Two Component Signaling Systems Part B 1st Edition by Melvin I Simon ,Brian Crane ,Alexandrine Crane ISBN 0123738520 978-0123738523

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ISBN 10:0123738520

ISBN 13:978-0123738523

Author:Melvin I Simon ,Brian Crane ,Alexandrine Crane

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 in Two-Component Signaling Systems, Part B include:

• Structural Approaches
• Reconstitution of Heterogeneous Systems
• Intracellular Methods and Assays
• Genome-Wide Analyses of Two-Component Systems

• Presents detailed protocols
• Includes troubleshooting tips

Table of contents: 

Introduction to Off-Premise Catering Management

Bacterial Quorum Sensing

The V. harveyi AI-2 Signal Transduction Pathway

Regulation of the LuxPQ Receptor Complex by AI-2

Expression of Wild-Type and Mutant LuxPQp

Purification of LuxP, LuxQp, and LuxPQp

Crystallization of LuxPQp Complexes

Functional Analysis

Conclusions

Acknowledgments

Section II: Reconstitution of Heterogeneous Systems

Liposome-Mediated Assembly of Receptor Signaling Complexes

Abstract

Introduction

Results—Biochemical Activity of Liposome-Assembled Receptor Fragments

Methods

Conclusion

Acknowledgment

Analyzing Transmembrane Chemoreceptors Using In Vivo Disulfide Formation Between Introduced Cysteines

Abstract

Introduction

Disulfide Formation In Vivo: Applications and Limitations

Oxidation Reagents

Oxidation Treatments That Preserve In Vivo Function

Experimental Designs

Procedures

Analysis

Closing Comments

Acknowledgments

Using Nanodiscs to Create Water-Soluble Transmembrane Chemoreceptors Inserted in Lipid Bilayers

Abstract

Introduction

Developing a Protocol for Producing Nanodisc-Embedded Protein

Preparation of Nanodisc-Embedded Chemoreceptor

Preparation of Cytoplasmic Membranes with High Tar-6H Content

Receptor Purification

Preparation of Receptor-Containing Nanodiscs

Analysis of Receptor-Containing Nanodiscs

Acknowledgments

Assays for CheC, FliY, and CheX as Representatives of Response Regulator Phosphatases

Abstract

Introduction

Assays

Phosphate Release Assay

Pulldowns

Acknowledgments

Genetic Dissection of Signaling Through the Rcs Phosphorelay

Abstract

Overview

Flowchart of Testing: Signaling Inputs

Analysis of the Regulation of a Target Gene

Analysis of Signaling via the Rcs Phosphorelay

RcsC-Dependent Signaling

RcsA-Dependent Signaling: Increased RcsA Synthesis or Stability

Determining Whether a Strain Carries a lon Mutation or is Phenotypically Lon−

Conclusions

Acknowledgments

Section III: Intracellular Methods and Assays

In Vivo Measurement by FRET of Pathway Activity in Bacterial Chemotaxis

Abstract

Introduction

FRET

FRET Measurement of the Interaction Between CheY-YFP and CheZ-CFP in a Population of Bacteria Fixed to a Microscope Cover Slip

FRET Measurement of the Interaction Between CheY-YFP and CheZ-CFP in Single Bacteria Fixed to a Microscope Cover Slip

BRET Measurement of the Interaction Between YFP-CheY and -CheZ-RLUC in a Population of Bacteria Swimming in a Cuvette

Comparison of Different Approaches and Application to Other Two-Component Systems

In Vivo and In Vitro Analysis of the Rhodobacter sphaeroides Chemotaxis Signaling Complexes

Abstract

Introduction

In Vitro Analysis of Signaling by the Kinase Cluster

Genomic Replacements with Fluorescent Protein Fusions for Studying Protein Localization

Assessing the Functionality of the Fluorescent Protein Fusions

Summary

In Vivo Crosslinking Methods for Analyzing the Assembly and Architecture of Chemoreceptor Arrays

