Replacement of the Bacillus subtilis subtilisin structural gene with an in vitro-derived deletion mutation

Abstract

The entire subtilisin structural gene from Bacillus subtilis I168 has been cloned, and its nucleotide sequence has been determined. When expressed on a high-copy-number shuttle vector, a 5-fold increase in serine protease activity was observed. The DNA sequence of the gene is 80% homologous to the Bacillus amyloliquefaciens subtilisin structural gene, and the translated mature coding sequence is 85% homologous to the published protein sequence of subtilisin BPN'. The chloramphenicol resistance determinant of a plasmid integrated at the subtilisin locus was mapped by PBS1 transduction and was found to be linked to glyB (83%) and argC (60%), but not with metC or purB. The chromosomal locus containing the wild-type subtilisin allele was replaced with an in vitro-derived allele of the gene (Δapr-684) that contained a 684-base-pair deletion. The technique used for introducing the deletion is a variation of the gene replacement methods used in Saccharomyces cerevisiae and Escherichia coli. When used in B. subtilis, deletion mutants could be directly screened among the transformants. Physiological characterization of the Δapr-684 mutation revealed no discernible effect on the formation of heat-resistant endospores, but strains carrying the mutation produced only 10% of wild-type serine protease activity. A model is presented that outlines the pathway for plasmid integration and deletion formation in B. subtilis.