Belt and braces: Two escape ways to maintain the cassette reservoir of large chromosomal integrons.

Integrons are adaptive devices that capture, stockpile, shuffle and express gene cassettes thereby sampling combinatorial phenotypic diversity. Some integrons called sedentary chromosomal integrons (SCIs) can be massive structures containing hundreds of cassettes. Since most of these cassettes are non-expressed, it is not clear how they remain stable over long evolutionary timescales. Recently, it was found that the experimental inversion of the SCI of Vibrio cholerae led to a dramatic increase of the cassette excision rate associated with a fitness defect. Here, we question the evolutionary sustainability of this apparently counter selected genetic context. Through experimental evolution, we find that the integrase is rapidly inactivated and that the inverted SCI can recover its original orientation by homologous recombination between two insertion sequences (ISs) present in the array. These two outcomes of SCI inversion restore the normal growth and prevent the loss of cassettes, enabling SCIs to retain their roles as reservoirs of functions. These results illustrate a nice interplay between gene orientation, genome rearrangement, bacterial fitness and demonstrate how integrons can benefit from their embedded ISs. Author summary: Bacteria have a great capacity to adapt to a wide range of stresses, such as the presence of antibiotics. The molecular mechanisms leading to this adaptation very often result in antimicrobial resistance phenomena responsible for the frequent failure of antibiotic treatments. Integrons are genetic systems that are largely involved in these adaptive responses to stress. They can carry up to 300 cassettes encoding different adaptive functions. It is therefore important to understand how such a large number of cassettes can be maintained during evolution. Here, we elucidate the question of integron maintenance by showing that bacteria can use two distinct escape ways to counter a too high cassette excision rate that could lead to an irreversible loss of functions. Firstly, by selecting mutated inactive integrases and secondly by selecting rearrangements within the integron that place the cassettes in a configuration where they become weakly excised. The presence of homopolymers in the integrase gene, as hotspots for replication sliding, favors the first escape solution, while the second is made possible by the presence of repeated insertion sequences integrated into the cassettes and allowing homologous recombination events. This work reveals the full power of the integron system maintaining its adaptive cassette reservoir. [ABSTRACT FROM AUTHOR]