INTEGRATIVE VECTORS FOR CONSTRUCTING SINGLE-COPY TRANSCRIPTIONAL FUSIONS BETWEEN BACILLUS-SUBTILIS PROMOTERS AND VARIOUS REPORTER GENES ENCODING HEAT-STABLE ENZYMES

Here, we report on the construction of three integrative plasmids for Bacillus subtilis (Bs) allowing in vitro construction of transcription al fusions. These plasmids contain a neomycin- or tetracycline-resista nce cassette and one of three promoterless genes: bgaB (encoding beta- galactosidase), cat (chloramphericol acetyltransferase), or xylE (cate chol 2,3-dioxygenase). All cassettes are flanked by the 3'- and 5'-end s of the amyE gene (encoding alpha-amylase) allowing integration of th ese cassettes at the amyE locus of the Bs chromosome. For propagation and selection in Escherichia coli, the plasmids contain the pBR322 ori gin of DNA replication and the beta-lactamase-encoding bla gene. Four unique restriction sites can be used for insertion of restriction frag ments carrying promoter fragments. All three reporter genes express he at-stable enzymes (stable up to at least 50 degrees C for 30 min) as s hown here. We would like to point to the modular nature of these plasm ids where the three reporter genes and the two resistance cassettes ca n be combined in any permutation. The versatility of the promoter-prob e vectors was demonstrated by the integration of the promoters of the dnaK and groE operons of Bs and following their heat-inducible express ion.