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 transcriptional fusions. These plasmids contain a neomycin- or tetracycline-resistance cassette and one of three promoterless genes: bgaB (encoding beta-galactosidase), cat (chloramphenicol acetyltransferase), or xylE (catechol 2,3-dioxygenase). All cassettes are flanked by the 3'- and 5'-ends of the amyE gene (encoding alpha-amylase) allowing integration of these cassettes at the amyE locus of the Bs chromosome. For propagation and selection in Escherichia coli, the plasmids contain the pBR322 origin of DNA replication and the beta-lactamase-encoding bla gene. Four unique restriction sites can be used for insertion of restriction fragments carrying promoter fragments. All three reporter genes express heat-stable enzymes (stable up to at least 50 degrees C for 30 min) as shown here. We would like to point to the modular nature of these plasmids where the three reporter genes and the two resistance cassettes can be combined in any permutation. The versatility of the promoter-probe vectors was demonstrated by the integration of the promoters of the dnaK and groE operons of Bs and following their heat-inducible expression.