Article

Development and use of a selectable, broad-host-range reporter transposon for identifying environmentally regulated promoters in bacteria

Department of Microbiology, University of Colorado Denver School of Medicine, Aurora, CO 80045, USA.
FEMS Microbiology Letters (Impact Factor: 2.72). 03/2009; 291(2):143-50. DOI: 10.1111/j.1574-6968.2008.01430.x
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ABSTRACT This report describes the development and use of TnKnXSp, a selectable broad-host-range reporter transposon with a promoterless aphA gene. Insertion of TnKnXSp into the chromosome of a kanamycin-susceptible bacterium confers resistance to kanamycin only if aphA is transcribed from an active promoter adjacent to the insertion site. We designed TnKnXSp as a tool for identifying environmentally regulated promoters in bacteria and developed general methods for initial characterization of any TnKnXSp integrant. To identify putative iron-regulated promoters in Corynebacterium diphtheriae, we constructed TnKnXSp integrants and identified a subgroup that expressed kanamycin resistance under low-iron, but not high-iron, conditions. We characterized representative integrants with this phenotype, located the TnKnXSp insertion in each, and demonstrated that transcription of aphA was repressed under high-iron vs. low-iron growth conditions. We also demonstrated that TnKnXSp can be used in bacteria other than C. diphtheriae, including Escherichia coli and Bacillus subtilis. Our findings validate TnKnXSp as a useful tool for identifying environmentally regulated promoters in bacteria.

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