Biochemical characterization of a SusD-like protein involved in β-1,3-glucan utilization by an uncultured cow rumen .

In ruminants, the rumen is a specialized stomach that is adapted to the breakdown of plant-derived complex polysaccharides through the coordinated activities of a diverse microbial community. Bacteroidota is a major phylum in this bovine rumen microbiota. They contain several clusters of genes called polysaccharide utilization loci (PULs) that encode proteins working in concert to capture, degrade, and transport polysaccharides. Despite the critical role of SusD-like proteins for efficient substrate transport, they remain largely unexplored. Here, we present the biochemical characterization of a SusD-like protein encoded by a β-glucan utilization locus from an metagenomic clone previously isolated by functional screening of the bovine rumen microbiome. In this study, we show that clone 41O1 can grow on laminaritriose, cellotriose, and a mixture of cellobiosyl-cellobiose and glucosyl-cellotriose as sole carbon sources. Based on this, we used various analyses to investigate the binding ability of 41O1_SusD-like towards these oligosaccharides and the corresponding polysaccharides. We observed a clear binding affinity for β-1,6 branched β-1,3-glucans (laminarins, yeast β-glucan) and laminaritriose. Comparison of the AlphaFold2 model of 41O1_SusD-like with its closest structural homologs highlights a similar pattern of substrate recognition. In particular, three tryptophan residues are shown to be crucial for laminarin recognition. In the context of the cow rumen, we discuss the possible substrates targeted by the 41O1_PUL, such as the (1,3;1,4)-β-d-glucans present in cereal grains or the β-1,3- and (1,3;1,6)-β-d-glucans that are components of the cell wall of ruminal yeasts.IMPORTANCEThe rumen microbiota can majorly impact overall animal health, feed efficiency, and release of harmful substances into the environment. This microbiota is involved in the fermentation of organic matter to provide the host with valuable and assimilable nutrients. Bacteroidota efficiently captures, breaks down, and imports complex polysaccharides through the concerted action of proteins encoded by polysaccharide utilization loci (PULs). Within this system, SusD-like protein has proven necessary for the active internalization of the substrate. Nevertheless, the vast majority of SusD-like proteins characterized to date originate from cultured bacteria. With regard to the diversity and importance of uncultured bacteria in the rumen, further studies are required to better understand the role of polysaccharide utilization loci in ruminal polysaccharide degradation. Our detailed characterization of the 41O1_SusD-like therefore contributes to a better understanding of the carbohydrate metabolism of an uncultured from the cow rumen.