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.

  • Review Article
  • Published:

Popping the cork: mechanisms of phage genome ejection

Nature Reviews Microbiology volume 11, pages 194–204 (2013)Cite this article

Subjects

Key Points

  • The double-stranded DNA genomes of most tailed phages are contained within the capsid at a concentration of ∼500 mg per ml.

  • The DNA inside the capsid is in the B form, arranged on a closely packed but imperfect hexagonal lattice. Packaging is thought to occur from the outside of the spool inwards. The energy for packaging DNA into the capsid is derived from the hydrolysis of ATP by terminase, a packaging nanomotor. Terminase can generate a force of up to 100 pN.

  • At this concentration, DNA is highly condensed, and much of the water that normally hydrates the DNA and its counterions must be removed in order to package a complete genome. Water removal during the packaging process occurs by reverse osmosis, the energy for which is derived from the activity of terminase.

  • Only about 10% of the energy stored in the packaged phage DNA is due to bending DNA more tightly than its persistence length. Most of the energy internal to the capsid is due to the dehydrated state of the DNA, which is reflected by internal osmotic pressures that reach tens of atmospheres.

  • The continuum mechanics model posits that the energy stored in the packaged DNA is used to drive ejection of the phage genome. This model is supported by the experimental suppression of DNA ejection through the application of an increased external osmotic pressure.

  • The hydrodynamic model of phage genome ejection proposes that following opening of the exit channel, water diffuses through the capsid shell to neutralize the osmotic imbalance, and DNA is pushed out by the hydrostatic pressure gradient across the tail. The data used to support the continuum mechanics model for in vitro ejection is also fully consistent with the hydrodynamic model.

  • According to the continuum mechanics model, during infection of a bacterial cell, the osmotic pressure of the cytoplasm opposes phage DNA ejection driven by forces internal to the phage capsid; only about half the genome, at most, can enter the cell by this process. Various ad hoc secondary mechanisms have been proposed to complete the infection process. The hydrodynamic model has no such problem, as continued water flow up the osmotic gradient (from the growth medium, through the capsid and into the cytoplasm) provides the necessary force for complete genome transport into the cell.

  • A general mechanism of phage genome ejection in vivo can be proposed, in which the width of the exit channel determines whether hydrodynamic flow can facilitate genome ejection. When the channel is wide enough for water and ions to pass through at the same time as the double-stranded DNA genome, then hydrodynamic flow is sufficient; this is likely to be the pathway used by most phage types. However, if the exit channel is only wide enough for double-stranded DNA alone, then energy-requiring motors are necessary to translocate the infecting phage genome into the cell.

Abstract

Sixty years after Hershey and Chase showed that nucleic acid is the major component of phage particles that is ejected into cells, we still do not fully understand how the process occurs. Advances in electron microscopy have revealed the structure of the condensed DNA confined in a phage capsid, and the mechanisms and energetics of packaging a phage genome are beginning to be better understood. Condensing DNA subjects it to high osmotic pressure, which has been suggested to provide the driving force for its ejection during infection. However, forces internal to a phage capsid cannot, alone, cause complete genome ejection into cells. Here, we describe the structure of the DNA inside mature phages and summarize the current models of genome ejection, both in vitro and in vivo.

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

Figure 1: Genome internalization by representative phage types.
Figure 2: DNA structures inside phage capsids.
Figure 3: DNA structural transitions during genome ejection.
Figure 4: Genome packaging requires reverse osmosis.
Figure 5: Models of DNA ejection.
Figure 6: Channel width dictates the ejection mechanism.

Similar content being viewed by others

References

  1. Kallmeyer, J., Pockalny, R., Adhikaria, R. R., Smith, D. C. & D'Hondt, S. Global distribution of microbial abundance and biomass in subseafloor sediment. Proc. Natl Acad. Sci. USA 109, 16213–16216 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Suttle, C. A., Marine viruses — major players in the global ecosystem. Nature Rev. Microbiol. 5, 801–812 (2007).

    Article  CAS  Google Scholar 

  3. Hershey, A. D. & M. Chase, M. Independent functions of viral protein and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36, 39–56 (1952).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Avery, O. T., McLeod, C. M. & McCarty, M. Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Induction of transformation by a desoxyribonucleic acid fraction isolated from Pneumococcus type III. J. Exp. Med. 79, 137–158 (1944).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hershey, A. D. An upper limit to the protein content of the germinal substance of bacteriophage T2. Virology 1, 108–127 (1955).

    Article  CAS  PubMed  Google Scholar 

  6. Miller, E. S. et al. Bacteriophage T4 genome. Microbiol. Mol. Biol. Rev. 67, 86–156 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Rossman, M. G. & Rao, V. B. Viruses: sophisticated biological machines. Adv. Exp. Med. Biol. 726, 1–3 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  9. 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  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  11. MacKay, D. J. & Bode, V. C. Events in lambda injection between phage adsorption & DNA entry. Virology 72, 154–166 (1976).

    Article  CAS  PubMed  Google Scholar 

  12. 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). This study examines the rates of phage T7 genome internalization catalysed by both E. coli RNA polymerase and virion proteins that are ejected into the infected cell.

