Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Resource
  • Published:

Nuclease–NTPase antiphage defence systems use conserved molecular features to control bacterial immunity

Nature Microbiology volume 11, pages 1424–1436 (2026) Cite this article

Subjects

Abstract

Bacteria encode diverse defence systems, including restriction–modification and CRISPR–Cas, that cleave nucleic acid to protect against phage infection. Bioinformatic analyses demonstrate that many recently identified antiphage defence operons comprise a nuclease and NTPase protein, suggesting that additional nucleic acid-targeting systems remain to be understood. Here we develop large-scale comparative cell biology and biochemical approaches to analyse 16 nuclease–NTPase systems and define molecular features that control antiphage defence. Purification, biochemical characterization and in vitro reconstitution of nucleic acid degradation demonstrates that protein–protein complex formation is a shared feature of multigene nuclease–NTPase systems. We show that PaAbpAB, BtHachiman and EcPD-T4-8 system nucleases use highly degenerate recognition site preferences to enable broad nucleic acid degradation, and the Azaca system exhibits specific phage targeting through the recognition of modified phage genomic DNA. Our results uncover principles of antiphage defence system function and highlight the mechanistic diversity of nuclease–NTPase systems in bacterial immunity.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Nuclease–NTPase systems broadly defend against DNA phage infection.
The alternative text for this image may have been generated using AI.
Fig. 2: Complex formation is a widespread feature of nuclease–NTPase systems.
The alternative text for this image may have been generated using AI.
Fig. 3: Nuclease–NTPase systems have distinct nucleic acid cleavage patterns.
The alternative text for this image may have been generated using AI.
Fig. 4: Nuclease–NTPase systems exhibit degenerate cleavage site motifs.
The alternative text for this image may have been generated using AI.
Fig. 5: A detailed reference for nuclease–NTPase antiphage defence systems that encode a PD(D/E)XK nuclease.
The alternative text for this image may have been generated using AI.
Fig. 6: A detailed reference for nuclease–NTPase antiphage defence systems that encode PLD-like nucleases and Toprim/OLD nucleases.
The alternative text for this image may have been generated using AI.

Similar content being viewed by others

Data availability

Processed data used to generate the MP plots in Fig. 2 are included, and the raw images are available upon request. Sequencing data generated from this study are available at the NCBI Gene Expression Omnibus under accession no. GSE303276. Information required to reanalyse the sequencing data are available via Zenodo at https://doi.org/10.5281/zenodo.18686340 (ref. 65). Plasmids for the nuclease–NTPase operons used in this study will be deposited to Addgene. All other data are available in this Resource or its Supplementary Information. Source data are provided with this paper.

References

  1. Roberts, R. J. et al. A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Nucleic Acids Res. 31, 1805–1812 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Loenen, W. A. M., Dryden, D. T. F., Raleigh, E. A., Wilson, G. G. & Murray, N. E. Highlights of the DNA cutters: a short history of the restriction enzymes. Nucleic Acids Res. 42, 3–19 (2014).

    Article  CAS  PubMed  Google Scholar 

  3. Hille, F. et al. The biology of CRISPR–Cas: backward and forward. Cell 172, 1239–1259 (2018).

    Article  CAS  PubMed  Google Scholar 

  4. Makarova, K. S. et al. Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. Nat. Rev. Microbiol. 18, 67–83 (2020).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Tesson, F. et al. Systematic and quantitative view of the antiviral arsenal of prokaryotes. Nat. Commun. 13, 2561 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Payne, L. J. et al. PADLOC: a web server for the identification of antiviral defence systems in microbial genomes. Nucleic Acids Res. 50, W541–W550 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556–1569 (2022).

    Article  CAS  PubMed  Google Scholar 

  10. Rousset, F. et al. Phages and their satellites encode hotspots of antiviral systems. Cell Host Microbe 30, 740–753 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat. Microbiol. 7, 1568–1579 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yasui, R., Washizaki, A., Furihata, Y., Yonesaki, T. & Otsuka, Y. AbpA and AbpB provide anti-phage activity in Escherichia coli. Genes Genet. Syst. 89, 51–60 (2014).

    Article  CAS  PubMed  Google Scholar 

  13. Sather, L. M. et al. A broadly distributed predicted helicase/nuclease confers phage resistance via abortive infection. Cell Host Microbe 31, 343–355 (2023).

