We selected 12/14 bacterial species ( Figure 1 A) from the list of the most common/frequent 75−89 species in the human gut (). Moreover, the selection of 14 species was based in part on carbohydrate utilization abilities of a larger pool of ∼350 strains that were screened on the same platform as shown in Figure 1 A (K. Urs, N.A.P., and E.C.M., unpublished data). Based on the in vitro assays, our synthetic microbiota (SM) is not biased toward mucin-degrading bacteria, as only 4/14 species possess this ability ( Figure 1 A).

A kanamycin (Km)-resistant wild-type C. rodentium strain (DBS120) and a luciferase-expressing strain of C. rodentium (DBS100; resistant to ampicillin (Amp)) were used (). Each mouse was gavaged with 0.2 mL of culture grown aerobically overnight at 37°C (∼10CFU grown in Luria-Bertani broth without antibiotics). The exact same culture was used to gavage all mice in a single experiment to rule out effects of growth variation on pathogenesis. For experiments with luciferase expressing C. rodentium, mice were fed the FF diet for nearly the same duration (39 days instead of 42) as for the other experiment (Experiment 2B, Figure 5 ), prior to infecting them with C. rodentium. For the experiment with germfree (GF) mice, two groups of mice were separately pre-fed the FR and FF diets for ∼4 weeks prior to infection with luciferase-expressing C. rodentium or the wild-type C. rodentium.

On day 14 after colonization ( Figure 1 B), mice were randomly assigned to groups by a technician, who was not aware of the details of the treatment groups. The mice were sometimes caged separately even within individual groups. For dietary oscillations, mice from their respective cage were transferred to a different cage with another diet. Bedding was replaced in each cage before the mice were transferred. To minimize the potential for circadian effects, the oscillation was carried out at nearly the same time of the day (±1.0 hr between different days) and fecal samples were collected just prior to their transfer to another cage containing a different diet. The fecal samples were immediately stored at −20°C until further use.

Fiber-free (FF) and Prebiotic (Pre) diets were sterilized by gamma irradiation and the Fiber-rich (FR) diet (LabDiet 5010; autoclavable rodent diet) was sterilized by autoclaving. The FF diet was manufactured by Teklad/Envigo (WI, USA) and, as previously described (TD.140343) (), consisted of a modified version of Harlan TD.08810 in which starch and maldodextrin were replaced with glucose. The Pre diet was a new formulation based on the FF diet with 2.1% of a purified polysaccharide mixture ( Table S1 ) added along with 10% cornstarch (each replacing an equivalent amount of glucose).

Identities and culture purity of the bacterial species in the synthetic gut microbiota were confirmed by sequencing their 16S rRNA genes, followed by comparison to sequences in public databases. Bacteria were grown in their respective media ( Table S1 ) for community assembly or in vitro growth evaluation on carbohydrates. Each individual bacterial member of the SM was grown anaerobically (atmosphere 85% N, 10% H, 5% CO) in its respective medium ( Table S1 ) at 37°C with final absorbance (600nm) readings ranging from about 0.5 to 1.0. Bacterial cultures were mixed in equal volumes and each individual inoculum sealed in its own tube with anaerobic headspace. Each mouse was gavaged with 0.2 mL of this mixture (freshly prepared each day) for three consecutive days at nearly the same time of the day.

All animal experiments followed protocols approved by the University of Michigan, University Committee for the Use and Care of Animals. Germfree male and female wild-type Swiss Webster mice were colonized at 8−9 weeks of age and none of these mice were involved in any previous experiments/treatments. Mice were housed alone or in groups as appropriate for gender, litter and diet requirements and provided ad libitum with autoclaved distilled water and the diets described below.

Method Details

Experimental Design A total of four gnotobiotic animal experiments (Experiments 1−4; also mentioned in figure legends) were performed – details of the experimental replication are provided in the corresponding figure legends. Both male and female mice were randomly used depending on the availability of animals. Gnotobiotic Experiment 1 contained 2 male mice in Fiber-rich (FR) group, 2 male mice in Fiber-free (FF) group and 1 male mouse in Prebiotic (Pre) group; all other animals in Gnotobiotic Experiment 1 were females. Gnotobiotic Experiment 2A and 2B had all male mice. All animals in Gnotobiotic Experiment 3 were females. Gender details of the animals in gnotobiotic Experiment 4 are shown in Figure 6 (both males and females were used). For infection with wild-type C. rodentium in germfree (GF) mice, all male mice were used. Gender details of GF mice used for infection with luciferase-expressing C. rodentium are included in Figure S7 (both males and females were used). Finally, all GF mice used for measurement of the colonic mucus layer ( Figure 4 C) were females. The researchers were not blinded to the identities of the treatment groups; however, the technician who assigned individual gnotobiotic animals to different treatment groups was not aware of the experimental details. Measurements of the colonic mucus layer were single blinded (see details below in the relevant section). The pathologist who devised the inflammation-scoring rubric was not blinded, and the pathologist who performed the histology scoring and the technician who performed electron microscopy were blinded for the identities of the treatment groups (see below for details of the methods). No data were excluded from the final analysis. Kamada et al., 2012 Kamada N.

Kim Y.-G.

Sham H.P.

Vallance B.A.

Puente J.L.

Martens E.C.

