Methods in Methane Metabolism: Methanogenesis

✍ Scribed by Amy Rosenzweig, Stephen W. Ragsdale

• Publisher: Academic Press
• Year: 2011
• Tongue: English
• Leaves: 401
• Series: Methods in Enzymology 494
• Edition: 1
• Category: Library

✦ Synopsis

Produced by microbes on a large scale, methane is an important alternative fuel as well as a potent greenhouse gas. This volume focuses on microbial methane metabolism, which is central to the global carbon cycle. Both methanotrophy and methanogenesis are covered in detail. Topics include isolation and classification of microorganisms, metagenomics approaches, biochemistry of key metabolic enzymes, gene regulation and genetic systems, and field measurements. The state of the art techniques described here will both guide researchers in specific pursuits and educate the wider scientific community about this exciting and rapidly developing field.Topics include isolation and classification of microorganisms, metagenomics approaches, biochemistry of key metabolic enzymes, gene regulation and genetic systems, and field measurements. The state of the art techniques described here will both guide researchers in specific pursuits and educate the wider scientific community about this exciting and rapidly developing field.

✦ Table of Contents

Introduction......Page 38
Preparation of oxygen scrubbers
......Page 39
Preparation of 200mL of anoxic medium by boiling
......Page 42
The vacuum-vortex method
......Page 45
Anoxic, aseptic use of a syringe
......Page 46
Growth of methanogens on agar medium, the bottle plate
......Page 47
Growth of methanogens in a Hungate roll tube
......Page 48
Preparation of a 3-L amount of anoxic medium
......Page 49
Aseptic transfer of sterile medium
......Page 53
Use of an Anoxic Chamber......Page 55
References......Page 58
Genetic Methods for Methanosarcina Species
......Page 60
Advantages and disadvantages of Methanosarcina as model Archaea
......Page 61
Liposome-Mediated Transformation......Page 65
Deleting Genes......Page 66
Using pMP44......Page 69
Using pJK301 and pMR55......Page 70
Construction of Methanosarcina Ptet strains
......Page 71
Testing gene essentiality on solid medium
......Page 73
Testing gene essentiality in broth medium
......Page 74
Reporter gene fusions using pAB79 and derivatives
......Page 75
References......Page 78
Genetic Systems for Hydrogenotrophic Methanogens
......Page 80
Introduction......Page 81
Liquid media preparation......Page 83
Solid media preparation......Page 88
Glycerol stock culture preparation......Page 90
Genetic markers
......Page 91
Shuttle vectors......Page 92
Integration plasmid and gene replacement mutagenesis
......Page 94
Reporter genes......Page 96
Transformation Methods......Page 97
Transformation of M. voltae by protoplast regeneration
......Page 98
Polyethylene glycol (PEG)-mediated transformation of M. maripaludis
......Page 101
Markerless mutagenesis
......Page 103
Random mutagenesis with ethylmethanesulfonate
......Page 105
Selection for auxotrophic mutants
......Page 106
Transposon approaches in Methanococcus
......Page 107
References......Page 108
Molecular Tools for Investigating ANME Community Structure and Function
......Page 111
Introduction
......Page 112
Sample processing and DNA extraction
......Page 114
Quantifying mcrA copy number using dye assay chemistry
......Page 115
Detecting ANME Proteins in Marine Sediments......Page 119
Protein digestion and HPLC separation......Page 120
Tandem mass spectrometry and peptide identification
......Page 123
References......Page 124
Studying Gene Regulation in Methanogenic Archaea
......Page 127
Introduction......Page 128
M. maripaludis culture conditions......Page 130
Reverse transcription (cDNA synthesis)......Page 131
Quantitative PCR and data analysis......Page 132
Analyzing Gene Expression Using uidA Reporter Gene Fusions in M. acetivorans
......Page 133
Construction of the reporter plasmid......Page 134
