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Comparative genomics of rumen methanogens
A thesis presented in partial fulfillment of the requirements for the
degree of Doctor of Philosophy in Biochemistry
At Massey University, Palmerston North, New Zealand
Yang Li
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Abstract
Methane (CH4) emissions from agriculture represent around 9% of global anthropogenic greenhouse gas emissions. The single largest source of this CH4 is animal enteric fermentation, predominantly from ruminant livestock, where it is produced mainly in their fermentative forestomach (or reticulo-rumen) by a group of archaea known as methanogens.
In order to reduce CH4 emissions from ruminants, it is necessary to understand the role of methanogenic archaea in the rumen, and to identify their distinguishing characteristics that can be used to develop CH4 mitigation technologies. To gain insights into the role of methanogens in the rumen environment, two methanogens have been isolated from ovine rumen and their genomes were sequenced: methanogenic archaeon ISO4-H5 of the order Methanomassiliicoccales and Methanobrevibacter sp. D5 of Methanobrevibacter gottschalkii clade.
Genomic analysis suggests ISO4-H5 is an obligate hydrogen-dependent methylotrophic methanogen, able to use methanol and methylamines as substrates for methanogenesis. Like other organisms within this order, ISO4-H5 does not possess genes required for the first six steps of hydrogenotrophic methanogenesis. Comparison between the genomes of different members of the order Methanomassiliicoccales revealed strong conservation in energy metabolism, particularly in genes of the methylotrophic methanogenesis pathway, as well as in the biosynthesis and use of pyrrolysine. Unlike members of Methanomassiliicoccales from human sources, ISO4-H5 does not contain the genes required for production of coenzyme M (CoM), and requires external supply of CoM to survive.
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Acknowledgements
It was my privilege to work under the guidance of Dr. Graeme Attwood, Dr. Sinead Leahy and Associate Professor Jasna Rakonjac, I am grateful for their contant guidance and unflinching support, they are a source of inspiration and motivation. This thesis would not be possible without them.
I am grateful to the New Zealand Agricultural Greenhouse Gas Research Centre and Massey University for the financial support and the opportunity they gave me to conduct a truly meaningful research project contributing towards CH4 mitigation. I would also like to thank AgResearch Ltd. for providing the excellent facility for me to conduct my research at the Grasslands Research Centre. My gratitude also to the Pastoral Greenhouse Gas Research Consortium (PGgRc) for kindly providing the methanogen cultures.
The experience and skills I have learned in my time here are invaluable, but the best thing that happened during my PhD is finding my better, wiser half, Jana Muller, who is also a PhD student here in AgResearch. She and my family provided me the strength to finish this long journey, which often seems never ending.
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Table of contents
Page
Abstract ii
Acknowledgements iii
Table of contents v
List of Tables x
List of Figures xvii
Abbreviations xxii
1
1.1. Greenhouse gases and global warming 1
1.2. New Zealand agriculture and associated CH4 emissions 1
1.3. The rumen environment and CH4 formation 2
1.4. CH4 mitigation strategies 5
1.5. Rumen methanogens 9
1.6. Methanogenesis 11
1.7 Methanogen Genomes 15
1.8. Research questions 18
19
2.1. Materials 19
2.1.1. Media 19
2.1.2. Media additives 20
