Series Editor......Page 1
Copyright......Page 2
Contributors......Page 3
Preface......Page 7
Methods in Enzymology......Page 9
Abstract......Page 38
Introduction......Page 39
Assay of PPi-PFK with Fru 6-P as phosphoryl acceptor......Page 41
Assay of PPi-PFK with sedoheptulose-1,7-bisphosphate or ribulose-1,5-bisphosphate......Page 42
Assay of fructosebisphosphate aldolase activity by using the recombinant PPi-PFK......Page 43
Brief Overview of Current Knowledge of PPi-PFK in Microorganisms......Page 44
PPi-PFK from Mc. capsulatus Bath......Page 45
Transcriptional organization of pfp and hpp genes in Mc. capsulatus Bath and their phylogeny......Page 46
Conclusion......Page 47
References......Page 48
Genes and Enzymes of Ectoine Biosynthesis in Halotolerant Methanotrophs......Page 52
Introduction......Page 53
PCR-Based Approach for Identification of the Ectoine Biosynthesis Genes in Methanotrophs......Page 54
Transcriptional Regulation of the Ectoine Biosynthesis Genes......Page 59
Diaminobutyric acid aminotransferase......Page 60
DABA acetyltransferase......Page 61
Ectoine synthase......Page 62
Aspartate kinase......Page 63
Acknowledgments......Page 64
References......Page 65
Facultative and Obligate Methanotrophs: How to Identify and Differentiate Them......Page 68
Introduction......Page 69
Culture conditions......Page 70
Registration of growth dynamics on methane......Page 71
Detection of genes encoding methane monooxygenase......Page 72
Substrate Utilization Tests......Page 74
Plating on complex organic media......Page 75
Whole-cell hybridization with fluorescent probes......Page 76
16S rRNA gene clone library analysis......Page 78
Quantification of methane monooxygenase-coding genes during growth on an alternate substrate......Page 79
References......Page 80
Approaches for the Characterization and Description of Novel Methanotrophic Bacteria......Page 82
Broad Level Systematic Diversity......Page 83
Requirements related to the Bacteriological Code......Page 85
Enrichment and isolation......Page 86
Chemotaxonomy......Page 89
Informative sequence-based analyses and genomic comparisons......Page 92
Concluding Remarks......Page 93
References......Page 96
Methylococcus capsulatus (Bath): From Genome to Protein Function, and Vice Versa......Page 100
Introduction......Page 101
Mapping of the Outer Membrane Proteome of M. capsulatus......Page 102
Prediction of beta-barrel proteins......Page 103
Subfractionation......Page 104
Multiple spots......Page 105
Analyzing (in vitro-generated) horizontal multiple spot patterns in 2DE gels......Page 106
Inner Membrane Protein Complexes Studied with Blue-Native Polyacrylamide Gel Electrophoresis (BN-PAGE)......Page 107
Detergent extraction of the M. capsulatus inner membrane protein complexes......Page 108
The first-dimension BN-PAGE......Page 109
The second-dimension SDS-PAGE......Page 110
Visualization and Identification of M. capsulatus c-type Heme Proteins......Page 111
Identification of c-type heme proteins......Page 113
References......Page 114
Functional Metagenomics of Methylotrophs......Page 117
Introduction......Page 118
Sample collection and cell labeling......Page 119
DNA extraction, isopycnic centrifugation, and labeled DNA recovery......Page 120
Phylogenetic markers......Page 121
Characterizing genomes at high sequence coverage......Page 123
Characterizing genomes at low sequence coverage......Page 125
RNA isolation......Page 126
Read quality assessment......Page 127
Postprocessing of alignment outputs......Page 128
Metatranscriptome coverage and specificity......Page 129
Conclusions and Future Perspectives......Page 131
References......Page 132
Genetic Systems for Moderately Halo(alkali)philic Bacteria of the Genus Methylomicrobium......Page 135
Introduction......Page 136
Routine genetic manipulations......Page 137
Efficient conjugal transformation of Methylomicrobium strains......Page 142
Construction of M. alcaliphilum 20Z mutant strains......Page 144
Validation of the pCM66 plasmid-based promoter-probe vector......Page 145
Construction of pDO4-based expression vectors for use in M. alcaliphilum 20Z......Page 146
Microarray design......Page 148
Validation of microarray platform......Page 149
References......Page 152
Development of a System for Genetic Manipulation of the Facultative Methanotroph Methylocella silvestris BL2......Page 155
Introduction......Page 156
Growth of M. silvestris......Page 158
Gene Deletion by Electroporation of Linear DNA......Page 159
Construction of Linear DNA for Gene Deletion by Homologous Recombination......Page 160
Efficiency of Gene Deletion......Page 163
Complementation......Page 164
Phenotype of the Isocitrate Lyase Deletion Mutant......Page 165
Conclusions......Page 166
References......Page 167
Mutagenesis of Soluble Methane Monooxygenase......Page 170
Introduction......Page 171
Bacteriological Growth Media and Antibiotics......Page 172
Design of mutants......Page 174
Mutagenesis......Page 175
Expression Hosts and Conjugation......Page 176
Confirmation of the mutant genotype......Page 179
