Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase

Abstract

Deletion of individual antibiotic resistance genes found within the variable region of integrons is demonstrated. Evidence for gene duplications and rearrangements resulting from the insertion of gene units at new locations is also presented. Deletion, duplication, and rearrangement occur only in the presence of the integron-encoded DNA integrase. These events are precise and involve loss or gain of one or more complete insert units or gene cassettes. This confirms the recent definition of gene cassettes as consisting of the gene coding sequences, all except the last 7 bases of the 59-base element found at the 3' end of the gene, and the core site located 5' to the gene (Hall et al., Mol. Microbiol. 5:1941-1959, 1991 and demonstrates that individual gene cassettes are functional units which can be independently mobilized. Both deletions and duplications can be generated by integrase-mediated cointegrate formation followed by integrase-mediated resolution involving a different pair of sites. However, deletion occurs 10 times more frequently than duplication, and we propose that the majority of deletion events are likely to involve integrase-dependent excision of the gene unit to generate a circular gene cassette. The implications of these findings in understanding the evolution of integrons and the spread of antibiotic resistance genes in bacterial populations is discussed.

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

“…The IntI gene and the cassette-borne genes are divergently transcribed from opposite DNA strands. The integrase, IntI, of an integron is the only protein known to be essential for movement of its cassettes (Martinez and de la Cruz, 1988;Collis and Hall, 1992a). There are potentially three types of integrons encoding integrases, which are about 50% identical by amino acid sequence (Sundströ m et Arakawa et al, 1995;K.…”

“…The targeting was demonstrated with circular cassettes as recombination substrates (Collis et al, 1993;Hall and Collis, 1995). These circles, which are intermediates in the transfer of cassettes, are formed by intramolecular recombination between the flank sites of a cassette (Collis and Hall, 1992a;1992b). Cassette transfer also occurs by formation of plasmid fusions (co-integrates) that are subsequently resolved by recombination at other integron sites than those used in the fusion step (Martinez and de la Cruz, 1988;Recchia et al, 1994).…”

Gene cassettes: a new class of mobile element

“…However, in certain integrons a duplication of 19 bp of the 5'-CS sequence alters the sequence such that translation can potentially be initiated within the 5'-CS at an ATG that is preceded by an RBS leading to a short N-terminal extension of the protein encoded by the first cassette (Wohlleben et al, 1991), and the product of the aacC7 gene has an N-terminal sequence that indicates that translation commences at this position. However, the aacC7 gene is also expressed when the cassette is located downstream in a cassette array, indicating that a suitable initiation site is also present within the cassette (Collis & Hall, 1992a). Furthermore, the level of expression of the aacA4 and aadA2 genes, measured by the level of antibiotic resistance conferred, does not appear to differ when the cassette abuts a 5'-CS that does or does not contain the 19 bp duplication (C. M. Collis & R. M. Hall, unpublished observations).…”

“…In other experimental systems this preference has not been observed. Rather, events involving two 59-base element recombination sites have generally predominated (Martinez & de la Cruz, 1990;Hall e t al., 1991 ;Collis & Hall, 1992a). While this type of event is clearly important to effect the excision of any cassette that is not located in the first position (i.e.…”

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

“…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 cassettes, indicating that it is indeed part of an integron.…”