Deletion of aprA and nprA genes for alkaline protease A and neutral protease A from Bacillus thuringiensis :: effect on insecticidal crystal proteins

The aprA gene encoding alkaline protease A (AprA) was cloned from Bacillus thuringiensis subsp. kurstaki, and the cloned gene was used to construct aprA-deleted (aprA1) strains of B. thuringiensis. An aprA I strain of B. thuringiensis that contained the wild-type gene for neutral protease A (aprA(+)) displayed levels of extracellular proteolytic activity that were similar to those of an aprA(+) nprA(+) strain. However, when EDTA was included in the protease assay to inhibit NprA activity the aprA lnprA(+) strain displayed only 2% of the extracellular proteolytic activity of the aprA(+) nprA(+) strain. A strain that was deleted for both aprA and nprA (aprA lnprA3 strain) failed to produce detectable levels of proteolytic activity either in the presence or absence of EDTA in the assay. Compared with the aprA(+) nprA(+) strain the aprA lnprA(+) strain yielded 10% more full-length CrylBb crystal protein and the aprAlnprA3 strain yielded 25% more full-length CrylBb protein. No significant differences were seen in the 50% lethal dose of CrylBb protein from aprA(+) nprA(+) and aprAlnprA3 strains against three species of lepidopteran insects. These results suggest that enhanced yield of certain crystal proteins can be obtained by deletion of the genes aprA and nprA which are the major extracellular proteases of B. thuringiensis. (C) 2000 Elsevier Science B.V. All rights reserved.