Direct visualization of yeast mannan metabolism in bovine-adapted Bacteroides thetaiotaomicron strains at the single cell level using fluoresceinamine - yeast mannan conjugates

The rumen of beef cattle houses a diverse community of microorganisms that impact feed digestion, nutrient accessibility, host health, and waste production. Fibrolytic enzymes, probiotics, and prebiotics are promising candidates for next-generation feed additives, with the goal of inducing beneficial changes to the rumen microbiome. Current methods to investigate the mechanistic interactions between bacteria and feed glycans are “indirect” and lack the required sensitivity to validate probiotic-prebiotic application. To address these limitations, I have extended fluorescent glycan conjugates (FGCs) to visualize polysaccharide uptake in bacterial isolates and complex ecosystems at the single-cell level. Yeast α-mannan (YM) was conjugated to 6-aminofluorescein and fed to pure cultures of Bacteroides thetaiotaomicron VPI-5482, a well-studied intestinal symbiont that metabolizes YM, and closely related bovine-adapted bacterial isolates. Uptake of FGCs, coupled to complementary genomic and transcriptomic analysis, provided direct evidence of individual genotypes endowed with YM metabolic potential in pure and complex culture.This work was supported by funding from the Beef and Cattle Research Council (Grant: FDE.13.15 & FDE.14.17)

Single cell fluorescence imaging of glycan uptake by intestinal bacteria

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.</p

Metabolism of a hybrid algal galactan by members of the human gut microbiome

Native porphyran is a hybrid of porphryan and agarose. As a common element of edible seaweed, this algal galactan is a frequent component of the human diet. Bacterial members of the human gut microbiota have acquired polysaccharide utilization loci (PULs) that enable the metabolism of porphyran or agarose. However, the molecular mechanisms that underlie the deconstruction and use of native porphyran remains incompletely defined. Here, we have studied two human gut bacteria, porphyranolytic Bacteroides plebeius and agarolytic Bacteroides uniformis, that target native porphyran. This reveals an exo-based cycle of porphyran depolymerization that incorporates a keystone sulfatase. In both PULs this cycle also works together with a PUL-encoded agarose depolymerizing machinery to synergistically reduce native porphyran to monosaccharides. This provides a framework for understanding the deconstruction of a hybrid algal galactan, and insight into the competitive and/or syntrophic relationship of gut microbiota members that target rare nutrients.</p