Вопросы вирусологии. 2021; 66: 434-441
Картирование ДНК в капсиде гигантского бактериофага phiEL (Caudovirales: Myoviridae: Elvirus) с помощью аналитической электронной микроскопии
https://doi.org/10.36233/0507-4088-80Аннотация
Введение. Гигантские phiKZ-подобные бактериофаги имеют внутри капсида уникальное белковое образование – внутреннее тело (ВТ), на которое навита суперскрученная ДНК. Стандартные подходы, используемые в криоэлектронной микроскопии (криоЭМ), не позволяют отличить эту структуру от окружающей её молекулы нуклеиновой кислоты фага. Ранее нами разработан аналитический подход для визуализации комплексов ДНК с белком на срезах бактериальных клеток Escherichia coli с использованием в качестве маркёра химического элемента фосфора. В настоящем исследовании мы адаптировали данную методику к значительно более мелким объектам – капсидам phiKZ-подобных бактериофагов.
Материал и методы. В исследовании применялись методы электронной микроскопии: аналитическая (АЭМ) (спектроскопия характеристических потерь энергии электронами, СХПЭЭ) и криоЭМ (сравнение изображений образцов с низкой и высокой дозой электронного облучения). Результаты. Мы изучили упаковку молекулы ДНК внутри капсидов гигантских бактериофагов phiEL из семейства Myoviridae, инфицирующих Pseudomonas aeruginosa. Построены карты распределения фосфора, показавшие несимметричное расположение ДНК внутри капсида.
Обсуждение. Мы разработали и применили методику визуализации ВТ с использованием высокоуглового темнопольного детектора (HAADF) и аналитического подхода СПЭМ-СХПЭЭ. Картирование распределения фосфора посредством СХПЭЭ и результаты криоЭМ выявили белковую структуру внутри капсида фагов phiEL в виде ВТ, размер которого был оценён с помощью теоретических расчётов.
Заключение. Разработанная методика может применяться для исследования распределения фосфора в других ДНК- или РНК-содержащих вирусах при сравнительно низких содержаниях искомого элемента.
Список литературы
1. Ochman H., Lawrence J., Groisman E. Lateral gene transfer and the nature of bacterial innovation. Nature. 2000; 405(6784): 299–304. https://doi.org/10.1038/35012500
2. Duplessis C.A., Biswas B. A review of topical phage therapy for chronically infected wounds and preparations for a randomized adaptive clinical trial evaluating topical phage therapy in chronically infected diabetic foot ulcers. Antibiotics. 2020; 9(7): 377. https://doi.org/10.3390/antibiotics9070377
3. Sharma R., Pielstick B., Bell K., Nieman T., Stubbs O., Yeates E., et al. A Novel, Highly Related Jumbo Family of Bacteriophages That Were Isolated Against Erwinia. Front. Microbiol. 2019; 10: 1533. https://doi.org/10.3389/fmicb.2019.01533
4. Fokine A., Kostyuchenko V.A., Efimov A.V., Kurochkina L.P., Sykilinda N.N., Robben J., et al. A three-dimensional cryo-electron microscopy structure of the bacteriophage ϕKZ head. J. Mol. Biol. 2005; 352(1): 117–24. https://doi.org/10.1016/j.jmb.2005.07.018
5. Sokolova O.S., Shaburova O.V., Pechnikova E.V., Shaytan A.K., Krylov S.V., Kiselev N.A., et al. Genome packaging in EL and Lin68, two giant phiKZ-like bacteriophages of P. aeruginosa. Virology. 2014; 468–470: 472–8. https://doi.org/10.1016/j.virol.2014.09.002
6. Hertveldt K., Lavigne R., Pleteneva E., Sernova N., Kurochkina L., Korchevskii R., et al. Genome comparison of Pseudomonas aeruginosa large phages. J. Mol. Biol. 2005; 354(3): 536–45. https://doi.org/10.1016/j.jmb.2005.08.075
7. Mesyanzhinov V.V., Robben J., Grymonprez B., Kostyuchenko V.A., Bourkaltseva M.V., Sykilinda N.N., et al. The genome of bacteriophage phiKZ of Pseudomonas aeruginosa. J. Mol. Biol. 2002; 317(1): 1–19. https://doi.org/10.1006/jmbi.2001.5396
8. Thomas J.A., Rolando M.R., Carroll C.A., Shen P.S., Belnap D.M., Weintraub S.T., et al. Characterization of Pseudomonas chlororaphis myovirus 201ϕ2-1 via genomic sequencing, mass spectrometry, and electron microscopy. Virology. 2008; 376(2): 330–8. https://doi.org/10.1016/j.virol.2008.04.004
