Identification of a Bacillus subtilis secretion mutant using a β-galactosidase screening procedure

Summary: High-level synthesis of exportable β-galactosidase (LacZ) fusion proteins in Bacillus subtilis results in a lethal phenotype, and has been suggested as a tool for the selection of secretion mutants. We tested a plasmid-based, inducible lacZ fusion gene system for this purpose, but frequent mutations in cis. which reduced expression of the fusion gene, forced abandonment of the induction-selection strategy. Instead, after modification of the indicator plasmid, a screening procedure for increased basal LacZ activity levels was adopted. This led to the identification of a conditional B. subtilis secretion mutant after nitrosoguanidine mutagenesis. At 42°C, but not at 30°C, this mutant displayed extreme growth retardation when the LacZ fusion protein was produced, and was also defective in the secretion of subtilisin Carlsberg. The processing kinetics and secretion of a subtilisin Carlsberg-alkaline phosphatase fusion derivative were found to be defective specifically at the non-permissive temperature. The secretion defect was not linked to the secA/div locus.