Table S2. Number of Orfs Annotated in Each Subsystem in the MG-RAST Program (Meyer Et Al

Fig.S1Enzymatic assay to measure CMCase (a), β-glucosidase (b) and xylanase (c) activities in clones of the metagenomic library. One aliquot of concentrate supernatant obtained from the consortium (Souza, 2012) was used as a control for CMCase and xylanase activities

Fig.S2Presence of genes coding for carbohydrate-active enzymes in different fosmid clones from the metagenomic library. The presence of each gene was investigated by PCR using the primers listed in Table 2. Proteins of the glycosylhydrolase (GH) family are identified

MG-RAST level 1 distribution
Subsystems / All contigs / 12 selected contigs
Clustering-based subsystems / 111 / 19
Carbohydrates / 89 / 39
Miscellaneous / 73 / 26
Amino Acids and Derivatives / 61 / 8
Cofactors, Vitamins, Prosthetic Groups, Pigments / 56 / 5
Membrane Transport / 41 / 11
Protein Metabolism / 35 / 2
Respiration / 26 / 5
Virulence, Disease and Defense / 26 / 7
RNA Metabolism / 25 / 5
Nucleosides and Nucleotides / 19 / 6
Stress Response / 18 / 5
Fatty Acids, Lipids, and Isoprenoids / 17 / 2
DNA Metabolism / 16 / 4
Regulation and Cell Signaling / 16 / 2
Iron Acquisition and Metabolism / 14 / 4
Cell Wall and Capsule / 13 / 1
Dormancy and Sporulation / 13 / 0
Motility and Chemotaxis / 12 / 1
Sulfur Metabolism / 12 / 4
Metabolism of Aromatic Compounds / 9 / 1
Phages, Prophages, Transposable elements, Plasmids / 6 / 2
Phosphorus Metabolism / 5 / 0
Cell Division and Cell Cycle / 3 / 2
Secondary Metabolism / 2 / 0
Nitrogen Metabolism / 1 / 0
Total of ORFs / 719 / 161

Table S2. Number of ORFs annotated in each subsystem in the MG-RAST program (Meyer et al., 2008).

Table S3. Distribution of ORFs in categories within the Clustered-based subsystem in the MG-RAST program (Meyer et al., 2008).

MG-RAST Level 2 distribution
Categories within Clustered Based Subsystem / All contigs / 12 selected contigs
Uncharacterised / 79 / 14
Protein export? / 7 / 0
TldD cluster / 6 / 1
Fatty acid metabolic cluster / 4 / 0
Putative associate of RNA polymerase sigma-54 factor rpoN / 4 / 1
Ribosome-related cluster / 4 / 0
Cell Division / 3 / 1
Hypothetical protein possible functionally linked with Alanyl-tRNAsynthetase / 3 / 0
Lysine, threonine, methionine, and cysteine / 3 / 1
Nucleotidyl-phosphate metabolic cluster / 3 / 0
Biosynthesis of galactoglycans and related lipopolysaccharides / 2 / 1
Hypothetical in Lysine biosynthetic cluster / 2 / 0
Carbohydrates / 1 / 1
Chemotaxis, response regulators / 1 / 0
Clustering-based subsystems / 1 / 0
Cytochrome biogenesis / 1 / 0
D-tyrosyl-tRNA(Tyr) deacylase (EC 3.1.-.-) cluster / 1 / 0
DNA metabolism / 1 / 0
Hypothetical Related to DihydroorateDehydrogenase / 1 / 0
Hypothetical lipase related to Phosphatidate metabolism / 1 / 1
Methylamine utilization / 1 / 1
Probably GTP or GMP signaling related / 1 / 0
Pyruvatekinase associated cluster / 1 / 0
Ribosomal Protein L28P relates to a set of uncharacterised proteins / 1 / 0
Translation / 1 / 0
Two related proteases / 1 / 1
Proteosome related / 1 / 0

Table S4.Distribution of ORFs in subcategories within the category Sugar utilization in Thermotogales in the Clustered-based subsystem in the MG-RAST program (Meyer et al., 2008).

