IS 26 -Mediated Precise Excision of the IS 26 - aphA1a Translocatable Unit

Abstract

ABSTRACT We recently showed that, in the absence of RecA-dependent homologous recombination, the Tnp26 transposase catalyzes cointegrate formation via a conservative reaction between two preexisting IS 26 , and this is strongly preferred over replicative transposition to a new site. Here, the reverse reaction was investigated by assaying for precise excision of the central region together with a single IS 26 from a compound transposon bounded by IS 26 . In a recA mutant strain, Tn 4352 , a kanamycin resistance transposon carrying the aphA1a gene, was stable. However, loss of kanamycin resistance due to precise excision of the translocatable unit (TU) from the closely related Tn 4352 B, leaving behind the second IS 26 , occurred at high frequency. Excision occurred when Tn 4352 B was in either a high- or low-copy-number plasmid. The excised circular segment, known as a TU, was detected by PCR. Excision required the IS 26 transposase Tnp26. However, the Tnp26 of only one IS 26 in Tn 4352 B was required, specifically the IS 26 downstream of the aphA1a gene, and the excised TU included the active IS 26 . The frequency of Tn 4352 B TU loss was influenced by the context of the transposon, but the critical determinant of high-frequency excision was the presence of three G residues in Tn 4352 B replacing a single G in Tn 4352. These G residues are located immediately adjacent to the two G residues at the left end of the IS 26 that is upstream of the aphA1a gene. Transcription of tnp26 was not affected by the additional G residues, which appear to enhance Tnp26 cleavage at this end. IMPORTANCE Resistance to antibiotics limits treatment options. In Gram-negative bacteria, IS 26 plays a major role in the acquisition and dissemination of antibiotic resistance. IS 257 (IS 431 ) and IS 1216 , which belong to the same insertion sequence (IS) family, mobilize resistance genes in staphylococci and enterococci, respectively. Many different resistance genes are found in compound transposons bounded by IS 26 , and multiply and extensively antibiotic-resistant Gram-negative bacteria often include regions containing several antibiotic resistance genes and multiple copies of IS 26 . We recently showed that in addition to replicative transposition, IS 26 can use a conservative movement mechanism in which an incoming IS 26 targets a preexisting one, and this reaction can create these regions. This mechanism differs from that of all the ISs examined in detail thus far. Here, we have continued to extend understanding of the reactions carried out by IS 26 by examining whether the reverse precise excision reaction is also catalyzed by the IS 26 transposase.