    Article  CAS  PubMed  Google Scholar 

  13. de Frutos, M., Letellier, L. & Raspaud, E. DNA ejection from bacteriophage T5: analysis of the kinetics and energetics. Biophys. J. 88, 1364–1370 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Raspaud, E., Forth, T., São-José, C., Tavares, P. & de Frutos, M. A kinetic analysis of DNA ejection from tailed phages revealing the prerequisite activation energy. Biophys. J. 93, 3999–4005 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Chiaruttini, N. et al. Is the in vitro ejection of bacteriophage DNA quasistatic? A bulk to single virus study. Biophys. J. 99, 447–455 (2010). A quantitative kinetic and energetic analysis, using both bulk and single-molecule approaches, of phage T5 adsorption and genome ejection.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Anderson, T. F. in Phage and the Origins of Molecular Biology (eds Cairns, J., Stent, G. S. & Watson, J. D.) 63–78 (Cold Spring Harbor Laboratory Press, 1966)

    Google Scholar 

  17. Hershey, A. D. in Phage and the Origins of Molecular Biology (eds Cairns, J., Stent, G. S. & Watson, J. D.) 100–108 (Cold Spring Harbor Laboratory Press, 1966)

    Google Scholar 

  18. Stent, G. S. Molecular Biology of Bacterial Viruses (W. H. Freeman, 1963).

    Google Scholar 

  19. Ore, A. & Pollard, E. Physical mechanism of bacteriophage injection. Science 124, 430–432 (1956).

    Article  CAS  PubMed  Google Scholar 

  20. Zárybnický, V. Mechanism of T-even DNA ejection. J. Theor. Biol. 22, 33–42 (1969).

    Article  PubMed  Google Scholar 

  21. Bloomfield, V. A. DNA condensation by multivalent cations. Biopolymers 44, 269–282 (1998).

    Article  Google Scholar 

  22. Grigorieff, N. & Harrison, S. C. Near-atomic resolution reconstructions of icosahedral viruses from electron cryo-microscopy. Curr. Opin. Struct. Biol. 21, 265–273 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Smith, D. E. et al. The bacteriophage straight Φ29 portal motor can package DNA against a large internal force. Nature 413, 748–752 (2001). The first single-molecule phage experiment, demonstrating the strength of a viral DNA-packaging motor.

    Article  CAS  PubMed  Google Scholar 

  24. Raspaud, E., Durand, D. & Livolant, F. Interhelical spacing in liquid crystalline spermine and spermidine–DNA precipitates. Biophys. J. 88, 392–403 (2005).

    Article  CAS  PubMed  Google Scholar 

  25. Todd, B. A., Parsegian, V. A., Shirahata, A., Thomas, T. J. & Rau, D. C. Attractive forces between condensed DNA double helices. Biophys. J. 94, 4775–4782 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Casjens, S. R. The DNA-packaging nanomotor of tailed bacteriophages. Nature Rev. Microbiol. 9, 647–657 (2011).

    Article  CAS  Google Scholar 

  27. Chemla, Y. R. et al. Mechanism of force generation of a viral DNA packaging motor. Cell 122, 683–692 (2005).

    Article  CAS  PubMed  Google Scholar 

  28. Moffitt, J. R. et al. Intersubunit coordination in a homomeric ring ATPase. Nature 457, 446–450 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Fuller, D. N., Raymer, D. M., Kottadiel, V. I., Rao, V. B. & Smith, D. E. Single phage T4 DNA packaging motors exhibit large force generation, high velocity, and dynamic variability. Proc. Natl Acad. Sci. USA 104, 16868–16873 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Fuller, D. N. et al. Measurements of single DNA molecule packaging dynamics in bacteriophage λ reveal high forces, high motor processivity, and capsid transformations. J. Mol. Biol. 373, 1113–1122 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Rickgauer, J. P. et al. Portal motor velocity and internal force resisting viral DNA packaging in bacteriophage φ29. Biophys. J. 94, 159–167 (2008).

    Article  CAS  PubMed  Google Scholar 

  32. Black, L. W. & Silverman, D. J. Model for DNA packaging into bacteriophage T4 heads. J. Virol. 28, 643–655 (1978).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Chattoraj, D. K. & Inman, R. B. Location of DNA ends in P2, 186, P4, and lambda bacteriophage heads. J. Mol. Biol. 87, 11–22 (1974).

    Article  CAS  PubMed  Google Scholar 

  34. Saigo, K. Polar DNA ejection in bacteriophage T7. Virology 65, 120–127 (1975).

    Article  CAS  PubMed  Google Scholar 

  35. Shaw, A. R. & Davison, J. Polarized injection of the bacteriophage T5 chromosome. J. Virol. 30, 933–935 (1979).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Tavares, P., Lurz, R., Stiege, A., Ruckert, B. & Trautner, T. A. Sequential headful packaging and fate of the cleaved DNA ends in bacteriophage SPP1. J. Mol. Biol. 264, 954–967 (1996).

    Article  CAS  PubMed  Google Scholar 

  37. González-Huici, V., Salas, M. & Hermoso, J. M. The push–pull mechanism of bacteriophage Φ29 DNA injection. Mol. Microbiol. 52, 529–540 (2004).

