TAC–TIC, a high-throughput genetics method to identify triggers or blockers of bacterial toxin–antitoxin systems

Toxin–antitoxin systems (TAs) are abundant in bacterial chromosomes and can arrest growth under stress, but usually remain inactive. TAs have been increasingly implicated in halting the growth of infected bacteria from bacteriophages or foreign genetic elements1,2 to protect the population (abortive infection, Abi). The vast diversity and abundance of TAs and other Abi systems3 suggest they play an important immunity role, yet what allows them to sense attack remains largely enigmatic. Here, we describe a method called toxin activation–inhibition conjugation (TAC–TIC), which we used to identify gene products that trigger or block the toxicity of phage-defending tripartite retron-TAs4. TAC–TIC employs high-density arrayed mobilizable gene-overexpression libraries, which are transferred into cells carrying the full TA system or only its toxic component, on inducible vectors. The double-plasmid transconjugants are then pinned on inducer-containing agar plates and their colony fitness is quantified to identify gene products that trigger a TA to inhibit growth (TAC), or that block it from acting (TIC). TAC–TIC is optimized for the Singer ROTOR pinning robot, but can also be used with other robots or manual pinners, and allows screening tens of thousands of genes against any TA or Abi (with toxicity) within a week. Finally, we present a dual conjugation donor/cloning strain (Escherichia coli DATC), which accelerates the construction of TAC–TIC gene-donor libraries from phages, enabling the use of TAC–TIC for identifying TA triggers and antidefense mechanisms in phage genomes. This protocol describes toxin activation–inhibition conjugation (TAC–TIC), a reverse genetics screening approach that can be used to identify triggers or blockers of bacterial toxin–antitoxin or phage immunity systems.