A xylose-inducible Bacillus subtilis integration vector and its application.
The construction of a xylose-inducible expression vector is described. This vector allows the integration of any gene, coding for its authentic protein, at the amyE locus of Bacillus subtilis (Bs). The controlable expression cassette consists of the repressor-encoding gene and the promoter of the Bacillus megaterium-derived operon for xylose utilization, sandwiched between the 5'- and 3'-ends of amyE. This thereby allows insertion of in vitro constructed transcriptional fusions at the amyE locus of the Bs chromosome. The versatility of this expression system was tested by fusing three different heat-shock genes to the xylose-inducible promoter and following their expression by Western immunoblot analysis. Whereas no increase in the amount of heat-shock protein could be detected under non-inducing conditions when compared to the isogenic wild-type strain, the three proteins were strongly induced after addition of xylose, depending on the gene. To determine the tightness and the induction factor of the system more accurately, the bgaB gene encoding a heat-stable beta-galactosidase (beta Gal) was analyzed. The background activity of beta Gal increased by a factor of at least 200 after addition of xylose. The system is not subject to catabolite, but rather to glucose repression.
Kim
L
L
Institute of Genetics, University of Bayreuth, Germany.
Mogk
A
A
Schumann
W
W
D001412:Bacillus subtilis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D014994:Xylose / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics
D004269:DNA, Bacterial; D014994:Xylose; D001616:beta-Galactosidase