A recombinase-based selection of differentially expressed bacterial genes.
Bacterial genes are often differentially expressed in response to specific environmental conditions. We have devised a method to identify regulated bacterial promoters, such that transient promoter expression leads to a permanent and selectable change in bacterial phenotype. This system consists of a promoterless derivative of cre, the phage P1 recombinase, carried on a plasmid, and two chromosomal loxP sites, the targets of the Cre recombinase. The loxP sites flank npt, conferring kanamycin resistance, and sacB, which confers sensitivity to sucrose, allowing positive selection for both the presence and absence of this chromosomal cassette. Fusion of active promoters to cre induces recombination of the loxP sites and deletion of intervening DNA, allowing selection on media containing sucrose, while inactive promoters fail to induce recombination and so remain resistant to kanamycin. We tested the system in Salmonella typhimurium using a known regulated promoter, that from the araBAD operon, and found it to be a sensitive indicator of gene expression over a wide range of promoter induction. We then used this system to identify S. typhimurium genes that are specifically expressed when bacteria interact with cultured epithelial cells and identified a novel DNA fragment, not found in E. coli, which might represent part of a new pathogenicity island.
Altier
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C
Department of Microbiology, North Carolina State University College of Veterinary Medicine, 4700 Hillsborough St., Raleigh, NC, USA. craig_altier@ncsu.edu
Suyemoto
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M
D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D019426:Integrases / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011995:Recombination, Genetic; D012486:Salmonella typhimurium / Q000235:genetics; D014170:Transformation, Genetic; D014407:Tumor Cells, Cultured; D014764:Viral Proteins
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases