Quantifying the adaptive power of the mobile integron

Mobile integrons are widespread and clinically relevant drivers of antibiotic resistance. Acting as genetic platforms, they allow bacteria to capture, express and re-order mobile antibiotic resistance gene cassettes, whose expression levels depend on their distance from the promoter, located at the integration site. Cassettes can be integrated and excised by the integrase enzyme, produced by the bacteria in time of stress. It has been hypothesized that integrons allow bacteria to adapt quickly to changing antibiotic pressures by reshuffling the cassette order, and therefore their resistance levels, such that integrons provide ‘adaptation on demand’. To test this ‘adaptation on demand’ hypothesis, I used a custom three-cassette integron system transformed in both E. coli and P. aeruginosa. I first investigated the effect of cassette position on cassette expression at the phenotypic, transcriptional and translational level and confirmed a transcriptional origin of the cassette expression gradient, which was either strengthened or reduced by additional translational effects. I then combined experimental evolution and whole genome sequencing to quantify the evolvability benefits of cassette shuffling against increasing concentrations of antibiotics. By comparing strains with and without a functional integrase and focusing on two different cassettes and antibiotics, I observed striking differences in population survival rates and evolutionary pathways depending on the cassette and on the antibiotic. On one hand, with the antibiotic gentamicin and the aadB cassette, I saw an increase in evolvability and identified extensive cassettes rearrangements as well as cassette duplications in the populations with a functional integrase. On the other hand, with piperacillin and blaVEB-1, integrase activity was linked with decreased evolvability and a much lower rearrangement frequency, but I observed unexpected integron-mediated recombinations in the plasmid conjugation machinery. These results highlight the key impact of antibiotics and cassette characteristics on the potential integrase shuffling benefits and provide novel insights on the dynamics controlling cassettes and integrase prevalence in the environment. In my experiments, high cassette mobility was necessary to compensate for the high fitness cost of integrase expression, or the integrase was a burden for bacteria survival. Moreover, I showed integrase activity was not limited to cassette shuffling or cassette acquisition but may also promote cassette duplication and beneficial off-target recombination, with far-reaching consequences on plasmid and antibiotic resistance evolution.