Multi-interface licensing of protein import into a phage nucleus (original) (raw)

Data availability

All genotypes from sequenced ΦKZ phages are included in Supplementary Table 1. Source data are available for Figs. 1–4 and Extended Data Figs. 1 and 3–6 in Supplementary Figs. 1 and 2. The Protein Data Bank was queried when running DALI from the DALI web server (http://ekhidna2.biocenter.helsinki.fi/dali/), and can can be accessed here: https://www.rcsb.org/.

References

  1. Chaikeeratisak, V. et al. Assembly of a nucleus-like structure during viral replication in bacteria. Science 355, 194–197 (2017).
    Article CAS PubMed Central MATH ADS PubMed Google Scholar
  2. Chaikeeratisak, V. et al. The phage nucleus and tubulin spindle are conserved among large Pseudomonas phages. Cell Rep. 20, 1563–1571 (2017).
    Article CAS PubMed Central MATH PubMed Google Scholar
  3. Mendoza, S. D. et al. A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases. Nature 577, 244–248 (2020).
    Article CAS MATH ADS PubMed Google Scholar
  4. Malone, L. M. et al. A jumbo phage that forms a nucleus-like structure evades CRISPR-Cas DNA targeting but is vulnerable to type III RNA-based immunity. Nat. Microbiol. 5, 48–55 (2020).
    Article CAS PubMed Google Scholar
  5. Laughlin, T. G. et al. Architecture and self-assembly of the jumbo bacteriophage nuclear shell. Nature 608, 429–435 (2022).
    Article CAS PubMed Central MATH ADS PubMed Google Scholar
  6. Prichard, A. et al. Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY. Cell Rep. 42, 112432 (2023).
    Article CAS PubMed Central MATH PubMed Google Scholar
  7. Weintraub, S. T. et al. Global proteomic profiling of Salmonella infection by a giant phage. J. Virol. 93, e01833-18 (2019).
    Article CAS PubMed Central MATH PubMed Google Scholar
  8. Jacquemot, L. et al. Therapeutic potential of a new jumbo phage that infects Vibrio coralliilyticus, a widespread coral pathogen. Front. Microbiol. 9, 2501 (2018).
    Article PubMed Central MATH PubMed Google Scholar
  9. Enustun, E. et al. Identification of the bacteriophage nucleus protein interaction network. Nat. Struct. Mol. Biol. 30, 1653–1662 (2023).
    Article CAS PubMed Central MATH PubMed Google Scholar
  10. Nieweglowska, E. S. et al. The ϕPA3 phage nucleus is enclosed by a self-assembling 2D crystalline lattice. Nat. Commun. 14, 927 (2023).
    Article CAS PubMed Central MATH ADS PubMed Google Scholar
  11. Heymann, J. B. et al. The mottled capsid of the Salmonella giant phage SPN3US, a likely maturation intermediate with a novel internal shell. Viruses 12, 910 (2020).
    Article CAS PubMed Central PubMed Google Scholar
  12. Lin, D. H. & Hoelz, A. The structure of the nuclear pore complex (an update). Annu. Rev. Biochem. 88, 725–783 (2019).
    Article CAS PubMed Central MATH PubMed Google Scholar
  13. Fossati, A. et al. Next-generation proteomics for quantitative jumbophage-bacteria interaction mapping. Nat. Commun. 14, 5156 (2023).
    Article CAS PubMed Central MATH ADS PubMed Google Scholar
  14. Enustun, E. et al. A phage nucleus-associated RNA-binding protein is required for jumbo phage infection. Nucleic Acids Res. 52, 4440–4455 (2024).
    Article CAS PubMed Central MATH PubMed Google Scholar
  15. Mirdita, M. et al. ColabFold: making protein folding accessible to all. Nat. Methods 19, 679–682 (2022).
    Article CAS PubMed Central MATH PubMed Google Scholar
  16. Putzeys, L. et al. Refining the transcriptional landscapes for distinct clades of virulent phages infecting Pseudomonas aeruginosa. Microlife 5, uqae002 (2024).
    Article CAS PubMed Central PubMed Google Scholar
  17. Yakunina, M. et al. A non-canonical multisubunit RNA polymerase encoded by a giant bacteriophage. Nucleic Acids Res. 43, 10411–10420 (2015).
    CAS PubMed Central MATH PubMed Google Scholar
  18. Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 63, 493–500 (2024).
    Article MATH ADS Google Scholar
  19. Gerovac, M. et al. Non-genetic messenger RNA silencing reveals essential genes in phage-host interplay. Preprint at bioRxiv https://doi.org/10.1101/2024.07.31.605949 (2024).
  20. Morgan, C. et al. An essential and highly selective protein import pathway encoded by nucleus-forming phage. Proc. Natl Acad. Sci. USA 121, e2321190121 (2024).
    Article CAS PubMed Central PubMed Google Scholar
  21. Qiu, D., Damron, F. H., Mima, T., Schweizer, H. P. & Yu, H. D. PBAD-based shuttle vectors for functional analysis of toxic and highly regulated genes in Pseudomonas and Burkholderia spp. and other bacteria. Appl. Environ. Microbiol. 74, 7422–7426 (2008).
    Article CAS PubMed Central ADS PubMed Google Scholar
  22. Choi, K.-H. & Schweizer, H. P. mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa. Nat. Protoc. 1, 153–161 (2006).
    Article CAS PubMed Google Scholar
  23. Choi, K.-H. et al. Genetic tools for select-agent-compliant manipulation of Burkholderia pseudomallei. Appl. Environ. Microbiol. 74, 1064–1075 (2008).
    Article CAS MATH ADS PubMed Google Scholar
  24. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
    Article CAS PubMed Central MATH PubMed Google Scholar
  25. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
    Article CAS MATH PubMed Google Scholar
  26. Holm, L., Laiho, A., Törönen, P. & Salgado, M. DALI shines a light on remote homologs: one hundred discoveries. Protein Sci. 32, e4519 (2023).
    Article CAS PubMed Central PubMed Google Scholar

