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

Abstract

Comparison of the structure-activity relationships of the integron-associated recombination sites attI3 and attI1 reveals common features Incorporation of gene cassettes into integrons occurs by IntI-mediated site-specific recombination between a 59-base element (59-be) site in the cassette and an attI site in the integron. While the 59-be sites share common features and are recognized by several different IntI recombinases, the sequences of attI sites are not obviously related and are preferentially recognized by the cognate IntI. To determine the features of attI sites that are required for recombination proficiency, the structure-activity relationships of a second attI site, the attI3 site from the class 3 integron, were examined. The attI3 site was confined to within a region consisting of 68 bp from the integron backbone and 15 bp from the adjacent cassette. This region includes four IntI3-binding sites, as assessed by gel shift and methylation interference studies. Two of the binding sites are inversely oriented and constitute a simple site that includes the recombination crossover point. The two additional binding sites appear to be directly oriented and one of them is essential for efficient recombination of the attI3 site with a 59-be, but not for recombination with a second full-length attI3 site, which occurs at 100-fold lower frequency. The fourth site enhances attI3 with 59-be recombination 10-fold. The finding that the organization and overall properties of attI3 are very similar to those of attI1 indicates that these features are likely to be common to all attI sites. INTRODUCTIONGene cassettes are small mobile elements that generally include a single gene and a recombination site known as a 59-be (59-base element) (Hall et al., 1991;Recchia & Hall, 1995. They are normally found in an integron in arrays of one, a few or many cassettes at a specific position which corresponds to the integron-associated attI recombination site (Stokes & Hall, 1989;Hall & Collis, 1995Hall, 2002). The attI site lies adjacent to the intI gene which produces the IntI site-specific recombinase responsible for the movement of gene cassettes. Incorporation of cassettes into an integron occurs by IntI-mediated recombination between the 59-be in a circular cassette and the attI site in the integron (Collis et al., 1993;Hall & Collis, 1995;Collis et al., 2002a). In contrast, excision of cassettes from an integron can occur via either recombination between two 59-be or between the attI site and a 59-be (Collis & Hall, 1992b;Bunny et al., 1995;Collis et al., 2002a;Drouin et al., 2002;Hansson et al., 2002).The IntI recombinases encoded by integrons form a distinct family within the tyrosine recombinase superfamily (Nunes-Düby et al., 1998) and contain an additional motif that appears to be a signature for IntI family members (Messier & Roy, 2001;Nield et al., 2001). Over 20 distinct IntI have been identified to date (Collis et al., 2002b), and in most cases, the intI gene is in close proximity to one or more identifiable gene c...

Novel integrons and gene cassettes from a Cascadian submarine gas-hydrate-bearing core

“…80 bases upstream of the predicted start of intI, a sequence was found possessing features found in both attI1 and attI3 (Fig. ) (Collis & Hall, ). These features included a simple site with a strong match to the consensus sequence for these regions and two regions comprising a direct repeat of a type seen in attI1 , attI3, and attI sites recorded from environmental samples and comprised likely IntI‐binding domains (Gravel et al ., ; Nield et al ., ; Collis & Hall, ).…”

Integron Involvement in Environmental Spread of Antibiotic Resistance

“…GCs are usually promoterless and require the Pc promoter for their expression as in an operon. The consequence of this system is that the last integrated cassette is the closest to the Pc promoter (Collis et al, 1993; Collis and Hall, 2004), leading to the highest level of expression in the integron.…”

Integrons and gene cassettes: hotspots of diversity in bacterial genomes

“…The features required for a functional attI site other than a short, simple site sequence at the cassette insertion point cannot be deduced from examination of the DNA sequence—see, for example, Refs. 13, 23, 25, and 28. The architecture of these sites has been examined experimentally only for class 1 5,29–31 and class 3, 28 both of which include a simple site consisting of inversely oriented IntI binding sites and two further IntI binding sites, one that is necessary for site activity and one that enhances it.…”