title: 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.
authors.affiliation: Department of Microbiology, University of Colorado Denver School of Medicine, Aurora, CO 80045, USA.
authors.forename: Jennifer K
authors.initials: JK
authors.lastname: Spinler
authors.forename: Sheryl L W
authors.initials: SL
authors.lastname: Zajdowicz
authors.forename: Jon C
authors.initials: JC
authors.lastname: Haller
authors.forename: Diana Marra
authors.initials: DM
authors.lastname: Oram
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Gill
authors.forename: Randall K
authors.initials: RK
authors.lastname: Holmes
mesh_terms: D001419:Bacteria / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D007501:Iron / Q000378:metabolism; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; C075586:DtxR protein, Corynebacterium diphtheriae; D007501:Iron