2.1.3. Methanogen and bacteria strains 22
2.1.4. General materials 22
2.1.5. RNA purification materials 23
2.2. Methods 23
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2.2.2 Microscopy 25
2.2.3. Purification of methanogens 26
2.2.4. DNA extraction 27
2.2.5. PCR 28
2.2.6. Agarose gel electrophoresis 29
2.2.7. PCR product purification 29
2.2.8. DNA quantification 29
2.2.9. Sequencing of PCR products 29
2.2.10. Genome sequencing 30
2.2.11. Quality assessment 30
2.2.12. Genome annotation 31
2.2.13. Primer design 31
2.2.14. Genome gap closure 31
2.2.15. Pulsed field Gel Electrophoresis (PFGE) 32
2.2.16. Inference of phylogenetic divergence 32
2.2.17. Prediction of RNAs 33
2.2.18. Prediction of signal peptides and secretome 34
2.2.19. Prediction of adhesin-like proteins 34
2.2.20. Prediction of horizontal transfer regions and genes 34 2.2.21. Prediction of non-ribosomal peptide synthase (NRPS) domains and substrates 37 2.2.22. Prediction of insertion sequence (IS) elements 37 2.2.23. Prediction of Clustered Regularly Interspaced Short Palindromic Repeat
(CRISPR) 37
2.2.24. Metabolic pathway mapping 37
2.2.25. Codon frequency, amino acid usage, BLAST matrix and Pan-core plot 37
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2.2.27. Gene homology analysis 38
2.2.28. Metabolite analyses 38
2.2.29. Transcriptome analysis of methanogenic archaeon ISO4-H5 39 43
3.1. Introduction 43
3.2. Results 44
3.2.1. Genome sequencing results and assembly 44
3.2.2. Genome properties 45
3.2.4. Metabolic pathway reconstruction 59
3.3. Discussion 89
3.4. Conclusions 105
107
4.1. Introduction 107
4.2. Results 108
4.2.1. Phylogenetic relationship of members of the Methanomassiliicoccales isolated from
gut environments 108
4.2.2. Genome characteristics 111
4.2.3. Pyrrolysine usage 118
4.2.4. Secretome 121
4.2.5. Synteny 122
4.2.6. Comparative analysis of gene families 125
4.2.7. Comparative analysis of metabolic profiles predicted from genomic sequences 133
4.3. Discussion 148
4.4. Conclusions 169
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5.1. Introduction 171
5.2. Results 172
5.2.1. Isolation of ISO4-H5 172
5.2.2. Growth of S. dextrinosolvens H5 176
5.2.3. NMR analysis of SSPGMS-supplemented ISO4-H5 culture 178
5.2.4. Substrate utilisation 185
5.2.5. Analysis of ISO4-H5 gene expression during growth on different methanogenic
substrates and under high or low H2 191
5.2.6. Statistical analyses 195
5.2.7. Differential gene expression in ISO4-H5 199
5.2.8 Differential gene expressions in S. dextrinosolvens H5 208 5.2.9. Differential gene expression in R. flavefaciens FD1 209 5.2.10. Associations between the ISO4-H5 and R. flavefaciens FD1, S. dextrinosolvens
H5 transcriptomes 210
5.3. Discussion 214
229
6.1. Introduction 229
6.2. Results 232
6.2.1. Cell morphology 232
6.2.2. Genome sequencing results and assembly 232
6.2.3. Phylogenetic relationship of sequenced members of Methanobrevibacter 237 6.2.4. Genome properties of Methanobrevibacter sp. D5 and other sequenced
Methanobrevibacter spp. 241
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6.3. Discussion 285
6.4 Conclusion 300
303
7.1. Rationale 303
7.2. Advancements since the start of this thesis 304
7.3. Summary of thesis results 305
7.4. Relevance of thesis findings to CH4 formation in the rumen 306
7.5. Future perspectives 310
References 314
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List of Tables
Page
Table 1.1. Rumen methanogen genomes from Genomes OnLine Database (GOLD). 17 Table 2.1. The final concentrations or volumes of additives to a 10 mL culture of BY medium
in Hungate tubes 25
Table 2.2. Primers used for PCR amplification of 16S rRNA gene 28 Table 2.3. Primers used for PCR amplification of ISO4-H5 putative prophage region 29
