Bacterial Secretion Systems: Methods and Protocols (Methods in Molecular Biology, 2715)

✍ Scribed by Laure Journet (editor), Eric Cascales (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 607
• Category: Library

✦ Synopsis

This second edition details new and updated protocols that cover techniques used to study secretion systems. Chapters focus on identifying and localizing the different subunits, defining interactions within subunits, monitoring conformational changes, purifying and imaging of large complexes, defining the assembly pathway by fluorescence microscopy and the role of energy during assembly and/or secretion, identifying secreted effectors as well as using reporters to follow effector transport. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Bacterial Secretion Systems: Methods and Protocols- Second Edition aims to be a useful and practical guide to new researchers and experts looking to expand their knowledge.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Identification of Protein Secretion Systems in Bacterial Genomes Using MacSyFinder Version 2
1 Introduction
2 Materials
2.1 Sequence Data
2.2 Predefined Models Available in TXSScan
2.3 Software
3 Methods
3.1 Compilation of Available Information on the System
3.1.1 Identification and Classification of the Components of the Secretion System
3.1.2 Extraction of HMM Protein Profiles from Databanks
3.1.3 Establishing the Model of the Genetic Architecture
3.1.4 Defining the Quorum of Components
3.2 Formulation of the Model
3.2.1 Defining the Model in an XML Text File: Example 1- The T1SS
3.2.2 Defining the Model in an XML Text File: Example 2-The T9SS
3.3 Running MacSyFinder
3.3.1 Organizing the Models Data or Installing the Macsy-Models from a Repository
3.3.2 Identification of the Secretion Systems
3.4 Finding the Most Relevant Information from MacSyFinder´s Output Files
3.5 Optimization, Validation, and Public Sharing of the Macsy-Models
3.5.1 Optimization
3.5.2 Validation
3.5.3 Sharing the Macsy-Models
4 Notes
References
Chapter 2: Protein Sorting Prediction
1 Introduction
2 Three Approaches to Prediction
3 Algorithms for Prediction
4 Performance of Prediction Methods
5 Recognition of Signal Peptides
6 Prediction of Secretion Without Signal Peptides
7 Prediction of Transmembrane Topology
8 Prediction of Cell Wall-Binding Motifs
9 Multi-category Predictors
10 Discussion
References
Chapter 3: Cell Fractionation
1 Introduction
2 Materials
2.1 Cell Fractionation
2.2 Proteins Solubilization
3 Methods
3.1 Cell Fractionation/Spheroplasts Formation
3.2 Protein Solubilization (See Note 11)
4 Notes
References
Chapter 4: Components Subcellular Localization: Identification of Lipoproteins Using Globomycin and Radioactive Palmitate
1 Introduction
2 Materials
2.1 3H-Palmitate (C16:0) Labeling of E. coli Bacterial Cultures
2.2 Inhibition of Lsp by Globomycin
2.3 Immune Precipitation of Lipoproteins
2.4 Tris-Tricine SDS Gel Electrophoresis
3 Methods
3.1 3H-Palmitate Labeling of E. coli Cultures
3.2 Inhibition of Lsp by Globomycin
3.3 Immune Precipitation of Lipoproteins
3.4 Tris-Tricine SDS Gel Electrophoresis
4 Notes
References
Chapter 5: Components Subcellular Localization: Identification of Lipoproteins Using Alkyne Fatty Acids and Click Chemistry
1 Introduction
2 Materials
2.1 Labeling of E. coli Cells with Alkyne Fatty Acids
2.2 Protein Preparation for Fluorescent Labeling of Lipoproteins
2.3 Phospholipid Preparation for Fluorescent Labeling
2.3.1 Total Phospholipid Extraction from Bacteria
2.3.2 Phospholipid Isolation by Thin Layer Chromatography (TLC)
2.3.3 PL Extraction from TLC Plate
2.3.4 Estimation Quantity of PE in PL Extract
2.4 Click Chemistry Reaction for Protein Labeling and Phospholipid Labeling (See Note 6)
2.4.1 For Protein Labeling
2.4.2 For Phospholipid Labeling
2.5 Enzymatic Activity Test
