The Bacillus subtilis spoIIG operon encodes both sigma E and a gene necessary for sigma E activation.

A sporulation-specific sigma factor of Bacillus subtilis (sigma E) is formed by a proteolytic activation of a precursor protein (P31). Synthesis of the precursor protein is shown to be abolished in B. subtilis mutants with plasmid insertions as far as 940 base pairs upstream of the P31 structural gene (sigE), and processing of P31 to sigma E is blocked by a deletion in this upstream region. These results substantiate the view that sigE is the distal member of a 2-gene operon and demonstrate that the upstream gene (spoIIGA) is necessary for sigma E formation.     

Mutational analysis of the precursor-specific region of Bacillus subtilis sigma E.

sigma E is a sporulation-specific sigma factor of Bacillus subtilis that is formed from an inactive precursor protein (pro-sigma E) by the removal of 27 to 29 amino acids from the pro-sigma E amino terminus. By using oligonucleotide-directed mutagenesis, sequential deletions were constructed in the precursor-specific region of sigE and analyzed for their effect on the gene product's activity, ability to accumulate, and susceptibility to conversion into mature sigma E. The results demonstrated that the first 17 residues of the pro sequence contribute to silencing the sigma-like activity of pro-sigma E and that the amino acids between positions 12 and 17 are also important for its conversion into sigma E. Deletions that remove 21 or more codons from sigE reduce sigma E activity in cells which carry it, presumably by affecting pro-sigma E stability. A 26-codon deletion results in a gene whose product is not detectable in B. subtilis by either reporter gene activity or Western blot (immunoblot) assay. The primary structure as well as the size of the pro region of sigma E contributes to the protein's stability. The placement of additional amino acids into the pro region reduces the cell's ability to accumulate pro-sigma E. Additional sigE mutations revealed that the amino acids normally found at the putative processing site(s) of pro-sigma E are not essential to the processing reaction; however, a Glu residue upstream of these sites (position 25) was found to be important for processing. These last results suggest that the pro-sigma E processing apparatus does not recognize the actual site within pro-sigma E at which cleavage occurs but rater sequence elements that are upstream of this site.     

Exchange of precursor-specific elements between Pro-sigma E and Pro-sigma K of Bacillus subtilis.

sigma E and sigma K are sporulation-specific sigma factors of Bacillus subtilis that are synthesized as inactive proproteins. Pro-sigma E and pro-sigma K are activated by the removal of 27 and 20 amino acids, respectively, from their amino termini. To explore the properties of the precursor-specific sequences, we exchanged the coding elements for these domains in the sigma E and sigma K structural genes and determined the properties of the resulting chimeric proteins in B. subtilis. The pro-sigma E-sigma K chimera accumulated and was cleaved into active sigma K, while the pro-sigma K-sigma E fusion protein failed to accumulate and is likely unstable in B. subtilis. A fusion of the sigE "pro" sequence to an unrelated protein (bovine rhodanese) also formed a protein that was cleaved by the pro-sigma E processing apparatus. The data suggest that the sigma E pro sequence contains sufficient information for pro-sigma E processing as well as a unique quality needed for sigma E accumulation.     

Influence of spo mutations on sigma E synthesis in Bacillus subtilis.

Bacillus subtilis mutants blocked at the same stage of development (stage II) as strains with mutations in the structural gene for sigma E (sigE[spoIIGB]) were analyzed immunologically for sigma E and its precursor protein, P31. Mutations at spoIIL, spoIIN, and spoIIJ loci but not at the spoIIM locus significantly reduced P31 formation. Mutations at the spoIIAA, spoIIAC, spoIIEA, spoIIEB, and spoIIEC loci did not affect P31 synthesis but blocked its processing into sigma E. These results demonstrate a requirement for at least eight stage II gene products in the developmental pathway which leads to sigma E and brings to 11 the number of stage II genes (including spoIIGA, spoIIGB, and spoIIF) now known to be needed for sigma E formation.     

Isolation and sequence analysis of dacB, which encodes a sporulation-specific penicillin-binding protein in Bacillus subtilis.

