Mikrobiol. Z. 2020; 82(4):80-93.
doi: https://doi.org/10.15407/microbiolj82.04.080

Morphological Biodiversity of Bacteriophages from Lysed Batch Culture
of Recombinant Escherichia coli BL21(DE3)

A.I. Kushkina, F.I. Tovkach

Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine

Sporadic phage infections (phage lysis) of bacterial cultures are omnipresent and lead to dramatic financial losses in biotechnological industry. Investigations of such cases of industrial phage contaminations have both practical and fundamental interests. Aim. It was found that the phage population assaulted a batch culture of an industrial recombinant derivative of E. coli BL21(DE3) was attenuated that manifested in producing pinprick-type plaques and their inability to propagation in subsequent passages. Because of this, the goals of the present research were to evaluate biodiversity and scrutinize the possible virion structural defects of the attenuated phage population prior to pure phage lines isolation. Methods. The anion exchange chromatography (AEC) was chosen as the principal method allowing to get a comparative phage population profile based on the virion net surface charge, as well as to treat the 5-liter virion-contained sample and collect high-quality concentrated and separated phage fractions for further analysis. Virion morphology was examined by transmission electron microscopy, phage identification was conducted by restriction analysis, and sizes of virion DNA were evaluated by pulse field gel electrophoresis. Statistical analysis was applied to estimate phage particles linear dimensions. Results and conclusions. The isolate consisted of a mix of two phages belonging to Myoviridae (A2-morphotype) and Siphoviridae (B1-morphotype) families. By restriction analysis, the main portion of this phage pool (about 99% of all virions) was identified as the primary population of myophage Lw1, which possessed its own intra-population biodiversity (heterogeneity). It consisted of a major and two minor subpopulations that differed by phage capsid size and shape. The subpopulation III consisted of aberrant tubby-phages with triprolate (expanded at both sides) capsids having low aspect ratio. Abbreviations: AEC – anion exchange chromatography, TEM – transmission electron microscopy.

Keywords: bacteriophages, E. coli BL21(DE3), biodiversity, phage morphology, aberrant T4-like capsid.

