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.

Cassette recombination dynamics within chromosomal integrons are regulated by toxin-antitoxin systems.

Integrons are adaptive bacterial devices that rearrange promoter-less gene cassettes into variable ordered arrays under stress conditions, thereby sampling combinatorial phenotypic diversity. Chromosomal integrons often carry hundreds of silent gene cassettes, with integrase-mediated recombination leading to rampant DNA excision and integration, posing a potential threat to genome integrity. How this activity is regulated and controlled, particularly through selective pressures, to maintain such large cassette arrays is unknown. Here, we show a key role of promoter-containing toxin-antitoxin (TA) cassettes as systems that kill the cell when the overall cassette excision rate is too high. These results highlight the importance of TA cassettes regulating the cassette recombination dynamics and provide insight into the evolution and success of integrons in bacterial genomes.

Integron cassettes integrate into bacterial genomes via widespread non-classical attG sites.

Integrons are genetic elements involved in bacterial adaptation which capture, shuffle and express genes encoding adaptive functions embedded in cassettes. These events are governed by the integron integrase through site-specific recombination between attC and attI integron sites. Using computational and molecular genetic approaches, here we demonstrate that the integrase also catalyses cassette integration into bacterial genomes outside of its known att sites. Once integrated, these cassettes can be expressed if located near bacterial promoters and can be excised at the integration point or outside, inducing chromosomal modifications in the latter case. Analysis of more than 5 × 105 independent integration events revealed a very large genomic integration landscape. We identified consensus recombination sequences, named attG sites, which differ greatly in sequence and structure from classical att sites. These results unveil an alternative route for dissemination of adaptive functions in bacteria and expand the role of integrons in bacterial evolution.

Unbridled Integrons: A Matter of Host Factors.

Integrons are powerful recombination systems found in bacteria, which act as platforms capable of capturing, stockpiling, excising and reordering mobile elements called cassettes. These dynamic genetic machineries confer a very high potential of adaptation to their host and have quickly found themselves at the forefront of antibiotic resistance, allowing for the quick emergence of multi-resistant phenotypes in a wide range of bacterial species. Part of the success of the integron is explained by its ability to integrate various environmental and biological signals in order to allow the host to respond to these optimally. In this review, we highlight the substantial interconnectivity that exists between integrons and their hosts and its importance to face changing environments. We list the factors influencing the expression of the cassettes, the expression of the integrase, and the various recombination reactions catalyzed by the integrase. The combination of all these host factors allows for a very tight regulation of the system at the cost of a limited ability to spread by horizontal gene transfer and function in remotely related hosts. Hence, we underline the important consequences these factors have on the evolution of integrons. Indeed, we propose that sedentary chromosomal integrons that were less connected or connected via more universal factors are those that have been more successful upon mobilization in mobile genetic structures, in contrast to those that were connected to species-specific host factors. Thus, the level of specificity of the involved host factors network may have been decisive for the transition from chromosomal integrons to the mobile integrons, which are now widespread. As such, integrons represent a perfect example of the conflicting relationship between the ability to control a biological system and its potential for transferability.