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Supplementary information
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Supplementary Materials and methods
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Fluorescent in situ hybridization (FISH) analysis
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A new oligonucleotide probe was designed to hybridise with GD/7 using the ARB software
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package (Ludwig et al, 2004). The suggested oligonucleotide probe, Ccat62, AGC AAG CTC
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TTC ATC GTC CGT, was found to match 243 sequences (1 Proteobacterium, 242
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Clostridiales, 239 Lachnospiraceae; 5 hits (all within the genus Coprococcus) were found for
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cultured bacteria) with the probe match function of the ribosomal database (Cole et al, 2009).
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Accessibility of the probe to the 16S rRNA molecule was checked (Behrens et al, 2003). The
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Ccat62 probe was synthesized and labelled with Cy3 dye (MWG-Biotech, Germany).
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Hybridisation conditions are 55oC overnight in hybridisation buffer (Franks et al, 1998);
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neither lysozyme nor formamide are used) followed by washing in salt buffer (0.9 M NaCl,
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20 mM Tris HCl; pH 7.2). The probe was validated against a panel of 24 gut bacterial isolates
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described previously (Aminov et al, 2006). The panel of strains used were selected to give
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broad coverage across the major bacterial groups present in the gut. The stains included
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representatives of the two major phyla of Firmicutes and Bacteroidetes. The strains were
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Streptococcus gordonii (DL1); Streptococcus bovis (26); Enterococcus faecalis (JH2-2);
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Faecalibacterium prausnitzii (A2-165); Bifidobacterium adolescentis (L2-32);
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Bifidobacterium longum (NCIMB 8809); Bifidobacterium infantis (DSM 20088);
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Eubacterium hallii (L2-7); Coprococcus eutactus (L2-50); Eubacterium rectale (A1-86);
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Roseburia intestinalis (DSM 14610T); Bacteroides vulgatus (DSM 1447T); Bacteroides
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thetaiotaomicron (B5482); Eubacterium cylindroides (T2-87); Lactococcus lactis (M41363);
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Lactobacillus acidophilus (A274); Megasphaera elsdenii (LC1); Escherichia coli (MG1655);
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Ruminococcus albus (SY3); Ruminococcus flavefaciens (17); Ruminococcus bromii (L2-63);
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Collinsella aerofaciens (DSM3979); Anaerostipes caccae (DSM 14662T) and Desulfovibrio
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piger (DSM 749). All 23 strains failed to hybridize with the Ccat62 probe whilst the C. catus
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strain was positive.
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The validated Ccat62 probe was employed to determine the abundance of the C. catus group
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in faecal samples from 10 healthy volunteers (5 females and 5 males) between 24 and 60
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years old. The volunteers had not taken antibiotics or other drugs known to influence the fecal
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microbiota for more than three months prior to the start of the study and ethical approval was
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obtained from Grampian Research Ethics Committee (project number 00/00133). Nine of the
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volunteers consumed a Western style diet and one volunteer consumed a vegetarian diet.
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Faecal samples were prepared for FISH as described previously (Walker et al, 2005).
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Formaldehyde treated samples from each donor were applied to wells on gelatin coated slides
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in triplicate and 25 fields of view were counted per well. The samples were hybridized with
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the fluorescently-labelled probe. The abundance of the C. catus group was estimated using the
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Ccat62 probe as a proportion of the total count using the Eub338 probe (Amann et al, 1995).
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Positive cells were viewed by epifluorescence microscopy (Leica, Germany).
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pduQ primer design
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For propanediol pathway PduP, it was difficult to find highly specific regions and it was
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therefore decided to also design primers for pduQ, which had a gene region very distinct from
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other related genes, as it is located next to pduP and displays similar levels of identity in
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bacteria carrying the pathway (analysed in R. inulinivorans A2-194, R. obeum ATCC 29174,
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R. obeum A2-162, Ruminococcus sp. SR1/5, Ruminococcus sp. 5_1_39B FAA, R. torques
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L2-14, R. gnavus ATCC 29149 and Clostridium sp. D5, data not shown), but is often absent
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in other bacteria carrying genes with lower similarity to pduP (no strong matches to pduQ
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gene found in Desulfosporosinus sp. strain OT, E. hallii DSM 3353, Fusobacterium ulcerans
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ATCC 49185, Fusobacterium varium ATCC 27725, Bacullus thermoglucosidasius C56-
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YS93, Clostridium beijerinckii NCIMB 8052, Clostridium botulinum B Erklund 17B,
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Ruminococcus torques ATCC 27756, Bacteroides thetaiotaomicron VPI-5482; pduQ only
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found in Akkermansia muciniphila BAA835; data not shown). Primers were also designed
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and validated to amplify across both genes (pduP and pduQ, Table 1 and Table S1).
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16S rRNA gene primer design for Negativicutes
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Negativicutes primers were designed by manual inspection of sequence alignments and
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specificity was checked using the ProbeMatch function of the Ribosome Database project
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(Cole et al, 2009). Primer specificity was validated with 16S rRNA amplicons of
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Megasphaera elsdenii ATCC 25940, Veillonella parvula L59, Anaerovibrio lipolytica ATCC
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33276, Bacteroides thetaiotaomicron VPI-5482, Bifidobacterium adolescentis DSM 20083,
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Collinsella aerofaciens DSM 3979, Clostridium acetobutylicum DSM 792, Faecalibacterium
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prausnitzii A2-165, Ruminococcus bromii L2-63, Eubacterium hallii L2-7, Eubacterium
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rectale A1-86, Ruminococcus obeum A2-162, Eubacterium cylindroides T2-87 and
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Desulfovibrio piger DSM 749. Any amplification detected with non-target strains was at least
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104-fold lower than target strains. The PCR amplification protocol was the same as described
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in the main manuscript with 30 s extension time.
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References
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Aminov RI, Walker AW, Duncan SH, Harmsen HJM, Welling GW, Flint HJ. (2006).
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Molecular diversity, cultivation, and improved detection by fluorescent in situ hybridization
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of a dominant group of human gut bacteria related to Roseburia spp. or Eubacterium rectale.
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Appl Environ Microbiol 72: 6371-6376.
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Amann RI, Ludwig W, Scheifer KH. (1995). Phylogenetic identification and in situ detection
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of individual microbial cells without cultivation. Microbiol Rev 59: 143-169.
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Behrens S, Ruhland C, Inacio J, Huber H, A. Fonseca, Spencer-Martins I, et al. (2003). In situ
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accessibility of small-subunit rRNA of members of the domains Bacteria, Archaea, and
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Eucarya to Cy3 labelled oligonucleotide probes. Appl Environ Microbiol 69: 1748-1758.
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Cole JR, Wang Q, Cardenas W, Fish J, Chai B, Farris RJ, et al. (2009). The Ribosomal
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Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res
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37: D141-145.
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Franks AH, Harmsen HJM, Raangs GC, Jansen GJ, Welling GW. (1998). Variations of
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bacterial populations in human faeces quantified by fluorescent in situ hybridization with
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group-specific 16S rRNA-targeted probes. Appl Environ Microbiol 64: 3336-3345.
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Ludwig W, Strunck O, Westram R, Richter L, Meier H, Yadhukumar A et al. (2004). ARB: a
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software environment for sequence data. Nucleic Acids Res 32: 1363-1371.
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Walker AW, Duncan SH, McWilliam Leitch EC, Child MW, Flint HJ. (2005). pH and peptide
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supply can radically alter bacterial populations and short-chain fatty acid ratios within
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microbial communities from the human colon. Appl Environ Microbiol 71: 3692-3700.
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Titles and legends to supplementary figures
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Figure S1: Phylogenetic tree of deduced protein sequence of propanol dehydrogenase pduQ.
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Database matches to R. inulinivorans (ABC25529) of at least 70% as well as the top hits
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below this cut-off (Thermoanaeraobacterium saccharolyticum YP_006390853,55%; T.
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xylanolyticum YP_004471776, 56%; C. celatum ZP_19296596, 58%; C. methylpentosum
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ZP_03705304, 51%; Akkermansia muciniphila YP_001878706, 58% are shown. Number of
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hits within the metagenomic dataset of Qin et al (2010) with at least 55% identity are
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indicated to the right (sequences with at least 95% identity were grouped; for a list of all hits
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see Supplementary Table S7). Sequences from clone library analysis of a human faecal
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sample (≥95% identity grouped) are shaded. Grey tree branches indicate gene with lower
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identity to R. inulinivorans assumed not to be bona-fide pdQ genes.
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Titles to supplementary tables
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Table S1: Validation of degenerate primers.
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Table S2: Genome organisation of succinate pathway genes.
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Table S3: Methylmalonyl-CoA decarboxylase (MmdA) gene clone library analysis.
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Table S4: Genome organisation of acrylate pathway genes.
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Table S5: Metagenomic sequence hits from Qin et al (2010) to LcdA.
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Table S6: Metagenomic sequence hits from Qin et al (2010) to PduP.
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Table S7: Metagenomic sequence hits from Qin et al (2010) to PduQ.
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