The prevalence of toxin/antitoxin (TA) systems in almost all genomes suggests they evolve rapidly. Here we show that an antitoxin from a type V system (GhoS, an endoribonuclease specific for the mRNA of the toxin GhoT) can be converted into a novel toxin (ArT) simply by adding two mutations. In contrast to GhoS, which increases growth, the new toxin ArT decreases growth dramatically in Escherichia coli. Transmission electron microscopy analysis revealed that the nucleoid in ArT-producing cells is concentrated and appears hollow. Whole-transcriptome profiling revealed ArT cleaves 50 additional transcripts, which shows that the endoribonuclease activity of GhoS has been broadened as it was converted to ArT. Furthermore, we evolved an antitoxin for the new toxin ArT from two unrelated antitoxin templates, the protein-based antitoxin MqsA and RNA-based antitoxin ToxI, and showed that the evolved MqsA and ToxI variants are able to counteract the toxicity of ArT. In addition, the de novo TA system was found to increase persistence, a phenotype commonly associated with TA systems. Therefore, toxins and antitoxins from disparate systems can be interconverted.