Characterization of the phd-doc and ccd Toxin-Antitoxin Cassettes from Vibrio Superintegrons

Toxin-antitoxin (TA) systems have been reported in the genomes of most bacterial species, and their role when located on the chromosome is still debated. TA systems are particularly abundant in the massive cassette arrays associated with chromosomal superintegrons (SI). Here, we describe the characterization of two superintegron cassettes encoding putative TA systems. The first is the phd-docSI system identified in Vibrio cholerae N16961. We determined its distribution in 36 V. cholerae strains and among five V. metschnikovii strains. We show that this cassette, which is in position 72 of the V. cholerae N16961 cassette array, is functional, carries its own promoter, and is expressed from this location. Interestingly, the phd-docSI system is unable to control its own expression, most likely due to the absence of any DNA-binding domain on the antitoxin. In addition, this SI system is able to cross talk with the canonical P1 phage system. The second cassette that we characterized is the ccdVfi cassette found in the V. fischeri superintegron. We demonstrate that CcdBVfi targets DNA-gyrase, as the canonical CcBF toxin, and that ccdVfi regulates its expression in a fashion similar to the ccdF operon of the conjugative plasmid F. We also establish that this cassette is functional and expressed in its chromosomal context in V. fischeri CIP 103206T. We tested its functional interactions with the ccdABF system and found that CcdAVfi is specific for its associated CcdBVfi and cannot prevent CcdBF toxicity. Based on these results, we discuss the possible biological functions of these TA systems in superintegrons.