Role of ISKpn7and Deletions inblaKPCGene Expression

Abstract

ABSTRACTThe carbapenemase-encodingblaKPCgene, which is rapidly spreading in Gram-negative rods, is located on a Tn3-based transposon, Tn4401, which carries a polymorphic region giving rise to five isoforms (a,b,c,d, ande) that is located immediately upstream of theblaKPCgene and thus likely involved in its expression. Using 5′ rapid amplification of cDNA ends (5′RACE), we identified three potential promoter sequences (P1, P2, and P3) upstream of theblaKPCgene, of which only P1 (absent from isoformscandd) and P2 (present in all isoforms, with a −35 box located inside the right inverted repeat of ISKpn7) were shown to be true promoters involved in expression. One representative of each different promoter combination of Tn4401, i.e., P2 alone (isoformc), P1-P2 (isoforma), and P1-P2-P3 (isoformb), was cloned into anEscherichia coliplasmid vector. Using reverse transcription-PCR (RT-PCR), the highest level of expression was obtained with isoforma(P1 and P2), which is also the most commonly encountered form in enterobacterial clinical isolates, followed by isoformsb(P1, P2, and P3) andc(P2 only). These differences in expression led to slight differences in MIC values of carbapenems.In silicoanalysis of the DNA sequence of isoformbrevealed a stem-loop structure that is likely responsible for strong stops observed in 5′RACE experiments and for decreased expression compared to that with isoforma(P1 and P2). In addition, such structures could also be at the origin for the deletions observed in isoformsaandc. Taken together, these results indicate that the P1 and P2 promoters both contribute to the expression of theblaKPCgene and that the construct with the highest level of expression is that possessing isoforma, which is also the most commonly encountered form in clinical isolates.