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

Nature Microbiology volume 7, pages 1028–1040 (2022)Cite this article

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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.

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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.

References

  1. Bernheim, A. et al. Prokaryotic viperins produce diverse antiviral molecules. Nature 589, 120–124 (2021).

  2. Cohen, D. et al. Cyclic GMP-AMP signalling protects bacteria against viral infection. Nature 574, 691–695 (2019).

    Article  CAS  Google Scholar 

  3. Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359, eaar4120-18 (2018).

    Article  Google Scholar 

  4. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084 (2020).

    Article  CAS  Google Scholar 

  5. Harms, A., Brodersen, D. E., Mitarai, N. & Gerdes, K. Toxins, targets, and triggers: an overview of toxin-antitoxin biology. Mol. Cell 70, 768–784 (2018).

    Article  CAS  Google Scholar 

  6. Song, S. & Wood, T. K. A primary physiological role of toxin/antitoxin systems is phage inhibition. Front. Microbiol. 11, 1895 (2020).

    Article  Google Scholar 

  7. Page, R. & Peti, W. Toxin-antitoxin systems in bacterial growth arrest and persistence. Nat. Chem. Biol. 12, 208–214 (2016).

    Article  CAS  Google Scholar 

  8. Ronneau, S. & Helaine, S. Clarifying the link between toxin-antitoxin modules and bacterial persistence. J. Mol. Biol. 431, 3462–3471 (2019).

    Article  CAS  Google Scholar 

  9. Yamaguchi, Y. & Inouye, M. Regulation of growth and death in Escherichia coli by toxin–antitoxin systems. Nat. Rev. Microbiol. 9, 779–790 (2011).

    Article  CAS  Google Scholar 

  10. LeRoux, M., Culviner, P. H., Liu, Y. J., Littlehale, M. L. & Laub, M. T. Stress can induce transcription of toxin-antitoxin systems without activating toxin. Mol. Cell 79, 280–292.e8 (2020).

    Article  CAS  Google Scholar 

  11. Christensen, S. K., Pedersen, K., Hansen, F. G. & Gerdes, K. Toxin–antitoxin loci as stress-response-elements: ChpAK/MazF and ChpBK cleave translated RNAs and are counteracted by tmRNA. J. Mol. Biol. 332, 809–819 (2003).

    Article  CAS  Google Scholar 

  12. Fineran, P. C. et al. The phage abortive infection system, ToxIN, functions as a protein-RNA toxin-antitoxin pair. Proc. Natl Acad. Sci. USA 106, 894–899 (2009).

    Article  CAS  Google Scholar 

  13. Guegler, C. K. & Laub, M. T. Shutoff of host transcription triggers a toxin-antitoxin system to cleave phage RNA and abort infection. Mol. Cell 81, 2361–2373 (2021).

    Article  CAS  Google Scholar 

  14. Koga, M., Otsuka, Y., Lemire, S. & Yonesaki, T. Escherichia coli rnlA and rnlB compose a novel toxin-antitoxin system. Genetics 187, 123–130 (2011).

    Article  CAS  Google Scholar 

  15. Makarova, K. S., Wolf, Y. I., Snir, S. & Koonin, E. V. Defense islands in bacterial and archaeal genomes and prediction of novel defense systems. J. Bacteriol. 193, 6039–6056 (2011).

    Article  CAS  Google Scholar 

  16. Makarova, K. S., Wolf, Y. I. & Koonin, E. V. Comparative genomics of defense systems in archaea and bacteria. Nucleic Acids Res. 41, 4360–4377 (2013).

    Article  CAS  Google Scholar 

  17. Jankevicius, G., Ariza, A., Ahel, M. & Ahel, I. The toxin-antitoxin system DarTG catalyzes reversible ADP-ribosylation of DNA. Mol. Cell 64, 1109–1116 (2016).

    Article  CAS  Google Scholar 

  18. Lawarée, E. et al. DNA ADP-ribosylation stalls replication and is reversed by recf-mediated homologous recombination and nucleotide excision repair. Cell Rep. 30, 1373–1384.e4 (2020).

    Article  Google Scholar 

  19. Schuller, M. et al. Molecular basis for DarT ADP-ribosylation of a DNA base. Nature 596, 597–602 (2021).

    Article  CAS  Google Scholar 

  20. Zaveri, A. et al. Depletion of the DarG antitoxin in Mycobacterium tuberculosis triggers the DNA-damage response and leads to cell death. Mol. Microbiol. 114, 641–652 (2020).

    Article  CAS  Google Scholar 

  21. De Souza, R. F. & Aravind, L. Identification of novel components of NAD-utilizing metabolic pathways and prediction of their biochemical functions. Mol. Biosyst. 8, 1661–1677 (2012).

    Article  Google Scholar 

  22. Rack, J. G. M., Perina, D. & Ahel, I. Macrodomains: structure, function, evolution, and catalytic activities. Annu. Rev. Biochem. 85, 431–454 (2016).

    Article  CAS  Google Scholar 

  23. Tromans-Coia, C. et al. TARG1 protects against toxic DNA ADP-ribosylation. Nucleic Acids Res. https://doi.org/10.1093/nar/gkab771 (2021).