Creation of the M. acetivorans reporter strain and determining GUS activity
......Page 135
Isolation of mutants with defects in regulatory pathways of M. acetivorans
......Page 136
Creating Random Disruption Mutants of M. maripaludis by In Vitro Transposon Mutagenesis
......Page 137
In vitro transposition and transformation of M. maripaludis
......Page 140
References......Page 141
Growth of Methanogens Under Defined Hydrogen Conditions
......Page 147
Introduction......Page 148
General considerations......Page 149
The gas delivery system......Page 150
General considerations......Page 152
The medium delivery system......Page 153
References......Page 154
Preparation of [Fe]-Hydrogenase from Methanogenic Archaea
......Page 155
Introduction......Page 156
Oxygen, Light, and Copper Sensitivity of [Fe]-Hydrogenase and FeGP Cofactor
......Page 160
Cultivation of M. marburgensis under nickel-limiting conditions
......Page 161
Purification of [Fe]-hydrogenase from M. marburgensis
......Page 162
Characterization of the purified [Fe]-hydrogenase
......Page 163
Extraction of FeGP cofactor from [Fe]-hydrogenase
......Page 164
Purification of FeGP cofactor
......Page 165
Characterization of purified FeGP cofactor
......Page 166
Heterologous overproduction of apoenzyme in E. coli
......Page 167
Reconstitution of the holoenzyme from the FeGP cofactor and apoenzyme
......Page 169
Assay of the holoenzyme......Page 170
Acknowledgments......Page 171
References......Page 172
Assay of Methylotrophic Methyltransferases from Methanogenic Archaea
......Page 174
Introduction......Page 175
Preparation of Anoxic Cell Extracts of M. barkeri
......Page 176
Isolation and Resolution of MtmBC Complexes
......Page 178
Assay for a component protein of a methyltransferase system using diluted extracts
......Page 180
Assay of CoM during methylation by purified methylamine methyltransferases
......Page 183
Assay of Methylthiol:CoM Methyltransferases
......Page 185
Spectrophotometric assay of methylamine:cob(I)alamin methyltransferases
......Page 187
Monitoring cognate corrinoid protein methylation by HPLC
......Page 189
Spectral Assay of Methylcobalamin:CoM Methyltransferases
......Page 190
Acknowledgments......Page 191
References......Page 192
Methyl-Coenzyme M Reductase from Methanothermobacter marburgensis
......Page 194
Introduction......Page 195
Equipment......Page 197
Cell Growth: Basic Procedure......Page 199
Whole-Cell Experiments......Page 202
MCRred1......Page 206
MCRred1......Page 207
Activation of MCR: Conversion of MCRox1 into MCRred1m
......Page 210
Activity assay......Page 211
Activity assay for methane oxidation......Page 212
Different MCR Forms......Page 213
Materials......Page 215
7,7-Dithiodiheptanoic acid......Page 217
(+)-N,N-(7,7-Dithiodiheptanoyl)bis(O-phospho-l-threonine)
......Page 218
N-7-Mercaptoheptanoyl-O-phospho-l-threonine (HS-CoB)
......Page 219
References......Page 220
Methods for Analysis of Acetyl-CoA Synthase: Applications to Bacterial and Archaeal Systems
......Page 223
Introduction......Page 224
Acetyl-CoA synthesis and cleavage
......Page 225
Requirement for reductive activation
......Page 229
Acetyltransferase Reactions......Page 230
Reagent list for acetyltransferase assay
......Page 232
Reversed-phase HPLC analyses......Page 234
Calculation of acetyltransferase initial rate
......Page 236
Acetyl-CoA Synthesis Reactions......Page 239
Procedure for acetyl-CoA synthesis assay
......Page 242
Gas mixture preparation......Page 244
The Acetyl-CoA Carbonyl:CO Exchange Reaction
......Page 245
Procedure for CO exchange assay......Page 246
Concluding Remarks......Page 247
Acknowledgments......Page 248
References......Page 249
Acetate Kinase and Phosphotransacetylase
......Page 252
Introduction......Page 253
Assay of activity......Page 254
Purification from M. thermophila......Page 255
Purification of M. thermophila Acetate Kinase Produced in Escherichia coli
