The DarTG toxin-antitoxin system provides phage defense 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 TA systems that protect E. coli MG1655 against phage, we searched for those frequently encoded near known phage defense genes in bacterial genomes. Two of the systems tested provide strong protection against phage infection and are homologs of DarTG, a recently discovered family of TA systems whose biological functions and natural activating conditions were unclear. We demonstrate that phage infection triggers the release of DarT toxin, a DNA ADP-ribosyltransferase, to modify viral DNA and prevent replication, thereby blocking the production of mature virions. Phages can evolve 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.

Mapping the landscape of anti-phage defense mechanisms in the E. coli pangenome

“…TA systems are extremely prevalent in bacterial genomes and MGEs, but their functions remain poorly understood 28 . A handful of TA systems have been found to provide anti-phage defense through an Abi mechanism 29,30,30,31 . Our selection yielded four different systems that were recognizable as TA-like in nature.…”

The evolution of a counter-defense mechanism in a virus constrains its host range

“…TifA is analogous to the co-operonic gene dmd , which encodes a direct inhibitor of RnlA, the toxin of the rnlAB system in E. coli . Additionally, the nearby and conserved gene 61.2 (now adfA ) was recently found to overcome the type IV TA system darTG (LeRoux et al, 2021). Thus, this region of the T4 genome may be an anti-TA island, similar to the anti-RM and anti-CRISPR islands found in other phages (Pinilla-Redondo et al, 2020).…”

“…pAH150- toxIN(K55A) was then constructed via round-the-horn PCR of pAH150- toxIN with SS-29/30. CRIM insertion of these plasmids into the attB ′″ locus of E. coli MG1655 was then performed as previously described (LeRoux et al, 2021) using the pINT-ts helper plasmid. Single insertions were confirmed by PCR and Sanger sequencing.…”

“…Consequently, RnlAB only protects E. coli against T4 phage lacking dmd (gene 61.5 ). More recently, T-even phages including T4 were shown to encode an inhibitor (AdfA, encoded by gene 61.2 in T4) of DarT toxins (LeRoux et al, 2021). In Pectobacterium atrosepticum , the type III TA system toxIN, involving an endoribonuclease toxin and RNA antitoxin, protects against infection by several phages, including <ΔTE and <ΔA2.…”

Beyond protein modification: the rise of non-canonical ADP-ribosylation

“…The observation that in pathogenic E. coli and cyanobacteria DarTG is often found to be inserted in an operon structure containing a type I restriction modification system suggested a link of DarTG to antiphage response and immunity [ 75 , 76 ]. Indeed, recently a role of DarT in antiphage defence was uncovered, where DarT is thought to modify invading viral DNA, thereby preventing its replication and consequently the production of virions, as a mechanism to control phage infections [ 82 ].…”