Letter | Published:

The bacteriophage ϕ29 tail possesses a pore-forming loop for cell membrane penetration

Nature volume 534, pages 544547 (23 June 2016) | Download Citation


Most bacteriophages are tailed bacteriophages with an isometric or a prolate head attached to a long contractile, long non-contractile, or short non-contractile tail1. The tail is a complex machine that plays a central role in host cell recognition and attachment, cell wall and membrane penetration, and viral genome ejection. The mechanisms involved in the penetration of the inner host cell membrane by bacteriophage tails are not well understood. Here we describe structural and functional studies of the bacteriophage ϕ29 tail knob protein gene product 9 (gp9). The 2.0 Å crystal structure of gp9 shows that six gp9 molecules form a hexameric tube structure with six flexible hydrophobic loops blocking one end of the tube before DNA ejection. Sequence and structural analyses suggest that the loops in the tube could be membrane active. Further biochemical assays and electron microscopy structural analyses show that the six hydrophobic loops in the tube exit upon DNA ejection and form a channel that spans the lipid bilayer of the membrane and allows the release of the bacteriophage genomic DNA, suggesting that cell membrane penetration involves a pore-forming mechanism similar to that of certain non-enveloped eukaryotic viruses2,3,4. A search of other phage tail proteins identified similar hydrophobic loops, which indicates that a common mechanism might be used for membrane penetration by prokaryotic viruses. These findings suggest that although prokaryotic and eukaryotic viruses use apparently very different mechanisms for infection, they have evolved similar mechanisms for breaching the cell membrane.

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Electron Microscopy Data Bank

Data deposits

The atomic coordinates and structure factor files have been deposited into the Protein Data Bank (PDB) under accession numbers 5FB4, 5FB5 and 5FEI. The electron microscopy maps have been deposited into the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-6556, EMD-6557 and EMD-6558.


  1. 1.

    Bacteriophage observations and evolution. Res. Microbiol. 154, 245–251 (2003)

  2. 2.

    et al. Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore. PLoS Pathog. 10, e1004294 (2014)

  3. 3.

    , , & The VP4 peptide of hepatitis A virus ruptures membranes through formation of discrete pores. J. Virol. 88, 12409–12421 (2014)

  4. 4.

    et al. NMR structure of a viral peptide inserted in artificial membranes: a view on the early steps of the birnavirus entry process. J. Biol. Chem. 285, 19409–19421 (2010)

  5. 5.

    et al. Assembly of a tailed bacterial virus and its genome release studied in three dimensions. Cell 95, 431–437 (1998)

  6. 6.

    & in Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics (eds , & ) 859–867 (American Society for Microbiology, 1993)

  7. 7.

    et al. Conservation of the capsid structure in tailed dsDNA bacteriophages: the pseudoatomic structure of ϕ29. Mol. Cell 18, 149–159 (2005)

  8. 8.

    , & ϕ29 family of phages. Microbiol. Mol. Biol. Rev. 65, 261–287 (2001)

  9. 9.

    et al. Structural changes of bacteriophage ϕ29 upon DNA packaging and release. EMBO J. 25, 5229–5239 (2006)

  10. 10.

    et al. DNA poised for release in bacteriophage ϕ29. Structure 16, 935–943 (2008)

  11. 11.

    et al. Crystal and cryoEM structural studies of a cell wall degrading enzyme in the bacteriophage ϕ29 tail. Proc. Natl Acad. Sci. USA 105, 9552–9557 (2008)

  12. 12.

    et al. Crystallographic insights into the autocatalytic assembly mechanism of a bacteriophage tail spike. Mol. Cell 34, 375–386 (2009)

  13. 13.

    et al.Shared catalysis in virus entry and bacterial cell wall depolymerization. J. Mol. Biol. 387, 607–618 (2009)

  14. 14.

