Structure of a Bacillus subtilis bacteriophage SPO1 gene encoding RNA polymerase sigma factor.

Gene 28 of Bacillus subtilis bacteriophage SPO1 codes for a regulatory protein, a sigma factor known as sigma gp28, that binds to the bacterial core RNA polymerase to direct the recognition of phage middle gene promoters. middle promoters exhibit distinctive and conserved nucleotide sequences in two regions centered about 10 and 35 base pairs upstream from the start point of mRNA synthesis. Here we report the cloning of gene 28 and its complete nucleotide sequence. We infer that sigma gp28 is a 25,707-dalton protein of 220 amino acids. Neither the nucleotide sequence of gene 28 nor the inferred amino acid sequence of sigma gp28 exhibits extensive homology to the gene or protein sequence of Escherichia coli sigma factor.     

Bacillus subtilis sigma factor sigma 29 is the product of the sporulation-essential gene spoIIG.

Evidence is presented that the sporulation-essential locus spoIIG codes for both sigma 29 and a structurally related protein, P31. This demonstrates that at least one specific Bacillus subtilis RNA polymerase binding protein provides a critical function in endospore formation. spoIIG-specific RNA is present in B. subtilis cultures that are synthesizing P31 and sigma 29 and is absent in those that are not. A monoclonal antibody specific for an antigenic determinant on P31/sigma 29 detected crossreacting proteins (P25/P21) but not P31 or sigma 29 in a Spo- B. subtilis strain with a mutation at the spoIIG locus (spoIIG41). The appearance of P25 and P21 occurs in this mutant at a time when P31 and sigma 29 would normally appear and suggests that they are homologous proteins. Transformation of the spoIIG41 strain with plasmid DNA carrying the structural gene for spoIIG complements the Spo- phenotype and results in the synthesis of P31, sigma 29, P25, and P21 at the appropriate times during sporulation. In Escherichia coli, the cloned spoIIG sequence encoded a protein that reacted with the anti-P31/sigma 29 monoclonal antibody and had the electrophoretic mobility of authentic P31.     

Heterogeneity of RNA polymerase in Bacillus subtilis: evidence for an additional sigma factor in vegetative cells.

Preparations of Bacillus subtilis RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from vegetatively growing cells contain small amounts of an activity (B. subtilis RNA polymerase holoenzyme II) that shows a unique promoter specificity with T7 bacteriophage DNA as compared with the normal B. subtilis holoenzyme (holoenzyme I) and lacks the normal sigma subunit [Jaehning, J. A., Wiggs, J. L. & Chamberlin, M. J. (1979) Proc. Natl. Acad. Sci. USA 76, 5470-5474]. By heparin-agarose chromatography we have obtained holoenzyme II fractions that have no detectable holoenzyme I activity as judged by their failure to utilize promoter sites for holoenzyme I on any template we have tested. These fractions are far more active with B. subtilis DNA than with T7 DNA or other heterologous templates. This high degree of specificity has allowed identification of plasmids containing cloned fragments of B. subtilis DNA that bear strong promoter sites for holoenzyme II. These promoter sites are not used at all by B. subtilis RNA polymerase holoenzyme I. The specificity of holoenzyme II is dictated by a peptide of Mr 28,000 as judged by copurification of the peptide with specific holoenzyme II activity and by reconstitution of the holoenzyme II promoter specificity when the isolated peptide is added to B. subtilis core polymerase. Hence the 28,000 Mr peptide appears to be a sigma factor that determines a promoter specificity distinct from that of RNA polymerase holoenzyme I and all other known bacterial RNA polymerases.     

Two developmental genes encoding sigma factor homologs are arranged in tandem in Bacillus subtilis.