2.6 Protein Gel Electrophoresis
2.6.1 Common Material for SDS-PAGE
2.6.2 Tris-Tricine SDS-PAGE
2.6.3 Tris-Glycine SDS-PAGE
2.7 In-Gel Fluorescent Detection of Lipoproteins or Lipopeptides
3 Methods
3.1 Alkyne Fatty Acid Labeling of E. coli
3.1.1 For Protein Labeling
3.1.2 For Phospholipid Labeling
3.2 Protein Preparation for Fluorescent Labeling of Lipoproteins
3.3 Phospholipid Preparation for Fluorescent Labeling In Vitro
3.3.1 Total Phospholipid Extraction from Bacteria
3.3.2 Phospholipid Isolation by Thin-Layer Chromatography (TLC)
3.4 Enzyme Activity Test with Alkyne PE from PL Extract
3.5 Click Chemistry Reaction
3.5.1 For Protein Labeling
3.5.2 For Phospholipid Labeling
3.6 Gel Electrophoresis
3.6.1 For Protein Labeling
3.6.2 For Lipopeptide In Vitro Labeling Using Alkyne Phospholipid
4 Notes
References
Chapter 6: Defining Membrane Protein Localization by Isopycnic Density Gradients
1 Introduction
2 Materials
2.1 Membrane Purification
2.2 Sucrose Density Fractionation
2.3 Isolation of Membranes After Density Fractionation
3 Methods
3.1 Membrane Purification
3.2 Sucrose Density Fractionation
3.3 Isolation of Membranes After Density Fractionation
4 Notes
References
Chapter 7: Components Subcellular Localization: Cell Surface Exposure
1 Introduction
2 Materials
2.1 Cell Surface Labeling Based on a Modification of Primary Amines
2.2 Cell Surface Labeling Based on a Modification of Sulfhydryls
2.3 Cell Surface Proteolysis
2.4 Whole-Cell Dot Blot Assay
2.5 SpyTag/SpyCatcher Labeling
3 Methods
3.1 Cell Surface Labeling Based on a Modification of Primary Amines
3.2 Cell Surface Labeling Based on a Modification of Sulfhydryls
3.3 Cell Surface Proteolysis
3.4 Whole-Cell Dot Blot Assay
3.5 SpyTag/SpyCatcher Labeling
4 Notes
References
Chapter 8: Probing Protein Topology and Conformation by Limited Proteolysis
1 Introduction
2 Material
2.1 Cell Growth and Spheroplast Preparation
2.2 Protease Accessibility Assay
2.3 Sample Analysis by SDS-PAGE and Immunodetection
3 Method
3.1 Cell Growth and Spheroplast Preparation (See Note 4)
3.2 Protease Accessibility (See Notes 9 and 10)
3.3 Sample Analysis by SDS-PAGE and Immunodetection
4 Notes
References
Chapter 9: Exploring Uniform, Dual, and Dynamic Topologies of Membrane Proteins by Substituted Cysteine Accessibility Method (...
1 Introduction
1.1 Membrane Protein Topology and Topogenesis
1.2 Method of Choice
1.3 Justifying SCAM Legacy and Advantages
1.4 Application of SCAM
1.5 Overview and General Rationale of Topology Mapping Using SCAM
1.5.1 SCAMTM
1.5.2 Developing of a Working Topology Model and Selection of Diagnostic Cysteines for SCAM
1.5.3 Mutation Strategy, Host and Vector Selections, and Construction of Plasmids Expressing Single-Cysteine Derivatives
1.5.4 Cell Growth and Regulated Expression of Single-Cysteine Derivatives
1.5.5 General Protocol for SCAM
1.5.6 Application of SCAM in the Identification of Mixed and Dual Topologies
2 Materials
2.1 Construction of Plasmids Expressing Single-Cysteine Derivatives
2.2 Growth of E. coli Strains
2.3 SCAMTM
2.4 Membrane Protein Solubilization
2.5 Immunoprecipitation (IP)
2.6 SDS-PAGE and Western Blotting
3 Methods
3.1 Labeling with Maleimide Derivatives
3.2 Sample Solubilization
3.3 Isolation of Derivatized Target Proteins
3.4 SDS-PAGE, Western Blot Analysis, and Staining with Avidin-HRP
3.5 Data Analysis and Interpretation
4 Notes
References
Chapter 10: Preparation of Uniformly Oriented Inverted Inner (Cytoplasmic) Membrane Vesicles from Gram-Negative Bacterial Cells
1 Introduction
1.1 Cytoplasmic (Inner) Membrane of Gram-Negative Bacteria
1.2 In Vesiculo Veritas: An Application of ISO Vesicles
1.3 Experimental Validation of IM ISO Vesicles Orientation
1.4 Overview and Experimental Rationale for Preparation of Orientated Membrane Vesicles and Establishment of Their Sidedness A...
