IDENTIFICATION OF A BACILLUS-SUBTILIS SECRETION MUTANT USING A BETA-GALACTOSIDASE SCREENING-PROCEDURE

High-level synthesis of exportable beta-galactosidase (LacZ) fusion pr oteins in Bacillus subtilis results in a lethal phenotype, and has bee n suggested as a tool for the selection of secretion mutants. We teste d 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 f or increased basal LacZ activity levels was adopted. This led to the i dentification of a conditional B. subtilis secretion mutant after nitr osoguanidine mutagenesis. At 42 degrees C, but not at 30 degrees C, th is mutant displayed extreme growth retardation when the LacZ fusion pr otein was produced, and was also defective in the secretion of subtili sin Carlsberg. The processing kinetics and secretion of a subtilisin C arlsberg-alkaline phosphatase fusion derivative were found to be defec tive specifically at the non-permissive temperature. The secretion def ect was not linked to the secA/div locus.