Single cell fluorescence imaging of glycan uptake by intestinal bacteria

Hehemann, Jan‐Hendrik; Reintjes, Greta; Klassen, Leeann; Smith, Adam D.; Ndeh, Didier; Arnosti, Carol; Amann, Rudolf; Abbott, D. Wade

doi:10.1038/s41396-019-0406-z

Cited by 30 publications

(25 citation statements)

References 20 publications

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“…One such approach is fluorescently labeled polysaccharides (FLA-PS). FLA-PS were initially developed to demonstrate selfish uptake of marine polysaccharides in marine Bacteroidetes [ 30 ] and have also been applied to the gut bacterium Bt VPI-5482 to confirm that YM metabolism also occurs through a selfish mechanism [ 31 ]. Fluorescent glucose analogs have been recently used to study glucose uptake by rumen bacteria [ 32 ]; however, use of fluorescent polysaccharides in the rumen has been limited until now.…”

Section: Introductionmentioning

confidence: 99%

Quantifying fluorescent glycan uptake to elucidate strain-level variability in foraging behaviors of rumen bacteria

et al. 2021

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Gut microbiomes, such as the microbial community that colonizes the rumen, have vast catabolic potential and play a vital role in host health and nutrition. By expanding our understanding of metabolic pathways in these ecosystems, we will garner foundational information for manipulating microbiome structure and function to influence host physiology. Currently, our knowledge of metabolic pathways relies heavily on inferences derived from metagenomics or culturing bacteria in vitro. However, novel approaches targeting specific cell physiologies can illuminate the functional potential encoded within microbial (meta)genomes to provide accurate assessments of metabolic abilities. Using fluorescently labeled polysaccharides, we visualized carbohydrate metabolism performed by single bacterial cells in a complex rumen sample, enabling a rapid assessment of their metabolic phenotype. Specifically, we identified bovine-adapted strains of Bacteroides thetaiotaomicron that metabolized yeast mannan in the rumen microbiome ex vivo and discerned the mechanistic differences between two distinct carbohydrate foraging behaviors, referred to as “medium grower” and “high grower.” Using comparative whole-genome sequencing, RNA-seq, and carbohydrate-active enzyme fingerprinting, we could elucidate the strain-level variability in carbohydrate utilization systems of the two foraging behaviors to help predict individual strategies of nutrient acquisition. Here, we present a multi-faceted study using complimentary next-generation physiology and “omics” approaches to characterize microbial adaptation to a prebiotic in the rumen ecosystem.

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Section: Introductionmentioning

confidence: 99%

Quantifying fluorescent glycan uptake to elucidate strain-level variability in foraging behaviors of rumen bacteria

et al. 2021

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“…However, the substrate must penetrate the cell wall or be physically located near the cell (Allison et al, 2012). Some polysaccharides substrates could be directly taken up into the periplasm of ‘selfish’ organism without production of extracellular hydrolysis products (Reintjes et al, 2017, 2019; Hehemann et al, 2019). Dissolved enzymes, which belong to a kind of “living dead” realm (Baltar, 2018), may originate from active secretion by cell (Alderkamp et al, 2007), bacterial starvation (Albertson et al, 1990) and changes in cell permeability (Chróst, 1991).…”

Section: Introductionmentioning

confidence: 99%

Extracellular Enzyme Activity and Its Implications for Organic Matter Cycling in Northern Chinese Marginal Seas

Sun

et al. 2019

Front. Microbiol.

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Extracellular enzymes, initiating the degradation of organic macromolecules, are important functional components of marine ecosystems. Measuring in situ seawater extracellular enzyme activity (EEA) can provide fundamental information for understanding the biogeochemical cycling of organic matter in the ocean. Here we investigate the patterns of EEA and the major factors affecting the seawater EEA of Chinese marginal seas. The geographic distribution of EEA along a latitudinal transect was examined and found to be associated with dissolved organic carbon. Compared with offshore waters, inshore waters had higher enzyme activity. All the tested substrates were hydrolyzed at different rates and phosphatase, β-glucosidase and protease contributed greatly to summed hydrolysis rates. For any particular enzyme activity, the contribution of dissolved to total EEA was strongly heterogenous between stations. Comparisons of hydrolysis rates of the polymers and their corresponding oligomers suggest that molecule size does not necessarily limit the turnover of marine organic matter. In addition, several typical enzyme-producing clades, such as Bacteroidetes, Planctomycetes, Chloroflexi, Roseobacter, Alteromonas, and Pseudoalteromonas, were detected in the in situ environments. These enzyme-producing clades may be responsible for the production of different enzymes. Overall, each enzyme was found to flexibly respond to environmental conditions and were linked to microbial community composition. It is likely that this activity will profoundly affect organic matter cycling in the Chinese marginal seas.

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“…Generating oligosaccharides that are either blocked in the non-reducing end or the reducing end could, in a straightforward way, aid in providing unequivocal evidence of which end is targeted by a given glycosidase. Furthermore, FITC labeling has been shown useful for studying the uptake of specific carbohydrates by microbes 37 . The labeling strategy presented here provides new tools for such studies and could advance these by enabling studies of the directionality of glycan uptake.…”

Section: Discussionmentioning

confidence: 99%

Synthesis of glycoconjugates utilizing the regioselectivity of a lytic polysaccharide monooxygenase

Westereng

Kračun

Leivers

et al. 2020

Sci Rep

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Polysaccharides from plant biomass are the most abundant renewable chemicals on Earth and can potentially be converted to a wide variety of useful glycoconjugates. Potential applications of glycoconjugates include therapeutics and drug delivery, vaccine development and as fine chemicals. While anomeric hydroxyl groups of carbohydrates are amenable to a variety of useful chemical modifications, selective cross-coupling to non-reducing ends has remained challenging. Several lytic polysaccharide monooxygenases (LPMOs), powerful enzymes known for their application in cellulose degradation, specifically oxidize non-reducing ends, introducing carbonyl groups that can be utilized for chemical coupling. This study provides a simple and highly specific approach to produce oxime-based glycoconjugates from LPMO-functionalized oligosaccharides. The products are evaluated by HPLC, mass spectrometry and NMR. Furthermore, we demonstrate potential biodegradability of these glycoconjugates using selective enzymes.

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Single cell fluorescence imaging of glycan uptake by intestinal bacteria

Cited by 30 publications

References 20 publications

Quantifying fluorescent glycan uptake to elucidate strain-level variability in foraging behaviors of rumen bacteria

Quantifying fluorescent glycan uptake to elucidate strain-level variability in foraging behaviors of rumen bacteria

Extracellular Enzyme Activity and Its Implications for Organic Matter Cycling in Northern Chinese Marginal Seas

Synthesis of glycoconjugates utilizing the regioselectivity of a lytic polysaccharide monooxygenase

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