Diversity of bacterial type II toxin-antitoxin systems: a comprehensive search and functional analysis of novel families.

Type II toxin-antitoxin (TA) systems are generally composed of two genes organized in an operon, encoding a labile antitoxin and a stable toxin. They were first discovered on plasmids where they contribute to plasmid stability by a phenomenon denoted as 'addiction', and subsequently in bacterial chromosomes. To discover novel families of antitoxins and toxins, we developed a bioinformatics approach based on the 'guilt by association' principle. Extensive experimental validation in Escherichia coli of predicted antitoxins and toxins increased significantly the number of validated systems and defined novel toxin and antitoxin families. Our data suggest that toxin families as well as antitoxin families originate from distinct ancestors that were assembled multiple times during evolution. Toxin and antitoxin families found on plasmids tend to be promiscuous and widespread, indicating that TA systems move through horizontal gene transfer. We propose that due to their addictive properties, TA systems are likely to be maintained in chromosomes even though they do not necessarily confer an advantage to their bacterial hosts. Therefore, addiction might play a major role in the evolutionary success of TA systems both on mobile genetic elements and in bacterial chromosomes.

New toxins homologous to ParE belonging to three-component toxin-antitoxin systems in Escherichia coli O157:H7.

Type II toxin-antitoxin (TA) systems are considered as protein pairs in which a specific toxin is associated with a specific antitoxin. We have identified a novel antitoxin family (paaA) that is associated with parE toxins. The paaA-parE gene pairs form an operon with a third component (paaR) encoding a transcriptional regulator. Two paralogous paaR-paaA-parE systems are found in E. coli O157:H7. Deletions of the paaA-parE pairs in O157:H7 allowed us to show that these systems are expressed in their natural host and that PaaA antitoxins specifically counteract toxicity of their associated ParE toxin. For the paaR2-paaA2-parE2 system, PaaR2 and Paa2-ParE2 complex are able to regulate the operon expression and both are necessary to ensure complete repression. The paaR2-paaA2-parE2 system mediates ClpXP-dependent post-segregational killing. The PaaR2 regulator appears to be essential for this function, most likely by maintaining an appropriate antitoxin : toxin ratio in steady-state conditions. Ectopic overexpression of ParE2 is bactericidal and is not resuscitated by PaaA2 expression. ParE2 colocalizes with the nucleoid, while it is diffusely distributed in the cytoplasm when PaaA2 is coexpressed. This indicates that ParE2 interacts with DNA-gyrase cycling on DNA and that coexpression of PaaA2 antitoxin sequesters ParE2 away from its target by protein-protein complex formation.