Investigating the mechanism of nutrient acquisition by SusCD transport systems using cryoEM

The Gram-negative Bacteroidetes are abundant in the human microbiota, occupying niches devoid of easily accessible nutrients. Uptake of complex nutrient molecules, especially glycans, by these bacteria is facilitated by outer membrane SusCD protein complexes, comprising a barrel-shaped integral membrane transporter component (SusC) and a substrate-binding lipoprotein (SusD). Recent X-ray crystal structures show that these complexes exist as SusC2D2 dimers with SusD subunits tightly capping the exterior face of the SusC barrels, trapping substrate in a solvent excluded cavity. However, the mechanistic details of substrate capture and translocation remain unclear, as does the role and organisation of additional surface-exposed lipoproteins that are prevalent in glycan utilisation systems. Here we present cryoEM structures of three SusCD systems from important members of the human microbiota. Using the SusCD-like peptide-transporting RagAB complex from the keystone pathogen in human periodontitis, Porphyromonas gingivalis, SusD subunits are shown to function as lids, capable of opening and closing to alternately expose and occlude the substrate binding cavity to the extracellular space. For the levan utilisation system of the prominent model gut symbiont Bacteroides thetaiotaomicron, we demonstrate that the additional lipoproteins, a levan binding protein and an endo-levanase, assemble on the core transporter. The resulting octameric complex constitutes a stable outer membrane machine, which we name a utilisome, that presents all of the activities required for efficient levan capture. Such utilisomes are not restricted to levan transport, as demonstrated by accompanying structures of the equivalent complex for the dextran transporter of the same bacterium. Finally, cryoEM structures of the levan utilisome in the absence and presence of substrate reveal concerted conformational changes that rationalise the position of each component for efficient nutrient capture, as well as providing a direct demonstration of the mechanism of levan capture at the OM for the first time. All Bacteroidetes sequenced to date contain SusCD homologues and we anticipate that utilisome assemblies represent a general mechanism for the translocation of large nutrients across the outer membrane of these bacteria.