Abstract
Animal and bacterial cells use nucleotidyltransferase (NTase) enzymes to respond to viral infection and control major forms of immune signalling including cGAS-STING innate immunity and CBASS anti-phage defence1,2,3,4. Here we discover a family of bacterial defence systems, which we name Hailong, that use NTase enzymes to constitutively synthesize DNA signals and guard against phage infection. Hailong protein B (HalB) is an NTase that converts deoxy-ATP into single-stranded DNA oligomers. A series of X-ray crystal structures define a stepwise mechanism of HalB DNA synthesis initiated by a C-terminal tyrosine residue that enables de novo enzymatic priming. We show that HalB DNA signals bind to and repress activation of a partnering Hailong protein A (HalA) effector complex. A 2.0-Å cryo-electron microscopy structure of the HalA–DNA complex reveals a membrane protein with a conserved ion channel domain and a unique crown domain that binds the DNA signal and gates activation. Analysing Hailong defence in vivo, we demonstrate that viral DNA exonucleases required for phage replication trigger release of the primed HalA complex and induce protective host cell growth arrest. Our results explain how inhibitory nucleotide immune signals can serve as molecular guards against phage infection and expand the mechanisms NTase enzymes use to control antiviral immunity.
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Data availability
Coordinates and structure factors of the Hailong HalB–ODA complex and HalB mutant enzymes have been deposited in PDB under the accession codes 9DBH, 9DBI and 9DBJ. Coordinates and density maps of the HalA–ODA complex have been deposited in PDB and EMDB under the accession codes 9NYI and EMD-49920. Source data for Fig. 4b,j and Extended Data Figs. 2h and 7a,b are provided with this paper. All other data are available in the manuscript or the Supplementary Information.
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Acknowledgements
We thank members of the Kranzusch and Sorek laboratories for helpful comments and discussion. We thank D. Wassarman and S. Yamaguchi from the Kranzusch laboratory for assistance with LC–MS analysis, and M. Gilman from the Kruse laboratory for assistance with cryo-EM data collection and processing. Mass spectrometry was performed at the Harvard Taplin Mass Spectrometry Facility with assistance from R. Tomaino. 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 R.S. from the European Research Council (grant no. ERC-AdG GA 101018520), the Israel Science Foundation (MAPATS grant no. 2720/22), the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, the Deutsche Forschungsgemeinschaft (SPP 2330, grant no. 464312965), a research grant from the Estate of Marjorie Plesset and the Knell Family Center for Microbiology; a grant to A.C.K. from the National Institutes of Health (grant no. U19AI158028); grants to M.J. from the National Institutes of Health (grant no. 1DP2GM154152) and the G. Harold and Leila Y. Mathers Charitable Foundation; and a grant to J.d.M. from National Institutes of Health (grant no. 5R00DC019401) and the Howard Hughes Medical Institute. J.M.J.T. is supported by a Servier PhD Fellowship. S.J.H. is supported through a Cancer Research Institute Irvington Postdoctoral Fellowship (grant no. CRI3996), and J.C.C. is supported by a Helen Hay Whitney postdoctoral fellowship. X-ray data were collected through support from an agreement between the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357, and the Diamond Light Source, the UK’s national synchrotron science facility, located at the Harwell Science and Innovation Campus in Oxfordshire. X-ray data were additionally collected at the Center for Bio-Molecular Structure (CBMS) which is primarily supported by the NIH-NIGMS through a Center Core P30 Grant (no. P30GM133893), and by the DOE Office of Biological and Environmental Research (grant no. KP1607011). NSLS2 is a US DOE Office of Science User Facility operated under Contract No. DE-SC0012704. This publication resulted from the data collected using the beamtime obtained through NECAT BAG proposal no. 311950. Cryo-EM data were collected at the Harvard Cryo-EM Center for Structural Biology at Harvard Medical School. Imaging experiments were supported by the Microscopy Resources on the North Quad (MicRoN) core at Harvard Medical School.
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The study was designed and conceived by J.M.J.T. and P.J.K. Crystallography, biochemical experiments, bacterial toxicity assays and live cell imaging microscopy were performed by J.M.J.T. Phage challenge assays and escape mutant generation and sequencing were performed by S.M. and R.S. Cryo-EM data collection, analysis and model building were performed by J.M.J.T., J.C.C. and A.C.K. Flow cytometry collection and analysis were performed by J.M.J.T., D.S. and M.J. SEC-MALS was performed by S.J.H. Bioinformatics analysis was performed by J.M.J.T. and J.d.M. The manuscript was written by J.M.J.T. and P.J.K. All authors contributed to editing the manuscript and support the conclusions.