2 Materials
2.1 Cell Growth
2.2 Preparation of ISO Vesicles
2.3 SCAMTM
2.4 Membrane Protein Solubilization
2.5 Immunoprecipitation (IP)
2.6 SDS-PAGE and Western Blotting
3 Methods
3.1 Isolation of Inverted Membrane Vesicles
3.2 Labeling with Maleimide Derivatives
3.3 Sample Solubilization
3.4 Isolation of Derivatized Target Proteins
3.5 SDS-PAGE, Western Blot Analysis, and Staining with Avidin-HRP
3.6 Data Analysis and Interpretation
4 Notes
References
Chapter 11: Defining Membrane Protein Topology Using pho-lac Reporter Fusions
1 Introduction
2 Materials
2.1 Bacterial Growth Media, Strain, and Plasmid Construction
2.2 β-Galactosidase Assay
2.3 Phosphatase Assay
3 Methods
3.1 Selection of the pho-lac Fusion Sites Within the Target Membrane Protein
3.2 C-Terminal Fusion Approach
3.3 Nested Deletion Approach
3.4 Sandwich Fusion Approach
3.5 Analyzing Clones on Dual Substrate Plates
3.6 Growth of the Bacterial Culture for the Enzymatic Assays
3.7 Assay of β-galactosidase Activity
3.8 Assay of Phosphatase Activity
4 Notes
References
Chapter 12: Measure of Peptidoglycan Degradation Activity
1 Introduction
2 Material
2.1 Peptidoglycan Purification
2.2 Turbidimetric Analyses of Peptidoglycan Degradation
2.3 Peptidoglycan Labeling with Remazol Brilliant Blue
2.4 RBB-Labeled Peptidoglycan Degradation Assay
3 Methods
3.1 Peptidoglycan Purification
3.2 Turbidimetric Analyses of Peptidoglycan Degradation
3.3 Peptidoglycan Labeling with Remazol Brilliant Blue
3.4 RBB-Labeled Peptidoglycan Degradation Assay
4 Notes
References
Chapter 13: Protein-Protein Interaction: Bacterial Two Hybrid
1 Introduction
2 Materials
2.1 Equipment
2.2 Bacterial Media
2.3 Solutions for β-Galactosidase Assays
2.4 BACTH Reporter Strains, Plasmids, and Antibodies
3 Methods
3.1 General Methodology
3.2 Construction of BACTH Plasmids Encoding the Hybrid Proteins
3.2.1 Standard Cloning of Genes Encoding Proteins of Interest into BACTH Vectors
3.2.2 GatewayTM Cloning of the Genes Encoding the Proteins of Interest into BACTHGW Vectors
3.3 Analysis of Interactions by Screening Procedure on Indicator Plates
3.4 BACTH Screening of Interacting Partners: Selection Procedure on Minimal Medium
3.5 Quantification of Functional Complementation Between Hybrid Proteins by β-galactosidase Assays
3.6 Characterization of Hybrid Proteins by Western Blots
4 Notes
References
Chapter 14: Protein-Protein Interactions: Oxidative Bacterial Two Hybrid
1 Introduction
2 Materials
2.1 Equipment
2.2 Bacterial Media and Solutions
2.3 BACTH Reporter Media
2.4 Two-Hybrid Reporter Strains and Plasmids
3 Methods
4 Notes
References
Chapter 15: Protein-Protein Interactions: Yeast Two Hybrid
1 Introduction
2 Materials
2.1 Yeast Strain and Vectors: (Information Below Is According to [3])
2.2 Yeast Cultures and Yeast Transformation
2.3 Selective Media
2.4 Preparation of Yeast Cultures for Protein Extraction and Western Blot
2.5 Preparation of Yeast Protein Extracts
3 Methods
3.1 Gene Construction in pGBKT7 and pGADT7 Vectors
3.2 Preparation of Yeast Cultures for Yeast Transformation
