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

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.