Abstract P-47: Analysis of Phosphorus Distribution in Giant Bacteriophage Capsid by Electron Energy Loss Spectroscopy
Background: We have recently developed a method to visualize the distribution of DNA in the cytoplasm of bacteria by analytical electron microscopy (EM), using the Phosphorus signal (dsDNA contains two phosphate groups per each nucleotide pair), that was detected and mapped onto the image of the cel...
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| Language: | English |
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International Medical Research and Development Corporation
2021-06-01
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| Series: | International Journal of Biomedicine |
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| Online Access: | http://ijbm.org/articles/v11s1/ijbm_2021_11_s1_p47.pdf |
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| author | Tatiana Trifonova Andrey Moiseenko Olga Shaburova Maria Bourkaltseva Viktor Krylov Olga Sokolova |
| author_facet | Tatiana Trifonova Andrey Moiseenko Olga Shaburova Maria Bourkaltseva Viktor Krylov Olga Sokolova |
| author_sort | Tatiana Trifonova |
| collection | DOAJ |
| description | Background: We have recently developed a method to visualize the distribution of DNA in the cytoplasm of bacteria by analytical electron microscopy (EM), using the Phosphorus signal (dsDNA contains two phosphate groups per each nucleotide pair), that was detected and mapped onto the image of the cell (Danilova et al, 2020; Loiko et al, 2020). Here we applied this technique to study much smaller objects – the DNA packing inside bacteriophage heads. We studied phiEL, giant phiKZ-like bacteriophage of the Myoviridae family that infects Pseudomonas aeruginosa (Krylov et al, 2003). We have earlier demonstrated that this phage contains an ‘inner body’ inside its capsid, which is responsible for the specific DNA packing (Sokolova et al, 2014).
Methods: The phage propagation was performed as described before (Sokolova et al, 2014). A 3 ul sample of purified bacteriophage phiEL was applied to the glow-discharged carbon-coated copper grid and stained with freshly prepared ammonium Molybdate 2% aquatic solution for 30 sec. Grids were loaded into Gatan cooling holder and the temperature of the specimen was kept at -180°C.
EELS spectra and phosphorus elemental maps were obtained on JEOL2100 microscope, operating at 200 kV with the Gatan GIF Quantum ER spectrometer in STEM mode. Pixel size was set to 15-20 nm. STEM drift correction was applied after each 40-50 pixels. Each spectrum was obtained at a 6.0 mrad collection angle, 0.25 eV dispersion, and 132 eV energy shift. The spectra from different pixels were aligned to carbon K-edge.
Results: Phosphorus mapping inside and outside the bacteriophage capsid was performed (Fig. 1). Outside the capsid, the phosphorus signal was practically absent, which corresponds to the presence of DNA only inside the capsid. The distribution of phosphorus inside the capsid was uneven: the rectangular area in the middle of the capsid contained a weak signal, while a more intense signal was detected on the periphery. This can be explained by the presence of an ‘inner body’ inside (Fig. 1C).