A novel penicillin-binding protein (PBP 5*) with D,D-carboxypeptidase activity is synthesized by Bacillus subtilis, beginning at about stage III of sporulation. The complete gene (dacB) for this protein was cloned by immunoscreening of an expression vector library and then sequenced. The identity of dacB was verified not only by the size and cross-reactivity of its product but also by the presence of the nucleotide sequence that coded for the independently determined NH2 terminus of PBP 5*. Analysis of its complete amino acid sequence confirmed the hypothesis that this PBP is related to other active-site serine D,D-peptidases involved in bacterial cell wall metabolism. PBP 5* had the active-site domains common to all PBPs, as well as a cleavable amino-terminal signal peptide and a carboxy-terminal membrane anchor that are typical features of low-molecular-weight PBPs. Mature PBP 5* was 355 amino acids long, and its mass was calculated to be 40,057 daltons. What is unique about this PBP is that it is developmentally regulated. Analysis of the sequence provided support for the hypothesis that the sporulation specificity and mother cell-specific expression of dacB can be attributed to recognition of the gene by a sporulation-specific sigma factor. There was a good match of the putative promoter of dacB with the sequence recognized by sigma factor E (sigma E), the subunit of RNA polymerase that is responsible for early mother cell-specific gene expression during sporulation. Analysis of PBP 5* production by various spo mutants also suggested that dacB expression is on a sigma E-dependent pathway.     

Cloning of the flagellin gene from Bacillus subtilis and complementation studies of an in vitro-derived deletion mutation.

The flagellin promoter and structural gene from Bacillus subtilis I168 was cloned and sequenced. The amino-terminal protein sequence deduced from the coding sequence of the cloned gene was identical to that of the amino terminus of purified flagellin, indicating that the export of this protein is not directed by a posttranslationally processed N-terminal signal peptide. A sequence that was homologous to that of a consensus sigma 28 RNA polymerase recognition site lay upstream of the proposed translational start site. Amplification of this promoter region on a multicopy plasmid resulted in the formation of long, filamentous cells that accumulated flagellin intracellularly. The chromosomal locus containing the wild-type flagellin allele was replaced with a defective allele of the gene (delta hag-633) that contained a 633-base-pair deletion. Transport analysis of various flagellin gene mutations expressed in the hag deletion strain suggest that the extreme C-terminal portion of flagellin is functionally involved in export of the protein.     

Studies of the processing of the protease which initiates degradation of small, acid-soluble proteins during germination of spores of Bacillus species.

Three mutant forms of the protease (GPR) that initiates degradation of small, acid-soluble spore proteins (SASP) during germination of spores of Bacillus species have been generated. In one variant (GPR delta), the putative pro sequence removed in conversion of the GPR zymogen (termed P46) to the active enzyme (termed P41) was deleted. GPR delta was expressed in both Escherichia coli and Bacillus subtilis as a polypeptide of 41 kDa (P41) which was active both in vivo and in vitro. The other two variants had changes in the sequence around the site where the pro sequence is removed, making this sequence even more like that recognized and cleaved by GPR in its SASP substrates. One of these variants (GPRS) was synthesized as P46S in both B. subtilis and E. coli, but P46S was processed to P41S earlier in B. subtilis sporulation than was wild-type P46. The second variant (GPREI) was made as P46EI but underwent extremely rapid processing to P41EI in both E. coli and B. subtilis. Expression of elevated (> 100-fold) levels of GPR delta or GPREI blocked sporulation at the time of synthesis of glucose dehydrogenase. Expression of elevated levels of GPRS or low levels (< 20% of the wild-type level) of GPR delta or GPREI did not retard sporulation, but the SASP level in the resultant spores was greatly reduced. Prolonged incubation of P41 delta, P41EI, or wild-type P41, either in vivo or with purified proteins in vitro, resulted in a second self-cleavage event generating a 39-kDa polypeptide termed P39. The sequence in the P(41)-->P(39) cleavage site was also quite similar to that recognized and cleaved by GPR in SASP. Together, these results strongly support a model in which activation of GPR during sporulation by conversion of P(46) to P(41) is a self-processing event triggered by a change in the spore core environment (i.e., dehydration) which precludes attack of the active P(41) on its SASP substrates. However, in the first minutes of spore germination, rapid spore core hydration allows rapid attack of active GPR on SASP.     

A single amino acid substitution in sigma E affects its ability to bind core RNA polymerase.

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.