INTEGRATIVE VECTOR FOR CONSTRUCTING SINGLE-COPY TRANSLATIONAL FUSIONSBETWEEN REGULATORY REGIONS OF BACILLUS-SUBTILIS AND THE BGAB REPORTERGENE ENCODING A HEAT-STABLE BETA-GALACTOSIDASE

Here we report on the construction of two integrative plasmids for Bac illus subtilis allowing in vitro construction of translational fusions . Both plasmids contain two cassettes in tandem: the bgaB gene encodin g a heal-stable beta-galactosidase devoid of its own regulatory sequen ces and the first two codons followed by a neomycin-resistance gene fo r selection in B. subtilis. Both cassettes are flanked by the 3'- and 5'-end of the amyE gene (encoding alpha-amylase) allowing integration of both cassettes at the amyE locus of the B. subtilis chromosome. For propagation in Escherichia coli, the plasmids contain the pBR322 orig in of DNA replication and the beta-lactamase-encoding gene. Whereas on e vector needs a promoter, a Shine-Dalgarno sequence and the beginning of a gene fused in-frame to bgaB, the other one already carries a con stitutive promoter. The versatility of the gene fusion vectors was dem onstrated by the integration of the regulatory regions of the dnaK and the cat-86 genes. In the first case, heat-inducible expression was fo und, and by comparison with an operon fusion, it seems that the dnaK o peron is regulated at both the transcriptional and the posttranscripti onal level. In the second case, chloramphenicol-inducible regulation o f the gene fusion could be demonstrated.