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 authen tic protein, at the amyE locus of Bacillus subtilis (Bs). The controla ble expression cassette consists of the repressor-encoding gene and th e promoter of the Bacillus megaterium-derived operon for xylose utiliz ation, sandwiched between the 5'- and 3'-ends of amyE. This thereby al lows insertion of in vitro constructed transcriptional fusions at the amyE locus of the Bs chromosome. The versatility of this expression sy stem was tested by fusing three different heat-shock genes to the xylo se-inducible promoter and following their expression by Western immuno blot 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 af ter addition of xylose, depending on the gene. To determine the tightn ess and the induction factor of the system more accurately, the bgaB g ene 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.