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

Abstract

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.

Polysaccharide-Bacteria Interactions From the Lens of Evolutionary Ecology

“…In general, the expression of large enzymatic repertoires can impede growth, as less resources are allocated to biomass production (Basan et al, 2015). Therefore, we expect phenotypic heterogeneity to play a significant role in populations carrying large enzyme repertoires, such as the Verrucomicrobia that target fucoidan (Sichert et al, 2020) or Bacteroidetes that need to coordinate large number of PULs (Klassen et al, 2021a). Overall, the potential benefits of these forms of phenotypic heterogeneity represent an exciting frontier in the study of polysaccharide-bacteria interactions.…”

Approaches to Investigate Selective Dietary Polysaccharide Utilization by Human Gut Microbiota at a Functional Level

“…This study demonstrated that FLA-PS can be used to correlate metabolic phenotypes with genotypes and to differentiate between foraging strategies (Grondin et al, 2017). More recently, FLA-PS were successfully used in combination with FISH to detect and identify metabolically active cells in rumen community, respectively, and to quantify differences in glycan uptake rates between Bacteroides strains (Klassen et al, 2021).…”

“…Use of 2-NBDG and similar monosaccharide probes is limited, however, because transport proteins are typically unable to accommodate the bulky fluorophore and transport it into the cell. In contrast, fluorescently labeled polysaccharides (FLA-PS) (Glabe et al, 1983) provide more flexibility to study substrate uptake, and have been very successful when fluoresceinamine is used as the conjugate fluorophore (Figure 2D; Reintjes et al, 2017;Hehemann et al, 2019;Klassen et al, 2021). In contrast to methods that only label the reducing end, using a stochastic method to label hydroxyl groups of monosaccharide residues generates a library of related probes that are sampled by enzymes and transporters (Figure 2E); probes that are amenable to importation can be visualized by fluorescence microscopy (Figure 2F).…”

Effect of Cistanche deserticola on Rumen Microbiota and Rumen Function in Grazing Sheep

“…The rumen is a complex microbial anaerobic fermentation chamber that harbors one of the most diverse intestinal microbial communities of the animal kingdom ( Li et al, 2021 ). In terms of quantity and variety, bacteria are the dominant species in the rumen, and the catabolic potential of ruminal bacteria plays a vital role in the health and nutrition of their host ( Klassen et al, 2021 ). Higher microbial diversity in the mammalian gastrointestinal system often equates with stronger metabolic capacity and stability ( Li et al, 2019 ), such as Plateau pika ( Li et al, 2019 ).…”