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