Bacteriophage imaging : past, present and future

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY-NC-ND 4.0 https://creativecommons.org/licenses/by-nc-nd/4.0/ Bacteriophage imaging : past, present and future © 2018 Institut Pasteur. Published by Elsevier Masson SAS. Accepted version (Final draft) De Freitas Almeida, Gabriel; Leppänen, Miika; Maasilta, Ilari; Sundberg, LottaRiina De Freitas Almeida, G., Leppänen, M., Maasilta, I., & Sundberg, L.-R. (2018). Bacteriophage imaging : past, present and future. Research in Microbiology, 169(9), 488-494. https://doi.org/10.1016/j.resmic.2018.05.006 2018 Accepted Manuscript Bacteriophage imaging: past, present and future Gabriel MF. Almeida, Miika Leppänen, Ilari J. Maasilta, Lotta-Riina Sundberg PII: S0923-2508(18)30080-9 DOI: 10.1016/j.resmic.2018.05.006 Reference: RESMIC 3668 To appear in: Research in Microbiology Received Date: 31 January 2018 Revised Date: 16 May 2018 Accepted Date: 22 May 2018 Please cite this article as: G.M. Almeida, M. Leppänen, I.J Maasilta, L.-R. Sundberg, Bacteriophage imaging: past, present and future, Research in Microbiologoy (2018), doi: 10.1016/j.resmic.2018.05.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 1 Title: Bacteriophage imaging: past, present and future 1 2 Authors: Gabriel MF Almeidaa* , Miika Leppänena,b , Ilari J Maasiltab , Lotta-Riina Sundberga. 3 4 a Centre of Excellence in Biological Interactions, Department of Biological and Environmental 5 Science, Nanoscience Center, University of Jyväskylä, Survontie 9C, FI-40014, Jyväskylä, 6 Finland 7 b Department of Physics, Nanoscience Center, University of Jyväskylä, Survontie 9C, FI-8 40014, Jyväskylä, Finland 9 10 11 12 13 14 Email addresses: 15 Almeida GMF: [email protected] *Correspondence and reprints 16 Maasilta IJ: [email protected] 17 Leppänen M: [email protected] 18 Sundberg LR: [email protected] 19 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 2 Abstract 20 The visualization of viral particles only became possible after the advent of the electron 21 microscope. The first bacteriophage images were published in 1940 and were soon followed 22 by many other publications that helped to elucidate the structure of the particles and their 23 interaction with the bacterial hosts. As sample preparation improved and new technologies 24 were developed, phage imaging became important approach to morphologically classify 25 these viruses and helped to understand its importance in the biosphere. In this review we 26 discuss the main milestones in phage imaging, how it affected our knowledge on these 27 viruses and recent developments in the field. 28 29 Keywords: bacteriophage ; phage ; virus ; imaging ; structure ; microscopy 30 31 32 33 34 35 36 37 38 39 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 3 Earlier years (1940-1948) 40 Although the existence of viruses was known since the end of the 19th 41 century, the true nature of the so called “contagium vivum fluidum”, and whether it was 42 liquid or particulated, remained unknown for many years [1]. Direct imaging of viral 43 particles only became possible after the advent of the transmission electron microscope, 44 allowing the determination of viral morphological characteristics. These machines were 45 developed in the late 1930s by two different groups: one working at the Siemens & Halske 46 laboratory company in Germany and another working at the University of Toronto in 47 Canada. Ernst Ruska led the German team while James Hillier led the Canadian team, in 48 development processes based on a concept that was already old by 1930, as mentioned by 49 Hillier several years later. While Ernst Ruska developed a model for Siemens in Europe, 50 Hillier and Prebus got a model working in America, and by the end of the thirties both teams 51 had fully functional machines [2,3]. Helmut Ruska, Ernst’s brother, was part of the team that 52 used the German “hypermicroscope” to image a virus for the first time in 1938. The virus in 53 question was ectromelia, a large DNA virus from the Poxviridae family, capable of infecting 54 mice [4]. 