    Article  CAS  PubMed  Google Scholar 

  38. 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  PubMed  Google Scholar 

  39. Tang, J. et al. DNA poised for release in bacteriophage Φ29. Structure 16, 935–943 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Liu, X. et al. Structural changes in a marine podovirus associated with release of its genome into Prochlorococcus. Nature Struct. Mol. Biol. 17, 830–836 (2010).

    Article  CAS  Google Scholar 

  41. 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  PubMed  PubMed Central  Google Scholar 

  42. Jiang, W. et al. Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus. Nature 439, 612–616 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. North, A. C. & Rich, A. X-ray diffraction studies of bacterial viruses. Nature 191, 1242–1245 (1961).

    Article  CAS  PubMed  Google Scholar 

  44. Richards, K. E., Williams, R. C. & Calendar, R. Mode of DNA packaging within bacteriophage heads. J. Mol. Biol. 78, 255–259 (1973).

    Article  CAS  PubMed  Google Scholar 

  45. Earnshaw, W. C. & Casjens, S. R. DNA packaging by the double-stranded DNA bacteriophages. Cell 21, 319–331 (1980).

    Article  CAS  PubMed  Google Scholar 

  46. Earnshaw, W. C. & Harrison, S. C. DNA arrangement in isometric phage heads. Nature 268, 598–602 (1977).

    Article  CAS  PubMed  Google Scholar 

  47. Earnshaw, W. C., King, J., Harrison, S. C. & Eiserling, F. A. The structural organization of DNA packaged within the heads of T4 wild-type, isometric and giant bacteriophages. Cell 14, 559–568 (1978).

    Article  CAS  PubMed  Google Scholar 

  48. Cerritelli, M. E. et al. Encapsidated conformation of bacteriophage T7 DNA. Cell 91, 271–280 (1997).

    Article  CAS  PubMed  Google Scholar 

  49. Agirrezabala, X. et al. Maturation of phage T7 involves structural modification of both shell and inner core components. EMBO J. 24, 3820–3829 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Leiman, P. G. et al. The structures of bacteriophages K1E and K1-5 explain processive degradation of polysaccharide capsules and evolution of new host specificities. J. Mol. Biol. 371, 836–849 (2007).

    Article  CAS  PubMed  Google Scholar 

  51. Choi, K. H. et al. Insight into DNA and protein transport in double-stranded DNA viruses: the structure of bacteriophage N4. J. Mol. Biol. 378, 726–736 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Chang, J. T. et al. Visualizing the structural changes of bacteriophage epsilon15 and its Salmonella host during infection. J. Mol. Biol. 402, 731–740 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Dai, W. et al. Three-dimensional structure of tropism switching Bordetella bacteriophage. Proc. Natl Acad. Sci. USA 107, 4347–4352 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Comolli, L. R. et al. Three-dimensional architecture of the bacteriophage Φ29 packaged genome and elucidation of its packaging process. Virology 371, 267–277 (2008).

    Article  CAS  PubMed  Google Scholar 

  55. 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  PubMed  Google Scholar 

  56. Effantin, G., Boulanger, P., Neumann, E., Letellier, L. & Conway, J. F. Bacteriophage T5 structure reveals similarities with HK97 and T4 suggesting evolutionary relationships. J. Mol. Biol. 361, 993–1002 (2006).

    Article  CAS  PubMed  Google Scholar 

  57. Olson, N. H., Gingery, M., Eiserling, F. A. & Baker, T. S. The structure of isometric capsids of bacteriophage T4. Virology 279, 385–391 (2001).

    Article  CAS  PubMed  Google Scholar 

  58. Fokine, A. et al. Molecular architecture of the prolate head of bacteriophage T4. Proc. Natl Acad. Sci. USA 101, 6003–6008 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Duda, R. L., Hendrix, R. W., Huang, W. M. & Conway, J. F. Shared architecture of bacteriophage SPO1 and herpesvirus capsids. Curr. Biol. 16, R11–R13 (2006).

    Article  CAS  PubMed  Google Scholar 

  60. Leforestier, A. & Livolant, F. The bacteriophage genome undergoes a succession of intracapsid phase transitions upon DNA ejection. J. Mol. Biol. 396, 384–395 (2010). This important study characterizes the structure of the DNA remaining in the phage T5 capsid during genome ejection.

    Article  CAS  PubMed  Google Scholar 

  61. Petrov, A. S. & Harvey, S. C. Structural and thermodynamic principles of viral packaging. Structure 15, 21–27 (2007).

    Article  CAS  PubMed  Google Scholar 

  62. Petrov, A. S., Boz, M. B. & Harvey, S. C. The conformation of double-stranded DNA inside bacteriophages depends on capsid size and shape. J. Struct. Biol. 160, 241–248 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Fuller, D. N. et al. Ionic effects on viral DNA packaging and portal motor function in bacteriophage. Proc. Natl Acad. Sci. USA 104, 11245–11250 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Evilevitch, E. et al. Effects of salt concentrations and bending energy on the extent of ejection of phage genomes. Biophys. J. 94, 1110–1120 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Wu, D., Van Valen, D., Hu, Q. & Phillips, R. Ion-dependent dynamics of DNA ejections for bacteriophage λ. Biophys. J. 99, 1101–1109 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Aubrey, K. L., Casjens, S. R. & Thomas, G. J. Jr. Secondary structure and interactions of the packaged dsDNA genome of bacteriophage P22 investigated by Raman difference spectroscopy. Biochemistry 31, 11835–11842 (1992).

