Abstract
Microbes in the intestines of mammals degrade dietary glycans for energy and growth. The pathways required for polysaccharide utilization are functionally diverse; moreover, they are unequally dispersed between bacterial genomes. Hence, assigning metabolic phenotypes to genotypes remains a challenge in microbiome research. Here we demonstrate that glycan uptake in gut bacteria can be visualized with fluorescent glycan conjugates (FGCs) using epifluorescence microscopy. Yeast α-mannan and rhamnogalacturonan-II, two structurally distinct glycans from the cell walls of yeast and plants, respectively, were fluorescently labeled and fed to Bacteroides thetaiotaomicron VPI-5482. Wild-type cells rapidly consumed the FGCs and became fluorescent; whereas, strains that had deleted pathways for glycan degradation and transport were non-fluorescent. Uptake of FGCs, therefore, is direct evidence of genetic function and provides a direct method to assess specific glycan metabolism in intestinal bacteria at the single cell level.
Highlights
Glycan utilization by intestinal Bacteroidetes is critical for digestion of dietary carbohydrates. This host–symbiont interaction is facilitated by cognate PUL systems that endow bacterial strains for consumption of specific glycans
Despite the remarkable progress that has been made in cataloging microbiome composition from diverse sources [16] and defining the molecular basis of PUL function [2], the field still is lacking methods to rapidly assign metabolic phenotypes to genotypes in microbial communities at the single cell level
Left panel: super-resolution structured illumination microscopy (SR-SIM) single cell images of B. theta cells labeled with DAPI, Nile Red, and fluorescent glycan conjugates (FGCs)
Summary
Glycan utilization by intestinal Bacteroidetes is critical for digestion of dietary carbohydrates This host–symbiont interaction is facilitated by cognate PUL systems that endow bacterial strains for consumption of specific glycans. Cells that were selected for fluorescence intensity line profiling are indicated with a green arrow and white line for profile. Blue: DAPI; Green: FLA; and Red: Nile Red. c Quantification of FLA-YM uptake by B. theta and mutant B. theta (BtMAN1/2/3) strains over time using flow cytometry. (2) B. theta shows significant increase in cellular fluorescence after 72 h incubation with FLA-YM. (3) In a B. theta strain with all three YM PULs deleted (BtΔMAN1/2/3) showed a low increase in fluorescence after 72 h incubation with FLA-YM. (4) Bar graph showing the mean fluorescence intensity of B. theta (white) and BtΔMAN1/2/3 (dashed) incubated with YM-FLA and a control (B. theta incubated with unlabeled YM, black) over time. Statistical differences were calculated by Welch’s t-test (ns: no significant difference, ***P < 0.001)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
