Aims Given the extreme toxicity of some of the toxins of toxin-antitoxin (TA) systems, we were curious how the cell silences toxins, if the antitoxin is inactivated or independent toxins are obtained via horizontal gene transfer. Methods and Results Growth curves of Escherichia coli K12 BW25113 harbouring plasmid pCA24N to produce RalR, MqsR, GhoT or Hha toxins, showed toxin inactivation after 3 h. Sequencing plasmids from these cultures revealed toxin inactivation occurred primarily due to consistent deletions in the promoter. The lack of mutation in the structural genes was corroborated by a bioinformatics analysis of 1000 E. coli genomes which showed both conservation and little variability in the four toxin genes. For those strains that lacked a mutation in the plasmid, single nucleotide polymorphism analysis was performed to identify that chromosomal mutations iraM and mhpR inactivate the toxins GhoT and MqsR/GhoT respectively. Conclusion We find that the RalR (type I), MqsR (type II), GhoT (type V) and Hha (type VII) toxins are inactivated primarily by a mutation that inactivates the toxin promoter or via the chromosomal mutations iraM and mhpR. Significance and Impact of the Study This study demonstrates toxins of TA systems may be inactivated by mutations that primarily affect the toxin gene promoter instead of the toxin structural gene.