Cloning, characterization, and expression of the spoVB gene of Bacillus subtilis

Mutation of the spoVB gene in Bacillus subtilis causes the production of spores containing a defective cortex and unable to acquire heat resistance. The spoVB locus is highly linked to another spo locus, spoIIIF, characterized by a single mutation (I. L. Lamont and J. Mandelstam, J. Gen. Microbiol. 130:1253-1261, 1984). A 18-kb DNA region overlapping the spoIIIF-spoVB region was cloned in successive steps starting from a Tn917 insertion in the nic locus. The exact location of the spollIF and spoVB loci was defined with various integrative plasmids carrying subfragments of that region. DNA sequencing established that spollIF and spoVB are a single monocistronic locus encoding a 518-amino-acid polypeptide with features of an integral membrane protein. The precise location of the spoIIIF590 and spoVB91 mutations in that unique open reading frame was determined, and both mutations were sequenced. A null mutation was engineered in the spoIIIF-spoVB locus and led to a typical spoVB phenotype, identical to the phenotype created by either spoIIIF590 or spoVB9l, suggesting that the original spollIF mutant contained a secondary mutation arresting sporulation at an earlier stage. A transcriptional spoVB-lacZ fusion was constructed, and its expression was found to be directly dependent on RNA polymerase containing SE. A null mutation of spoVB had no effect on expression of sspB and cotA, members of the 7G- and rK-controlled regulons respectively, while expression of cotC, a member of the latest known mother cell regulon, was delayed and strongly reduced. These results are consistent wit