Profiling the intragenic toxicity determinants of toxin–antitoxin systems: revisiting hok/Sok regulation

Type I toxin–antitoxin systems (T1TAs) are extremely potent bacterial killing systems difficult to characterize using classical approaches. To assess the killing capability of type I toxins and to identify mutations suppressing the toxin expression or activity, we previously developed the FASTBAC-Seq (Functional AnalysiS of Toxin–Antitoxin Systems in BACteria by Deep Sequencing) method in Helicobacter pylori. This method combines a life and death selection with deep sequencing. Here, we adapted and improved our method to investigate T1TAs in the model organism Escherichia coli. As a proof of concept, we revisited the regulation of the plasmidic hok/Sok T1TA system. We revealed the death-inducing phenotype of the Hok toxin when it is expressed from the chromosome in the absence of the antitoxin and recovered previously described intragenic toxicity determinants of this system. We identified nucleotides that are essential for the transcription, translation or activity of Hok. We also discovered single-nucleotide substitutions leading to structural changes affecting either the translation or the stability of the hok mRNA. Overall, we provide the community with an easy-to-use approach to widely characterize TA systems from diverse types and bacteria.