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

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Abstract
The entire subtilisin structural gene from B. subtilis I168 was cloned, and its nucleotide sequence was 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 B. 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 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 (.DELTA.apr-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 .DELTA.apr-684 mutation revealed no discernable 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.