    Article  CAS  PubMed  Google Scholar 

  14. Bari, S. M. et al. A unique mode of nucleic acid immunity performed by a multifunctional bacterial enzyme. Cell Host Microbe 30, 570–582 (2022).

    Article  CAS  PubMed  Google Scholar 

  15. Ernits, K. et al. The structural basis of hyperpromiscuity in a core combinatorial network of type II toxin–antitoxin and related phage defense systems. Proc. Natl Acad. Sci. USA 120, e2305393120 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Cheng, R. et al. A nucleotide-sensing endonuclease from the Gabija bacterial defense system. Nucleic Acids Res. 49, 5216–5229 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Antine, S. P. et al. Structural basis of Gabija anti-phage defence and viral immune evasion. Nature 625, 360–365 (2024).

    Article  CAS  PubMed  Google Scholar 

  18. Li, J. et al. Structures and activation mechanism of the Gabija anti-phage system. Nature 629, 467–473 (2024).

    Article  CAS  PubMed  Google Scholar 

  19. Yang, X.-Y. et al. Molecular basis of Gabija anti-phage supramolecular assemblies. Nat. Struct. Mol. Biol. 31, 1243–1250 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Tuck, O. T. et al. Genome integrity sensing by the broad-spectrum Hachiman antiphage defense complex. Cell 187, 6914–6928 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Cui, Y. et al. Bacterial Hachiman complex executes DNA cleavage for antiphage defense. Nat. Commun. 16, 2604 (2025).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Knizewski, L., Kinch, L. N., Grishin, N. V., Rychlewski, L. & Ginalski, K. Realm of PD-(D/E)XK nuclease superfamily revisited: detection of novel families with modified transitive meta profile searches. BMC Struct. Biol. 7, 40 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Orlowski, J. & Bujnicki, J. M. Structural and evolutionary classification of Type II restriction enzymes based on theoretical and experimental analyses. Nucleic Acids Res. 36, 3552–3569 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Yang, W. Nucleases: diversity of structure, function and mechanism. Q. Rev. Biophys. 44, 1–93 (2011).

    Article  PubMed  Google Scholar 

  25. Gottlin, E. B., Rudolph, A. E., Zhao, Y., Matthews, H. R. & Dixon, J. E. Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate. Proc. Natl Acad. Sci. USA 95, 9202–9207 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ipsaro, J. J., Haase, A. D., Knott, S. R., Joshua-Tor, L. & Hannon, G. J. The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis. Nature 491, 279–283 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Aravind, L. Toprim—a conserved catalytic domain in type IA AND II topoisomerases DnaG-type primases OLD family nucleases AND RecR proteins. Nucleic Acids Res. 26, 4205–4213 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Raney, K. D., Byrd, A. K. & Aarattuthodiyil, S. in DNA Helicases and DNA Motor Proteins Vol. 767 (ed. Spies, M.) 17–46 (Springer, 2013).

  29. Byrd, A. K. & Raney, K. D. Superfamily 2 helicases. Front. Biosci. 17, 2070–2088 (2012).

    Article  Google Scholar 

  30. Fairman-Williams, M. E., Guenther, U.-P. & Jankowsky, E. SF1 and SF2 helicases: family matters. Curr. Opin. Struct. Biol. 20, 313–324 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Gorbalenya, A. E. & Koonin, E. V. Helicases: amino acid sequence comparisons and structure-function relationships. Curr. Opin. Struct. Biol. 3, 419–429 (1993).

    Article  CAS  Google Scholar 

  32. Longo, L. M. et al. On the emergence of P-loop NTPase and Rossmann enzymes from a beta-alpha-beta ancestral fragment. eLife 9, e64415 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Maffei, E. et al. Systematic exploration of Escherichia coli phage–host interactions with the BASEL phage collection. PLoS Biol. 19, e3001424 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Stokar-Avihail, A. et al. Discovery of phage determinants that confer sensitivity to bacterial immune systems. Cell 186, 1863–1876 (2023).