55 The first bacteriophage micrographs appeared on the literature in 1940, in two 56 papers published at the same issue of the Naturwissenschaften journal. In one paper 57 Helmut Ruska imaged infected bacterial cells and was able to show virus adsorption, cell 58 lysis and resistant bacterial cells (Fig.1a). Phages were described as small round particles, 59 and crystalloid structures were seen and hypothesized to be centers for genesis of viral 60 proteins [5]. In the other paper Pfankuch and Kausche, also working at the Siemens & 61 Halske laboratory, analyzed purified phage suspensions and described the viruses as small 62 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 4 rounded corpuscles that aggregate in higher concentrations [6]. Both papers mention 63 particle destruction by electron irradiation. It is now believed that the phages seen at the 64 time were T7 coliphages. Translated reprints of both articles were published in 2011 [7]. 65 Following these publications, phage images spread in Europe causing excitement, reaching 66 even Felix d´Herelle, one of the discoverers of these viruses. Helmut Ruska continued to be 67 an important influence on phage imaging. In the early forties he described some phage 68 particles obtained from bacterial lysates as being club-like, possessing distinct heads and 69 tails (probably these were T4-like phages) ; reported at least four phage morphotypes ; 70 proposed a morphological classification for viruses and even introduced the term “phage” 71 as an abbreviation to the term bacteriophage [7-10]. 72 Meanwhile Luria and Anderson used the commercial version of Hillier’s 73 microscope to analyze unstained Escherichia coli and Staphylococcus phages in New York. In 74 their first paper on the subject it is mentioned that phage imaging can “offer favorable 75 possibilities for the identification of the virus particles through a study of the reaction 76 between the individual particles and the bacterial cell under the microscope” [11]. 77 Interestingly, the imaging papers published by German authors including Ruska were 78 mentioned, showing that despite the Second World War scientific information was still 79 flowing from Europe to America. Luria and Anderson described coliphages as extremely 80 constant particles composed of a round head and a much thinner tail, with the heads not 81 being homogeneous in their composition but consisting of a pattern of granules. Adsorption 82 and cell lysis were visually described, but due to the lack of knowledge on virus biology and 83 genetics at the time, some of the speculations on their mechanisms have later proven to be 84 wrong (such as mentioning that adsorption could happen by either head or tail, and that 85 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 5 phage reproduction might take place at the cell wall). Imaging of Staphylococcal phages was 86 mentioned to be harder, but particles containing heads and tails, able to adsorb to the host 87 cells, were also detected. In their conclusions Luria and Anderson highlighted the interest of 88 finding constant and relatively elaborate structural differentiation of macromolecular 89 entities, and mentioned that the correspondence between particle size determined from 90 microscopy to that obtained from indirect methods of measurement was remarkable. They 91 also hypothesized that electron microscopy could also have an impact for genetics, since 92 genes are also macromolecular entities and had been indirectly measured before [11]. The 93 phages described in this study were later classified as T2 (T-even type) [7,12]. 94 One year later Luria, Delbrück and Anderson published another paper on 95 phage imaging [13], mentioning in the introduction the revival of interest in phages and the 96 advantage of using these organisms as models. Images were taken from crude or partially 97 purified viral suspensions, and also from dried drops of bacterial and phage mixtures for 98 studying interaction between both. Besides typical tailed phage visualizations, a rounded 99 phage without tail was described. It was mentioned that differential centrifugation 100 mechanically inactivated one of the tailed phages (as noted by broken tails in the 101 micrographs). Different multiplicities of infection were tested, which showed an agreement 102 between the numbers of visible adsorbed particles to infective titers obtained by titrations. 