Núñez G. Regulated virulence controls the ability of a pathogen to compete with the gut microbiota. Sample size estimations were performed as follows in consultation with a statistician. Based on previous studies it was assumed an effect size (ratio of mean difference to within group standard deviation) of 3 would be reasonable for readouts such as mucus layer measurements, enzyme assays and measurement of transcript changes. With 3 animals in each group and a 5% significance level, two-sided, this would yield a power of 78% for the t test. Therefore, for some of the feeding groups (those alternated between different diets), 3 animals were used. However, for other feeding groups that were more important for the central research question of the study (e.g., constant feeding on Fiber-rich (FR) and Fiber-free (FF) diets), at least 4 animals were used to obtain higher power. For C. rodentium infection experiments, in most cases 5 animals per group were used based on results of our previous study ().

Sample Processing for Animal Experiments 2 asphyxiation followed by cervical dislocation. The gastrointestinal tracts were quickly removed. The colons were gently separated, by cutting at the cecum-colon junction and the rectum, and immediately preserved in Carnoy’s fixative (dry methanol:chloroform:glacial acetic acid in the ratio 60:30:10) with slight modifications to a previous protocol ( Johansson and Hansson, 2012 Johansson M.E.V.

Hansson G.C. Preservation of mucus in histological sections, immunostaining of mucins in fixed tissue, and localization of bacteria with FISH. All animals were killed using COasphyxiation followed by cervical dislocation. The gastrointestinal tracts were quickly removed. The colons were gently separated, by cutting at the cecum-colon junction and the rectum, and immediately preserved in Carnoy’s fixative (dry methanol:chloroform:glacial acetic acid in the ratio 60:30:10) with slight modifications to a previous protocol (). Note that the Carnoy’s fixative was made fresh with anhydrous methanol, chloroform and glacial acetic acid. The colons were fixed in Carnoy’s solution for 3 hr followed by transfer to fresh Carnoy’s solution for 2−3 hr. The colons were then washed in dry methanol for 2 hr, placed in cassettes and stored in fresh dry methanol at 4°C until further use. Cecal contents from each animal were divided into replicates; instantly flash-frozen in liquid nitrogen and were stored at −80°C until further use. Immediately after squeezing out the cecal contents, the cecal tissues were transferred in separate screw-cap tubes and were rapidly flash-frozen in liquid nitrogen, followed by their storage at −80°C until further use. Lengths of colons were measured immediately after fixation in Carnoy’s solution by photographing the colons in a reference cassette of identical size, followed by length measurement in ImageJ.

Purification of Mucin O-Glycans Martens et al. (2008) Martens E.C.

Chiang H.C.

Gordon J.I. Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont. 4 were added to final concentrations of 0.1 M and 1 M, respectively. This solution was incubated at 65°C for 18 hr to promote selective release of O-linked glycans (mucin O-glycans and GAGs) from mucin glycopeptides by alkaline β-elimination. The pH was subsequently decreased to 7.0 with HCl and the neutralized mixture centrifuged at 21,000 x g for 30 min at 4°C, and then filtered through a 0.22 μm filter (Millipore) to remove remaining insoluble material. The filtrate was exhaustively dialyzed (1 kDa cutoff) against deionized distilled H 2 O to remove salts and contaminating small molecules. The collected mucosal glycans were further fractionated using anion exchange chromatography by passing them twice over a DEAE-Sepharose (Sigma) column (325 mL bed volume; equilibrated in 50 mM Tris 7.4; gravity flow). The flow through (neutral fraction) was collected and the column washed with 1L of 50 mM Tris, pH 7.4. This fraction was used in all growth experiments and was further prepared by dialyzing against ddH 2 O (1 kDa cutoff), lyophilized and resuspended in ddH 2 O at 20 mg/ml. Mucin O-glycans were purified from porcine gastric mucus as previously described in, albeit with several modifications. Porcine gastric mucin (Type III, Sigma, USA) was suspended at 2.5% w/v in 100 mM Tris (pH 7.4): the mixture was immediately autoclaved for 5 min to increase solubility and reduce potential contaminating glycoside hydrolase and polysaccharide lyase activity, then cooled to 55°C. Proteinase K (Invitrogen, USA) was added to a final concentration of 0.1 mg/ml and the suspension was incubated at 55°C for 16−20 hr with slow shaking. The proteolyzed solution was subsequently centrifuged at 21,000 x g for 30 min at 4°C to remove insoluble material, and NaOH and NaBHwere added to final concentrations of 0.1 M and 1 M, respectively. This solution was incubated at 65°C for 18 hr to promote selective release of O-linked glycans (mucin O-glycans and GAGs) from mucin glycopeptides by alkaline β-elimination. The pH was subsequently decreased to 7.0 with HCl and the neutralized mixture centrifuged at 21,000 x g for 30 min at 4°C, and then filtered through a 0.22 μm filter (Millipore) to remove remaining insoluble material. The filtrate was exhaustively dialyzed (1 kDa cutoff) against deionized distilled HO to remove salts and contaminating small molecules. The collected mucosal glycans were further fractionated using anion exchange chromatography by passing them twice over a DEAE-Sepharose (Sigma) column (325 mL bed volume; equilibrated in 50 mM Tris 7.4; gravity flow). The flow through (neutral fraction) was collected and the column washed with 1L of 50 mM Tris, pH 7.4. This fraction was used in all growth experiments and was further prepared by dialyzing against ddHO (1 kDa cutoff), lyophilized and resuspended in ddHO at 20 mg/ml.

