Methods in enzymology. V.422 Part A, Two-component signaling systems. /

Front Cover 1
Two-Component Signaling Systems, Part A 4
Copyright Page 5
Table of Contents 6
Contributors to Volume 422 10
Volumes in Series 14
Section I: Computational Analyses of Sequences and Sequence Alignments 38
Chapter 1: Comparative Genomic and Protein Sequence Analyses of a Complex System Controlling Bacterial Chemotaxis 40
Introduction 40
Bioinformatics Tools and Resources for Identifying and Analyzing Chemotaxis Components 43
Defining MCP Membrane Topology 46
Diversity of Input (Sensory) Domains in MCPs 48
HAMP Domain Identification 50
MCP Signaling Domain 51
MCP Pentapeptide Tether 51
The CheA Histidine Kinase: Domain Organization, Conservation, and Diversity 52
The CheY Response Regulator: Big Problems of the Small Protein 57
CheB and CheR 57
CheC and CheX 58
CheD 59
CheZ 61
CheW and CheV 63
References 64
Chapter 2: Two-Component Systems in Microbial Communities: Approaches and Resources for Generating and Analyzing Metagenomic Data Sets 69
Introduction 69
Generating Metagenomic Data 71
Assembly of Environmental Sequence Data 74
Gene Prediction in Environmental Sequence Data 75
Analysis of Two-Component System Genes in Environmental Sequence Data 76
Acknowledgments 81
References 81
Chapter 3: Identification of Sensory and Signal-Transducing Domains in Two-Component Signaling Systems 84
Introduction 84
Computational Tools for Domain Identification 86
Sequence Analysis of Histidine Kinases 89
Sequence Analysis of Response Regulators 97
Sequence Analysis of Prokaryotic Signal Transducers 101
Functional Annotation of Multidomain Proteins 104
References 106
Chapter 4: Features of Protein-Protein Interactions in Two-Component Signaling Deduced from Genomic Libraries 112
Introduction 112
Identifying Coupled Columns 115
Predicting Protein-Protein Interaction 129
Summary 135
Acknowledgments 135
References 135
Chapter 5: Sporulation Phosphorelay Proteins and Their Complexes: Crystallographic Characterization 139
Introduction 139
Methods 141
Insights from Structural Analysis 147
Conclusion 157
Acknowledgments 157
References 157
Chapter 6: Control Analysis of Bacterial Chemotaxis Signaling 160
Introduction 160
Basic Concepts in Dynamics and Mathematical Modeling 161
Robustness and Steady-State Sensitivity Analysis 164
Constructing and Interpreting a Bode Plot 166
Primer on Integral Feedback Control 169
Noise Filtering and the Kalman Filter 172
Future Perspectives and Further Information 175
References 176
Chapter 7: Classification of Response Regulators Based on Their Surface Properties 178
Introduction 178
Classification of the Receiver Domain of RRs Using Protein Interaction Surfaces 179
Modeling and Subclassification of Receiver Domains of OmpR Subfamily RRs in B. subtilis and E. coli 181
Modeling and Subclassification of the Receiver Domain of RRs in V. vulnificus 195
References 205
Section II: Biochemical and Genetic Assays of Individual Components of Signaling Systems 208
Chapter 8: Purification and Assays of Rhodobacter capsulatus RegB-RegA Two-Component Signal Transduction System 210
Introduction 210
Expression and Purification of RegB 212
Expression and Purification of RegA 216
RegB Kinase and Phosphotransfer Assays 217
Acknowledgment 219
References 219
Chapter 9: Purification and Reconstitution of PYP-Phytochrome with Biliverdin and 4-Hydroxycinnamic Acid 221
Introduction 221
Vector Construct 222
Prepation of 4-Hydroxycinnamic Acid Anhydride and Biliverdin 222
Overexpression and Reconstitution of apo-Ppr with Chromophores 222
Purification of Ppr Reconstituted with Chromophores 223
Spectroscopic Measurements of holo-Ppr, Ppr-BV, and Ppr-pCA 224
In Vitro Autophosphorylation of Ppr 225
