Elsevier S & T

Two-Component Signaling Systems, Part A

Simon, Melvin I.

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Table of contents

Table of Contentsv
Contributors to Volume 422ix
Volumes in Seriesxiii
Section I: Computational Analyses of Sequences and Sequence Alignments1
Chapter 1: Comparative Genomic and Protein Sequence Analyses of a Complex System Controlling Bacteri3
Introduction3
Bioinformatics Tools and Resources for Identifying and Analyzing Chemotaxis Components6
Defining MCP Membrane Topology9
Diversity of Input (Sensory) Domains in MCPs11
HAMP Domain Identification13
MCP Signaling Domain14
MCP Pentapeptide Tether14
The CheA Histidine Kinase: Domain Organization, Conservation, and Diversity15
The CheY Response Regulator: Big Problems of the Small Protein20
CheB and CheR20
CheC and CheX21
CheD22
CheZ24
CheW and CheV26
References27
Chapter 2: Two-Component Systems in Microbial Communities: Approaches and Resources for Generating a32
Introduction32
Generating Metagenomic Data34
Assembly of Environmental Sequence Data37
Gene Prediction in Environmental Sequence Data38
Analysis of Two-Component System Genes in Environmental Sequence Data39
Acknowledgments44
References44
Chapter 3: Identification of Sensory and Signal-Transducing Domains in Two-Component Signaling Syste47
Introduction47
Computational Tools for Domain Identification49
Sequence Analysis of Histidine Kinases52
Sequence Analysis of Response Regulators60
Sequence Analysis of Prokaryotic Signal Transducers64
Functional Annotation of Multidomain Proteins67
References69
Chapter 4: Features of Protein-Protein Interactions in Two-Component Signaling Deduced from Genomic75
Introduction75
Identifying Coupled Columns78
Predicting Protein-Protein Interaction92
Summary98
Acknowledgments98
References98
Chapter 5: Sporulation Phosphorelay Proteins and Their Complexes: Crystallographic Characterization102
Introduction102
Methods104
Insights from Structural Analysis110
Conclusion120
Acknowledgments120
References120
Chapter 6: Control Analysis of Bacterial Chemotaxis Signaling123
Introduction123
Basic Concepts in Dynamics and Mathematical Modeling124
Robustness and Steady-State Sensitivity Analysis127
Constructing and Interpreting a Bode Plot129
Primer on Integral Feedback Control132
Noise Filtering and the Kalman Filter135
Future Perspectives and Further Information138
References139
Chapter 7: Classification of Response Regulators Based on Their Surface Properties141
Introduction141
Classification of the Receiver Domain of RRs Using Protein Interaction Surfaces142
Modeling and Subclassification of Receiver Domains of OmpR Subfamily RRs in B. subtilis and E. coli144
Modeling and Subclassification of the Receiver Domain of RRs in V. vulnificus158
References168
Section II: Biochemical and Genetic Assays of Individual Components of Signaling Systems171
Chapter 8: Purification and Assays of Rhodobacter capsulatus RegB-RegA Two-Component Signal Transduc173
Introduction173
Expression and Purification of RegB175
Expression and Purification of RegA179
RegB Kinase and Phosphotransfer Assays180
Acknowledgment182
References182
Chapter 9: Purification and Reconstitution of PYP-Phytochrome with Biliverdin and 4-Hydroxycinnamic184
Introduction184
Vector Construct185
Prepation of 4-Hydroxycinnamic Acid Anhydride and Biliverdin185
Overexpression and Reconstitution of apo-Ppr with Chromophores185
Purification of Ppr Reconstituted with Chromophores186
Spectroscopic Measurements of holo-Ppr, Ppr-BV, and Ppr-pCA187
In Vitro Autophosphorylation of Ppr188
Acknowledgments188
References188
Chapter 10: Oxygen and Redox Sensing by Two-Component Systems That Regulate Behavioral Responses: Be190
Introduction190
Assays of Oxygen and Redox Sensing: General Considerations194