The sporulation-essential gene spoIIG of the Gram-positive bacterium Bacillus subtilis encodes the sporulation-specific sigma factor sigma 29(sigma E). We report here the initial characterization of a gene, referred to as ORF3, located immediately downstream of the spoIIG gene. The results indicate that ORF3 encodes a sigma homolog, whose expression is highly regulated during development. Analysis of the ORF3 nucleotide sequence reveals an open reading frame encoding a polypeptide of 260 amino acid residues (molecular mass of 30.1 kDa). Its predicted amino acid sequence shows significant similarity to that of other RNA polymerase sigma factor sequences. S1 nuclease mapping experiments indicate that ORF3 is initially cotranscribed with spoIIG from about 1 to 4 hr into the sporulation process and that later on ORF3 is transcribed independently from a new site located between spoIIG and ORF3. The role of ORF3 was investigated by constructing a deletion mutation in its structural gene. The mutant exhibits normal growth but is unable to produce heat-resistant spores. We propose that the ORF3 gene product is a sigma factor or a related peptide essential for sporulation at a late stage of development.     

DNA polymerase III gene of Bacillus subtilis.

The Bacillus subtilis dnaF (polC) gene that codes for the alpha subunit of the DNA polymerase III holoenzyme has been sequenced. It consists of 4005 base pairs coding for 1335 amino acids (from the start to the stop codon), giving a molecular weight of 151,273. A mutation (azp-12) that confers resistance to the antimicrobial drug 6-(p-hydroxyphenylazo)-uracil is due to a single base change at nucleotide 3523, from TCA to GCA, resulting in a change of the 1175th amino acid, serine, to alanine. It is in the active site and located at the C-terminal part of the enzyme. The amino acid composition in an N-terminal domain has 26% homology to the epsilon subunit coded by the dnaQ gene of Escherichia coli, which is a 3'----5' proofreading exonuclease, supporting an earlier observation that this function is an integral part of the polymerase molecule in B. subtilis.     

Chromosomal location of a structural gene for the RNA polymerase sigma factor in Escherichia coli.

A set of F' strains of Escherichia coli K-12 partially diploid for various chromosomal segments has been examined for possible gene dosage effects in the synthesis of sigma factor of the DNA-dependent RNA polymerase (RNA nucleotidyltransferase; nucleoside-triphosphate:RNA nucleoti-dyltransferase, EC 2.7.7.6). It was found that all F' strains diploid for the dnaG region synthesize sigma at rates two to three times higher than other F' or F- strains. Moreover, strains of Salmonella typhimurium harboring these F' plasmids produce E. coli sigma in addition to Salmonella sigma. This has been shown on the basis of the finding that Salmonella sigma can be precipitated with antiserum against E. coli RNA polymerase but is distinguishable from E. coli sigma in its mobility in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. E. coli sigma polypeptides thus produced seem to be stable in cells of S. typhimurium. These results indicate that a structural gene for sigma (rpoD) is located at the metC-argG region, probably near the dnaG locus (66 min on the current genetic map of E. coli).     

Spo0A binds to a promoter used by sigma A RNA polymerase during sporulation in Bacillus subtilis.

Examination of the effects of 56 single-base-pair substitutions in the spoIIG promoter and studies of the interaction of the spo0A product (Spo0A) with this promoter in vitro demonstrated that Spo0A acts directly to enable this promoter to be used by sigma A-associated RNA polymerase (EC 2.7.7.6). The spoIIG operon from Bacillus subtilis is transcribed during sporulation by a form o RNA polymerase containing sigma A, the primary sigma factor in vegetative cells. The spoIIG promoter is unusual in that it contains sequences that are similar to those found at the -10 and -35 regions of promoters that are used by sigma A-associated RNA polymerase, but these sigma A-like recognition sequences are separated by 22 base pairs rather than the typical 17 or 18 base pairs. We found that single-base-pair substitutions in the around the -35-like sequence, and substitutions in a region upstream from this position, around position -87, reduced promoter activity. DNase I protection and electrophoretic gel mobility shift assays were used to demonstrate that Spo0A binds specifically to these regions in vitro. Evidently, the -35-like sequence is part of a Spo0A binding site and therefore is possibly not a sigma A-recognition sequence. These results support a model in which Spo0A activates the spoIIG promoter after the onset of endospore formation.     

A gene from Escherichia coli affecting the sigma subunit of RNA polymerase.