Abstract

Introduction

Use of a Lysine-Targeted Crosslinker to Probe Receptor–Receptor Interactions in Cells

Use of Cys-Targeted Crosslinking to Probe for the Trimer-of-Dimers Geometry in Cellular Chemoreceptor Assemblies

Intracytoplasmic Disulfide Crosslinks

A Trifunctional Cys-Targeted Crosslinker

TMEA Competition Assay: A Tool for Assessing the Trimer-Forming Ability of Mutant Receptors

Exchange Assay: Dynamic Changes in Trimer Composition as a Consequence of Changes in the Receptor Population

Concluding Remarks

Acknowledgments

A “Bucket of Light” for Viewing Bacterial Colonies in Soft Agar

Abstract

Viewing Colonies Grown in Soft Agar

Building a Bucket of Light

Acknowledgments

Phenotypic Suppression Methods for Analyzing Intra- and Inter-Molecular Signaling Interactions of Chemoreceptors

Abstract

Introduction

Genetic Analyses of Chemoreceptors

Balancing Suppression: Methylation-Independent Chemoreceptors

Conformational Suppression within Receptor Molecules

Conformational Suppression Between Receptor Molecules

Acknowledgments

Single-Cell Analysis of Gene Expression by Fluorescence Microscopy

Abstract

Introduction

Transcriptional Reporters

Measuring Cellular Fluorescence by Microscopy

Agarose Pads

Fluorescence Microscopy and Image Acquisition

Image Analysis

Concluding Remarks

Section IV: Genome-Wide Analyses of Two-Component Systems

Two-Component Systems of Mycobacterium tuberculosis—Structure-Based Approaches

Abstract

Introduction

Orphan TCS Proteins

Information from Crystal Structures

Structural Genomics as a Driving Force

Domain Boundary Definitions

Protein Production as a Source of Material for Structural Studies and In Vitro Inhibition Assays

Crystallographic Studies

Information on Solution Structure from Small-Angle X-Ray Scattering

Structural Information Relating to Regulation Mechanisms

Transcriptomic Analysis of ArlRS Two-Component Signaling Regulon, a Global Regulator, in Staphylococcus aureus

Abstract

Introduction

Construction of an arlR Allelic Replacement Mutant in S. aureus

Purification of Total RNA From Wild Type and arlR Mutant Strains

cDNA Synthesis, cDNA Fragmentation, and Labeling

Microarray Analysis

Quantitative Real-Time RT-PCR Analysis

Acknowledgments

Global Analysis of Two-Component Gene Regulation in H. pylori by Mutation Analysis and Transcriptional Profiling

Abstract

Introduction

Functional Analysis of Essential Response Regulators of H. pylori

Characterization of the Regulons Controlled by the H. pylori Two-Component Systems

Design of the Experiment for Transcriptional Profiling

Validation of the Data

Concluding Remarks

Phosphotransfer Profiling: Systematic Mapping of Two-Component Signal Transduction Pathways and Phosphorelays

Abstract

Overview

Detailed Protocols

Interpretation and Analysis

Phosphorelays and Histidine Phosphotransferases

Concluding Remarks

Identification of Histidine Phosphorylations in Proteins Using Mass Spectrometry and Affinity-Based Techniques

Abstract

Introduction

Sample Fractionation

Phosphoprotein Enrichment

Gel Separation

Mass Spectrometry

Phosphopeptide Enrichment

Identification of Phosphohistidine in a Model Protein

Phosphorylation and Digestion of HPr

IMAC Conditions

MALDI-TOF MS Conditions

Enrichment of His-Phosphorylated Peptides

Selectivity for Phosphorylated Histidine

Detection of His-Phosphorylated Peptides

Specificity for Phosphohistidines

Differential Hydrolysis of Phosphohistidines

Summary and Conclusions

Subject Index

Publisher Summary

Author Index

Publisher Summary

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Tags: Melvin I Simon, Brian Crane, Alexandrine Crane, Two, Component, Signaling