A general method for fusion of the Escherichia coli lacZ gene to chromosomal genes in Bacillus subtilis.
A series of plasmids has been constructed that can be used to fuse the beta-galactosidase gene (lacZ) of Escherichia coli to chromosomal genes of Bacillus subtilis. Insertion of the lacZ gene is facilitated by the use of a selectable chloramphenicol acetyl-transferase (cat) gene. The latter is included, along with the lacZ gene, in a single DNA fragment or 'cartridge' that can be removed from the plasmid with a variety of different restriction endonucleases. Methods applicable to any cloned B. subtilis gene are described that enable the lac-cat cartridge to be inserted at specific sites, or at random, directly into the B. subtilis chromosome in a single step. These single-copy chromosomal fusions can be readily transferred, by selection for chloramphenicol resistance, to a temperate phage such as phi 105, to permit a more extensive genetic analysis of the expression of the target gene. Alternatively, the lac-cat cartridge and flanking DNA sequences can be transferred into different genetic backgrounds by transformation. These techniques have been used to construct, in a single step, lac fusions to genes in the sporulation operons spoIIA and spoVA.
D001412:Bacillus subtilis / Q000235:genetics* / Q000502:physiology; D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D005696:Galactosidases / Q000235:genetics; D005798:Genes, Bacterial; D005818:Genetic Engineering; D010957:Plasmids; D014176:Protein Biosynthesis; D013171:Spores, Bacterial; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics*
D005696:Galactosidases; D001616:beta-Galactosidase