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Visualization of Carbohydrate Uptake Using Fluorescent Polysaccharides.

Greta Reintjes1, Leeann Klassen2, D Wade Abbott2

  • 1Max Planck Institute for Marine Microbiology, Department of Molecular Ecology, Bremen, Germany. greta.reintjes@mpi-bremen.de.

Methods in Molecular Biology (Clifton, N.J.)
|May 6, 2023
PubMed
Summary

Researchers developed fluorescently labeled polysaccharides to study carbohydrate-bacterial interactions and hydrolysis. This method allows visualization and quantification in various environments, enabling in situ metabolic phenotyping of bacterial cells.

Keywords:
Carbohydrate utilizationEpifluorescence microscopyFlow cytometryFluorescent polysaccharidesGlycanHeterotrophy

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Area of Science:

  • Microbiology
  • Biochemistry
  • Analytical Chemistry

Background:

  • Carbohydrate-bacterial interactions are crucial in many biological processes.
  • Visualizing and quantifying these interactions is challenging in complex microbial communities.
  • Understanding carbohydrate metabolism is key to microbial ecology and biotechnology.

Purpose of the Study:

  • To develop a method for fluorescently labeling polysaccharides for studying carbohydrate-bacterial interactions.
  • To establish a protocol for visualizing and quantifying these interactions in diverse microbial settings.
  • To introduce a novel approach for in situ metabolic phenotyping of bacterial cells.

Main Methods:

  • Generation of polysaccharides conjugated to fluoresceinamine.
  • Incubation of fluorescent probes in bacterial cultures and environmental microbial communities.
  • Visualization via fluorescence microscopy and quantification using flow cytometry.
  • In situ metabolic phenotyping using fluorescently activated cell sorting (FACS) coupled with omics analysis.

Main Results:

  • Successfully generated fluorescently labeled polysaccharide probes.
  • Demonstrated visualization of bacterial-polysaccharide interactions.
  • Quantified carbohydrate hydrolysis rates in cultures and complex communities.
  • Established a novel FACS-based method for bacterial metabolic phenotyping.

Conclusions:

  • Fluorescently labeled polysaccharides are effective tools for studying carbohydrate-bacterial interactions.
  • The developed methods allow for robust quantification of carbohydrate hydrolysis.
  • The novel FACS-omics approach enables high-throughput in situ metabolic phenotyping of bacteria.