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Applied and Environmental Microbiology
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Environmental Microbiology

Use of a Fluorescent Analog of Glucose (2-NBDG) To Identify Uncultured Rumen Bacteria That Take Up Glucose

Junyi Tao, Courtney McCourt, Halima Sultana, Corwin Nelson, John Driver, Timothy J. Hackmann
Volker Müller, Editor
Junyi Tao
aDepartment of Animal Sciences, University of Florida, Gainesville, Florida, USA
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Courtney McCourt
aDepartment of Animal Sciences, University of Florida, Gainesville, Florida, USA
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Halima Sultana
aDepartment of Animal Sciences, University of Florida, Gainesville, Florida, USA
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Corwin Nelson
aDepartment of Animal Sciences, University of Florida, Gainesville, Florida, USA
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John Driver
aDepartment of Animal Sciences, University of Florida, Gainesville, Florida, USA
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Timothy J. Hackmann
aDepartment of Animal Sciences, University of Florida, Gainesville, Florida, USA
bDepartment of Animal Science, University of California, Davis, California, USA
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Volker Müller
Goethe University Frankfurt am Main
Roles: Editor
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DOI: 10.1128/AEM.03018-18
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ABSTRACT

Few characteristics are more important to a bacterium than the substrates it consumes. It is hard to identify what substrates are consumed by bacteria in natural communities, however, because most bacteria have not been cultured. In this study, we developed a method that uses fluorescent substrate analogs, cell sorting, and DNA sequencing to identify substrates taken up by bacteria. We deployed this method using 2[N-(7-nitrobenz-2-oxa-1,2-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG), a fluorescent glucose analog, and bacteria of the bovine rumen. This method revealed over 40 different bacteria (amplicon sequence variants [ASVs]) from the rumen that take up glucose. Nearly half of these ASVs represent previously uncultured bacteria. We attempted to grow these ASVs on agar media, and we confirmed that nearly two-thirds resisted culture. In coculture experiments, the fluorescent label of 2-NBDG was not transferred to nontarget bacteria by cross-feeding. Because it is not affected by cross-feeding, our method has an advantage over stable isotope probing. Though we focus on glucose, many substrates can be labeled with the fluorophore NBD. Our method represents a new paradigm for identifying substrates used by uncultured bacteria. It will help delineate the niche of bacteria in their environment.

IMPORTANCE We introduce a method for identifying what substrates are consumed by bacteria in natural communities. Our method offers significant improvement over existing methods for studying this characteristic. Our method uses a fluorescently labeled substrate which clearly labels target bacteria (glucose consumers in our case). Previous methods use isotope-labeled substrates, which are notorious for off-target labeling (due to cross-feeding of labeled metabolites). Our method can be deployed with a variety of substrates and microbial communities. It represents a major advance in connecting bacteria to the substrates they take up.

FOOTNOTES

    • Received 17 December 2018.
    • Accepted 30 January 2019.
    • Accepted manuscript posted online 1 February 2019.
  • Supplemental material for this article may be found at https://doi.org/10.1128/AEM.03018-18.

  • Copyright © 2019 American Society for Microbiology.

All Rights Reserved.

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Use of a Fluorescent Analog of Glucose (2-NBDG) To Identify Uncultured Rumen Bacteria That Take Up Glucose
Junyi Tao, Courtney McCourt, Halima Sultana, Corwin Nelson, John Driver, Timothy J. Hackmann
Applied and Environmental Microbiology Mar 2019, 85 (7) e03018-18; DOI: 10.1128/AEM.03018-18

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Use of a Fluorescent Analog of Glucose (2-NBDG) To Identify Uncultured Rumen Bacteria That Take Up Glucose
Junyi Tao, Courtney McCourt, Halima Sultana, Corwin Nelson, John Driver, Timothy J. Hackmann
Applied and Environmental Microbiology Mar 2019, 85 (7) e03018-18; DOI: 10.1128/AEM.03018-18
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KEYWORDS

DNA sequencing
bacteria
flow cytometry
rumen
substrates

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