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  1. Studier FW, Moffatt BA. Use of bacteriophage T7 RNA polymerase to direct selective highlevel expression of cloned genes. J Mol Biol. 1986; 189:113–30. https://doi.org/10.1016/0022-2836(86)90385-2
  2. Li S, Liu L, Zhu J, Zou L, Li M, Cong Y, et al. Characterization and genome sequencing of a novel coliphage isolated from engineered Escherichia coli. Intervirology. 2010; 53:211–20. https://doi.org/10.1159/000299063
  3. Kushkina AI, Tovkach FI, Comeau AM, Kostetskii IE, Lisovski I, Ostapchuk AM, et al. Complete genome sequence of Escherichia phage Lw1, a new member of the RB43 group of pseudo T-Even bacteriophages. Genome Announc. 2013; 1(6):e00743–13. https://doi.org/10.1128/genomeA.00743-13
  4. Kropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP. Enumeration of bacteriophages by bouble agar overlay plaque assay. In: Clokie MR, Kropinski AM, Editors. Methods in molecular biology, vol. 501. Bacteriophages. Humana Press; 2009. https://doi.org/10.1007/978-1-60327-164-6_7
  5. Tikhonenko TI, Koudelka IA, Borishpolets ZI. Phage concentration and purification by the column chromatography method. Mikrobiologiia. 1963; 32:723–6.
  6. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press; 1989.
  7. Serwer P, Pichler ME. Electrophoresis of bacteriophage T7 and T7 capsids in agarose gels. J Virol. 1978; 28:917–28. https://doi.org/10.1128/JVI.28.3.917-928.1978
  8. Serwer P, Hayes SJ, Thomas JA, Griess GA, Hardies SC. Rapid determination of genomic DNA length for new bacteriophages. Electrophoresis. 2007; 28:1896–902. https://doi.org/10.1002/elps.200600672
  9. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Meth. 2012; 9:671–5. https://doi.org/10.1038/nmeth.2089
  10. Sturges HA. The choice of a class interval. J Am Stat Assoc. 1926; 21:65–6. https://doi.org/10.1080/01621459.1926.10502161
  11. Zwillinger D. CRC standard mathematical tables and formulae. Chapman and Hall/CRC; 2002. p. 220. https://doi.org/10.1201/9781420035346
  12. Boy de la Tour E, Kellenberger E. Aberrant forms of the T-even phage head. Virology. 1965; 27:222–5. https://doi.org/10.1016/0042-6822(65)90163-7
  13. Eiserling FA, Geiduschek EP, Epstein RH, Metter EJ. Capsid size and deoxyribonucleic acid length: the petite variant of bacteriophage T4. J Virol. 1970; 6:865–76. https://doi.org/10.1128/JVI.6.6.865-876.1970
  14. Ackermann HW. Frequency of morphological phage descriptions in the year 2000. Arch Virol. 2001; 146:843–57. https://doi.org/10.1007/s007050170120
  15. Fokine A, Chipman PR, Leiman PG, Mesyanzhinov VV, et al. Molecular architecture of the prolate head of bacteriophage T4. Proc Nat Acad Sci. 2004; 101:6003–8. https://doi.org/10.1073/pnas.0400444101
  16. Olson NH, Gingery M, Eiserling FA, Baker TS. The structure of isometric capsids of bacteriophage T4. Virology. 2001; 279:385–91. https://doi.org/10.1006/viro.2000.0735
  17. Mosig G, Carnighan JR, Bibring JB, Cole R, Bock HGO, Bock S. Coordinate variation in lengths of deoxyribonucleic acid molecules and head lengths in morphological variants of bacte riophage T4. J Virol. 1972; 9:857–71. https://doi.org/10.1128/JVI.9.5.857-871.1972
  18. Doermann AH, Eiserling FA, Boehner L. Genetic control of capsid length in bacteriophage T4 I. Isolation and preliminary description of four new mutants. J Virol. 1973; 12:374–85. https://doi.org/10.1128/JVI.12.2.374-385.1973
  19. Cummings DJ, Chapman VA, DeLong SS, Couse NL. Structural aberrations in T-even bacteriophage. III. Induction of “lollipops” and their partial characterization. Virology. 1973; 54:245–61. https://doi.org/10.1016/0042-6822(73)90134-7
  20. Keller B, Dubochet J, Adrian M, Maeder M, Wurtz M, Kellenberger E. Length and shape variants of the bacteriophage T4 head: mutations in the scaffolding core genes 68 and 22. J Virol. 1988; 62:2960–9. https://doi.org/10.1128/JVI.62.8.2960-2969.1988
  21. Moody MF. The shape of the T-even bacteriophage head. Virology. 1965; 26:567–76. https://doi.org/10.1016/0042-6822(65)90319-3
  22. Cummings DJ, Couse NL, Forrest GL. Structural defects of T-even bacteriophages. In: Smith KM, Lauffer MA, Bang FB, Editors. Advances in virus research. Academic Press; 1970. p. 1–41. https://doi.org/10.1016/S0065-3527(08)60020-2
  23. Eiserling FA. T4 structure and initiation of infection. Structure of the T4 virion. In: Mathews CK, Kutter EM, Mosig G, Berget PB, Editors. Bacteriophage T4, ASM 1983, p.11–24.
  24. Mosig G. Coordinate variation in density and recombination potential in T4 phage particles produced at different times after infection. Genetics. 1963; 48:1195–200.
  25. Lane T, Serwer P, Hayes SJ, Frederick E. Quantized viral DNA packaging revealed by rotating gel electrophoresis. Virology. 1990; 174:472–8. https://doi.org/10.1016/0042-6822(90)90101-V
  26. Miller ES, Kutter E, Mosig G, Arisaka F, Kunisawa T, Rüger W. Bacteriophage T4 genome. Microbiol Mol Biol Rev. 2003; 67:86–156. https://doi.org/10.1128/MMBR.67.1.86-156.2003
  27. Paulson JR, Lazaroff S, Laemmli UK. Head length determination in bacteriophage T4: the role of the core protein P22. J Mol Biol. 1976; 103:155–74. https://doi.org/10.1016/0022-2836(76)90057-7
  28. Smith KO, Trousdale M. Multiple-tailed T4 bacteriophage. J Bacteriol. 1965; 90:796–802. https://doi.org/10.1128/JB.90.3.796-802.1965
  29. Pfau CJ, Holt SC. Multitailed T2 bacteriophage. J Virol. 1967; 1:1087–8. https://doi.org/10.1128/JVI.1.5.1087-1088.1967
  30. Ackermann HW, Dauguet C, Paterson WD, Popoff M, Rouf MA, Vieu JF. Aeromonas bacteriophages: reexamination and classification. Ann Inst Pasteur Virol. 1985; 136:175–99. https://doi.org/10.1016/S0769-2617(85)80044-7
  31. Voelker R, Sulakvelidze A, Ackermann HW. Spontaneous tail length variation in a Salmonella myovirus. Virus Res. 2005; 114:164–6. https://doi.org/10.1016/j.virusres.2005.05.006
  32. Rao VB, Black LW. Structure and assembly of bacteriophage T4 head. Virol J. 2010; 7:356. https://doi.org/10.1186/1743-422X-7-356
  33. Kramberger P, Honour RC, Herman RE, Smrekar F, Peterka M. Purification of the Staphylococcus aureus bacteriophages VDX-10 on methacrylate monoliths. J Virol Meth. 2010; 166:60–4. https://doi.org/10.1016/j.jviromet.2010.02.020
  34. Korol N, Van den Bossche A, Romaniuk L, Noben JP, Lavigne R, Tovkach F. Experimental evidence for proteins constituting virion components and particle morphogenesis of bacteriophage ZF40. FEMS Microbiol Lett. 2016; 363–6. https://doi.org/10.1093/femsle/fnw042
  35. Joshi A, Siddiqi JZ, Rao GR, Chakravorty M. MB78, a virulent bacteriophage of Salmonella typhimurium. J Virol. 1982; 41:1038–43. https://doi.org/10.1128/JVI.41.3.1038-1043.1982