A T7 promoter-specific, inducible protein expression system for Bacillus subtilis.
The adaptation and application of the Escherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression in Bacillus subtilis is reported. The expression cassette used in Bacillus subtilis was tightly regulated and T7 RnA polymerase (T7 RNAP)appeared 30 minutes after induction. The efficiency of T7 promoter-specific gene expression in B.subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation of E. coli beta-galactosidase, as well as a 1,4-beta-glucosidase from Thermoanaerobacter brockii in B. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. The alpha-amylase of Thermactinomyces 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 chi-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation.
Conrad
B
B
Institut fur Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany.
Savchenko
R S
RS
Breves
R
R
Hofemeister
J
J
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014764:Viral Proteins; D014994:Xylose / Q000494:pharmacology; D000516:alpha-Amylases / Q000096:biosynthesis / Q000235:genetics; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000235:genetics
D014764:Viral Proteins; D014994:Xylose; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D000516:alpha-Amylases; D001617:beta-Glucosidase; D001616:beta-Galactosidase