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Single cell fluorescence imaging of glycan uptake by intestinal bacteria.

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 ?-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.

SUBMITTER: Hehemann JH

PROVIDER: S-EPMC6776043 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Single cell fluorescence imaging of glycan uptake by intestinal bacteria.

Hehemann Jan-Hendrik JH Reintjes Greta G Klassen Leeann L Smith Adam D AD Ndeh Didier D Arnosti Carol C Amann Rudolf R Abbott D Wade DW

The ISME journal 20190401 7

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 rhamnog ...[more]

PMID: 30936421

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Project description:Magnetotactic bacteria (MTB) form intracellular chain-assembled nanocrystals of magnetite or greigite termed magnetosomes. The characterization of magnetosome crystals requires electron microscopy due to their nanoscopic sizes. However, electron microscopy does not provide phylogenetic information for MTB. We have developed a strategy for the simultaneous and rapid phylogenetic and biomineralogical characterization of uncultured MTB at the single-cell level. It consists of four steps: (i) enrichment of MTB cells from an environmental sample, (ii) 16S rRNA gene sequencing of MTB, and (iii) fluorescence in situ hybridization analyses coordinated with (iv) transmission or scanning electron microscopy of the probe-hybridized cells. The application of this strategy identified a magnetotactic Gammaproteobacteria strain, SHHR-1, from brackish sediments collected from the Shihe River estuary in Qinhuangdao City, China. SHHR-1 magnetosomes are elongated prismatic magnetites which can be idealized as hexagonal prisms. Taxonomic groups of uncultured MTB were also identified in freshwater sediments from Lake Miyun in northern Beijing via this novel coordinated fluorescence and scanning electron microscopy method based on four group-specific rRNA-targeted probes. Our analyses revealed that major magnetotactic taxonomic groups can be accurately determined only with coordinated scanning electron microscopy observations on fluorescently labeled single cells due to limited group coverage and specificity for existing group-specific MTB fluorescence in situ hybridization (FISH) probes. Our reported strategy is simple and efficient, offers great promise toward investigating the diversity and biomineralization of MTB, and may also be applied to other functional groups of microorganisms.IMPORTANCE Magnetotactic bacteria (MTB) are phylogenetically diverse and biomineralize morphologically diverse magnetic nanocrystals of magnetite or greigite in intracellular structures termed magnetosomes. However, many uncultured MTB strains have not been phylogenetically identified or structurally investigated at the single-cell level, which limits our comprehensive understanding of the diversity of MTB and their role in biomineralization. We developed a fluorescence-coupled electron microscopy method for the rapid phylogenetic and biomineralogical characterization of uncultured MTB at the single-cell level. Using this novel method, we successfully identified taxonomic groups of several uncultured MTB and one novel magnetotactic Gammaproteobacteria strain, SHHR-1, from natural environments. Our analyses further indicate that strain SHHR-1 forms elongated prismatic magnetites. Our findings provide a promising strategy for the rapid characterization of phylogenetic and biomineralogical properties of uncultured MTB at the single-cell level. Furthermore, due to its simplicity and generalized methodology, this strategy can also be useful in the study of the diversity and biomineralization properties of microbial taxa involved in other mineralization processes.

Dataset Information

Single cell fluorescence imaging of glycan uptake by intestinal bacteria.

Publications

Single cell fluorescence imaging of glycan uptake by intestinal bacteria.

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