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R.S. is a scientific cofounder and advisor of BiomX and Ecophage. A.C.K. is a cofounder and consultant for Tectonic Therapeutic and Seismic Therapeutic and for the Institute for Protein Innovation, a non-profit research institute. M.J. declares outside interest in Evozyne and DEM BioPharma. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Structural and biochemical analysis of HalB.
a, Structure guided multiple sequence alignment of HalB protein homologues from indicated bacterial species. Shading indicates degree of residue conservation. b,c, Protein structural homology of HalB WT N-terminal lobe (b) and HalB catalytically inactive mutant (DDAA) C-terminal lobe (c) against all entries in PDB showing the DALI Z score of the top 30 and 150 hits respectively. HalB DDAA was used in (c) as the structure of the mutant protein resolves additional residues in the C-terminal tail that are not visible in the wildtype structure. d, R. bacterium QY30 HalB was expressed as an N-terminal 6×His-SUMO2 fusion and purified by Ni-NTA and separated from His-SUMO2 by size exclusion chromatography. e, Coomassie-stained SDS-PAGE analysis of fully purified HalB from R. bacterium QY30. f, Overview of tetrameric HalB crystal structure with one protomer in blue and three protomers in different shades of grey. Numbered boxes highlight dimerization interface described in detail in (h). g, Size exclusion chromatography with multi-angle static light scattering (SEC-MALS) analysis of purified HalB confirms HalB tetramerization. h, Detailed view of interacting residues as in (f) showing dimerization interface between HalB protomers. i, Urea-PAGE analysis of HalB WT and LEFE mutant ODA synthesis. j, Size exclusion chromatography overlay of HalB WT and LEFE mutant showing a rightward shift indicating a loss of tetrameric complex formation. k, Bacterial growth assay of E. coli expressing HalA with HalB active site and dimerization mutants. LEFE, L24E and F67E double mutant. l, Urea-PAGE analysis of HalB substrate and metal specificity using NTPs and α32P-labeled dATP and NTPs as indicated. m, Coomassie-stained SDS-PAGE analysis of ODA synthesis with purified HalB WT and catalytically inactive mutant (DDAA) given indicated µM concentrations of dNTP. n, Urea-PAGE analysis of ODA synthesis over a 24 h period with purified HalB given α32P-dNTP, the indicated µM concentrations of dNTP, and treated with proteinase K. Expression of HalB used in this figure was from R. bacterium QY30. Data shown are representative of at least three independent experiments.
Extended Data Fig. 2 Mechanism of ODA synthesis.
a, Urea-PAGE analysis of ODA synthesis with purified HalB and α32P-dATP supplemented with different ddNTP substrates to induce chain termination and determine specificity to adenine. b, Schematic of LC-MS workflow to determine the chemical composition of ODA. c, Left, LC-MS analysis of purified HalB WT or DDAA mutant incubated with dNTP. Right, Characterization of the extracted signal molecule by LC-MS in negative mode. Formate and chloride ions formed the major adducts [dA+formate]− and [dA+Cl]− respectively observed with deoxyadenosine. d, Uncropped Urea-PAGE analysis of ODA cleavage from the HalB–ODA complex using α32P-labeled dATP treated with proteinase K and E. coli soluble lysate fraction as in Fig. 2. α32P-labeled dAMP made with apyrase-treated dATP and nuclease P1 were used as controls to visualize single nucleotide product. e, Schematic depicting process of biochemical fraction of E. coli BL21 cell lysate to enrich for HalB–ODA cleavage activity using heparin IEX or ammonium sulfate precipitation followed by phenyl hydrophobic interaction. f, Urea-PAGE analysis of ODA cleavage from the HalB–ODA complex using fractions obtained after S200 size-exclusion chromatography. Active fractions used for mass spectrometry analysis are highlighted in bold. g, Summary of mass spectrometry results from both fractionation methods. HIC, hydrophobic-interaction chromatography; IEX, ion exchange. h, List of enriched candidates shared between both purification schemes. Data shown as mean for two independent experiments. i, Urea-PAGE analysis of ODA cleavage from the HalB–ODA complex using α32P-labeled dATP treated with purified reconstitution of candidate proteins. Two common contaminating ribosomal proteins (RplP and RplE) and one catalytically inactive protein due to a frameshift mutation (Rph) were excluded from further analysis. j, Urea-PAGE analysis of ODA cleavage from the HalB–ODA complex using α32P-labeled dATP treated with purified H-NS and StpA. k, Overview of tetrameric HalB catalytically inactive mutant crystal structure with one protomer in light blue and three protomers in different shades of grey. l, R. bacterium QY30 HalB catalytically inactive mutant was expressed as an N-terminal 6×His-SUMO2 fusion and purified by Ni-NTA and separated from 6×His-SUMO2 by size exclusion chromatography. m, Coomassie-stained SDS-PAGE analysis of fully purified HalB catalytically inactive mutant. n, Size exclusion chromatography with multi-angle static light scattering analysis of purified HalB catalytically inactive mutant. o, Overview of tetrameric HalB R164A crystal structure with one monomer in dark blue and three monomers in different shades of grey. p, Coomassie-stained SDS-PAGE analysis of fully purified HalB R164A. q, Overview of HalB and residue R164 in an open conformation prior to ODA synthesis and an active confirmation during ODA synthesis. r, Detailed view of 1) HalB residues stabilizing incoming dATP substrate and 2) polder omit map contoured at 5.5 σ of Y227 and non-hydrolyzable dATP. HalB adenine discrimination occurs through residues T129 that coordinate sequence-specific contacts with the adenine nucleobase Watson-Crick edge, and L60 and R128 that restrict guanine and pyrimidine base recognition. Detailed view of 3) newly synthesized ODA bound to HalB C-terminal tyrosine residue and 4) polder omit map contoured at 3.5 σ of dAMP bound to Y227. Expression of HalB used in this figure was from R. bacterium QY30. Data shown are representative of at least three independent experiments.
