An ATP Binding Cassette Transporter Mediates the Uptake of α-(1,6)-Linked Dietary Oligosaccharides in Bifidobacterium and Correlates with Competitive Growth on These Substrates

Abstract

The molecular details and impact of oligosaccharide uptake by distinct human gut microbiota (HGM) are currently not well understood. Non-digestible dietary galacto- and gluco-α-(1,6)-oligosaccharides from legumes and starch, respectively, are preferentially fermented by mainly bifidobacteria and lactobacilli in the human gut. Here we show that the solute binding protein (BlG16BP) associated with an ATP binding cassette (ABC) transporter from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 binds α-(1,6)-linked glucosides and galactosides of varying size, linkage, and monosaccharide composition with preference for the trisaccharides raffinose and panose. This preference is also reflected in the α-(1,6)-galactoside uptake profile of the bacterium. Structures of BlG16BP in complex with raffinose and panose revealed the basis for the remarkable ligand binding plasticity of BlG16BP, which recognizes the non-reducing α-(1,6)-diglycoside in its ligands. BlG16BP homologues occur predominantly in bifidobacteria and a few Firmicutes but lack in other HGMs. Among seven bifidobacterial taxa, only those possessing this transporter displayed growth on α-(1,6)-glycosides. Competition assays revealed that the dominant HGM commensal Bacteroides ovatus was out-competed by B. animalis subsp. lactis Bl-04 in mixed cultures growing on raffinose, the preferred ligand for the BlG16BP. By comparison, B. ovatus mono-cultures grew very efficiently on this trisaccharide. These findings suggest that the ABC-mediated uptake of raffinose provides an important competitive advantage, particularly against dominant Bacteroides that lack glycan-specific ABC-transporters. This novel insight highlights the role of glycan transport in defining the metabolic specialization of gut bacteria.

Keywords: ABC transporter; Bifidobacterium; carbohydrate-binding protein; crystal structure; gut microbiota; isomalto-oligosaccharide; oligosaccharide uptake; probiotic; raffinose; surface plasmon resonance (SPR).

FIGURE 1.

Chemical structures of the oligosaccharide ligands of BlG16PB. RFOs, α-(1,6)-galactosides: n = 0, raffinose; n = 1, stachyose; n = 2, verbascose; IMOs, α-(1,6)-glucosides): m = 0, isomaltose; m = 1, isomaltotriose through to m = 5, isomaltoheptaose.

FIGURE 2.

Ligand binding to BlG16BP as analyzed by surface plasmon resonance. A, reference and baseline-corrected sensograms depicting binding of raffinose (0.24 μm-1 mm) to BlG16BP at 25 °C. B, relative response as function of raffinose concentration (triangles), isomaltotriose (squares), and isomaltose (diamonds) and 1:1 binding model fits to the data (solid lines).

FIGURE 3.

Binding energetics of panose and raffinose to BlG16BP analyzed by ITC. A, representative heat traces of 2 mm panose and raffinose titrated into 108 μm BlG16BP (see “Experimental Procedures”). B, integrated binding isotherms from panel A and single site binding model fits shown as solid lines. The experiments were performed at 25 °C in 20 mm citrate-phosphate buffer, pH 7.0.

FIGURE 4.

Shown is a ribbon representation of the overall structure of BlG16BP in complex with panose (A) and raffinose (B) and the binding protein from S. pneumoniae TIGR4 (RafE, PDB code 2i58) in complex with raffinose (C). The SBPs consist of an N-terminal domain (Domain 1, brown) and a larger C-terminal domain (Domain 2, green). The two domains are linked by hinge regions shown in light blue. Shown is a close-up of the binding sites of BlG16BP in complex with panose (D) and raffinose (E) and RafE in complex with raffinose (F), which is shown in a slightly tilted view compared with D and E for clarity. The dashed black line in panels A and B illustrates a flexible loop between residues Ser-86 and Leu-97, which could not be modeled due to missing electron density. A σA-weighted difference electron density map (coefficients mF_obs − DF_calc) was calculated without phase information from the ligand and contoured at 3σ as a light blue mesh. Yellow dashes depict polar interactions to protein atoms or water (red spheres). Polar interactions were identified with PyMOL using a threshold of 3.1 Å. Main chain atoms are omitted for clarity unless they participate in polar interactions.