......Page 256
Catalytic mechanism of acetate kinase from M. thermophila
......Page 257
Measurement of acetate in biological fluids
......Page 258
Purification from M. thermophila......Page 259
Purification of M. thermophila phosphotransacetylase produced in E. coli
......Page 260
Catalytic mechanism of phosphotransacetylase from M. thermophila
......Page 261
References......Page 262
Sodium Ion Translocation and ATP Synthesis in Methanogens
......Page 265
Introduction......Page 266
M. barkeri......Page 269
M. thermautotrophicus DeltaH
......Page 270
Na+ Transport in Cell Suspensions......Page 271
Assay for determination of internal Na+ concentration in the steady state of methanogenesis
......Page 272
Counting radioactivity......Page 273
Silicon oil centrifugation......Page 274
Determining the intracellular volume......Page 276
Determining the membrane potential of the cell......Page 277
Determining the pH gradient of the cell......Page 278
Cell cultivation......Page 280
Assay for determination of ATP synthesis coupled to methanogenesis
......Page 281
DeltapH-driven ATP-synthesis
......Page 282
Measurement of ATP......Page 283
Assay for ATP hydrolysis......Page 284
References......Page 285
Proton Translocation in Methanogens
......Page 288
Introduction......Page 289
Procedure......Page 291
Analysis of the H2-dependent electron transport chain
......Page 292
Procedure......Page 293
Procedure......Page 294
Preparation of the heterodisulfide (CoM-S-S-CoB)
......Page 295
Analysis of the F420H2-dependent electron transport chain
......Page 297
Purification and reduction of coenzyme F420
......Page 298
Analysis of the ferredoxin-dependent electron transport chain
......Page 299
Analysis of the Fd:heterodisulfide oxidoreductase (coupled reaction of Ech hydrogenase, F420-nonreducing hydrogenase and heterodisulfide reductase)
......Page 300
Preparation of CO-dehydrogenase/acetyl-CoA synthase
......Page 301
Preparation of washed inverted vesicles
......Page 302
Procedure......Page 304
Reaction conditions
......Page 306
Procedure......Page 307
References......Page 308
Measuring Isotope Fractionation by Autotrophic Microorganisms and Enzymes
......Page 312
Introduction......Page 313
General assay considerations......Page 314
Setting up the enzyme assay......Page 316
Dissolved inorganic carbon (CO2, HCO3-, CO3-2)
......Page 317
Methane......Page 319
Multicarbon compounds
......Page 320
General assay considerations......Page 321
Setting up the culture assay......Page 322
Calculations......Page 323
Acknowledgments......Page 325
References......Page 326
2-Oxoacid Metabolism in Methanogenic CoM and CoB Biosynthesis
......Page 331
Chemistry of 2-Oxoacids......Page 332
Biosynthesis of Coenzyme M......Page 334
Synthesis of 2-Sulfopyruvate
......Page 337
Discussion of CoM Biosynthesis......Page 338
Coenzyme B Biosynthesis......Page 339
Homocitrate Synthase......Page 340
Homocitrate Synthase Enzymology......Page 341
Substrate Specificity Determinants in HCS
......Page 342
Chemical Synthesis of 2-Oxoacids......Page 343
Chemical Synthesis of Homocitrate and Its Analogs
......Page 344
Homoaconitase Enzymology......Page 345
Chemical Synthesis of Homoaconitate and Its Analogs
......Page 346
Enzymology of Homoisocitrate Dehydrogenase
......Page 347
Chromatographic Analysis of 2-Oxoacids......Page 348
Acknowledgments......Page 351
References......Page 352
Biomethanation and Its Potential
......Page 357
Introduction......Page 358
Hydrolysis......Page 359
Fermentation......Page 361
Fermentation of amino acids......Page 362
Acetogenesis......Page 363
Methanogenesis......Page 365
Biochemical Methane Potential......Page 368
Theoretical potential......Page 369
Practical potential......Page 370
Biogas Applications......Page 372
Anaerobic digestion of solid waste......Page 373
Anaerobic treatment of wastewater......Page 375
Biomethanation-Global Aspects......Page 376
References......Page 377