103 Micrographs also confirmed the eclipse period, allowed the observation of several steps of 104 the phage infection cycle, and showed long E. coli cells (mentioned as “not unusual” in 105 young broth cultures of the strain used). It was seen that new viruses were liberated from 106 the interior of the bacterial cell, but it was not possible to determine where inside the 107 bacteria the viruses are produced (deep interior or inner surfaces). The absence of bacterial 108 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 6 components of size comparable to viruses released by lysed cells was used to explain why 109 crude suspensions, differential centrifugation and filtrations can be used successfully for 110 phage work. It was also noted, unexpectedly at the time as pointed out by the authors, that 111 adsorbed particles remained at the cell surface. This was considered to be the finding of 112 greatest consequence, and the most plausible theory chosen to explain was that only one 113 particle enters the cell and then makes the bacteria impermeable to other viruses (an 114 analogy to monospermic eggs fecundation was made, with the caution to mention that 115 there was no conclusive data to fully support it). This imaging paper also helped to test and 116 eliminate three theories concerning phages that existed at the time: 1) no phage aggregates 117 were seen, contradicting an idea that some phages would normally bind to larger unspecific 118 carriers (such as bacterial debris) ; 2) the homogeneity of particle size disproved that there 119 was a reversible equilibrium between small and large viral particles ; 3) and the consistency 120 in progeny morphology when the same host was infected with different phages debunked a 121 proposition that bacterial cells could contain a precursor of the phage particle, which upon 122 infection would be converted to viruses. These three theories were based on indirect 123 measurements made by diffusion on differential filtration, by sedimentation rate in 124 ultracentrifugation, or as an analogy to proteolytic enzymes and its precursors, and were all 125 disproved by direct imaging on the electron microscope. There was also a discussion on the 126 common practice at the time of considering viruses to be molecules, warning that “such a 127 terminology should not prejudice our views regarding the biological status of the viruses, 128 which has yet to be elucidated” [13]. 129 Improvements on sample preparation (1948 onwards) 130 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 7 A next advance on phage imaging was the introduction of contrast to the 131 samples. By using chromium vapor to cover the preparations, Wyckoff was able to obtain 132 more information on height and shape of the particles. In 1948 he used the technique to 133 study T coliphages, chosen for their distinct shapes and for the ease of working with their 134 hosts when compared to opaque staphylococci or mucoid and capsular streptococci. Two 135 papers were published. The first was based on imaging phage plaques on samples obtained 136 from solid media, using the embedded replica technique [14] (Fig.1b). Elongated E. coli were 137 seen on young cultures and plaque characteristics were described and shown to differ 138 between phages. The second paper focused on micrographs prepared from liquid samples 139 [15]. Purified T4 preparations were used to describe phage morphology, and infected liquid 140 cultures used for showing cells undergoing lysis with phages within and around their limits. 141 Variation on the structure of phage heads and their contents was mentioned, and a 142 correlation between grainy content inside the head and stages of maturation was made. 143 Bacterial contents release by lysis were described, and their “conversion” to phages in 144 favorable instances was noted as the most impressive result of the paper, hinted to be 145 crucial in understanding how phages multiply. 