Download references

Acknowledgements

J.B.-D. is supported by the National Institutes of Health (nos. R01 AI171041 and R01 AI167412). C.K. received support from the UCSF Discovery Fellowship. We thank Bondy–Denomy laboratory members for input into this work, including I. Fedorova for help in running FastTree. We additionally thank D. Agard, E. Nieweglowska, C. Gross and A. Davidson for vital input.

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA, USA
    Claire Kokontis, Timothy A. Klein, Sukrit Silas & Joseph Bondy-Denomy
  2. Quantitative Biosciences Institute, University of California San Francisco, San Francisco, CA, USA
    Joseph Bondy-Denomy

Authors

  1. Claire Kokontis
    You can also search for this author inPubMed Google Scholar
  2. Timothy A. Klein
    You can also search for this author inPubMed Google Scholar
  3. Sukrit Silas
    You can also search for this author inPubMed Google Scholar
  4. Joseph Bondy-Denomy
    You can also search for this author inPubMed Google Scholar

Contributions

C.K. and J.B.-D. conceived the project and designed experiments. C.K. executed phage genetic screens, sequencing, bacterial genetics, fluorescence microscopy, Imp1–Nlp2 biochemical experiments and associated data analysis. T.A.K. executed Imp1–6 biochemical experiments. S.S. provided assistance with sequencing and analysis of phage mutants. J.B.-D. and C.K. wrote the manuscript, and all authors edited it. J.B.-D. supervised experiments and procured funding.

Corresponding author

Correspondence toJoseph Bondy-Denomy.

Ethics declarations

Competing interests

J.B.-D. is a scientific advisory board member of SNIPR Biome and Excision Biotherapeutics, a consultant to LeapFrog Bio and a scientific advisory board member and cofounder of Acrigen Biosciences and ePhective Therapeutics. S.S. is cofounder and equity holder in BillionToOne, Inc. and a scientific advisory board member for Junevity, Inc. The remaining authors declare no competing interests. The Bondy–Denomy laboratory received past research support from Felix Biotechnology.

Peer review

Peer review information

Nature thanks Alexander Hynes, Ulrich Kubitscheck and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

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

Extended data figures and tables

Extended Data Fig. 1 imp1 and imp2 are required for protein import.

