Metabolic labeling of Escherichia coli genomic DNA with erythrosine-11-dUTP for functional imaging via correlative microscopy.
Microsc Res Tech
; 83(8): 937-944, 2020 Aug.
Article
em En
| MEDLINE
| ID: mdl-32233103
ABSTRACT
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 of Escherichia coli K-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 of E. coli at 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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA Bacteriano
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Escherichia coli K12
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Nucleotídeos de Desoxiuracil
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Microscopia Eletrônica de Transmissão
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Eritrosina
Idioma:
En
Revista:
Microsc Res Tech
Assunto da revista:
DIAGNOSTICO POR IMAGEM
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Japão