Table 2.4. Bioinformatics software used in this study 35
Table 3.1. Genome assembly summary 44
Table 3.2. General features of the ISO4-H5 genome 46
Table 3.3. Predicted rRNA and ncRNA genes of ISO4-H5 48
Table 3.4. Predicted tRNAs in the ISO4-H5 genome 48
Table 3.5. Codon usage of ISO4-H5 50
Table 3.6. Genes within the ISO4-H5 genome predicted to incorporate pyrrolysine in their
encoded proteins 52
Table 3.7. IS elements of ISO4-H5 53
Table 3.8. CRISPR spacer homology 54
Table 3.9. Predicted horizontal gene transfer regions of ISO4-H5 55
Table 3.10. Adhesin-like proteins 57
Table 3.11. Genes involved in central carbon metabolism 65
Table 3.12. Genes encoding amino acid biosynthesis 67
Table 3.13. Genes involved in cofactor biosynthesis 73
Table 3.14. Genes involved in lipid biosynthesis 77
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Table 3.17. DNA recombination and repair 82
Table 3.18. ISO4-H5 RNA polymerase genes 82
Table 3.19. ISO4-H5 genes involved in aminoacyl-tRNA biosynthesis 83 Table 3.20. ISO4-H5 genes involved in tRNA modification 84
Table 3.21. Translation factors in the ISO4-H5 genome 84
Table 3.22. ISO4-H5 genes involved in secretion 85
Table 3.23. ISO4-H5 genes involved in protein folding and stability 85
Table 3.24. ISO4-H5 genes involved in protein degradation 86 Table 3.25. ISO4-H5 genes involved in regulatory protein interaction 87
Table 3.26. ISO4-H5 genes potentially involved in nitrogen fixation 88 Table 3.27. ISO4-H5 genes involved in oxidative stress 88 Table 4.1. General genome properties of sequenced members of Methanomassiliicoccales 112
Table 4.2. Non-coding RNAs in Methanomassiliicoccales genomes 113 Table 4.3. Predicted tRNAs of Methanomassiliicoccales 114
Table 4.4. Amber codon utilisation in Methanomassiliicoccales 1199 Table 4.5. Predicted secretome of Methanomassiliicoccales 121 Table 4.6. Gene content of Regions 1 and 2 in ISO4-H5 and ISO4-G11 genomes 125
Table 4.7. ISO4-H5 conserved gene families and novel gene families classified by COGs
functional categories 129
Table 4.8. Gene families conserved only in rumen Methanomassiliicoccales by functional
category 131
Table 4.9. Gene families conserved only in ovine rumen Methanomassiliicoccales by
functional category 131
Table 4.10. Amino acid biosynthesis summary 146
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Table 5.2. Metabolite concentrations estimated by 1H NMR spectroscopy 181 Table 5.3. Experimental set up of high and low H2 co-cultures of ISO4-H5 enrichment grown
with methanol or methylamine 192
Table 5.4. Quality of RNA extracted from ISO4-H5 co-cultures 194
Table 5.5. Reads aligned to ISO4-H5, S. dextrinosolvens H5 and R. flavefaciens FD1 genomes
using Rockhopper 195
Table 5.6. ISO4-H5 genes differentially expressed between Trt2 (high H2) and Trt3 (low H2)
200 Table 5.7. Genes with 2-fold expression differences in Trt4 compared with other treatments
204 Table 5.8. Genes with 2-fold expression differences in Trt1 compared with other treatments 206
Table 5.9. Genes differentially expressed between Trt2 and Trt3 209 Table 6.1. Methanobrevibacter species and their nutritional requirements 230
Table 6.2. Genome assembly summary of Methanobrevibacter sp. D5 from enrichment culture
H6 233
Table 6.3. Genome assembly summary of Methanobrevibacter sp. D5 234
Table 6.4. Phylogenetic divergence of Methanobrevibacter spp. indicated by the % identity of
16S rRNA gene. 237
Table 6.5. General features of the Methanobrevibacter sp. D5 genome 241 Table 6.6. General genome properties of sequenced species of the genus Methanobrevibacter 244
