Rosenzweig / Ragsdale Methods in Methane Metabolism, Part B

Methanotrophy
1. Auflage 2011
ISBN: 978-0-12-386906-7
Verlag: Elsevier Science & Techn.
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, Band Volume 495, 360 Seiten, Web PDF

Reihe: Methods in Enzymology

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

Autoren/Hrsg.

Rosenzweig, Amy

Ragsdale, Stephen W.

Inhaltsverzeichnis

1;Front Cover;1
2;Methods in Methane Metabolism, Part B: Methanotrophy;4
3;Copyright Page;5
4;Contents;6
5;Contributors;12
6;Preface;16
7;Volume in series;18
8;Chapter 1: Characterization of the Recombinant Pyrophosphate-Dependent 6-Phosphofructokinases from Methylomicrobium alcaliphilum 20Z andMethylococcus capsulatus Bath;48
8.1;1. Introduction;49
8.2;2. Methods;51
8.3;3. Brief Overview of Current Knowledge of PPi-PFK in Microorganisms;54
8.4;4. Properties of PPi-PFK from Methanotrophs ;55
8.5;5. Conclusion ;57
8.6;Acknowledgments;58
8.7;References;58
9;Chapter 2: Genes and Enzymes of Ectoine Biosynthesis in Halotolerant Methanotrophs;62
9.1;1. Introduction;63
9.2;2. PCR-Based Approach for Identification of the Ectoine Biosynthesis Genes in Methanotrophs ;64
9.3;3. Transcriptional Regulation of the Ectoine Biosynthesis Genes;69
9.4;4. Key Enzymes of Ectoine Biosynthesis;70
9.5;5. Conclusion;74
9.6;Acknowledgments;74
9.7;References;75
10;Chapter 3: Facultative and Obligate Methanotrophs: How to Identifyand Differentiate Them;78
10.1;1. Introduction;79
10.2;2. Identification of Methanotrophic Capabilities in Novel Isolates ;80
10.3;3. Substrate Utilization Tests;84
10.4;4. Tests for Culture Purity;85
10.5;References;90
11;Chapter 4: Approaches for the Characterization and Description of Novel Methanotrophic Bacteria;92
11.1;1. Introduction to Aerobic Methanotroph Characterization for Systematic Research and Other Purposes;93
11.2;2. Broad Level Systematic Diversity;93
11.3;3. Methodological Approaches;96
11.4;4. Concluding Remarks;103
11.5;References;106
12;Chapter 5: Methylococcus capsulatus (Bath): From Genome to Protein Function, and Vice Versa;110
12.1;1. Introduction;111
12.2;2. Mapping of the Outer Membrane Proteome of M. capsulatus;112
12.3;3. Inner Membrane Protein Complexes Studied with Blue-Native Polyacrylamide Gel Electrophoresis (BN-PAGE);117
12.4;4. Visualization and Identification of M. capsulatus c-type Heme Proteins;121
12.5;Acknowledgments;124
12.6;References;124
13;Chapter 6: Functional Metagenomics of Methylotrophs;128
13.1;1. Introduction;129
13.2;2. Enrichment for Specific Functional Types Using SIP;130
13.3;3. Creating a Metagenomic Database and Linking It to Functionality;132
13.4;4. Ultrashort Read-Based Metatranscriptomics;137
13.5;5. Conclusions and Future Perspectives;142
13.6;Acknowledgments;143
13.7;References;143
14;Chapter 7: Genetic Systems for Moderately Halo(alkali)philic Bacteria of the Genus Methylomicrobium;146
14.1;1. Introduction;147
14.2;2. Methods;148
14.3;3. Outlook;163
14.4;Acknowledgments;163
14.5;References;163
15;Chapter 8: Development of a System for Genetic Manipulation of the Facultative Methanotroph Methylocella silvestris BL2;166
15.1;1. Introduction;167
15.2;2. Growth of M. silvestris;169
15.3;3. Introduction of Plasmid DNA by Conjugation and Electroporation;170
15.4;4. Gene Deletion by Electroporation of Linear DNA;170
15.5;5. Preparation of Competent Cells for Electroporation of M. silvestris;171
15.6;6. Construction of Linear DNA for Gene Deletion by Homologous Recombination ;171