The RNA polymerase sigma subunits of Escherichia coli K, E. coli C, and Salmonella typhimurium can be resolved by electrophoresis. Using this technique, we have analyzed Salmonella strains carrying F' plasmids from E. coli K in order to map the gene for the sigma factor. Partial diploid analyses show the location of the sigma gene at 62-66 min on the E. coli genetic map. This gene is cotransducible with toIC and dnaG, at 66 min.     

Sporulation-specific sigma factor sigma 29 of Bacillus subtilis is synthesized from a precursor protein, P31.

Evidence is presented that a sporulation-essential sigma factor of Bacillus subtilis, sigma 29, is synthesized as an inactive precursor (P31) and that its activation occurs by a developmentally regulated cleavage of 29 amino acids from the P31 amino terminus. A pulse-chase experiment demonstrated that sigma 29 was derived from a preexisting protein, with appearance of radioactively labeled sigma 29 paralleling the disappearance of labeled P31. The disappearance of pulse-labeled P31 did not occur when the experiment was done with a B. subtilis strain carrying a mutation in a locus (spoIIE) required for sigma 29, but not P31, synthesis. Microsequencing of sigma 29 protein revealed that its amino terminus originates at amino acid 30 of the P31 amino acid sequence. In order to test whether a proteolytic event alone could activate P31 to a protein with sigma 29-like properties, a fusion protein (P31*) containing most of P31 was overproduced in Escherichia coli and converted in vitro into a protein with the electrophoretic mobility of sigma 29 by limited treatment with Staphylococcus aureus V8 protease. Protease-treated P31*, but not untreated P31*, was capable of directing B. subtilis core RNA polymerase to specifically initiate RNA synthesis at a sigma 29-recognized promoter in vitro.     

Heterogeneity of the principal sigma factor in Escherichia coli: the rpoS gene product, sigma 38, is a second principal sigma factor of RNA polymerase in stationary-phase Escherichia coli.

The rpoS gene of Escherichia coli encodes a putative RNA polymerase sigma factor that is considered to be the central regulator of gene expression in stationary phase. The gene product (sigma 38) was overproduced using the cloned rpoS gene and purified to homogeneity. Reconstituted RNA polymerase holoenzyme (E sigma 38) was found to recognize in vitro a number of typical sigma 70-type promoters, including the lacUV5 and trp promoters. Some, however, were recognized exclusively or preferentially by E sigma 70, whereas at least one, fic, was favored by E sigma 38. Thus E. coli promoters can be classified into three groups: the first group is recognized by E sigma 70 and E sigma 38, but the second and third groups are recognized substantially by either E sigma 70 or E sigma 38 alone. In contrast to other minor sigma factors, sigma 38 shares a set of amino acid sequences common among the principal sigma factors of eubacteria and is therefore a member of the RpoD-related protein family. The intracellular level of sigma 38 was demonstrated to increase in vivo upon entry into stationary phase. These results together indicate that sigma 38 is a second principal sigma factor in stationary-phase E. coli.     

Catabolite-resistant sporulation (crsA) mutations in the Bacillus subtilis RNA polymerase sigma 43 gene (rpoD) can suppress and be suppressed by mutations in spo0 genes.

The catabolite-resistant sporulation (crsA) mutation is able to overcome the repressive effect of glucose on sporulation in Bacillus subtilis. Three chromosomal crsA mutations, crsA1, crsA4, and crsA47, were transferred by the "gene conversion" process to B. subtilis plasmid pRPD11, which consists of the entire wild-type rpoD coding sequence, encoding the major sigma 43 factor of B. subtilis RNA polymerase, and pUB110. By DNA sequence analysis we showed that all three crsA mutations were identical two-base changes, CCT (proline) to TTT (phenylalanine), within the rpoD coding sequence. Furthermore, the crsA47 mutation restored spo0J and spo0K sporulation to wild-type levels and partially improved the sporulation efficiencies of spo0B, spo0D, and spo0F. Extragenic suppressors (scr) of crsA47 included mutations in spo0A, spo0D, spo0F, and spo0K plus other mutations that have not been specifically identified. Thus sigma 43 appears to be involved in catabolite repression by glucose, to interact either directly or indirectly with spo0 gene products, and to play an important role in the initiation of spore development in B. subtilis.