MG-RAST Level 4 distribution
Subcategories within the category Sugar utilization in Thermotogales / All contigs / 12 selected contigs
Alpha-L-fucosidase (EC 3.2.1.51) / 1 / 1
Alpha-N-arabinofuranosidase (EC 3.2.1.55) / 1 / 1
Alpha-galactosidase (EC 3.2.1.22) / 2 / 1
Beta-galactosidase (EC 3.2.1.23) / 1 / 1
Beta-glucosidase (EC 3.2.1.21) / 5 / 5
Beta-hexosaminidase (EC 3.2.1.52) / 2 / 2
Beta-xylosidase (EC 3.2.1.37) / 1 / 1
Endo-1,4-beta-xylanase A precursor (EC 3.2.1.8) / 2 / 2
Fructokinase (EC 2.7.1.4) / 1 / 1
Glycerol kinase (EC 2.7.1.30) / 1 / 0
Glycerol-3-phosphate ABC transporter, permease protein UgpA (TC3.A.1.1.3) / 2 / 0
Glycerol-3-phosphate ABC transporter, permease protein UgpE (TC3.A.1.1.3) / 1 / 0
Ribulose-phosphate 3-epimerase (EC 5.1.3.1) / 1 / 0
Transketolase (EC 2.2.1.1) / 1 / 0
Xylose ABC transporter, permease component / 1 / 1
Xylose ABC transporter, substrate-binding component / 2 / 2

Table S5. Distribution of ORFs in categories within the Carbohydrate subsystem in the MG-RAST program (Meyer et al., 2008).

MG-RAST Level 2 distribution
Categories within the Carbohydrate subsystem / All contigs / 12 selected contigs
Uncharacterised / 25 / 18
Central carbohydrate metabolism / 19 / 4
Di- and oligosaccharides / 18 / 17
CO2 fixation / 17 / 2
Monosaccharides / 14 / 11
Sugar alcohols / 12 / 2
Fermentation / 9 / 1
One-carbon Metabolism / 9 / 0
Aminosugars / 6 / 6
Organic acids / 3 / 0
Polysaccharides / 3 / 2
Glycoside hydrolases / 1 / 1

Table S6. Distribution of ORFs in categories within the Amino Acids and Derivatives subsystem in the MG-RAST program (Meyer et al., 2008).

MG-RAST Level 2 distribution
Categories within Amino Acids and Derivatives subsystem / All contigs / 12 selected contigs
Alanine, serine, and glycine / 11 / 2
Arginine; urea cycle, polyamines / 7 / 0
Aromatic amino acids and derivatives / 8 / 2
Branched-chain amino acids / 14 / 1
Glutamine, glutamate, aspartate, asparagine; ammonia assimilation / 3 / 0
Histidine Metabolism / 3 / 1
Lysine, threonine, methionine, and cysteine / 19 / 2
Proline and 4-hydroxyproline / 3 / 0

Table S1. Number of ORFs classified by the MG-RAST program (Meyer et al., 2008) using the KEGG database.

Domain / All contigs / 12 selected contigs
Archaea / 1 / 0
Bacteria / 749 / 199
Eukaryota / 3 / 1
Unassigned / 9 / 4
Phylum / All contigs / 12 selected contigs
Actinobacteria / 14 / 8
Bacteroidetes / 4 / 2
Chlorobi / 1 / 0
Chloroflexi / 2 / 0
Chrysiogenetes / 1 / 0
Deinococcus-Thermus / 6 / 3
Firmicutes / 414 / 152
Fusobacteria / 3 / 3
Planctomycetes / 2 / 0
Proteobacteria / 296 / 25
Spirochaetes / 1 / 1
Synergistetes / 1 / 0
Thermotogae / 4 / 4
Verrucomicrobia / 1 / 1
750 / 199
Classes within the Firmicutes phylum
All contigs / 12 selected contigs
Bacilli / 38 / 19
Clostridia / 376 / 133
Classes within the Proteobacteria phylum
All contigs / 12 selected contigs
Alphaproteobacteria / 32 / 4
Betaproteobacteria / 45 / 9
Deltaproteobacteria / 13 / 1
Epsilonproteobacteria / 2 / 0
Gammaproteobacteria / 204 / 11

Table S7. Comparison of amino acid sequences of ORFs in Contig 31 against the Non-redundant protein database (nr) at NCBI.

Contig gene / Coverage (%) / Identity (%) / Closest genus at NCBI / Corresponding protein
1 / 71 / 86 / Clostridium / HD superfamilyphosphohydrolases
2 / 99 / 99 / Clostridium / Hypothetical protein
3 / 95 / 99 / Clostridium thermocellum / Transposase
4 / 99 / 100 / Clostridium / Putative transposase
5 / 99 / 93 / Clostridium clarifavum / Transposase
6 / 98 / 98 / Clostridium stercorarium / Hypothetical protein
7 / 33 / 42 / Paenibacillus / AraC family transcriptional regulator
8 / 81 / 31 / Clostridiumintestinalis / Hypothetical protein
9 / 90 / 76 / Ruminoclostridium / Hypothetical protein
10 / - / - / no hit / no hits
11 / 90 / 41 / Paenibacillus / AraC family transcriptional regulator
12 / 93.2 / 67 / Bacillus / N-acetylmuramoyl-L-alanine amidase
13 / 98 / 55 / Robinsoniella / bgaB; beta-galactosidase [EC:3.2.1.23]
14 / 99 / 79 / Clostridiumcellulosi / Transport protein
15 / 97 / 53 / Robinsoniella / AXY8; alpha-L-fucosidase 2 [EC:3.2.1.51]
16 / 98 / 53 / Clostridiales / Hypothetical protein
17 / 99 / 46 / Paenibacillus / Hypothetical protein
18 / 88 / 60 / Robinsoniella / Hypothetical protein
19 / 95 / 67 / Robinsoniella / lplC; putative aldouronate transport system permease protein
20 / 94 / 74 / Robinsoniella / lplB; putative aldouronate transport system permease protein
21 / 99 / 68 / Robinsoniella / FUCA; alpha-L-fucosidase [EC:3.2.1.51]
22 / 97 / 65 / Robinsoniella / Hypothetical protein
23 / 99 / 66 / Clostridiales / Trimethylaminecorrinoid protein
24 / 96 / 62 / Lachnospiraceae bacterium / yicL; alpha-D-xylosidexylohydrolase [EC:3.2.1.177]
25 / 99 / 54 / Clostridiales bacterium / bglX; beta-glucosidase [EC:3.2.1.21]
26 / no hit / no hits
27 / 98 / 78 / Paenibacillus / yteR; unsaturated rhamnogalacturonylhydrolase [EC:3.2.1.172]
28 / 99 / 86 / Clostridium / E3.2.1.22B; alpha-galactosidase [EC:3.2.1.22]
29 / 99 / 77 / Clostridium / PGAM; 2,3-bisphosphoglycerate-dependent phosphoglyceratemutase [EC:5.4.2.11]
30 / 98 / 47 / Caudiprolobacter / Hypothetical protein
31 / 97 / 51 / Treponema / Multidrug transporter MATE

Table S8. Comparison of amino acid sequences of ORFs in Contig 61 against the Non-redundant protein database (nr) at NCBI.

Contig gene / Coverage (%) / Identity (%) / Closest genus at NCBI / Corresponding protein
1 / 71 / 48 / Thermoanaerobacter / Integrase
2 / 98 / 92 / Bacillus / ARSC2; arsenate reductase [EC:1.20.4.1]
3 / 97 / 80 / Clostridium / TC.ACR3; arsenite transporter, ACR3 family
4 / 99 / 86 / Clostridium / Redox-active disulfide protein 2
5 / 99 / 86 / Clostridium / Predicted permease
6 / 94 / 79 / Clostridium / arsR; ArsR family transcriptional regulator
7 / 99 / 86 / Clostridium stercorarim / Transposase
8 / 64 / 73 / Enterococcus / Recombinase
9 / 90 / 74 / Clostridium / Hypothetical protein
10 / 98 / 58 / Clostridium / Hypothetical protein
11 / 66 / 47 / Turicibacter / Transposase
12 / 95 / 57 / Bacillus / Transposase
13 / 66 / 62 / Bacillus / no hits
14 / 89 / 37 / Sporolactobacillus / Transcriptional Regulator
15 / 89 / 62 / Haloplasma / PTS-EI.PTSI; phosphotransferase system, enzyme I, PtsI [EC:2.7.3.9]
16 / 92 / 54 / Paenibacillus / PTS beta-glucoside transporter subunit
17 / 98 / 57 / VIrginibacillus / Hypothetical protein
18 / No hits
19 / 99 / 68 / Bacillus / Thioredoxinreductase
20 / no hits
21 / 86 / 44 / Haloplasma / Beta-glucosidase
22 / 99 / 54 / Acholeplasmabrassicae] / Predicted membrane protein, putative toxin regulator
23 / 91 / 45 / Listeria / Hypothetical protein
24 / 69 / 61 / Bacillus / Hypothetical protein
25 / 97 / 71 / Haloplasma / hupB; DNA-binding protein HU-beta
26 / 91 / 44 / Bacillus / Hypothetical protein
27 / 97 / 60 / Alkaliphilus / Multidrug transporter MatE
28 / 99 / 56 / Haloplasma / PepD; dipeptidase D [EC:3.4.13.-]
29 / 95 / 62 / Bacillus / Hypothetical protein
30 / 94 / 60 / Flavobacterium / Hypotetical protein
31 / 94 / 51 / Haloplasma / Sulfatase
32 / 92 / 27 / Rhodopirelulla / pgsA; CDP-diacylglycerol--glycerol-3-Phosphate 3-phosphatidyltransferase [EC:2.7.8.5]
33 / 85 / 32 / Clostridium / Glycoside hydrolase