Toxins of toxin/antitoxin systems are inactivated primarily through promoter mutations

Abstract

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.

Strategies to Investigate Membrane Damage, Nucleoid Condensation, and RNase Activity of Bacterial Toxin–Antitoxin Systems

“…Conversely, cloning the Lpt coding sequence into a pET11 vector and subsequent transformation of E. coli C41 (DE3) pLysS cells allowed us to eliminate the toxic effect of the basal expression and to obtain the expected phenotype upon IPTG induction [20]. Remarkably, the toxic phenotype observed in E. coli C41 cells upon induction disappeared after few days from transformation with the recombinant pET plasmid, probably due to the selection of clones with mutations affecting the expression or activity of the toxin [26]. This phenomenon was not investigated further, but we managed to overcome the phenotype loss by using freshly transformed cells in each experiment.…”

Mechanisms of Tolerance and Resistance to Chlorhexidine in Clinical Strains of Klebsiella pneumoniae Producers of Carbapenemase: Role of New Type II Toxin-Antitoxin System, PemIK

“…This PemI/PemK TA system is composed of a 258 bp antitoxin gene (pemI) and a 333 bp toxin gene (pemK). To confirm that this system is a TA system, pemI/pemK and pemK genes alone were cloned into the overexpression vector pCA24N, widely used in the literature to overexpress TA systems [28,29], and transformed into the cured plasmid strain ST846-OXA48CA CP (i.e., lacking plasmids and therefore the plasmid that encodes the PemI/PemK TA system). The toxicity of this TA system was tested by growth curves overexpressing both pemI/pemK and pemK ( Figure 2).…”

Evaluating the Potential for Cross-Interactions of Antitoxins in Type II TA Systems

“…Some investigations have documented an attenuation of chromosomally-encoded TA system toxicity. This has arisen either through sequence changes resulting in lower affinity for interaction with the cellular target [68][69][70][71][72] or by attenuation of expression via changes in the promoter regions [73][74][75]. A loss of function, termed "degeneration", has also been observed for some systems, such as a chromosomal CcdAB system in E. coli [62].…”