The molecular basis of cereal mixed-linkage ß-glucan utilization by the human gut bacterium Segatella copri.

Mixed-linkage ß(1,3)/ß(1,4)-glucan (MLG) is abundant in the human diet through the ingestion of cereal grains and is widely associated with healthful effects on metabolism and cholesterol levels. MLG is also a major source of fermentable glucose for the human gut microbiota (HGM). Bacteria from the family Prevotellaceae are highly represented in the HGM of individuals who eat plant-rich diets, including certain indigenous people and vegetarians in postindustrial societies. Here, we have defined and functionally characterized an exemplar Prevotellaceae MLG polysaccharide utilization locus (MLG-PUL) in the type-strain Segatella copri (syn. Prevotella copri) DSM 18205 through transcriptomic, biochemical, and structural biological approaches. In particular, structure-function analysis of the cell-surface glycan-binding proteins and glycoside hydrolases of the S. copri MLG-PUL revealed the molecular basis for glycan capture and saccharification. Notably, syntenic MLG-PULs from human gut, human oral, and ruminant gut Prevotellaceae are distinguished from their counterparts in Bacteroidaceae by the presence of a ß(1,3)-specific endo-glucanase from glycoside hydrolase family 5, subfamily 4 (GH5_4) that initiates MLG backbone cleavage. The definition of a family of homologous MLG-PULs in individual species enabled a survey of nearly 2000 human fecal microbiomes using these genes as molecular markers, which revealed global population-specific distributions of Bacteroidaceae- and Prevotellaceae-mediated MLG utilization. Altogether, the data presented here provide new insight into the molecular basis of ß-glucan metabolism in the HGM, as a basis for informing the development of approaches to improve the nutrition and health of humans and other animals.

A Low-Volume, Parallel Copper-Bicinchoninic Acid (BCA) Assay for Glycoside Hydrolases.

The quantitation of liberated reducing sugars by the copper-bicinchoninic acid (BCA) assay provides a highly sensitive method for the measurement of glycoside hydrolase (GH) activity, particularly on soluble polysaccharide substrates. Here we describe a straightforward method adapted to low-volume polymerase chain reaction (PCR) tubes that enables the rapid, parallel determination of GH kinetics in applications ranging from initial activity screening and assay optimization to precise Michaelis-Menten analysis.

Configured for the Human Gut Microbiota: Molecular Mechanisms of Dietary ß-Glucan Utilization.

The ß-glucans are a disparate group of structurally diverse polysaccharides, whose members are widespread in human diets as components of the cell walls of plants, algae, and fungi (including yeasts), and as bacterial exopolysaccharides. Individual ß-glucans from these sources have long been associated with positive effects on human health through metabolic and immunological effects. Remarkably, the ß-configured glucosidic linkages that define these polysaccharides render them inaccessible to the limited repertoire of digestive enzymes encoded by the human genome. As a result, the various ß-glucans become fodder for the human gut microbiota (HGM) in the lower gastrointestinal tract, where they influence community composition and metabolic output, including fermentation to short chain fatty acids (SCFAs). Only recently, however, have the specific molecular systems that enable the utilization of ß-glucans by select members of the HGM been fully elucidated by combined genetic, biochemical, and structural biological approaches. In the context of ß-glucan structures and their effects on human nutrition and health, we summarize here the functional characterization of individual polysaccharide utilization loci (PULs) responsible for the saccharification of mixed-linkage ß(1→3)/ß(1→4)-glucans, ß(1→6)-glucans, ß(1→3)-glucans, ß(1→2)-glucans, and xyloglucans in symbiotic human gut bacteria. These exemplar PULs serve as well-defined biomarkers for the prediction of ß-glucan metabolic capability in individual bacterial taxa and across the global human population.