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.

  • Article
  • Published:

Structural dynamics of bacteriophage P22 infection initiation revealed by cryo-electron tomography

Nature Microbiology volume 4, pages 1049–1056 (2019)Cite this article

Subjects

Abstract

For successful infection, bacteriophages must overcome multiple barriers to transport their genome and proteins across the bacterial cell envelope. We use cryo-electron tomography to study the infection initiation of phage P22 in Salmonella enterica serovar Typhimurium, revealing how a channel forms to allow genome translocation into the cytoplasm. Our results show free phages that initially attach obliquely to the cell through interactions between the O antigen and two of the six tailspikes; the tail needle also abuts the cell surface. The virion then orients perpendicularly and the needle penetrates the outer membrane. The needle is released and the internal head protein gp7* is ejected and assembles into an extracellular channel that extends from the gp10 baseplate to the cell surface. A second protein, gp20, is ejected and assembles into a structure that extends the extracellular channel across the outer membrane into the periplasm. Insertion of the third ejected protein, gp16, into the cytoplasmic membrane probably completes the overall trans-envelope channel into the cytoplasm. Construction of a trans-envelope channel is an essential step during infection of Gram-negative bacteria by all short-tailed phages, because such virions cannot directly deliver their genome into the cell cytoplasm.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: Tomograms reveal P22 intermediates at different stages of infection.
Fig. 2: P22 binds obliquely to the cell surface.
Fig. 3: Intermediate structures during commitment to infection.
Fig. 4: The 3D structure of the trans-envelope channel.
Fig. 5: Channel formed by E protein-defective particles.

Similar content being viewed by others

Data availability

The structures derived from cryo-ET and subtomogram averaging were deposited in the Electron Microscopy Data Bank under accession codes EMDB-9006, EMDB-9007, EMDB-9008, EMDB-9009 and EMDB-9010.

References

  1. Ackermann, H. W. Bacteriophage observations and evolution. Res. Microbiol. 154, 245–251 (2003).

    Article  CAS  Google Scholar 

  2. Casjens, S. R. & Molineux, I. J. Short noncontractile tail machines: adsorption and DNA delivery by podoviruses. Adv. Exp. Med. Biol. 726, 143–179 (2012).

    Article  CAS  Google Scholar 

  3. Davidson, A. R., Cardarelli, L., Pell, L. G., Radford, D. R. & Maxwell, K. L. Long noncontractile tail machines of bacteriophages. Adv. Exp. Med. Biol. 726, 115–142 (2012).

    Article  CAS  Google Scholar 

  4. Leiman, P. G. & Shneider, M. M. Contractile tail machines of bacteriophages. Adv. Exp. Med. Biol. 726, 93–114 (2012).

    Article  CAS  Google Scholar 

  5. Hu, B., Margolin, W., Molineux, I. J. & Liu, J. Structural remodeling of bacteriophage T4 and host membranes during infection initiation. Proc. Natl Acad. Sci. USA 112, E4919–E4928 (2015).

    Article  CAS  Google Scholar 

  6. Taylor, N. M. I. et al. Structure of the T4 baseplate and its function in triggering sheath contraction. Nature 533, 346–352 (2016).

    Article  CAS  Google Scholar 

  7. Farley, M. M., Tu, J., Kearns, D. B., Molineux, I. J. & Liu, J. Ultrastructural analysis of bacteriophage Phi29 during infection of Bacillus subtilis. J. Struct. Biol. 197, 163–171 (2017).

    Article  CAS  Google Scholar 

  8. Xu, J., Gui, M., Wang, D. & Xiang, Y. The bacteriophage ϕ29 tail possesses a pore-forming loop for cell membrane penetration. Nature 534, 544–547 (2016).

    Article  CAS  Google Scholar 

  9. Kemp, P., Garcia, L. R. & Molineux, I. J. Changes in bacteriophage T7 virion structure at the initiation of infection. Virology 340, 307–317 (2005).