Table 6.7. Non-coding RNAs predicted in Methanobrevibacter spp. genomes 247 Table 6.8. Predicted tRNAs in Methanobrevibacter spp. genomes 247
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Table 6.10. CRISPR spacer homology of Methanobrevibacter sp. D5 251 Table 6.11. IS elements of Methanobrevibacter sp. D5 252
Table 6.12. Predicted secretome in Methanobrevibacter species 254 Table 6.13. Adhesin-like protein prediction of Methanobrevibacter sp. D5 256
Table 6.14. Conserved and novel genes of Methanobrevibacter genomes by functional
categories 262
Table 6.15. Methanobrevibacter gene families uniquely conserved in the rumen by functional
category 263
Table 6.16. Gene families uniquely conserved in the Mbb. gottschalkii clade by functional
category 264
Table 6.17. D5 genes predicted to be involved in secretion 281
Table 6.18. D5 genes involved in oxidative stress 282
Table A.3.1. Manual functional annotation of the methanogenic archaeon isolate ISO4-H5 predicted ORFs. Table excludes hypothetical proteins 383
Table A.4.1. Percentage codon usage in Methanomassiliicoccales genomes 393 Table A.4.2. CRISPR associated genes in Methanomassiliicoccales genomes analysed 395 Table A.4.3. Predicted pyrrolysine usage in the ISO4-G11 genome 396
Table A.4.4. Predicted pyrrolysine usage in the ISO4-G1 genome 397 Table A.4.5. Predicted pyrrolysine usage in the BRNA1 genome 398
Table A.4.6. Predicted pyrrolysine usage in the MpT1 genome 399 Table A.4.7. Predicted pyrrolysine usage in the RumEn M1 genome 400 Table A.4.8. Predicted pyrrolysine usage in the RumEn M2 genome 405
Table A.4.9. Predicted pyrrolysine usage in 1R26 genome 407 Table A.4.10. Domains of predicted secretome in the Methanomassiliicoccales 409
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Table A.4.12. Gene families unique to ISO4-H5 421
Table A.4.13. Gene families unique to ISO4-G1 426
Table A.4.14. Table A.4.14. Gene families unique to ISO4-G11 431
Table A.4.15. Genes involved in methanogenesis and energy generation in
Methanomassiliicoccales 436
Table A.4.16. Genes involved in central carbon metabolism in Methanomassiliicoccales 442 Table A.4.17. Genes involved in amino acid metabolism in Methanomassiliicoccales 445
Table A.4.18. Genes involved in nucleotide biosynthesis in Methanomassiliicoccales 450 Table A.4.19. Genes involved in cell replication in Methanomassiliicoccales 452 Table A.4.20. Genes involved in cofactor biosynthesis in Methanomassiliicoccales 454
Table A.4.21. Genes involved in secretion 45656
Table A.5.1. T-test of spectra bins in sample (paired) and log transformed with P-value
threshold of 0.05 45757
Table A.5.2. T-test of spectra bins in control (paired) and log transformed with P-value
threshold of 0.05 45858
Table A.5.3. Gene expressions of ISO4-H5 459
Table A.5.4. Gene expressions of Succinivibrio dextrinosolvens H5 464
Table A.5.5. Gene expressions of Ruminococcus flavefaciens FD1 466
Table A.5.6. FD1 genes with 2-fold expression difference in Trt2 and Trt3 475
Table A.6.1. Manual functional annotation of the Methanobrevibacter sp. D5 predicted ORFs
478
Table A.6.2. Percentage codon usage in Methanobrevibacter genomes 490
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Table A.6.4. Domains of predicted secretome in the Methanobrevibacter genomes analysed 495
Table A.6.5. Predicted Methanobrevibacter sp. D5 genes within the Methanobrevibacter core
genome 502
Table A.6.6. Predicted Mbb. ruminantium M1T genes conserved within the Mbb. ruminantium
clade 508
Table A.6.7. Table A.6.7. Predicted Methanobrevibacter sp. AbM4 genes conserved within the
Mbb. wolinii clade 510
Table A.6.8. Predicted Methanobrevibacter sp. D5 genes unique to the Methanobrevibacter sp.
