Integrative vector for constructing single-copy translational fusions between regulatory regions of Bacillus subtilis and the bgaB reporter gene encoding a heat-stable β-galactosidase

• Published in: FEMS microbiology letters 1997-05, Vol.150 (1), p.49-54
• Main Authors: Stoß, Oliver, Mogk, Axel, Schumann, Wolfgang
• Format: Article
• Language: English
• Subjects: alpha-Amylases - genetics; amyE; Bacillus subtilis; Bacillus subtilis - genetics; Base Sequence; beta-Galactosidase - genetics; beta-Lactamases - genetics; bgaB; cat-86; Chloramphenicol - pharmacology; Chloramphenicol O-Acetyltransferase - genetics; Escherichia coli - genetics; Escherichia coli Proteins; Gene Expression Regulation, Bacterial - genetics; Genes, Bacterial - genetics; Genes, Reporter - genetics; Genetic Vectors - genetics; Geobacillus stearothermophilus - enzymology; Geobacillus stearothermophilus - genetics; Heat shock; Hot Temperature; HSP70 Heat-Shock Proteins - genetics; Integration vector; Kanamycin Kinase; Molecular Sequence Data; Phosphotransferases (Alcohol Group Acceptor) - genetics; Protein Biosynthesis; Recombinant DNA; Recombinant Fusion Proteins - genetics; Regulatory Sequences, Nucleic Acid - genetics
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1016/S0378-1097(97)00095-5

Here we report on the construction of two integrative plasmids for Bacillus subtilis allowing in vitro construction of translational fusions. Both plasmids contain two cassettes in tandem: the bgaB gene encoding a heat-stable β-galactosidase devoid of its own regulatory sequences and the first two codons followed by a neomycin-resistance gene for selection in B. subtilis. Both cassettes are flanked by the 3′- and 5′-end of the amyE gene (encoding α-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 origin of DNA replication and the β-lactamase-encoding gene. Whereas one 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 constitutive promoter. The versatility of the gene fusion vectors was demonstrated by the integration of the regulatory regions of the dnaK and the cat-86 genes. In the first case, heat-inducible expression was found, and by comparison with an operon fusion, it seems that the dnaK operon is regulated at both the transcriptional and the posttranscriptional level. In the second case, chloramphenicol-inducible regulation of the gene fusion could be demonstrated.