Large-scale production of cultures expressing mutant enzymes......Page 180
References......Page 181
Single Cell Methods for Methane Oxidation Analysis......Page 183
Introduction......Page 184
Evaluation of RedoxSensor Green dye performance......Page 185
Evaluation of the physiological state of cells by flow cytometry......Page 186
Application of the FCRS for detection of actively metabolizing microbes in natural samples......Page 190
Overview......Page 192
RRI protocol......Page 193
Overview......Page 195
Microwell array chips......Page 196
Observation chamber preparation......Page 197
Respiration measurements......Page 198
References......Page 199
Methanotroph Outer Membrane Preparation......Page 201
Introduction......Page 202
Isolation of OM and associated proteins......Page 204
Isolation of loosely associated cell-surface proteins......Page 206
Biotin labeling of intact cells......Page 207
References......Page 209
Overexpression and Purification of the Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath)......Page 211
Introduction......Page 212
Overproduction of pMMO......Page 213
A flow reactor for the overproduction of pMMO......Page 215
Optimal conditions for the culturing of the bacteria......Page 216
Isolation of pMMO-enriched membranes......Page 217
Isolation and Purification of pMMO from the pMMO-Enriched Membranes......Page 218
Solubilization of the pMMO-enriched membranes by dodecyl beta-d-maltoside and fractionation of the detergent-protein.........Page 219
Methods of pMMO-purification employed by other research groups......Page 222
Chemical composition of the purified pMMO-detergent complex......Page 223
Specific activities of the purified pMMO......Page 225
References......Page 226
Metal Reconstitution of Particulate Methane Monooxygenase and Heterologous Expression of the pmoB Subunit......Page 228
Introduction......Page 229
M. capsulatus (Bath) growth conditions......Page 230
pMMO activity assays......Page 231
Metal removal with EDTA......Page 232
Metal removal with cyanide......Page 233
Metal reconstitution......Page 234
Design of vectors......Page 236
Isolation and purification of inclusion bodies......Page 237
Refolding of inclusion bodies......Page 238
Copper assays......Page 239
Activity assays of the soluble pmoB domains......Page 240
References......Page 242
Particulate Methane Monooxygenase from Methylosinus trichosporium OB3b......Page 244
Introduction......Page 245
Bacterial Cells Expressing pMMO......Page 246
Culture medium......Page 247
Activity assay of pMMO in bacterial cells......Page 248
Application for methanol production......Page 249
Cell disruption......Page 252
Protein analysis......Page 253
Solubilization to water phase using detergents......Page 254
Purification using column chromatography......Page 255
Crystallization of pMMO......Page 256
References......Page 257
Production, Isolation, Purification, and Functional Characterization of Methanobactins......Page 259
Introduction: Copper, Siderophores, Chalkophores, and Methanobactin......Page 260
Methanotroph Growth and Optimizing Methanobactin Production......Page 263
Initial methanobactin capture and concentration......Page 265
UV-Vis detection of methanobactin......Page 267
Purification of methanobactin......Page 268
Mass spectral analysis......Page 269
Assessing methanobactins based on toxicity and growth......Page 270
Methanobactin solubilization studies with copper minerals......Page 271
MMO gene expression assays for assessing Cu uptake......Page 272
References......Page 275
A Simple Assay for Screening Microorganisms for Chalkophore Production......Page 278
Introduction......Page 279
Preparation of Cu-CAS solution......Page 280
Preparation of Cu-CAS agar plates......Page 282
Fe-CAS Assay for Detecting Nonspecific Binding of Copper from Cu-CAS by a Siderophore......Page 283
Preparation of liquid Fe-CAS......Page 284
Preparation of Fe-CAS agar plates......Page 286
Acknowledgments......Page 287
References......Page 288
Isolation of Methanobactin from the Spent Media of Methane-Oxidizing Bacteria......Page 290
Isolation of Methanobactin from the Spent Media of MOB......Page 291
Method 1: Centrifugation and filtration......Page 292
Method 2: Tangential flow or hollow fiber filtration......Page 293
Method 2: Dianion HP-20......Page 294
Purification of mb......Page 296
Sample Variability......Page 297
References......Page 299
Measurements of Biosphere-Atmosphere Exchange of CH4 in Terrestrial Ecosystems......Page 301
Introduction......Page 302
Closed chamber technique......Page 304
Chamber effects on environmental conditions......Page 305
Gas mixing......Page 306
Spatiotemporal variations......Page 307
Sampling procedure......Page 308
Flux calculations......Page 310
Dynamic chamber technique......Page 312
Determination of different emission pathways (plant-mediated transport, bubbles, gas diffusion)......Page 313
Micrometeorological Measurements of CH4 Exchange......Page 314
References......Page 315
Author Index......Page 318
Subject Index......Page 331