Regulated expression of heterologous genes in Bacillus subtilis using the Tn10 encoded tet regulatory elements.
The Escherichia coli-derived tet regulatory elements from Tn10 have been used to construct vectors allowing the regulated, inducible, high-level expression of foreign genes in Bacillus subtilis. While the wild-type tet promoters are inactive in B. subtilis, a synthetic mutant tet sequence with improved promoter consensus sequences and upstream poly A blocks shows activity in B. subtilis. The expression of an indicator cat gene is inducible by sublethal amounts of tetracycline, indicating that the Tet repressor protein and the tet operator sequences are functional. However, the inducibility and maximal expression are not sufficient in this construct. To improve these properties a tet operator sequence was placed between the -35 and -10 boxes of the B. subtilis-derived very strong xyl promoter. In the presence of a tetR gene this construct is about 100-fold inducible and has high promoter strength, but some basal expression. This is avoided by placing a second tet operator downstream resulting in no detectable basal expression at the expense of reduced inducibility. Using the system with a single tet operator inducible expression of glucose dehydrogenase from B. megaterium was obtained at a very high level, and inducible expression of human single-chain urokinase-like plasminogen activator was achieved at the same level as in E. coli. Unlike in E. coli, the product was not degraded up to 4 h after induction in B. subtilis. These results demonstrate that the regulated expression vector described here should be very useful for production of foreign gene products from B. subtilis cultures.
Institut für Mikrobiologie und Biochemie, Friedrich-Alexander Universität Erlangen-Nürnberg, Federal Republic of Germany.
M
M
Geissendörfer
W
W
Hillen
D001410:Bacillus megaterium / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D016384:Consensus Sequence; D004251:DNA Transposable Elements; D005343:Fibrinolytic Agents / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D042843:Glucose 1-Dehydrogenase; D005948:Glucose Dehydrogenases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D010960:Plasminogen Activators / Q000235:genetics; D011401:Promoter Regions, Genetic; D011815:R Factors; D012097:Repressor Proteins / Q000235:genetics*; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D005343:Fibrinolytic Agents; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D005948:Glucose Dehydrogenases; D042843:Glucose 1-Dehydrogenase; D015500:Chloramphenicol O-Acetyltransferase; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator