Samuel and Gordon. 10.1073/pnas.0602187103. |
Supporting Table 1
Supporting Figure 5
Supporting Table 2
Supporting Figure 6
Supporting Table 3
Supporting Table 4
Supporting Figure 7
Supporting Figure 8
Supporting Methods
Fig. 5. Clusters of Orthologous Group (COG) categorization of B. thetaiotaomicron genes up- or down-regulated in vivo in the presence of M. smithii. All genes designated by GeneChip analysis as being significantly (P < 0.05) up- or down-regulated in B. thetaiotaomicron/M. smithii biassociated mice compared with B. thetaiotaomicron monoassociated mice have been placed into COGs (see Table 1 for a list of regulated genes in each category).
Fig. 6. Effect of cocolonization with D. piger (Dp) on the B. thetaiotaomicron (Bt) transcriptome. (A) qRT-PCR analysis of the expression of selected B. thetaiotaomicron genes in the ceca of B. thetaiotaomicron/M. smithii (Ms) or B. thetaiotaomicron/D. piger biassociated mice vs. B. thetaiotaomicron monoassociated animals. *, P < 0.05. (B) B. thetaiotaomicron glycoside hydrolase genes whose expression is significantly different (P < 0.05) in the presence of D. piger compared with monoassociated controls. Fold-difference was defined by GeneChip. Each column represents data obtained from a cecal sample harvested from an individual mouse.
Fig. 7. Effects of M. smithii on glycan foraging by B. thetaiotaomicron. GC-MS analysis of neutral and amino sugars present in the cecal contents of GF, B. thetaiotaomicron/M. smithii biassociated, and monoassociated mice (n = 4 per group). Mean values ± SEM are plotted.
Fig. 8. Preferential consumption of formate by M. smithii during in vitro culture. Growth of M. smithii in a batch fermentor containing complex methanogen medium (MBC), supplemented with formate and acetate, under a constant stream of H2/CO2 gas (4:1) is shown. Aliquots were taken periodically to measure optical density (OD600) and levels of organic acids (ionization chromatography).
Supporting Materials and Methods
Culture Conditions.
Bacteroides thetaiotaomicron VPI-5482 [American Type Culture Collection (ATCC) 29148] was cultured anaerobically in TYG (1% tryptone/0.5% yeast extract/0.2% glucose) medium, whereas Methanobrevibacter smithii PS (ATCC 35061) was grown in 125-ml serum bottles (Bellco Glass) containing 15 ml of Methanobrevibacter complex medium (MBC; similar to medium 1 in ref. 1) supplemented with 3 g/liter formate, 3 g/liter acetate, and 0.3 ml of a freshly prepared, anaerobic solution of filter-sterilized 2.5% Na2S. The remaining volume in the bottle (headspace) contained a 4:1 mixture of H2 and CO2: the headspace was rejuvenated every 1-2 d. M. smithii also was cultured in a BioFlor-110 batch culture fermentor with dual fermentation vessels, each containing 750 ml of supplemented MBC, at 37°C under a constant flow of H2/CO2 (4:1). One hour before inoculation, 7.5 ml of sterile 2.5% Na2S solution was added to the 1.2-liter culture vessel, followed by half of the contents of a serum bottle culture that had been harvested on day 5 of growth. The flow rate of the fermentor was maintained at 0.1 liter/h (agitation setting, 250 rpm). Sterile 2.5% Na2S solution (1 ml) was added daily. Aliquots were removed from each vessel at specified times during growth for measurement of OD600 and analysis of metabolites. Desulfovibrio piger (ATCC 29098) was cultured anaerobically in Postgate’s Medium B (2).Defining the Density of Colonization.
Luminal contents were manually extruded from the cecum and the distal half of the colon immediately after killing, flash-frozen in liquid nitrogen, and stored at –80°C. Cells in an aliquot of frozen luminal contents were lysed with bead beating in 2 ml of RLT buffer (Qiagen, Valencia, CA; 5 min in a Biospec Mini Bead-beater set on maximum). Genomic DNA (gDNA) then was recovered by using the Qiagen DNeasy kit and its accompanying protocol. Quantitative PCR (qPCR) was performed by using an Mx3000 real-time PCR system (Stratagene). Reaction mixtures (25 ml) contained SYBRGreen Supermix (Bio-Rad), 300 nM 16S rRNA gene-specific primers (see below), and 10 ng of gDNA from cecal contents or microbial DNA purified from monocultures (used as standards). Amplification conditions were 55°C for 2 min and 95°C for 15 min, followed by 40 cycles of 95°C (15 sec), 55°C (45 sec), 72°C (30 sec), and 86°C (20 sec). Primer pairs targeted 16S rRNA genes from B. thetaiotaomicron [Bt.1F, 5'-ATAGCCTTTCGAAAGRAAGAT-3'; Bt.1R, 5'-CCAGTATCAACTGCAATTTTA-3'; 500-bp product (3)], M. smithii (Msm.1F, 5'-TGAGATGTCCGGCGTTGAA-3'; Msm.1R, 5'- AAGCCATGCAAGTCGAACGA-3'; 458-bp product), or D. piger (Dp.1F, 5'-CTAGGGTGTTCTAATCATCATCCTAC-3'; Dp.1R, 5'-TTGAGTTTCAGCCTTGCGACC-3'; 481-bp product).Quantitative RT-PCR (qRT-PCR).
qRT-PCR analyses were performed using methods similar to the qPCR assay described above, with the exception that each reaction contained 10 ng of cDNA template and uracil-DNA glycosidase (0.01 units/ml). Data were normalized to either 16S rRNA (microbial transcripts) or L32 mRNA (host transcripts) (DDCT method) before comparing treatment groups. Primers are listed in Table 4. All amplicons were 100–150 bp.Biochemical Analysis of Cecal Metabolites.
Cecal samples were freeze-dried at –35°C for 4 d (yield 5-10 mg/dry weight) then homogenized at 1°C in 0.4 ml of a solution containing 0.2 M NaOH with 1 mM EDTA. An 80-ml aliquot of the homogenate was removed, heated at 80°C for 20 min, and then neutralized by adding 80 ml of 0.25 M HCl/100 mM Trizma base ("alkaline extract"). For "acidic extracts," a 60-ml aliquot was removed, mixed with 20 ml of 0.7 M HCl, heated at 80°C for 20 min, and then neutralized with 40 ml of 100 mM Trizma base. All extracts were stored at –20°C. Succinate, lactate and NADH, and NAD+ were quantified by using pyridine nucleotide-coupled assays, as described by Passonneau and Lowry (4).Measurement of Organic Acids.
A 60-ml aliquot of the extracted sample (cecal contents or serum) was mixed together with 20 ml of N-tert-butyldimethylsilyl-N-methyltrifluoracetamide (MTBSTFA; Sigma) at room temperature. An aliquot (2 ml) of the resulting derivatized material was injected into a gas chromatograph (Hewlett Packard 6890) coupled to a mass spectrometer detector (Agilent Technologies 5973). Analyses were completed using DB-5MS (60 m, 0.25 mm i.d., 0.25 mm film coating; P. J. Cobert, St. Louis, MO) and electronic impact (70 eV) for ionization. A linear temperature gradient was used. The initial temperature of 80°C was held for 1 min, then increased to 280°C (15°C/min) and maintained at 280°C for 5 min. The source temperature and emission current were 200°C and 300 mA, respectively. The injector and transfer line temperatures were 250°C. Quantitation was completed in selected ion monitoring acquisition mode by comparison to labeled internal standards [formate was also compared to acetate-13C1,d2]. The m/z ratios of monitored ions were as follows: 103 (formic acid), 117 (acetic acid), 131 (propionic acid), 145 (butyric acid), 121 ([2H2]- and [1-13C]acetate), 136 ([2H5]propionate), and 149 ([13C4]butyrate).Organic acids in in vitro cultures were analyzed by using a Dionex 600X Ion Chromatograph (IC). The analytes were separated on a Dionex AS11-HC column and detected with a Dionex ED50 electrochemical detector using suppressed conductivity with multistep gradient program and 1.5–60 mM potassium hydroxide as the eluent. The eluent was generated by a Dionex EG40 Eluent Generator equipped with a Dionex Potassium Hydroxide EluGen cartridge. The IC was calibrated from 0.5 to 10 ppm for all analytes. Detection limits using this method are 0.1 ppm for the six organic anions.
Statistical Analyses.
Pairwise comparisons were made by using unpaired Student’s t test. One-way ANOVA followed by Tukey’s post hoc multiple comparison test was used to determine statistical significance of differences observed between three or more groups of mice.1. Kayar, S. R., Fahlman, A., Lin, W. C. & Whitman, W. B. (2001) J. Appl. Physiol. 91, 2713–2719.
2. Postgate, J. R., Kent, H. M., Robson, R. L. & Chesshyre, J. A. (1984) J. Gen. Microbiol. 130, 1597–1601.
3. Matsuki, T., Watanabe, K., Fujimoto, J., Miyamoto, Y., Takada, T., Matsumoto, K., Oyaizu, H. & Tanaka, R. (2002) Appl. Environ. Microbiol. 68, 5445–5451.
4. Passonneau, J. V. & Lowry, O. H. (1993) Enzymatic Analysis: A Practical Guide (Humana, Totawa, NJ).