Human Gut Bacteroidetes Can Utilize Yeast Mannan Through a Selfish Mechanism

ARTICLE doi:10.1038/nature13995

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism

Fiona Cuskin1,2*, Elisabeth C. Lowe1*, Max J. Temple1*, Yanping Zhu1,2, Elizabeth A. Cameron3, Nicholas A. Pudlo3, Nathan T. Porter3, Karthik Urs3, Andrew J. Thompson4, Alan Cartmell5, Artur Rogowski1, Brian S. Hamilton6, Rui Chen7, Thomas J. Tolbert8, Kathleen Piens9, Debby Bracke9, Wouter Vervecken9, Zalihe Hakki5, Gaetano Speciale5, Jose L. Muno¯z-Muno¯z1, Andrew Day1, Maria J. Pen˜a2, Richard McLean10, Michael D. Suits11, Alisdair B. Boraston11, Todd Atherly12, Cherie J. Ziemer12{, Spencer J. Williams5, Gideon J. Davies4, D. Wade Abbott2,10, Eric C. Martens3 & Harry J. Gilbert1,2

Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall a-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast a-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of a-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a ‘selfish’ model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.

The microbial community in the human large bowel, the microbiota1,2, from Saccharomyces cerevisiae13, Schizosaccharomyces pombe and the is central to the health and nutrition of its host3–6. Glycan utilization is pathogenic yeast Candida albicans (Fig. 1b and Extended Data Fig. 1a, b). a key evolutionary driver underpinning the structure1,2 of this micro- B. thetaiotaomicron mutants lacking MAN-PUL2 or MAN-PUL1/2/3 cosm1,2, with the Bacteroidetes playing a dominant role in this process. were unable to grow on yeast mannan in vitro, (Extended Data Fig. 1c). The genomes of Bacteroidetes contain polysaccharide utilization loci In gnotobiotic mice fed a diet lacking glycans the B. thetaiotaomicron (PULs)7 that encode the apparatus required to utilize complex carbohy- mutant DMAN-PUL1/2/3 outcompeted the wild-type bacterium, whereas drates, with each PUL orchestrating the degradation of a specific glycan. wild-type B. thetaiotaomicron was the dominant species in rodents fed The microbiota contains a cohort of bacteria that target a-mannosidic a yeast-mannan-rich diet, (Fig. 2a). These data underscore the impor- linkages (based on sequence similarity to glycoside hydrolase families tance of MAN-PUL1/2/3 when B. thetaiotaomicron is exposed to yeast (GHs) that are populated by a-mannosidases and a-mannanases)8,9, mannan in vivo, while the intriguing dominance of the mutant strain indicating that a-mannose-containing glycans, such as those from yeast in animals fed a polysaccharide-free diet is further considered in Sup- and other fungal a-mannans, aresignificant nutrients forthese microbes plementary Information section 3.2. To explore whether B. thetaiotao- (see Supplementary Information section 1.0). Furthermore, these glycans micron degraded a-mannan and HMNG13 by distinct enzyme systems, are implicated in the immunopathology of the inflammatory bowel disease, the PULs activated bya HMNG, Man8GlcNAc2,wereevaluated.Asingle 10,11 Crohn’s disease (Supplementary Information section 2.0). The genome PUL was activated by Man8GlcNAc2, which was distinct from MAN- of Bacteroides thetaiotaomicron, a dominant member of the microbiota, PUL1/2/3 (Fig. 1c), demonstrating that degradation, and thus utiliza- encodes 36 proteins predicted to display a-mannosidase or a-mannanase tion, of a-mannan and HMNG are carried out by different PULs. activity12. Here we unravel the mechanism by which B. thetaiotaomicron Analysis of the growth profiles of 29 human gut Bacteroidetes species metabolizes the major a-mannose-containing glycans presented to the revealed that nine species metabolized S. cerevisiae a-mannan, with 33 large bowel. The data also show that B. thetaiotaomicron expresses a out of 34 strains of B. thetaiotaomicron growing on the glycan (Fig. 1d). specific yeast a-mannan degrading system that is distinct from the high These data show that B. thetaiotaomicron, along with some of its phy- mannose mammalian N-glycan (HMNG) depolymerizing apparatus. logenetically related neighbours, dominates yeast mannan metabolism in the Bacteroidetes phylum of the microbiota. Within fully sequenced Yeast a-mannan utilization by B. thetaiotaomicron genomes of 177 microbiota members, the presence of GHs integral to B. thetaiotaomicron utilizes a-mannan as a sole carbon source and tran- a-mannan degradation (GH38, GH92 and GH76) are restricted to five scriptional studies identified three B. thetaiotaomicron PULs (MAN-PUL1, species of Bacteroides and three of Parabacteroides; no Firmicutes con- MAN-PUL2andMAN-PUL3;Fig. 1a) that were activated bya-mannan tained this grouping of glycoside hydrolases8.

1Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle-upon-Tyne NE2 4HH, UK. 2Complex Carbohydrate Research Center, The University of Georgia, 315 Riverbend Road, Athens, Georgia 30602, USA. 3Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109 USA. 4Department of Chemistry, University of York, York YO10 5DD, UK. 5School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia. 6Interdisciplinary Biochemistry Graduate Program, Indiana University, 800 E. Kirkwood Avenue, Bloomington, Indiana 47405, USA. 7Department of Chemistry, Indiana University, 800 E. Kirkwood Avenue, Bloomington, Indiana 47405, USA. 8Department of Pharmaceutical Chemistry, University of Kansas School of Pharmacy, 2095 Constant Avenue, Lawrence, Kansas 66047, USA. 9Oxyrane, 9052 Ghent, Belgium. 10Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, Alberta T1J 4B1, Canada. 11Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 5C2, Canada. 12USDA, Agricultural Research Service, National Laboratory for Agriculture and the Environment, Ames, Iowa 50011, USA. *These authors contributed equally to this work. {Deceased.

8JANUARY2015|VOL517|NATURE|165 ©2015 Macmillan Publishers Limited. All rights reserved RESEARCH ARTICLE a Figure 1 | B. thetaiotaomicron PULs involved in yeast a-mannan metabolism, and utilization of MAN-PUL1 (BT2620–32) BT2620 BT2622 BT2623 BT2629 BT2631BT2632 the glycan in Bacteroidetes. a, Genes encoding GH97 GH67 GH76 HTCS regulator GH92 GH76 GH125 known or predicted functionalities are colour- BT2630 coded and, where appropriate, annotated MAN-PUL2 (BT3773–92) according to their CAZy family. SGBP; surface BT3773 BT3774 BT3775–BT3778 glycan binding protein. b, Structures of yeast GH92 GH38/CBM32 (S. cerevisiae) mannan and HMNG. c, Cells were GT32 grown on media containing glycans as the sole BT3780 BT3781 BT3782 BT3784 BT3791 BT3792 carbon source (n 5 3 separate cultures per GH130 GH125 GH76 GH92 HTCS sensor/regulator GH76 substrate), and susC transcripts derived from PULs BT3783 that encode GH76 and/or, GH92 enzymes were MAN-PUL3 (BT3853–62) BT3858 BT3862 quantified by qPCR. The fold change relative to SARP/OmpR GH92 GH99 glucose-grown cells is shown on the y axis and the x axis shows the genes probed. Inset: levels of susC Phosphatase Regulator SusC-like Glycoside hydrolase b transcripts derived from MAN-PUL1/2/3 when Unknown SusD-like SGBP Glycosyl transferase B. thetaiotaomicron was cultured on mannan

~60 residues comprising only the a-1,6-Man backbone or ~150 residues ~100–150 residues1 P P Linkage and sugar key: 6 Candida albicans mannan. Data are averages and α-1,2 α-1,3 standard deviations of three biological replicates. α-1,6 β -1,4 d, Strains of 29 different bacterial species of β -1,2 Bacteroidetes (number of strains in parentheses) Asn Mannose were inoculated into minimal media containing S. cerevisiae C. albicans S. pombe Mammalian high GlcNAc P Phosphate S. cerevisiae mannan as the sole carbon source α-mannan α-mannan α-mannan mannose N-glycan Galactose (HMNG) (n 5 2 replicate cultures per strain). Growth 100 B. xylanisolvens 97 B. ovatus measured at 48 h at A . Inset: phylogeny of c 60 1,000 d 1.25 95 B. ﬁnegoldii 600nm Unbranched 100 B. caccae α-1,6-mannan 100 B. thetaiotaomicron Bacteroidetes species with bootstrap numbers Glucose B. fragilis 50 Chitobiose C. albicans mannan 56 B. salyersiae 100 B. nordii indicated; organisms growing on mannan α-mannan 1.00 98 B. intestinalis 100 100 B. cellulosilyticus connected by red lines. The non-Bacteroides genera Man8GlcNAc2 100 B. oleiciplenus 40 B. ﬂuxus 99 B. uniformis were Odoribacter (O), Parabacteroides (P) and 0.75 100 B. eggerthii 100 B. stercoris 30 96 B. clarus Dysgonomonas (D). 10 B. plebeius 90 B. massiliensis 100 B. vulgatus 20 100 B. dorei 0.50 100 D. gadei 81 D. mossii 96 O. splanchnicus B. intestinihominis Fold change vs glucose 10 1 BT2626 BT3788 BT3854 100 P. distasonis absorbance (600 nM) Normalized maximum P. merdae PUL1 PUL2 PUL3 0.25 100 100 P. johnsonii 100 P. gordonii 0 100 P. goldsteinii P. copri

0 BT3983BT2626BT3788BT3854BT2952BT4088BT3519BT1040BT1042BT1047BT2107BT1875BT2202BT3297BT4081BT0455BT4958BT1774 ) ) ) ) ) ) 4) 5) 9) 4) 3) 1) 1) 3) 4) 1) 1) (5) 19) (3 (1 (1) ( (3) ( ( ( ( (1) ( (7 34) ( (1 (1) (1) (2) (1) (3 (2 ( (2 (16 i (12) (10) (1 i (56 i s ( i ii n ae s ni e s hi dii is us ri is ei si n ns us rus rt lis rdi n is s cus onii ro tus rsi ca cu la rei ol uxus nalis ns o co m ad tei susC-like or other sentinel gene probed ic at ti g fl gi ti ie beius ni ac a .n ple .g mo lye merdaeohnsoc ly ne fr es B for isolve stasoni B. cB. doegge B. ssil ici D D. olds B. ovasa P. B. int a le lanch taom an . vulgdi P. j B. losi B. B. fi B. pleB. ster p P. gord io B. B llu B. B. uniestinihomin P. g a P. .m B. o t .s et ce B in O th B. xyl B. B. B.

Structure of the B. thetaiotaomicron a-mannan PULs described in Extended Data Fig. 3c, d. In addition to BT3862, other B. thetaiotaomicron MAN-PUL1 and MAN-PUL2 display significant surface enzymes are required to partially expose the a-1,6-Man-backbone. synteny (Supplementary Table 1) while MAN-PUL3 displays no organ- Screening for such enzymes revealed an a-mannosidase, BT2199, that izational similarity to the other two loci (Fig. 1a). Characterization of mediates very limited extracellular removal of side chains (Extended the 15 enzymes encoded by the mannan PULs revealed that these loci Data Fig. 4a) and thus facilitates limited cleavage of the mannan back- orchestrate a-mannan degradation from diverse yeasts and possibly bone by the surface endo-a-1,6-mannanases BT2623 and BT3792. The other fungi. For example, MAN-PUL1 contains an a-galactosidase, activity profiles of BT2199 and the surface endo-acting mannanases BT2620, which targets a-galactosyl linkages absent in S. cerevisiae mannan are consistent with the observed production of large oligosaccharides but present in other fungal a-mannans such as the yeast Schizosac- by non-growing cells of B. thetaiotaomicron incubated with yeast man- charomyces pombe14 (Extended Data Fig. 1d), explaining why inactivation nan (Extended Data Fig. 4b, c). of MAN-PUL1 affects growth of B. thetaiotaomicron on this polysac- The cellular location of the key a-mannan hydrolysing enzymes charide (Extended Data Fig. 1b). Functional diversity is also evident in (Fig. 2b, c) indicates that the polysaccharide was degraded primarily MAN-PUL2, which, in addition to its catabolic role, encodes glycosyl- in the periplasm where the side chains were removed by the synergistic transferases that mediate synthesis of the trisaccharide Man-a-1,3- action of a-mannosidases and sugar-6-monophosphatases15 (Fig. 2d, e, [Man-a-1,6]-Man (Extended Data Fig.2). Thus, MAN-PUL2 comprises Extended Data Fig. 5, Supplementary Tables 2 and 3 and Supplemen- a unique example of the co-regulation of related catabolic and biosyn- tary Information section 4.0). The broad specificity of BT3774 enabled thetic functions within a single PUL13. the a-mannosidase to play a key role in the removal of the uncharged side chains, being the only enzyme capable of removing the sterically Mannan degradation at the cell surface and periplasm restricted a-1,2-Man units linked to the a-mannan backbone (Fig. 3 and The enzymatic degradation of a-mannan is restricted through steric Extended Data Figs 4f and 5) as well as cleaving the Man-1-phosphate constraints imposed through the side chains appended to the back- linkage, a critical step in the removal of the phosphorylated branches bone (Supplementary Table 2 and Extended Data Figs 1e and 4a). (Fig. 2d). The importance of BT3774 in a-mannan catabolism was con- Critically, the a-1,6-Man-backbone is not accessible to the endo-a- sistent with the growth profile of the B. thetaiotaomicron mutant Dbt3774 1,6-mannanases until the side chains have been removed, consistent (Extended Data Fig. 1d). with the topography of the substrate binding cleft of these enzymes Hydrolysis of the a-1,6-Man backbone of a-mannan by B. thetaio- (Extended Data Fig. 3a, b). The MAN-PUL3-encoded endo-a-1,2- taomicron was mediated by surface and periplasmic ‘retaining’ endo- mannosidase BT3862 released a limited proportion of the terminal a-1,6-mannanases (Extended Data Fig. 4e). The surface endo-mannanases Man-a-1,3-Man disaccharides, thereby assisting in exposing the back- BT2623 and BT3792 (Fig. 2b, c) generated large oligosaccharides, while bone. The structural basis for the specificity displayed by BT3862 is the periplasmic mannanases BT2631 and BT3782 produced small limit

a b 100 c BT3792 (GH76) * 75 * 0 h 16 h RP * * * BT3792 50 BT3774 25

Percentage of BT3774 (GH38)

competing strain BT2630 0 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 Days post-colonization BT3858 Glycan-free diet Wild type Glycan-free diet + mannan water ΔMAN-PUL1/2/3 Bread diet d e

a b Mnn4 h jk Mnn4 uncapped c i BT2623 BT2631 Man Man 5 BT3783 + Mnn4 BT3782 g BT2629 + Mnn4 3 Man4 d Man uncapped f 6 BT3792 107 )

e –1 6 a) Man -(P-Man) g) Man5-P-Man 10 h k BT3774 + Mnn4 8 2 j 105 min

–1 4 b) Man8-P-Man h) Man5-P2 10

(M 103 Man RNaseB M 5 Man6 c) Man -P*-Man 2 Man 7 / K 10 Man 8 Man i) Man5-P 7 9 cat 1 Relative fluorescence units 10 d) Man -(P-Man) k 6 2 10 0 –1 Man BT2629 + j) Man4-P 10 5 e) Man -P Log RNaseB 6 2 10–2 345678 f) Man -P*-Man k) Man3-P 6 Manno-oligosaccharide D.P.

Figure 2 | Mannan PULs enable colonization of gnotobiotic mice; key type B. thetaiotaomicron on the bread diet compared to the glycan-free diet biochemical and cellular features of the encoded enzymes. a, Colonization (P # 0.05) are indicated with an asterisk . b, Fluorescent- and light-microscopy of gnotobiotic mice (n 5 5) by wild type B. thetaiotaomicron (red) and a images of B. thetaiotaomicron incubated with polyclonal antibodies against mutant (black) lacking MAN-PUL1/2/3 (DMAN-PUL1/2/3). On day 0 mice BT3792 and BT3774. c, Western blots of B. thetaiotaomicron cells cultured on were gavaged with ,108 colony-forming units (c.f.u.) of 50:50 of the yeast mannan that were untreated with proteinase K (0 h) or incubated with two B. thetaiotaomicron strains and then fed a control diet lacking 2mgml21 proteinase K for 16 h. RP, recombinant protein. Blots were B. thetaiotaomicron-digestible glycans (green). Left, after 7 days the control diet probed with antibodies against the B. thetaiotaomicron enzymes. Localization was supplemented with 1% YM in drinking water (pink shading). Middle, blots and microscopy images (b, c) are representative data from three biological identical treatment as to the left panel except no mannan was included after replicates. d, Phosphorylated high mannose N-glycan MNN4 and RNAase day 7 (shaded green). At day 21 mannan in the water was switched between B incubated with BT2629 (GH92 a-mannosidase), BT3783 (phosphatase) or the groups in the left and middle panels, indicated by the colour panels BT3774 (GH38 a-mannosidase) and the products analysed by capillary (pink 5 mannan, green 5 no mannan). Right, mice fed the control diet (shaded electrophoresis. Man5 to Man9 (subscript numbers refer to the number of Man green) were switched to a diet containing leavened bread (blue). The average units in the N-glycans) lack phosphate and mannose-1-phosphate groups. abundance of the mannan utilization mutant (black) in mice fed the bread diet P* indicates the N-glycan contains a single phosphate, which is in the following compared to the corresponding time points in mice fed the glycan-free diet structure: Man-a-1-phosphate-a-6-Man-a-1,2-Man. e, Catalytic efficiency (mean 6 s.e.m., 81% 6 1.2% in animals fed the bread diet versus 90% 6 1.7% in of the endo-a-1,6-mannanases against a-1,6-mannooligosaccharides mice fed the glycan-free diet; P 5 0.00005 by unpaired Student’s t-test). Time (see Supplementary Information Table 4 for full data). D.P., degree of points at which there was a significant difference between the mutant and wild polymerization of the mannooligosaccharides. products (Extended Data Fig. 6a–c). Consistent with these product pro- endo-acting N-acetylglucosaminidase, which cleaves the oligosaccharide files, the periplasmic enzymes were ,100- to 1,000-fold more active from its polypeptide (Extended Data Fig. 7c, d). The released HMNG is than the surface mannanases against oligosaccharides with a degree heldonthesurface ofB. thetaiotaomicronthroughthe mannose-binding of polymerization (d.p.) ,6, whereas against large oligosaccharides protein BT3986, while the SusD homologue BT3984, by recognizing (d.p. $ 6) the periplasmic and surface enzymes displayed broadly GlcNAc at the reducing end of the glycan (Extended Data Fig. 7e, f and similar activities (Fig. 2f and Supplementary Table 4). The linear and Supplementary Table 5), probably orientates the glycan into the outer hence unadorned a-1,6-mannooligosaccharides generated in the peri- membrane porin (SusC homologue BT3983) for transport into the periplasm were hydrolyzed by the exo-a-1,6-mannosidases BT2632 and plasm, where the periplasmic a-mannosidases hydrolyse the oligosac- BT3781 (Extended Data Fig. 6d, e and Supplementary Table 2), as pre- charide into a trisaccharide that is degraded by enzymes that are not viously proposed16. The inability of the backbone-cleaving enzymes to encoded by HMNG-PUL. A model for the enzymatic degradation of attack wild-type yeast a-mannan indicates that these glycoside hydro- HMNG is displayed in Fig. 4. The importance of HMNG-PUL in the lases cannot accommodate a-1,6-linked Man decorated at O2 (side chains metabolism of the N-glycan is consistent with the reduced growth of are appended a-1,2 to the mannan backbone) in the active site or prox- the Dbt3993 mutant (lacking the extra-cellular s factor regulator of imal subsites, consistent with the structures of the substrate binding HMNG-PUL) cultured on Man8GlcNAc2 (Extended Data Fig. 7g). clefts of BT3792 and BT3781 (Extended Data Fig. 3a–f). Yeast a-mannan degradation by B. thetaiotaomicron A B. thetaiotaomicron PUL for HMNG catabolism Substrate promiscuity is a hallmark of the a-mannan degradation appa- HMNG-PUL encodes four enzymes and two surface glycan binding ratus of B. thetaiotaomicron, as steric constraints limit enzymatic access proteins (Extended Data Fig. 7a). BT3990 and BT3991 target a-1,2-Man to the glycan side chains, and consequently the polysaccharide backbone. and a-1,3-Manlinkages, respectively,in HMNGs17. The terminal undec- Accordingly, B. thetaiotaomicron uniquely uses surface a-mannosidase(s), orated a-1,6-Man exposed by BT3990 is hydrolysed by BT3994, which probably BT2199, to generate limited, but sufficient, side chain removal requires GlcNAc at the reducing end for activity, producing Man-a- for the surface endo-acting enzymes to mediate infrequent cleavage of 1,6-Man-b1,4-GlcNAc (Extended Data Fig. 7b). BT3987 is a surface the backbone. The B. thetaiotaomicron mannan-degrading apparatus

Figure 3 | Model of yeast mannan deconstruction by B. thetaiotaomicron. Boxes show examples of bonds cleaved by the endo-a- 1,2-mannosidase BT3862, by the a-1,3- and a-1,2- 1 BT3862 1 P P mannosidase activities displayed by BT2629 and 6 6 BT3792/ BT3784, respectively, and the Man-1-P and BT2623 1 a-1,2-Man linkages targeted by BT3774. Extended P 1 1 6 Data Fig. 3 shows 3D structures (cartoons, P P 6 6 colour-ramped from the amino terminus (blue) to the carboxy terminus (red)) of the enzymes that 1 P play a key role in mannan degradation. In this 6 model limited degradation occurs at the surface Surface Constitutively expressed and the bulk of glycan degradation occurs in the endo-α-1,6-mannanases α-mannosidases and endo-α-1,2 BT3792 and BT2623 (GH76) periplasm. SusC-like proteins mediate transport Outer membrane mannosidase BT3862 (GH99) across the outer membrane7. SusC

Periplasm

BT2631/ 1 BT2630/ 1 BT3782 P P BT2629/ BT3783 6 6 BT3784 BT3774

1 P P 6 6

α -1,6-endo-mannanases BT3774 (GH38) and α-1,2/3-mannosidases BT2631 and BT3782 (GH76) α-1,6-mannosidases mannose-6 phosphatases BT2629 and BT3784 (GH92) BT2632 and BT2630 and BT3783 BT3781 (GH125) α-1,2 Mannose α-1,3 GlcNAc α-1,3-mannosidase α -1,6 P Phosphate BT3858 (GH92) β-1,4 is optimized to minimize nutrient loss, illustrated by the observation that mannan-backbone-using strains of Bacteroides on intact S. cerevisiae no oligosaccharides were released into the culture supernatant when a-mannan (Fig. 5). It should be emphasized, however, that the degra- B.thetaiotaomicron wascultured ona-mannan(Extended Data Fig. 4c). dation of at least some polysaccharides is mediated through synergistic Endo-a-1,6-mannanases are central to the hierarchical degradation interactions between different members of the microbiota18,19, illustrat- of yeast mannan, reflecting their location and substrate specificities. ing the diverse mechanisms by which nutrients are used in this micro- The surface endo-acting enzymes generate large oligosaccharides min- bial community. imizing extracellular metabolism and loss to other microbiota residents. This work provide insights into the adaptation of the microbiota to The periplasmic endo-a-1,6-mannanases generate numerous short oli- yeast domestication in the human diet reflecting the regular consump- gosaccharides, maximizing the substrate available to the periplasmic tion of yeast-leavened bread, fermented beverages and products such exo-acting a-1,6-mannosidases. This ‘selfish’ model is consistent with as soy sauce. B. thetaiotaomicron, and a limited number of other micro- the inability of B. thetaiotaomicron to support growth of mannose- and biota-derived Bacteroidetes, have evolved a complex machinery to digest and metabolize yeast cell-wall mannans. Phylogenetic analysis suggests that this trait penetrated the human gut Bacteroidetes at least twice, α-1,2 α-1,3 once each in the Bacteroides and Parabacteroides. It is also possible, α-1,6 however, that close relatives of B. thetaiotaomicron have gained parts of β-1,4 this complex catabolic trait via separate events. Analysis of 250 human Mannose metagenomic samples revealed a-mannan-degrading PULs closely GlcNAc GH18 endo-β-N- related to those from B. thetaiotaomicron in a majority (62%) of sub- acetylglucosaminidase jects (Extended Data Fig. 8) making it more common than culturally- (BT3987) 20 Asn SGBP restricted traits like red algal porphyran degradation , but less common D than plant cell-wall xyloglucan degrading PULs, identified in divergent C Bacteroides species21,22 (see Supplementary Information section 3.3). The a-mannan degrading capacity of B. thetaiotaomicron is consistent with the bacterium’s ‘glycan generalist’ strategy7,12,23, enabling the microorganism to thrive in the competitive environment of the microbiota, where the omnivorous diet of the host requires rapid adap- tion to the nutrients presented to the distal gut. Many of the organisms α-1,6-mannosidase α-1,3-mannosidase α-1,2-mannosidase GH92 (BT3994) GH92 (BT3991) GH92 (BT3990) in the microbiota produce enzymes that attack the major components of the human diet such as starch and pectins. An additional survival Figure 4 | Model of HMNG depolymerization by B. thetaiotaomicron. The strategy for some members of the microbiota, such as B. thetaiotaomi- interaction of mannose with the surface glycan binding protein (SGBP) is cron, is the targeting of low-abundance, highly complex dietary glycans shown, while the binding of the SusD homologue (D) to the reducing end GlcNAc directs the N-glycan into the SusC-like (C) porin. In both the yeast that are not metabolized by most other organisms, exemplified here by mannan and the HMNG models the majority of glycan degradation occurs in yeast mannan. With respect to the human host the degradation of the periplasm and not on the surface. The enzymes probably involved in the a-mannan by B. thetaiotaomicron may also be relevant to the health- periplasmic degradation of the trisaccharide generated by the HMNG-PUL- promoting effects of the human microbiota24. This work provides encoded system are addressed in Supplementary Information section 4.3. insights into a sophisticated a-mannan degrading apparatus that exists

168|NATURE|VOL517|8JANUARY2015 ©2015 Macmillan Publishers Limited. All rights reserved ARTICLE RESEARCH a b 7. Martens, E. C., Koropatkin, N. M., Smith, T. J. & Gordon, J. I. Complex glycan Mannan Mannose

–1 –1 catabolism by the human gut microbiota: the Bacteroidetes Sus-like paradigm. 100 Bc alone 45 Bt in Bt/Bc mix J. Biol. Chem. 284, 24673–24677 (2009). Bt in Bt/Bc mix Bc in Bt/Bc mix Bc in Bt/Bc mix 35 8. El Kaoutari, A., Armougom, F., Gordon, J. I., Raoult, D. & Henrissat, B. The 75 abundance and variety of carbohydrate-active enzymes in the human gut 25 25 microbiota. Nature Rev. Microbiol. 11, 497–504 (2013). 15 9. Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M. & Henrissat, B. The 5 carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42, 0 0 Fold change in c.f.u. ml t al al y Fold change in c.f.u. ml l l D490–D495 (2014). ti ti ar rt n tia tia Star en Sta n ne on 10. Konrad, A. et al. Immune sensitization to yeast antigens in ASCA-positive patients o nen Stati expon Stationary with Crohn’s disease. Inflamm. Bowel Dis. 10, 97–105 (2004). exp expo y expone ate rl L ate 11. Mpofu, C. M. et al. Microbial mannan inhibits bacterial killing by macrophages: a Early Ea L possible pathogenic mechanism for Crohn’s disease. Gastroenterology 133, 120 Bc Bc 120 1487–1498 (2007). Bt 100 Bt 100 12. Xu, J. et al. A genomic view of the human–Bacteroides thetaiotaomicron symbiosis. 80 80 Science 299, 2074–2076 (2003). 60 60 13. Martens, E. C., Chiang, H. C. & Gordon, J. I. Mucosal glycan foraging enhances 40 40 20 20 fitness and transmission of a saccharolytic human gut bacterial symbiont. Cell Percentage (%) Percentage (%) 0 0 Host Microbe 4, 447–457 (2008). l ia ial ry 14. Ballou, C. E., Ballou, L. & Ball, G. Schizosaccharomyces pombe glycosylation mutant tial t ntial nt Start Start na nen onary ne ne tio with altered cell surfaceproperties.Proc.Natl Acad. Sci.USA 91, 9327–9331 (1994). o po ta ponen Stati x xpo S xp e e y 15. Raschke, W. C., Kern, K. A., Antalis, C. & Ballou, C. E. Genetic control of yeast mannan y ex te te e arl La Earl La E structure. Isolation and characterization of mannan mutants. J. Biol. Chem. 248, c d 4660–4666 (1973). 16. Gregg, K. J. et al. Analysis of a new family of widely distributed metal-independent –1 Mannan –1 Mannose 175 Bx alone 150 alpha-mannosidases provides unique insight into the processing of N-linked 150 Bt in Bt/Bx mix Bt in Bt/Bx mix glycans. J. Biol. Chem. 286, 15586–15596 (2011). 125 Bx in Bt/Bx mix 100 Bx in Bt/Bx mix 21 100 17. Zhu, Y. et al. Mechanistic insights into a Ca -dependent family of a-mannosidases 75 50 in a human gut symbiont. Nature Chem. Biol. 6, 125–132 (2010). 50 18. Rakoff-Nahoum, S., Coyne, M. J. & Comstock, L. E. An ecological network of 25 0 0 polysaccharide utilization among human intestinal symbionts. Curr. Biol. 24, l Fold change in c.f.u. ml ry Fold change in c.f.u. ml tial ia 40–49 (2014). art ial tart n na S St ent o ne nent nential ti o tionary 19. Ze, X., Duncan, S. H., Louis, P. & Flint, H. J. Ruminococcus bromii is a keystone on o po ta xp S xp Sta e species for the degradation of resistant starch in the human colon. ISME J. 6, e e ex e exp at Early Lat 1535–1543 (2012). Early L 20. Hehemann, J. H. et al. Transfer of carbohydrate-active enzymes from marine 120 120 bacteria to Japanese gut microbiota. Nature 464, 908–912 (2010). 100 Bx Bx Bt 100 Bt 80 21. Larsbrink, J. et al. A discrete genetic locus confers xyloglucan metabolism in select 80 60 human gut Bacteroidetes. Nature 506, 498–502 (2014). 60 40 22. Martens, E. C., Kelly, A. G., Tauzin, A. S. & Brumer, H. The devil lies in the details: how 40

Percentage (%) 20 variations in polysaccharide fine-structure impact the physiology and evolution of

Percentage (%) 20 0 gut microbes. J. Mol. Biol. 426, 3851–3865 (2014). l 0 art tia ry l 23. Martens, E. C. et al. Recognition and degradation of plant cell wall polysaccharides ntial rt St en na tia ial Sta en ent by two human gut symbionts. PLoS Biol. 9, e1001221 (2011). on one atio tionary xp St on on ta e xp xp S 24. Everard, A., Matamoros, S., Geurts, L., Delzenne, N. M. & Cani, P. D. Saccharomyces e rly te exp La rly te e Ea La boulardii administration changes gut microbiota and reduces hepatic steatosis, Ea low-grade inflammation, and fat mass in obese and type 2 diabetic db/db mice. Figure 5 | Bacteroides co-culture sharing experiments. a–d, B. MBio. 5, e01011–e01014 (2014). thetaiotaomicron was co-cultured with Bacteroides cellulosilyticus WH2 (a, b) Supplementary Information is available in the online version of the paper. or with Bacteroides xylanisolvens NLAE-zI-p352 (c, d) with either mannan (a, c) or mannose (b, d) as the sole carbon source. Each non-mannan user was Acknowledgements This work was supported by grants from the European Research Council (G.J.D., Glycopoise; H.J.G., No. 322820), The Wellcome Trust (H.J.G., also cultured on mannan independently. The upper graph in each panel depicts 21 21 WT097907AIA), BBSRC (M.J.T., G.J.D.; BB/G016127/1), US Department of Energy the c.f.u. ml of each strain, relative to the c.f.u. ml at inoculation. Total (DOE) Bioenergy Research Center (BESC) supported by the Office of Biological and c.f.u. ml21 was determined by colony counts, and the proportion of each bacteria Environmental Research in the DOE Office of Science (M.J.P.) and the National was determined by qPCR of marker genes from genomic DNA (shown in the Institutes of Health (E.C.M. and T.J.T., GM090080). Gnotobiotic mouse experiments lower graph of each panel). Error bars represent s.d. of three biological replicates. were supported by a subsidy from the University of Michigan Medical School Host Microbiome Initiative, Agriculture and Agri-Food Canada, AgriFlex (D.W.A., #2510), Bc, B. cellulosilyticus;Bt,B. thetaiotaomicron;Bx,B. xylanisolvens. Canadian Institute of Health Research operating grant (A.B.B., MOP-68913), Australian Research Council; Mizutani Foundation (S.J.W.). We thank the staff of the Diamond Light Source for the provision of beamline facilities. We would also like to thank various within widespread members of the human microbiota, thereby revealing members of ICaMB for providing the yeast strains used in this work. We were greatly the impact made by the historical domestication of yeast and other saddened by the passing of C.Z. during the course of this work. dietary fungi on the structure of this microbial consortium. Author Contributions Enzyme characterization: F.C., M.J.T., J.L.M.-M. and D.W.A. Capillary electrophoresis: D.B., K.P. and W.V. E.C.L. created gene deletion strains and Online Content Methods, along with any additional Extended Data display items determined phenotypes with F.C. E.C.L., F.C. and A.R. performed enzyme localisation. and Source Data, are available in the online version of the paper; references unique F.C. and E.C.L. carried out the co-culturing experiments. Gene expression analysis: to these sections appear only in the online paper. E.A.C., N.A.P. and E.C.M. Growth analysis on purified mannans and HMNG: E.C.L., N.A.P., K.U. and E.C.M. Characterization of HMNG binding proteins: Y.Z. Characterization of the Received 24 January; accepted 22 October 2014. Dbt3774 mutant: A.D. Phylogenetic reconstruction and metagenomic analysis: E.C.M. Gnotobiotic mouse experiments: E.A.C., N.A.P., N.T.P. and E.C.M. Purification of HMNG: 1. Arumugam, M. et al. Enterotypes of the human gut microbiome. Nature 473, T.J.T., B.S.H. and R.C. Isolation and genomic analysis of pig gut strains: T.A., C.J.Z. A.C. 174–180 (2011). and G.S. performed NMR experiments on GH76 and M.J.P. on GT products. Z.H. and 2. Ba¨ckhed, F., Ley, R. E., Sonnenburg, J. L., Peterson, D. A. & Gordon, J. I. G.S. synthesized substrates. Crystallographic studies by A.J.T., G.J.D., M.D.S., A.B.B. and Host-bacterial mutualism in the human intestine. Science 307, 1915–1920 R.M. Experiments designed by F.C., E.C.L., G.J.D., S.J.W., D.W.A., E.C.M. and H.J.G. The (2005). manuscript was written primarily by H.J.G. and E.C.M. with contributions from S.J.W., 3. Arpaia, N. et al. Metabolites produced by commensal bacteria promote peripheral G.J.D. and D.W.A. E.C.L. and E.C.M. prepared the figures. regulatory T-cell generation. Nature 504, 451–455 (2013). Author Information The protein crystal structures reported in this study have been 4. Flint, H. J., Bayer, E. A., Rincon, M. T., Lamed, R. & White, B. A. Polysaccharide deposited under the following PDB accession codes: 4C1R (BT3783-Mg binary utilization by gut bacteria: potential for new insights from genomic analysis. complex); 4C1S (BT3792); and 4UTF (BxGH99/Man-IFG/mannobiose ternary Nature Rev. Microbiol. 6, 121–131 (2008). complex). Reprints and permissions information is available at www.nature.com/ 5. Kau, A. L., Ahern, P. P., Griffin, N. W., Goodman, A. L. & Gordon, J. I. Human nutrition, reprints. The authors declare no competing financial interests. Readers are welcome the gut microbiome and the immune system. Nature 474, 327–336 (2011). to comment on the online version of the paper. Correspondence and requests 6. Round, J. L. & Mazmanian, S. K. The gut microbiota shapes intestinal immune for materials should be addressed to H.J.G. ([email protected]), responses during health and disease. Nature Rev. Immunol. 9, 313–323 (2009). E.C.M. ([email protected]) or D.W.A. ([email protected]).

METHODS glucose-6-phosphate, generated from the released mannose, is oxidized by glucose- 6-phosphate dehydrogenase with concomitant reduction of NADP1 to NADPH, Producing recombinant proteins for biochemical assays. DNAs encoding the which was monitored at 340 nm using an extinction coefficient of 6223 M21 cm21. mature forms of the enzymes used in this study were amplified by PCR using The polysaccharide substrates used were S. cerevisiae a-mannans from wild-type appropriate primers. The amplified DNAs were cloned into NcoI/XhoI, Nco/ or appropriate mutant strains that produce variants of the polysaccharide (Extended BamHI, NdeI/XhoI or NdeI/BamHI restricted pET21a or pET28a, as appropriate. Data Fig. 5); these mannans were purified from stationary-phase cultures of the The encoded recombinants generally contained a C-terminal His -tag, although, 6 yeast grown in yeast extract peptone dextrose medium, as described previously15. where appropriate, the His-tag was located at the N-terminus of the protein. The The a-1,6-mannooligosaccharides, which were also used as substrates, were gen- GH92 enzymes encoded by HMNG-PUL were cloned in a previous study17.To erated as follows: 1 g a-mannan from the S. cerevisiae mutant mnn2 (comprises the express the recombinant genes encoding the mannan degrading enzymes, Escherichia a-1,6-mannan backbone with no side chains) was digested to completion with the coli strains BL21(DE3) or TUNER, containing appropriate recombinant plasmids, endo-mannanase BT3792. The products were freeze-dried and the small mannoo- were cultured to mid-exponential phase in Luria Bertani broth at 37 C. This was u ligosacchardes (d.p. 2 to 5) were purified on two P2 Bio-gel columns set up in series, followed by the addition of 1 mM (strain BL21(DE3)) or 0.2 mM (TUNER) iso- while the large oligosaccharides, with a d.p. of 6 to 8, were fractionated on two P4 propyl b-D-galactopyranoside (IPTG) to induce recombinant gene expression, and 21 Bio-gel columns also in series. The columns were run at 0.2 ml min in distilled the culture was incubated for a further 5 h at 37 uC or 16 h at 16 uC, respectively. water. The 5-ml fractions were evaluated by thin-layer chromatography (TLC) and . The recombinant proteins were purified to 90% electrophoretic purity by immo- those containing the same oligosaccharide were pooled. The activity of the endo- bilized metal ion affinity chromatography using Talon, a cobalt-based matrix, and a 25 mannanases against -mannans was determined in 50 mM Na-HEPES buffer eluted with 100 mM imidazole, as described previously . (pH 7.5) containing an appropriate concentration of the polysaccharide (ranging Producing recombinant proteins BT3783, BT3792 and BxGH99 for crystal- from 0.1–6 mg ml21) and 1 mg ml21 BSA. Reactions were incubated for 30 min at lization. pET28a expression vectors encoding mature BT3783 (residues 27 to 314) 37 uC and, at regular time intervals, 500-ml aliquots were removed and the amount and BT3792 (residues 155 to 514) were cloned into pET28a via NheI and XhoI of reducing sugar was quantified deploying the dinitrosalicylic acid reagent28 and a sites. The gene encoding the B. xylanisolvens homologue of GH99 (BxGH99) was standard curve of mannose in the reaction conditions used. TLC was also used to constructed from synthesized oligonucleotide fragments (Genscript) and also sub- provide a qualitative profile of the mannooligosaccharides generated by the GH76 cloned into pET28a using NdeI and XhoI restriction sites. Plasmids encoding BT3783 endo-mannanases from these reactions. Around 4 ml of the reaction was spotted on and BT3792 were transformed into E. coli BL21(DE3) Star chemically competent silica gel TLC plates and the plates were developed in butanol:acetic acid:water 21 cells, and grown in LB broth at 37 uC supplemented with 50 mgml kanamycin. 2:1:1 and carbohydrate products detected by spraying with 0.5% orcinol in 10% Production of recombinant BT3783 was induced by the addition of 0.2 mM IPTG sulfuric acid and heating to 100 uC for 10 min. Substrate-depletion assays were at a culture OD600 5 0.6, and incubation at 16 uC for 16 h. BT3792 was produced used to determine the activity of the endo-mannanases against a-1,6-mannooli- 26 using the auto-induction method by shaking inoculated 0.5-l cultures supple- gosaccharides. In brief, 50 mM of the oligosaccharides in 50 mM sodium phosphate 21 mented with 50 mgml kanamycin for 36 h at 37 uC and then 48 h at 20 uC. E. coli buffer (pH 7.5), containing 0.1 mg ml21 BSA (NaP buffer), was incubated with an BL21 (DE3) cells harbouring the BxGH99-encoding plasmid were cultured in 0.5 l appropriate concentration of enzyme (10 to 500 nM). Aliquots were removed at 21 ZYM-5052 auto-induction media26, supplemented with 50 mgml kanamycin, at regular intervals for up to 1 h and, after boiling for 10 min to inactivate the enzyme, 37 uC for 8 h, with induction occurring overnight at 16 uC. the amount of the substrate mannooligosaccharide remaining was quantified To purify BT3783 and BT3792, cells were collected by centrifugation and rup- by high-performance anion-exchange chromatography (HPAEC) using standard tured by chemical lysis procedure at 4 uC. In brief, cells were resuspended in 25 ml methodology. In brief, the reaction products were bound to a Dionex CarboPac of a solution consisting of 7% (w/v) sucrose, 50 mM Tris-HCl (pH 7.5), for 5 min. PA-200 column equilibrated with 100 mM NaOH. Mannose and mannooligosac- Lysozyme (Sigma-Aldrich; 10 mg) was then added and stirred for 10 min. A solu- charides were eluted with a 0–200 mM sodium acetate gradient in 100 mM NaOH tion (50 ml) consisting of 0.6% (w/v) Triton-X, 0.6% (w/v) deoxycholate, 20 mM at a flow rate of 0.25 ml min21, using pulsed amperometric detection. The data 29 Tris-HCl (pH 7.5) was added and stirring continued, after which 5 mM MgCl2 was were used to determine catalytic efficiency (kcat/KM) as described previously .To added followed by addition of DNase (Sigma-Aldrich) to a final concentration of determine the activity of the GH99 endo-a-1,2-mannosidase BT3862, the enzyme 21 8.5 mgml . The resulting solutions were then centrifuged for 45 min at 13,000g was incubated with Man-a-1,3-Man-a-1,2-Man-a-1,2-Man-1-CH3. and, in com- Similarly, cells harbouring BxGH99 were collected and resuspended in 50 mM bination with the a-1,2-mannosidase BT3990, mannose release was monitored NaH2PO4 (pH 8.0), 300 mM NaCl, and lysed by sonication. using the continuous assay described above (BT3990 is active on the product of BT3783 clarified cell lysates were purified by nickel Sepharose affinity chro- BT3862, Man-a-1,2-Man-a-1,2-Man-1-CH3). The activity of BT3862 against aryl- matography by stepwise elution with imidazole. Positive fractions identified by mannosidases was as described previously30. A strategy deploying 2-aminobenzamide SDS gel electrophoresis were dialysed into 20 mM Tris (pH 8.0) and concentrated (2-AB)-labelled glycans at the reducing end, using the Sigma GlycoProfile 2-AB to 11.25 mg ml21 with an Amicon stirred ultrafiltration unit model 8200. BT3792 labelling kit as per the manufacturer’s instructions, was used to determine the clarified cell lysates were purified using nickel-affinity chromatography, and catalytic efficiency of BT3994. The enzyme (100 nM) was incubated with 5 mM positive fractions were buffer exchanged by dialysis into 25 mM Tris-HCl (pH 8.0) Man5GlcNAc2 (generated by treating Man9GlcNAc2 with BT3990 an a-1,2- before further purification by anion exchange chromatography. Anion exchange mannosidases) or Man3GlcNAc2 (generated from Man5GlcNAc2 by treating with purified BT3792 buffered in 25 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 2 mM BT3991, a-1,3-mannosidase17) in NaP buffer for up to 1 h. Aliquots were removed dithiothreitol was concentrated using a stirred cell Amicon with a 10 kDa cutoff to at regular intervals and the amount of glycan remaining was determined using 20 mg ml21. For BxGH99 soluble lysate (isolated by centrifuging at 13,000g) was HPAEC and a fluorometric detector. A similar strategy, employing labelled glycans applied to a NiSO4-charged 5 ml HiTrap chelating column (GE Healthcare), pre- in combination with HPAEC and fluorescence detection was used to measure the equilibrated in the same buffer. Protein was eluted in an imidazole gradient, dia- endo-b-N-acetylglucosaminidase activity of BT3987 against Man9GlcNAc2. The lysed, concentrated, and further purified on an S75 16/60 gel filtration column (GE) activity of the enzyme was also evaluated against glycoproteins using matrix-assisted pre-equilibrated in25 mM Na-HEPES (pH 7.0), 100 mM NaCl, 1 mM dithiothreitol. laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry as Binding studies. Affinity gel electrophoresis was used to screen for potential described below. BT2630 and BT3783 were assayed for phosphatase activity using glycan binding proteins, following the method described in ref. 27 with the target the EnzChek Phosphate Assay kit to detect the release of phosphate from appro- polysaccharide at 1 mg ml21. The binding of proteins to their ligands was quantified priate phosphorylated sugars. Prior to these assays the two proteins were treated by isothermal titration calorimetry (ITC), as described previously27.Titrationswere with EDTA and the chelating agent was removed using a PD10 column. The assays carried out in 50 mM Na-HEPES buffer,pH 7.5 at25 uC.The reaction cellcontained were carried out in 50 mM Na-HEPES buffer (pH 7.5) containing 1 mM of MgCl2 protein at 50 mM, while the syringe contained either the oligosaccharide at 10 mM or another divalent ion. In this discontinuous assay aliquots were removed at or polysaccharide at 3–5 mg ml21. The titrations were analysed using Microcal regular intervals, the enzyme was inactivated by boiling and phosphate release Origin version 7.0 software to derive n, Ka, and DH values, while DS was calculated determined. using the standard thermodynamic equation, RTlnKa 5 DG 5 DH 2 TDS. The specificity of enzymes against phosphorylated and neutral high mannose Glycoside hydrolase and phosphatase assays. a-Mannosidase activity was deter- N-glycans was assessed by capillary electrophoresis. The methodology was essen- mined by the continuous monitoring of mannose release using a linked enzyme tially that described in ref. 31. The two substrates were MNN4 (phosphorylated assay system purchased from Megazyme International (mannose/fructose/glu- high mannose N-glycans released from glycoproteins expressed by a genetically cose detection kit). The reaction was carried out at 37 uC in 50 mM Na-HEPES modified strain of Yarrowia lipolytica) and N-glycan from RNAase B (primarily 1 buffer (pH 7.0) containing 2 mM MgCl2, 1 mM ATP and 1 mM NADP , excess high mannose N-glycans), which were released from their respective glycopro- concentrations of linker enzymes (hexokinase, phosphoglucose isomerase and teins with PNGase F. The N-glycans were labelled at the reducing end with 8- glucose-6-phosphate dehydrogenase) and 1 mg ml21 BSA. Through linker enzymes aminopyrene-1,3,6-trisulfonic acid32, and incubated overnight at 30 uC with

,2 mM of enzyme in 10 mM Na-Hepes buffer (pH 7.0) containing 2 mM CaCl2. appearance of the anomeric a- and b-proton signals. Signals were assigned by two- The reactions were analysed by capillary electrophoresis with laser-induced fluor- dimensional NMR analysis (HSQC). escence detection (CE-LIF) using an ABI 3130 capillary DNA sequencer as described Bacteroides culture and whole-cell assays. B. thetaiotaomicron was cultured previously32. anaerobically at 37 uC in minimal media containing an appropriate carbon source, Mannosyl-transferase assays. BT3775 and BT3776 at 30 mM were screened for or in TYG (tryptone yeast extract glucose medium) as described previously21. activity using mannose and all possible a-mannobiose acceptors at a concentra- Growth curves presented in the paper are averages of six biological replicates. tion of 10 mM. Reactions were performed in 10 mM Na-Hepes buffer (pH 7.5), B. thetaiotaomicron was grown in 5 ml minimal media on 0.5% w/v S. cerevisiae 10 mM MnCl2, 50 mM GDP-Mannose at 37 uC over time course intervals of mannan (Sigma) or glucose as the sole source to mid exponential phase (OD600nm 0 min, 5 min, 15 min, 30 min, 60 min and 16 h. Reactions were stopped by boiling 0.6–0.8). Cells were harvested by centrifugation, 5000g for 10 min at room tem- for 10 min. To explore the synergy between the two GT32 glycosyltransferases, perature and washed in 5 ml PBS (pH 7.1) before being resuspended in 500 mlPBS. reactions were performed with BT3775 and BT3776 simultaneously using man- Cells (50 ml) were assayed against yeast mannan (10 mg ml21)at37uC for 16 h. nose as an acceptor, and in a step-wise progression with BT3775 and mannose as Assays were analysed by thin layer chromatography, 5 ml of eachsample was spotted the acceptor, followed by BT3776 on the purified products of the initial BT3775 onto silica plates and resolved in butanol/acetic acid/water buffer. The plates were reaction. Enzyme concentrations were maintained in both conditions at 30 mM. dried and sugars visualized by orcinol/sulfuric acid heated to 70 uC. MALDI-TOF mass spectrometry of reaction products. To permethylate N-glycan Cellular localization. Cultures of B. thetaiotaomicron (100 ml) were grown in fragments and mannooligosaccharides appropriate enzyme reactions were freeze- minimal media on yeast mannan (0.5% w/v) as a sole carbon source, to mid dried and suspended in dry dimethylsulfoxide (DMSO; 200 ml). Oligoglycans were exponential growth phase (OD600nm 0.6–0.8). Cells were harvested by centrifu- per-O-methylated using standard methods33. Freshly prepared sodium hydroxide gation and washed in 10 ml PBS before being resuspended in 5 ml of the buffer. base in dry DMSO (300 ml) and then iodomethane (150 ml) was added to each The cells were split into four 1 ml aliquots. To 3 of the aliquots 2 mg ml21 Proteinase sample. The tube was purged with nitrogen and vortexed vigorously. Permethylated K was added and incubated at 37 uC for 1–16 h, the fourth sample was left as an oligosaccharides (PMOs) were extracted in 2 ml water. After removing excess untreated control also for 16 h. Following incubation with the protease the samples iodomethane (CH3I), 2 ml dichloromethane was added and vortexed to extract were centrifuged at 5000g for 10 min and the supernatant discarded. The cell lipophilic components. The aqueous layer was removed after centrifugation. This pellets were resuspended in 1 ml PBS and the proteins precipitated by the addition was repeated five more times. After the final rinse, the dichloromethane layer was of 200 ml trichloroacetic acid and incubation on ice for 30 min. The precipitated transferred into a fresh tube and dried down under N2 stream. The samples were proteins were pelleted by centrifugation and washed 4 times in 1 ml ice-cold then dehydrated and dissolved in 50% aqueous methanol and loaded onto C18 acetone. The protein pellets were resuspended in 250 ml Laemmli buffer and sub- reverse phase resin (100 mg ml21, Resprep SPE Cartridges) and eluted with acet- jected to SDS/PAGE. Gels were transferred to Whatman Protran BA 85 nitrocel- onitrile (AcN). AcN washes were dried down under N2 and dissolved in 20 ml lulose membrane. Proteins of interest were detected using anti-sera raised against methanol. Equal volumes of the sample and 2% DHBA in aqueous methanol, which the corresponding protein. The secondary antibody used was a chicken anti-rat was used as the matrix, were combined in a separate microfuge tube and loaded on conjugated to horseradish peroxidase. Antibodies were detected by chemi-lumin- the MALDI-TOF mass spectrometry plate. MALDI-TOF mass spectrometry ana- escence using Biorad Clarity Western ECL Substrate. lyses were performed with an AB4700 MS instrument (Applied Biosystems) in Immunofluorescence microscopy. B. thetaiotaomicron suspensions (OD600nm 5 positive ion, reflector mode with 100 shots taken per spectrum. PMO mass values 0.8) in PBS (pH 7.0) were applied to clean Eppendorf tubes, fixed by an equal were queried online (http://www.expasy.org/tools/glycomod/) by GlycoMod tool volume of 23 formalin (9% formaldehyde in PBS), and rocked for 90 min at 25 uC. that can predict possible oligosaccharide structures that occur on proteins from The cells were then pelleted by centrifugation for 3 min at 7000g and washed twice their experimentally determined masses. with 1 ml of PBS. The bacterial cell pellet was resuspended in 1 ml of blocking NMR analysis of glycosyltransferase reaction products. The mannooligosac- solution (2% goat serum, 0.02% NaN3 in PBS) and incubated at 4 uC for 16 h. After charide products were separated by size exclusion chromatography on a Superdex- incubation cells were centrifuged again at 7000g and the supernatant discarded. 75 HR10/30 column using a Dionex Ultimate 3000 HPLC equipped with a ShodexRI- For labelling, the bacteria were incubated with 0.5 ml of primary rat IgG (1/500 101 refractive index detector. The column was eluted with water and fractions were dilution of IgG in blocking solution) for 2 h at 25 uC. The cells were then pelleted, collected and freeze-dried. The lyophilized fraction containing the trisaccharide by centrifugation, washed in 1 ml of PBS and resuspended in 0.4 ml goat anti-rat was dissolved in D2O (0.3 ml, 99.9%; Cambridge Isotope Laboratories) and one- IgG Alexa-Fluor 488 (Sigma), diluted 1/500 in blocking solution, and incubated 1 h and two-dimensional NMR spectra were recorded at 298 K with a Varian Inova at 25 uC in the dark. The cells were then pelleted, washed with PBS and resus- NMR spectrometer operating at 600 MHz equipped with a 3 mm NMR cold probe. pended in 50 ml of PBS containing ProLong Gold antifade reagent (Life Tech- The homonuclear (gCOSY, TOCSY and NOESY) and the hetereonuclear (gHSQC nologies). Labelled bacterial cells were mounted onto glass slides and secured with and gHMBC) experiments were recorded using standard Varian pulse programs. coverslips. Fluorescence was visualized using a Leica SP2 UV microscope (Leica Chemical shifts were measured relative to internal acetone (dH 2.225). Data were Microsystems, Heidelberg, GmbH) with 363 NA 1.32 lens. Alexa-Fluor 488-labelled processed using MestReNova software (Universidad de Santiago de Compostela, bacteria were viewed under an ultraviolet microscope view and compared with Spain). Interglycosidic scalar couplings observed in the gHMBC spectra were used bright-field phase-contrast of the same image. to determine the sequence and glycosidic linkages of the mannosyl residues in the Constructing mutants of the yeast mannan and HMNG PULs in B. thetaio- a-Man-(1,3)-[a-Man-(1,6)]-Man trisaccharide product. taomicron. The inactivation of the HMNG-PUL extra-cellular factor regulator Linkage analysis of GT32 glycosyltransferase mannooligosaccharide pro- was described previously13. The other mutants deployed in this study, PUL knock- ducts. Overnight incubations were boiled for 5 min and then centrifuged to pellet outs and single gene clean deletions, were introduced by allelic exchange using the denatured protein. A 200 mg aliquot of the sample was suspended in 200 ml DMSO pExchange vector as described in ref. 35 The use of quantitative reverse transcrip- and magnetically stirred for 5 days. The sample was then permethylated accord- tase PCR to quantify appropriate transcripts followed the methods described in ing to the method in ref. 34. Two hundred microlitres of the NaOH base were ref. 13. added and, after 10 min, 100 mlCH3I were added and the sample was stirred for Gnotobiotic mice experiments. All animal experiments were approved by the 40 min. An additional 200 ml base and 100 mLCH3I were then added and stirring University Committee on Use and Care of Animals at the University of Michigan was continued for 40 min. The reaction was worked up by addition of 2 ml H2O, and were supervised by a veterinarian. Germ-free mouse experiments were con- removal of excess CH3I by sparging with N2, and CH2Cl2 extraction. The per- ducted in a total of 24, 6–8-week-old male and female Swiss Webster mice (each methylated material was hydrolyzed using 2 M trifluroacetic acid (TFA) (2 h in a was considered to be a single biological replicate in its respective experiment and sealed tube at 121 uC), reduced with NaBD4, and acetylated using Ac2O/TFA. The treatment group). Mice were randomly assigned into groups by a technician who resulting partially methylated alditol acetates were analysed on a Hewlett Packard was not familiar with the project. The investigators were not blinded to the iden- 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ion- tities of the treatment groups during the experiment and no data were excluded ization mode); separation was performed on a 30 m Supelco 2330 bonded phase from the final analysis. For in vivo gene expression studies, mice were randomly fused silica capillary column. grouped into three groups containing three animals each, and then subjected to Stereochemistry of endo-a-1,6-mannanases. A solution of BT3792 (25 mg, three different dietary regiments. Two groups were fed a gamma-irradiated custom approximately 400 mmole) in buffered D2O (0.25 ml, 50 mM phosphate-citrate, diet (glycan-free diet) that contained only glucose as the available carbohydrate as 200 mM NaCl, pD 6.0 (deuterium ion concentration is 1 mM)) was added to a well as cellulose, a polysaccharide that cannot be degraded by B. thetaiotaomicron, solution of 4-nitrophenyl a-D-mannopyranosyl-1,6-a-D-mannopyranoside (4.1 mg, as a non-digestible fibre supplement36. One of the two groups maintained on this 0.0086 mmol) in D2O (0.75 ml, 50 mM phosphate-citrate, 200 mM NaCl, pD 6.0) diet was provided with purified a-mannan (1% w/v) in drinking water (Harlan- at 22 uC. The course of the reaction was monitored by 1H NMR (500 MHz) to Teklad). The third group was fed a custom version of the glycan-free diet in which identify the stereochemistry of the reaction by analysing the relative timing of the the glucose was replaced with dried/crumbed bread (50% w/w of final diet) that

©2015 Macmillan Publishers Limited. All rights reserved RESEARCH ARTICLE had been produced using yeast as a leavening agent (Zingerman’s Bake House, SCALA38. BT3783 and BT3792 structures were solved by molecular replacement Ann Arbor, MI). Mice were pre-fed on each dietary condition for five days, colo- with the program Phaser MR40 using the coordinates of apo-BT3783 (PDB: nized with B. thetaiotaomicron by oral gavage (,108 c.f.u. per animal) and main- 3MPR) and the coordinates of Lin0763 from Listeria innocua (PDB: 3K7X). tained for an additional 5 days before euthanizing and collection of caecal contents The asymmetric unit of BT3783 and BT3792 contain four and two protein mole- for gene expression analysis. For the in vivo competition experiment, wild-type and cules, respectively. The structures were subsequently improved with cycles of mannan PUL triple mutant strains were each labelled with a unique 24 base pair manual building with COOT41 and refinement with REFMAC38. Five per cent (bp) oligonucleotide tag, which is contained in a pNBU2-based chromosomal of the reflections were flagged as ‘free’ to monitor the refinement process. The integration vector and quantifiable by qPCR, as previously described13. Each strain structure of BT3783 extends from residues 26–309 with a magnesium ion coor- was grown overnight in TYG medium and combined at approximately equal amounts dinated in the putative active centre. The structure of BT3792 runs continuously before being gavaged into mice as described above. For competition experiments, from residues 151–525. Due to a lack of continuous density corresponding to this five mice were used in each group (chosen based on the sample size used in polypeptide region, residues 274–290 are absent in the model of BT3783. BT3792 previous studies to observe significant changes in competitive index18,29) and fed structural refinement required amplitude correction for twinning. The final refined varying regimens of the three diets described above for a total period of 38 days. All structures were validated with the aid of Molprobity42. Final BT3783 and BT3792 germfree mice used in the competition experiment were pre-fed the glycan-free structures featured, respectively, 94.8 and 99.6% of all modelled residues within the diet for 1 week before colonization. Ramachandran ‘favoured’ region, with a further 4.2 and 0.4% within the ‘additional Bacteroides co-culturing experiments. B. thetaiotaomicron, B. xylanisolvens allowed’ region. NLAE-zl p352 and B. cellulosilyticus WH2 were cultured in minimal media con- Diffraction data for the BxGH99-Man-IFG-mannobiose ternary complex were taining glucose (0.5% w/v) as a carbon source to mid exponential growth phase collected at beamline I03 (l 5 0.97625 A˚ , data collected at 100 K) of the Diamond 39 43 (OD600 0.7–0.8) . Cells were collected by centrifugation and washed twice with Light Source, Didcot, UK. The data sets were processed using XDS and AIMLESS 5 ml of PBS to remove any residual glucose. The washed cells were resuspended in (also within CCP438). Phases were derived from a previously solved (native) BxGH99 5 ml of fresh minimal media with no carbon source. Minimal media (10 ml) atomic model, with initial refinement conducted using REFMAC38. Calculated containing mannose or yeast mannan as the carbon source was inoculated with electron density maps were visually assessed for evidence of ligand-binding, with equal volumes of B. thetaiotaomicron/B. xylanisolvens or B. thetaiotaomicron/B. subsequent completed atomic models, featuring appropriately assigned ligand cellulosilyticus. A control culture of minimal media containing yeast mannan was coordinates, refined to convergence through numerous cycles of REFMAC38 and also inoculated with either B. xylanisolvens or B. cellulosilyticus. All cultures were additional manual correction using COOT41. The final BxGH99 ternary structure set up in triplicate. Samples (1 ml) were taken at the point of inoculation, early featured, respectively, 96.5 and 96.9% of all modelled residues within the Ramachandran exponential, late exponential and stationary phase of growth. Serial dilutions of ‘favoured’ region, with a further 2.3 and 1.9% within the ‘additional allowed’ region. each sample were plated on to rich media and incubated for 2 days before colony Data collection and refinement statistics for all structures are presented in Sup- counts were recorded. Proportions of B. thetaiotaomicron, B. xylanisolvens and plementary Table 6. B.cellulosilyticus per sample were determined by quantitative PCR from genomic Experimental group size and statistical analysis of data. For all quantitative DNA using unique marker genes for each strain. enzyme assays at least three technical replicates were performed unless specif- Searches of human gut metagenomic data sets for mannan PULs. A search of ically stated in the Table legends. Standard errors of the mean are stated for kinetic 250 different healthy and diseased (Crohn’s disease and ulcerative colitis) human parameters in Supplementary Tables 2–4. For Supplementary Table 5 the kinetic metagenomic samples was conducted exactly as described previously36. Probes parameters are reported 6 the error of the fit of the data using linear regression. corresponding to the three B. thetaiotaomicron mannan PULs were first checked For isothermal titration calorimetry experiments three technical replicates were against database of complete and draft microbial genome sequences to identify performed for each titration. Standard errors of the mean are shown in Sup- regions that were present in species other than B. thetaiotaomicron, or the porcine plementary Table 5. In the gnotobiotic mice experiments displayed in Fig. 2a five B. xylanisolvens strains in the case of MAN-PUL1. In the case of MAN-PUL2 and mice were assigned to each diet and the error bars for the two bacterial strains MAN-PUL3, the entire sequence was determined to be specific for sequenced represent standard errors of the mean. Statistical significance of the persistence of B. thetaiotaomicron isolates; whereas, MAN-PUL1 was trimmed to a region con- different strains of B. thetaiotaomicron in the same mice was analysed by unpaired taining 15.059 kilobases (kb) that were unique to B. thetaiotaomicron and porcine Student’s t-test. For the microbial sharing experiment detailed in Fig. 4 the error B. xylanisolvens. The three probes used correspond to the following base pair coor- bars represent standard deviations of the mean from three biological replicates. dinates in the B. thetaiotaomicron VPI-5482 genome sequences: MAN-PUL1 25. Charnock, S. J. et al. Key residues in subsite F play a critical role in the activity (3262908..3277966), MAN-PUL2 (4893415..4928241) and MAN-PUL3 (5012915.. of Pseudomonas fluorescens subspecies cellulosa xylanase A against 5031551). xylooligosaccharides but not against highly polymeric substrates such as xylan. Crystallization. BT3783 and BT3792 were crystallized using the sitting drop vapour J. Biol. Chem. 272, 2942–2951 (1997). diffusion methodat 18 uC. BT3783 crystals were obtained by mixing equal volumes 26. Studier, F. W. Protein production by auto-induction in high density shaking of purified, recombinant BT3783 protein at a concentration of 11.25 mg ml21 with cultures. Protein Expr. Purif. 41, 207–234 (2005). 27. Szabo, L. et al. Structure of a family 15 carbohydrate-binding module in complex mother liquor solution consisting of 15% (w/v) polytheylene glycol 4,000, 5% (1/-)- with xylopentaose. Evidence that xylan binds in an approximate 3-fold helical 2-methyl-2,4-pentanediol (v/v), 3 mM D-mannose, and 200 mM MgCl2. Crystals conformation. J. Biol. Chem. 276, 49061–49065 (2001). of BT3792 were obtained by mixing equal volumes of purified recombinant BT3792 28. Miller, G. L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. at a concentration of 20 mg ml21 buffered in in 25 mM Tris-HCl (pH 8.0), 500 mM Anal. Chem. 31, 426–428 (1959). NaCl, 2 mM dithiothreitol with a reservoir solution consisting of 5% glycerol (v/v), 29. Charnock, S. J. et al. The topology of the substrate binding clefts of glycosyl 24% (w/v) polyethylene glycol monomethyl ether (average M 2,000), 0.25 M sodium hydrolase family 10 xylanases are not conserved. J. Biol. Chem. 273, w 32187–32199 (1998). acetate, and Bis-Tris-HCl (pH 5.5). Large, thin plate crystals of BT3792 developed 30. Thompson, A. J. et al. Structural and mechanistic insight into N-glycan processing over a period of 5 days to a week. BT3792 crystals were cryoprotected in crystallization by endo-a-mannosidase. Proc. Natl Acad. Sci. USA 109, 781–786 (2012). solutions supplemented with 25% ethylene glycol and cryo-cooled directly in a N2 31. Stewart, T. S., Mendershausen, P. B. & Ballou, C. E. Preparation of a stream at 2160 uC before diffraction experiments. mannopentaose, mannohexaose, and mannoheptaose from Saccharomyces BxGH99 crystals were grown at 19 uC using hanging-drop vapour diffusion cerevisiae mannan. Biochemistry 7, 1843–1854 (1968). 21 32. Laroy, W., Contreras, R. & Callewaert, N. Glycome mapping on DNA sequencing with equal volumes of protein (30 mg ml ) and reservoir solution consisting of equipment. Nature Protocols 1, 397–405 (2006). 0.1 M sodium acetate pH 5.1, 20% w/v polyethylene glycol monomethyl ether 33. Ciucanu, I. Per-O-methylation reaction for structural analysis of carbohydrates by (average Mw 2,000), 2.0% low molecular weight poly-c-glutamic acid. Ligand mass spectrometry. Anal. Chim. Acta 576, 147–155 (2006). complex formation as a ternary with Man-isofagamine and a-1,2-mannobiose, 34. Anumula, K. R. & Taylor, P. B. A comprehensive procedure for preparation of was achieved by soaking native BxGH99 crystals in mother liquor supplemented partially methylated alditol acetates from glycoprotein carbohydrates. Anal. Biochem. 203, 101–108 (1992). with approximately 10 mM of respective ligand solutions for a period of 30 min 35. Koropatkin, N. M., Martens, E. C., Gordon, J. I. & Smith, T. J. Starch catabolism by a before flash cooling in liquid N2. Crystals were cryo-protected by the stepwise prominent human gut symbiont is directed by the recognition of amylose helices. addition of ethylene glycol, supplemented with appropriate ligand(s), to a final Structure 16, 1105–1115 (2008). concentration of 20% v/v. 36. Larsbrink, J. et al. A discrete genetic locus confers xyloglucan metabolism in select Data collection, structure solution and refinement. Diffraction data from both human gut Bacteroidetes. Nature 506, 498–502 (2014). BT3783 and BT3792 crystals were collected at the Canadian Lightsource beam- 37. Leslie, A. W. & Powell, H. in Evolving Methods for Macromolecular Crystallography ˚ (eds Read, J. R. & Sussman, J. L.) Ch. 4, 41–51 (Springer, 2007). line 08B1-1 (CMCF-BM) in oscillation mode (l 5 0.97961 and 0.98005 A, with 38. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta data collected at 100 K, respectively). The BT3783 and BT3792 data sets were Crystallogr. D 67, 235–242 (2011). processed with iMosflm37,38 and XDS39, respectively, and both were scaled with 39. Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010).

40. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 42. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular (2007). crystallography. Acta Crystallogr. D 66, 12–21 (2010). 41. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of 43. Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Coot. Acta Crystallogr. D 66, 486–501 (2010). Acta Crystallogr. D 69, 1204–1214 (2013).

Extended Data Figure 1 | The role of specific B. thetaiotaomicron PULs and e, Enzymes at 1 mMat37uC were incubated with either undecorated a-1,6- enzymes in utilization of mannan from S. cerevisiae and other yeast species. mannan (derived from mnn2 mutant of S. cerevisiae) (lanes 1–3) or mannan a, Growth of wild-type B. thetaiotaomicron on Candida albicans mannan and from S. pombe (lanes 4–9). Lanes 1 and 4, the mannans incubated in the absence glucose. b, Growth of wild-type B. thetaiotaomicron and the mutant lacking of the enzymes; lanes 2 and 6, mannans incubated with the periplasmic MAN-PUL1 and MAN-PUL3 (DMAN-PUL1/3) on Schizosaccharomyces mannanase BT3782; lanes 3 and 7, mannans incubated with the surface pombe a-mannan. c, Growth of wild-type B. thetaiotaomicron, and the mannanase BT3792; lane 5, S. pombe mannan incubated with the GH97 B. thetaiotaomicron mutants lacking MAN-PUL2 (DMAN-PUL2), or all three a-galactosidase BT2620; lanes 8 and 9, S. pombe mannan incubated with mannan PULs (DMAN-PUL1/2/3) on S. cerevisiae a-mannan. d, The growth BT2620/BT3782 and BT2620/BT3792, respectively. Lane 10 galactose profile of wild-type B. thetaiotaomicron and the B. thetaiotaomicron mutant standard; lane 11 a-1,6-mannooligosaccharides: mannose (M1), mannobiose lacking bt3774 (Dbt3774)onS. cerevisiae mannan. In panels a, b, c and d, each (M2), mannotriose (M3) and mannotetraose (M4). point on the growth curve represents the mean of three biological replicates.

