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Replicative resolution of integron cassette insertion.

ABSTRACT: Site-specific recombination catalyzed by tyrosine recombinases follows a common pathway consisting of two consecutive strand exchanges. The first strand exchange generates a Holliday junction (HJ), which is resolved by a second strand exchange. In integrons, attC sites recombine as folded single-stranded substrates. Only one of the two attC site strands, the bottom one, is efficiently bound and cleaved by the integrase during the insertion of gene cassettes at the double-stranded attI site. Due to the asymmetry of this complex, a second strand exchange on the attC bottom strand (bs) would form linearized abortive recombination products. We had proposed that HJ resolution would rely on an uncharacterized mechanism, probably replication. Using an attC site carried on a plasmid with each strand specifically tagged, we followed the destiny of each strand after recombination. We demonstrated that only one strand, the one carrying the attC bs, is exchanged. Furthermore, we show that the recombination products contain the attC site bs and its entire de novo synthesized complementary strand. Therefore, we demonstrate the replicative resolution of single-strand recombination in integrons and rule out the involvement of a second strand exchange of any kind in the attC×attI reaction.

SUBMITTER: Loot C

PROVIDER: S-EPMC3458562 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Replicative resolution of integron cassette insertion.

Loot Céline C Ducos-Galand Magaly M Escudero José Antonio JA Bouvier Marie M Mazel Didier D

Nucleic acids research 20120626 17

Site-specific recombination catalyzed by tyrosine recombinases follows a common pathway consisting of two consecutive strand exchanges. The first strand exchange generates a Holliday junction (HJ), which is resolved by a second strand exchange. In integrons, attC sites recombine as folded single-stranded substrates. Only one of the two attC site strands, the bottom one, is efficiently bound and cleaved by the integrase during the insertion of gene cassettes at the double-stranded attI site. Due ...[more]

PMID: 22740653

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Project description:BACKGROUND:Integrons 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. RESULTS:We 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. CONCLUSIONS:This 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.

Project description:Integrons are bacterial genetic elements able to capture and express genes contained within mobile gene cassettes. Gene cassettes are expressed via a Pc promoter and can be excised from or integrated into the integron by integrase IntI. Although the mechanisms of gene cassette integration and excision are well known, the kinetics and modes of gene cassette shuffling leading to new gene cassette arrays remain puzzling. It has been proposed that under antibiotic selective pressure, IntI-mediated rearrangements can generate integron variants in which a weakly expressed gene cassette moves closer to Pc, thus leading to higher-level resistance. To test this hypothesis, we used an integron with four gene cassettes, intI1-aac(6')-Ib-dfrA15-aadA1-catB9, and applied selective pressure with chloramphenicol, resistance to which is encoded by catB9. Experiments were performed with three different Pc variants corresponding to three IntI1 variants. All three integrases, even when not overexpressed, were able to bring catB9 closer to Pc via excision of the dfrA15 and aadA1 gene cassettes, allowing their host bacteria to adapt to antibiotic pressure and to grow at high chloramphenicol concentrations. Integrase IntI1(R32_H39), reported to have the highest recombination activity, was able, when overexpressed, to trigger multiple gene cassette rearrangements. Although we observed a wide variety of rearrangements with catB9 moving closer to Pc and leading to higher chloramphenicol resistance, "cut-and-paste" relocalization of catB9 to the first position was not detected. Our results suggest that gene cassette rearrangements via excision are probably less cost-effective than excision and integration of a distal gene cassette closer to Pc.Integrons are bacterial genetic elements able to capture and express gene cassettes. Gene cassettes are expressed via a Pc promoter; the closer they are to Pc, the more strongly they are expressed. Gene cassettes can be excised from or integrated into the integron by integrase IntI. The kinetics and modes of gene cassette shuffling, leading to new gene cassette arrays remain puzzling. We used an integron with 4 antibiotic resistance gene cassettes and applied selective pressure with the antibiotic for which resistance was encoded by cassette 4. All IntI variants were able to bring cassette 4 closer to Pc. Rearrangements occur via excision of the previous gene cassettes instead of cut-and-paste relocalization of the fourth gene cassette.

Dataset Information

Replicative resolution of integron cassette insertion.

Publications

Replicative resolution of integron cassette insertion.

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