  24. Otsuka, Y. & Yonesaki, T. Dmd of bacteriophage T4 functions as an antitoxin against Escherichia coli LsoA and RnlA toxins. Mol. Microbiol. 83, 669–681 (2012).

    Article  CAS  Google Scholar 

  25. McCorquodale, D. J. & Warner, H. R. in The Bacteriophages (ed. Calendar, R.) 439–475 (Springer, 1988); https://doi.org/10.1007/978-1-4684-5424-6_10

  26. Ando, Y. et al. ELTA: enzymatic labeling of terminal ADP-ribose. Mol. Cell 73, 845–856.e5 (2019).

    Article  CAS  Google Scholar 

  27. Srikant, S., Guegler, C. K. & Laub, M. T. The evolution of a counter-defense mechanism in a virus constrains its host range. Preprint at bioRxiv https://doi.org/10.1101/2022.04.14.488369 (2022).

  28. Petrov, V. M., Ratnayaka, S., Nolan, J. M., Miller, E. S. & Karam, J. D. Genomes of the T4-related bacteriophages as windows on microbial genome evolution. Virol. J. 7, 292 (2010).

    Article  Google Scholar 

  29. Warner, H. R., Drong, R. F. & Berget, S. M. Early events after infection of Escherichia coli by bacteriophage T5. Induction of a 5’-nucleotidase activity and excretion of free bases. J. Virol. 15, 273–280 (1975).

    Article  CAS  Google Scholar 

  30. Pinilla-Redondo, R. et al. Discovery of multiple anti-CRISPRs highlights anti-defense gene clustering in mobile genetic elements. Nat. Commun. 11, 5652 (2020).

    Article  CAS  Google Scholar 

  31. Bondy-Denomy, J. et al. Prophages mediate defense against phage infection through diverse mechanisms. ISME J. 10, 2854–2866 (2019).

    Article  Google Scholar 

  32. Dedrick, R. M. et al. Prophage-mediated defence against viral attack and viral counter-defence. Nat. Microbiol. 2, 16251 (2017).

    Article  CAS  Google Scholar 

  33. Rousset, F., Dowding, J., Bernheim, A., Rocha, E. P. C. & Bikard, D. Prophage-encoded hotspots of bacterial immune systems. Preprint at bioRxiv https://doi.org/10.1101/2021.01.21.427644 (2021).

  34. Hallez, R. et al. New toxins homologous to ParE belonging to three-component toxin–antitoxin systems in Escherichia coli O157:H7. Mol. Microbiol. 76, 719–732 (2010).

    Article  CAS  Google Scholar 

  35. Peltier, J. et al. Type I toxin-antitoxin systems contribute to the maintenance of mobile genetic elements in Clostridioides difficile. Commun. Biol. 3, 718 (2020).

    Article  CAS  Google Scholar 

  36. Yao, J. et al. Type II toxin/antitoxin system ParESO /CopASO stabilizes prophage CP4So in Shewanella oneidensis. Environ. Microbiol. 20, 1224–1239 (2018).

    Article  CAS  Google Scholar 

  37. Chen, I.-M. A. et al. IMG/M v.5.0: an integrated data management and comparative analysis system for microbial genomes and microbiomes. Nucleic Acids Res. 47, D666–D677 (2019).

    Article  CAS  Google Scholar 

  38. Steinegger, M. & Söding, J. MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets. Nat. Biotechnol. 35, 1026–1028 (2017).

    Article  CAS  Google Scholar 

  39. Bobonis, J. et al. Bacterial retrons encode tripartite toxin/antitoxin systems. Preprint at bioRxiv https://doi.org/10.1101/2020.06.22.160168 (2020).

  40. Chung, C. T., Niemela, S. L. & Miller, R. H. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl Acad. Sci. USA 86, 2172–2175 (1989).

    Article  CAS  Google Scholar 

  41. Haldimann, A. & Wanner, B. L. Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria. J. Bacteriol. 183, 6384–6393 (2001).

    Article  CAS  Google Scholar 

  42. Blomfield, I. C., Vaughn, V., Rest, R. F. & Eisenstein, B. I. Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol. Microbiol. 5, 1447–1457 (1991).

    Article  CAS  Google Scholar 

  43. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    Article  CAS  Google Scholar 

  44. Duong, M. M., Carmody, C. M., Ma, Q., Peters, J. E. & Nugen, S. R. Optimization of T4 phage engineering via CRISPR/Cas9. Sci. Rep. 10, 18229 (2020).

    Article  CAS  Google Scholar 

  45. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

    Article  CAS  Google Scholar 

Download references

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).

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Authors and Affiliations

  1. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Michele LeRoux, Sriram Srikant, Gabriella I. C. Teodoro, Tong Zhang, Megan L. Littlehale & Michael T. Laub

  2. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

    Shany Doron & Rotem Sorek

  3. Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA

    Mohsen Badiee & Anthony K. L. Leung

  4. Department of Molecular Biology and Genetics, Department of Genetic Medicine, Department of Oncology, School of Medicine, Johns Hopkins University, Baltimore, MD, USA

    Anthony K. L. Leung

  5. Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA

    Michael T. Laub

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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.

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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. c–d, 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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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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