    Article  CAS  PubMed  Google Scholar 

  35. Banh, D. V. et al. Author correction: bacterial cGAS senses a viral RNA to initiate immunity. Nature 625, E3 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Richmond-Buccola, D. et al. A large-scale type I CBASS antiphage screen identifies the phage prohead protease as a key determinant of immune activation and evasion. Cell Host Microbe 32, 1074–1088 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Lopatina, A., Tal, N. & Sorek, R. Abortive infection: bacterial suicide as an antiviral immune strategy. Annu. Rev. Virol. 7, 371–384 (2020).

    Article  CAS  PubMed  Google Scholar 

  38. Tang, D. et al. DUF4297 and HerA form abortosome to mediate bacterial immunity against phage infection. Mol. Cell 85, 1176–1188 (2025).

    Article  CAS  PubMed  Google Scholar 

  39. Sasaki, T. et al. Phage single-stranded DNA-binding protein or host DNA damage triggers the activation of the AbpAB phage defense system. mSphere 8, e0037223 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Huo, Y. et al. Structural and biochemical insights into the mechanism of the Gabija bacterial immunity system. Nat. Commun. 15, 836 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. An, Q. et al. Molecular and structural basis of an ATPase–nuclease dual-enzyme anti-phage defense complex. Cell Res. 34, 545–555 (2024).

    Article  CAS  PubMed  Google Scholar 

  42. Rish, A. D. et al. Architecture remodeling activates the HerA–DUF anti-phage defense system. Mol. Cell 85, 1189–1201 (2025).

    Article  CAS  PubMed  Google Scholar 

  43. Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493–500 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Hooper, M. M. et al. Phage-encoded factor stimulates DNA degradation by the Hna anti-phage defense system. Preprint at bioRxiv https://doi.org/10.1101/2025.11.12.688083 (2025).

  45. Loeff, L., Walter, A., Rosalen, G. T. & Jinek, M. DNA end sensing and cleavage by the Shedu anti-phage defense system. Cell 188, 721–733 (2025).

    Article  CAS  PubMed  Google Scholar 

  46. Gu, Y. et al. Bacterial Shedu immune nucleases share a common enzymatic core regulated by diverse sensor domains. Mol. Cell 85, 523–536 (2025).

    Article  CAS  PubMed  Google Scholar 

  47. Weigele, P. & Raleigh, E. A. Biosynthesis and function of modified bases in bacteria and their viruses. Chem. Rev. 116, 12655–12687 (2016).

    Article  CAS  PubMed  Google Scholar 

  48. Lau, R. K. et al. Structure and mechanism of a cyclic trinucleotide-activated bacterial endonuclease mediating bacteriophage immunity. Mol. Cell 77, 723–733 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. J. 17, 10–12 (2011).

    Article  Google Scholar 

  50. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Lehman, I. R. & Pratt, E. A. On the structure of the glucosylated hydroxymethylcytosine nucleotides of coliphages T2, T4, and T6. J. Biol. Chem. 235, 3254–3259 (1960).

    Article  CAS  PubMed  Google Scholar 

  52. Hossain, A. A. et al. DNA glycosylases provide antiviral defence in prokaryotes. Nature 629, 410–416 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Robins, W. P., Meader, B. T., Toska, J. & Mekalanos, J. J. DdmABC-dependent death triggered by viral palindromic DNA sequences. Cell Rep. 43, 114450 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Getz, L. J. et al. Antiviral defence is a conserved function of diverse DNA glycosylases. Preprint at bioRxiv https://doi.org/10.1101/2025.10.29.685425 (2025).

  55. Hobbs, S. J. et al. Phage anti-CBASS and anti-Pycsar nucleases subvert bacterial immunity. Nature 605, 522–526 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Wang, X. et al. Cryptic prophages help bacteria cope with adverse environments. Nat. Commun. 1, 147 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Mazzocco, A., Waddell, T. E., Lingohr, E. & Johnson, R. P. Enumeration of bacteriophages using the small drop plaque assay system. Methods Mol. Biol. 501, 81–85 (2009).

    Article  CAS  PubMed  Google Scholar 

  58. Zhou, W. et al. Structure of the human cGAS–DNA complex reveals enhanced control of immune surveillance. Cell 174, 300–311.e11 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Liu, H. & Naismith, J. H. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 8, 91 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Young, G. et al. Quantitative mass imaging of single biological macromolecules. Science 360, 423–427 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).