Bacterial Growth Assays in a Custom Carbohydrate Array Martens et al., 2011 Martens E.C.

Lowe E.C.

Chiang H.

Pudlo N.A.

Wu M.

McNulty N.P.

Abbott D.W.

Henrissat B.

Gilbert H.J.

Bolam D.N.

Gordon J.I. Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts. 2 , 5% CO 2 and 85% N 2 ) and were allowed to equilibrate with the anaerobic atmosphere for ∼3−4 hr. Growth assays for all carbohydrates were carried out in non-adjacent duplicates and all growth arrays contained two non-adjacent water only negative controls that were checked to ascertain that other medium components without added carbohydrates did not yield detectable growth. The cultures for the inoculation were grown overnight in their respective minimal media (MM)/regular growth media at 37°C under an anaerobic atmosphere (10% H 2 , 5% CO 2 and 85% N 2 ). MM for some bacterial species were used from previous studies, and for other members of the synthetic microbiota, MM with novel formulations were devised (see All species except Desulfovibrio piger were evaluated in a custom carbohydrate array (n = 2 replicate cultures per glycan); D. piger failed to grow in any of the tested minimal media, but is not predicted to have extensive carbohydrate-degrading capacity based on its small genomic complement of only 30 carbohydrate active enzymes. The custom carbohydrate array was formulated according to, but with a few modifications included in the following protocol: flat bottom 96-well plates (Costar) were used, to which 100 μL of a 2x concentrated solution (prepared in Milli-Q water) of each of the sterilized carbohydrate stocks ( Table S1 ) were added. The plates were then transferred to the anaerobic chamber (10% H, 5% COand 85% N) and were allowed to equilibrate with the anaerobic atmosphere for ∼3−4 hr. Growth assays for all carbohydrates were carried out in non-adjacent duplicates and all growth arrays contained two non-adjacent water only negative controls that were checked to ascertain that other medium components without added carbohydrates did not yield detectable growth. The cultures for the inoculation were grown overnight in their respective minimal media (MM)/regular growth media at 37°C under an anaerobic atmosphere (10% H, 5% COand 85% N). MM for some bacterial species were used from previous studies, and for other members of the synthetic microbiota, MM with novel formulations were devised (see Table S1 for compositions of all growth media used in this study). MM were pre-reduced in the anaerobic chamber overnight by loosening the lids of the glass bottles containing the MM. 1 mL of the culture was centrifuged and the pellet was recovered – note that the centrifugation was performed inside the anaerobic chamber. The pellet was washed 2 times in the respective MM in order to remove carried over carbohydrates from the culture media and was then resuspended in 1 ml, 2x concentrated MM without any carbohydrates. This 1 mL culture was used to inoculate 50 mL of 2x concentrated MM without carbohydrates at a 1:50 ratio. 100 μL of the resulting cultures were then added to the individual wells of the carbohydrate solutions in the 96-well plates, resulting in 200 μL of final volumes. A gas permeable, optically clear polyurethane membrane (Diversified Biotech, USA) was then used to seal the well plates under the same anaerobic atmosphere. Next, the well plates were loaded in a Biostack automated plate-handling device (Biotek Instruments, USA) placed inside the anaerobic chamber, which was coupled with a Powerwave HT absorbance reader (Biotek Instruments, USA). 600 ) at an interval of 10 min over 96 hr for all species, except for Akkermansia muciniphila, for which the absorbance was measured over 144 hr, owing to its relatively slow growth on mucin O-glycans ( Absorbance values were measured at 600nm (A) at an interval of 10 min over 96 hr for all species, except for Akkermansia muciniphila, for which the absorbance was measured over 144 hr, owing to its relatively slow growth on mucin O-glycans ( Figure S1 B). To construct the heatmap containing relative growth values ( Figure 1 A), absorbance data of all bacterial species were normalized as follows: only carbohydrate growth assays for which both replicate cultures produced an increase in absorbance of more than 0.1 were scored as positive (all other values were set to 0). Next, the maximum change in absorbance was normalized within each individual species by setting its best growth to 1.0 and normalizing all other positive growths to this maximum value (normalized values were thus between 0 and 1.0). Finally, growth on each substrate was normalized across species by setting the maximum (previously normalized) growth value on that substrate to 1.0 and then adjusting the growth values for other strains on that same substrate relative to the maximum value, yielding final normalized values between 0 and 1.0. Both raw and normalized values are provided in Table S1 600 values between 0.45−0.6). Cultures of B. caccae were grown separately on glucose and mucin O-glycan as the carbon sources (10 mg/ml final concentration for both sugars) to mid-log phase (OD values between 0.7−0.8). Cultures of both species were treated with RNAprotect (QIAGEN, USA) according to the manufacturer’s instructions. The RNAprotect treated bacterial pellets were stored at −80°C until extraction of RNA. Two replicate cultures per glycan (with closely matching ODs) were performed for each of the two bacterial species. To perform RNA-Seq analysis on pure cultures, A. muciniphila and Bacteroides caccae were grown anaerobically in their respective minimal media ( Table S1 ). A. muciniphila was grown separately on two different substrates N-acetylglucosamine (5 mg/ml final concentration) and purified mucin O-glycans (10 mg/ml final concentration). Cultures of A. muciniphila were grown to mid-log phase (Avalues between 0.45−0.6). Cultures of B. caccae were grown separately on glucose and mucin O-glycan as the carbon sources (10 mg/ml final concentration for both sugars) to mid-log phase (OD values between 0.7−0.8). Cultures of both species were treated with RNAprotect (QIAGEN, USA) according to the manufacturer’s instructions. The RNAprotect treated bacterial pellets were stored at −80°C until extraction of RNA. Two replicate cultures per glycan (with closely matching ODs) were performed for each of the two bacterial species.

