Abstract
BackgroundIntegrons are genetic elements able to integrate and disseminate genes as cassettes by a site-specific recombination mechanism. These elements contain a gene coding for an integrase that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a gene cassette. Integron integrases (IntIs) bind specifically to the bottom strand of attC sites. The extrahelical bases resulting from folding of attC bottom strands are important for the recognition by integrases. These enzymes are directly involved in the accumulation and formation of new cassette arrangements in the variable region of integrons. Thus, it is important to better understand interactions between IntIs and their substrates.ResultsWe compared the ability of five IntIs to carry out excision of several cassettes flanked by different attC sites. The results showed that for most cassettes, IntI1 was the most active integrase. However, IntI2*179E and SonIntIA could easily excise cassettes containing the attCdfrA1 site located upstream, whereas IntI1 and IntI3 had only a weak excision activity for these cassettes. Analysis of the secondary structure adopted by the bottom strand of attCdfrA1 has shown that the identity of the extrahelical bases and the distance between them (A-N7-8-C) differ from those of attCs contained in the cassettes most easily excisable by IntI1 (T-N6-G). We used the attCdfrA1 site upstream of the sat2 gene cassette as a template and varied the identity and spacing between the extrahelical bases in order to determine how these modifications influence the ability of IntI1, IntI2*179E, IntI3 and SonIntIA to excise cassettes. Our results show that IntI1 is more efficient in cassette excision using T-N6-G or T-N6-C attCs while IntI3 recognizes only a limited range of attCs. IntI2*179E and SonIntIA are more tolerant of changes to the identity and spacing of extrahelical bases.ConclusionsThis study provides new insights into the factors that influence the efficiency of cassette excision by integron integrases. It also suggests that IntI2 and SonIntIA have an evolutionary path that is different from IntI1 and IntI3, in their ability to recognize and excise cassettes.
Highlights
Integrons are genetic elements able to integrate and disseminate genes as cassettes by a sitespecific recombination mechanism
Comparative excision activities of IntI1, IntI3, IntI2*179E, SonIntIA and VchIntIA on cassettes containing different attC sites In order to determine why some cassettes are excised by several integron integrase (IntI) while others are poorly excised, we compared the efficiency of five IntIs in excision of cassettes flanked by different attI and attC sites
Nineteen clones (pLQ423 to pLQ431 and pLQ437 to pLQ446 (Table 1) containing various resistance gene cassettes cloned into pACYC184 were used to compare the recombination activity of IntI1, IntI3, IntI2*179E, SonIntIA and VchIntIA by qualitative excision tests (QL-ETs)
Summary
Integrons are genetic elements able to integrate and disseminate genes as cassettes by a sitespecific recombination mechanism. These elements contain a gene coding for an integrase that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a gene cassette. The extrahelical bases resulting from folding of attC bottom strands are important for the recognition by integrases These enzymes are directly involved in the accumulation and formation of new cassette arrangements in the variable region of integrons. IntIs can share as little as 35% sequence identity, indicating a long evolutionary history for these enzymes Their catalytic domain is similar to that of other members of the tyrosine recombinase family and contains the conserved residues: Arg146-Lys171-His277-Arg280-His/Trp303 and the nucleophilic tyrosine, Tyr312 (coordinates are those of IntI1). Integration of cassettes occurs preferentially by recombination of the attC site in a closed-circular cassette with the attI site of an integron [3] while excision of a cassette, generating a circular form, occurs preferentially by recombination between two attC sites, one of them associated with the upstream cassette [4]
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