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Abstract

Retrons are prokaryotic genetic elements that contain reverse transcriptases (RTs) and produce small multi-copy single-stranded DNA (msDNA). First identified 40 years ago, their function remained enigmatic until recently, when it was established that they defend against phages through abortive infection (Abi) (Bobonis et al., 2020). They form tripartite toxin-antitoxin (TA) systems where the toxin effector (RcaT) is inhibited by an antitoxin complex composed of a cognate RT bound to its msDNA (Bobonis et al., 2022). RcaT toxicity can be triggered by proteins that perturb or modify the msDNA, which are often carried by phages. At the same time, phages carry anti-retron mechanisms that block the activity of RcaT to prevent Abi, thereby bypassing bacterial defence.

Despite these advances, what fraction and which components of the phage genome are sensed by retrons or are used by the phage to deactivate retrons remain largely unknown. To address this, I combined a recently developed high-throughput reverse genetics method called TAC-TIC (Toxin Activation-Inhibition Conjugation) with the construction of single-gene genome-wide overexpression libraries, to systematically screen two model phages, T5 and P1vir, for retron triggers and blockers. Applying TAC-TIC across different retrons, I found that both phages encode multiple potential triggers and blockers within their genome, independent of infection outcome. This challenges the current paradigm of phage defence, which assumes that infection success is determined by the presence of a single key trigger and the absence of a blocker in the phage genome. For example, the essential DNA exonuclease of phage T5 triggers multiple retrons by degrading msDNA during phage infection, whereas gain-of-function mutations in the T5.035 phosphatase enable the phage to evade retron defence. The presence of multiple blockers in its genome is not enough for T5 to circumvent retron defence, and abolishing the DNA exonuclease activity is not enough to silence retron activation. Finally, by expanding TAC-TIC screening beyond model phages, I helped to reveal an exquisite diversity and abundance of RcaT retron blockers in environmental viruses. In toto, I propose that bacterial defence is dependent on the dynamic equilibrium between the ability of retrons to detect phage-encoded triggers and the strength of the multitude of phage-encoded retron blockers that bypass immunity.