146 Focusing on phage tails and the controversy concerning their role in the life 147 cycle of phages at the time, Fraser and Williams used the freeze-drying technique to prepare 148 T3 and T7 phages (believed to be tailless until then) for microscopy [16]. The technique was 149 used for its minimal preparative distortion, and purified phages were freeze-dried for 150 comparative analysis to air dried samples. Freeze-drying made clearer that phages are not 151 spherical but geometrical, and short appendages (“stubby tails”) were detected on the 152 phages that were thought to be tailless at the time. The first result was taken as support of 153 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 14 particles on an epifluorescence microscope. Although this technique does not provide any 292 structural details, its simplicity in sample preparation and equipment requirements made it 293 the most common technique for enumerating viruses from the environment [53-54]. This 294 method revealed that TEM analysis is not only more time consuming for this purpose but 295 also tends to underestimate viral abundance. A variation of the technique, consisting of 296 stained particles treated with Dnase I, has been applied for indirect evaluation of phage 297 capsid structural deformity [55]. 298 Moving away from electrons: helium ion microscopy 299 The scanning helium ion microscope (HIM) is a recent advance in imaging. 300 Instead of using electrons, imaging is based on the use of a positively charged helium ion 301 beam [56]. Helium beam allows higher image resolution (close to 0.5nm), larger depth of 302 focus and dispenses conductive coating of biological samples, this last advantage being 303 important for imaging of fragile sub-nanometer structures and for avoiding artifacts or 304 masking generated by coating. Biological samples, including a bacterium, were imaged by 305 HIM for the first time in 2013 [57]. In 2017 the nanoscale imaging capacity of the HIM was 306 used to investigate plaques formed by T4 infection on E. coli bacterial lawns in order to test 307 the applicability of helium ion microscopy to phage-host interaction studies [58] (Fig.1e). 308 The samples were directly prepared from pieces of double layered agar containing the 309 bacteria lawn with phage plaques, so the imaging could be made on viral plaques as they 310 naturally occurs. Various stages of T4 infection could be seen by imaging different spots 311 within and around a plaque, since the infection spreads radially from the ground zero, with 312 no cells in the center and newly infected cells on the edges. It was possible to obtain high 313 resolution images of burst cells, cells with multiple phages attached, phages with normal 314 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 15 morphology and phages with already contracted tails. Icosahedral head shape, widening in 315 the tail end due to the baseplate structure, and tail fibers attached to the bacterial cell wall 316 were also visualized in detail. A large number of elongated E. coli cells, also mentioned in 317 older phage imaging papers [13,14], were also seen, hinting that these mutants may be 318 more common than previously thought. Another characteristic of the HIM was also tested in 319 the samples mentioned above. By increasing the ion current, it is possible to mill (cut) the 320 material at specific locations. Cross sections of bacterial cells, of phage particles and 321 removal of agar substrate pieces were all demonstrated by the milling process. Comparing 322 to other imaging techniques, HIM imaging appears to be more straightforward to use and 323 provides the opportunity to image whole colonies or plaques or other types of complex 324 microbial samples directly on their substrate, with sub-nanometer resolution, with no need 325 for metal coating [58]. 