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• W2494559098 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. 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. The gastrointestinal tract hosts a highly diverse microbial community referred to as the human gut microbiota (HGM) 3The abbreviations used are: HGM, human gut microbiota; ABC, ATP binding cassette; BlG16BP, B. lactis glycoside α-1,6 binding protein; IMO, isomalto-oligosaccharides; ITC, isothermal titration calorimetry; RFO, raffinose family oligosaccharides; SBP, solute binding protein; SPR, surface plasmon resonance; PDB, Protein Data Bank; r.m.s.(d.), root mean square (deviation). (1Eckburg P.B. Bik E.M. Bernstein C.N. Purdom E. Dethlefsen L. Sargent M. Gill S.R. Nelson K.E. Relman D.A. Diversity of the human intestinal microbial flora.Science. 2005; 308: 1635-1638Crossref PubMed Scopus (5537) Google Scholar). This community, which is established shortly after birth, develops rapidly to form one of the most densely populated ecological niches in nature by the age of 2–3 years. Despite millions of years of co-evolution with mammalian hosts (2Ley R.E. Hamady M. Lozupone C. Turnbaugh P.J. Ramey R.R. Bircher J.S. Schlegel M.L. Tucker T.A. Schrenzel M.D. Knight R. Gordon J.I. Evolution of mammals and their gut microbes.Science. 2008; 320: 1647-1651Crossref PubMed Scopus (2398) Google Scholar), only recently has the profound impact of the gut microbiota on various aspects of human health, including metabolic and immune-disorders, colon cancer, and brain function, as well as rate of aging been established (3Sommer F. Bäckhed F. The gut microbiota: masters of host development and physiology.Nat. Rev. Microbiol. 2013; 11: 227-238Crossref PubMed Scopus (2086) Google Scholar, 4Cryan J.F. Dinan T.G. Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour.Nat. Rev. Neurosci. 2012; 13: 701-712Crossref PubMed Scopus (2487) Google Scholar5Nicholson J.K. Holmes E. Kinross J. Burcelin R. Gibson G. Jia W. Pettersson S. Host-gut microbiota metabolic interactions.Science. 2012; 336: 1262-1267Crossref PubMed Scopus (2899) Google Scholar). Evidence is rapidly accumulating that advantage can be taken of the gut microbiota in diagnosis, treatment, and prevention of diseases and disorders. To realize this potential it is important to discern the complex metabolic interactions among different microbiota taxa, which promote a healthy composition and prevent imbalance (dysbiosis) associated with disease (6Brown K. DeCoffe D. Molcan E. Gibson D.L. Diet-induced dysbiosis of the intestinal microbiota and the effects on immunity and disease.Nutrients. 2012; 4: 1095-1119Crossref PubMed Scopus (434) Google Scholar). Diet is a key effector of the composition of the HGM (7David L.A. Maurice C.F. Carmody R.N. Gootenberg D.B. Button J.E. Wolfe B.E. Ling A.V. Devlin A.S. Varma Y. Fischbach M.A. Biddinger S.B. Dutton R.J. Turnbaugh P.J. Diet rapidly and reproducibly alters the human gut microbiome.Nature. 2014; 505: 559-563Crossref PubMed Scopus (5570) Google Scholar, 8Muegge B.D. Kuczynski J. Knights D. Clemente J.C. González A. Fontana L. Henrissat B. Knight R. Gordon J.I. Diet drives convergence in gut microbiome functions across mammalian phylogeny and within humans.Science. 2011; 332: 970-974Crossref PubMed Scopus (1215) Google Scholar), and the metabolism of glycans has been highlighted as pivotal in maintaining a healthy bacterial community (9Koropatkin N.M. Cameron E.A. Martens E.C. How glycan metabolism shapes the human gut microbiota.Nat. Rev. Microbiol. 2012; 10: 323-335Crossref PubMed Scopus (863) Google Scholar). Raffinose family oligosaccharides (RFOs), containing α-(1,6)-galactosides (10Mäkeläinen H. Hasselwander O. Rautonen N. Ouwehand A.C. Panose, a new prebiotic candidate.Lett. Appl. Microbiol. 2009; 49: 666-672Crossref PubMed Scopus (41) Google Scholar), are abundant in soybeans and other legumes and seeds (11Andersen K.E. Bjergegaard C. Møller P. Sørensen J.C. Sørensen H. Compositional variations for α-galactosides in different species of Leguminosae, Brassicaceae, and barley: a chemotaxonomic study based on chemometrics and high-performance capillary electrophoresis.J. Agric. Food Chem. 2005; 53: 5809-5817Crossref PubMed Scopus (30) Google Scholar, 12Jukanti A.K. Gaur P.M. Gowda C.L. Chibbar R.N. Nutritional quality and health benefits of chickpea (Cicer arietinum L.): a review.Br. J. Nutr. 2012; 108: S11-S26Crossref PubMed Scopus (570) Google Scholar), but are non-digestible by humans. Similarly, isomalto-oligosaccharides (IMOs), comprising α-(1,6)-gluco-oligosaccharides derived from breakdown of starch and the bacterial exopolysaccharide dextran (13Ao Z. Simsek S. Zhang G. Venkatachalam M. Reuhs B.L. Hamaker B.R. Starch with a slow digestion property produced by altering its chain length, branch density, and crystalline structure.J. Agric. Food Chem. 2007; 55: 4540-4547Crossref PubMed Scopus (210) Google Scholar, 14Swennen K. Courtin C.M. Delcour J.A. Non-digestible oligosaccharides with prebiotic properties.Crit. Rev. Food Sci. Nutr. 2006; 46: 459-471Crossref PubMed Scopus (260) Google Scholar), are resistant to degradation by human digestive enzymes. Both of these classes of α-(1,6)-oligosaccharides selectively boost the counts of bifidobacteria and gut-adapted lactobacilli in vitro and in vivo (10Mäkeläinen H. Hasselwander O. Rautonen N. Ouwehand A.C. Panose, a new prebiotic candidate.Lett. Appl. Microbiol. 2009; 49: 666-672Crossref PubMed Scopus (41) Google Scholar, 15Ishizuka S. Iwama A. Dinoto A. Suksomcheep A. Maeta K. Kasai T. Hara H. Yokota A. Synbiotic promotion of epithelial proliferation by orally ingested encapsulated Bifidobacterium breve and raffinose in the small intestine of rats.Mol. Nutr. Food Res. 2009; 53: S62-S67Crossref PubMed Scopus (15) Google Scholar), but the molecular basis of this preferential enumeration is unclear. Bifidobacteria are strictly saccharolytic, but in contrast to other commensals of the HGM most of their glycoside hydrolases are intracellular, and their genomes encode only a few extracellular hydrolases targeting polysaccharides (16Ventura M. O'Connell-Motherway M. Leahy S. Moreno-Munoz J.A. Fitzgerald G.F. van Sinderen D. From bacterial genome to functionality; case bifidobacteria.Int. J. Food Microbiol. 2007; 120: 2-12Crossref PubMed Scopus (68) Google Scholar, 17Flint H.J. Bayer E.A. Plant cell wall breakdown by anaerobic microorganisms from the mammalian digestive tract.Ann. N.Y. Acad. Sci. 2008; 1125: 280-288Crossref PubMed Scopus (148) Google Scholar). Instead, bifidobacteria rely on their specialized carbohydrate transport systems for uptake of available oligosaccharides (18Flint H.J. Duncan S.H. Scott K.P. Louis P. Interactions and competition within the microbial community of the human colon: links between diet and health.Environ. Microbiol. 2007; 9: 1101-1111Crossref PubMed Scopus (440) Google Scholar). ABC transporters are the most common class of glycan transporters in bifidobacteria (19Pokusaeva K. Fitzgerald G.F. van Sinderen D. Carbohydrate metabolism in Bifidobacteria.Genes Nutr. 2011; 6: 285-306Crossref PubMed Scopus (475) Google Scholar). ABC transporters use the free energy of ATP hydrolysis via two intracellular nucleotide binding domains to energize the uptake of a variety of molecules. Extracellular solute binding proteins (SBPs) are responsible for capture of ligands, which are subsequently translocated through a pore formed by two transmembrane domains (20ter Beek J. Guskov A. Slotboom D.J. Structural diversity of ABC transporters.J. Gen. Physiol. 2014; 143: 419-435Crossref PubMed Scopus (249) Google Scholar). Although some substrate recognition is conferred by the transmembrane domains, SBPs largely define the specificity and the affinity of bacterial ABC importers (21Berntsson R.P. Smits S.H. Schmitt L. Slotboom D. J Poolman B. A structural classification of substrate-binding proteins.FEBS Lett. 2010; 584: 2606-2617Crossref PubMed Scopus (386) Google Scholar). Oligosaccharide transporters are likely to play a pivotal role in the highly competitive and extremely densely populated gut niche, as the intracellular (or periplasmic) accumulation of oligosaccharides precludes loss to competing organisms. Despite their importance, molecular insight for most oligosaccharide transporters in the gut niche and especially those from bifidobacteria is lacking. Growth of the probiotic bacterium Bifidobacterium animalis subsp. lactis Bl-04 on the α-(1,6)-galactosides melibiose, raffinose, and stachyose and the α-(1,6)-glucosides isomaltose and panose (Fig. 1) differentially induced the expression of a locus encoding three glycoside hydrolases and an ABC transporter (22Andersen J.M. Barrangou R. Abou Hachem M. Lahtinen S.J. Goh Y.J. Svensson B. Klaenhammer T.R. Transcriptional analysis of oligosaccharide utilization by Bifidobacterium lactis Bl-04.BMC Genomics. 2013; 14: 312Crossref PubMed Scopus (61) Google Scholar). In the present study, we determined the affinities of BlG16BP and the SBP associated to this ABC transporter to a range of α-(1,6)-oligosaccharides and established its specificity for galactosides and glucosides sharing a common α-(1,6)-diglycoside motif. The structures of the SBP in complex with the two preferred ligands raffinose and panose provided insight into the structural basis for this specificity. Genetic and phylogenetic analyses show that close homologues of BlG16BP are conserved within most bifidobacteria and a few strains of Firmicutes, consistent with preferential fermentation of α-(1,6)-oligosaccharides by these taxonomic groups of the HGM. Competition assays between B. ovatus, from the dominant HGM genus Bacteroides, and B. animalis subsp. lactis provided evidence that ABC-mediated oligosaccharide uptake confers an advantage in competition for raffinose, the preferred carbohydrate ligand for the identified ABC transporter. This first biochemical and structural study of an α-(1,6)-oligosaccharide transport protein presented here provides a basic understanding of the versatility of ABC transporter associated SBPs in glycan capture and provides evidence for a competitive advantage associated with ABC-mediated glycan transport. Ligand preference of BlG16BP was explored using SPR. No binding to the monosaccharides fructose, galactose, and glucose was observed. The SBP was specific for α-(1,6)-glucosides (IMO and panose) and α-(1,6)-galactosides (RFO) (Fig. 1) as judged by the lack of binding to the β-glycosides cellobiose, xylo-oligosaccharides, β-galacto-oligosaccharides, β-fructo-oligosaccharides, or malto-oligosaccharides. The binding kinetics rate constants were too fast to be modeled, and the equilibrium dissociation constants (Table 1) were determined from steady state sensograms (Fig. 2). The highest affinity was measured toward the trisaccharides panose and raffinose (Table 1) with KD values of 9 μm and 21 μm, respectively. Reducing the ligand size to a disaccharide resulted in a substantial affinity reduction (∼35-fold for the melibiose compared with raffinose and 120-fold for isomaltose as compared with panose). By contrast, the change in affinity with increasing oligosaccharide size was clearly different between the RFO and the IMO. Thus, the presence of two additional α-(1,6) galactosyl moieties at the non-reducing end of