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 subtilus. Our findings validate TnKnXSp as a useful tool for identifying environmentally regulated promoters in bacteria.

Phage Conversion and the Role of Bacteriophage and Host Functions in Regulation of Diphtheria Toxin Production by Corynebacterium diphtheriae

“…These site-specific integration vectors permit a single copy of a cloned gene to be introduced into the chromosome of C. diphtheriae for complementation tests or other purposes (Oram et al 2007). In 2009, Spinler et al developed a broad host-range reporter transposon with a selectable but promoterless aphA gene that is useful as a tool to select for and identify environmentally regulated promoters in bacteria, including iron-regulated promoters in C. diphtheriae (Spinler et al 2009). During the last decade, therefore, techniques for performing genetic manipulations that have been available for many years in model bacterial systems like E. coli or Bacillus subtilis have become available for routine use in C. diphtheriae.…”

Corynebacterium diphtheriae Proteome Adaptation to Cell Culture Medium and Serum

“…The manganese superoxide dismutase (sodA) DIP2261 also showed a high abundance in all three samples. SodA is involved in cell viability and knock-out mutants of Corynebacterium melassecola showed increased sensitivity to superoxide radicals [42]. Based on the abundance of each protein a multiple sample test using the ANOVA algorithm was applied.…”

“…In the first cluster are proteins with a high expression in RPMI 1640 and lower expression rates in BHI and FCS represented. Two proteins of this cluster, DIP0550 and DIP0620, are under the regulation of DtxR [39,42], which indicates iron starvation under these conditions.…”