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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, bacteriophages. To identify such TA systems, we searched bioinformatically for those frequently encoded near known phage defence genes in bacterial genomes. This search identified homologues 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 ADP-ribosyltransferase, 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 defence 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 defence may be a common function for TA systems and reveal the mechanism by which DarTG systems inhibit phage infection.

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Fig. 1: TA systems encoded near known phage defence elements also defend against phage.
Fig. 2: DarTG systems provide phage defence via an abortive infection mechanism.
Fig. 3: DarTG inhibits phage DNA replication by ADP-ribosylating viral DNA.
Fig. 4: Activated DarT inhibits RNA synthesis and the timing of phage protein production.
Fig. 5: RB69 and SECϕ18 escape DarTG-mediated defence by two distinct mechanisms.
Fig. 6: Model for DarTG-mediated defence against phage.

Data availability

The bioinformatic analysis was performed using protein sequences available in the Integrated Microbial Genomes (IMG) database (https://img.jgi.doe.gov/). DarTG1 and DarTG2 were identified in Escherichia coli C7 (NCBI accession GCA_001901425.1) and Escherichia coli 2-460-02_S4_C3 (NCBI accession GCA_000704545.1). Sequencing data are available on NCBI (BioProject PRJNA776027). Sequencing data were aligned to reference genomes of MG1655 (accession number CP025268.1), RB69 (accession number NC_004928), SECϕ18 (accession number LT960609) or T5 (accession number NC_005859.1). All other source data have been deposited to Mendeley Data V1 at https://doi.org/10.17632/v9bmr549nf.1. Source data are provided with this paper.

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Acknowledgements

M.L. was supported by a postdoctoral fellowship from the Charles A. King Trust Postdoctoral Research Fellowship Program, Bank of America, N.A., Co-Trustees. This work was funded by an NIH grant to M.T.L. (R01GM082899), who is also an Investigator of the Howard Hughes Medical Institute; the Sagol Weizmann-MIT Bridge Program (M.T.L. and R.S.); and grants to R.S.: the European Research Council (grant ERC-CoG 681203), the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, and the German Research Council (DFG) priority program SPP 2330 (grant SO 1611/2). A.K.L.L. was supported by an NIH grant (R01GM104135).

Author information

Authors and Affiliations

Authors

Contributions

M.L. performed all experiments. S.S. helped with the construction of phage gene deletions and plaque assays, and with design and interpretation of phage evolution experiments. G.I.C.T. helped with strain construction, bacterial and phage growth and survival assays, and DNA extraction and sequencing. T.Z. helped with the cysteine and methionine incorporation assays. M.L.L. helped with bacterial and phage growth and survival experiments. M.B. and A.K.L.L. supplied biochemical reagents and controls and helped with design of ELTA assays. S.D. and R.S. performed a bioinformatic analysis of TA proximity to known defence elements. M.L. and M.T.L. designed experiments, analysed data, prepared figures and wrote the manuscript.

Corresponding author

Correspondence to Michael T. Laub.

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R.S. is a scientific cofounder and advisor of BiomX and Ecophage.

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Extended data

Extended Data Fig. 1 Sequence alignment of DarT homologs.

Full multiple sequence alignments of representative DarT homologs corresponding to Fig. 1f.

Extended Data Fig. 2 Sequence alignment of DarG homologs.

Full multiple sequence alignments of representative DarG homologs corresponding to Fig. 1f.

Extended Data Fig. 3 DarTG systems provide phage defense via an abortive infection mechanism.

Growth curves for strains with the indicated plasmid-encoded TA system after infection with RB69 (a) or T5 (b) phage at varying multiplicities of infection (MOI). The mean and S.D. of 6-12 technical replicates are presented; data are an independent biological replicate for data presented in Fig. 2a, b.

Source data

Extended Data Fig. 4 DAPI-staining detects phage particles and changes in DNA compaction in infected cells.

a, Snapshots taken of cultures of E. coli MG1655 cells containing DAPI 10-20 min after addition of the indicated phage. b, Time-lapse series of DAPI-stained E. coli MG1655 cells on agarose pads containing 100 µg/mL carbenicillin (top) or untreated (bottom). Lysed cells are indicated with red arrows. cd, Fluorescence intensity profiles of individual cells are plotted for DarTG1 (c) or DarT*G1 (d) cells. Profiles for 18-20 representative cells are shown for each condition and are grouped into diffuse (left), bimodal (middle), or asymmetric (right). Scale bars, 4 µM.

Source data

Extended Data Fig. 5 Less DNA is recovered from infected cells when a DarTG2 system is present.

Total DNA extracted from cells containing darTG2 or darT*G2 and infected with T5 for the times indicated. The intensity of the band at each time post-infection relative to the pre-infection band is reported at the bottom. Two independent replicates are shown.

Source data

Extended Data Fig. 6 Activated DarT inhibits the timing of phage protein production.

Protein synthesis rates as measured by 35S-labeled cysteine and methionine incorporation at various time points after infection for E. coli bearing the indicated plasmids and infected with either RB69 at MOI 5 (a) or treated with 300 µg/mL rifampicin (rif) (b). Data shown are representative of 2 independent biological replicates.

Source data

Extended Data Fig. 7 Gene 61.2 is not essential in T4.

Plaque assays of the wild-type T4, or variants with either a premature stop-codon in gp61.2 (L68*) or a large deletion of residues 65-196 (T4 61.2∆65–196) on E. coli with an active or inactive version of the DarTG1 system.

Supplementary information

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Dot blot.

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Autoradiograph image.

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LeRoux, M., Srikant, S., Teodoro, G.I.C. et al. The DarTG toxin-antitoxin system provides phage defence by ADP-ribosylating viral DNA. Nat Microbiol 7, 1028–1040 (2022). https://doi.org/10.1038/s41564-022-01153-5

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