The DarTG toxin-antitoxin system provides phage defence by ADP-ribosylating viral DNA

Abstract

Toxin-antitoxin (TA) systems are broadly distributed, yet poorly conserved, genetic elements whose biological functions are unclear and controversial. Some TA systems may provide bacteria with immunity to infection by their ubiquitous viral predators, the bacteriophage. To identify such TA systems, we searched bioinformatically for those frequently encoded near known phage defense genes in bacterial genomes. This search identified homologs of DarTG, a recently discovered family of TA systems whose biological functions and natural activating conditions were unclear. Representatives from two different subfamilies, DarTG1 and DarTG2, strongly protected E. coli MG1655 against different phages. We demonstrate that for each system, infection with either RB69 or T5 phage, respectively, triggers release of the DarT toxin, a DNA ADPribosyltransferase, that then modifies viral DNA and prevents replication, thereby blocking the production of mature virions. Further, we isolated phages that have evolved to overcome DarTG defense either through mutations to their DNA polymerase or to an anti-DarT factor, gp61.2, encoded by many T-even phages. Collectively, our results indicate that phage defense may be a common function for TA systems and reveal the mechanism by which DarTG systems inhibit phage infection.

Discovery of phage determinants that confer sensitivity to bacterial immune systems

“…Such mutations allowed phages to escape the systems Lamassu, Hachiman, ietAS , Retron-Eco8, ShosTA, Borvo, and some AVAST systems (Figure 3, Figure 6). Previous studies on the anti-phage defense systems AbiK (Bouchard and Moineau, 2004; Wang et al, 2011), AbiQ (Samson et al, 2013b), ApbA/B (Yasui et al, 2014), DarTG (LeRoux et al, 2022), Tin (Mosig et al, 1997), and Nhi (Bari et al, 2022) have also reported phage escape via mutation in one of these core replication proteins. Thus, diverse defense systems have evolved to sense or target the core determinants of phage replication.…”

“…Some of the phage mutants we isolated may not escape by mutating the component identified by the defense system, but rather evade immunity via other means. It is possible that some mutations could allow the phage to overcome the effect of the system, as proposed, for example, in the case of the DarTG system, which installs ADP-ribose on phage DNA in response to infection (LeRoux et al, 2022). Phages mutated in their DNA polymerase could tolerate the effect of the DarTG system, and it was suggested that these mutations allowed the polymerase to replicate DNA despite the DNA modifications made by the DarTG system (LeRoux et al, 2022).…”

“…It is possible that some mutations could allow the phage to overcome the effect of the system, as proposed, for example, in the case of the DarTG system, which installs ADP-ribose on phage DNA in response to infection (LeRoux et al, 2022). Phages mutated in their DNA polymerase could tolerate the effect of the DarTG system, and it was suggested that these mutations allowed the polymerase to replicate DNA despite the DNA modifications made by the DarTG system (LeRoux et al, 2022). Another possibility is that the mutation in the phage has a “gain of function” effect, generating a protein that actively inhibits the defense system.…”

“…Another possibility is that the mutation in the phage has a “gain of function” effect, generating a protein that actively inhibits the defense system. Such mutants were described in two recent studies in which mutations switched a phage anti-CBASS or anti-DarTG protein from an inactive to an active form, enabling phages to overcome the CBASS and DarTG systems, respectively (Huiting et al, 2022; LeRoux et al, 2022). Another study has identified phage mutations that allowed phages to overcome the ToxIN system by evolving a non-coding RNA acting as an antitoxin to block the ToxN protein (Blower et al, 2012).…”

Cryo-EM structure of the RADAR supramolecular anti-phage defense complex

“…While most bacteria carry at least one restriction enzyme and about half of them encode CRISPR-Cas as part of their immune arsenal, other defense systems are sparsely distributed in microbial genomes (Payne et al, 2021; Tesson et al, 2022). Over sixty anti-phage defense systems have been discovered in the past few years (Doron et al, 2018; Gao et al, 2020; LeRoux and Laub, 2022; Millman et al, 2022; Rousset et al, 2022) and while the mechanisms of action of a minority of them have been determined (Bernheim et al, 2021; Garb et al, 2021; Johnson et al, 2022; LeRoux et al, 2022; Millman et al, 2020; Tal et al, 2022; Whiteley et al, 2019), the vast majority remain unexplored. It is estimated that an average bacterial genome contains more than five defense systems, with some bacteria encoding as many as 57 such systems (Tesson et al, 2022).…”