A CONSERVED SOLUTE BINDING PROTEIN RECOGNIZES Α-(1,6)-OLIGOSACCHARIDES AND PROVIDES EVIDENCE OF ATP- TRANSPORTER MEDIATED UPTAKE OF THESE DIETARY GLYCANS IN BIFIDOBACTERIUM
Ejby M., Fredslund F., Andersen J., Svensson B., Slotboom D., Abou Hachem M.
Technical University of Denmark, Denmark
Introduction-Glycan utilization plays a key role in establishing and modulating the composition of the highly diverse and dynamic microbial community referred to as the human gut microbiota. Oligosaccharide transporters are likely to play an important role in the competition for energy sources, as the intracellular (or periplasmic) accumulation of oligosaccharides prevents loss to competing organisms. Despite the competitive advantages of specialized oligosaccharide-uptake by gut bacteria, molecular insight for most oligosaccharide transporters in the gut niche is largely unexplored. Here we characterize the solute binding protein (BlG16BP) of an α-(1,6)-oligosaccharide ABC-transporter from Bifidobacterium animalis subsp. lactis Bl-04 and discuss the utilization preference of α-(1,6)-oligosaccharides derived from diet and the role of this ABC-transporter in competition within other gut microbiota taxa.
Methods-The gene encoding the solute binding protein BlG16BP (Balac_1599) was cloned and the resulting recombinant protein was produced in Escherichia coli and purified to electrophoretic homogeneity. The Affinities of immobilized BlG16BP towards oligosaccharide ligands were measured using surface plasmon resonance (SPR). Binding energetics of raffinose and panose were also measured using isothermal titration calorimetry. The structures of BlG16BP in complex with either panose or raffinose were determined to a maximum resolution of 1.4 Å by X-ray crystallography using single-wavelength anomalous diffraction (SAD) experimental phasing of selenomethionine labelled BlG16BP in complex with raffinose.
Results- BlG16BP is able to recognize α-(1,6)-linked glucosides and galactosides of varying size, glycosidic linkage and monosaccharide composition, with a preference for trisaccharides. This preference corresponds well to the in vivo uptake preference of Bifidobacterium animalis subsp. lactis Bl-04. Crystal structures of BlG16BP in complex with the tri-saccharides raffinose and panose provide an explanation for this remarkable plasticity. BlG16BP recognizes the non-reducing α-(1,6)-diglycoside motif in its ligands, while an open binding-site architecture lacking further interactions allows promiscuous ligand accommodation beyond this motif.
Discussion- Diet is a major effector of the composition of the gut microbiota and the metabolism of glycans has been highlighted as extremely important in maintaining a healthy bacterial community. Raffinose family oligosaccharides (RFO), containing α-(1,6)-galactosides are abundant in soybeans and other legumes and seeds, but are non-digestible by humans. Similarly, isomalto-oligosaccharides (IMO) comprising α-(1,6)-gluco-oligosaccharides derived from breakdown of starch and the bacterial exopolysaccharide dextran are resistant to degradation by humans. Both these classes of α-(1,6)-oligosaccharides are selectively fermented by bifidobacteria and gut-adapted lactobacilli. Homologs of BlG16BP occurs predominantly in gut adapted bifidobacteria and distinct Firmicutes providing a possible explanation for the selective utilizations of α-(1,6)-linked glucosides and galactosides by these taxa. These findings highlight the role of oligosaccharide-transport in defining the metabolic specialisation of gut bacteria and offer a framework for design of prebiotics based on mapping of the optimal ligands for oligosaccharide transport systems.
Keywords: Glycan utilization, Bifidobacteria, Carbohydrate transport, Prebiotics, Probiotics, Molecular microbiology
Ejby M., et al. (2016). A conserved solute binding protein recognizes A-(1,6)-oligosaccharides and provides evidence of ATP-transporter mediated uptake of these dietary glycans in bifidobacterium. Conference Proceedings of IPC2016. Paper presented at the International Scientific Conference on Probiotics and Prebiotics, Budapest (p. 91.). IPC2016