Metabolic labeling of Escherichia coli genomic DNA with erythrosine-11-dUTP for functional imaging via correlative microscopy
Metabolic labeling of Escherichia coli genomic DNA with erythrosine-11-dUTP for functional imaging via correlative microscopy
复制标题
使用 erythrosine-11-dUTP 代谢标记大肠杆菌基因组 DNA，通过相关显微镜进行功能成像
DOI：
10.1002/jemt.23487
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发表时间：
2020
影响因子：
2.5
通讯作者：
Nakabayashi Seiichiro
中科院分区：
文献类型：
--
作者：
Loukanov Alexandre;Nikolova Svetla;Filipov Chavdar;Nakabayashi Seiichiro
微生物基因组荧光代谢标记技术是一种先进的成像技术，可用于观察和研究单个细胞或组织中的天然形状、结构变化、功能以及核酸的追踪。我们试图通过相关的光电子显微镜观察大肠杆菌K-12（胸苷需要突变株，菌株N4316）的冰包埋增殖细胞的完整类核内新合成的DNA。为此，合成了赤藓红-11-dUTP，并将其用作外源性胸苷底物的修饰类似物，用于代谢掺入细菌染色体。在细胞内聚合酶反应过程中形成的荧光基因组DNA引起最小的细胞停滞和E.在一定的控制条件下。通过落射荧光显微镜以典型的红色发射颜色观察染色的细胞。将其进一步冰包埋，并使用希尔伯特微分对比透射电子显微镜进行检查。在高分辨率下，与未标记的类核相比，标记的类核的超微结构显示出显著更高的电子密度。染色体中的增强对比区域归因于赤藓红染料中碘含量的存在。该标记方法可能是一种强有力的策略，以揭示自然DNA复制的结构和动态变化，包括体内新合成的核酸与细胞的生理状态之间的关系。
The fluorescent metabolic labeling of microorganisms genome is an advanced imaging technique to observe and study the native shapes, structural changes, functions, and tracking of nucleic acids in single cells or tissues. We have attempted to visualize the newly synthesized DNA within the intact nucleoid of ice‐embedded proliferating cells ofEscherichia coliK‐12 (thymidine‐requiring mutant, strain N4316) via correlative light‐electron microscopy. For that purpose, erythrosine‐11‐dUTP was synthesized and used as a modified analog of the exogenous thymidine substrate for metabolic incorporation into the bacterial chromosome. The formed fluorescent genomic DNA during in cellulo polymerase reaction caused a minimal cellular arrest and cytotoxicity ofE. coliat certain controlled conditions. The stained cells were visualized in typical red emission color via an epifluorescence microscope. They were further ice‐embedded and examined with a Hilbert differential contrast transmission electron microscopy. At high‐resolution, the ultrastructure of tagged nucleoid appeared with significantly higher electron dense in comparison to the unlabeled one. The enhanced contrast areas in the chromosome were ascribed to the presence of iodine contents from erythrosine dye. The presented labeling approach might be a powerful strategy to reveal the structural and dynamic changes in natural DNA replication including the relationship between newly synthesized in vivo nucleic acid and the physiological state of the cell.