Temporal Assay for Aerotaxis197
Spatial-Gradient Capillary Assay for Aerotaxis203
Using a Capillary to Determine the Preferred Partial Pressure of Oxygen for Bacteria205
Spatial-Gradient Soft Agar Plate Assays for Aerotaxis208
Spatial Assays for Redox Taxis214
Temporal Assay for Redox Taxis216
Disulfide Cross-Linking In Vivo to Elucidate the Structure of Aer218
Site-Directed Mutagenesis for Cysteine Replacement219
Disulfide Cross-Linking in the Cytosol Using Copper Phenanthroline220
Differentiating Intra- from Interdimeric Disulfide Bonds221
In Vivo Cross-Linking Using Bifunctional Sulfhydryl-Reactive Linkers223
Determining the Boundaries of Transmembrane Segments in Receptors224
Accessibility Studies in Membrane Vesicles224
Acknowledgments227
References228
Chapter 11: Two-Component Signaling in the Virulence of Staphylococcus aureus: A Silkworm Larvae-Pat233
Introduction233
Silkworm Larvae Infection Model234
Pathogenicity-Related Genes That Can Be Identified in the Silkworm Infection Assay235
Identification of Genes Involved in the Killing of Silkworm Larvae by Bacteria236
Identification of an SA0614 Response Regulator Mutant by Monitoring CPZ Sensitivity and Ability to K237
Silkworm Larvae Infection Assay238
Measurement of the Number of Bacteria in Silkworm Hemolymph239
Defect in Cell Wall Integrity of the SA0614 Response Regulator Mutant241
Detergent and Lysozyme Sensitivity Test241
Melanization-Inducing Activity of Bacterial Peptidoglycan242
References242
Chapter 12: TonB System, In Vivo Assays and Characterization245
Introduction245
Selection For and Against the tonB Gene246
Precautions for Experiments Where TonB System Proteins Are Expressed from Plasmids248
Phenotypic Assays for the TonB System249
Mechanistically Informative Assays255
Potentially Mechanistically Informative Assays262
Acknowledgments265
References265
Chapter 13: Biochemical Characterization of Plant Ethylene Receptors Following Transgenic Expression270
Introduction270
Transgenic Expression of Ethylene Receptors in Yeast273
Histidine Kinase Activity274
Isolation of Receptors for Use in Ethylene-Binding Assays277
Ethylene-Binding Activity280
Considerations When Working with Mercuric Perchlorate283
Acknowledgments285
References285
Chapter 14: Structure of SixA, a Histidine Protein Phosphatase of the ArcB Histidine-Containing Phos288
Introduction288
Overall Structure290
Conservation of Catalytic Machinery and Active Site293
Sequence Analysis of SixA Homologs295
Eukaryotic Histidine Phosphatases302
Acknowledgments302
References302
Chapter 15: Triggering and Monitoring Light-Sensing Reactions in Protein Crystals305
Light-Regulated Histidine Kinases: A Brief Introduction305
Microbial Rhodopsins306
PAS/GAF/LOV Domains307
Photoreceptors and Kinetic Crystallography: A Near Perfect Match310
Kinetic Crystallography: Two Alternative Strategies311
Trapping Intermediates for X-Ray Crystallography313
Structural Interpretation of Kinetic Crystallography Results315
Optical Properties of Protein Crystals317
Mounting Crystals318
Design of a Single Crystal Microspectrophotometer320
Challenges of Recording UV-Visible Absorption Spectra in Crystals322
Leaking Light Introduces Spectral and Kinetic Artifacts324
Fluorescence Measurements327
Microspectrophotometry: Summary and Warning331
Light Activation of Photoreceptor Crystals331
Aligning the Activating Light Beam and the Crystal Position333
Summary and Outlook334
References334
Chapter 16: Synthesis of a Stable Analog of the Phosphorylated Form of CheY: Phosphono-CheY338
Introduction338
Protocols345
Acknowledgments350
References350
Chapter 17: Application of Fluorescence Resonance Energy Transfer to Examine EnvZ/OmpR Interactions352
Introduction352
