Development and use of a selectable, broad-host-range reporter transposon for identifying environmentally regulated promoters in bacteria.
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.
Spinler
Jennifer K
JK
Department of Microbiology, University of Colorado Denver School of Medicine, Aurora, CO 80045, USA.
Zajdowicz
Sheryl L W
SL
Haller
Jon C
JC
Oram
Diana Marra
DM
Gill
Ronald E
RE
Holmes
Randall K
RK
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
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; C075586:DtxR protein, Corynebacterium diphtheriae; D007501:Iron