    Article  CAS  PubMed  Google Scholar 

  67. Overman, S. A. et al. Conformation and interactions of the packaged double-stranded DNA of bacteriophage T7. Biospectroscopy 4, S47–S56 (1998).

    Article  CAS  PubMed  Google Scholar 

  68. Panja, D. & Molineux, I. J. Dynamics of bacteriophage genome ejection in vitro and in vivo. Phys. Biol. 7, 045006 (2010). A comprehensive thermodynamic analysis of the pressures in a mature phage capsid.

    Article  PubMed  Google Scholar 

  69. Vafabakhsh, R. & Ha, T. Extreme bendability of DNA less than 100 base pairs long revealed by single-molecule cyclization. Science 337, 1097–1101 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Qiu, X. et al. Salt-dependent DNA-DNA spacings in intact bacteriophage λ reflect relative importance of DNA self-repulsion and bending energies. Phys. Rev. Lett. 106, 028102 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Saenger, W. Principles of Nucleic Acid Structure.. (Springer, 1984).

    Book  Google Scholar 

  72. Leikin, S., Parsegian, V. A. & Rau, D. C. Hydration forces. Annu. Rev. Phys. Chem. 44, 369–395 (1993).

    Article  CAS  PubMed  Google Scholar 

  73. Brovchenko, I., Krukau, A., Oleinikova, A. & Mazur, A. K. Water clustering and percolation in low hydration DNA shells. J. Phys. Chem. B. 111, 3258–3266 (2007).

    Article  CAS  PubMed  Google Scholar 

  74. Wolf, B. & Hanlon, S. Structural transitions of deoxyribonucleic acid in aqueous electrolyte solutions. II. The role of hydration. Biochemistry 14, 1661–1670 (1975).

    Article  CAS  PubMed  Google Scholar 

  75. Evilevitch, A., Castelnovo, M., Knobler, C. M. & Gelbart, W. M. Measuring the force ejecting DNA from phage. J. Phys. Chem. B 108, 6838–6843 (2004).

    Article  CAS  Google Scholar 

  76. Grayson, P. et al. The effect of genome length on ejection forces in bacteriophage lambda. Virology 348, 430–436 (2006). A quantitative experimental test of theories about the internal pressures in phage λ virions, using osmotic suppression of genome ejection.

    Article  CAS  PubMed  Google Scholar 

  77. São-José, C., de Frutos, M., Raspaud, E., Santos, M. A. & Tavares, P. Pressure built by DNA packing inside virions: enough to drive DNA ejection in vitro, largely insufficient for delivery into the bacterial cytoplasm. J. Mol. Biol. 374, 346–355 (2007). This study extends the observations of reference 76 by investigating osmotic suppression of genome ejection in phage SPP1.

    Article  CAS  PubMed  Google Scholar 

  78. Leforestier, A. et al. Bacteriophage T5 DNA ejection under pressure. J. Mol. Biol. 384, 730–739 (2008). This paper shows that genome ejection from phage T5 virions is not fully explained by the continuum mechanics model.

    Article  CAS  PubMed  Google Scholar 

  79. Kindt, J., Tzlil, S., Ben-Shaul, A. & Gelbart, W. M. DNA packaging and ejection forces in bacteriophage. Proc. Natl Acad. Sci. USA 98, 13671–13674 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Tzlil, S., Kindt, J. T., Gelbart, W. M. & Ben-Shaul, A. Forces and pressures in DNA packaging and release from viral capsids. Biophys. J. 84, 1616–1627 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Purohit, P. K. et al. Forces during bacteriophage DNA packaging and ejection. Biophys. J. 88, 851–866 (2005).

    Article  CAS  PubMed  Google Scholar 

  82. Purohit, P. K., Kondev, J. & Phillips, R. Mechanisms of DNA packaging in viruses. Proc. Natl Acad. Sci. USA 100, 3173–3178 (2005).

    Article  CAS  Google Scholar 

  83. Inamdar, M. M., Gelbart, W. M. & Phillips, R. Dynamics of DNA ejection from bacteriophages. Biophys. J. 91, 411–420 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Evilevitch, A., Lavelle, L., Knobler, C. M., Raspaud, E. & Gelbart, W. M. Osmotic pressure inhibition of DNA ejection from phage. Proc. Natl Acad. Sci. USA 100, 9292–9295 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Löf, D., Schillén, K., Jönsson, B. & Evilevitch, A. Forces controlling the rate of DNA ejection from phage λ. J. Mol. Biol. 368, 55–65 (2007).

    Article  CAS  PubMed  Google Scholar 

  86. Molineux, I. J. Fifty-three years since Hershey and Chase; much ado about pressure but which pressure is it? Virology 344, 221–229 (2006).