Extended Data Fig. 3 HalA substrate binding specificity.
a, R. bacterium QY30 HalA was expressed as an N-terminal 6×His fusion with untagged HalB and purified by Ni-NTA in the presence of DDM detergent and separated by size exclusion chromatography in the presence of GDN detergent. b, Coomassie-stained SDS-PAGE analysis of fully purified HalA. c, Electrophoretic mobility shift assay of HalA–ODA complex formation with indicated FAM-labeled (abbreviated as F) DNA substrates. Expression of HalA used in this figure was from R. bacterium QY30. Data shown are representative of at least three independent experiments.
Extended Data Fig. 4 Cryo-EM data processing for the HalA–ODA complex.
a, Cryo-EM data processing scheme. To facilitate visual comparison, each volume’s hand was flipped wherever necessary to match the hand of the final reconstruction. b, Left, example motion-corrected micrograph, subjected to a 5-Å long-pass filter. Right, the same image subjected to CryoSPARC’s Micrograph Denoiser. Particles present in the final stack are circled in green. c, Example 2D class averages from the particle curation stage. d, Gold-standard Fourier shell correlation (GSFSC) curves after FSC-mask auto-tightening, as produced by CryoSPARC. The horizontal black line represents the FSC = 0.143 threshold. e, Local resolution of the final map (unsharpened and without density modification).
Extended Data Fig. 5 Structural analysis of HalA.
a, Isolated HalA protomer showing regions involved in membrane interaction and formation of the ion channel (transmembrane domain α6-α10) and regions involved in ODA binding (pentapeptide repeat domain). b, Isolated HalA tetrameric ion channel region used for structural homolog comparison. c,d, Protein structural homology of HalA transmembrane domain against all entries in PDB showing the DALI Z score and FoldSeek E value of the top 130 and 1200 hits respectively. Entries with annotated ion channel activity are highlighted. e, HalA comparison to structurally related and well-characterized ion channel proteins, and corresponding PDB entry IDs are in parentheses. 2TM domain of HalA and ion channel proteins were used to highlight their structural similarities within the ion selectivity filter and transmembrane regions. Highlighted 2TM domains are shown as individual proteins next to the full structure, or overlayed altogether with HalA. f, Detailed view of HalA interacting residues required for oligomerization. g, Detailed view of the ion channel comparing the closed state (cryo-EM structure) and AlphaFold model of HalA. The AlphaFold model shows a wider conformation of the ion conduction pathway, suggestive of conformational rearrangements that would lead to an open state in the absence of ODA. h, Detailed view of HalA residues involved in recognition of ODA binding specificity. Sequence-specific interactions occur between HalA residues T83, T104, and T124 with base dA5; S141 and N143 with base dA3; and side-chains W26, K109, and F139 facilitate additional interactions that control selective ODA recognition and restrict guanine and pyrimidine base recognition.
Extended Data Fig. 6 HalA sequence analysis and growth assay.
a, Structure guided multiple sequence alignment of HalA protein homologs from indicated bacterial species. Shading indicates degree of residue conservation. b, Bacterial growth assay of E. coli expressing HalA mutants required for channel assembly with and without HalB. Expression of HalA used in this figure was from R. bacterium QY30. Data shown are representative of at least three independent experiments.