    Article  CAS  Google Scholar 

  10. Kemp, P., Gupta, M. & Molineux, I. J. Bacteriophage T7 DNA ejection into cells is initiated by an enzyme-like mechanism. Mol. Microbiol. 53, 1251–1265 (2004).

    Article  CAS  Google Scholar 

  11. Chang, C. Y., Kemp, P. & Molineux, I. J. Gp15 and gp16 cooperate in translocating bacteriophage T7 DNA into the infected cell. Virology 398, 176–186 (2010).

    Article  CAS  Google Scholar 

  12. Hu, B., Margolin, W., Molineux, I. J. & Liu, J. The bacteriophage T7 virion undergoes extensive structural remodeling during infection. Science 339, 576–579 (2013).

    Article  CAS  Google Scholar 

  13. Israel, V. E proteins of bacteriophage P22. I. Identification and ejection from wild-type and defective particles. J. Virol. 23, 91–97 (1977).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Molineux, I. J. & Panja, D. Popping the cork: mechanisms of phage genome ejection. Nat. Rev. Microbiol. 11, 194–204 (2013).

    Article  CAS  Google Scholar 

  15. Susskind, M. M. & Botstein, D. Molecular genetics of bacteriophage P22. Microbiol. Rev. 42, 385–413 (1978).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Hryc, C. F. et al. Accurate model annotation of a near-atomic resolution cryo-EM map. Proc. Natl Acad. Sci. USA 114, 3103–3108 (2017).

    Article  CAS  Google Scholar 

  17. Lander, G. C. et al. The P22 tail machine at subnanometer resolution reveals the architecture of an infection conduit. Structure 17, 789–799 (2009).

    Article  CAS  Google Scholar 

  18. Chang, J., Weigele, P., King, J., Chiu, W. & Jiang, W. Cryo-EM asymmetric reconstruction of bacteriophage P22 reveals organization of its DNA packaging and infecting machinery. Structure 14, 1073–1082 (2006).

    Article  CAS  Google Scholar 

  19. Lander, G. C. et al. The structure of an infectious P22 virion shows the signal for headful DNA packaging. Science 312, 1791–1795 (2006).

    Article  CAS  Google Scholar 

  20. Tang, J. et al. Peering down the barrel of a bacteriophage portal: the genome packaging and release valve in p22. Structure 19, 496–502 (2011).

    Article  CAS  Google Scholar 

  21. Olia, A. S., Prevelige, P. E. Jr, Johnson, J. E. & Cingolani, G. Three-dimensional structure of a viral genome-delivery portal vertex. Nat. Struct. Mol. Biol. 18, 597–603 (2011).

    Article  CAS  Google Scholar 

  22. Steinbacher, S. et al. Crystal structure of P22 tailspike protein: interdigitated subunits in a thermostable trimer. Science 265, 383–386 (1994).

    Article  CAS  Google Scholar 

  23. Steinbacher, S. et al. Crystal structure of phage P22 tailspike protein complexed with Salmonella sp. O-antigen receptors. Proc. Natl Acad. Sci. USA 93, 10584–10588 (1996).

    Article  CAS  Google Scholar 

  24. Steinbacher, S. et al. Phage P22 tailspike protein: crystal structure of the head-binding domain at 2.3 A, fully refined structure of the endorhamnosidase at 1.56 A resolution, and the molecular basis of O-antigen recognition and cleavage. J. Mol. Biol. 267, 865–880 (1997).

    Article  CAS  Google Scholar 

  25. Olia, A. S., Casjens, S. & Cingolani, G. Structure of phage P22 cell envelope-penetrating needle. Nat. Struct. Mol. Biol. 14, 1221–1226 (2007).

    Article  CAS  Google Scholar 

  26. Pintilie, G., Chen, D. H., Haase-Pettingell, C. A., King, J. A. & Chiu, W. Resolution and probabilistic models of components in cryoEM maps of mature P22 bacteriophage. Biophys. J. 110, 827–839 (2016).

