Methods in Methane Metabolism, Part B (eBook)

Front Cover 1
Methods in Methane Metabolism, Part B: Methanotrophy 4
Copyright Page 5
Contents 6
Contributors 12
Preface 16
Volume in series 18
Chapter 1: Characterization of the Recombinant Pyrophosphate-Dependent 6-Phosphofructokinases from Methylomicrobium alcaliphilum 20Z andMethylococcus capsulatus Bath 48
1. Introduction 49
2. Methods 51
3. Brief Overview of Current Knowledge of PPi-PFK in Microorganisms 54
4. Properties of PPi-PFK from Methanotrophs 55
5. Conclusion 57
Acknowledgments 58
References 58
Chapter 2: Genes and Enzymes of Ectoine Biosynthesis in Halotolerant Methanotrophs 62
1. Introduction 63
2. PCR-Based Approach for Identification of the Ectoine Biosynthesis Genes in Methanotrophs 64
3. Transcriptional Regulation of the Ectoine Biosynthesis Genes 69
4. Key Enzymes of Ectoine Biosynthesis 70
5. Conclusion 74
Acknowledgments 74
References 75
Chapter 3: Facultative and Obligate Methanotrophs: How to Identifyand Differentiate Them 78
1. Introduction 79
2. Identification of Methanotrophic Capabilities in Novel Isolates 80
3. Substrate Utilization Tests 84
4. Tests for Culture Purity 85
References 90
Chapter 4: Approaches for the Characterization and Description of Novel Methanotrophic Bacteria 92
1. Introduction to Aerobic Methanotroph Characterization for Systematic Research and Other Purposes 93
2. Broad Level Systematic Diversity 93
3. Methodological Approaches 96
4. Concluding Remarks 103
References 106
Chapter 5: Methylococcus capsulatus (Bath): From Genome to Protein Function, and Vice Versa 110
1. Introduction 111
2. Mapping of the Outer Membrane Proteome of M. capsulatus 112
3. Inner Membrane Protein Complexes Studied with Blue-Native Polyacrylamide Gel Electrophoresis (BN-PAGE) 117
4. Visualization and Identification of M. capsulatus c-type Heme Proteins 121
Acknowledgments 124
References 124
Chapter 6: Functional Metagenomics of Methylotrophs 128
1. Introduction 129
2. Enrichment for Specific Functional Types Using SIP 130
3. Creating a Metagenomic Database and Linking It to Functionality 132
4. Ultrashort Read-Based Metatranscriptomics 137
5. Conclusions and Future Perspectives 142
Acknowledgments 143
References 143
Chapter 7: Genetic Systems for Moderately Halo(alkali)philic Bacteria of the Genus Methylomicrobium 146
1. Introduction 147
2. Methods 148
3. Outlook 163
Acknowledgments 163
References 163
Chapter 8: Development of a System for Genetic Manipulation of the Facultative Methanotroph Methylocella silvestris BL2 166
1. Introduction 167
2. Growth of M. silvestris 169
3. Introduction of Plasmid DNA by Conjugation and Electroporation 170
4. Gene Deletion by Electroporation of Linear DNA 170
5. Preparation of Competent Cells for Electroporation of M. silvestris 171
6. Construction of Linear DNA for Gene Deletion by Homologous Recombination 171
7. Electroporation 174
8. Efficiency of Gene Deletion 174
9. Case Study: Deletion of Isocitrate Lyase 175
10. Complementation 175
11. Phenotype of the Isocitrate Lyase Deletion Mutant 176
12. Conclusions 177
References 178
Chapter 9: Mutagenesis of Soluble Methane Monooxygenase 182
1. Introduction 183
2. Bacteriological Growth Media and Antibiotics 184
3. Mutagenesis and Subcloning 186
4. Expression Hosts and Conjugation 188
5. Analysis of Mutants 191
Acknowledgments 193
References 193
Chapter 10: Single Cell Methods for Methane Oxidation Analysis 196
1. Introduction 197
2. Methods 198
Acknowledgment 212
References 212
Chapter 11: Methanotroph Outer Membrane Preparation 214
1. Introduction 215
2. Isolation of OMs 217
3. Enrichment of Integral and Tightly Associated OMPs 219
4. Extraction of Surface-Associated Proteins 219
5. Biotin Labeling of Surface-Exposed OMPs 220
References 222
Chapter 12: Overexpression and Purification of the Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath) 224
1. Introduction 225
2. Overproduction of pMMO 226
3. Isolation and Purification of pMMO from the pMMO-Enriched Membranes 231
4. Characterization of the Purified pMMO-Detergent Complex 236
Acknowledgments 239
References 239
Chapter 13: Metal Reconstitution of Particulate Methane Monooxygenase and Heterologous Expression of the pmoB Subunit 242
1. Introduction 243
2. Preparation of apo pMMO Membranes and Metal Reconstitution 244
3. Soluble Domain Constructs of the pmoB Subunit 250
Acknowledgments 256
References 256
Chapter 14: Particulate Methane Monooxygenase from Methylosinus trichosporium OB3b 258
1. Introduction 259
2. Bacterial Cells Expressing pMMO 260
3. Membrane-Bound Form of pMMO 266
4. Purification of Detergent-Solubilized pMMO 268
5. Summary 271
Acknowledgments 271
References 271
Chapter 15: Production, Isolation, Purification, and Functional Characterization of Methanobactins 274
1. Introduction: Copper, Siderophores, Chalkophores, and Methanobactin 275
2. Methanotroph Growth and Optimizing Methanobactin Production 278
3. Methanobactin Isolation and Purification 280
4. Studying Methanobactins in Pseudonatural Environments 286
Acknowledgments 290
References 290
Chapter 16: A Simple Assay for Screening Microorganisms for Chalkophore Production 294
1. Introduction 295
2. Cu-CAS Assay for Chalkophore Detection 296
3. Fe-CAS Assay for Detecting Nonspecific Binding of Copper from Cu-CAS by a Siderophore 299
4. Conclusions 303
Acknowledgments 303
References 304
Chapter 17: Isolation of Methanobactin from the Spent Media of Methane-Oxidizing Bacteria 306
1. Introduction 307
2. Isolation of Methanobactin from the Spent Media of MOB 307
3. Purification of mb 312
4. Sample Variability 313
Acknowledgments 315
References 315
Chapter 18: Measurements of Biosphere-Atmosphere Exchange of CH4 in Terrestrial Ecosystems 318
1. Introduction 319
2. Chamber Measurements of CH4 Exchange 321
3. Micrometeorological Measurements of CH4 Exchange 331
Acknowledgment 332
References 332
Author Index 336
Subject Index 350
Color Plates 358