9. Krylov V.N., Smirnova T.A., Minenkova I.B., Plotnikova T.G., Zhazikov I.Z., Khrenova E.A. Pseudomonas bacteriophage contains an inner body in its capsid. Can. J. Microbiol. 1984; 30(6): 758–62. https://doi.org/10.1139/m84-116
10. Wu W., Thomas J., Naiqian C., Black L., Steven A.C. Bubblegrams reveal the inner body of bacteriophage phiKZ. Science. 2012; 335(6065): 182. https://doi.org/10.1126/science.1214120
11. Yakunina M., Artamonova T., Borukhov S., Makarova K.S., Severinov K., Minakhin L. A non-canonical multisubunit RNA polymerase encoded by a giant bacteriophage. Nucleic Acids res. 2015; 43(21): 10411–20. https://doi.org/10.1093/nar/gkv1095
12. Danilova Y.A., Belousova V.V., Moiseenko A.V., Vishnyakov I.E., Yakunina M.V., Sokolova O.S. Maturation of Pseudo-Nucleus Compartment in P. aeruginosa, Infected with Giant phiKZ Phage. Viruses. 2020; 12(10): 1197. https://doi.org/10.3390/v12101197
13. Matsko N., Klinov D., Manykin A., Demin V., Klimenko S. Atomic force microscopy analysis of bacteriophages phiKZ and T4. J. Electron. Microsc. (Tokyo). 2001; 50(5): 417–22. https://doi.org/10.1093/jmicro/50.5.417
14. Fontana J., Jurado K.A., Cheng N., Ly N.L., Fuchs J.R., Gorelick R.J., et al. Distribution and Redistribution of HIV-1 Nucleocapsid Protein in Immature, Mature, and Integrase-Inhibited Virions: a Role for Integrase in Maturation. J. Virol. 2015; 89(19): 9765–80. https://doi.org/10.1128/JVI.01522-15
15. Wu W., Leavitt J.C., Cheng N., Gilcrease E.B., Motwani T., Teschke C.M., et al. Localization of the houdinisome (Ejection Proteins) inside the bacteriophage P22 virion by bubblegram imaging. mBio. 2016; 7(4): e01152–16. https://doi.org/10.1128/mBio.01152-16
16. Wu W., Newcomb W.W., Cheng N., Aksyuk A., Winkler D.C., Steven A.C. Internal Proteins of the Procapsid and Mature Capsids of Herpes Simplex Virus 1 Mapped by Bubblegram Imaging. J. Virol. 2016; 90(10): 5176–86. https://doi.org/10.1128/JVI.03224-15
17. Shebanova A., Ismagulova T., Solovchenko A., Baulina O., Lobakova E., Ivanova A., et al. Versatility of the green microalga cell vacuole function as revealed by analytical transmission electron microscopy. Protoplasma. 2017; 254(3): 1323–40. https://doi.org/10.1007/s00709-016-1024-5
18. Scotuzzi M., Kuipers J., Wensveen D.I., De Boer P., Hagen K.C.W., Hoogenboom J.P., et al. Multi-color electron microscopy by element- guided identification of cells, organelles and molecules. Sci. Rep. 2017; 7: 45970. https://doi.org/10.1038/srep45970
19. Allard-Vannier E., Hervé-Aubert K., Kaaki K., Blondy T., Shebanova A., Shaitan K.V., et al. Folic acid-capped PEGylated magnetic nanoparticles enter cancer cells mostly via clathrin-dependent endocytosis. Biochim. Biophys. Acta Gen. Subj. 2017; 1861(6): 1578–86. https://doi.org/10.1016/j.bbagen.2016.11.045
20. Loiko N., Danilova Y., Moiseenko A., Kovalenko V., Tereshkina K., Tutukina M., et al. Morphological peculiarities of the DNA-protein complexes in starved Escherichia coli cells. PLoS One. 2020; 15(10): e0231562. https://doi.org/10.1371/journal.pone.0231562
21. Bazett-Jones D.P., Ottensmeyer F.P. Phosphorus distribution in the nucleosome. Science. 1981; 211(4478): 169–70. https://doi.org/10.1126/science.7444457
22. Ottensmeyer F.P., Andrew J.W. High-resolution microanalysis of biological specimens by electron energy loss spectroscopy and by electron spectroscopic imaging. J. Ultrastruct. Res. 1980; 72(3):336–48. https://doi.org/10.1016/s0022-5320(80)90069-6
23. Aronova M.A., Kim Y.C., Harmon R., Sousa A.A., Zhang G., Leapman R.D. Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST). J. Struct. Biol. 2007; 160(1): 35–48. https://doi.org/10.1016/j.jsb.2007.06.008
24. Nevsten P., Evilevitch A., Wallenberg R. Chemical mapping of DNA and counter-ion content inside phage by energy-filtered TEM. J. Biol. Phys. 2012; 38(2): 229–40. https://doi.org/10.1007/s10867-011-9234-8
25. Sambrook J., Fritsch E.F., Maniatis T. Molecular Cloning: a Laboratory Manual. New York: Cold Spring Harbor Laboratory Press; 1989.