    Article  CAS  Google Scholar 

  27. Andres, D. et al. Tailspike interactions with lipopolysaccharide effect DNA ejection from phage P22 particles in vitro. J. Biol. Chem. 285, 36768–36775 (2010).

    Article  CAS  Google Scholar 

  28. Berget, P. B. & Poteete, A. R. Structure and functions of the bacteriophage P22 tail protein. J. Virol. 34, 234–243 (1980).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. King, J., Lenk, E. V. & Botstein, D. Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. II. Morphogenetic pathway. J. Mol. Biol. 80, 697–731 (1973).

    Article  CAS  Google Scholar 

  30. Lenk, E., Casjens, S., Weeks, J. & King, J. Intracellular visualization of precursor capsids in phage P22 mutant infected cells. Virology 68, 182–199 (1975).

    Article  CAS  Google Scholar 

  31. Botstein, D., Waddell, C. H. & King, J. Mechanism of head assembly and DNA encapsulation in Salmonella phage p22. I. Genes, proteins, structures and DNA maturation. J. Mol. Biol. 80, 669–695 (1973).

    Article  CAS  Google Scholar 

  32. Strauss, H. & King, J. Steps in the stabilization of newly packaged DNA during phage P22 morphogenesis. J. Mol. Biol. 172, 523–543 (1984).

    Article  CAS  Google Scholar 

  33. Conlin, C. A., Trun, N. J., Silhavy, T. J. & Miller, C. G. Escherichia coli prlC encodes an endopeptidase and is homologous to the Salmonella Typhimurium opdA gene. J. Bacteriol. 174, 5881–5887 (1992).

    Article  CAS  Google Scholar 

  34. Conlin, C. A., Vimr, E. R. & Miller, C. G. Oligopeptidase A is required for normal phage P22 development. J. Bacteriol. 174, 5869–5880 (1992).

    Article  CAS  Google Scholar 

  35. Jin, Y. et al. Bacteriophage P22 ejects all of its internal proteins before its genome. Virology 485, 128–134 (2015).

    Article  CAS  Google Scholar 

  36. Kastowsky, M., Gutberlet, T. & Bradaczek, H. Molecular modelling of the three-dimensional structure and conformational flexibility of bacterial lipopolysaccharide. J. Bacteriol. 174, 4798–4806 (1992).

    Article  CAS  Google Scholar 

  37. Goldman, R. C. & Leive, L. Heterogeneity of antigenic-side-chain length in lipopolysaccharide from Escherichia coli 0111 and Salmonella Typhimurium LT2. Eur. J. Biochem. 107, 145–153 (1980).

    Article  CAS  Google Scholar 

  38. Palva, E. T. & Makela, P. H. Lipopolysaccharide heterogeneity in Salmonella typhimurium analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Eur. J. Biochem. 107, 137–143 (1980).

    Article  CAS  Google Scholar 

  39. Baxa, U. et al. Interactions of phage P22 tails with their cellular receptor, Salmonella O-antigen polysaccharide. Biophys. J. 71, 2040–2048 (1996).

    Article  CAS  Google Scholar 

  40. Israel, V. A model for the adsorption of phage P22 to Salmonella typhimurium. J. Gen. Virol. 40, 669–673 (1978).

    Article  CAS  Google Scholar 

  41. Israel, V., Rosen, H. & Levine, M. Binding of bacteriophage P22 tail parts to cells. J. Virol. 10, 1152–1158 (1972).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Tu, J. G. et al. Dual host specificity of phage SP6 is facilitated by tailspike rotation. Virology 507, 206–215 (2017).

    Article  CAS  Google Scholar 

  43. Leavitt, J. C. et al. The tip of the tail needle affects the rate of DNA delivery by bacteriophage P22. PLoS ONE 8, e70936 (2013).

    Article  CAS  Google Scholar 

  44. Israel, V. Role of the bacteriophage P22 tail in the early stages of infection. J. Virol. 18, 361–364 (1976).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Perez, G. L., Huynh, B., Slater, M. & Maloy, S. Transport of phage P22 DNA across the cytoplasmic membrane. J. Bacteriol. 191, 135–140 (2009).

