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


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.

SUBMITTER: Spinler JK 

PROVIDER: S-EPMC2772819 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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

Spinler Jennifer K JK   Zajdowicz Sheryl L W SL   Haller Jon C JC   Oram Diana Marra DM   Gill Ronald E RE   Holmes Randall K RK  

FEMS microbiology letters 20090201 2


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  ...[more]

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