiation of sproulation. Here we report that several base substitutions in the ctc promoter differentially affect the utilization of the promoter by E sigma 37 or E sigma 29.

The RNA polymerase sigma factor sigma H is essential for the onset of endospore formation in Bacillus subtilis. sigma H also is required for several additional stationary-phase-specific responses, including the normal expression of several genes that are required for the development of competence for DNA uptake. It is necessary to identify the genes that are transcribed by sigma H RNA polymerase (E sigma H) in order to understand the role of this sigma factor during the transition from exponential growth to stationary phase. Feavers et al. (Mol. Gen. Genet. 211:465-471, 1988) proposed that citG, the structural gene for fumarase, is transcribed from two promoters, one of which (citGp2 [P2]) may be used by E sigma H. It is likely that the citGp2 promoter is used by E sigma H because we found that this promoter was used accurately in vitro by E sigma H and directed expression of xylE in vivo. This xylE expression was dependent on spo0H, the structural gene for sigma H, and was independent of the citGp1 promoter. Comparison of the nucleotide sequences of several sigma H-dependent promoters showed that these sequences were similar at two regions approximately 10 and 35 base pairs upstream from the start points of transcription. These sequences may signal recognition of these promoters by E sigma H. Primer extension analyses were used to examine transcription from three sigma H-dependent promoters during growth and sporulation. The citGp2 promoter appeared to be active during the middle and late stages of exponential growth, whereas activation of the spoIIA promoter was delayed until after the end of exponential growth. Evidently, promoters used by E sigma H can display different temporal patterns of expression.

The discovery of secondary sigma factors in Bacillus subtilis that enable RNA polymerase to transcribe cloned sporulation genes in vitro has led to the proposal that the appearance of new sigma factors during sporulation directs RNA polymerase to the different temporal classes of sporulation genes. One sigma factor, which appears 2 h after the initiation of sporulation, is sigma E (formerly sigma 29). Mutations that inactivate the structural gene for sigma E prevent transcription from promoter G4. To determine whether sigma E-RNA polymerase interacts with the G4 promoter in vivo, we examined the effects of six single-base-pair substitutions in the G4 promoter on its utilization in vivo and in vitro by sigma E-RNA polymerase. The mutations in the G4 promoter affected utilization of the promoter in vivo in the same way that they affected its utilization in vitro by purified sigma E-RNA polymerase; therefore, we conclude that this polymerase interacts directly with the G4 promoter in vivo. The effects of these mutations also support the model in which sigma E-RNA polymerase utilizes promoters by interacting with two distinct sets of nucleotides located 10 and 35 base pairs upstream from the start point of transcription.

The program of gene expression that underlies endospore formation by Bacillus subtilis may be controlled in part by a sporulation-induced form of RNA polymerase, E sigma 29. The nucleotide sequences of four promoters, which are known to be recognized by E sigma 29, are highly conserved at two regions, 10 bp and 35 bp upstream from the start point of transcription. We have used oligonucleotide-directed mutagenesis to construct several base substitutions in the ctc promoter from B. subtilis to test the role of the highly conserved sequences in utilization of the promoter by E sigma 29. In vitro transcription analysis demonstrated that the conserved nucleotides at positions -15, -14 and -12 affect the utilization of the promoter by E sigma 29. These and previous results support a model in which E sigma 29 recognizes its cognate promoters by interacting with nucleotides near the -10 and -35 regions. We also examined the effects of these base substitutions on utilization of the promoter by two other forms of RNA polymerase from B. subtilis, E sigma 37 and E sigma 32.