15.7;7. Electroporation;174
15.8;8. Efficiency of Gene Deletion;174
15.9;9. Case Study: Deletion of Isocitrate Lyase;175
15.10;10. Complementation;175
15.11;11. Phenotype of the Isocitrate Lyase Deletion Mutant;176
15.12;12. Conclusions;177
15.13;References;178
16;Chapter 9: Mutagenesis of Soluble Methane Monooxygenase;182
16.1;1. Introduction;183
16.2;2. Bacteriological Growth Media and Antibiotics ;184
16.3;3. Mutagenesis and Subcloning;186
16.4;4. Expression Hosts and Conjugation;188
16.5;5. Analysis of Mutants;191
16.6;Acknowledgments;193
16.7;References;193
17;Chapter 10: Single Cell Methods for Methane Oxidation Analysis;196
17.1;1. Introduction;197
17.2;2. Methods;198
17.3;Acknowledgment;212
17.4;References;212
18;Chapter 11: Methanotroph Outer Membrane Preparation;214
18.1;1. Introduction;215
18.2;2. Isolation of OMs;217
18.3;3. Enrichment of Integral and Tightly Associated OMPs;219
18.4;4. Extraction of Surface-Associated Proteins;219
18.5;5. Biotin Labeling of Surface-Exposed OMPs;220
18.6;References;222
19;Chapter 12: Overexpression and Purification of the Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath);224
19.1;1. Introduction;225
19.2;2. Overproduction of pMMO;226
19.3;3. Isolation and Purification of pMMO from the pMMO-Enriched Membranes ;231
19.4;4. Characterization of the Purified pMMO-Detergent Complex;236
19.5;Acknowledgments;239
19.6;References;239
20;Chapter 13: Metal Reconstitution of Particulate Methane Monooxygenase and Heterologous Expression of the pmoB Subunit;242
20.1;1. Introduction;243
20.2;2. Preparation of apo pMMO Membranes and Metal Reconstitution;244
20.3;3. Soluble Domain Constructs of the pmoB Subunit;250
20.4;Acknowledgments;256
20.5;References;256
21;Chapter 14: Particulate Methane Monooxygenase from Methylosinus trichosporium OB3b;258
21.1;1. Introduction;259
21.2;2. Bacterial Cells Expressing pMMO;260
21.3;3. Membrane-Bound Form of pMMO;266
21.4;4. Purification of Detergent-Solubilized pMMO;268
21.5;5. Summary;271
21.6;Acknowledgments;271
21.7;References;271
22;Chapter 15: Production, Isolation, Purification, and Functional Characterization of Methanobactins;274
22.1;1. Introduction: Copper, Siderophores, Chalkophores, and Methanobactin ;275
22.2;2. Methanotroph Growth and Optimizing Methanobactin Production;278
22.3;3. Methanobactin Isolation and Purification;280
22.4;4. Studying Methanobactins in Pseudonatural Environments;286
22.5;Acknowledgments;290
22.6;References;290
23;Chapter 16: A Simple Assay for Screening Microorganisms for Chalkophore Production;294
23.1;1. Introduction;295
23.2;2. Cu-CAS Assay for Chalkophore Detection;296
23.3;3. Fe-CAS Assay for Detecting Nonspecific Binding of Copper from Cu-CAS by a Siderophore;299
23.4;4. Conclusions;303
23.5;Acknowledgments;303
23.6;References;304
24;Chapter 17: Isolation of Methanobactin from the Spent Media of Methane-Oxidizing Bacteria;306
24.1;1. Introduction;307
24.2;2. Isolation of Methanobactin from the Spent Media of MOB;307
24.3;3. Purification of mb;312
24.4;4. Sample Variability;313
24.5;Acknowledgments;315
24.6;References;315
25;Chapter 18: Measurements of Biosphere-Atmosphere Exchange of CH4 in Terrestrial Ecosystems;318
25.1;1. Introduction;319
25.2;2. Chamber Measurements of CH4 Exchange;321
25.3;3. Micrometeorological Measurements of CH4 Exchange;331
25.4;Acknowledgment;332
25.5;References;332
26;Author Index;336
27;Subject Index;350
28;Color Plates;358