    Article  CAS  PubMed  Google Scholar 

  62. Cater, K. et al. A novel Staphylococcus podophage encodes a unique lysin with unusual modular design. mSphere 2, e00040-17 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Summer, E. J. Preparation of a phage DNA fragment library for whole genome shotgun sequencing. Methods Mol. Biol. 502, 27–46 (2009).

    Article  CAS  PubMed  Google Scholar 

  64. Crooks, G. E., Hon, G., Chandonia, J.-M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Ragucci, A. E. et al. Nuclease–NTPase systems use shared molecular features to control bacterial anti-phage defense. Zenodo https://doi.org/10.5281/zenodo.18686340 (2026).

  66. Li, H., Tan, Y., Basu, D., Corbett, K. D. & Zhang, D. Unveiling the multifaceted domain polymorphism of the Menshen antiphage system. Nucleic Acids Res. 53, gkaf357 (2025).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to members of the Kranzusch Laboratory for helpful comments and discussion. We thank the Center for Macromolecular Interactions at Harvard Medical School and the Dana-Farber Cancer Institute Molecular Biology Core Facility. We thank R. Sorek (Weizmann Institute of Science), L. Marraffini (The Rockefeller University) and G. Atkinson (Lund University) for sharing reagents. The work was funded by grants to P.J.K. from the Pew Biomedical Scholars programme, the Burroughs Wellcome Fund PATH programme, The G. Harold and Leila Y. Mathers Charitable Foundation, The Mark Foundation for Cancer Research, the Cancer Research Institute, the Parker Institute for Cancer Immunotherapy and the National Institutes of Health (grant no. 1DP2GM146250-01), grants to A.S.Y.L. from The G. Harold and Leila Y. Mathers Charitable Foundation and the National Institutes of Health (grant no. R35GM142527) and grants to V.H. from the Knut and Alice Wallenberg Foundation (project grant no. 2020-0037), the Swedish Research Council (Vetenskapsrådet) grants (grant nos. 2021-01146 and 2024-06059) and the Göran Gustafsson Foundation for Research in Natural Sciences and Medicine (the Göran Gustafsson Prize). J.M.G is supported by a Ford Foundation Predoctoral Fellowship and the National Institutes of Health (grant no. 5TL1TR002543).

Author information

Author notes

  1. These authors contributed equally: Adelyn E. Ragucci, Sadie P. Antine.

Authors and Affiliations

  1. Department of Microbiology, Harvard Medical School, Boston, MA, USA

    Adelyn E. Ragucci, Sadie P. Antine, Ethan M. Leviss, Sarah E. Mooney & Philip J. Kranzusch

  2. Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA, USA

    Adelyn E. Ragucci, Sadie P. Antine, Ethan M. Leviss, Jasmine M. Garcia, Amy S. Y. Lee & Philip J. Kranzusch

  3. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Jasmine M. Garcia & Amy S. Y. Lee

  4. Department of Experimental Medical Science and NanoLund, Lund University, Lund, Sweden

    Lena Shyrokova & Vasili Hauryliuk

  5. Institute of Technology, University of Tarfu, Tartu, Estonia

    Vasili Hauryliuk

  6. Parker Institute for Cancer Immunotherapy, Dana-Farber Cancer Institute, Boston, MA, USA

    Philip J. Kranzusch

Authors
  1. Adelyn E. Ragucci
    View author publications

    Search author on:PubMed Google Scholar

  2. Sadie P. Antine
    View author publications

    Search author on:PubMed Google Scholar

  3. Ethan M. Leviss
    View author publications

    Search author on:PubMed Google Scholar

  4. Sarah E. Mooney
    View author publications

    Search author on:PubMed Google Scholar

  5. Jasmine M. Garcia
    View author publications

    Search author on:PubMed Google Scholar

  6. Lena Shyrokova
    View author publications

    Search author on:PubMed Google Scholar

  7. Vasili Hauryliuk
    View author publications

    Search author on:PubMed Google Scholar

  8. Amy S. Y. Lee
    View author publications

    Search author on:PubMed Google Scholar

  9. Philip J. Kranzusch
    View author publications

    Search author on:PubMed Google Scholar

Contributions

The study was designed and conceived by A.E.R., S.P.A. and P.J.K. The phage challenge experiments were performed by A.E.R and S.E.M. Protein purification and biochemical assays were performed by A.E.R, S.P.A, E.M.L. and S.E.M. Bacterial growth assays were performed by S.E.M and E.M.L. MP and AlphaFold3 modelling was performed by S.P.A. Sequencing analysis was performed by J.M.G and A.S.Y.L., and L.S. and V.H. performed initial phage defence assays with the Toprim-TA system. The figures were prepared by A.E.R., with contributions from S.P.A and E.M.L. The paper was written by A.E.R. and P.J.K. All authors contributed to editing the paper and support the conclusions.