raffinose in verbascose abolished the binding within the tested ligand concentration range, whereas the affinity for the α-(1,6)-glucosides with a degree of polymerization of 3–7 was similar and only ∼14-fold lower than for the preferred trisaccharide panose (Table 1). The binding affinity of BlG16BP for raffinose was essentially unchanged in the pH range 5.5–8.0 (data not shown).TABLE 1Binding parameters of BlG16BP measured by SPRCarbohydrateKDaDissociation constants (KD) are the means of triplicates with the S.E.RmaxbThe maximum binding level from the fits to a one-binding-site model.χ2cχ2, statistical goodness of the fit to a one-site-binding model.μmMelibiose729 ± 70240.22Raffinose21 ± 4350.12Stachyose327 ± 11370.27Verbascose>4000550.38Isomaltose1060 ± 73190.06Isomaltotriose126 ± 3330.70Panose9 ± 0350.11Isomaltotetraose94 ± 1420.05Isomaltopentaose103 ± 1510.07Isomaltohexaose104 ± 1560.06Isomaltoheptaose143 ± 1690.04a Dissociation constants (KD) are the means of triplicates with the S.E.b The maximum binding level from the fits to a one-binding-site model.c χ2, statistical goodness of the fit to a one-site-binding model. Open table in a new tab The thermodynamic parameters and binding stoichiometry for panose and raffinose were determined using isothermal titration calorimetry (ITC) and revealed a 1:1 binding driven by a favorable enthalpy change, which was offset by a large unfavorable binding entropy (Table 2 and Fig. 3). The affinity trend and the magnitude of the binding constants were in good agreement with the SPR data.TABLE 2Thermodynamic parameters of panose and raffinose binding to BlG16BP measured by isothermal titration calorimetryOligosaccharideKDΔGΔH−TΔSnμmkJ/molkJ/molkJ/molPanose17.5 ± 0.3−27.1−63.7 ± 0.536.50.70 ± 0.01Raffinose27 ± 2−26.1−46.3 ± 0.120.20.60 ± 0.02 Open table in a new tab Crystal structures of BlG16BP in complex with the two preferred ligands were solved to a maximum resolution of 1.4 Å (Table 3). BlG16BP adopts a canonical SBP-fold (cluster B type SBP according to structural classification; Ref. 21Berntsson R.P. Smits S.H. Schmitt L. Slotboom D. J Poolman B. A structural classification of substrate-binding proteins.FEBS Lett. 2010; 584: 2606-2617Crossref PubMed Scopus (386) Google Scholar), which comprises two domains of different size joined by a tripartite hinge region with the ligand binding site located at the domain interface (Fig. 4, A and B). Domain 1 (1–162; 327–373) is formed by six α-helices and five β-strands, two of which form a part of the hinge region and continue into the larger C-terminal domain (Domain 2). The loop spanning Ser-86 to Leu-93 in Domain 1 was not ordered, as judged by a poor electron density map, and no model is included for this region The unmodeled region corresponds to Pro-40–Phe-47 of the maltose-binding protein (MBP) from Escherichia coli, which is located at the interface between MBP and the transmembrane MalG domain in the pretranslocation state of the ABC-transporter (23Oldham M.L. Chen S. Chen J. Structural basis for substrate specificity in the Escherichia coli maltose transport system.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 18132-18137Crossref PubMed Scopus (61) Google Scholar). Domain 2 (167–322; 390–437) consists of 11 α-helices and 3 β-strands. The hinge region comprises two short β-strands arranged in an anti-parallel β-sheet spanning the two domains (163–166; 323–326) and the loop 372–375 (Fig. 4). A DALI structure comparison search (24Holm L. Rosenström P. Dali server: conservation mapping in 3D.Nucleic Acids Res. 2010; 38: W545-W549Crossref PubMed Scopus (3053) Google Scholar) against the Protein Data Bank (PDB) identified the raffinose-binding protein RafE 4Substrate-binding protein component of the raffinose uptake system from S. pneumonia. from the Streptococcus pneumoniae (PDB code 2i58; Z-score = 39.0; r.m.s.d. = 2.5 Å for 363 aligned Cα atoms and 23% sequence identity) as the structurally most related orthologue to BlG16BP. The second best hit is the arabinoxylo-oligosaccharide-specific SBP from the same organism (25Ejby M. Fredslund F. Vujicic-Zagar A. Svensson B. Slotboom D.J. Abou Hachem M. Structural basis for arabinoxylo-oligosaccharide capture by the probiotic Bifidobacterium animalis subsp. lactis Bl-04.Mol. Microbiol. 