D5 genome 511
Table A.6.9. Genes involved in methanogenesis and energy generation in Methanobrevibacter
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List of Figures
Page
Figure 1.1. The thermodynamic relationship between H2 concentration and VFA production
from cellulose and starch 4
Figure 1.2. Methanogenesis pathways 11
Figure 3.1. ISO4-H5 genome assembly and representation 45 Figure 3.2. Functional classification of the ISO4-H5 predicted genes based on the clusters of
orthologous proteins (COG) database 47
Figure 3.3. The predicted codon and amino acid usage of ISO4-H5 51
Figure 3.4. Classification of ISO4-H5 signal peptides and their associated ORFs 58 Figure 3.5. Methylotrophic methanogenesis of ISO4-H5 59
Figure 3.6. Methanogenesis genes of ISO4-H5 61
Figure 3.7. ISO4-H5 central carbon metabolism 65
Figure 3.8. Biosynthesis of arginine in ISO4-H5 69
Figure 3.9. Incomplete biosynthesis pathway of methionine in ISO4-H5 71
Figure 3.10. Absence of a complete CoM biosynthesis pathway in ISO4-H5 73
Figure 3.11. Phospholipid biosynthesis in ISO4-H5 75
Figure 3.12. De novo nucleotide biosynthesis in ISO4-H5 79 Figure 4.1. Phylogenic relationship of members of the order Methanomassiliicoccales 111 Figure 4.2. Codon usage heatmap of the Methanomassiliicoccales genomes analysed 115
Figure 4.3. Amino acid usage map of the Methanomassiliicoccales genomes analysed 117 Figure 4.4. Gene organisation of the pyrrolysine operon in Methanomassiliicoccales genomes
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Figure 4.6. Gene synteny plots for completed genomes of Methanomassiliicoccales 123 Figure 4.7. Genome map comparison of members of the order Methanomassiliicoccales 124
Figure 4.8. Order level pan-genome and core-genome plot of Methanomassiliicoccales
genomes 126
Figure 4.9. BLASTP matrix illustrating the number of conserved protein families between
Methanomassiliicoccales genomes 127
Figure 4.10. Conserved and novel gene families among the 11 Methanomassiliicoccales
genomes 128
Figure 4.11. Chart displaying the COG classification of predicted core-genome for members
of Methanomassiliicoccales 13030
Figure 4.12. NRPS of ISO4-G1 1333
Figure 4.13. Methanogenesis pathway from methyl compounds by Methanomassiliicoccales
134 Figure 4.14. Phylogenic tree of methanogens based on McrA/MrtA amino acid sequences 136
Figure 4.15. Conservation of a hypothetical protein associated with the Mrt operon in
Methanomassiliicoccales 137
Figure 4.16. ClustalW alignment of predicted protein amino acid sequence of conserved
hypothetical protein located between the fpoJ and fpoK genes 139 Figure 4.17. ClustalW alignment of predicted protein amino acid sequence of NiFe-binding
motif in hydrogenase large subunits 140
Figure 4.18. CoM biosynthesis pathway in the Methanomassiliicoccales genomes analysed 140
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Figure 5.1. Stimulation of CH4 production in ISO4-H5 enrichment cultures triplicates by
glucose or pectin supplementation 172
Figure 5.2. Isolation procedure for ISO4-H5 174
Figure 5.3. Transmission electron micrograph (TEM) of methanogenic archaeon ISO4-H5 and
S. dextrinosolvens H5 175
Figure 5.4. Comparison of growth between pure and enrichment cultures of ISO4-H5 176 Figure 5.5. Growth of S. dextrinosolvens H5 on varying sources of carbohydrate and its growth
medium supplementation to ISO4-H5 culture 177
Figure 5.6. NMR analysis of medium composition before and after growth of ISO4-H5 179
Figure 5.7. Statistical analysis of paired control and sample replicates 180 Figure 5.8. Spectra bins that were reduced significantly post-growth 181
Figure 5.9. One-dimensional peak alignments 182