FIGURE 5.

Representative organization of α-(1,6)-galactoside/glucoside utilization loci in bifidobacteria and other HGM taxa in addition to Streptococcus strains that colonize the oral cavity and lungs in humans. The loci are aligned with respect to the ABC transport system (light gray) and consisting of SBP and two adjacent transmembrane domain permease genes (Perm). Glycoside hydrolases are white, and raffinose-specific α-galactosidases of GH36 subfamily 1 (GH36_I) are in bold. Transcriptional regulators are dark gray, and hypothetical genes are black. The consensus organization found in most bifidobacteria is shown on top followed by the organization found in B. animalis subsp. lactis. Distinct B. longum and B. breve strains have a longer insertion in the locus.

FIGURE 6.

Phylogenetic tree of BlG16BP homologues. A, radial cladogram of the homologues of BlG16BP, which were identified based on the co-localization of their genes with those encoding GH36 enzymes. The cladogram is divided into three subtrees: subtree 1 (blue background), subgroup 2 (green background), subtrees 3 (red background). Subtree 1 contains α-(1,6)-galactoside/glucoside-specific sequences including BlG16BP and functionally related homologues from Streptococcus in addition to Lactobacillus sequences specific for raffinose family α-(1,6)-galactosides. In agreement with this functional assignment, genes encoding sequences in subtree 1 are co-localized with genes of α-galactosidases (GH36_I) targeting raffinose family α-(1,6)-galactosides. Most sequences belonging to subtree 2 are distinguished by co-localization of their genes with putative α-N-acetylgalactosaminidase genes, which suggests a functional role in metabolism of host O-glycans. The specificities of subtree 3 SBPs are unknown. The branches from subtree 1 are populated by sequences from genera adapted to the human lung, oral cavity, and gut ecological niches. B, phylogram representation of the tree to show pylogentic distances.

FIGURE 7.

Uptake of raffinose family α-(1,6)-galactosides by B. animalis subsp. lactis Bl-04. A, growth curve of B. animalis subsp. lactis Bl-04 supplemented with a 0.5% (w/v) mixture of melibiose, raffinose, and stachyose in equal amounts. The numbers indicate sampling time points for oligosaccharide analysis of culture supernatants. B, high performance anion exchange chromatography with per amperometric detection analysis of the fermentation supernatants with the same numbering as in panel A. The peaks corresponding to melibiose (M), raffinose (R), and stachyose (S) are indicated.

FIGURE 8.

Competition co-cultures between B. ovatus and B. animalis subsp. lactis Bl-04. B. animalis subsp. lactis Bl-04 and B. ovatus were co-cultured on 0.5% (w/v) raffinose, and the proportion of B. ovatus as compared with the total colony forming units in the co-cultures was determined by plating on selective media and viable counts.

FIGURE 9.

Uptake and metabolism model of α-(1,6)-galactoside/glucoside by B. animalis subsp. lactis and the other bifidobacteria possessing a similar system (see Fig. 6). BlG16BP (Balac_1599), which is anchored to the extracellular side of the cell membrane by a lipid anchor, captures α-(1,6)-galacto-oligosaccharides (RFO) and α-(1,6)-gluco-oligosaccharides (IMO), which are translocated to the cytoplasm by the ABC transport system (Balac_1598, Balac_1597, and Balac_1610). The α-galactosidase (Balac_1601) and the α-(1,6)-glucosidase are responsible for the degradation of α-(1,6)-galactosides and α-(1,6)-glucosides, respectively, based on transcriptional data (22) and homology to characterized counterparts. α-(1,4)-Glucosidases and sucrose phosphorylases are possible candidates for the degradation of the maltose moiety of panose and the sucrose moiety of raffinose, respectively, into glucose and galactose, which is converted into glucose 1-phosphate via the Lelior pathway before entering the bifid shunt. Leloir refers to the Leloir pathway a metabolic pathway for catabolism of galactose.