Diversity of Class 1 Integron Gene Cassette Rearrangements Selected under Antibiotic Pressure

Abstract

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.

Importance: 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.

FIG 1

Construction of integrons 1S and 2S. Integrons 1S and 2S were obtained by assembly PCR. To construct integron 1S, fragment B+C (2.2 kb) was first obtained and then coupled with fragment A. To construct integron 2S, fragments D and E were fused. Integron 1S was 3.9 kb, and integron 2S 1.9 kb. The primers used for assembly are represented by dotted lines and indicated by numbers (see Table 1S in the supplemental material), and the primers used for catB9 detection are represented by black arrows (Fwd is located in pSU38ΔtotlacZ).

FIG 2

Example of multiple gene cassette rearrangements observed under Cm selection pressure, starting with DH5α plus p1S plus pBad-intI1*_{R32_H39}. Integron 1 represents the initial integron intI1-aac(6′)-Ib-dfrA15-aadA1-catB9; the intI1 gene is represented by a white arrow, the aac(6′)-Ib gene cassette by a gray arrow, the dfrA15 gene cassette by a cross-hatched arrow, the aadA1 gene cassette by a stippled arrow, and catB9 gene cassette by a black arrow. For example, integron 2 harbors the gene cassette catB9 (black arrow), and integron 3 harbors the two gene cassettes aac(6′)-Ib and catB9 (gray arrow followed by a black arrow). In this assay, after gene cassette rearrangement, all colonies harbored at least 2 different integrons. The different rearrangements are numbered arbitrarily from 3 to 16. The letters (A to O) correspond to integron combinations observed within a given strain.