Acknowledgments 225
References 225
Chapter 10: Oxygen and Redox Sensing by Two-Component Systems That Regulate Behavioral Responses: Behavioral Assays and Structural Studies of Aer Using In Vivo Disulfide Cross-Linking 227
Introduction 227
Assays of Oxygen and Redox Sensing: General Considerations 231
Temporal Assay for Aerotaxis 234
Spatial-Gradient Capillary Assay for Aerotaxis 240
Using a Capillary to Determine the Preferred Partial Pressure of Oxygen for Bacteria 242
Spatial-Gradient Soft Agar Plate Assays for Aerotaxis 245
Spatial Assays for Redox Taxis 251
Temporal Assay for Redox Taxis 253
Disulfide Cross-Linking In Vivo to Elucidate the Structure of Aer 255
Site-Directed Mutagenesis for Cysteine Replacement 256
Disulfide Cross-Linking in the Cytosol Using Copper Phenanthroline 257
Differentiating Intra- from Interdimeric Disulfide Bonds 258
In Vivo Cross-Linking Using Bifunctional Sulfhydryl-Reactive Linkers 260
Determining the Boundaries of Transmembrane Segments in Receptors 261
Accessibility Studies in Membrane Vesicles 261
Acknowledgments 264
References 265
Chapter 11: Two-Component Signaling in the Virulence of Staphylococcus aureus: A Silkworm Larvae-Pathogenic Agent 270
Introduction 270
Silkworm Larvae Infection Model 271
Pathogenicity-Related Genes That Can Be Identified in the Silkworm Infection Assay 272
Identification of Genes Involved in the Killing of Silkworm Larvae by Bacteria 273
Identification of an SA0614 Response Regulator Mutant by Monitoring CPZ Sensitivity and Ability to Kill Silkworm Larvae 274
Silkworm Larvae Infection Assay 275
Measurement of the Number of Bacteria in Silkworm Hemolymph 276
Defect in Cell Wall Integrity of the SA0614 Response Regulator Mutant 278
Detergent and Lysozyme Sensitivity Test 278
Melanization-Inducing Activity of Bacterial Peptidoglycan 279
References 279
Chapter 12: TonB System, In Vivo Assays and Characterization 282
Introduction 282
Selection For and Against the tonB Gene 283
Precautions for Experiments Where TonB System Proteins Are Expressed from Plasmids 285
Phenotypic Assays for the TonB System 286
Mechanistically Informative Assays 292
Potentially Mechanistically Informative Assays 299
Acknowledgments 302
References 302
Chapter 13: Biochemical Characterization of Plant Ethylene Receptors Following Transgenic Expression in Yeast 307
Introduction 307
Transgenic Expression of Ethylene Receptors in Yeast 310
Histidine Kinase Activity 311
Isolation of Receptors for Use in Ethylene-Binding Assays 314
Ethylene-Binding Activity 317
Considerations When Working with Mercuric Perchlorate 320
Acknowledgments 322
References 322
Chapter 14: Structure of SixA, a Histidine Protein Phosphatase of the ArcB Histidine-Containing Phosphotransfer Domain in Escherichia Coli 325
Introduction 325
Overall Structure 327
Conservation of Catalytic Machinery and Active Site 330
Sequence Analysis of SixA Homologs 332
Eukaryotic Histidine Phosphatases 339
Acknowledgments 339
References 339
Chapter 15: Triggering and Monitoring Light-Sensing Reactions in Protein Crystals 342
Light-Regulated Histidine Kinases: A Brief Introduction 342
Microbial Rhodopsins 343
PAS/GAF/LOV Domains 344
Photoreceptors and Kinetic Crystallography: A Near Perfect Match 347
Kinetic Crystallography: Two Alternative Strategies 348
Trapping Intermediates for X-Ray Crystallography 350
Structural Interpretation of Kinetic Crystallography Results 352
Optical Properties of Protein Crystals 354
Mounting Crystals 355
Design of a Single Crystal Microspectrophotometer 357
Challenges of Recording UV-Visible Absorption Spectra in Crystals 359
Leaking Light Introduces Spectral and Kinetic Artifacts 361
Fluorescence Measurements 364