700 700 Man1,3 Man

600 BT3775 600 BT3775

500 BT3775+BT3776 500 BT3775+BT3776

400 400

300 300 Signal (nC) Signal Signal (nC) Signal

200 200

100 100

0 0 156 174 192 210 228 246 264 282 300 318 336 354 372 390 408 426 444 462 480 498 516 534 552 570 588 606 624 642 660 678 696 714 732 750 768 786 804 156 175 194 213 232 251 270 289 308 327 346 365 384 403 422 441 460 479 498 517 536 555 574 593 612 631 650 669 688 707 726 745 764 783 802 Retention time (sec) Retention time (sec)

e f α-Man-(1→3)-Man α-Man-(1→3)-[α -Man-(1→6)]-Man

T-α-Man-(1→3)- α -Manr T-α-Man-(1→3)- 2 β α -Manr -Manr T-α-Man-(1→6)- 1 2 β -Manr 3 1 4 3

5.20 5.15 5.10 5.05 5.00 4.95 4.90 4.85 5.20 5.15 5.10 5.05 5.00 4.95 4.90 4.85 ppm ppm gh α3 Terminal Mannose Terminal Mannose

1600000 3-linked Mannose 1.4e+07

1400000 1.2e+07

1200000 1e+07

1000000 α3 α6 8000000 800000

Abundance 6000000 Abundance 600000 4000000 3,6-linked Mannose 400000

2000000 200000

9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 Time Time Extended Data Figure 2 | Product profiling of GT32 glycosyltransferases correspond to the a-anomer and b-anomer of the mannose at the reducing end, encoded by MAN-PUL2. a, HPAEC of biosynthetic reactions using mannose respectively; peak 2 corresponds to the terminal a-mannosyl residue linked to as an acceptor and GDP-a-Man as the donor. Mannobiose is formed in the O3 of the mannose at the reducing end. f, NMR analysis of the a-1,3,( a-1,6)- presence of BT3775 (black) and mannotriose with BT3775 and BT3776 (red). mannotriose BT3776 product. The numbering of the peaks are the same as The blue trace is a mannose standard. b, HPAEC of biosynthetic reactions with in e. Peak 4 corresponds to the terminal a-mannosyl residue linked to O6 of the a-1,3-mannobiose as the acceptor and GDP-a-Man as the donor. BT3775 is 3,6-linked mannose at the reducing end. g, Alditol-acetate linkage analysis of not capable of extending mannobiose (black). In the presence of BT3775 and mannobiose produced by BT3775 from mannose. h, Alditol-acetate linkage BT3776 mannotriose is produced (red). The blue trace is an a-1,3-mannobiose analysis of branched (a-1,3),(a-1,6)-mannotriose produced by BT3776 from standard. c, d, MALDI-TOF analysis of the reaction products of a-1,3-mannobiose. The green circles indicate the mannose residues present in BT3775 1 BT3776 using mannose (c) and a-1,3-mannobiose (d)asan carbohydrates identified by HPAEC, MALDI-TOF and NMR. acceptor. e, NMR analysis of the a-1,3-mannobiose substrate. Peaks 1 and 3

BT3792 GH76 endo-α1,6-mannanase

a b

D258

D259

BT3862 GH99 endo-α 1,2-mannosidase cd R295/R291

E333/E329

-2 -1 +2

E336/E332

H63/H64

W126W126/W125W125

BT3781 GH125 exo- α 1,6-mannosidase

R109/R64 E174/E129 W107/W62 D110/D65 K176/K131 R234/R188

E174/E129 N348/N302

D241/D195 R481/R405

H396/H350 E439/E393

BT3783 mannose-6-phosphatase g h

F214/W307

D280/H359

D178/D272

E51/E162

H142/H236

Extended Data Figure 3 | The structures of enzymes that play a key role in 12 subsite are coloured bright green. e, Overlay of BT3781 (green; PDB code yeast mannan degradation. a, Overlay of the hydrophobic conserved residues 2P0V) with the substrate and catalytic residues of the Clostridium perfringens in the predicted substrate-binding cleft of BT3792 (yellow), BT2949 (cyan) and GH125 a-mannosidase CpGH125 (cyan; PDB code 3QT9), in which the the Listeria protein Lin0763 (green; PDB code 3K7X), and the predicted ligand 6-S-a-D-mannopyranosyl-6-thio-a-D-mannopyranose (Man-S-Man) is catalytic aspartates. b, Solvent representation of BT3792 in which the predicted shown in yellow. f, Solvent-exposed surface of BT3781 in the vicinity of the catalytic residues, Asp258 and Asp259, are coloured green. c, Overlay of active site in which the catalytic residues (Glu 174 and Glu 439) are depicted BT3862 (cyan) with a homologue of the enzyme from B. xylanisolvens, BxGH99 in green. The position of Man-S-Man is based on the overlay shown in e. (green; PDB code 4UTF) in complex with Man-a-1,3-isofagomine and a-1,2- g, Overlay of BT3783 with the catalytic and substrate binding residues of a mannobiose (Man residues coloured yellow and isofagomine pink). d, Solvent- tyrosyl-DNA phosphodiesterase (PDB code 4GYZ) in complex with Mg21 exposed surface of the substrate binding cleft of the BxGH99 (teal) ligand (slate-blue sphere) and phosphate (coloured orange). h, A region of the solvent- complex overlaid with BT3862 (grey). The subsites are numbered with the accessible surface of BT3783 in which the catalytic residues are coloured green. catalytic residues, Glu 333 and Glu 336, coloured red and the solvent exposed The figure was prepared using PyMOL. A detailed description of the structures O2 of Man bound at the 22 subsite and O1 and O6 of the Man located at the of these proteins is provided in Supplementary Information section 5.0.

a b GH92 GH92/GH76 M1 M1 M2 M2 M3 M3 M4 M4

1 2 3 1 2 3

132 c d

2.0 M1 M2M

M2 M3M M4M4 1.5 M3 M4 1.0

0.5 Absorbance 600 nm 600 Absorbance

0.0 0h 2h 6h 8h 10h 12h 4h 24h 31h 36h 48h 0 10 20 30 40 50 60 Time (h)

SH1α e f

M1 0 min

P PH1α H1’α M3 8 min M4

13 min

18 min 1 2 3 4

P H1’β PH1β 30 min

1 day

HO HO OH HO OH OH HO O HO O HO O HO H1' HO H1' GH76 HO H1' O OH O OH O OH α O O endo- -1,6-mannosidase O HO HO HO HO OH HO H1 Bt3792 HO H1 H OpNP OH 1 substrate, S product product α-anomer (P-α) β-anomer (P-β)

Extended Data Figure 4 | The degradation of yeast mannan by were analysed by TLC. In all panels the samples were chromatographed with B. thetaiotaomicron in culture and the selected enzymes expressed by the the following a-1,6-mannooligosaccharides: mannose, M1; mannobiose, M2; bacterium, and the stereochemistry of the reaction catalysed by GH76 endo- mannotriose, M3; mannotetraose, M4. d, The absorbance of the culture used a-1,6-mannanases. a, GH92 a-mannosidases at high concentrations (50 mM) in c. e, BT3792 (GH76) endo-a-1,6-mannosidase is a retaining glycoside were incubated with yeast mannan for 5 h in the absence (labelled GH92) hydrolase. Enzymatic hydrolysis of 4-nitrophenyl a-D-mannopyranosyl-1,6-a- 1 or in the presence (GH92/GH76) of the endo-a-1,6-mannanase BT3782. D-mannopyranoside (S) was monitored by H-NMR spectroscopy (500 MHz). The GH92 a-mannosidases in this example were BT2199 (1), BT2130 The stacked spectra show the reaction progress over time. SH1a is the anomeric (2) and BT3773 (3). The GH76 endo-a-1,6-mannanase only releases proton of the reducing end mannopyranoside of the substrate, and SH19a is the mannooligosaccharides in the presence of BT2199; see also Supplementary anomeric proton of the non-reducing end mannopyranoside. The reaction Information section 4.1. b, B. thetaiotaomicron was grown on yeast proceeds with the initial formation of the product, the a-anomer of a-1,6- mannan or glucose. Yeast mannan was incubated with no bacterium (1), mannobiose (P-a, peaks PH1a and PH19a), which slowly mutarotates to the B. thetaiotaomicron previously cultured on yeast mannan (2) and b-anomer (P-b, peaks PH1b and PH19b). f, TLC analysis of S. cerevisiae mannan B. thetaiotaomicron grown on glucose (3). The cells were incubated for 5 h at incubated without enzyme (lane 1), BT3774 (lane 2), BT3792 (lane 3), and 37 uC with the polysaccharide without a nitrogen source and thus were not BT3774 and BT3792 (lane 4). M1–M4 are a-mannnooligosaccharide standards growing. The products released by the B. thetaiotaomicron cells, analysed by numbered according to their d.p. GH76 mannanase BT3792 does not attack the TLC, were mediated by the activity of enzymes presented on the surface of backbone of S. cerevisiae mannan unless the side chains are first removed by B. thetaiotaomicron, and not through the action of periplasmic mannanases the GH38 a-mannosidase BT3774, confirming that this enzyme cleaves the and mannosidases. The black box highlights very low levels of high molecular mannose a-1,2-linked to the mannan backbone. The data in a, b and c are weight mannooligosaccharides generated by the cells incubated in yeast representative of two biological replicates, while the data in f are representative mannan. c, B. thetaiotaomicron was cultured for up to 48 h (stationary phase) of two technical replicates. on yeast mannan. The supernatant of the culture at the time points indicated

1.4 1 1.3 native mannan P Enzyme Activity (μmoles 1.2 B. theta 6 mannose min-1) 1.1 1.0 B. ovatus 0.9 B. xylanisolvens BT3774 0.09 0.8 0.7 1 BT3784 0.22 0.6 P 0.5

6 BT2629 0.067 Absorbance (600 nm) 0.4 Wild-type mannan 0.3 BT3958 ND 0.2 0.1 0.0 -0.1 0 500 1000 1500 2000 2500 3000 Time (min)

1 1.4 P 1.3 Mnn1 mannan 6 1.2 Enzyme Activity (μmoles 1.1 mannose min-1) 1.0 0.9 0.8 1 BT3774 0.28 0.7 P 0.6 6 BT3784 0.46 0.5

mnn1 Absorbance (600 nm) 0.4 BT2629 0.17 0.3 0.2 BT3958 ND 0.1 0.0 -0.1 0 500 1000 1500 2000 2500 3000 Time (min)

1 1.4 P 1.3 Mnn5 mannan 6 Enzyme Activity (μmoles 1.2 1.1 -1 mannose min ) 1.0 0.9 BT3774 0.44 0.8 1 0.7 P BT3784 0.061 0.6 6 0.5

BT2629 0.021 Absorbance (600 nm) 0.4 mnn5 0.3 0.2 BT3958 ND 0.1 0.0 -0.1 0 500 1000 1500 2000 2500 3000 Time (min)

1.4 1.3 Mnn2 mannan 1.2 Enzyme Activity (μmoles 1.1 mannose min-1) 1.0 0.9 BT3774 ND 0.8 mnn2 0.7 0.6 α-1,2 BT3784 ND 0.5

α-1,3 Absorbance (600 nm) 0.4 α-1,6 BT2629 ND 0.3 0.2 β-1,4 BT3958 ND 0.1 Mannose 0.0 -0.1 GlcNAc 0 500 1000 1500 2000 2500 3000 P Phosphate Time (min)