Citrobacter rodentium quantification To determine the CFUs of C. rodentium, freshly collected fecal samples were weighed and homogenized in cold phosphate-buffered saline and were plated on LB agar plates with 50 μg/ml Km (for strain DBS120) or 200 μg/ml Amp (for strain DBS100) at serial dilutions up to 10−9. The plates were incubated aerobically overnight at 37°C. Killing of E. coli HS, the only facultative anaerobe in our SM, to Km and Amp was confirmed by plating it on LB agar with Km or Amp.

Extraction of Nucleic Acids DNA from fecal samples was isolated using the MoBio PowerSoil Isolation Kit (MoBio Laboratories, USA) adapted for use in the epMotion 5075 TMX or the DNA extraction protocol used for Collinsella aerofaciens (mentioned below). DNA was extracted from the bacterial pure cultures using DNeasy Blood & Tissue Kit (QIAGEN, USA), except that the following bead beating and phenol−chloroform extraction protocol, was employed to better extract DNA from C. aerofaciens: 1−2 mL of the overnight grown culture was centrifuged and the resulting pellet was combined with acid-washed glass beads (212−300 μm; Sigma-Aldrich, USA), 500 μl Buffer A (200 mM NaCl, 200 mM Tris, 20 mM EDTA), 210 μl SDS (20% w/v, filter-sterilized) and 500 μl phenol:chloroform:isoamyl alcohol (25:24:1, pH 8.05; Thermo Fisher Scientific, USA). A Mini-BeadBeater-16 (Biospec Products, USA) was used to disrupt the bacterial cells for 5 min at room temperature, which was followed by cooling the samples for 1−2 min on wet ice. The samples were then centrifuged and the aqueous phase was recovered. An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added to the aqueous phase and was mixed with the aqueous phase by gentle inversion. After centrifugation (12,000 rpm, 4°C, 3 min), the aqueous phase was recovered. Next, 500 μl of pure chloroform was added to the aqueous phase, mixed by inversion and the tubes were centrifuged (12,000 rpm, 4°C, 3 min). The aqueous phase was transferred into fresh tubes and 1 volume of −20°C chilled 100% isopropanol and 1/10 volume 3 M sodium acetate (pH 5.2) were added to the aqueous phase. The samples were mixed by gentle inversion and incubated at −20°C for 1 hr, centrifuged for 20 min (12,000 rpm, 4°C) and the supernatants were discarded. The pellets were washed in 70% ethanol (v/v, prepared in nuclease-free water), air-dried and then resuspended in nuclease-free water. The resulting DNA extracts were purified by using DNeasy Blood & Tissue Kit (QIAGEN, USA). Sonnenburg et al., 2006 Sonnenburg J.L.

Chen C.T.L.

Gordon J.I. Genomic and metabolic studies of the impact of probiotics on a model gut symbiont and host. RNA was extracted from cecal contents using a standard phenol−chloroform method with bead beating as mentioned earlier (), but with a few modifications: 1 mL of RNAprotect (QIAGEN, USA) stored at room temperature was added to 200−300 mg of cecal contents stored at −80°C, followed by thawing the cecal samples on wet ice. After the cecal contents were thawed, 250 μl acid-washed glass beads (212−300 μm; Sigma-Aldrich, USA) were added to the samples. Next, 500 μl of a solution of Buffer A (200 mM NaCl, 200 mM Tris, 20 mM EDTA), 210 μl of 20% SDS (filter sterilized) and 500 μl of phenol:chloroform:isoamyl alcohol (125:24:1, pH 4.3; Fischer Scientific, USA) were added to the samples. The mixture was then bead beaten (instrument same as above) for 5 min and centrifuged at 4°C (3 min at 13000 rpm). The aqueous phase was recovered and mixed with 500 μl of the aforementioned phenol:chloroform:isoamyl alcohol solution. Afterward, the mixture was centrifuged again at 4°C (3 min at 13,000 rpm) and the aqueous phase was recovered. 1/10 volume of a 3M sodium acetate (pH: 5.2) and 1 volume of −20°C chilled ethanol were added to the aqueous phase. The resulting solution was then mixed by gentle inversion and incubated for 20 min on ice. Afterward, the mixture was centrifuged at 4°C (20 min at 13,000 rpm). The pellet was recovered and washed twice in 500 μl of cold 70% ethanol. The mixture was centrifuged at 4°C (5 min at 13,000 rpm) and the RNA pellet was recovered, air-dried and then resuspended in nuclease-free water. The RNA extracts were then purified using an RNeasy Mini kit (QIAGEN, USA) according to the manufacturer’s protocol. During extraction of RNA from the cecal contents, a portion (∼100−200 μl) of homogenized material was removed immediately after bead beating and stored at −80°C for extraction of DNA. To extract DNA from these cecal-content derived samples, DNA extraction protocol described above (used for C. aerofaciens) was used, except that bead beating and inclusion of glass beads were skipped. RNA was extracted from the RNAprotect-treated cell pellets of bacterial pure cultures of B. caccae using RNeasy Protect Bacteria Mini Kit. For extraction of RNA from RNAprotect-treated cell pellets of A. muciniphila, the RNA extraction protocol used for cecal contents (see above) was used, except that the samples were not treated with RNAprotect after thawing. RNA was extracted from cecal tissue by thawing the samples in the presence of RNAprotect (as described above for cecal contents), followed by homogenization (OMNI International) involving metal beads. RNA was then extracted with Trizol (Invitrogen, USA) according to manufacturer’s instructions. All RNA extracts were subjected to digestion of DNA using TURBO DNase (Ambion, USA) according to the manufacturer’s instructions.

Bioluminescence Imaging and Transmission Electron Microscopy For bioluminescence imaging of the luciferase-expressing Citrobacter rodentium, GI tracts were removed and luminal contents were gently flushed with a syringe by passing phosphate-buffered saline (PBS) through the colon. The GI tracts were then cut open flat and rinsed in PBS to remove loosely attached luminal contents. Bioluminescence was visualized (identical exposure across all samples) and photographed using the IVIS200, Xenogen system. The colonic tissue sections showing highest luciferase intensity (from both Fiber-rich (FR) and Fiber-free (FF) diet fed colonized mice) were then immediately fixed in 2.5% glutaraldehyde prepared in 0.1 M Sorensen’s buffer (pH 7.4). Thereafter, the samples were treated with 1% osmium tetroxide in 0.1 M Sorensen’s buffer and were sequentially dehydrated in graded alcohols and propylene oxide, followed by infiltration in Spurrs or Epon. Ultrathin sections of the tissue samples were made using a diamond knife, stained and were visualized with a transmission electron microscope (Philips CM-100).

Laser Capture Microdissection To perform laser capture microdissection (LCM) on colonic thin sections, 4 and 3 fecal masses were analyzed for the Fiber-rich (FR) and Fiber-free (FF) groups, respectively. Colonic thin sections that were deposited on microscope slides were deparaffinized in xylene followed by dehydration by isopropanol (see details in the immunofluorescence staining protocol below). The sections were stored overnight in a container with Drierite dessicant (Drierite, USA). LCM was carried out using a Veritas Microdissection instrument (Arcturus, USA). DNA was extracted from the microdissected samples using the Arcturus Pico Pure DNA extraction kit and the accompanying protocol. In order to perform Ilumina sequencing, 16S rRNA genes were amplified from the LCM-derived samples using a low biomass-optimized touch down PCR protocol as follows: denaturation at 95°C for 2 min; a total of 20 cycles with a touch-down program: denaturation at 95°C for 20 s, extension at 72°C for 5 min, annealing starting at 60°C for 15 s which decreased 0.3°C per cycle; a total of 20 cycles: extension at 72°C for 5 min, annealing at 55°C 15 s and extension at 72°C for 5 min; final extension at 72°C for 5 min. Note that a 5 min extension was used in order to reduce chimera development. Library preparation and sequencing were carried out using similar protocols described for fecal and cecal samples (see below).

Illumina Sequencing and Data Analysis Kozich et al. (2013) Kozich J.J.

Westcott S.L.

Baxter N.T.

Highlander S.K.

Schloss P.D. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Kozich et al. (2013) Kozich J.J.

Westcott S.L.

Baxter N.T.

Highlander S.K.

Schloss P.D. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. PCR and library preparation were performed by the University of Michigan Microbial Systems Molecular Biology Lab as described by. The V4 region of the 16S rRNA gene was amplified using the dual-index primers described bywith a few modifications to the PCR assay, which are included in the following protocol. Each of these dual-index primers contains an Illumina adaptor, an 8-nt index sequence, a 10-nt pad sequence, a 2-nt linker, and the V4 primers F515 and R806. These primer sequences are listed in Table S2 . For the PCR assays, 5 μL of each of the 4 μM primers, 0.15 μL AccuPrime High Fidelity Taq polymerase (Thermo Fisher Scientific, USA), 2 μL of 10x AccuPrime PCR II buffer (Thermo Fisher Scientific, USA), 11.85 μL of sterile PCR-grade water and 1 μL of the DNA template were mixed. The PCR cycles started with a 2 min of denaturation at 95°C, followed by 30 cycles each consisting of 95°C for 20 s, 55°C for 15 s and 72°C for 5 min, followed by a final step of 72°C for 10 min. The amplicons were normalized to the lowest concentration of the pooled plates using a SequalPrep normalization plate kit (Thermo Fisher Scientific, USA). A KAPA Library Quantification kit for Illumina platforms (Kapa Biosystems, USA) was used to determine the library’s concentration and an Agilent Bioanalyzer high-sensitivity DNA analysis kit (Agilent, USA) was employed to determine the amplicon size. The amplicons were sequenced using an Illumina MiSeq with a MiSeq Reagent 222 kit V2 (Illumina, USA). The libraries were prepared following the Illumina protocol for 2nM libraries: ‘Preparing Libraries for Sequencing on the MiSeq’ (part 15039740, Rev. D). Schloss et al., 2009 Schloss P.D.

Westcott S.L.

Ryabin T.

Hall J.R.

Hartmann M.

Hollister E.B.

Lesniewski R.A.

Oakley B.B.

Parks D.H.

Robinson C.J.

et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Edgar et al., 2011 Edgar R.C.

Haas B.J.

Clemente J.C.

Quince C.