326 We are continuing to apply helium ion microscopy to study phages and phage-327 bacteria interactions. We have developed protocols to avoid agar collapse during 328 preparations, and gained experience with different types of organisms. In Fig.2 we present a 329 few images as examples of our latest phage-bacteria imaging. Sample preparation was made 330 according to Leppänen et al 2017 [58]. 331 Conclusions and perspectives 332 From its origins almost eighty years ago to today, phage imaging has improved 333 immensely (Fig.1) and helped to understand much of these intriguing and important 334 organisms. The earlier years of the electron microscope development resulted in James 335 Hillier and Ernst Ruska sharing the 1960 Albert Lasker Award for Basic Medical Research for 336 their contribution to the development of the first electron microscopes, and Ernst Ruska 337 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 16 receiving a Nobel Prize in 1986 for his fundamental work in electron optics and for the 338 design of the first electron microscope [2,3]. It is also possible to see in the first Luria, 339 Delbruck and Anderson phage imaging papers [13] their interest in basic molecular biology 340 that led to the shared Nobel Prize in 1969 related to replication mechanisms and genetic 341 structure of viruses. In 2017, Dubochet, Frank and Henderson were awarded a Nobel Prize 342 in chemistry for developing cryo-EM, a method that has had a significant impact in high-343 resolution imaging and consequently in three-dimensional structure determination of 344 biomolecules and viruses. Electron microscopy in all its variations and other imaging 345 techniques were crucial for better understanding phages, from structural details to 346 interaction with hosts and diversity. Imaging has contributed to the knowledge that phages 347 are the most abundant organisms in the biosphere, are crucial in regulating global 348 biochemical cycles, have had an important role as models for molecular biology studies and 349 are a viable alternative to treat bacterial diseases by the use of phage therapy. 350 It is hard to imagine how imaging techniques will improve in the next decades, 351 and what knowledge will be gained from their use. However, it can be expected that there 352 will always be attempts to improve existing equipment and technologies, and to create new 353 ones. From a technical point of view, advances in the ability to see in more detail at 354 molecular or atomic resolution, at shorter time scales, and close to native conditions may be 355 the main motivations [59]. From a biological point of view, there is a high interest in single-356 cell live imaging, which can also be applied in combination to single-virus and single-357 molecule imaging. It has been advancing in relation to time resolution (changes detected in 358 milliseconds) and sensitivity (detection of few photons per pixel), but still requires the use of 359 light microscopy and fluorescent labels [60]. Latest developments in AFM include the High-360 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 17 Speed AFM, which allow the following of single molecules dynamics in real time, with 361 potential to be applied to viruses [61]. Mega-electron-volt (MeV) ion beams have been 362 recently used for imaging cells, and the capacity of penetrating through several microns of 363 biological tissue with little deflection (thus maintaining spatial resolution) can also be useful 364 for viral infection studies [62]. Current advances in state of the art microscopy are based on 365 the use of quantum mechanics for photoionization microscopy. A quantum magnetic 366 resonance microscope approach was recently used to image copper complexes in solution, 367 by a non-invasive and non-interfering process that could perhaps in time be applied to living 368 cells [63]. A combination of adaptive optics to lattice light-sheet microscopy (AO-LLSM) was 369 used to visualize cellular processes tri-dimensionally recently [64]. Its non-invasive imaging 370 of events at different scales has potential to be adapted to the study of phage infected cells. 371 As technology gets more advanced sample preparation steps and sample modifications prior 372 to imaging may decrease, optimally leading to analysis of samples close to their native state 373 by higher definition approaches. 