The toxin–antitoxin cassettes as selecting forces ensuring accumulation and maintenance of cassette functions in chromosomal integrons

Integrons are bacterial adaptive devices containing promoterless gene cassettes that recombine into expression spot under stress conditions. Integrons come in two types, the small Mobile Integrons (MIs) well known for their pivotal role in multidrug resistance dissemination among most Gram-negative pathogens; and the large ones, called Sedentary Chromosomal Integrons (SCIs) are an almost inexhaustible source of cassettes for MIs. It remains unclear how a second order of selection operates on silent cassette of SCIs to maintain such large cassette arrays. Through inactivation of promoter-containing toxin–antitoxin (TA) cassettes, using multiplexed CRISPR base editing, we demonstrated a key role of these special cassettes as abortive systems that kill the cell when the cassette excision rate is too high. These results highlight the importance of toxin–antitoxin cassettes acting as selective forces ensuring cassette accumulation and maintenance in large integrons and provide insight into the evolution and success of integrons in bacterial genomes.