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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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  • FIG 1
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    FIG 1

    Outline of our method of identifying uncultured rumen bacteria that take up glucose with 2-NBDG.

  • FIG 2
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    FIG 2

    Microscopic image of mixed rumen bacteria incubated in 2-NBDG. Images show cells before cell sorting (A, C, and E) and after two sequential rounds of sorting (C, D, and F). Cells labeled with 2-NBDG are green. Images are from one of two independent experiments.

  • FIG 3
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    FIG 3

    Flow cytometry histogram showing bacteria labeled with 2-NBDG and their separation by fluorescence activated cell sorting (FACS). Shown are cells before sorting (A), after one round of sorting (B), and after two rounds (C). Over 95% purity was achieved. Panels B and C present results of a postsort analysis. SYTO 62 was added to stain DNA. Histograms are from 1 of 15 independent experiments.

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    FIG 4

    Violin plot of the 10 most abundant bacteria that take up 2-NBDG. A single-sided Wilcoxon signed-rank test was performed to test if abundance was >0 after accounting for impurity from unsorted samples. Each observation represents 1 of 15 independent experiments. See Fig. S3 for the full list of bacteria.

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    FIG 5

    Microscope image that shows absence of cross-feeding of 2-NBDG between Streptococcus equinus JB1 (coccus) and Anaerovibrio lipolyticus 5S (crescent). Cocultures of S. equinus JB1 and A. lipolyticus 5S were given 2-NBDG (5 mM). S. equinus JB1 takes up 2-NBDG and is labeled green, whereas A. lipolyticus 5S remains unlabeled; the fluorescent group in 2-NBDG is not transferred from the former to the latter. Images are from one of two independent experiments.

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  • TABLE 1

    Diet fed to rumen fluid donor cows

    ParameterAlfalfa hayConcentrate supplementa,b
    Amt fed (kg of dry matter/day)8.35.8
    Composition (g/100 g of dry matter)
        Organic matter90.792.2
        Neutral detergent fiberc37.418.5
        Acid detergent fiber31.712.9
        Crude protein18.815.2
        Starch1.539.7
        Ether extract2.34.5
    • ↵a Shiloh finisher, Corbett’s feed.

    • ↵b Ingredients included corn, molasses, soybean meal or cottonseed hulls, vitamins, minerals, and fat (inclusion rates of ingredients are proprietary).

    • ↵c Analyzed with heat-stable amylase and expressed exclusive of residual ash.

Additional Files

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    • Supplemental file 1 -

      Flow cytometry histogram showing that 2-NBDG is not taken up by dead cells (Fig. S1); analysis of α- and β-diversity of samples (Fig. S2); heat map for relative abundance of bacteria that are present in FACS-sorted cells and take up 2-NBDG (Fig. S3); violin plot of ten most abundant bacteria identified by anaerobic culture using RGA or RA medium (Fig. S4); flow cytometry histogram showing stability of 2-NBDG label in bacterial cells under aerobic conditions (Fig. S5); analysis of α- and β-diversity for two different DNA extraction methods (Fig. S6).

      PDF, 1.2M

    • Supplemental file 2 -

      Percentages of rumen bacteria gated as positive for 2-NBDG during cell sorting (Data Set S1); relative abundances of amplicon sequence variants in all sample types (Data Set S2); sequences and assigned taxonomy of amplicon sequence variants (Data Set S3); matches between previously cultured strains and amplicon sequence variants that take up 2-NBDG (Data Set S4).

      XLSX, 1.6M

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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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