Extended Data Fig. 7 Flow cytometry and live cell imaging analysis of HalA activation.
a,b, Flow cytometry quantification of DiBAC4-positive cells (a) and PI-positive cells (b) from E. coli expressing HalA or HalAB treated with glucose, polymyxin B, or induced with arabinose for the indicated times. c, Left, gating strategy: bacterial cells were selected using side scatter height versus forward scatter height (SSC-H vs. FSC-H) and side scatter height versus width (SSC-H vs. SSC-W). Right, representative plots from cells treated with polymyxin B, or cells expressing arabinose-inducible plasmids containing HalA or HalAB. Flow cytometry data shown are collected from 100,000 events and are representative of at least two independent experiments. d,e, Live cell imaging analysis of membrane depolarization using DiBAC4 in E. coli containing plasmids expressing Hailong defence system and infected with (d) WT or escape mutant phage SECφ4 at a calculated multiplicity of infection of 10 or 40, or (e) no phage infection. Scale bars, 5 µm. f, Cellular localization of HalA visualized in E. coli expressing EGFP-HalA and periplasimic-mCherry. Scale bars, 1 µm. Expression of HalA and HalAB used in this figure was from R. bacterium QY30. Live cell imaging data shown are representative of at least three independent experiments.
Extended Data Fig. 8 Identification and structural characterization of Hailong phage escape mutants.
a, Full analysis of isolated phage escape mutants. Left, sequenced genes containing the indicated point mutations are highlighted in light green within the SECφ4 genome. Right, representative plaque assays and heatmap illustrating fold defence of E. coli expressing Hailong from E. coli STEC1178 and challenged with WT SECφ4 phage and SECφ4 escape mutant phages. b, Multiple sequence alignment of SECφ4 escape mutant gp43 proteins from indicated phage and bacterial homologs. Shading indicates degree of residue conservation. c, AlphaFold modeled structure of SECφ4 Exo (left) and comparison with human EXO5 (right) with (PDB ID: 7LW9) and without DNA (PDB ID: 7LW7). Boxes highlight nuclease active site. d, Detailed view of nuclease active residues in SECφ4 Exo (left) and human EXO5 (right).
Extended Data Fig. 9 Phylogenetic and biochemical analysis of SECφ4 Exo.
a, Phylogenetic analysis of ~1,700 SECφ4 Exo sequence homologs obtained using NCBI BLAST. b, Genera of phages encoding SECφ4 Exo. c, SECφ4 gp43 DNA exonuclease (Exo) was expressed as an N-terminal 6xHis-SUMO2 fusion and purified by Ni-NTA and separated by size exclusion chromatography. d, Coomassie-stained SDS-PAGE analysis of fully purified SECφ4 Exo. e, Analysis of 5 nt fluorescein-labeled deoxynucleotide substrates (F-dA, dA-F, dC-F), 5 nt single-stranded RNA (rA-F), or 20 bp thymidine-labeled double-stranded DNA (dA-dT-F). Direction of fluorescein (labeled as green F) tagged to the oligonucleotide indicates either a 5′-tagged DNA (indicating a free 3′ end) or a 3′-tagged DNA (indicating a free 5′ end). Data shown are representative of at least three independent experiments. f, AlphaFold modeled structure of SECφ4 Exo with escape mutant residues within scaffolding regions.
Supplementary information
Supplementary Fig. 1
Uncropped SDS–PAGE and Urea–PAGE gel images. a, Uncropped SDS–PAGE gels. Red boxes indicate cropped regions used in the indicated figures. b, Uncropped Urea–PAGE gels. Red boxes indicate cropped regions used in the indicated figures.
Supplementary Table 1
Sequence information of HalB homologs. List of IMG Gene IDs and amino acid sequences of RbHalB homologs obtained from IMG/MER.
Supplementary Table 2
Taxonomy information of HalB homologs. List of accession codes and taxonomy IDs of RbHalB homologs obtained from IMG/MER.
Supplementary Table 3
Summary of data collection, phasing and refinement and validation statistics.
Supplementary Table 4
Taxonomy information of phage Exo homologs. List of accession codes and taxonomy IDs of SECφ4 phage Exo homologs obtained from NCBI.
Supplementary Video 1
Structural model of HalA activation. Cryo-EM structure of ODA-bound HalA in a closed conformation and an AlphaFold modelled apo structure of HalA showing a wider diameter along the channel, suggesting a more open conformation. Video was prepared in PyMOL using morphing tool to illustrate transition between HalA–ODA and apo HalA.
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Tan, J.M.J., Melamed, S., Cofsky, J.C. et al. A DNA-gated molecular guard controls bacterial Hailong anti-phage defence. Nature 643, 794–800 (2025). https://doi.org/10.1038/s41586-025-09058-z
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DOI: https://doi.org/10.1038/s41586-025-09058-z
