pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis

pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis. It carries the chloramphenicol resistance g ene (cat) from Tn9, which is not transcribed in either host by lack of a pr omoter. The cat gene is preceded by a Shine-Dalgarno sequence functional in E. coli but not in B. subtilis. Deleted pGR71 plasmids were obtained in B. subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety. These mutant plasmids expressed chloramphenicol acetylt ransferase (CAT), conferring on B, subtilis resistance to high chlorampheni col concentrations. CAT expression peaked at the early postexponential phas e of B, subtilis growth. The transcription initiation site of cat, determin ed by primer extension, was located downstream of a putative promoter seque nce within the pUB110 moiety. N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids. The cat ribosome-binding site, functional in E. coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B. subtilis 16S rRNA, explaining the production of native enzyme.