374 375 376 377 378 379 380 381 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 18 Conflict of interests 382 There is no conflict of interests between the authors. 383 384 Acknowledgements 385 This work was supported by the Finnish Centre of Excellence Program of the Academy 386 of Finland; the CoE in Biological Interactions 2012–2017 (#252411), by the Academy of 387 Finland (grants #266879 and #304615), by the Jane and Aatos Erkko Foundation, and by the 388 Academy of Finland FIRI2014 infrastructure programme. 389 390 391 392 393 394 395 396 397 398 399 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 19 References 400 [1] Lustig A, Levine A. One hundred years of virology. J Virol 1992;66:4629–31. 401 [2] Oransky I. James Hillier. Lancet 2007;369:1162. 402 [3] Kruger DH, Schneck P, Gelderblom HR. Helmut Ruska and the visualisation of viruses. 403 Lancet 2000;355:1713–7. 404 [4] von Borries B, Ruska E, Ruska H. Bakterien und Virus in übermikroskopischer Aufnahme. 405 Klin Wochenschr. 1938;17:921–51938;17:921–5. 406 [5] Ruska H. Über die Sichtbarmachung der bakteriophagen Lyse im Übermikroskop. 407 Naturwissenschaften 1940;28:45–6. 408 [6] Peankuch E, Kausche GA. Isolierung und übermikroskopische Abbildung eines 409 Bakteriophagen. Naturwissenschaften 1940;28:46. 410 [7] Ackermann H-W. The first phage electron micrographs. Bacteriophage 2011;1:225–7. 411 [8] Ruska H. Morphologische Befunde bei der bakteriophagen Lyse. Arch Gesamte 412 Virusforsch 1942:345–87. 413 [9] Ruska H. Versuch zu einer Ordnung der Virusarten. Arch Gesamte Virusforsch 414 1943;2:480–98. 415 [10] Ruska H. Ergebnisse der Bakteriophagenforschung und ihre Deutung nach 416 morphologischen Befunden. Ergeb Hyg Bakteriol Immunforsch Exp Ther 1943, p. 437–98. 417 [11] Luria SE, Anderson TF. The identification and characterization of bacteriophages with 418 the electron microscope. Proc Natl Acad Sci U S A 1942;28:127–130.1. 419 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 20 [12] Sharma S, Chatterjee S, Datta S, Prasad R, Dubey D, Prasad RK, et al. Bacteriophages 420 and its applications: an overview. Folia Microbiol (Praha) 2017;62:17–55. 421 [13] Luria SE, Delbrück M, Anderson TF. Electron Microscope Studies of Bacterial Viruses. J 422 Bacteriol 1943;46:57–77. 423 [14] Wyckoff R. The electron microscopy of developing bacteriophage. I. Plaques on solid 424 media. Biochim Biophys Acta 1948;2:27–37. 425 [15] Wyckoff R. The electron microscopy of developing bacteriophage. II. Growth of T4 in 426 liquid culture. Biochim Biophys Acta 1948;2:246–53. 427 [16] Fraser D, Williams RC. Details of frozen-dried T3 and T7 bacteriophages as shown by 428 electron microscopy. J Bacteriol 1953;65:167–70. 429 [17] Williams RC, Fraser D. Morphology of the seven T-bacteriophages. J Bacteriol 430 1953;66:458–64. 431 [18] Brenner S, Horne RW. A negative staining method for high resolution electron 432 microscopy of viruses. Biochim Biophys Acta 1959;34:103–10. 433 [19] Brenner S, Streisinger G, Horne RW, Champe SP, Barnett L, Benzer S, et al. Structural 434 components of bacteriophage. J Mol Biol 1959;1:IN9-IN21. 435 [20] Bradley DE, Kay D. The Fine Structure of Bacteriophages. J Gen Microbiol 1960;23:553–436 63. 437 [21] Ackermann HW. 5500 Phages examined in the electron microscope. Arch Virol 438 2007;152:227–43. 439 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 21 [22] Bradley DE. Ultrastructure of bacteriophage and bacteriocins. Bacteriol Rev 440 1967;31:230–314. 441 [23] McCamish J, Mickelson JC. Staining of bacteriophages for light microscopy. Appl 442 Microbiol 1971;21:149. 443 [24] Wendelschafer-Crabb G, Erlandsen SL, Walker DH. Conditions critical for optimal 444 visualization of bacteriophage adsorbed to bacterial surfaces by scanning electron 445 microscopy. J Virol 1975;15:1498–503. 446 [25] Broers AN, Panessa BJ, Gennaro JF. High-resolution scanning electron microscopy of 447 bacteriophages 3C and T4. Science 1975;189:637–9. 