A General Method for Fusion of the Escherichia coli lacZ Gene to Chromosomal Genes in Bacillus subtilis

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Abstract
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 .vphi.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.