Overexpression of EnvZ and Preparation of Spheroplasts354
Protein Purification and Fluorescent Labeling of OmpR355
Fluorescence Resonance Energy Transfer357
OmpR Has a Higher Affinity for EnvZ Than OmpR~P357
Concluding Remarks359
Acknowledgments359
References359
Chapter 18: Gene Promoter Scan Methodology for Identifying and Classifying Coregulated Promoters361
Introduction361
Challenge of Identifying Promoter Features Governing Gene Transcription363
GPS Methodology as an Integrated Algorithm365
Exploring Targets of Regulation of a Response Regulator Using GPS368
Technical Specifications of GPS373
Uncovering Promoter Profiles Regulated by Response Regulator PhoP Using GPS378
Conclusions380
Acknowledgments381
References382
Chapter 19: Targeting Two-Component Signal Transduction: A Novel Drug Discovery System386
Introduction386
Differential Growth Assay387
High-Throughput Genetic System391
Acknowledgments394
References394
Chapter 20: The Essential YycFG Two-Component System of Bacillus subtilis396
Introduction396
Construction of Conditional Mutants397
Transposon Mutagenesis to Identify Regulatory Elements400
Constructing In-Frame Deletions in the yyc Operon403
Studying Interactions between the YycG Kinase and Its Regulatory Proteins408
Subcellular Localization Studies411
Concluding Remarks415
Acknowledgments415
References415
Section III: Physiological Assays and Readouts419
Chapter 21: Isolation and Characterization of Chemotaxis Mutants of the Lyme Disease Spirochete Borr421
Introduction421
Borrelia burgdorferi Mutagenesis422
Chemotaxis and Motility Analysis423
Materials and Methods424
Acknowledgments435
References436
Chapter 22: Phosphorylation Assays of Chemotaxis Two-Component System Proteins in Borrelia burgdorfe438
Introduction439
Regulation of CheY-P439
Materials and Methods442
Acknowledgments446
References446
Chapter 23: Regulation of Respiratory Genes by ResD-ResE Signal Transduction System in Bacillus subt448
Introduction448
Oxygen Limitation and ResDE-Dependent Transcription449
Stimulatory Effect of NO on ResDE-Dependent Transcription451
Phosphorylation Assay Using Full-Length ResE453
In Vivo Effect of alpha-CTD Alanine Substitutions on ResDE-Dependent Transcription455
In Vitro Effect of alpha-CTD Alanine Substitutions on ResDE-Dependent Transcription458
Acknowledgments461
References462
Chapter 24: Detection and Measurement of Two-Component Systems That Control Dimorphism and Virulence465
Introduction465
Experimental Approaches468
Acknowledgment485
References485
Chapter 25: Using Two-Component Systems and Other Bacterial Regulatory Factors for the Fabrication o488
Using Two-Component Signal Transduction Systems in Synthetic Biology Approaches489
Using the NRI/NRII System to Build a Synthetic Genetic Clock491
Fabrication of Synthetic Genetic Clock493
Functions of Individual Clock Modules496
Improved Procedures for Fabrication of Synthetic Genetic Modules and Integration of These Modules in508
Fabricating Genetic Modules511
References512
Author Index513
Subject Index541

Book details

Vendor Elsevier S & T
SKU 9780123738516
ISBN-13 9780080548715
Author Simon, Melvin I.
Category Science
Subject Molecular Biology

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Multicellular organisms must be able to adapt to cellular events to accommodate prevailing conditions. Sensory-response circuits operate by making use of a phosphorylation control mechanism known as the "two-component system."

Sections include:
Computational Analyses of Sequences and Sequence Alignments
Biochemical and Genetic Assays of Individual Components of Signaling Systems
Physiological Assays and Readouts

* Presents detailed protocols
* Includes troubleshooting tips

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