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

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 IS26, 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 IS26 from a compound transposon bounded by IS26. In a recA mutant strain, Tn4352, 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 Tn4352B, leaving behind the second IS26, occurred at high frequency. Excision occurred when Tn4352B 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 IS26 transposase Tnp26. However, the Tnp26 of only one IS26 in Tn4352B was required, specifically the IS26 downstream of the aphA1a gene, and the excised TU included the active IS26. The frequency of Tn4352B 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 Tn4352B replacing a single G in Tn4352. These G residues are located immediately adjacent to the two G residues at the left end of the IS26 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.

Targeted conservative formation of cointegrates between two DNA molecules containing IS26 occurs via strand exchange at either IS end

“…Theoretically, TU excision would require strand exchange between one inner and one outer IS end of the transposon, that is, between two left or two right IS termini, but only the left IS 26 (as shown in Table ) was required to produce active Tnp26 for excision to occur. This is consistent with cis action of Tnp26, and it was predicted that the left ends of the two IS 26 in Tn 4352 B were involved in the reaction (Harmer and Hall, ). To examine this prediction, mutations were introduced at either the left, right or both outer ends of Tn 4352 B as shown in Table .…”

“…In all cases, the two substituted right terminal bases were present. Hence, the TU had been formed from the wild‐type left‐hand IS 26 and the aphA1a ‐containing central fragment, and the sequence of the amplicon derived from the excised TU using primers aphA1‐A and aphA1‐B as described previously (Harmer and Hall, ) confirmed this. Hence, the outer right end is not required for the IS 26 ‐mediated excision of a TU from Tn 4352 B.…”

“…The excision of a TU from Tn 4352 B, which also involves a reaction between two like IS 26 ends, also required the terminal bases. However, in this case the reaction depends on the presence of two additional G residues adjacent to the GG at the left terminus of IS 26 (Harmer and Hall, ), and these residues appear to alter the DNA structure to overcome the constraints that normally prevent this reaction when two IS 26 are present in the same DNA molecules…”

“…The plasmids used in this study are listed in Table . pRMH977, R388, R388::IS 26 , R388::Tn 4352B and pUC19::Tn tet (D) have been described previously (Partridge and Hall, ; Harmer et al ., ; Harmer and Hall, ). pRMH977 derivatives containing an IS 26 with mutated terminal bases (pRMH1002, pRMH1003, pRMH1004) were generated by site‐directed mutagenesis as described previously (Harmer et al ., ) using the primers listed in Table .…”

Real time monitoring of Aeromonas salmonicida evolution in response to successive antibiotic therapies in a commercial fish farm

“…The frequency of pS28‐3 (which only confers resistance to florfenicol) declined in 2015, while plasmid pS121‐1b, a pS121‐1a variant devoid of the floR‐ containing region, was first detected the same year. The deletion of genomic regions flanked by IS26 elements has been regularly reported (Harmer et al ., ; He et al ., ) and support the hypothesis that pS121‐1b emerged directly from pS121‐1a through the excision of the floR ‐containing region. Our observation thus demonstrated that the exposure to antibiotic treatment has a direct, within‐year effect on the MGE prevalence in the pathogen's population, and that this prevalence is presumably connected to independent MGE acquisition patterns for different subclonal groups of the original sensitive clone.…”