a, Efficiency of plating (EOP) of WT ΦKZ on EcoRI fusions, quantified as the number of plaque forming units (PFU)/mL on PAO1 expressing the indicated EcoRI fusion relative to PFU/mL on a nontargeting EcoRI strain. Closed circles indicate plaques that could be counted. Open circles indicate that no plaques were observed, and an arbitrary value of 1 plaque was recorded to calculate a non-zero limit of detection. Variation in plaquing efficiencies and limits of detection on a given strain result from different titers of the phage stock when the test was done. Bar heights represent the mean EOP between replicates where plaques could be counted. EcoRI-Nlp1 n = 5 independent biological replicates, Imp2[EcoRI-Nlp1] n = 4, EcoRI-Nlp2 n = 5, EcoRI-Nlp3 n = 3, EcoRI-Nlp4 n = 3, EcoRI-Imp2 n = 3, Imp1[EcoRI-Imp2] n = 4, EcoRI-Imp1ΦPA3 n = 3, EcoRI-TopA n = 3, EcoRI-Imp1ΦKZ n = 2. b-d, Plaque assays with WT ΦKZ or WT 14-1 (EcoRI-sensitive phage) (b) or WT ΦKZ and mutant phage (c, d) spotted in 10-fold serial dilutions on a lawn of PAO1 expressing indicated sfCherry2 fusions or an empty vector control (EV), with or without expression of a phage gene in trans from the bacterial attTn7 site (Tn7::impX) (c, d). e, Representation of EOP of WT ΦKZ and EcoRI-Nlp1 resistant mutant phages on PAO1 expressing EcoRI-Nlp1-4. EOP was calculated as the number of PFU/mL on the EcoRI-Nlp strain relative to PFU/mL on the non-targeting dead EcoRI-Nlp1 (dEcoRI-Nlp1) strain. EOP for each phage/strain pair is colored by mean EOP between three independent biological replicates. f, Imp2 phylogenetic tree. g, Plaque assays with WT ΦPA3 or mutant phage spotted in 10-fold serial dilutions on a lawn of PAO1 expressing indicated sfCherry2 fusions. Plaque assays were performed two (b, g) or three (c, d) independent times in biological replicates with similar results. Please see Supplementary Data 1 for source data underlying graphical representations (a, e).

Extended Data Fig. 2 Mutations in imp1 and imp2 decrease nuclear protein localization.

a-e, Representative images of live-cell fluorescence microscopy of PAO1 expressing the indicated sfCherry2 fusions, infected with WT or indicated mutant ΦKZ (EcoRI+WT ΦKZ, n = 118 cells. EcoRI-Nlp2+WT ΦKZ, n = 102. EcoRI-Nlp2+imp1 E310G ΦKZ, n = 230. EcoRI-Nlp1+WT ΦKZ, n = 69. EcoRI-Nlp1+imp2 K45N, n = 58). Scale bars, 1 µm. “Excluded” refers to localization of sfCherry2-fused proteins outside of the phage nucleus. Microscopy was performed as in Fig. 1c and replicated two (EcoRI-Nlp1+imp2 K45N) or three (EcoRI+WT ΦKZ, EcoRI-Nlp2+WT ΦKZ, EcoRI-Nlp2+imp1 E310G ΦKZ, EcoRI-Nlp1+WT ΦKZ) independent times in biological replicates with similar results.

Extended Data Fig. 3 Imp1-mNG localizes early near the site of genome injection and remains with the phage nucleus.

a, Live-cell fluorescence microscopy of PAO1 expressing Imp1-mNeonGreen (Imp1-mNG) from the attTn7 site, infected with WT ΦKZ (Top eight panels, n = 154 cells; scale bar, 1 µm) or uninfected (bottom panel, n = 82 cells; scale bar, 2 µm). b, Live-cell time-lapse fluorescence microscopy of PAO1 attTn7::Imp1-mNG infected with WT ΦKZ. n = 102 cells; scale bar, 1 µm. “t = ” indicates time in minutes after injected phage DNA is first seen as puncta at the cell pole. Microscopy was performed three independent times in biological replicates with similar results. c, Plaque assays with the indicated WT or mutant phage spotted in 10-fold serial dilutions on a lawn of PAO1 expressing the indicated sfCherry2 fusions, with or without expression of the appropriate phage gene in trans from the bacterial attTn7 site (Tn7::ImpX). Plaque assays were performed as in Fig. 1b and replicated two independent times in biological replicates with similar results.

Extended Data Fig. 4 imp1 and imp3 mutational analysis.

a, Plaque assays with WT ΦKZ or WT 14-1 (EcoRI-sensitive phage) spotted in 10-fold serial dilutions on a lawn of PAO1 expressing the indicated sfCherry2 fusions. b, Top output Imp1 model from AlphaFold 2, with mutated residues from isolated imp1 mutant phages color coded by the EcoRI selection from which they were isolated. c, Overlay of all five output Imp1 models from AlphaFold 2, colored by confidence scores. d, Surface map of electrostatic potential (semi-transparent overlay) of the top Imp1 predicted structural model from the same view (top), or rotated 90° view (bottom). Several mutated residues are indicated. e, Plaque assays with the indicated WT or mutant phage spotted on PAO1 expressing the indicated sfCherry2 fusions, with or without Imp3 or the Imp3 operon (p18-imp3-orf60-orf61) expressed in trans. Plaque assays were performed as in Fig. 1b and replicated two (a) or three (e, using 1 or 2 mM IPTG to induce expression from the attTn7 site) independent times in biological replicate with similar results.

