1. Metagenome Analysis: a case study Analysis of a thermophilic terephthalate-degrading syntrophic community Thanos Lykidis

2. Two major metabolic reconstruction questions: degradation of TA and subsequent methanogenesis

3. Our goal Who is there? (Phylogenetic diversity) What are they doing? (Metabolic diversity)

4. Bacterial 16S rRNA gene clone libraries Firmicutes: Desulfotomaculum Delta: dechlorinating clone group Thermotogae Delta: Syntrophaceae OP5 Spiro: SP/WWE1 Clostridia: Pepto/Acidamino Other Firmicutes Bacteroidales Other Deltaproteobacteria AC1 Delta: Desulfomonile Synergistes Chlorobi Caldithrix Chloroflexi Other Spirochaetes marine group A Betaproteobacteria OP9/JS1 Epsilon proteobacteria Gammaproteobacteria OP8 Actinobacteria Other Bacteria Day 221 Day 280 Day 346 Day 430 Pelotomaculum spp. Candidate phylum: OP5 Thermotogae WWE1

6. Which contigs are coming from OP5? Check the 16S RNAS

14. Estimation of the OP5 genome size

16. Based on the content of the OP5 bin in single copy COGs (90/180) we predict that we have covered approximately 50% of the genome. Therefore, we predict that the OP5 genome will be approximately 2.8 Mb and contain 3200 genes.

18. OP5 gene distribution

19. 1.4 Mb of sequence has been binned to OP5. Phylopithia analysis predicts the existence of 215 scaffolds in the OP5 bin the longest bin is 58.9 kb and 40 scaffolds longer than 10kb. A total of 1613 genes are predicted to originate from the OP5 phylum Analysis of the genes classified in the OP5 bin indicates the existence of the non mevalonate pathway for isoprenoid biosynthesis, the Sec system for protein translcation, components of peptidoglycan biosynthesis, and the biosynthesis of CoA, NAD, pyridoxal, the transport of thiamine OP5 has a butyryl-CoA:acetoacetate CoA-transferase alpha subunit (tadcc30460) (EC 2.8.3.9) that participates in butyrate synthesis. OP5 also has representatives of the phosphotransacetylase butyrate kinase pathway for butyrate formation

23. What is the coverage of an isolate genome? 57% of the isolate Pelotomaculum genome is Represented in the metagenome

26. TA degradation; metal/cofactor-independent decarboxylases

27. Non-oxidative reversible aromatic decarboxylase/carboxylases

31. C B D

32. Tadcc27178 IMG top hits

33. Tadcc27178 NCBI top hits Firmicutes; Lactobacillales; Symbiobacterium Firmicutes; Clostridia; Clostridiales; Heliobacteriaceae

34. Tadcc27166 IMG top hits

35. Tadcc27171 IMG top hits

36. Checking the assembly

41. BcrC/BadD/HgdB: benzoyl-CoA reductase/2-hydroxyglutaryl-CoA dehydratase subunit Activator of 2-hydroxyglutaryl-CoA dehydratase (HSP70-class ATPase domain) [ligase] (putative CoA-substrate-specific enzyme activase) COG1234COG1234 - Metal-dependent hydrolases of the beta-lactamase superfamily III COG0689COG0689 - RNase PH ISChy7, transposase COG0796COG0796 - Glutamate racemase COG0860COG0860 - N-acetylmuramoyl-L-alanine amidase ISChy7, transposase [Carboxydothermus hydrogenoformans Z-2901] COG2452COG2452 - Predicted site-specific integrase-resolvase COG3583COG3583 - Uncharacterized protein conserved in bacteria hypothetical protein COG2109COG2109 - ATP:corrinoid adenosyltransferase COG0084COG0084 - Mg-dependent DNase COG0143COG0143 - Methionyl-tRNA synthetase Peletomaculum sp Benzoyl-CoA reductase and ligase??

42. Methanogenesis A) HydrogenotrophicB) Acetoclastic

43. 16S rRNA gene-based Phylogeny (Day 346)

44. Hydrogenotrophic methanogens (mcrA)

45. Methyl coenzyme M reductase (alpha subunit)- based phylogeny tree Methanomicrobiales Methanosarcinales

46. TA Pelotomaculum Methanolinea Methanosaeta CH 4 OP5 Ac CO 2 H2H2 But CH 4 CO 2 H2H2 Syntrophus