    Article  CAS  PubMed  Google Scholar 

  87. Grayson, P. & Molineux, I. J. Is phage DNA “injected” into cells - biologists and physicists can agree. Curr. Opin. Microbiol. 10, 401–409 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Lemay, S. G., Panja, D. & Molineux, I. J. Role of osmotic and hydrostatic pressures in bacteriophage genome ejection. Preprint at arXiv [online] (2012).

  89. Grayson, P., Han, L., Winther, T. & Phillips, R. Real-time observations of single bacteriophage λ DNA ejections in vitro. Proc. Natl Acad. Sci. USA 104, 14652–14657 (2007). This work examines genome ejection using single phage λ virions.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Mangenot, S., Hochrein, M., Radler, J. & Letellier, L. Real-time imaging of DNA ejection from single phage particles. Curr. Biol. 15, 430–435 (2005). The first single-particle experiments on phage DNA ejection in vitro . Phage T5 DNA is shown to leave the virion at rates of up to ∼75 kb per second.

    Article  CAS  PubMed  Google Scholar 

  91. Fang, P. A. et al. Visualization of bacteriophage T3 capsids with DNA incompletely packaged in vivo. J. Mol. Biol. 384, 1384–1399 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Koch, A. L. The biophysics of Gram-negative periplasmic space. Crit. Rev. Microbiol. 24, 23–59 (1998).

    Article  CAS  PubMed  Google Scholar 

  93. Whatmore, A. M. & Reed, R. H. Determination of turgor pressure in Bacillus subtilis: a possible role for K+ in turgor regulation. J. Gen. Microbiol. 136, 2521–2526 (1990).

    Article  CAS  PubMed  Google Scholar 

  94. Pruss, G., Goldstein, R. N. & Calendar, R. In vitro packaging of satellite phage P4 DNA. Proc. Natl Acad. Sci. USA 71, 2367–2371 (1974).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Zavriev, S. K. & Shemyakin, M. F. RNA polymerase-dependent mechanism for the stepwise T7 phage DNA transport from the virion into E. coli. Nucleic Acids Res. 10, 1635–1652 (1982).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Moffatt, B. A. & Studier, F. W. Entry of bacteriophage T7 DNA into the cell and escape from host restriction. J. Bacteriol. 170, 2095–2105 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. García, L. R. & Molineux, I. J. Rate of translocation of bacteriophage T7 DNA across the membranes of Escherichia coli. J. Bacteriol. 177, 4066–4076 (1995).

    Article  PubMed  PubMed Central  Google Scholar 

  98. García, L. R. & Molineux, I. J. Translocation and cleavage of bacteriophage T7 DNA by the type I restriction enzyme EcoKI in vivo. Proc. Natl Acad. Sci. USA 96, 12430–12435 (1999).

    Article  PubMed  PubMed Central  Google Scholar 

  99. Kazmierczak, K. & Rothman-Denes, L. in The Bacteriophages 2nd edn Ch. 21 (ed. Calendar, R.) 302–314 (Oxford Univ. Press, 2006).

    Google Scholar 

  100. Gonzalez-Huici, V., Salas, M. & Hermoso, J. M. Requirements for Bacillus subtilis bacteriophage Φ29 DNA ejection. Gene 374, 19–25 (2006).

    Article  CAS  PubMed  Google Scholar 

  101. Alcorlo, M., Gonzalez-Huici, V. & Hermoso, J. M. The phage φ29 membrane protein p16.7, involved in DNA replication, is required for efficient ejection of the viral genome. J. Bacteriol. 189, 5542–5549 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Lanni, Y. T. First-step-transfer deoxyribonucleic acid of bacteriophage T5. Bacteriol. Rev. 32, 227–242 (1968).

    CAS  PubMed  PubMed Central  Google Scholar 

  103. McCorquodale, D. J. & Warner, H. R. in The Bacteriophages 1st edn Vol. 1 (ed. Calendar, R.) 439–475 (Plenum, 1988).

    Book  Google Scholar 

  104. Filali Maltouf, A. F. & Labedan, B. Host cell metabolic energy is not required for injection of bacteriophage T5 DNA. J. Bacteriol. 153, 124–133 (1983).

    CAS  PubMed  PubMed Central  Google Scholar 

  105. 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  PubMed  Google Scholar 

  106. 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  PubMed  Google Scholar 

  107. Molineux, I. J. No syringes please, ejection of T7 DNA from the virion is enzyme-driven. Mol. Microbiol. 40, 1–8 (2001).

    Article  CAS  PubMed  Google Scholar 

  108. Molineux, I. J. in The Bacteriophages 2nd edn Ch. 20 (ed. Calendar, R.) 277–301 (Oxford Univ. Press, 2006).

    Google Scholar 

  109. García, L. R. & Molineux, I. J. Transcription-independent DNA translocation of bacteriophage T7 DNA into Escherichia coli. J. Bacteriol. 178, 6921–6929 (1996).

    Article  PubMed  PubMed Central  Google Scholar 

  110. Struthers-Schlinke, J. S., Robins, W. P., Kemp, P. & Molineux, I. J. The internal head protein gp16 of bacteriophage T7 controls DNA ejection from the virion. J. Mol. Biol. 301, 35–45 (2000).