3.3 PEG/LiAc-Mediated Transformation of Yeast (Small-Scale Transformation of Bait and Prey Plasmids) (See Note 15)
3.4 Selection of Transformants
3.5 Testing for Protein-Protein Interactions
3.6 Preparation of Yeast Cultures for Protein Extraction (See Note 28)
3.7 Preparation of Yeast Protein Extracts and Western Blot Analysis
4 Notes
References
Chapter 16: Protein-Protein Interactions: Bimolecular Fluorescence Complementation and Cytology Two Hybrid
1 Introduction
1.1 Bimolecular Fluorescence Complementation (BiFC)
1.1.1 Cytology-Based Two Hybrid
2 Materials
2.1 Bimolecular Fluorescence Complementation
2.2 Cytology-Based Two Hybrid
3 Methods
3.1 Bimolecular Fluorescence Complementation
3.2 Cytology-Based Two Hybrid
4 Notes
References
Chapter 17: Bacterial One- and Two-Hybrid Assays to Monitor Transmembrane Helix Interactions
1 Introduction
1.1 Monitoring TMH Homotypic Interactions
1.2 Monitoring TMH Heterotypic Interactions
2 Material
2.1 Monitoring TMH Homotypic Interactions: The TOXCAT Assay
2.2 Monitoring TMH Heterotypic Interactions: The GALLEX Assay
3 Methods
3.1 Monitoring TMH Homotypic Interactions: The TOXCAT Assay
3.2 Monitoring TMH Heterotypic Interactions: The GALLEX Assay
4 Notes
References
Chapter 18: Protein-Protein Interactions: Co-immunoprecipitation
1 Introduction
2 Materials
2.1 Materials for Co-IP by Cross-Linking
2.1.1 Cross-Linking of the Bacterial Cells (See Note 1)
2.1.2 Preparation of Bacterial Cell Extracts (See Note 4)
2.1.3 Protein Sample Preclearing
2.1.4 Coupling of Antibodies to Protein A Sepharose Beads
2.1.5 Purification and Isolation of Protein Complexes
2.1.6 TrueBlot for Protein Detection of Co-IP Complexes
2.2 Materials for Co-IP Without Cross-Linking
3 Methods
3.1 Methods for Co-IP by Cross-Linking
3.1.1 Cross-Linking of the Sample
3.1.2 Preparation of Bacterial Cell Extracts
3.1.3 Protein Sample Preclearing and Coupling of Antibody to Protein A/G Beads
3.1.4 Purification and Isolation of Protein Complexes
3.1.5 TrueBlot for Protein Detection of Co-IP Complexes
3.2 Methods for Co-IP Without Cross-Linking
3.2.1 Bacterial Culture Collection
3.2.2 Cell Lysis and Preparation of Bacterial Cell Extracts
3.2.3 Immunoprecipitation
4 Notes
References
Chapter 19: Protein-Protein Interaction: Tandem Affinity Purification in Bacteria
1 Introduction
2 Materials
2.1 Engineering of a TAP-Tag Translational Fusion and Verification of Production of Hybrid Protein by Western Blot
2.2 Preparation of the Protein Extract
2.3 Tandem Affinity Purification
2.4 Trichloroacetic Acid Precipitation
3 Methods
3.1 Verification of Expression of TAP-Tag Translational Fusion by Western Blot
3.2 Preparation of the Protein Extract
3.3 Tandem Affinity Purification
3.4 Trichloroacetic Acid Precipitation
3.5 Analysis by SDS-PAGE and Mass Spectrometry
4 Notes
References
Chapter 20: In Vivo Site-Directed and Time-Resolved Photocrosslinking of Envelope Proteins
1 Introduction
2 Materials
2.1 Biogenesis of the Autotransporter EspP: Site-Directed and Time-Resolved Photocrosslinking in Cells Metabolically Labeled w...
