Non‐palindromic attI sites of integrons are capable of site‐specific recombination with one another and with secondary targets

Abstract

SummaryGenes borne on cassettes are mobile owing to sitespecific recombination systems called integrons, which have created various combinations of antibiotic resistance genes in R-plasmids. In these processes, the palindromic site, attC (59-base element), at cassette junctions has been proposed as being essential. Excised and circularized cassettes have been found to integrate with preference for an attI site at one end of the conserved sequence in integrons. In this work, we give evidence that recombination is possible in the absence of the highly organized attC sites between the more simply organized attI sites. Furthermore, at a very low frequency representing the background in our recombination assay, we observed cross-overs between attI and secondary sites. To characterize recombination excluding the attC sites, we have used naturally occurring attI variants and constructed mutants. The cross-over point was identified between a guanine and a thymine in attI using point mutations. Progressive deletions showed the extent of attI and identified two important regions in the conserved sequence 5Ј of the cross-over point. A region 27-36 bp 5Ј of attI influenced recombination with attC sites only, whereas a sequence 9-14 bp 5Ј of the cross-over point in attI was important for recombination with both attI and attC. Recombination between attI and secondary sites could allow fusion of the conserved sequence encoding the integron site-specific recombinase to new sequences.

Comparison of the structure–activity relationships of the integron-associated recombination sites attI3 and attI1 reveals common features

“…The properties of the attI3 site identified in this study were very similar to those of the attI1 site (Recchia et al, 1994;Hansson et al, 1997;Collis et al, 1998;Gravel et al, 1998;Partridge et al, 2000). Four IntI3-binding sites were detected in gel shift assays and the positions of three determined directly using methylation interference.…”

“…Two attI1 sites are able to recombine (Hansson et al, 1997;Partridge et al, 2000), but the frequency is about 100-fold lower than for attI1 with 59-be events. To facilitate detection of these events with attI3, a conjugative plasmid that contains only the full-length attI3 site (pRMH961; Fig.…”

“…1a) further enhances recombination efficiency. The simple site is sufficient for recombination with a second complete attI1 site (Hansson et al, 1997;Partridge et al, 2000).…”

“…IntI recombinases also appear to differ from other tyrosine recombinases in that they catalyse only a single strand exchange which occurs either between the G and TT in the right hand core site (vertical arrow in Fig. 1) or between the AA and C on the complementary strand (Hansson et al, 1997;Stokes et al, 1997). By analogy with the XerCD system (Colloms et al, 1996), strand exchange on the complementary strand seems more likely.…”

“…1b). Only the attI1 site from class 1 integrons has been characterized (Recchia et al, 1994;Hansson et al, 1997;Collis et al, 1998;Gravel et al, 1998;Hall et al, 1999;Partridge et al, 2000). Detailed studies have revealed that attI1 includes four IntI-binding sites each beginning with GTTRRRY or variations thereof ( Fig.…”