26. Печникова Е.В., Кирпичников М.П., Соколова О.С. Радиационные повреждения в криомикроскопии: всегда ли во вред? Природа. 2015; (3): 25–9.
27. Mishyna M., Volokh O., Danilova Ya., Gerasimova N., Pechnikova E., Sokolova O.S. Effects of radiation damage in studies of protein-DNA complexes by cryo-EM. Micron. 2017; 96: 57–64. https://doi.org/10.1016/j.micron.2017.02.004
28. Petrov A.S., Harvey S.C. Packaging double-helical DNA into viral capsids: structures, forces, and energetics. Biophys. J. 2008; 95(2):497–502. https://doi.org/10.1529/biophysj.108.131797
29. Буркальцева М.В., Крылов В.Н., Плетенева Е.А., Шабурова О.В., Крылов С.В., Волкарт Г., и др. Феногенетическая характеристика группы гигантских φKZ-подобных бактериофагов Pseudomonas aeruginosa. Генетика. 2002; 38(11): 1470–9.
30. Bagrov D.V., Glukhov G.S., Moiseenko A.V., Karlova M.G., Litvinov D.S., Zaitsev P.А., et al. Structural characterization of β-propiolactone inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles. Microsc. Res. Tech. 2021. https://doi.org/10.1002/jemt.23931
Problems of Virology. 2021; 66: 434-441
DNA mapping in the capsid of giant bacteriophage phiEL (Caudovirales: Myoviridae: Elvirus) by analytical electron microscopy
https://doi.org/10.36233/0507-4088-80Abstract
Introduction. Giant phiKZ-like bacteriophages have a unique protein formation inside the capsid, an inner body (IB) with supercoiled DNA molecule wrapped around it. Standard cryo-electron microscopy (cryo-EM) approaches do not allow to distinguish this structure from the surrounding nucleic acid of the phage. We previously developed an analytical approach to visualize protein-DNA complexes on Escherichia coli bacterial cell slices using the chemical element phosphorus as a marker. In the study presented, we adapted this technique for much smaller objects, namely the capsids of phiKZ-like bacteriophages.
Material and methods. Following electron microscopy techniques were used in the study: analytical (AEM) (electron energy loss spectroscopy, EELS), and cryo-EM (images of samples subjected to low and high dose of electron irradiation were compared).
Results. We studied DNA packaging inside the capsids of giant bacteriophages phiEL from the Myoviridae family that infect Pseudomonas aeruginosa. Phosphorus distribution maps were obtained, showing an asymmetrical arrangement of DNA inside the capsid.
Discussion. We developed and applied an IB imaging technique using a high angle dark-field detector (HAADF) and the STEM-EELS analytical approach. Phosphorus mapping by EELS and cryo-electron microscopy revealed a protein formation as IB within the phage phiEL capsid. The size of IB was estimated using theoretical calculations.
Conclusion. The developed technique can be applied to study the distribution of phosphorus in other DNA- or RNA-containing viruses at relatively low concentrations of the element sought.