2013; 90: 1100-1112Crossref PubMed Scopus (51) Google Scholar) (PDB code 4c1u; Z-score = 37.0; r.m.s.d. = 2.4 Å for 354 aligned Cα atoms and 23% sequence identity).TABLE 3Data collection and refinement statistics of the complex structure of BlG16BP with raffinose and panose and a selenomethionine derivativeBlG16BP raffinoseBlG16BP panoseBlG16BP SeMetPDB IDWavelength (Å)0.97970.97730.9805Resolution range (Å)57-1.37 (1.41-1.37)23-1.76 (1.82-1.76)43.4-2.019 (2.091-2.019)Space groupP 21 21 21P 21 21 21P 21 21 21Unit cell (Å)55.54 90.82 146.7554.88 91.33 142.0555.39 90.95 145.53Unique reflectionsaValues in the parentheses are for the highest resolution shell.151,108 (10,787)70,949 (6,995)48,992 (4,766)MultiplicityaValues in the parentheses are for the highest resolution shell.6.2 (3.4)3.4 (3.2)4.1 (4.1)Completeness (%)aValues in the parentheses are for the highest resolution shell.96.9 (94.1)99 (99)100 (99)R-measaValues in the parentheses are for the highest resolution shell.0.08 (0.775)0.098 (0.764)0.136 (0.477)Mean I/σ(I)aValues in the parentheses are for the highest resolution shell.12.8 (2.1)10.65 (2.12)11.9 (3.53)Wilson B-factor (Å2)13.317.714.0R-factor0.1320.1580.166R-free0.1670.2050.210Number of atomsMacromolecules6,0306,0315,767Ligands878168Water1,114987853Protein residuesr.m.s. bonds (Å)0.0130.0050.012r.m.s. angles (°)1.350.971.26Ramachandran favored (%)989898Ramachandran outliers (%)000Clash score1.662.322.28Average B-factor (Å2)18.420.015Macromolecules (Å2)16.318.614.1Ligands (Å2)16.714.88.9Water (Å2)30.02922a Values in the parentheses are for the highest resolution shell. Open table in a new tab The crystal structures of BlG16BP in complex with raffinose and panose showed well defined densities for the bound oligosaccharides in the ligand binding site that features a deep but open pocket large enough to accommodate a trisaccharide. Three aromatic residues from Domain 2 provide stacking interactions to each glycosyl ring in a trisaccharide ligand, whereas polar contacts are provided by both domains of BlG16BP. The non-reducing end glycosyl unit of both ligands (galactosyl in raffinose and glucosyl in panose) stacks onto Phe-392 (defined as position 1) and makes polar contacts to Asp-394, Asn-109, and His-395 (Fig. 4, D and E). Interestingly Asp-394 is able to form hydrogen bonds to both the equatorial C4-OH of the nonreducing end glucosyl in panose and the axial C4-OH of the galactosyl in raffinose, thus allowing for an equivalent mode of ligand binding at position 1 (Fig. 4, D and E). The glucosyl moiety of raffinose and panose at position 2 stacks onto Tyr-291 and makes polar contacts to Lys-58, Glu-60, and Asp-326. Although the pyranose rings at positions 1 and 2 can be overlaid almost perfectly, the planes of the terminal glycosyl moieties residues at position 3 are almost orthogonal to each other. Thus, the glucosyl moiety of panose stacks onto Trp-216 with almost parallel planes of the sugar ring and the indole side chain (Fig. 4, D and E). By contrast, the fructosyl residue in raffinose has a smaller area of van der Waals contact with Trp-216, with the planes of the furanose and indole rings being almost orthogonal to each other (Fig. 4, D and E). Homologues of BlG16BP are encoded by genomes of several species within