Harmonization of techniques associated with ruminal ... · Harmonization of techniques associated...

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


Gene/protein

functions

Genome/

metagenom

e

sequence

data

Phenotype

Transcriptomics

Proteomics

Metabolomics

Mutagenesis

Fu

nctio

nal g

en

om

ics

Handelsman et al,

2004

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


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


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


Vaccine targets Vaccine targets

572

337

96

47

24

≥ 1 TMM

Unique to

methanogens

Conserved in

methanogens

ORFs ≤ 4 TMMs

ORFs ≥ 4 TMMs


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?


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



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




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)


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


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)


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


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


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


-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


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


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

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