References
1. Ochman H., Lawrence J., Groisman E. Lateral gene transfer and the nature of bacterial innovation. Nature. 2000; 405(6784): 299–304. https://doi.org/10.1038/35012500
2. Duplessis C.A., Biswas B. A review of topical phage therapy for chronically infected wounds and preparations for a randomized adaptive clinical trial evaluating topical phage therapy in chronically infected diabetic foot ulcers. Antibiotics. 2020; 9(7): 377. https://doi.org/10.3390/antibiotics9070377
3. Sharma R., Pielstick B., Bell K., Nieman T., Stubbs O., Yeates E., et al. A Novel, Highly Related Jumbo Family of Bacteriophages That Were Isolated Against Erwinia. Front. Microbiol. 2019; 10: 1533. https://doi.org/10.3389/fmicb.2019.01533
4. Fokine A., Kostyuchenko V.A., Efimov A.V., Kurochkina L.P., Sykilinda N.N., Robben J., et al. A three-dimensional cryo-electron microscopy structure of the bacteriophage ϕKZ head. J. Mol. Biol. 2005; 352(1): 117–24. https://doi.org/10.1016/j.jmb.2005.07.018
5. Sokolova O.S., Shaburova O.V., Pechnikova E.V., Shaytan A.K., Krylov S.V., Kiselev N.A., et al. Genome packaging in EL and Lin68, two giant phiKZ-like bacteriophages of P. aeruginosa. Virology. 2014; 468–470: 472–8. https://doi.org/10.1016/j.virol.2014.09.002
6. Hertveldt K., Lavigne R., Pleteneva E., Sernova N., Kurochkina L., Korchevskii R., et al. Genome comparison of Pseudomonas aeruginosa large phages. J. Mol. Biol. 2005; 354(3): 536–45. https://doi.org/10.1016/j.jmb.2005.08.075
7. Mesyanzhinov V.V., Robben J., Grymonprez B., Kostyuchenko V.A., Bourkaltseva M.V., Sykilinda N.N., et al. The genome of bacteriophage phiKZ of Pseudomonas aeruginosa. J. Mol. Biol. 2002; 317(1): 1–19. https://doi.org/10.1006/jmbi.2001.5396
8. Thomas J.A., Rolando M.R., Carroll C.A., Shen P.S., Belnap D.M., Weintraub S.T., et al. Characterization of Pseudomonas chlororaphis myovirus 201ϕ2-1 via genomic sequencing, mass spectrometry, and electron microscopy. Virology. 2008; 376(2): 330–8. https://doi.org/10.1016/j.virol.2008.04.004
9. Krylov V.N., Smirnova T.A., Minenkova I.B., Plotnikova T.G., Zhazikov I.Z., Khrenova E.A. Pseudomonas bacteriophage contains an inner body in its capsid. Can. J. Microbiol. 1984; 30(6): 758–62. https://doi.org/10.1139/m84-116
10. Wu W., Thomas J., Naiqian C., Black L., Steven A.C. Bubblegrams reveal the inner body of bacteriophage phiKZ. Science. 2012; 335(6065): 182. https://doi.org/10.1126/science.1214120
11. Yakunina M., Artamonova T., Borukhov S., Makarova K.S., Severinov K., Minakhin L. A non-canonical multisubunit RNA polymerase encoded by a giant bacteriophage. Nucleic Acids res. 2015; 43(21): 10411–20. https://doi.org/10.1093/nar/gkv1095
12. Danilova Y.A., Belousova V.V., Moiseenko A.V., Vishnyakov I.E., Yakunina M.V., Sokolova O.S. Maturation of Pseudo-Nucleus Compartment in P. aeruginosa, Infected with Giant phiKZ Phage. Viruses. 2020; 12(10): 1197. https://doi.org/10.3390/v12101197
13. Matsko N., Klinov D., Manykin A., Demin V., Klimenko S. Atomic force microscopy analysis of bacteriophages phiKZ and T4. J. Electron. Microsc. (Tokyo). 2001; 50(5): 417–22. https://doi.org/10.1093/jmicro/50.5.417
14. Fontana J., Jurado K.A., Cheng N., Ly N.L., Fuchs J.R., Gorelick R.J., et al. Distribution and Redistribution of HIV-1 Nucleocapsid Protein in Immature, Mature, and Integrase-Inhibited Virions: a Role for Integrase in Maturation. J. Virol. 2015; 89(19): 9765–80. https://doi.org/10.1128/JVI.01522-15
