ISCR28 is associated with initial armA capture and provides promoter activity for its expression

Over two decades after its initial detection in Klebsiella pneumoniae, the aminoglycoside resistance gene armA is now widely disseminated, having been reported in 16 genera from eight families, predominantly in Klebsiella spp. (Enterobacteriaceae) and Acinetobacter spp. (Moraxellaceae). Here, we identified a 243-bp remnant of the insertion sequence common region (ISCR) 28 containing a 2-bp truncation at the origin of insertion (oriIS) end (∆ISCR28) in the intergenic region between insertion sequence Escherichia coli 28 (ISEc28) and armA within the original armA dissemination vehicle, transposon Tn1548, extending the inferred mobilization history of armA. Comparative analysis of genetic contexts in 17 representative sequences from different hosts revealed conservation of ∆ISCR28 in all sequences, implicating ISCR28 in the initial capture of armA, and suggesting, under a parsimonious evolutionary model, that this event preceded its association with ISEc28 and ISEc29. A rapid amplification of cDNA ends (5'-RACE) experiment confirmed a transcription start site of armA located 146 bp upstream of the start codon, and showed that the promoter region P_CR28-1 lies within the right arm of ISCR28 and is required for armA expression. The tandem array ISCR1-ISEc28-∆ISCR28 extending to the downstream insertion sequence (IS)26 was more conserved during transmission than the IS26-ISCR1 region. Analysis of 8-bp target site duplications flanking IS26 indicated that downstream IS26 in Tn1548 co-transferred with armA, whereas the upstream IS26 did not. Collectively, these findings clarify the role of ISCR28 in mediating armA expression and initiating its mobilization.