Structural and functional characterisation of the dextran utilisome from Bacteroides thetaiotaomicron.

Bacteroides thetaiotaomicron (B. theta) is a model Bacteroidota of the healthy human gut microbiota and a specialist in glycan utilisation. Like other Bacteroides, B. theta has many highly regulated polysaccharide utilisation loci (PUL) that encode outer membrane (OM) TonB-dependent transporters (SusC), closely associated "lid" lipoproteins (SusD), and additional surface-exposed lipoproteins (SLPs) that bind and partially degrade specific glycans derived from host cells, diet, or other microbiota members. The canonical starch PUL products are thought to form a dynamic complex in the presence of starch. However, other PULs form stable complexes in the absence of substrate (recently named "utilisomes"), with additional surface lipoproteins tightly associated with the core SusCD complex. In this study, we characterised the B. theta dextran utilisome, with a SusCDdex core and an associated glycoside hydrolase (GHdex) and surface glycan binding protein (SBGPdex). Via X-ray crystallography we solved high-resolution structures of SBGPdex in isolation and SusDdex and GHdex bound to dextran oligosaccharides. We used isothermal titration calorimetry (ITC) to quantify ligand binding of wild type and mutant SLPs. We further used single particle cryo-EM of the catalytically inactive dextran utilisome to visualise open and closed states of the complex. Three occupied dextran binding sites were observed across SusCdex, SusDdex and GHdex, with substrate observed in both open and closed states of SusDdex. 3D variability analysis showed a minority of particles in the process of SusDdex lid closure. Together our work defines commonalities and differences across utilisomes dedicated to the import of simple glycans.