Conclusion: Thus, our results justify the possibility of using the analytical EM technique to study the distribution of DNA by mapping Phosphorus in biological nano-objects at relatively low content of the element. |
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| id | doaj.art-1116cce4ee424988b5c5ed53ef27b85f |
| institution | Directory Open Access Journal |
| issn | 2158-0510 2158-0529 |
| language | English |
| last_indexed | 2024-12-22T00:50:35Z |
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| series | International Journal of Biomedicine |
| spelling | doaj.art-1116cce4ee424988b5c5ed53ef27b85f2022-12-21T18:44:26ZengInternational Medical Research and Development CorporationInternational Journal of Biomedicine2158-05102158-05292021-06-0111Suppl_1333310.21103/IJBM.11.Suppl_1.P47Abstract P-47: Analysis of Phosphorus Distribution in Giant Bacteriophage Capsid by Electron Energy Loss SpectroscopyTatiana Trifonova0Andrey Moiseenko1Olga Shaburova2Maria Bourkaltseva3Viktor Krylov4Olga Sokolova5Lomonosov Moscow State University, Moscow, RussiaLomonosov Moscow State University, Moscow, Russia; Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow, RussiaI.I. Mechnikov Research Institute of Vaccines and Sera, Moscow, RussiaI.I. Mechnikov Research Institute of Vaccines and Sera, Moscow, RussiaI.I. Mechnikov Research Institute of Vaccines and Sera, Moscow, RussiaLomonosov Moscow State University, Moscow, RussiaBackground: We have recently developed a method to visualize the distribution of DNA in the cytoplasm of bacteria by analytical electron microscopy (EM), using the Phosphorus signal (dsDNA contains two phosphate groups per each nucleotide pair), that was detected and mapped onto the image of the cell (Danilova et al, 2020; Loiko et al, 2020). Here we applied this technique to study much smaller objects – the DNA packing inside bacteriophage heads. We studied phiEL, giant phiKZ-like bacteriophage of the Myoviridae family that infects Pseudomonas aeruginosa (Krylov et al, 2003). We have earlier demonstrated that this phage contains an ‘inner body’ inside its capsid, which is responsible for the specific DNA packing (Sokolova et al, 2014). Methods: The phage propagation was performed as described before (Sokolova et al, 2014). A 3 ul sample of purified bacteriophage phiEL was applied to the glow-discharged carbon-coated copper grid and stained with freshly prepared ammonium Molybdate 2% aquatic solution for 30 sec. Grids were loaded into Gatan cooling holder and the temperature of the specimen was kept at -180°C. EELS spectra and phosphorus elemental maps were obtained on JEOL2100 microscope, operating at 200 kV with the Gatan GIF Quantum ER spectrometer in STEM mode. Pixel size was set to 15-20 nm. STEM drift correction was applied after each 40-50 pixels. Each spectrum was obtained at a 6.0 mrad collection angle, 0.25 eV dispersion, and 132 eV energy shift. The spectra from different pixels were aligned to carbon K-edge. Results: Phosphorus mapping inside and outside the bacteriophage capsid was performed (Fig. 1). Outside the capsid, the phosphorus signal was practically absent, which corresponds to the presence of DNA only inside the capsid. The distribution of phosphorus inside the capsid was uneven: the rectangular area in the middle of the capsid contained a weak signal, while a more intense signal was detected on the periphery. This can be explained by the presence of an ‘inner body’ inside (Fig. 1C). Conclusion: Thus, our results justify the possibility of using the analytical EM technique to study the distribution of DNA by mapping Phosphorus in biological nano-objects at relatively low content of the element.http://ijbm.org/articles/v11s1/ijbm_2021_11_s1_p47.pdfanalytical emeelspseudomonas aeruginosaphage phieldna packing |
| spellingShingle | Tatiana Trifonova Andrey Moiseenko Olga Shaburova Maria Bourkaltseva Viktor Krylov Olga Sokolova Abstract P-47: Analysis of Phosphorus Distribution in Giant Bacteriophage Capsid by Electron Energy Loss Spectroscopy International Journal of Biomedicine analytical em eels pseudomonas aeruginosa phage phiel dna packing |
| title | Abstract P-47: Analysis of Phosphorus Distribution in Giant Bacteriophage Capsid by Electron Energy Loss Spectroscopy |
| title_full | Abstract P-47: Analysis of Phosphorus Distribution in Giant Bacteriophage Capsid by Electron Energy Loss Spectroscopy |
| title_fullStr | Abstract P-47: Analysis of Phosphorus Distribution in Giant Bacteriophage Capsid by Electron Energy Loss Spectroscopy |
| title_full_unstemmed | Abstract P-47: Analysis of Phosphorus Distribution in Giant Bacteriophage Capsid by Electron Energy Loss Spectroscopy |
| title_short | Abstract P-47: Analysis of Phosphorus Distribution in Giant Bacteriophage Capsid by Electron Energy Loss Spectroscopy |
| title_sort | abstract p 47 analysis of phosphorus distribution in giant bacteriophage capsid by electron energy loss spectroscopy |
| topic | analytical em eels pseudomonas aeruginosa phage phiel dna packing |
| url | http://ijbm.org/articles/v11s1/ijbm_2021_11_s1_p47.pdf |
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