Figure S1 – The Mollicute bloom occurs in conventionally-raised wild-type C57BL/6J mice as well as in mice without an intact innate or adaptive immune system. Wild-type (+/+), MyD88 −/−, or Rag1 −/− C57BL/6J mice were weaned onto a standard low-fat polysaccharide-rich (CHO) or high-fat/high-sugar (Western) diet. 16S rRNA gene sequence-based surveys were performed; sequences were aligned (NAST; DeSantis et al., 2006), and inserted into an ARB neighbor-joining tree (Ludwig et al., 2004). Asterisks indicate significant differences (Student’s t-test p<0.001).
Figure S2 – Mice with diet-induced obesity that are switched to a FAT-R or CARB-R diet exhibit stabilization of weight, decreased caloric intake and reduced adiposity. (A) Weight gain (g) and (B) percentage epidydymal fat-pad weight to body weight in wild type C57BL/6J mice that were initially weaned onto a Western diet for 8 weeks, and then maintained on the Western diet, or switched to a FAT-R or CARB-R diet for four weeks (n=5-6 mice/treatment group). Weight was monitored during the four-week period. (C) Chow consumption (kcal/d) is decreased in mice switched to a FAT-R or CARB-R diet. Data are represented as mean±SEM. Asterisks indicate significant differences (ANOVA of FAT-R or CARB-R versus Western, *p<0.05, **p<0.01, ***p<0.0001).
Figure S3 – Switching from a Western to FAT-R or CARB-R diet results in a division-wide increase in the relative abundance of Bacteroidetes, and a decrease in the relative abundance of Mollicutes. UniFrac-based analysis of bacterial community membership shows an impact of diet on gut microbial ecology: cecal communities analyzed from two families of C57BL/6J wild-type mice (Table S3
) generally cluster based on host diet (Western, FAT-R, and CARB-R). The average relative abundance (% of total 16S rRNA gene sequences) of bacterial lineages within the cecal microbiota of all mice fed a Western, FAT-R, or CARB-R diet is displayed as pie charts. Black boxes indicate nodes that were reproduced in >50% of all jackknife replications (n=126 sequences were randomly re-sampled). Asterisks indicate cecal samples that were analyzed by whole community shotgun sequencing.
Figure S4 – Taxonomic assignments of metagenomic sequencing reads from seven cecal microbiome datasets based on BLAST homology searches, and by alignment of 16S rRNA gene fragments. (A) The cecal microbiome is dominated by sequences homologous to Bacteria. Sequencing reads were trimmed based on quality and vector sequence and the resulting datasets were used as queries against the NCBI non-redundant database (e-value<10−5
). Sequences were assigned to the lowest taxonomic group that would include all significant hits, using MEGAN (Huson et al., 2007
). Pie charts are shown for each individual dataset and for the average of all datasets. Colors indicate assignments to bacteria (red), archaea (green), eukarya (yellow), viruses (blue), sequences that could not be confidently assigned to a group (purple), and sequences with no significant BLASTX matches (orange). (B)
Relative abundance of microbiome sequences homologous to genomes from four bacterial divisions: Bacteroidetes (red), Proteobacteria (yellow), Actinobacteria (orange), and Firmicutes (blue). All divisions observed at >1% relative abundance are shown. (C)
Relative abundance of microbiome sequences homologous to genomes from bacterial classes within the Firmicutes division: Bacilli (dark blue), Clostridia (yellow), and Mollicutes (light blue). (D)
Taxonomic assignments of 16S rRNA gene fragments obtained from cecal microbiome datasets. 16S rRNA gene fragments were identified by querying the Ribosomal Database Project (RDP) database (version 9.33; BLASTN e-value < 10−5
; Cole et al., 2005
). 16S rRNA gene fragments were aligned with NAST (DeSantis et al.
, 2006) and added to an ARB neighbor-joining tree (Ludwig et al.
, 2004). 16S rRNA gene fragments from the Bacteroidetes (red), Proteobacteria (yellow), Verrucomicrobia (green), Mollicutes (light blue), and other Firmicutes (dark blue) are shown.
Figure S5 - Assembly of metagenomic sequence data reveals physical linkage between the Mollicute phosphotransferase system (PTS) and other genes involved in carbohydrate metabolism. The pooled mouse gut microbiome dataset was assembled using ARACHNE (n=7 combined datasets; Batzoglou et al., 2002
; see Tables S6
for assembly statistics). The contig length is shown as a solid black bar. Arrows indicate predicted proteins. Functional assignments were derived from the NCBI annotations and verified by BLASTP comparisons of each predicted protein with the STRING-extended COG database (von Mering et al., 2007
) and the KEGG database (Kanehisa et al., 2004
), in addition to Hidden Markov Model (HMM)-based protein domain searching with InterProScan (Mulder et al., 2005
). Contigs 23 and 73 are >98% identical over the region in pink (234/238 nucleotides): they are likely different ends of the same gene that were not joined due to the relatively stringent assembly parameters employed.
Figure S6 – Concentration of bacterial fermentation end-products in the ceca of Western, FAT-R, and CARB-R mice. Acetate and butyrate levels (μmol per g wet weight cecal contents) were measured by gas chromatography mass spectrometry. Lactate levels (mM per kg protein) were measured using established microanalytic methods (see Methods above). Data are represented as mean±SEM. Asterisks indicate significant differences (Student’s t-test of Western versus CARB-R, *p<0.05, **p<0.01).
Figure S7 – KEGG metabolic pathways significantly enriched in the human gut-derived Eubacterium dolichum
strain DSM 3991 genome relative to eight human gut-associated Firmicutes. Pathways whose relative representation is significantly different between the E. dolichum
genome and the pooled gut Firmicute genomes (n=8) were identified using a bootstrap comparison of the abundance of sequences assigned to all KEGG pathways (xipe version 2.4; confidence level = 0.98, sample size = 10,000; Rodriguez-Brito et al., 2006
). The relative abundance of all KEGG pathways with significantly different representation found at a relative abundance >0.6% in at least two microbiome datasets was transformed into a z-score and clustered by genome and pathway using a Euclidean distance metric (de Hoon et al.
, 2004). Enrichment (yellow) and depletion (blue) are defined as a relative abundance greater or less than the mean for all datasets (i.e. a z-score greater or less than zero, respectively). For full strain names see .
Figure S8 – STRING-based protein network analysis of the predicted E. dolichum
proteome. MetaGene (Noguchi et al., 2006
) was used to predict proteins from the E. dolichum
deep draft assembly. Proteins were subsequently assigned to COGs based on homology (BLASTP e-value<10−5
; STRING database version 7, von Mering et al., 2007
). Annotated COG interactions were used to organize the protein network, including interactions based on neighborhood, gene fusion, co-occurrence, homology, co-expression, experiments, databases, and text mining (Medusa Java
appet; Hooper and Bork, 2005). Nodes, each representing a different orthologous group, are colored as follows: green, present in all analyzed Firmicute genomes (including the mycoplasma); blue, present in all recently sequenced gut Firmicute genomes; red, present in the Western diet-associated cecal microbiome (based on BLAST homology searches, e-value<10−5
and the deposited annotations in the STRING database, version 7). 89% of the COGs found in the E.dolichum
genome were also found in the Western diet microbiome. Most of the COGs in green are involved in essential cellular functions such as transcription and translation (56% of the COG category assignments are to ‘Information storage and processing’). Some clusters of interest are highlighted, including the phosphotransferase system (PTS), the 2-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis (MEP), cell wall biosynthesis, ABC transporters, and V-type ATPases for H+
Supplementary Table 1: Protein, carbohydrate, and fat composition of various mouse chow diets
Supplementary Table 2: Percent weight of chow ingredients
Supplementary Table 3: 16S rRNA gene-sequence libraries
Supplementary Table 4: Nomenclature used to designate microbiome datasets obtained from the cecal microbiota of C57BL/6J mice
Supplementary Table 5: Microbiome sequencing statistics
Supplementary Table 6: Microbiome assembly statistics
Supplementary Table 7: Read placements in contigs and BLAST results
Supplementary Table 8: E.dolichum draft genome sequencing statistics