    Article  CAS  PubMed  Google Scholar 

  111. Roessner, C. A. & Ihler, G. M. Formation of transmembrane channels in liposomes during injection of λ DNA. J. Biol. Chem. 261, 386–390 (1986).

    CAS  PubMed  Google Scholar 

  112. Roessner, C. A., Struck, D. & Ihler, G. M. Injection of DNA into liposomes by bacteriophage λ. J. Biol. Chem. 258, 643–648 (1983).

    CAS  PubMed  Google Scholar 

  113. Böhm, J. et al. FhuA-mediated phage genome transfer into liposomes: a cryo-electron tomography study. Curr. Biol. 11, 1168–1175 (2001).

    Article  PubMed  Google Scholar 

  114. Simon, L. D. & Anderson, T. F. The infection of Escherichia coli by T2 and T4 bacteriophages as seen in the electron microscope. I. Attachment and penetration. Virology 32, 279–297 (1967).

    Article  CAS  PubMed  Google Scholar 

  115. Tarahovsky, Y. S., Khusainov, A. A., Deev, A. A. & Kim, Y. V. Membrane fusion during infection of E. coli cells by phage T4. FEBS Lett. 289, 18–22 (1991).

    Article  CAS  PubMed  Google Scholar 

  116. Liu, J., Chen, C. Y. Shiomi, D. Niki, H. & Margolin, W. Visualization of bacteriophage P1 infection by cryo-electron tomography of tiny Escherichia coli. Virology 417, 304–311 (2011).

    Article  CAS  PubMed  Google Scholar 

  117. Hu, B., Margolin, W., Molineux, I. J. & Liu, J. The bacteriophage T7 virion undergoes extensive structural remodeling during infection. Science 10 Jan 2013 (doi:10.1126/science.1231887).

  118. Labrie, S. J., Samson, J. E. & Moineau, S. Bacteriophage resistance mechanisms. Nature Rev. Microbiol. 8, 317–327 (2010).

    Article  CAS  Google Scholar 

  119. Newbold, J. E. & Sinsheimer, R. L. The process of infection with bacteriophage φX174. XXXII. Early steps in the infection process: attachment, eclipse and DNA penetration. J. Mol. Biol. 49, 49–66 (1970).

    Article  CAS  PubMed  Google Scholar 

  120. Parenchych, W. in RNA Phages (ed. Zinder, N.) 85–112 (Cold Spring Harbor Laboratory Press, 1975).

    Google Scholar 

  121. Zsigray, R. M., Miss, A. L. & Landman, O. E. Penetration of a bacteriophage into Bacillus subtilis: blockage of infection by deoxyribonuclease. J. Virol. 11, 69–77 (1973).

    CAS  PubMed  PubMed Central  Google Scholar 

  122. Santos, M. A., De Lencastre, H. & Archer, L. J. Homology between phages SPP1, 41c, 22a, ρ15 and SF6 of Bacillus subtilis. J. Gen. Virol. 65, 2067–2072 (1984).

    Article  CAS  PubMed  Google Scholar 

  123. Grinius, L. Nucleic acid transport driven by ion gradient across cell membrane. FEBS Lett. 113, 1–10 (1980).

    Article  CAS  PubMed  Google Scholar 

  124. Kalasauskaitė, E. V., Kadišaitė, D. L., Daugelavičius, R. J., Grinius, L. & Jasaitis, A. A. Studies on energy supply for genetic processes. Eur. J. Biochem. 130, 123–130 (1983).

    Article  PubMed  Google Scholar 

  125. Grinius, L. & Daugelavičius, R. Depolarization of Escherichia coli cytoplasmic membrane by bacteriophages T4 and lambda: evidence for induction of ion-permeable channels. Bioelectrochem. Bioenerg. 19, 235–245 (1988).

    Article  CAS  Google Scholar 

  126. Kuhn, A. & Kellenberger, E. Productive phage infection in Escherichia coli with reduced internal levels of the major cations. J. Bacteriol. 163, 906–912 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  127. Boulanger, P. & Letellier, L. Characterization of ion channels involved in the penetration of T4 DNA into Escherichia coli cells. J. Biol. Chem. 263, 9767–9775 (1988).

    CAS  PubMed  Google Scholar 

  128. Boulanger, P. & Letellier, L. Ion channels are likely to be involved in the two steps of phage T5 DNA penetration into Escherichia coli cells. J. Biol. Chem. 267, 3168–3172 (1992).

    CAS  PubMed  Google Scholar 

  129. Guihard, G., Benedetti, H. Besnard, M. & Letellier, L. Phosphate efflux through the channels formed by colicins and phage T5 in Escherichia coli cells is responsible for the fall in cytoplasmic ATP. J. Biol. Chem. 268, 17775–17780 (1993).

    CAS  PubMed  Google Scholar 

  130. Daugelavičius, R., Bamford, J. K. & Bamford, D. H. Changes in host cell energetics in response to bacteriophage PRD1 DNA entry. J. Bacteriol. 179, 5203–5210 (1997).