Knight R. UCHIME improves sensitivity and speed of chimera detection. Wickham, 2011 Wickham H. The split-apply-combine strategy for data. McNulty et al., 2013 McNulty N.P.

Wu M.

Erickson A.R.

Pan C.

Erickson B.K.

Martens E.C.

Pudlo N.A.

Muegge B.D.

Henrissat B.

Hettich R.L.

Gordon J.I. Effects of diet on resource utilization by a model human gut microbiota containing Bacteroides cellulosilyticus WH2, a symbiont with an extensive glycobiome. Raw sequences were analyzed using mothur (v1.33.3) (). The following control samples were included: 1) DNA extracted from the fecal samples collected from germfree mice, 2) a mixture of extracted DNA from pure cultures of the members of the synthetic microbiota (DNA samples from each strain were mixed in equal amounts) and 3) PBS negative controls during DNA extraction and PCR amplification. Following sequence barcode-trimming, sequences were aligned to a custom reference database, consisting of the V4 16S rRNA region from each of the 14 bacterial members and C. rodentium. UCHIME () was used to remove sequence chimeras. The R package ‘vegan’ was used to calculate the principal coordinates analysis (PCoA) from the Bray-Curtis dissimilarity index based on phylotype classification of the 14 bacterial members. Standard R commands and the R package ‘plyr’ () were used to generate median values of relative abundance or change in relative abundance over time, and the Wilcoxon signed-rank test (two-sample comparisons) or the Kruskal-Wallis test (multiple groups) was used to determine significance as indicated due to the nonparametric distribution of relative abundance data. R was used to visualize relative abundance of bacterial members in different groups, over time as streamplots, or in heatmaps. Change in relative abundance over time was determined by subtracting the relative abundance of the specified microbial member from the day prior within each animal, over time. Change in relative abundance followed a parametric distribution, and Student’s t tests were used to calculate significant differences in the change of relative abundance between diet groups. The R package ‘ggplots’ was used to generate heatmaps visualizing the Percent of Maximum Abundance (POMA) as previously described (). For this, the relative abundance of the different species was normalized by their maximum abundance observed for a given species across all time-points from the given animal. A detailed list of commands used to analyze the data, including the commands used in mothur, are included in https://github.com/aseekatz/mouse.fiber . Raw sequences have been deposited in the Sequence Read Archive under the study accession and Bioproject identifiers (SRA: SRP065682 and PRJNA300261).

Microbial RNA-Seq and CAZyme Annotation Martens et al., 2008 Martens E.C.

Chiang H.C.

Gordon J.I. Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont. Sonnenburg et al., 2005 Sonnenburg J.L.

Xu J.

Leip D.D.

Chen C.-H.

Westover B.P.

Weatherford J.

Buhler J.D.

Gordon J.I. Glycan foraging in vivo by an intestine-adapted bacterial symbiont. Microbial RNA-Seq was performed on pure cultures of A. muciniphila and B. caccae that were grown separately on mucin O-glycans and on the respective simple sugars (see above). For Bacteroides thetaiotaomicron, gene expression data from previous studies was utilized (). To perform RNA-Seq on cecal samples, 3 samples each (out of 4) were randomly selected from Fiber-rich (FR) and Fiber-free (FF) diet groups and all three samples each from the Prebiotic diet (Pre), FR−FF daily oscillation and Pre−FF daily oscillation groups were utilized. To remove ribosomal RNA, samples were subjected to Ribo-Zero rRNA Removal Kits (Bacteria) (Epicenter, Illumina, USA) according to the manufacturer’s instructions. The resulting residual mRNA concentrations were quantified using Qubit RNA Assay Kit (Life Technologies, USA). Libray preparation and sequencing of RNA-Seq libraries was carried out using the Illumina HiSeq platform and TruSeq adaptors. Samples were multiplexed in groups of 24 per lane (see Tables S4 S5 , and S6 for quantification of reads mapped to each sample). The resulting data in fastq file format were demultiplexed and mapped to the respective species genomes or community metagenomes using RPKM normalization and default parameters, and were further analyzed for fold-change and statistics (moderated t test with Benjamini-Hochberg correction) within the Arraystar software package (DNAStar, USA). Mapping reads to all genes in the 14 species community was intended to retain the contributions of community member abundance shifts while mapping only to individual genomes was intended to normalize abundance shifts of the same species between conditions and isolate gene expression changes between conditions. The diet-specific behavior of known B. thetaiotaomicron and B. ovatus genes involved in fiber polysaccharide degradation ( Table S6 ) was used as internal validation that biologically relevant changes in gene expression were indeed being detected. As stated above, three biological replicates were analyzed for each of the in vivo dietary conditions used.