Figure 5.10. TOCSY of pooled samples 182
Figure 5.11. HSQC of pooled samples 183
Figure 5.12. Identification of spectrum bin 8.71 as nicotinamide 184 Figure 5.13. Growth of ISO4-H5 pure cultures with nicotinamide and nicotinic acid
supplementation 185
Figure 5.14. Growth of ISO4-H5 pure cultures with varying methanol concentrations 186 Figure 5.15. Growth of ISO4-H5 pure cultures with methyl substrates and H2+CO2 187
Figure 5.16. Growth of ISO4-H5 pure cultures with methyl-sources and H2+CO2 188 Figure 5.17. Growth of ISO4-H5 pure culture with putative reducing potential-supplying
compound in absence of H2 189
Figure 5.18. Effect of ethanol and formate addition on CH4 formation in ISO4-H5 pure cultures 190
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Figure 5.20. ISO4-H5 pure culture supplementation 191
Figure 5.21. Growth of ISO4-H5 enrichment culture under high or low H2 with either methanol
or methylamine 193
Figure 5.22. Multivariate analysis based on relative abundance of transcripts in the ISO4-H5
transcriptome via Bray Curtis distance matrix 196
Figure 5.23. Multivariate analysis based on relative abundance of transcripts in the S. dextrinosolvens H5 transcriptome via Bray Curtis distance matrix 197
Figure 5.24. Multivariate analysis based on relative abundance of transcripts in the R. flavefaciens FD1 transcriptome via Bray Curtis distance matrix 198
Figure 5.25. Network diagram showing the associations between genes and treatments based
on Correspondence Analysis 199
Figure 5.26. Expression of genes involved in methanol utilizing methanogenesis under high
and low H2 conditions 202
Figure 5.27. Expression of ISO4-H5 genes involved in methylamine and methylthiol use 203
Figure 5.28. Network diagram showing associations between methanogenesis genes and
treatments based on Correspondence Analysis 205
Figure 5.29. Differentially expressed ISO4-H5 genes during growth under low and high H2
levels and different methyl sources 207
Figure 5.30. PCR amplifications flanking and within the AR505_0313 - AR505_0358 region
208 Figure 5.31. Regularised Canonical Correlation analysis of associations between ISO4-H5 and R. flavefaciens FD1, S. dextrinosolvens H5 transcriptomes 211
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Figure 5.33. Expression of genes involved in methanol utilizing methanogenesis between high
and low H2 condition 222
Figure 6.1. Transmission electron micrograph of negatively stained thin sections of
Methanobrevibacter sp. D5 232
Figure 6.2. Contig coverage and %G+C of enrichment culture H6 sequencing assembly 234 Figure 6.3. Contig coverage and %G+C of D5 pure culture sequencing assembly 235
Figure 6.4. Flow chart of D5 gap closure procedure 235
Figure 6.5. PFGE of Methanobrevibacter sp. D5 genomic DNA 236 Figure 6.6. 16S rRNA gene-based phylogenetic tree 238
Figure 6.7. Functional Genome Distribution tree based on of the Methanobrevibacter spp.
ORFeome 239
Figure 6.8. Pan-genome tree of sequenced type strains and rumen members of
Methanobrevibacter spp 240
Figure 6.9. Circular representation of the Methanobrevibacter sp. D5 genome 242
Figure 6.10. Functional classification Methanobrevibacter sp. D5 genes 243 Figure 6.11. Codon and amino acid usage of the Methanobrevibacter genomes analysed 249 Figure 6.12. Distribution of Methanobrevibacter sp. D5 signal peptides 253
Figure 6.13. Gene synteny plots for completed genomes of Methanobrevibacter spp 258 Figure 6.14. Pan-genome and core-genome plot of Methanobrevibacter genomes 259
Figure 6.15. BLASTP matrix illustrating the number of conserved protein families between
Methanobrevibacter genomes 260
Figure 6.16. Conserved and novel gene families among the 18 Methanobrevibacter genomes
