Single-site DNA methylation activates retron defence by enabling effector tetramerisation
Retrons are unique among bacterial defence systems in using DNA to sense phage infection. They achieve this by producing multicopy single-stranded DNA (msDNA) that monitors the activity of DNA-targeting phage proteins. Although we understand how extensive msDNA perturbations such as degradation activate retrons, activation by subtle chemical modifications remains enigmatic. Here, we used a structure-function approach to elucidate how single-site msDNA methylation activates Retron-Sen2. The cryo-EM structure of Retron-Sen2 features RcaT, the most prevalent retron effector, sequestered as an inactive dimer within a four-component assembly of previously undescribed architecture. Methylation by DNA adenine methyltransferase disrupts the RcaT-msDNA interaction and exposes a previously occluded interface that enables RcaT dimers to form tetramers, leading to retron disassembly. We show that RcaT dimer-dimer assembly is essential for its toxicity. To assess whether this is a conserved RcaT feature, we characterised additional homologues. RcaTs capable of tetramerisation or higher-order oligomerisation are toxic and form distinct phylogenetic clades from RcaTs that remain dimeric and are non-toxic. Together, our findings provide a paradigm for retron activation by single-site msDNA modifications and showcase how variation in an intrinsic protein property can diversify the toxicity of the same bacterial defence effector during evolution.