p-Nitrophenyl Glycoside-Based Enzyme Assays p-Nitrophenyl glycoside-based enzyme assays were carried out on cecal samples stored at −80°C. The cecal samples were thawed on wet ice and 500 μl buffer (50 mM Tris, 100 mM KCl, 10 mM MgCl 2 ; pH 7.25) was added to 22−67 mg of cecal contents. The buffer additionally contained the following additives: lysozyme (tiny amount of powder/100 mL buffer), TritonX (100 μl, 12%/100 mL buffer), DNases (tiny amount of powder/100 mL buffer) and protease inhibitor (one tablet of EDTA-free, Protease Inhibitor Cocktail, Roche, USA/100 mL buffer). After adding the buffer to cecal samples, the samples were sonicated with an ultrasonic processor for 45 s (9 cycles of 5 s sonication followed by a break of 10 s; 35% amplitude; using a tapered microtip of 3 mm) on ice. Sonicated samples were subjected to centrifugation (10,000 g, 10 min, 4°C). Supernatants (∼400 μl) were carefully pipetted and were stored at −20°C until further use. The following nitrophenyl-linked substrates (Sigma-Aldrich, USA) were employed: Potassium 4-nitrophenyl sulfate, 4-nitrophenyl α-D-galactopyranoside, 4-nitrophenyl N-acetyl-β-D-glucosaminide, 4-nitrophenyl β-D-glucopyranoside, p-nitrophenyl α-L-fucopyranoside and p-nitrophenyl β-D-xylopyranoside. Protein concentrations in the supernatants were determined using Pierce Microplate BCA Protein Assay Kit (Thermo Scientific, USA). Some samples were diluted with the buffer (same buffer as above) to obtain a homogeneous range of protein concentrations across all samples. 5 μg of total protein was used in the 150 μl reactions inside flat-bottom, 96-well plates (Costar) with 10 mM nitrophenyl-based substrate in the buffer (same buffer as above). Absorbance measurements (405 nm) were started immediately in a plate reader (Biotek, USA) at 37°C and absorbance values were recorded every minute for 6−12 hr duration depending on linearity of the kinetic curve. The enzyme activities were determined by plotting a standard curve of known concentrations of 4-nitrophenol and measuring the OD values at 37°C.

Thickness Measurements of the Colonic Mucus Layer Post Carnoy’s fixation, the methanol-stored colon samples (see above) were embedded in paraffin and thin sections (∼5 μm) were cut and deposited on glass slides. Alcian blue staining was performed by the following protocol: 1) deparaffinization and hydration to distilled water, 2) Alcian blue solution for 30 min, 3) washing in running tap water for 2 min, 4) rinsing in distilled water, 5) dehydration with 95% alcohol (2X changes) and treatment with absolute alcohol (2X changes), 3 min each, 6) clearance in xylene (3X changes), 3 min each 7) cover with coverslip. To measure the thickness of the colonic inner mucus layer, thousands of partially overlapping photographs were taken from nearly the entire length of each colon based from the Alcian blue stained slides after cross-validation using anti-Muc2 staining ( Figures 4 A and 4B main text). The images captured all of the available fecal masses of all mice, although this number was variable and there were generally fewer colonic fecal masses in mice fed the FF diet alone or in any combination. Image sample names were blinded by M.S.D. and M.W., and the thickness of the colonic sections were then measured by E.C.M. using ImageJ. Only regions in which the mucus layer was sandwiched between epithelium on one side and luminal contents on the other were used; care was taken to measure regions that represented the average thickness in each blinded image; 2−3 measurements per image were taken and averaged over the entire usable colon surface. See Figures 4 A and 4B for representative images in which the region measured as the inner mucus layer is delineated in both Alcian blue and anti-Muc2 staining. Measurements in Cr infected mice were conducted exactly as described for non-infected mice above, with the exception that only distal colon−rectal tissue was considered as this was uniformly a site at which inflammation was high and C. rodentium would be present. Since only sections in which luminal contents that could be visualized adjacent to the mucus layer were considered, only a few measurements were obtained for a single SM-colonized infected mouse fed the FF diet due to the fact that all mice in this group were extremely morbid and not eating.

qPCR In addition to Illumina sequencing of the 16S rRNA genes (V4 region), as a second approach to quantifying relative bacterial abundance in fecal samples, phylotype-specific bacterial primers were designed. The primers were designed against randomly selected genes that were checked for homology against the other 13 species in each case. These primer sequences are listed in Table S3 A. The primers were tested for specificities against the target strain by comparing the primer and target gene sequences against sequences in public databases. Moreover, specificity of each primer was validated by the following three approaches: 1) by quantitative PCR (qPCR) against target species genome and melting curve analysis (for a single peak), 2) by qPCR for each primer set against a non-target template comprising of genomic DNA from the 13 bacterial species in our synthetic microbiota, 3) by performing qPCR against DNA extracted from the fecal samples of germfree mice feeding on the Fiber-rich diet. qPCR was carried out in 384 wells (with each plate including known concentrations of template DNA included to plot a standard curve). The qPCR analyses were performed using KAPA SYBR FAST qPCR Kits (KAPA Biosystems, USA) on Applied Biosystems (ABI) Real Time PCR instrument (ABI, USA). The amount of DNA was quantified by plotting a standard curve of varying DNA concentrations of the target template.

Quantification of Short-Chain Fatty Acids Cecal samples stored at −80°C were used to quantify short-chain fatty acids (SCFAs). Samples were first thawed on wet ice. Then, an equivalent amount of Milli-Q water was added (100 ul per 100 mg of material) to cecal contents (≥0.05 g) and the samples were thoroughly homogenized by vortexing for 1 min. The samples were then centrifuged at 13,000 g for at least 3 min (or for a longer time depending on time required to obtain a tight pellet). The supernatant was pipetted and filtered through a 0.22 μm filter (Millex-gv 4mm SLGV004SL). Samples were kept on ice or frozen until quantification of SCFAs by high-performance liquid chromatography (HPLC). Some samples were diluted to obtain enough liquid to inject onto the HPLC, or in certain cases they were diluted so that they could be filtered. A Shimadzu HPLC with an Agilent HP-87X column was utilized for separating compounds, with a mobile phase of 0.01 N H 2 SO 4 , a flow rate of 0.6 ml/min, and a column temperature of 50°C. A UV detector set to a wavelength of 214 nm was used to measure concentrations.

