Visualization of the native shape of bodipy‐labeled DNA inEscherichia coliby correlative microscopy

Abstract

The native shape and intracellular distribution of newly synthesized DNA was visualized by correlative (light and electron) microscopy in ice embedded whole cells of Escherichia coli. For that purpose, the commercially available modified nucleoside triphosphate named BODIPY® FL-14-dUTP was enzymatically incorporated in vivo into the genome of E. coli mutant K12 strain, which cannot synthesize thymine. The successful incorporation of this thymidine analogue was confirmed first by fluorescence microscope, where the cells were stained in the typical for bodipy green color. Later the preselected labeled E. coli were observed by Hilbert Differential Transmission Electron Microscope (HDC TEM) and the distribution of elemental boron (contained in bodipy) was visualized at high-resolution by an electron spectroscopic imaging (ESI) technique. The practical detection limit of boron was found to be around 5 ∼ 10 mmol/kg in area of 0.1 μm , which demonstrated that ESI is a suitable approach to study the cytochemistry and location of labeled nucleic fragments within the cytoplasmic chromosomal area. In addition, the fine cellular fibrous and chromosomal ultrastructures were revealed in situ by combing of phase-plate HDC TEM and ESI. The obtained results conclude that the correlation between fluorescent microscopy with phase-plate HDC TEM and ESI is a powerful approach to explore the structural and conformation dynamics of DNA replication machinery in frozen cells close to the living state.

Fluorescent dATP for DNA Synthesis In Vivo

“…Subsequent bioorthogonal chemical reactions with fluorescent probes can then identify the locations of the modified nucleotides. − However, this approach has not been successfully used for live-animal imaging, due to the poor cellular uptake of the fluorescent probe and/or the high stringency of cellular kinases that tolerate only small modifications on the nucleoside analogue. − One way to overcome these obstacles is through the direct delivery of fluorescently-labeled nucleoside triphosphates (dNTPs) into cells by microinjection or electroporation. Previous studies demonstrated that modified uracil triphosphate (dUTP) derivatives conjugated to a fluorescent probe at the 5 position of the nucleobase can be metabolically incorporated into genomic DNA during S-phase. − It is unknown, however, if this approach is compatible with live-animal imaging or if purine-based derivatives can be used. In addition, the impact of the fluorescent probe itself on the biological fitness of the animals containing modified genomes has not been previously explored.…”

Active Uptake and Trafficking of Nucleoside Triphosphates In Vivo

“…Due to the high substrate specificity of cellular kinases, nucleoside phosphorylation is often the limiting step of metabolic incorporation. , Fluorescent nucleoside triphosphates could offer a solution to this problem: several studies have shown that especially C5 modifications for pyrimidines and C7 modifications for deazapurines are well tolerated by various polymerases. − In particular, installation of long flexible linkers in these positions allows for the modification to extend outside of the active site. , Fluorescent nucleoside triphosphates have successfully been investigated in eukaryotic cells to gain detailed insights into spatial organization and dynamics of nuclear processes − or as environment-sensitive viscosity probes . Visualization of DNA by fluorescent triphosphates was also successfully realized in Escherichia coli as well as Caenorhabditis elegans. , Due to the anionic character of triphosphates, which prevents passive diffusion over cellular membranes, these studies however rely on artificial, in part rather invasive delivery methods, such as microinjection, scratching, or bead loading . Various strategies for the delivery of biologically active nucleoside phosphates have been developed, which include masking as lipophilic prodrugs − or recombinant expression of a transporter .…”

“…19,20 Fluorescent nucleoside triphosphates have successfully been investigated in eukaryotic cells to gain detailed insights into spatial organization and dynamics of nuclear processes 21−24 or as environment-sensitive viscosity probes. 25 Visualization of DNA by fluorescent triphosphates was also successfully realized in Escherichia coli 26 as well as Caenorhabditis elegans. 27,28 Due to the anionic character of triphosphates, which prevents passive diffusion over cellular membranes, these studies however rely on artificial, in part rather invasive delivery methods, such as microinjection, scratching, or bead loading.…”

Light‐triggered Janus nanomotor for targeting and photothermal lysis of pathogenic bacteria

“…The advantage of this technique and in particular the mode of in‐focus phase‐contrast imaging is that it provides higher image contrast than the conventional one (Danev, Okawara, Usuda, Kametani, & Nagayama, 2002). Thus the captured micrographs of ice embedded whole cells exhibit the studied biological specimen close to its living state, while retaining all molecular constituents within the cell undisturbed (Loukanov, Emin, Singh, & Angelov, 2010; Loukanov, Mladenova, Toshev, & Nakabayashi, 2018; Loukanov, Nikolova, Filipov, & Nakabayashi, 2020). The images obtained by HDC‐TEM can display a valuable topographic features of the damaged bacteria thankful to the half plane π‐phase plate, which was inserted in the back focal plane of the objective lens.…”