Extended Data Fig. 5 Import of host protein TopA requires imp1, imp3, imp4, and imp5.

a, Overlay of all five output Imp3 models from AlphaFold 2, colored by confidence scores. b, Imp4 and Imp5 phylogenetic trees. c, Conservation of Imp homologs across nucleus-forming jumbo phages. d, Plaque assays with WT ΦKZ or WT 14-1 (EcoRI-sensitive phage) spotted in 10-fold serial dilutions on a lawn of PAO1 expressing the indicated sfCherry2 fusions. e, Plaque assays with the indicated WT or mutant phage spotted in 10-fold serial dilutions on a lawn of PAO1 expressing the indicated sfCherry2 fusions, with or without individual or combinations of Imp1-5 or the Imp3 operon (p18-imp3-orf60-orf61) expressed in trans. Plaque assays were performed as in Fig. 1b and in two (d) or three (e) independent biological replicates with similar results.

Extended Data Fig. 6 The Nlp2 C-terminal domain is predicted to bind at the Nlp2-specific Imp1 interface, and is sufficient for import into the phage nucleus.

a, Top left, top Imp1-Nlp2 model output from AlphaFold3 is displayed. Imp1 is colored in grey and Nlp2 is colored in purple, and Nlp2 N-terminal domain (NTD) and C-terminal domain (CTD) are indicated. All five models output from AlphaFold3 were aligned and are structurally similar, but only the first model is shown for clarity. Bottom left, top model overlayed with confidence scores, colored from high (blue) to low (red) confidence (ipTM = 0.74, pTM = 0.73). Right, boxed views show Imp1 residue positions mutated under selection with EcoRI-Nlp2 (pink) at the predicted interface between Imp1 and Nlp2. Non-carbon atoms are colored according to identity (oxygen in red, nitrogen in blue, sulfur in yellow). b, Plaque assays with WT ΦKZ or WT F8 (EcoRI-sensitive phage) spotted in 10-fold serial dilutions on a lawn of PAO1 expressing the indicated EcoRI fusions. FL, full length. NTD, N-terminal domain (residues 1-200). CTD, C-terminal domain (residues 201-482). Plaque assays were repeated three independent times in biological replicates with similar results. c, Model for how multiple cargo-specific Imp1 interfaces may facilitate cargo import into the phage nucleus through the nuclear wall (ChmA). Independent Imp1 interfaces are colored and shaped differently to indicate distinct cargo compatibilities. An Imp6 dimer is shown bound to Imp1, and a hypothetical interaction between Imp1 and Imp3 is also depicted. d, Nlp2 residues positioned at the predicted interface with Imp1 (residues 345-366, highlighted in blue) and Nlp3 proposed import signal (residues 77−9520, highlighted in yellow).

Supplementary information

Supplementary Figures

Supplementary Fig. 1 (pages 1–5): five display items showing uncropped and unprocessed blot and gel source data with protein ladders, with relevant samples identified and legends shown under the figures, along with any other additional notes. Black boxes around protein bands indicate how the image was cropped for the final figure. Supplementary Fig. 2 (pages 6–15): ten display items showing uncropped and unprocessed plate source images, with relevant samples identified. Black boxes within the plates and around phage plaques indicate how the image was cropped for the final figure.

Reporting Summary

Supplementary Table 1

Genotypes of all phage mutants isolated in this study, including nucleotide changes, amino acid substitutions and method of sequencing used to confirm the mutation.

Supplementary Data 1

Plaque counts, PFU ml−1 calculations and efficiency of plating calculations used to generate graphical representations in Extended Data 1a,e (one tab for each Extended Data figure).

Peer Review File

Supplementary Video 1

Time-lapse video of WT ΦKZ infection of PAO1 expressing Imp1–mNG; Imp1–mNG is coloured in green, with phage DNA stained with DAPI (blue). Time stamp indicates the progression of the time lapse in minutes.

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

Cite this article

Kokontis, C., Klein, T.A., Silas, S. et al. Multi-interface licensing of protein import into a phage nucleus.Nature (2025). https://doi.org/10.1038/s41586-024-08547-x

Download citation