    Article  PubMed  PubMed Central  Google Scholar 

  131. Jakutytė, L. et al. First steps of bacteriophage SPP1 entry into Bacillus subtilis. Virology 422, 425–434 (2012).

    Article  CAS  PubMed  Google Scholar 

  132. Grahn, A. M., Daugelavičius, R. & Bamford, D. H. Sequential model of phage PRD1 DNA delivery: active involvement of the viral membrane. Mol. Microbiol. 46, 1199–1209 (2002).

    Article  CAS  PubMed  Google Scholar 

  133. Labedan, B. & Goldberg, E. B. Requirement for membrane potential in injection of phage T4 DNA. Proc. Natl Acad. Sci. USA 76, 4669–4673 (1979).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. Labedan, B., Heller, K. B., Jasaitis, A. A., Wilson, T. H. & Goldberg, E. B. A membrane potential threshold for phage T4 DNA injection. Biochem. Biophys. Res. Comm. 93, 625–630 (1980).

    Article  CAS  PubMed  Google Scholar 

  135. McAllister, W. T. Bacteriophage infection: which end of the SP82G genome goes in first? J. Virol. 5, 194–198 (1970).

    CAS  PubMed  PubMed Central  Google Scholar 

  136. Labedan, B., Crochet, M., Legault-Demme, J. & Stevens, B. J. Location of the first step transfer fragment and single strand interruptions in T5st0 bacteriophage DNA. J. Mol. Biol. 75, 213–234 (1973).

    Article  CAS  PubMed  Google Scholar 

  137. Labedan, B. & Legault-Demare, J. Penetration into host cells of naked partially injected (post FST) DNA of bacteriophage T5. J. Virol. 12, 226–229 (1973).

    CAS  PubMed  PubMed Central  Google Scholar 

  138. Van Valen, D. et al. A single-molecule Hershey-Chase experiment. Curr. Biol. 22, 1339–1343 (2012). This study is the first to measure the rate of phage genome ejection using single phage virions.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  139. Elliott, J. & Arber, W. E. coli K-12 pel mutants, which block phage λ DNA injection, coincide with ptsM, which determines a component of a sugar transport system. Mol. Gen. Genet. 161, 1–8 (1978).

    Article  CAS  PubMed  Google Scholar 

  140. Kiino, D. R. & Rothman-Denes, L. B. Genetic analysis of bacteriophage N4 adsorption. J. Bacteriol. 171, 4595–4602 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  141. Baptista, C., Santos, M. A. & São-José, C. Phage SPP1 reversible adsorption to Bacillus subtilis cell wall teichoic acids accelerates virus recognition of membrane receptor YueB. J. Bacteriol. 190, 4989–4996 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  142. Geller, B. L., Ivey, R. G., Trempy, J. E. & Hettinger-Smith, B. Cloning of a chromosomal gene required for phage infection of Lactococcus lactis subsp. lactis C2. J. Bacteriol. 175, 5510–5519 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. Räisänen, L., Schubert, K., Jaakonsaari, T. & Alatossava, T. Characterization of lipoteichoic acids as Lactobacillus delbrueckii phage receptor components. J. Bacteriol. 186, 5529–5532 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  144. Räisänen, L. et al. Molecular interaction between lipoteichoic acids and Lactobacillus delbrueckii phages depends on D-alanyl and α-glucose substitution of poly(glycerophosphate) backbones. J. Bacteriol. 189, 4135–4140 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  145. Baumann, L., Benz, W. C. Wright, A. & Goldberg, E. B. Inactivation of urea-treated phage T4 by phosphatidylglycerol. Virology 41, 356–364 (1970).

    Article  CAS  PubMed  Google Scholar 

  146. Boulanger, P. et al. Phage T5 straight tail fiber is a multifunctional protein acting as a tape measure and carrying fusogenic and muralytic activities. J. Biol. Chem. 283, 13556–13564 (2008).

    Article  CAS  PubMed  Google Scholar 

  147. Hancock, R. E. W. & Braun, V. Nature of the energy requirement for the irreversible adsorption of bacteriophages T1 and Φ80 to Escherichia coli. J. Bacteriol. 125, 409–415 (1976).

    CAS  PubMed  PubMed Central  Google Scholar 

  148. Filali Maltouf, A. & Labedan, B. The energetics of the injection process of bacteriophage lambda DNA and the role of the ptsM/pel-encoded protein. Biochem. Biophys. Res. Comm. 130, 1093–1101 (1985).

    Article  CAS  PubMed  Google Scholar 

  149. Cvirkaitė-Krupovič, V., Krupovič, M. Daugelavičius, R. & Bamford, D. H. Calcium ion-dependent entry of the membrane-containing bacteriophage PM2 into its Pseudoalteromonas host. Virology 405, 120–128 (2010).

    Article  CAS  PubMed  Google Scholar 

  150. Molineux, I. J. Host-parasite interactions: recent developments in the genetics of abortive phage infections. New Biol. 3, 230–236 (1991).

    CAS  PubMed  Google Scholar 

  151. Snyder, L. Phage-exclusion enzymes: a bonanza of biochemical and cell biology reagents? Mol. Microbiol. 15, 415–423 (1995).

