A T7 PROMOTER-SPECIFIC, INDUCIBLE PROTEIN EXPRESSION SYSTEM FOR BACILLUS-SUBTILIS

The adaptation and application of the Escherichia coli T7 RNA polymera se system for regulated and promoter-specific gene expression in Bacil lus subtilis is reported. The expression cassette used in Bacillus sub tilis was tightly regulated and T7 RNA polymerase (T7 RNAP) appeared 3 0 min after induction. The efficiency of T7 promoter-specific gene exp ression in B. subtilis was studied using one secretory and two cytosol ic proteins of heterologous origin. The accumulation of E. coli P-gala ctosidase, as well as a 1,4-beta-glucosidase from Thermoanaerobacter b rockii in B. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. The alpha-amylase of The rmoactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10-20 h after T7 RNAP induction, but was also deposited in cellular fractions. The addition of rifampicin inhibited alpha-amylase secretion, but unexpect edly, after a short period, also prevented its further (intra)cellular accumulation.