15. Wu W., Leavitt J.C., Cheng N., Gilcrease E.B., Motwani T., Teschke C.M., et al. Localization of the houdinisome (Ejection Proteins) inside the bacteriophage P22 virion by bubblegram imaging. mBio. 2016; 7(4): e01152–16. https://doi.org/10.1128/mBio.01152-16
16. Wu W., Newcomb W.W., Cheng N., Aksyuk A., Winkler D.C., Steven A.C. Internal Proteins of the Procapsid and Mature Capsids of Herpes Simplex Virus 1 Mapped by Bubblegram Imaging. J. Virol. 2016; 90(10): 5176–86. https://doi.org/10.1128/JVI.03224-15
17. Shebanova A., Ismagulova T., Solovchenko A., Baulina O., Lobakova E., Ivanova A., et al. Versatility of the green microalga cell vacuole function as revealed by analytical transmission electron microscopy. Protoplasma. 2017; 254(3): 1323–40. https://doi.org/10.1007/s00709-016-1024-5
18. Scotuzzi M., Kuipers J., Wensveen D.I., De Boer P., Hagen K.C.W., Hoogenboom J.P., et al. Multi-color electron microscopy by element- guided identification of cells, organelles and molecules. Sci. Rep. 2017; 7: 45970. https://doi.org/10.1038/srep45970
19. Allard-Vannier E., Hervé-Aubert K., Kaaki K., Blondy T., Shebanova A., Shaitan K.V., et al. Folic acid-capped PEGylated magnetic nanoparticles enter cancer cells mostly via clathrin-dependent endocytosis. Biochim. Biophys. Acta Gen. Subj. 2017; 1861(6): 1578–86. https://doi.org/10.1016/j.bbagen.2016.11.045
20. Loiko N., Danilova Y., Moiseenko A., Kovalenko V., Tereshkina K., Tutukina M., et al. Morphological peculiarities of the DNA-protein complexes in starved Escherichia coli cells. PLoS One. 2020; 15(10): e0231562. https://doi.org/10.1371/journal.pone.0231562
21. Bazett-Jones D.P., Ottensmeyer F.P. Phosphorus distribution in the nucleosome. Science. 1981; 211(4478): 169–70. https://doi.org/10.1126/science.7444457
22. Ottensmeyer F.P., Andrew J.W. High-resolution microanalysis of biological specimens by electron energy loss spectroscopy and by electron spectroscopic imaging. J. Ultrastruct. Res. 1980; 72(3):336–48. https://doi.org/10.1016/s0022-5320(80)90069-6
23. Aronova M.A., Kim Y.C., Harmon R., Sousa A.A., Zhang G., Leapman R.D. Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST). J. Struct. Biol. 2007; 160(1): 35–48. https://doi.org/10.1016/j.jsb.2007.06.008
24. Nevsten P., Evilevitch A., Wallenberg R. Chemical mapping of DNA and counter-ion content inside phage by energy-filtered TEM. J. Biol. Phys. 2012; 38(2): 229–40. https://doi.org/10.1007/s10867-011-9234-8
25. Sambrook J., Fritsch E.F., Maniatis T. Molecular Cloning: a Laboratory Manual. New York: Cold Spring Harbor Laboratory Press; 1989.
26. Pechnikova E.V., Kirpichnikov M.P., Sokolova O.S. Radiatsionnye povrezhdeniya v kriomikroskopii: vsegda li vo vred? Priroda. 2015; (3): 25–9.
27. Mishyna M., Volokh O., Danilova Ya., Gerasimova N., Pechnikova E., Sokolova O.S. Effects of radiation damage in studies of protein-DNA complexes by cryo-EM. Micron. 2017; 96: 57–64. https://doi.org/10.1016/j.micron.2017.02.004
28. Petrov A.S., Harvey S.C. Packaging double-helical DNA into viral capsids: structures, forces, and energetics. Biophys. J. 2008; 95(2):497–502. https://doi.org/10.1529/biophysj.108.131797
29. Burkal'tseva M.V., Krylov V.N., Pleteneva E.A., Shaburova O.V., Krylov S.V., Volkart G., i dr. Fenogeneticheskaya kharakteristika gruppy gigantskikh φKZ-podobnykh bakteriofagov Pseudomonas aeruginosa. Genetika. 2002; 38(11): 1470–9.
30. Bagrov D.V., Glukhov G.S., Moiseenko A.V., Karlova M.G., Litvinov D.S., Zaitsev P.A., et al. Structural characterization of β-propiolactone inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles. Microsc. Res. Tech. 2021. https://doi.org/10.1002/jemt.23931
