Metagenome Analysis: a case study
Analysis of a thermophilic terephthalate-degradingsyntrophic community
Thanos Lykidis
Two major metabolic reconstruction questions:degradation of TA and subsequent methanogenesis
Our goal
• Who is there? (Phylogenetic diversity)
• What are they doing? (Metabolic diversity)
Bacterial 16S rRNA gene clone libraries
Firmicutes: DesulfotomaculumDelta: dechlorinating clone groupThermotogaeDelta: SyntrophaceaeOP5Spiro: SP/WWE1Clostridia: Pepto/AcidaminoOther FirmicutesBacteroidalesOther DeltaproteobacteriaAC1Delta: DesulfomonileSynergistesChlorobiCaldithrixChloroflexiOther Spirochaetesmarine group ABetaproteobacteriaOP9/JS1Epsilon proteobacteriaGammaproteobacteriaOP8ActinobacteriaOther Bacteria
Day 221Day 280
Day 346
Day 430
Pelotomaculum spp.
Candidate phylum: OP5
Thermotogae
WWE1
Which contigs are coming from OP5?Check the 16S RNAS
Estimation of the OP5 genome size
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 Mband contain 3200 genes.
OP5 gene distribution
1.4 Mb of sequence has been binned to OP5. Phylopithia analysis predicts the existence of 215 scaffolds in the OP5 binthe 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
What is the coverage of an isolate genome?
57% of the isolate Pelotomaculum genome isRepresented in the metagenome
TA degradation; metal/cofactor-independent decarboxylases
Non-oxidative reversible aromatic decarboxylase/carboxylases
C B D
Tadcc27178 IMG top hits
Tadcc27178 NCBI top hits
Firmicutes; Lactobacillales; Symbiobacterium
Firmicutes; Clostridia; Clostridiales; Heliobacteriaceae
Tadcc27166 IMG top hits
Tadcc27171 IMG top hits
Checking the assembly
Checking the assembly
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)
COG1234 - Metal-dependent hydrolases of the beta-lactamase superfamily III
COG0689 - RNase PH
ISChy7, transposase
COG0796 - Glutamate racemase
COG0860 - N-acetylmuramoyl-L-alanine amidase
ISChy7, transposase [Carboxydothermus hydrogenoformans Z-2901]
COG2452 - Predicted site-specific integrase-resolvase
COG3583 - Uncharacterized protein conserved in bacteria
hypothetical protein
COG2109 - ATP:corrinoid adenosyltransferase
COG0084 - Mg-dependent DNase
COG0143 - Methionyl-tRNA synthetase
Peletomaculum spBenzoyl-CoA reductase and ligase??
Methanogenesis
A) Hydrogenotrophic B) Acetoclastic
16S rRNA gene-based Phylogeny (Day 346)16S rRNA gene-based Phylogeny (Day 346)
Hydrogenotrophic methanogens (mcrA)
Methyl coenzyme M reductase (alpha subunit)-based phylogeny tree
Methanomicrobiales
Methanosarcinales
TA
Pelotomaculum
Methanolinea
Methanosaeta
CH4
OP5
Ac
Ac
CO2
CO2
H2
But
CH4
CO2H2
Syntrophus