Microspectrophotometry: Summary and Warning 368
Light Activation of Photoreceptor Crystals 368
Aligning the Activating Light Beam and the Crystal Position 370
Summary and Outlook 371
References 371
Chapter 16: Synthesis of a Stable Analog of the Phosphorylated Form of CheY: Phosphono-CheY 375
Introduction 375
Protocols 382
Acknowledgments 387
References 387
Chapter 17: Application of Fluorescence Resonance Energy Transfer to Examine EnvZ/OmpR Interactions 389
Introduction 389
Overexpression of EnvZ and Preparation of Spheroplasts 391
Protein Purification and Fluorescent Labeling of OmpR 392
Fluorescence Resonance Energy Transfer 394
OmpR Has a Higher Affinity for EnvZ Than OmpR~P 394
Concluding Remarks 396
Acknowledgments 396
References 396
Chapter 18: Gene Promoter Scan Methodology for Identifying and Classifying Coregulated Promoters 398
Introduction 398
Challenge of Identifying Promoter Features Governing Gene Transcription 400
GPS Methodology as an Integrated Algorithm 402
Exploring Targets of Regulation of a Response Regulator Using GPS 405
Technical Specifications of GPS 410
Uncovering Promoter Profiles Regulated by Response Regulator PhoP Using GPS 415
Conclusions 417
Acknowledgments 418
References 419
Chapter 19: Targeting Two-Component Signal Transduction: A Novel Drug Discovery System 423
Introduction 423
Differential Growth Assay 424
High-Throughput Genetic System 428
Acknowledgments 431
References 431
Chapter 20: The Essential YycFG Two-Component System of Bacillus subtilis 433
Introduction 433
Construction of Conditional Mutants 434
Transposon Mutagenesis to Identify Regulatory Elements 437
Constructing In-Frame Deletions in the yyc Operon 440
Studying Interactions between the YycG Kinase and Its Regulatory Proteins 445
Subcellular Localization Studies 448
Concluding Remarks 452
Acknowledgments 452
References 452
Section III: Physiological Assays and Readouts 456
Chapter 21: Isolation and Characterization of Chemotaxis Mutants of the Lyme Disease Spirochete Borrelia burgdorferi Using Allelic Exchange Mutagenesis, Flow Cytometry, and Cell Tracking 458
Introduction 458
Borrelia burgdorferi Mutagenesis 459
Chemotaxis and Motility Analysis 460
Materials and Methods 461
Acknowledgments 472
References 473
Chapter 22: Phosphorylation Assays of Chemotaxis Two-Component System Proteins in Borrelia burgdorferi 475
Introduction 476
Regulation of CheY-P 476
Materials and Methods 479
Acknowledgments 483
References 483
Chapter 23: Regulation of Respiratory Genes by ResD-ResE Signal Transduction System in Bacillus subtilis 485
Introduction 485
Oxygen Limitation and ResDE-Dependent Transcription 486
Stimulatory Effect of NO on ResDE-Dependent Transcription 488
Phosphorylation Assay Using Full-Length ResE 490
In Vivo Effect of alpha-CTD Alanine Substitutions on ResDE-Dependent Transcription 492
In Vitro Effect of alpha-CTD Alanine Substitutions on ResDE-Dependent Transcription 495
Acknowledgments 498
References 499
Chapter 24: Detection and Measurement of Two-Component Systems That Control Dimorphism and Virulence in Fungi 502
Introduction 502
Experimental Approaches 505
Acknowledgment 522
References 522
Chapter 25: Using Two-Component Systems and Other Bacterial Regulatory Factors for the Fabrication of Synthetic Genetic Devices 525
Using Two-Component Signal Transduction Systems in Synthetic Biology Approaches 526
Using the NRI/NRII System to Build a Synthetic Genetic Clock 528
Fabrication of Synthetic Genetic Clock 530
Functions of Individual Clock Modules 533
Improved Procedures for Fabrication of Synthetic Genetic Modules and Integration of These Modules into Chromosomal Landing Pads 545
Fabricating Genetic Modules 548
References 549
Author Index 550
Subject Index 578