    , , , & Multifunctional roles of a bacteriophage ϕ29 morphogenetic factor in assembly and infection. J. Mol. Biol. 378, 804–817 (2008)

  15. 15.

    et al.Phages have adapted the same protein fold to fulfill multiple functions in virion assembly. Proc. Natl Acad. Sci. USA 107, 14384–14389 (2010)

  16. 16.

    Using Situs for the integration of multi-resolution structures. Biophys. Rev. 2, 21–27 (2010)

  17. 17.

    , , & . HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nat. Methods 9, 173–175 (2011)

  18. 18.

    & Cell entry by non-enveloped viruses. Curr. Top. Microbiol. Immunol. 343, v–vii (2010)

  19. 19.

    et al. FisB mediates membrane fission during sporulation in Bacillus subtilis. Genes Dev. 27, 322–334 (2013)

  20. 20.

    & Structure of bacteriophage ϕ29 head fibers has a supercoiled triple repeating helix-turn-helix motif. Proc. Natl Acad. Sci. USA 108, 4806–4810 (2011)

  21. 21.

    et al. High-resolution comparative modeling with RosettaCM. Structure 21, 1735–1742 (2013)

  22. 22.

    , , & Rapid orientated cloning in a shuttle vector allowing modulated gene expression in Bacillus subtilis. FEMS Microbiol. Lett. 205, 91–97 (2001)

  23. 23.

    , , & Transcriptome analysis documents induced competence of Bacillus subtilis during nitrogen limiting conditions. FEMS Microbiol. Lett. 206, 197–200 (2002)

  24. 24.

    et al. Chemically functionalized carbon films for single molecule imaging. J. Struct. Biol. 185, 405–417 (2014)

  25. 25.

    3-D structures of macromolecules using single-particle analysis in EMAN. Methods Mol. Biol. 673, 157–173 (2010)

  26. 26.

    & Prevention of overfitting in cryo-EM structure determination. Nat. Methods 9, 853–854 (2012)

  27. 27.

    , , , & Molecular dynamics flexible fitting: a practical guide to combine cryo-electron microscopy and X-ray crystallography. Methods 49, 174–180 (2009)

  28. 28.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

  29. 29.

    A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008)

  30. 30.

    , , , & Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr. D 59, 2023–2030 (2003)

  31. 31.

    , & in International Tables for Crystallography Vol. F (eds & ) 507–510 (Kluwer Academic Publishers, 2000)

  32. 32.

    & The detection of sub-units within the crystallographic asymmetric unit. Acta Crystallogr. 15, 24–31 (1962)

  33. 33.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

  34. 34.

    et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

  35. 35.

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

  36. 36.

    , , & ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305–308 (1999)

  37. 37.

    & PrDOS: prediction of disordered protein regions from amino acid sequence. Nucleic Acids Res. 35, W460–W464 (2007)

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We thank L. Q. Zhang, N. Yan, H. T. Li, S. L. Fan, N. Gao, C. Z. Zhou, D. L. Anderson and M. G. Rossmann for support; the Tsinghua University Branch of the China National Center for Protein Sciences for the facility support; and the staff at the Shanghai Synchrotron Research Facility beam line BL17U for assistance with data collection. This work was supported by funds from the 973 program (2015CB910102), the National Natural Science Foundation of China (31470721 and 81550001), the Junior Thousand Talents Program of China (20131770418) and the Beijing Advanced Innovation Center for Structural Biology to Y.X.

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  1. Centre for Infectious Diseases Research, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Beijing Advanced Innovation Center for Structural Biology, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China

    • Jingwei Xu
    • , Miao Gui
    • , Dianhong Wang
    •  & Ye Xiang


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J.X. and Y.X. designed the research; J.X., M.G., D.W. and Y.X. performed the experiments; J.X. and Y.X. analysed the data and wrote the paper; and all authors contributed to the editing of the manuscript.

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The authors declare no competing financial interests.

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Correspondence to Ye Xiang.

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