A mutation in P23, the first gene in the RNA polymerase sigma A (sigma 43) operon, affects sporulation in Bacillus subtilis

Abstract

Mutations within P23, the first gene of the Bacillus subtilis SA operon, were not detrimental to vegetative growth or sporulation. One deletion of P23 resulted in a strain that sporulated earlier than the wild type. This aberrant phenotype may be due to the simultaneous deletion of a CH promoter from the FA operon.The Bacillus subtilis major vegetative RNA polymerase u factor (uA) is encoded by the sigA gene. The sigA gene has been cloned and sequenced (3,8; GenBank/EMBL accession no. X03897). It was found to lie within an operon with the dnaE (encodes DNA primase) and the P23 genes, the gene order being P23, dnaE, sigA (11).The organization of the B. subtilis CA operon is similar to that of the Escherichia coli Cr70 operon (11). The genes and gene products of dnaE and sigA are very homologous to their E. coli counterparts, dnaG and rpoD, respectively. In contrast, the deduced 23-kilodalton polypeptide product encoded by the P23 gene has no homology to E. coli ribosomal protein S21, which is the product of rpsU, the first gene in the r70 operon. A computer search of the Georgetown protein data bank did not reveal any significant homology of the P23 product to any known protein.The results of Wang and Doi (13) suggested that three polypeptides were produced from the P23 gene when the products were expressed as fusion proteins to 3-galactosidase from plasmids in B. subtilis and E. coli. Their results indicated that these polypeptides were all in the same phase and so would share the same C-terminal amino acids. The molecular masses of these polypeptides were deduced from sequence data as 23, 19, and 9 kilodaltons. Expression from the P23 and P19 ribosome-binding sites (RBSs) was observed during vegetative growth, but expression from the P9 RBS was observed only after the onset of sporulation. This pattern of expression was attributed to promoter switching between the three major promoters of the CrA operon (12). The operon is expressed during vegetative growth from the 9A-dependent P1 and P2 promoters located upstream from the P23 RBS. An additional promoter is located within the P23 gene between the P19 and P9 RBSs. Expression from this P3 promoter is induced at T2 and is dependent upon cH, the gene product of spoOH (2).As part of a continuing genetic characterization of the B. subtilis crA operon, we have mutated the P23 gene in order to attempt to identify its role during growth and sporulation.Both Figure 1 shows the predicted structure in the P23 gene region for the three different B. subtilis Cmr mutant derivatives DB226, DB227, and DB228. DB226 contains the cat gene inserted at the position of the XmnI site of P23. DB227 contains an XmnI-RsaI deletion of P23, replaced with the cat gene. DB228 contains a cat gene replacement of the HaeIIIRsaI fragment at the 3' end of the P23 gene. The integration was confirmed in each case by Southern hybridization. Hindlll-digested chromosomal DNA from each transformant was probed with plasmid pUC19-P23 (L. F. Wang and R. H. Doi, unpublished). This plasmid carries the com...

The sigma factors of Bacillus subtilis.

“…The P23 function is unknown. Mutagenesis studies in which P23 was inactivated by inserting a chloramphenicol acetyltransferase (CAT) gene into its interior revealed no obvious detrimental effect on growth or sporulation (343). One mutant in which P23 was deleted sporulated earlier than the wild-type strain; however, this deletion also removed one of the operon's H -dependent promoters, thus making the cause of the altered sporulation phenotype unclear (343).…”

Altering the level and regulation of the major sigma subunit of RNA polymerase affects gene expression and development in Bacillus subtilis

“…1) with two other genes, orf23 (previously P23) which encodes a nonessential protein of approx. 23 kDa with unknown function (Wang and Doi, 1986;Zuberi and Doi, 1990), and dnaG (formerly called dnaE) which encodes DNA primase (Wang eta/., 1985). The operon contains at least six promoters ( Fig.…”

Characterization of csh203::Tn917lac, a mutation in Bacillus subtilis that makes the sporulation sigma factor sigma-H essential for normal vegetative growth

“…1) contains three genes in the order orf23, dnaG, rpoD (32). orf23 (previously P23) encodes a nonessential protein of approximately 23 kDa with unknown function (32,43). dnaG (formerly called dnaE) encodes DNA primase (34).…”