Bifidobacterium. While the solvent-exposed residues are variable, the binding pocket residues of BlG16BP that make direct ligand contacts are invariant in Bifidobacterium (data not shown). An exception is the conservative substitution of Asn-109 to aspartic acid in some Bifidobacterium longum subsp. longum strains. The composition and conservation of loci encompassing orthologues of the BlG16BP gene within Bifidobacterium was performed. Genes encoding permease components of an ABC importer, transcriptional regulators, GH36 α-galactosidases that mediate degradation of soybean α-1,6-galactosides (RFO), oligo-α-1,6-glucosidases (glycoside hydrolase family 13, subfamily 31, GH13_31) that catalyze the hydrolysis of α-(1,6)-glucosides (IMO), were invariably co-localized with BlG16BP orthologues in human gut adapted taxa (Fig. 5). This organization defines the α-(1,6)-galactoside/glucoside utilization loci in Bifidobacterium with the exception of the insect gut-residing Bifidobacterium asteroides (Fig. 5). The occurrence of potential ABC transporters of α-(1,6)-glycosides in other taxa, particularly from the gut niche, was analyzed using a different strategy to overcome the low sequence conservation of SBPs. The genetic co-localization of an ABC transporter and an α-galactosidase of family GH36 was used as a discriminator to map the taxonomic distribution of BlG16BP homologues in putative α-(1,6)-galactoside utilization loci. Using this approach, 263 gene loci encoding GH36 enzymes (mainly α-galactosidases) together with an ABC transporter and enzymes predicted to be involved in the degradation of α-(1,6)-glucosides (e.g. α-(1,6)-glucosidases of GH13_31) or the sucrose moieties produced from RFO (e.g. sucrose phosphorylases of GH13_18 or sucrose hydrolases of GH32) were found. Most of the loci originated from the Firmicutes and Actinobacteria, including bifidobacteria species and other taxa typically associated with human hosts (Fig. 5). These SBPs populated three subtrees (1, 2, and 3) in this phylogenetic analysis (Fig. 6). BlG16BP and homologous sequences sharing the same genetic organization were in subtree 1. By contrast, subtree 2 SBPs co-localized with putative α-N-acetylgalactosaminidase genes (of GH36, data not shown), suggesting a role in uptake of α-N-acetyl-hexoseamine-containing glycans, e.g. from mucin. Subtree 3 is mostly populated by SBPs from marine or soil bacteria. The majority of bifidobacterial SBPs populate a single branch in subtree 1 together with BlG16BP (Fig. 6). A few sequences from the HGM Eubacterium rectale, Ruminococcus SR1/5, and Ruminococcus torques spp. (26Mahowald M.A. Rey F.E. Seedorf H. Turnbaugh P.J. Fulton R.S. Wollam A. Shah N. Wang C. Magrini V. Wilson R.K. Cantarel B.L. Coutinho P.M. Henrissat B. Crock L.W. Russell A. Verberkmoes N.C. Hettich R.L. Gordon J.I. Characterizing a model human gut microbiota composed of members of its two dominant bacterial phyla.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 5859-5864Crossref PubMed Scopus (501) Google Scholar, 27Wang L. Christophersen C.T. Sorich M.J. Gerber J.P. Angley M.T. Conlon M.A. Increased abundance of Sutterella spp., and Ruminococcus torques in feces of children with autism spectrum disorder.Mol. Autism. 2013; 4: 42Crossref PubMed Scopus (246) Google Scholar), cluster on an adjacent branch to bifidobacterial counterparts (Fig. 6). Another clade of BlG16BP homologues harbored sequences from acidophilus cluster lactobacilli (28Barrangou R. Azcarate-Peril M.A. Duong T. Conners S.B. Kelly R.M. Klaenhammer T.R. Global analysis of carbohydrate utilization by Lactobacillus acidophilus using cDNA microarrays.