Immunofluorescence Staining The immunofluorescence staining for Muc2 mucin was performed on the colonic thin sections after several modifications to the protocols from Johansson and Hansson, 2012 and an immunohistochemistry/tissue section staining protocol from BD Biosciences, USA ( http://www.bdbiosciences.com ). The sections were deparaffinized by dipping in 50 mL Falcon conical tubes filled with xylene (Sigma-Aldrich, USA) for 5 min, followed by transfer to another tube with fresh xylene for 5 min – care was taken to completely immerse the tissue material in the liquid (also in the subsequent steps). This was followed by two dehydration steps of 5 min each using 100% isopropanol contained in conical tubes. The slides were then washed by dipping in conical tubes containing Milli-Q water. The antigens were retrieved by placing the slides in a glass beaker with enough BD Retrievagen A (pH 6.0; BD Biosciences, USA) to cover the slides. The sections were then heated by microwaving and holding at about 89°C for 10 min (microwaving was repeated during this time, as required). The slides were then cooled for 20 min at room temperature. Afterward, the slides were washed 3 times with Milli-Q water. Excess liquid was gently blotted away and a PAP pen was used to draw a circle around the tissue area, in order to better hold liquid on the tissue area during subsequent steps. Blocking was performed by immersing the slides into blocking buffer (1:10 dilution of goat serum (Sigma, USA) in 1x Tris-buffered Saline (TBS; 500 mM NaCl, 50 mM Tris, pH 7.4)) and incubating them at room temperature for 1 hr. For the primary antibody staining, the tissue sections were covered in a 1:200 dilution Mucin 2 antibody (H-300) (original concentration: 200 μg/ml; Santa Cruz Biotechnology, USA) in the aforementioned blocking buffer and incubated for 2 hr at room temperature. After the incubation step, the excess liquid was blotted away and the slides were rinsed 3 times in 1x TBS (in conical tubes) for 5 min each. The secondary antibody staining was performed by covering the tissue sections with a 1:200 dilution of Alexa Fluor 488 conjugated goat anti-rabbit IgG antibody (original concentration: 2 mg/ml; Thermo Fisher Scientific, USA) in blocking buffer and the sections were incubated for 1 hr at room temperature in dark. The excess liquid was blotted away and the sections were rinsed twice for 5 min each using TBS. Next, the sections were stained for 5 min at room temperature in dark using a 10 μg/ml of DAPI solution diluted in 1x TBS (Sigma-Aldrich, USA). The sections were then rinsed with Milli-Q water and blotted dry. Finally, the sections were covered with ProLong Gold Antifade Mountant (Invitrogen, USA), covered with coverslips and the edges of the coverslips sealed with nail polish. The slides were kept at room temperature in dark for at least 24 hr and then visualized by Olympus BX60 upright fluorescence microscope (Olympus, USA).

ELISA for Fecal Lipocalin Chassaing et al., 2015 Chassaing B.

Koren O.

Goodrich J.K.

Poole A.C.

Srinivasan S.

Ley R.E.

Gewirtz A.T. Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome. Frozen fecal samples (−20°C) were used to determine the levels of fecal Lipocalin (LCN-2). The assays were performed within 30 days of sample collection. The samples were prepared as mentioned previously (), with a few modifications in the sample prepration protocol: fecal samples stored at −20°C were thawed on wet ice and 6.9−67.7 mg of samples were separated in fresh tubes, to which 0.5 mL of 1% (v/v) Tween 20 (Sigma-Aldrich, USA) prepared in PBS was added. To get a homogeneous suspension, the samples were vortexed for 20 min. The suspension was then centrifuged at 4°C for 10 min at 12000 rpm. Next, the supernatant was carefully recovered and stored at −20°C until the analysis. To measure the LCN-2 levels, a mouse Lipocalin-2/NGAL DuoSet ELISA kit (R & D Biosystems, USA) was employed and the manufacturer’s protocol was followed.

Tissue Histology To perform histology analyses on GI tracts of C. rodentium infected mice: first the intestinal segments (cecum and colon together) were fixed in Carnoy’s fixative for 3 hr, followed by transfer to fresh Carnoy’s fixative overnight. Next, the samples were washed in 100% methanol (2x) for 30 min each, which was followed by washing in 100% ethanol (2x) for 20 min each. The samples were then stored in 100% ethanol at 4°C until further use. After 100% ethanol washes, the intestinal tissue samples were divided into 3 sections for histology: cecum, ascending colon, and the descending colon/rectum. These sections were embedded, processed and cut by an experienced histology core (Washington University, USA), and then stained with hematoxylin and eosin (H and E) prior to analysis. An unblinded experienced pathologist (T.S.S.) examined the slides from each of the groups to determine a viable readout. The best readout was determined to be the extent of epithelial area showing crypt hyperplasia. After a scoring rubric was devised, an independent blinded evaluator (C.A.H.) then measured the total length of each intestinal segment with a ruler in millimeters. Areas of increased crypt hyperplasia were then determined by microscopy and the lengths of these areas were measured as a percentage of the total epithelial length on a single slide.