    Article  CAS  Google Scholar 

  46. Zhao, H. et al. Structure of a bacterial virus DNA-injection protein complex reveals a decameric assembly with a constricted molecular channel. PLoS ONE 11, e0149337 (2016).

    Article  Google Scholar 

  47. Botstein, D., Chan, R. K. & Waddell, C. H. Genetics of bacteriophage P22. II. Gene order and gene function. Virology 49, 268–282 (1972).

    Article  CAS  Google Scholar 

  48. Poteete, A. R. & King, J. Functions of two new genes in Salmonella phage P22 assembly. Virology 76, 725–739 (1977).

    Article  CAS  Google Scholar 

  49. Farley, M. M., Hu, B., Margolin, W. & Liu, J. Minicells, back in fashion. J. Bacteriol. 198, 1186–1195 (2016).

    Article  CAS  Google Scholar 

  50. Reeve, J. N. Bacteriophage infection of minicells: a general method for identification of “in vivo” bacteriophage directed polypeptide biosynthesis. Mol. Gen. Genet. 158, 73–79 (1977).

    Article  CAS  Google Scholar 

  51. Roozen, K. J., Fenwick, R. G. Jr. & Curtiss, R.3rd. Synthesis of ribonucleic acid and protein in plasmid-containing minicells of Escherichia coli K-12. J. Bacteriol. 107, 21–33 (1971).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  Google Scholar 

  53. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  Google Scholar 

  54. Mastronarde, D. N. & Held, S. R. Automated tilt series alignment and tomographic reconstruction in IMOD. J. Struct. Biol. 197, 102–113 (2017).

    Article  Google Scholar 

  55. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    Article  CAS  Google Scholar 

  56. Agulleiro, J. I. & Fernandez, J. J. Tomo3D 2.0—exploitation of advanced vector extensions (AVX) for 3D reconstruction. J. Struct. Biol. 189, 147–152 (2015).

    Article  Google Scholar 

  57. Hrabe, T. et al. PyTom: a python-based toolbox for localization of macromolecules in cryo-electron tomograms and subtomogram analysis. J. Struct. Biol. 178, 177–188 (2012).

    Article  CAS  Google Scholar 

  58. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Casjens for providing phage strains and amber mutant sequence data. We also thank M. M. Susskind and A. R. Poteete for phage and bacterial strains. This work was supported by grant nos. GM124378 and GM110243 to I.J.M. and J.L. C.W., J.T. and J.L. were also supported in part by grant no. AI087946 from the NIAID and grant no. AU-1714 from the Welch Foundation.

Author information

Author notes

  1. These authors contributed equally: Chunyan Wang, Jiagang Tu.

Authors and Affiliations

  1. Department of Microbial Pathogenesis, Yale University, New Haven, CT, USA

    Chunyan Wang & Jun Liu

  2. Microbial Sciences Institute, Yale University, West Haven, CT, USA

    Chunyan Wang & Jun Liu

  3. Department of Pathology and Laboratory Medicine, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX, USA

    Chunyan Wang, Jiagang Tu & Jun Liu

  4. LaMontagne Center for Infectious Disease, Institute for Cell and Molecular Biology, Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA

    Ian J. Molineux

Authors
  1. Chunyan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jiagang Tu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jun Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ian J. Molineux
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.L. and I.J.M. designed the research. C.W., J.T., I.J.M. and J.L. prepared the samples and collected and analysed the data. C.W., J.T., J.L. and I.J.M. wrote the manuscript.

Corresponding author

Correspondence to Ian J. Molineux.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Supplementary information

Supplementary Information

Supplementary Figures 1–4 and Supplementary Table 1.

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, C., Tu, J., Liu, J. et al. Structural dynamics of bacteriophage P22 infection initiation revealed by cryo-electron tomography. Nat Microbiol 4, 1049–1056 (2019). https://doi.org/10.1038/s41564-019-0403-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41564-019-0403-z

This article is cited by

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