Controlled expression of click beetle luciferase using a bacterial operator-repressor system.
The bioluminescent phenotype conferred by luciferase genes in a particular bacterium has demonstrated to be one of the most versatile and useful methods to detect microorganisms. Genetic constructions derived from miniTn5 vectors have been constructed for the introduction and stable maintenance of the click beetle luciferase gene, lucOR, in various Gram-negative bacteria. To attenuate the expression in the environment where the marked strain has to survive (and to allow sensitive detection when desired) a DNA fragment containing the repressor gene lacIq and a Ptrc::lucOR fusion was cloned onto a suicide plasmid. This construction is able to express high luciferase levels only when induced by IPTG. Matings between Escherichia coli containing the suicide transposon vector and different recipient bacteria gave transposition frequencies from 10(-7) to 10(-5). Strains with miniTn5-lucOR insertions showed luciferase activity induced by IPTG addition. The stringency of the regulation and the intensity of light emission depended on the tagged strain. This system allows stable maintenance of the marker and tight control of luciferase expression in the environment.
Enrique Vázquez
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Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Spain.
Cebolla
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Palomares
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D000818:Animals; D001517:Coleoptera / Q000201:enzymology* / Q000235:genetics; D004784:Environmental Monitoring / Q000379:methods; D015964:Gene Expression Regulation, Bacterial; D006090:Gram-Negative Bacteria / Q000235:genetics; D008156:Luciferases / Q000096:biosynthesis* / Q000235:genetics; D009875:Operator Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000235:genetics
D011994:Recombinant Proteins; D012097:Repressor Proteins; D008156:Luciferases