Extended Data Figure 5 | The activity of periplasmic a-mannosidases and (black), B. ovatus (red) and B. xylanisolvens (blue) on the glycans (each point the growth of different species of Bacteroides against yeast mannan. represents the mean growth of 3 separate cultures 6 s.d.). The porcine-derived Structures of the mannans derived from wild-type and mutants of S. cerevisiae. B. xylanisolvens strain shown here acquired MAN-PUL1 by lateral gene The tables adjacent to the different yeast structures depict the initial rate of transfer (Extended Data Fig. 9), explaining its capacity to degrade processed mannan hydrolysis by the four enzymes. The growth curves adjacent to the mannans. Vertical error bars represent standard deviation of three separate different mannan structures show the growth profile of B. thetaiotaomicron replicates in each condition.

a IP M1 M2 M3 M4

2.0 60 min

48 min 1.5 ) t

36 min /S

0 1.0 24 min ln (S ln 12 min 0.5 0 min 0.0 0 10 20 30 40 50 60 70 Time (min)

b IP M1 M2 M3 M4 3.5 3.0 60 min 2.5 ) 48 min t /S 2.0 0 36 min 1.5 ln (S ln 24 min 1.0 12 min 0.5

0 min 0.0 0 10 20 30 40 50 60 70 Time (min)

d c M1 M1

M2 M2

M3 M3

M4 M4

No enzyme BT3792 BT3782 (Surface) (Periplasmic) No enzyme BT2632 BT3781 Mannose (100 μM) e

Untreated mnn2 mannan

BT2632 vs mnn2 mannan

BT3781 vs mnn2 mannan

Extended Data Figure 6 | The activity of GH76 a-mannanases and GH125 50 mM sodium phosphate buffer, pH 7.0. The limit products were analysed by a-mannosidases a, b, BT3792 and BT3782, respectively, were incubated with TLC. a-1,6-Mannooligosaccharides are identified by their degree of = a-1,6-mannotetraose at a concentration KM. Substrate depletion was polymerization (M1, mannose; M2, mannobiose; M3, mannotriose; M4, measured using HPAEC and the rate (right of a and b) enabled kcat/KM to be mannotetraose); IP, injection peak. d, e, BT2632 and BT3781 at 100 nM were determined. c, BT3792 and BT3782 were incubated with unbranched yeast incubated with 1 mg ml21 of the debranched mannan for 1 h in the buffer mannan (derived from the S. cerevisiae mutant MNN2). The yeast mannan at described above. TLC analysis (d) and HPAEC traces (e) of the reactions are 0.1% was incubated with the two GH76 a-1,6-mannanases for 1 h at 37 uCin shown. The data in c and d are representative of two technical replicates.

a BT3992 BT3983 BT3984 BT3985 BT3986 BT3987 BT3988 BT3989 BT3990 BT3991 BT3993 BT3994 GH18 GH92 GH92 anti-s ECF-s GH92

SGBP Glycoside hydrolase Regulator SusD-like SusC-like Unknown

b 100 nM BT3994 + Man GlcNAc -AB 10 μM BT3994 + Man -AB

60 min 60 min 40 min 40 min 30 min 30 min 20 min 20 min 10 min 10 min 0 min 0 min

c 1

d 0h 16h RP

BT3990

2 Man GlcNAc 5 Man GlcNAc 6 g

0.6 WT Bt Δbt3993

0.5

0.4

0.3 e f 0.2 Absorbance (600 nm)

0.1

0.0 0 250 500 750 1000 1250 1500 1750 2000 2250 2500 Time (minutes)

Extended Data Figure 7 | HMNG deconstruction by B. thetaiotaomicron. with proteinase K (0 h) or incubated with 2 mg ml21 proteinase K for 16 h a, Structure of the HMNG PUL. Genes drawn to scale with their orientation (16 h). The lane labelled RP contained a purified recombinant form of BT3990. indicated. Genes encoding known or predicted functionalities are colour-coded The blots were probed with antibodies against BT3990. The data in d are and, where appropriate, are also annotated according to their CAZy glycoside representative of two biological replicates. e, f, Representative isothermal hydrolase (GH) family number. SGBP represents a surface glycan binding calorimetry titrations for BT3984 titrated with Gal-b1,4-GlcNAc (LacNAc; protein. b, BT3994 was incubated with a-1,6-mannotetraose (Man4)orthe 25 mM) (e), and for BT3986 titrated with mannose (50 mM) (f). The top half of high mannose N-glycan Man5GlcNAc2, with both oligosaccharides labelled each panel shows the raw isothermal calorimetry titration heats; the bottom with 2-aminobenzamide (AB). At the indicated time points aliquots were half, the integrated peak areas fitted using a single binding model by MicroCal removed and analysed by HPAEC using a fluorescence detection system. While Origin software. ITC was carried out in 50 mM Na-HEPES, pH 7.5 at 25 uC. Man5GlcNac2-AB was hydrolysed by BT3994, the enzyme was not active The affinities and thermodynamic parameters of binding are shown in against Man4-AB. c, Chicken ovalbumin was incubated with buffer (1) or 1 mM Supplementary Table 5. g, Growth profile of wild-type B. thetaiotaomicron of BT3987 (2) in 20 mM Na-HEPES buffer, pH 7.5, for 5 h at 37 uC, and the (WT Bt; black) and the mutant Dbt3993 (red), which lacks the extra-cellular soluble material was permethylated and analysed by MALDI-TOF mass factor s regulator gene of HMNG-PUL, cultured on Man8GlcNAc2 (each curve spectrometry. The high mannose N-glycans released are labelled. d, Western shows the mean 6 s.d. of 3 separate cultures). blot of B. thetaiotaomicron cells cultured on yeast mannan that were untreated

Extended Data Figure 8 | Metagenomic analysis of the occurrence of the in a single individual. The leftmost column summarizes the total mannan PUL yeast mannan PULs in humans. Abundance of Bacteroides mannan PULs in content in each person (annotated according to the colour key in the upper humans from a survey of metagenomic sequencing data from a total of 250 right corner). The mannan PUL frequency across all 250 samples is shown at adult human samples (211 healthy, 27 ulcerative colitis, 12 Crohn’s disease; see the bottom for each condition and is compared to the frequency of several other Methods for references). Data sets were individually queried by BLAST PULs implicated in xyloglucan and porphyran utilization. Graph at far right using either each entire mannan PUL (PULs 2,3) or a sub-fragment that was illustrates the variation in sequencing depth for each sample/study; black lines trimmed to eliminate cross-detection of other species genomes beyond show the average depth in megabase pairs (Mbp) for each study; the light grey B. thetaiotaomicron and porcine B. xylanisolvens (PUL1; see Methods for line shows the depth for each individual sample. additional search details). Each horizontal line represents the presence of a hit

Extended Data Figure 9 | Presence of a conjugative transposon (cTn) that porcine B. xylanisolvens strains. Although the draft genomes of the B. contains MAN-PUL1 in the genomes of porcine B. xylanisolvens strains xylanisolvens strains contains gaps in all five assemblies at the left side of the and the mannan presented to these organisms. a, Shown across the top is a MAN-PUL1, the right side insertion site was resolved in all genomes, schematic of a cTn that has been integrated into the 39 end of a tRNAphe gene suggesting that the B. xylanisolvens loci were also transferred by lateral transfer in B. thetaiotaomicron strain VPI-5482. Integration is mediated by a 22 bp at some point in the history of these strains. b, Forty-three different strains direct repeat sequence that is contained in tRNAphe and repeated again at the from five Bacteroidetes isolated from animal guts (each indicated with a solid other side of the cTn (right insertion site). The location of B. thetaiotaomicron circle) were inoculated into minimal media containing S. cerevisiae mannan as MAN-PUL1 is denoted within the larger cTn element using a colour scheme the sole carbon source. The growth of the cultures was measured over 48 h identical to Fig. 1a. The lower panel shows an expanded view of the MAN- by recording the optical density at 600 nm. c, TLC analysis of the products PUL1 locus in five sequenced strains of B. xylanisolvens from the faeces of pigs generated by incubating BT3774 and BT3792 with a-mannan extracted from fed a diet enriched with distillers grains that were fermented with yeast. the distillers grain fed to the pigs from which the B. xylanisolvens were isolated. A nearly identical copy (both by amino acid homology and syntenic The data are representative of two technical replicates. organization) of this genomic region is present in B. thetaiotaomicron and the

Extended Data Figure 10 | In vivo and in vitro expression of the mannan mannan in the media after 30 min exposure. The prototypic susC (BT3701) PULs. a, Level of susC-like transcripts derived from MAN-PUL1 (BT2626), involved in starch metabolism is shown for comparison. c, An identical MAN-PUL2 (BT3788) and MAN-PUL3 (BT3854) from B. thetaiotaomicron exposure experiment to that shown in b, except that glucose-grown in monocolonized gnotobiotic mice fed a glycan-free diet deficient in B. thetaiotaomicron cells were exposed to aqueous extracts of the cereal grain B. thetaiotaomicron-digestible glycans (red), the same diet with added yeast diet (natural diet) fed to mice before the experiment, or the digestible glycan- mannan (1% w/v in drinking water) as the only usable polysaccharide (green), free control diet (glycan-free diet) used as a base for all feeding treatments. and a diet containing 50% bread (blue). The levels of the susC transcripts were Exposure was conducted for 30 min to determine if any diet extract contained quantified relative to the same mRNA species in B. thetaiotaomicron cultured contaminating levels of mannan that could be detected by B. thetaiotaomicron in vitro on glucose minimal medium (MM-G). Note that in all cases, expression cells; inclusion of purified mannan (5 mg ml21) in addition to the of MAN-PUL genes is equally high in vivo. b, Transcription of the same glycan-free diet served as positive controls. In all panels, the results represent mannan susC-like genes in response to increasing concentrations of yeast the mean of 3 biological replicates and error bars represent s.d.

CORRIGENDUM doi:10.1038/nature14334 Corrigendum: Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism Fiona Cuskin, Elisabeth C. Lowe, Max J. Temple, Yanping Zhu, Elizabeth A. Cameron, Nicholas A. Pudlo, Nathan T. Porter, Karthik Urs, Andrew J. Thompson, Alan Cartmell, Artur Rogowski, Brian S. Hamilton, Rui Chen, Thomas J. Tolbert, Kathleen Piens, Debby Bracke, Wouter Vervecken, Zalihe Hakki, Gaetano Speciale, Jose L. Muno¯z-Muno¯z, Andrew Day, Maria J. Pen˜a, Richard McLean, Michael D. Suits, Alisdair B. Boraston, Todd Atherly, Cherie J. Ziemer, Spencer J. Williams, Gideon J. Davies, D. Wade Abbott, Eric C. Martens & Harry J. Gilbert

Nature 517, 165–169 (2015); doi:10.1038/nature13995 In this Article focusing on the selfish metabolism of yeast mannan by Bacteroidetes, we alsodescribed a polysaccharide utilizationlocus (PUL) responsible for the degradation of high mannose mammalian N-glycan (HMNG) but omitted to cite two relevant papers1,2, for which we apol- ogise. Both studies describe a model for the degradation of complex biantennary N-glycans by Bacteroidetes in which the degradative enzymes are encoded by PULs. These studies1,2 provide examples of how PULs can orchestrate N-glycan metabolism in addition to the HMNG PUL wedescribe in thisArticle. Inallthree papers itisproposed that N-glycan depolymerization occurs primarily in the periplasm.

1. Renzi, F. et al. The N-glycan glycoprotein deglycosylation complex (Gpd) from Capnocytophaga canimorsus deglycosylates human IgG. PLoS Pathog. 7, e1002118 (2011). 2. Nihira, T. et al. Discovery of b-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase involved in the metabolism of N-glycans. J. Biol. Chem. 288, 27366–27374 (2013).