Bacterial retrons encode phage-defending tripartite toxin–antitoxin systems

Abstract

Retrons are genetic retroelements, commonly found in bacterial genomes and recently repurposed as genome editing tools. Their encoded reverse transcriptase (RT) produces a multi-copy single-stranded DNA (msDNA). Despite our understanding of their complex biosynthesis, the function of msDNAs and therefore, the physiological role of retrons has remained elusive. We establish that the retron-Sen2 in Salmonella Typhimurium encodes a toxin, which we have renamed as RcaT (Retron cold-anaerobic Toxin). RcaT is activated when msDNA biosynthesis is perturbed and its toxicity is higher at ambient temperatures or during anaerobiosis. The RT and msDNA form together the antitoxin unit, with the RT binding RcaT, and the msDNA enabling the antitoxin activity. Using another E. coli retron, we establish that this toxin/antitoxin function is conserved, and that RT-toxin interactions are cognate.Altogether, retrons constitute a novel family of tripartite toxin/antitoxin systems..

Discovery of phage determinants that confer sensitivity to bacterial immune systems

“…A common theme among retron-interacting proteins found in this and other studies, including T7 SSB, T5 A1, Rac prophage DNA exonuclease, E. coli DNA methylase (Bobonis et al, 2022) and RecBCD, is that they all have the capacity or predicted capacity to interact with DNA. It is possible that interaction of these proteins with the retrongenerated ssDNA forms part of the basis for their sensing by the retron system.…”

“…The retron RT reverse transcribes the non-coding RNA to form a chimeric RNA-DNA molecule which, together with the RT, has been hypothesized to sense phage infection. Defense by retrons involves cell suicide or growth arrest in response to phage infection (abortive infection), which is carried out by the associated effector protein (Bobonis et al, 2022; Millman et al, 2020). Based on phage escaper data, it was previously shown that retron Ec48 “guards” the cellular RecBCD complex, such that when phages inhibit RecBCD, the retron becomes active and leads the cell to growth arrest (Millman et al, 2020).…”

“…Based on phage escaper data, it was previously shown that retron Ec48 “guards” the cellular RecBCD complex, such that when phages inhibit RecBCD, the retron becomes active and leads the cell to growth arrest (Millman et al, 2020). Studies on another retron, St85, demonstrated that phage proteins that degrade or modify the retron reverse-transcribed DNA activate the toxicity of the retron effector (Bobonis et al, 2022). However, it is unknown how other retrons sense phages.…”

“…Selected retrons from the tree are marked, with the predicted phage-encoded activator denoted. The triggering mechanism for the St85 retron is reported based on a previous study (Bobonis et al, 2022). (D) Expression of a phage-encoded ssDNA binding protein (SSB) activates toxicity in a strain that contains the retron Eco8 system.…”

“…However, studies over the past few years have revealed a plethora of additional defense mechanisms commonly employed by bacteria. These include phage restriction by prokaryotic Argonaute proteins (Garb et al, 2021; Koopal et al, 2022; Kuzmenko et al, 2020; Zaremba et al, 2021), production of small molecules that block phage propagation (Bernheim et al, 2021; Kever et al, 2022; Kronheim et al, 2018), depletion of molecules essential for phage replication (Garb et al, 2021; Hsueh et al, 2022; Ofir et al, 2021; Tal et al, 2022), systems that use small molecule signaling to activate immune effectors (Cohen et al, 2019; Ofir et al, 2021; Tal et al, 2021; Whiteley et al, 2019), retrons that involve reverse transcription of non-coding RNAs (Bobonis et al, 2022; Gao et al, 2020; Millman et al, 2020), and more (Doron et al, 2018; Gao et al, 2020; Goldfarb et al, 2015; Johnson et al, 2022; Millman et al, 2022; Ofir et al, 2018; Rousset et al, 2022; Vassallo et al, 2022).…”