Metagenomics and Methanogenesis in Ruminants Chris McSweeney CSIRO Livestock Industries
The metagenomics challenge unleashing the microbial world Our understanding of the microbial world based on pure cultures One or several cell types Majority of microbes in nature resistant to culture Complex and diverse mixtures
"the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species." US National Research Council 2007
A Global Problem IPCC 1.1 billion ruminants in the world 20-40 L methane /day Methane has 23 times more GWP carbon dioxide 21 million tonnes CO 2 equiv. Australian livestock greenhouse emissions = 11% of total CO 2 e 200-350 L methane/day
Role of methanogens in the rumen CH 4 CO 2 Fibre-degrading bacteria protozoa fungi Plant polysaccharides CO 2 CH 4 Soluble sugars NAD NADH A P B L S F H 2 CO 2 methanogens alternative routes of utilisation? CH 4 ~10% DE A P B CSIRO. absorbed across epithelium and utilised by the ruminant Courtesy of G. Attwood
Who s there (Identity)? linear representation of 16S rrna gene (~1550 bases) 5' 3' highly conserved variable highly variable EUKARYA BACTERIA 10 11-10 12 cells/ml > 90% uncultured Groupe 1 Groupe 2 Groupe 3 Groupe 4 Groupe 5 Groupe 6 Increasing variability ARCHAEA (methanogens)
Project elements (Meta)genomics/proteomics methanogens Cattle/sheep genetics High & Low emitters Mt Metagenomics alternative H 2 users in absence of methanogens Metagenomics marsupial forestomach Rumen methane and hydrogen measurement technologies H 2 is central Delivery Pre and Probiotics Bioactive Compounds Vaccine
Comparative metagenomics novel microbes underpinning lower methane emissions? High methane Proteobacteria Low methane Firmicutes Bacteroidetes Firmicutes Proteobacteria Comparative analysis Bacteroidetes Methanogens Methanogens
Alternative hydrogenotrophic pathways Cellulose Hemicellulose Starch Hexose CO 2 Formate Reductive acetogenesis: 4H 2 + 2CO 2 CH 3 COOH +H 2 O Pyruvate CO 2 Acetyl CoA Acetogens normal gut flora but diverse phylogenetically and metabolically Lactate Oxaloacetate CO 2 Acryl CoA Malate Butyrate Fumarate Acetate Rumen & forestomach samples Propionate Succinate CO 2 CO 2 CH 4 Enrichment cultures
The reductive acetogensis pathway reconstructed from metagenomic data (454 pyrosequencing) Wood-Ljungdahl acetyl-coa pathway 15 sequences 36 sequences 34 sequences 4 sequences 34 sequences 2 sequences 34 sequences 12 sequences
MEGAN assignment of reductive acetogen pathway genes to phylogenetic groups (NCBI taxonomy data) ACS: acetyl-coa-synthase CODH: carbon monoxide dehydrogenase cooc: accessory protein MTHFT: methyltetrahydrofolate methyltransferase CFeSP_large: corrinoid/iron-sulfur protein large subunit CFeSP_small: corrinoid/iron-sulfur id/i protein small subunit FDH: formate dehydrogenase FTHFS: formyl tetrahdrofolate synthase MTFHC/MTHFD: methyltetrahydrofolatecyclohydrolase MTHFR: methyltetrahydrofolate reductase PFOR: pyruvate ferroxidoreductase.
Rumen microbial responses to inhibition of methanogenesis timothy/p5 timothy/p5 + BCM-CD Bromochloromethane (BCM) methanogen inhibitor Low 0.5g/100kg live weight Adaption period to feed control Adaption to BCM BCM 14 days 8 days 18 days 8 days Day 1 2 3 4 5 6 7 8 Mid 2g/100kg g live weight A Chamber 1 out of chamber High 5g/100kg live weight B Chamber 2 out of chamber C out of chamber Chamber 1 D out of chamber Chamber 2 Collect rumen fluid.
Methane and hydrogen production Methane #501 #503 #418 (L/day) H BCM M BCM L BCM Control M H BCM M BCM L BCM Control H BCM M BCM L BCM Control Hydrogen
Barcoded pyrosequencing of 16S rdna in rumen samples 3 animals at 4 treatments (Control, Low, Mid & High) gdna & cdna 24 samples barcoded for Bacteria and Archaea* Filtered for length 300 550 bp ~257000 sequence reads Count 60000 Read length 50000 40000 30000 20000 10000 0 40 80 120 160 200 240 280 320 360 400 440 Length bp 480 520 560 600 640 680
Family level assignment Control Control BCM-high Acetic Propionic Butyric (mm) Control 31.0 8.1 5.4 +BCM (high) 32.0 12.7* 6.5 * BCM * * * * * *
Prevotella spp. 16S rdna placement Cellulose Hemicellulose Starch Hexose CO 2 Formate Pyruvate CO 2 Acetyl CoA Lactate Oxaloacetate CO 2 Acryl CoA Malate Butyrate Prevotella ruminicola Fumarate Acetate Propionate Succinate CO 2 CO 2 CH 4
Methanogen population monitoring mcra gene libraries & qpcr 10 Fold diffe erence 1 a 0.1 a 0.01 Low Mid High
Uncultured Rumen Methanogens Rumen cluster C Uncultured rumen methanogens 25% all sequences Janssen and Kirs (2008) AEM: 74, 3619
Microbial metagenomics: Genomes from metagenomes Putting the pieces together Bacteroides spp. Clostridium spp. Archaeon (RCC) Complete assembly of the archaeon Complete assembly of the archaeon Small low complex genome 1.46Mb Novel methanogenic pathway
Functional studies Hydrogenotrophy formate CO 2 H 2 MFR Lumazine (2,4-pteridinedione) - analogue of methanopterin H MPT 4 CHO - MFR CHO -H 4 MPT MFR 60.00 Methylotrophy Methylated compounds H 2 50.00 CH?H 4 MPT H 2 40.00 H 2 CH 2 =H 4 MPT CH 3 -H 4 MPT H 4 MPT methyltransferase CH 3 -S- CoM CH 4 μmole es 30.00 20.00 10.00 0.00 CH3-compounds BES Lum Mev HS- CoM + HS- CoB Methyl coenzyme M reductase ( mcra ) H 2 H 2 CoM-S-S- CoB CH 4 BES- Structural analogue of CoM Mevinolin (mevastatin) inhibitor cell wall synthesis
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