261 Figure 6.17. Chart displaying the COG classification of the predicted core-genome for
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Figure 6.18. Hydrogenotrophic methanogenesis pathway of Methanobrevibacter sp. D5 266 Figure 6.19. Central carbon metabolism of Methanobrevibacter sp. D5 269
Figure 6.20. Amino acid biosynthesis of Methanobrevibacter sp. D5 27070 Figure 6.21. Predicted phospholipid biosynthesis pathway in Methanobrevibacter sp. D5 273
Figure 6.22. De novo nucleotide biosynthesis in Methanobrevibacter sp. D5 277 Figure 6.23. Non-ribosomal surfactin synthase and NRPS of D5 280 Figure 6.24. Hydrogenotrophic methanogenesis pathway of Methanobrevibacter spp. 284 Figure 7.1. Theoretical ΔG’ of methanogenesis in relation to ATP synthesis in the rumen 311
Figure A.4.1. Nucleotide alignment of genome identity region 1 between H5 and
ISO4-G11 413
Figure A.4.2 Nucleotide alignment of genome identity region 2 between H5 and
ISO4-G11 414
Figure A.4.3. Nucleotide identity comparison of members of the order
Methanomassiliicoccales 415
Figure A.4.4. Nucleotide identity region between BRNA1 and ISO4-G11 416
Figure A.4.5. Conserved and novel gene families among the 11 Methanomassiliicoccales
genomes analysed 435
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Abbreviations
Non-standard abbreviations:
aa Amino acids (length of peptide chain or sequence identity) ANOVA Analysis of variance
ATP Adenosine triphosphate BES 2-bromoethanesulfonic acid
BLAST Basic Local Alignment Search Tool BLOSUM BLOcks SUbstitution Matrix BRIG BLAST Ring Image Generator CAI Codon adaptation index
CDS Coding DNA sequence
CH4 Methane
CO Carbon monoxide CO2 Carbon dioxide CoA Coenzyme A
CoB Coenzyme B
COG Clusters of Orthologous Groups CoM Coenzyme M
CRISPR Clustered regularly interspaced short palindromic repeat D2O Deuterated water
DEPC Diethylpyrocarbonate DNA Deoxyribonucleic acid
EDTA Ethylenediaminetetraacetic acid F390/420/430 Cofactor F390/420/430
FDR False discovery rate Fdx Ferredoxin
FGD Functional genome distribution GHGs Greenhouse gas
GIT Gastrointestinal
H4MPT Tetrahydromethanopterin HMM Hidden Markov model
xxiii IVOM Interpolated variable ordered motif
KEGG Kyoto Encyclopedia of Genes and Genomes KW Kruskal-Wallis rank sum test
M3MSP Methyl-3-methylthiopropionate M3SP Methylmercaptopropionate MCL Maximum Composite Likelihood MDS Multidimensional scaling
MF Methanofuran
MMIC Manawatu Microscopy and Imaging Centre mRNA Messenger RNA
N2 Nitrogen (gas or liquid)
NAD Nicotinamide adenine dinucleotide
NADP Nicotinamide adenine dinucleotide phosphate NCBI National Center for Biotechnology Information NMR Nuclear magnetic resonance
NRPS Non-ribosomal peptide synthase
NZ New Zealand
O2 Oxygen
ORB Origin recognition box ORF Open reading frame
PCoA Principal coordinate analysis PCR Polymerase chain reaction PFGE Pulsed field gel electrophoresis RCC Rumen Cluster C
RNA Ribonucleic acid snRNA Small nuclear RNA snoRNA Small nucleolar RNA
SSPGMS Succinivibrio spent pectin growth media supernatant TAE Tris acetate EDTA
TBE Tris borate EDTA
TE Tris EDTA
TEM Transmission electron micrograph TMH Transmembrane helix
xxiv tRNA Transfer RNA
UPGMA Unweighted pair group method with arithmetic mean UV Ultra violet
VFA Volatile fatty acid
Measurement Units:
oC Degree Celsius µg Microgram µL Microlitre µm Micrometer µM Micromolar bp Base pair h Hour kcal kilocalorie kb kilobase kDa kilodaltons kpa kilopascal kV kilovolts L Litre M Molar
Mb Million base pairs mg Milligram
MHz Mega hertz min Minutes mL Millilitre mm Millimeter mM Millimolar mV Millivolts ng Nanogram nm Nanometer ppm Parts per million s Seconds