Corresponding author

Correspondence to Philip J. Kranzusch.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Microbiology thanks Haidai Hu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Table 1 Nuclease and NTPase classes included in this study
Full size table

Extended Data Fig. 1 Nuclease and NTPase classes included in this study.

Summary of the nuclease and NTPase class included in each selected operon.

Extended Data Fig. 2 Mechanisms of defense of nuclease-NTPase systems.

a, Growth of control (sfGFP) or defense system expressing bacteria challenged with T4 phage at MOIs of 0.02 or 2. BmAzaca and EcMenshen result in abortive infection against T4 phage. b, Growth of control (sfGFP) or defense system expressing bacteria challenged with T5 phage at MOIs of 0.02 or 2. PsNhi-like results in abortive infection while EcMokosh I and AkUpx show a delayed collapse in culture at low T5 phage MOI. c, Growth of control (sfGFP) or EcOlokun expressing bacteria challenged with Bas39 phage at MOIs of 0.02 or 2. d, Growth of control (sfGFP) or EcMokosh II expressing bacteria challenged with SECΦ17 phage at MOIs of 0.05 or 5. EcMokosh II shows direct immunity to SECΦ17 phage. Data in a–d are representative of 3 independent experiments.

Source data

Extended Data Fig. 3 Purification of nuclease-NTPase system protein complexes.

a, Coomassie-stained 10% SDS-PAGE gel analysis of purified nuclease-NTPase protein complexes grouped by nuclease type demonstrates that all multi-gene operons co-purify. b–j, l–o, Size-exclusion chromatograms (16/600 S200 or 16/600 S300) for purified nuclease-NTPase complexes. Shading indicates which peaks were used for downstream analysis. k, Heparin ion exchange (IEX) chromatogram for purification of EcMokosh II. Data are representative of at least 2 independent experiments.

Source data

Extended Data Fig. 4 Nuclease-NTPase AlphaFold3 predictions.

a–i, AlphaFold3 predictions of nuclease-NTPase systems colored by plDDT with expected position error graphs shown to the right of each model.

Extended Data Fig. 5 Cleavage assay results of all 14 purified nuclease-NTPase systems with various nucleic acid substrates.

a–j, Nucleic acid cleavage and agarose gel analysis of in vitro cleavage of E. coli chromosomal DNA (a), phage Bas17 DNA (b), phage λ DNA (c), phage T5 DNA (d), phage T7 DNA (e), phage Bas65 DNA (f), phage SECΦ17 DNA (g), phage M13 DNA (h), phage SPO1 DNA (i), and phage MS2 RNA (j) by nuclease-NTPase systems. Data are summarized in Fig. 3c and are representative of at least 3 independent experiments. k, Quantification of the plasmid nicking activity of nuclease-NTPase systems. Error bars indicate the standard deviation of three replicates. Data are representative of 3 independent experiments.

Source data

Extended Data Fig. 6 Nuclease-NTPase complexes and nuclease active site residues are required for nucleic acid cleavage.

Agarose gel analysis of the ability of nuclease-NTPase systems and individual subunits to cleave phage and plasmid DNA. a–b, Analysis of PaAbpAB mutants and individual subunits demonstrates that complex formation is required, but the wildtype NTPase active site motifs are not required for cleavage. c, Analysis of BmAzaca demonstrates that conserved residues in ZacA are dispensable for DNA cleavage, but nuclease and NTPase mutant proteins are catalytically inactive. d–e, Analysis of BtHachiman demonstrates that complex formation is required, but the wildtype NTPase active site motifs are not required for cleavage. f, Analysis of EcMokosh II with M13 DNA demonstrates DNA cleavage is only inhibited when the nuclease active site is mutated. g, Analysis of EcPD-T4-1 demonstrates that a nuclease mutant is unable to cleave phage M13 DNA. h, Analysis of EcPD-T4-4 with MS2 RNA demonstrates RNA cleavage is only inhibited when the nuclease active site is mutated. i, Analysis of the ability of EcPD-T4-4 NTPase or nuclease alone to cleave MS2 RNA demonstrates that both proteins are required for cleavage. j–k, Analysis of EcPD-T4-8 demonstrates the nuclease active site and full-length protein sequence is required to cleave phage T4 DNA and plasmid DNA. Data in a–k are representative of at least 3 independent experiments.

