σ(E) is a sporulation-specific sigma factor of Bacillus subtilis that is synthesized from an inactive precursor protein (P31). The structural gene (sigE) for P31 was reengineered by oligonucleotide-directed mutagenesis to encode σ(E) directly. The sequence specifying the first amino acid of σ(E) (GGC) was placed immediately downstream of the initiating codon (ATG) of P31. The resulting sigE allele (sigEΔ84) encodes a σ(E)-like protein which differs from the 'processed product' by a single Met residue at its amino terminus. B. subtilis strains which carried this allele were Spo- and contained no detectable σ(E). The sigEΔ84 allele generated a product in Escherichia coli which, by quantitative Western immunoblot analysis, was present at 10 to 20% of the level of product (P31) obtained from a wild-type allele. A σ(E)-like product was also not detected in two B. subtilis strains with missense mutations in the sequence encoding the processed region of P31. These results suggest that σ(E) is a highly labile protein that is stabilized during its synthesis by an element of the precursor sequence. A mutant allele (sigEΔ48) which made an active σ(E)-like protein in B. subtilis was isolated. This gene specified a product in which five amino acids, not derived from the P31 processed region, were joined to P31 at a position eight amino acids upstream of the processing site. The sigEΔ48 product was not processed, but it activated the σ(E)-dependent spoIID promoter in vivo. The sigEΔ48 product therefore lost both an essential target for processing and a region which inhibited σ(E) activity. Cells which carried sigEΔ48 were Spo-. The basis of the sigEΔ48-dependent defect in sporulation is unknown, but the σ(EΔ48) activity appeared to persist beyond the time in development (4 h after onset of sporulation) when wild-type σ(E) activity declines. Thus, it may interfere with the proper regulation of late sporulation genes.
ASJC Scopus subject areas
- Molecular Biology