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 3816-3821Crossref PubMed Scopus (162) Google Scholar), e.g. Lactobacillus acidophilus and Lactobacillus crispatus together with Streptococcus spp., e.g. Streptococcus mutans, a human oral cavity commensal (29Loesche W.J. Role of Streptococcus mutans in human dental decay.Microbiol. Rev. 1986; 50: 353-380Crossref PubMed Google Scholar). To assay for oligosaccharide uptake preference, B. animalis subsp. lactis Bl-04 was grown in the presence of equal amounts of the RFOs melibiose, raffinose, and stachyose (Fig. 1). The depletion of oligosaccharides was monitored in the culture supernatant throughout the growth experiment. Raffinose was depleted first followed by stachyose and, thereafter, melibiose (Fig. 7). Growth of seven strains of bifidobacteria on raffinose or a mixture of IMOs was performed. All tested strains except Bifidobacterium bifidum, which lacks the genes encoding the BlG16BP ABC transporter, grew efficiently on both raffinose and the IMO mixture (Table 4). Moreover, B. ovatus from the dominant human commensal Bacteroidetes phylum, was able to grow equally well on these substrates (Table 4). This shows that growth on α-(1,6)-glycosides does not require ABC-mediated transport as Bacteroides lack this type of transporters.TABLE 4Growth of different bifidobacteria and B. ovatus on raffinose and a mixture of isomaltooligosaccharidesStrainsRaffinoseIMO mixB. animalis subsp. lactis Bl-041.8 ± 0.052.0 ± 0.16B. adolescentis ATCC 157032.4 ± 0.052.7 ± 0.05B. longum subsp. longum JCM 12171.7 ± 0.011.4 ± 0.05B. longum subsp. infantis ATCC 156970.8 ± 0.122.0 ± 0.15B. breve ATCC 156481.5 ± 0.311.6 ± 0.17B. dentium Bd11.1 ± 0.101.2 ± 0.08B. bifidum ATCC 29521NDNDB. ovatus ATCC 84831.9 ± 0.011.8 ± 0.13 Open table in a new tab We wanted to evaluate the ability of B. ovatus to compete with B. animalis lactis Bl-04 for raffinose, the preferred substrate for the BlG16BP ABC transporter. B. animalis lactis Bl-04 efficiently out-competed B. ovatus and accounted for ∼98% of the colony-forming units after 18 h of growth in cultures starting from a 50:50 mixture. This distribution persisted after a second round of co-culture (Fig. 8). The complexity and the density of the HGM are highest in the distal gut, reaching 1011 cells g−1 content (30Kleerebezem M. Vaughan E.E. Probiotic and gut lactobacilli and bifidobacteria: molecular approaches to study diversity and activity.Annu. Rev. Microbiol. 2009; 63: 269-290Crossref PubMed Scopus (232) Google Scholar). Humans consume large amounts of non-digestible glycans that reach the colon intact and exert a significant effect on the composition of the microbiota (9Koropatkin N.M. Cameron E.A. Martens E.C. How glycan metabolism shapes the human gut microbiota.Nat. Rev. Microbiol. 2012; 10: 323-335Crossref PubMed Scopus (863) Google Scholar). Uptake of glycan oligomers is likely to be a key adaptation strategy to the fierce competition as it allows further intracellular (or periplasmic) breakdown of these oligomers and precludes loss to competing taxa. The involvement of ABC transporters in uptake of non-digestible oligosaccharides in health-stimulating bacteria has been highlighted through several “omics” studies (22Andersen J.M. Barrangou R. Abou Hachem M. Lahtinen S.J. Goh Y.J. Svensson B. Klaenhammer T.R. Transcriptional analysis of oligosaccharide utilization by Bifidobacterium lactis Bl-04.BMC Genomics. 2013; 14: 312Crossref PubMed Scopus (61) Google Scholar, 31Gilad O. Jacobsen S. Stuer-Lauridsen B. Pedersen M.B. Garrigues C. Svensson B. Combined transcriptome and proteome analysis of Bifidobacterium animalis subsp. lactis BB-12 grown on xylo-oligosaccharides and a mode" @default.
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• W2494559098 title "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" @default.
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