    Article  CAS  PubMed  Google Scholar 

  152. Coren, J., Pierce, J. & Sternberg, N. Headful packaging revisited: the packaging of more than one DNA molecule into a bacteriophage P1 head. J. Mol. Biol. 249, 176–184 (1995).

    Article  CAS  PubMed  Google Scholar 

  153. Leffers, G. & Rao, V. B. A discontinuous headful packaging model for packaging less than headful length DNA molecules by bacteriophage T4. J. Mol. Biol. 258, 839–850 (1996).

    Article  CAS  PubMed  Google Scholar 

  154. Schrader, H. S. et al. Bacteriophage infection and multiplication occur in Pseudomonas aeruginosa starved for 5 years. Can. J. Microbiol. 43, 1157–1163 (1997).

    Article  CAS  PubMed  Google Scholar 

  155. Calendar, R. (ed.) The Bacteriophages 2nd edn (Oxford Univ. Press, 2006).

    Google Scholar 

  156. Black, L. W. & Thomas, J. A. Condensed genome structure. Adv. Exp. Med. Biol. 726, 469–487 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Most of the research on T7 DNA ejection that was carried out in the I.J.M. laboratory was supported by US National Institutes of Health grant GM32095. The authors also thank the entire phage community for many lively and insightful discussions.

Author information

Authors and Affiliations

  1. Molecular Genetics and Microbiology, Institute for Cell and Molecular Biology, The University of Texas at Austin, Austin, 78712, Texas, USA

    Ian J. Molineux

  2. Institute of Physics, Universiteit van Amsterdam, Science Park 904, Postbus 94485, Amsterdam, 1090 GL, The Netherlands

    Debabrata Panja

  3. Institute for Theoretical Physics, Universiteit Utrecht, Leuvenlaan 4, Utrecht, 3584 CE, The Netherlands

    Debabrata Panja

Authors
  1. Ian J. Molineux
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Debabrata Panja
    View author publications

    You can also search for this author in PubMed Google Scholar

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Related links

Related links

FURTHER INFORMATION

Ian J. Molineux's homepage

Debabrata Panja's homepage

Glossary

Lysogenic cycle

A cycle wherein a phage infects a cell and enters into a quiescent phase, during which few of its genes are expressed. The cycle is completed by the induction of the phage, when phage genes for lytic growth are derepressed.

Baseplate

A multiprotein complex at the head-distal end of long-tailed phages.

Transformation

The uptake of naked DNA by intact cells.

Brownian motion

The random movement of particles in a fluid, resulting from their collisions with fast-moving molecules in the fluid.

Interhelical distance

The distance between the centre of two adjacent helices within the DNA.

Tailed phages

Phages containing a double-stranded DNA genome in an icosahedral head in which one vertex is occupied by a protein tail.

Portal

The head–tail connector in a phage. During infection of a cell, the DNA packaged in the phage head is ejected through a channel in the centre of the portal.

B form

Pertaining to DNA: the normal Watson–Crick structure of DNA, with 10.4 bp per helical turn.

Inverse spool

A spool of line (here, DNA) wound from the outside to the inside.

Packaging motors

Enzymes (terminases) that convert ATP into mechanical movement to translocate DNA into the pre-formed phage prohead (the precursor structure to the head, or capsid, of a phage). Most terminases also cleave double-stranded DNA to complete the DNA-packaging process.

Toroid

A doughnut-shaped object (in mathematical terminology).

Persistence length

A mechanical property quantifying the bending rigidity of DNA (or any polymer). Molecules shorter than the persistence length are considered to be straight rods. The persistence length of long double-stranded DNA is usually described as ∼50 nm (∼150 bp), but for segments ≤150 bp, which are pertinent to DNA packaged in phage capsids or to cell biology in general, double-stranded DNA seems to be more flexible, having a persistence length of <100 bp.

Hydration energy

The energy expended in removing water molecules from ions.

Hydration layers

The layers or shells of water molecules surrounding a solute.

Reverse osmosis

The removal of water molecules from a solution through a membrane.

Monotonically

Continuously increasing or decreasing, but not necessarily at a constant rate.

Brownian ratchets

Nanomachines that extract useful work from chemical potentials and other microscopic non-equilibrium sources. They can be micro-fabricated or, in the context of this Review, proteins or protein complexes. The concept of Brownian ratchets derive from formal analyses by Feynman and others, who corrected the fallacies associated with an apparent perpetual-motion machine which, in violation of the laws of thermodynamics, was driven by Brownian motion.

Headful packaging

A phage DNA-packaging process in which a terminase cuts the DNA at a nonspecific sequence when the phage head is full. Some terminases cut at only a specific sequence, in a process called cos packaging (named for the cos sites of phage Ι).

Circularly permuted

Pertaining to a phage genome: containing more than a complete genome equivalent, owing to packaging from replicating concatameric DNA such that if the genome is considered to be ABCDE, the DNA actually packaged into individual phage progeny is, in turn, ABCDEA, BCDEAB, and so on.

Infective centres

Infected bacteria that will give rise to progeny phages.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Molineux, I., Panja, D. Popping the cork: mechanisms of phage genome ejection. Nat Rev Microbiol 11, 194–204 (2013). https://doi.org/10.1038/nrmicro2988

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrmicro2988

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