Source data

Extended Data Fig. 7 Nuclease-NTPase nuclease active site residues and conserved NTPase motifs are required for phage defense.

a, Heatmap displaying the log10 fold protection conferred by mutant nuclease-NTPase operons against T4, T4, or SECΦ17 phages. b, Plaque assays for bacteria expressing wild type (WT), nuclease, and NTPase mutant operons challenged with phage T4. Both WT EcPD-T4-1 and the nuclease mutant designed for biochemistry exhibit defense against T4 phage. Nuclease (**) and NTPase (*) mutants of the EcPD-T4-4 system no longer provide defense compared to WT EcPD-T4-4. The nuclease mutant of EcPD-T4-8 loses protection against T4 phage. WT and the ZacA*BC mutant of BmAzaca provide defense while nuclease (ZacAB*C) and NTPase (ZacABC*) mutants do not provide defense against T4. c, Plaque assays for bacteria expressing WT PsNhi-like as well as nuclease (*) and NTPase (**) mutants of this system challenged with T5 phage. Only WT PsNhi-like provides defense against T5. d, Plaque assays for bacteria expressing WT EcMokosh II as well as nuclease and NTPase mutants of this system challenged with SECΦ17 phage. WT EcMokosh II provides defense against SECΦ17 while a nuclease mutant (**) provides attenuated defense and NTPase mutant (*) loses defense. Data in a–d are representative of at least 3 independent experiments.

Extended Data Fig. 8 Protein titration analysis of nuclease-NTPase nucleic acid cleavage reactions.

a–g, Nucleic acid cleavage and agarose gel analysis of the ability of BtHachiman (a), EcMokosh II (b), PsNhi-like (c), PfOlokun (d), EcPD-T4-1 (e), EcPD-T4-4 (f), and EcPD-T4-8 (g) to cleave target nucleic acid at protein concentrations from 10 nM to 1 µM. PaAbpAB, EcPD-T4-1, EcPD_T4-4, and BmAzaca cleave DNA at concentrations ≤50 nM. BtHachiman, PsNhi-like, PfOlokun, EcPD-T4-8, and EcMokosh II cleave at 1 µM. Data are summarized in Fig. 4c and are representative of at least 3 independent experiments.

Source data

Extended Data Fig. 9 Sequencing analysis of T4 and plasmid DNA fragments incubated with nuclease-NTPase systems reveals degenerate cleavage motifs.

a, c, d, f, Fragment size and position graphs for PaAbpAB (a), BmAzaca (c), BtHachiman (d), and EcPD-T4-8 (f) cleavage reactions with phage T4 DNA reveals that each system cuts throughout the length of T4 DNA. b, e, g, WebLogos, consensus sequences, fragment size and position graphs for PaAbpAB (b), BtHachiman (e), EcPD-T4-8 (g) cleavage reactions with plasmid DNA. All three systems exhibit degenerate cleavage site motifs and cut throughout the length of plasmid DNA. All reactions were optimized such that fragment sizes would be between 150–350 bp for sequencing.

Supplementary information

Source data

Source Data Fig. 2 (download XLSX )

Raw MP data files.

Source Data Figs. 3 and 4 and Extended Data Figs. 3, 5, 6 and 8 (download PDF )

Uncropped gels.

Source Data Extended Data Fig. 2 (download XLSX )

Excel file containing the raw data used to generate growth assay graphs in Extended Data Fig. 2.

Source Data Extended Data Fig. 5 (download XLSX )

Excel file containing the raw data used to generate the graph displaying plasmid nicking activity in Extended Data Fig. 5k.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ragucci, A.E., Antine, S.P., Leviss, E.M. et al. Nuclease–NTPase antiphage defence systems use conserved molecular features to control bacterial immunity. Nat Microbiol 11, 1424–1436 (2026). https://doi.org/10.1038/s41564-026-02312-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41564-026-02312-8

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology