Trifonova T S, Moiseenko A V, Bourkaltseva M V, Shaburova O V, Shaytan A K, Krylov V N, Sokolova O S
FSAEI HE «People's Friendship University of Russia», Physical, Mathematical, and Natural Sciences Department; FSBEI HE «Lomonosov Moscow State University», Bioengineering Department, Biological Faculty.
FSBEI HE «Lomonosov Moscow State University», Bioengineering Department, Biological Faculty; FSBIS «N.N. Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences».
Vopr Virusol. 2022 Jan 8;66(6):434-441. doi: 10.36233/0507-4088-80.
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.
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).
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.
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.
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.
巨型类phiKZ噬菌体在衣壳内部具有独特的蛋白质结构，即内部体（IB），其周围缠绕着超螺旋DNA分子。标准的冷冻电子显微镜（cryo-EM）方法无法将这种结构与噬菌体周围的核酸区分开来。我们之前开发了一种分析方法，利用化学元素磷作为标记，在大肠杆菌细菌细胞切片上可视化蛋白质-DNA复合物。在本研究中，我们将该技术应用于更小的物体，即类phiKZ噬菌体的衣壳。
本研究采用了以下电子显微镜技术：分析电子显微镜（AEM）（电子能量损失谱，EELS）和冷冻电子显微镜（比较了低剂量和高剂量电子辐照样品的图像）。
我们研究了感染铜绿假单胞菌的肌尾噬菌体科巨型噬菌体phiEL衣壳内的DNA包装。获得了磷分布图，显示衣壳内DNA的不对称排列。
我们开发并应用了一种使用高角度暗场探测器（HAADF）和STEM-EELS分析方法的内部体成像技术。通过EELS和冷冻电子显微镜进行的磷映射揭示了噬菌体phiEL衣壳内作为内部体的蛋白质结构。使用理论计算估计了内部体的大小。
所开发的技术可应用于在相对低浓度的目标元素下研究其他含DNA或RNA病毒中磷的分布。