448 [26] Keller RW, Dunlap DD, Bustamante C, Keller DJ. Scanning tunneling microscopy images 449 of metal-coated bacteriophages and uncoated, double-stranded DNA. J Vac Sci Technol A 450 Vacuum, Surfaces, Film 1990;8. 451 [27] Kolbe WF, Ogletree DF, Salmeron MB. Atomic force microscopy imaging of T4 452 bacteriophages on silicon substrates. Ultramicroscopy 1992;42–44:1113–7. 453 [28] Alsteens D, Trabelsi H, Soumillion P, Dufrêne YF. Multiparametric atomic force 454 microscopy imaging of single bacteriophages extruding from living bacteria. Nat Commun 455 2013;4. 456 [29] Wikoff WR, Duda RL, Hendrix RW, Johnson JE. Crystallization and preliminary X-ray 457 analysis of the dsDNA bacteriophage HK97 mature empty capsid. Virology 1998 Mar 458 30;243(1):113-8. 459 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 22 [30] Abrescia NGA, Cockburn JJB, Grimes JM, Sutton GC, Diprose JM, Butcher SJ, et al. 460 Insights into assembly from structural analysis of bacteriophage PRD1. Nature 2004;432:68–461 74. 462 [31] Skinner MM, Zhang H, Leschnitzer DH, et al. Structure of the gene V protein of 463 bacteriophage f1 determined by multiwavelength x-ray diffraction on the selenomethionyl 464 protein. Proc Natl Acad Sci U S A. 1994;91(6):2071-2075. 465 [32] Welsh LC, Marvin DA, Perham RN. Analysis of X-ray diffraction from fibres of Pf1 466 Inovirus (filamentous bacteriophage) shows that the DNA in the virion is not highly ordered. 467 J Mol Biol. 1998;284(5):1265-1271. 468 [33] Rissanen I, Pawlowski A, Harlos K, Grimes JM, Stuart DI, Bamford JKH. Crystallization 469 and preliminary crystallographic analysis of the major capsid proteins VP16 and VP17 of 470 bacteriophage P23-77. Acta Crystallogr Sect F Struct Biol Cryst Commun 2012;68:580–3. 471 [34] Reddy HKN, Yoon CH, Aquila A, et al. Coherent soft X-ray diffraction imaging of 472 coliphage PR772 at the Linac coherent light source. Sci Data 2017;4. 473 [35] Reddy VS, Natchiar SK, Stewart PL, Nemerow GR. Crystal Structure of Human 474 Adenovirus at 3.5 A Resolution. Science. 2010;329(5995):1071-1075. 475 [36] Liu H, Jin L, Koh SBS, et al. Atomic structure of human adenovirus by Cryo-EM reveals 476 interactions among protein networks. Science. 2010;329(5995):1038-1043. 477 [37] Dent KC, Thompson R, Barker AM, Hiscox JA, Barr JN, Stockley PG, et al. The asymmetric 478 structure of an icosahedral virus bound to its receptor suggests a mechanism for genome 479 release. Structure 2013;21:1225–34. 480 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 23 [38] Jiang W, Baker ML, Jakana J, Weigele PR, King J, Chiu W. Backbone structure of the 481 infectious epsilon15 virus capsid revealed by electron cryomicroscopy. Nature 482 2008;451:1130–4. 483 [39] Taylor NMI, Prokhorov NS, Guerrero-Ferreira RC, Shneider MM, Browning C, Goldie KN, 484 et al. Structure of the T4 baseplate and its function in triggering sheath contraction. Nature 485 2016;533:346–52. 486 [40] Laanto E, Mäntynen S, De Colibus L, Marjakangas J, Gillum A, Stuart DI, et al. Virus 487 found in a boreal lake links ssDNA and dsDNA viruses. Proc Natl Acad Sci 2017:201703834. 488 [41] Zhao H, Li K, Lynn AY, Aron KE, Yu G, et al. Structure of a headful DNA-packaging 489 bacterial virus at 2.9 Å resolution by electron cryo-microscopy. Proc Natl Acad Sci 490 2017;114:3601–6. 491 [42] Stevens GB, Krüger M, Latychevskaia T, Lindner P, Plückthun A, Fink HW. Individual 492 filamentous phage imaged by electron holography. Eur Biophys J 2011;40:1197–201. 493 [43] Peralta B, Gil-Carton D, Castaño-Díez D, et al. Mechanism of Membranous Tunnelling 494 Nanotube Formation in Viral Genome Delivery. PLoS Biol. 2013;11(9). 495 [44] Chaikeeratisak V, Nguyen K, Khanna K, Brilot AF, Erb ML, Coker JKC, et al. Assembly of a 496 nucleus-like structure during viral replication in bacteria. Science (80- ) 2017;355:194–7. 497 [45] St-Pierre F, Endy D. Determination of cell fate selection during phage lambda infection. 498 Proc Natl Acad Sci 2008;105:20705–10. 499 [46] Zeng L, Skinner SO, Zong C, Sippy J, Feiss M, Golding I. Decision Making at a Subcellular 500 Level Determines the Outcome of Bacteriophage Infection. Cell 2010;141:682–91. 501 MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT