harmonization of techniques associated with ruminal ... · harmonization of techniques associated...
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Functional (meta)genomics Harmonization of techniques associated with
ruminal microbiome and metagenome analysis
Graeme Attwood
Joint RuminOmics/Rumen Microbial Genomics Network Workshop
Functional (meta)genomics methods and techniques
Joint RuminOmics/Rumen Microbial Genomics Network Workshop
Gene/protein
functions
Genome/
metagenom
e
sequence
data
Phenotype
Transcriptomics
Proteomics
Metabolomics
Mutagenesis
Fu
nctio
nal g
en
om
ics
Handelsman et al,
2004
Joint RuminOmics/Rumen Microbial Genomics Network Workshop
Fold up-regulated
3.06
2.82
4.56
9.25
2.02
2.21
14.9
3.71
2.98
10.4
4.60
21.73
2.16
9.09
2.90
3.87
3.46
22.69
3.99
5.84
5.43
3.75
2.23
2.07
2.10
2.75
Microarray analysis of
genes involved in
hemicellulose degradation
Xylan
Xylose
mRNA Labelled
cDNA
Glycosyl hydrolase
ORF 896 cel5A xsa43H xsa43A xyn10A 897 898 899 900 ORF 901 902 903 904 894 891
5.48 8.22 7.63 8.45
3.17 2.18
8.52
16.86
22.69 21.73
3.87 2.23 2.02 Fold
up r
egula
tion
ABC transporter Transcriptional regulator Conserved hypothetical
Joint RuminOmics/Rumen Microbial Genomics Network Workshop
Proteome analysis
Xylan Xylose
Secretome
Membrane
associated
Intracellular
Gel matching
Differentially expressed protein spots identified
Spots excised, digested, MALDI-TOF analysis
Peptide mass fingerprints
Identify proteins and corresponding genes
57 kDa carbohydrate
binding protein
ORF 0313 CBM2 CBM2 Cna B
137 kDa
endoxylanase
Xyn10B (6.9%) CBM9 GH 10
Signal peptide Cell wall binding repeat CBM = Carbohydrate binding module
Joint RuminOmics/Rumen Microbial Genomics Network Workshop
Outside Inside
xylose
arabinose 4-O-methyl
glucuronic acid
ferulic acid
G G G G G
Xyn10A,10E
Xsa43J
Est1C
Xyn10A, 10B, 10E
Xsa43J
Cel 5A, 5C
Cpb
Cel 5A, 5C
Cbp
Cbp
Xyn10B
G glucose
Outside Inside
Arf51A
Arf51B
Xyn10D
Xyn8A
Xsa43A
Xsa43D
Xsa43E
Xsa43G
Xsa43H
Xyl39A
Gh3A
Agu67A
Outside
G G G G G
Xyn10A,10E
Xsa43J
Est1C
Xyn10A, 10B, 10E
Xsa43J
Cel 5A, 5C
Cpb
Cel 5A, 5C
Cbp
Cbp
Xyn10B
xylose
arabinose 4-O-methyl
glucuronic acid
ferulic acid
G glucose
Leahy et al. (2010) PLoS ONE 5(1): e8926 Joint RuminOmics/Rumen Microbial Genomics Network Workshop
Comparison of methanogen genomes
244 150 47
33
127
FGD DBA
Biochemistry/
structure
58 40
Chemogenomics targets
Joint RuminOmics/Rumen Microbial Genomics Network Workshop
Vaccine targets Vaccine targets
572
337
96
47
24
≥ 1 TMM
Unique to
methanogens
Conserved in
methanogens
ORFs ≤ 4 TMMs
ORFs ≥ 4 TMMs
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Fwd
HFGDBAC
Formyl-MF
CO2
Formyl-H4MPT
Methenyl-H4MPT
Methylene-H4MPT
Methyl-H4MPT
Fdred?
Fdox?
MF
Fdox?
Ftr1
Ftr2
Formate FdhABD1
FdhABD2
FdhC
H2O
Mch
H2
Hmd Mtd
Mcr AGCDB
AtwA1, A2 MvhBAGD-HdrACBD
CH4
H4MPT
Na+
Na+
CoBS-SCoM
HSCoM
HSCoB
Methyl-S-CoM
HSCoM
Na+
MF
H4MPT
F420 H2
F420
Mer
H+
FrhBGDA
Nha
ADP+Pi H+/Na+
ATP
AhaHIK
ECFABD
H2
Fdox?
F420 H2
F420 H2
pool
F420 NADPH
+H+
FrhBGDA
NADP+
NpdG1,2
Fdred?
pool Eha / Ehb
complex
Capture of reductant Reduction of CO2 Energy conservation
Mtr
HGFAB
CDE
H2
F420 H2
F420
Fdred?
pool
F420
two NADPH-dependent
F420 dehydrogenase
(npdG1, 2) genes and
three NADP-dependent
alcohol dehydrogenase
(adh1, 2 and 3) genes
does this allow growth
on ethanol or
isopropanol via NADP+-
dependent oxidation of
the alcohol coupled to
F420 reduction of
methenyl-H4MPT to
methyl-H4MPT?
Joint RuminOmics/Rumen Microbial Genomics Network Workshop
Stimulation of M1 growth by alcohols
Methanol MeOH + H2
MeOH
H2
Ethanol
5 mM EtOH +
H2
10 mM
EtOH +
H2
H2
5 or 10 mM
EtOH
Joint RuminOmics/Rumen Microbial Genomics Network Workshop
Joint RuminOmics/Rumen Microbial Genomics Network Workshop
Locus tag Annotation Fold difference
Energy Metabolism - Formate Metabolism
mru0333 formate dehydrogenase alpha subunit FdhA 3.46
mru0334 formate dehydrogenase beta subunit FdhB 2.34
Energy Metabolism - Methanogenesis
mru1928 methyl-coenzyme M reductase beta subunit McrB 2.26
mru1926 methyl-coenzyme M reductase C subunit McrC 2.39
mru1927 methyl-coenzyme M reductase D subunit McrD 2.23
mru1925 methyl-coenzyme M reductase gamma subunit McrG 3.41
mru1919 tetrahydromethanopterin S-methyltransferase subunit A MtrA 2.02
mru1920 tetrahydromethanopterin S-methyltransferase subunit B MtrB 2.23
mru1921 tetrahydromethanopterin S-methyltransferase subunit C MtrC 3.23
mru1916 tetrahydromethanopterin S-methyltransferase subunit H MtrH 2.14
mru1907 methyl viologen-reducing hydrogenase gamma subunit MvhG 2.25
mru0344 tungsten formylmethanofuran dehydrogenase subunit A FwdA 2.12
Cell envelope - Cell surface
mru1222 adhesin-like protein 2.31
mru2173 adhesin-like protein 2.28
mru2134 adhesin-like protein 4.08
mru0076 adhesin-like protein 2.01
mru0828 adhesin-like protein with transglutaminase domain 2.14
mru1499 adhesin-like protein with transglutaminase domain 2.50
Fwd
HFGDBAC
CO2
Formyl-MF
Formyl-H4MPT
Methenyl-H4MPT
Methylene-H4MPT
Methyl-H4MPT
CH4
Fdred?
Fdox?
MF Fdox?
H4MPT
Ftr1
Ftr2
Formate F420
FdhABD1
FdhABD2
FdhC
H2O
Mch
H2
Hmd Mtd
Mcr AGCDB AtwA1, A2
MvhBAGD-HdrACBD
H4MPT
Na+
CoBS-SCoM
HSCoM
HSCoB Methyl-S-CoM
HSCoM
Na+
MF
F420 H2
F420
Mer FrhBGDA
H2
Fdox?
F420 H2
F420 H2
pool
Fdred?
pool Eha / Ehb
complex
Mtr
HGFA
BCDE
H2
F420 H2
F420
Fdred?
pool
Significantly up-regulated (>2 fold) in
co-culture vs monoculture
Formylmethanofuran
dehydrogenase (fwdA),
H4MPT methyltransferase
(mtrABCH)
Methyl CoM reductase (mcrBCDG),
Methyl viologen-reducing
hydrogenase (mvhG),
Formate utilisation (fdhAB)
Joint RuminOmics/Rumen Microbial Genomics Network Workshop
Sheep vary in their CH4 emissions per unit
of intake
9
10
11
12
13
14
15
16
17
18
CH
4 e
mis
sio
n (
g p
er
kg
DM
I)
Sheep
High CH4 emitters
Low CH4
emitters
Joint RuminOmics/Rumen Microbial Genomics Network Workshop
4 highest and 4 lowest methane emitting sheep, stomach tubed rumen content x 2 time points
Rumen microbial DNA
16S rRNA
Marker Gene Sequencing
(PCR Amplification)
Metagenomic
Sequencing
(0.84TB)
Rumen Microbial RNA
Metatranscriptomic
Sequencing
(0.25TB)
Sampling, nucleic acids extractions and
analyses
RNA extracted for transcript
analysis using a hot lysis-acid
phenol extraction method.
RNA concentration was
determined using Qubit and
quality checked via Bioanalyzer
DNA extracted from frozen
rumen contents using
“Repeated Bead Beating and
Column (RBB+C) purification”
method (Yu & Morrison, 2004)
HMW DNA, for large paired-
end insert libraries, prepared
using non-mechanical lysis
DNA extraction method
(Rosewarne et al, 2011)
Joint RuminOmics/Rumen Microbial Genomics Network Workshop
DNA extracted from frozen digesta using “Repeated Bead Beating and
Column (RBB+C) purification” method (ref)
HMW DNA, for large paired-end insert libraries, prepared using a non-
mechanical lysis DNA extraction method (ref)
RNA extracted for transcript analysis using a hot lysis-acid phenol
extraction method.
RNA concentration was determined using Qubit and quality checked via
Bioanalyzer
DNA and RNA extractions
Joint RuminOmics/Rumen Microbial Genomics Network Workshop
In vitro experimentation on methanogen
co-cultures
• Methanogens in the rumen usually experience H2 concentrations in the range
0.1 to 50 µM
• In the lab, methanogens grown by pumping with H2 + CO2 to 2 atm = ~770 µM
H2
• As methanogens grow, H2 is used, so over time H2 concentration falls in
cultures, until cultures are re-pumped with H2
• Controlling H2 at a low concentration is technically not possible without a H2-
producing partner
• Low H2 concentrations achieved by co-inoculating with the slow growing,
cellulose-degrading, H2-producing rumen bacterium Ruminococcus
flavefaciens FD1
• In these co-cultures, H2 is expected to be in the rumen relevant range ie 0.1 to
50 µM
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Co-culture set up and sample collections
• Methanobrevibacter ruminantium M1
8 x 50 ml methanogen mono or co-cultures with Ruminococcus
flavefaciens FD1 as hydrogen producer growing on cellulose
pumped with H2 + CO2 or N2 (FD1)
M1 grown on H2 + CO2 (hydrogenotrophic)
38oC with shaking
CH4, H2 measurements made daily
4 x 50ml samples collected for RNA, DNA, protein and VFAs at 2 time points
RNA extracted
VFAs analysed
rRNA depletion
RNA seq
DNA removed
cDNA synthesis
qPCR of specific transcripts
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-2
-1
0
1
2
3
4
M1 52hr M1 76hr M1+FD1High H2
52hr
M1+FD1High H2
76hr
M1+FD1Low H2
52hr
M1+FD1Low H2
76hr
Succinic
Formic
Acetic
0
1
2
3
4
FD1alone52hr
FD1alone76hr
Volatile Fatty Acids
Methanobrevibacter
ruminantium M1 in co-culture
with Ruminococcus
flavefaciens FD-1
0
5
10
15
20
25
30
0 24 48 72 96
M1
M1 + FD1 High H2
M1 + FD1 Low H2
Joint RuminOmics/Rumen Microbial Genomics Network Workshop
Cultured rumen methanogens available for
in vitro testing
Methanogen Strain Mode of methane formation
Methanobrevibacter spp.
(ruminantium group)
M1,
YLM1,
NT7*
hydrogenotrophs
Methanobrevibacter spp.
(gottschalkii group) SM9, D5 hydrogenotrophs
Methanobrevibacter sp. (wolinii clade) AbM4 hydrogenotroph
Methanobacterium formicicum BRM9 hydrogenotroph
Methanosphaera sp. ISO3-F5 methylotroph
Methanosarcina sp. CM1 aceticlastic
Rumen Cluster C ISO4-H5 methylotroph, co-culture with a
Succinivibrio sp.
H2 producer Preferred substrate
Ruminococcus flavefaciens FD-1 Cellulose degrader
Butyrivibrio proteoclasticus B316 Xylan degrader
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Acknowledgements
AgResearch Core funding
Bill Kelly
Sinead Leahy
Christina Moon
Eric Altermann
Adrian Cookson
Dragana Gagic
Suzanne Lambie
Jonathan Dunne
Zhanhao Kong
Carl Yeoman
Sandra Kittelmann
Henning Seedorf
Peter Janssen
Ron Ronimus
Neil Wedlock
John McEwan
Cesar Pinares-Patino
Joint Genome Institute
Edward Rubin
Weibing Shi
Christina Fan
Funding provided through the NZAGRC science programme
Research was funded by and carried out under contract to the Pastoral
Greenhouse Gas Research Consortium (PGgRc)
Funded by the New Zealand Government to support the objectives of the Livestock
Research Group of the Global Research Alliance on Agricultural Greenhouse Gases