Comprehensive Functional Analysis of the 18 Vibrio cholerae N16961 Toxin-Antitoxin Systems Substantiates Their Role in Stabilizing the Superintegron.

UNLABELLED: The role of chromosomal toxin-antitoxin (TA) systems, which are ubiquitous within the genomes of free-living bacteria, is still debated. We have scanned the Vibrio cholerae N16961 genome for class 2 TA genes and identified 18 gene pair candidates. Interestingly, all but one are located in the chromosome 2 superintegron (SI). The single TA found outside the SI is located on chromosome 1 and is related to the well-characterized HipAB family, which is known to play a role in antibiotic persistence. We investigated this clustering within the SI and its possible biological consequences by performing a comprehensive functional analysis on all of the putative TA systems. We demonstrate that the 18 TAs identified encode functional toxins and that their cognate antitoxins are able to neutralize their deleterious effects when expressed in Escherichia coli. In addition, we reveal that the 17 predicted TA systems of the SI are transcribed and expressed in their native context from their own promoters, a situation rarely found in integron cassettes. We tested the possibility of interactions between noncognate pairs of all toxins and antitoxins and found no cross-interaction between any of the different TAs. Although these observations do not exclude other roles, they clearly strengthen the role of TA systems in stabilizing the massive SI cassette array of V. cholerae. IMPORTANCE: The chromosomal toxin-antitoxin systems have been shown to play various, sometimes contradictory roles, ranging from genomic stabilization to bacterial survival via persistence. Determining the interactions between TA systems hosted within the same bacteria is essential to understand the hierarchy between these different roles. We identify here the full set of class 2 TAs carried in the Vibrio cholerae N16961 genome and found they are all, with a single exception, located in the chromosome 2 superintegron. Their characterization, in terms of functionality, expression, and possible cross-interactions, supports their main role as being the stabilization of the 176-cassette-long array of the superintegron but does not exclude dual roles, such as stress response elements, persistence, and bacteriophage defense through abortive infection mechanisms.