2.1.1 Plasmid Construction and Transformation of E. coli Cells
2.1.2 Expression of a EspP Variant Containing Bpa and Pulse-Chase Radiolabeling of Cells Using 35S-Labeled Amino Acids
2.1.3 Radiolabeling with 32P-Labeled Inorganic Phosphate and Expression of an EspP Variant Containing Bpa
2.1.4 Photocrosslinking
2.1.5 Immunoprecipitation and SDS-PAGE
2.2 Site-Directed Photocrosslinking of the Outer Membrane Lipoprotein DolP: Purification of a Photocrosslinked Partner Protein...
2.2.1 Expression of a DolPHis Variant Containing Bpa
2.2.2 Envelope Isolation, Solubilization, DolPHis Affinity Chromatography, and SDS-PAGE
2.2.3 MALDI-TOF Mass Spectrometry Analysis of Crosslinked Proteins
3 Method
3.1 Biogenesis of the Autotransporter EspP: Site-Directed and Time-Resolved Photocrosslinking in Cells Metabolically Labeled w...
3.1.1 Strategy Design and Plasmid Constructions to Overproduce Photoprobed EspP
3.1.2 Preparation of Cell Cultures for EspP Expression and Radiolabeling
3.1.3 Expression of Photoprobed EspP and Preparation of Cells for 35S-Pulse-Chase Labeling
3.1.4 Cell Labeling with 32P-Inorganic Phosphate, Expression of Photoprobed EspP and Photocrosslinking
3.1.5 35S-Pulse-Chase Labeling of Cells and Photocrosslinking
3.1.6 Immunoprecipitation of EspP and Analysis of Photocrosslinking Products
3.2 Site-Directed Photocrosslinking of the Outer Membrane Lipoprotein DolP: Purification of a Photocrosslinked Partner Protein...
3.2.1 Preparation of Cell Cultures Expressing the Photoprobed DolP Variant
3.2.2 DolPV52BpaHis Photocrosslinking and Protein Affinity Chromatography
3.2.3 Identification of Crosslinked Proteins and Mapping of the Interactions by MALDI-TOF Mass Spectrometry
4 Notes
References
Chapter 21: Identification of Protein Partners by APEX2 Proximity Labeling
1 Introduction
2 Materials
2.1 General Equipment
2.2 Solutions and Buffers
2.3 Labeling Reagents
2.4 Quenchers
2.5 Cell Lysis Reagents
2.6 Visualization and Enrichment of Biotinylated Proteins
3 Methods
3.1 Biotin-Phenol Incorporation
3.2 APEX2 Proximity Labeling
3.3 Cell Lysis
3.4 Biotinylation ``Fingerprint´´
3.5 Enrichment of Biotinylated Partners
3.6 Identification of Biotinylated Partners
4 Notes
References
Chapter 22: Blue Native PAGE Analysis of Bacterial Secretion Complexes
1 Introduction
2 Materials
2.1 Sample Preparation
2.1.1 Sample Preparation General Materials
2.1.2 Extraction of Membrane Proteins from Crude Bacterial Membrane Preparations
2.1.3 Membrane Fractionation by Sucrose Density Gradient Centrifugation
2.1.4 Immunoprecipitation of Membrane Protein Complexes
2.2 Blue Native PAGE
2.2.1 One-Dimensional BN PAGE Using Precast Mini Gels
2.2.2 Two-Dimensional BN/SDS PAGE
2.3 Protein Detection and Analysis
2.3.1 Colloidal Coomassie Staining
2.3.2 Silver Staining
2.3.3 Immunoblotting Using Dual-Color Detection
2.3.4 Preparation of BN PAGE-Separated Complexes for Analysis by Mass Spectrometry
3 Methods
3.1 Sample Preparation
3.1.1 Extraction of Membrane Proteins from Whole Bacterial Cells
3.1.2 Extraction of Membrane Proteins from Crude Bacterial Membrane Preparations
3.1.3 Preparation of Crude Membranes for Sucrose Density Gradient Centrifugation
3.1.4 Membrane Fractionation by Sucrose Density Gradient Centrifugation Using a Biocomp Gradient Station
3.1.5 Membrane Fractionation by Sucrose Density Gradient Centrifugation Using a Manual Sucrose Step Gradient
3.1.6 Preparing Membrane Fractions from Sucrose Gradient Fractionations for Downstream Experiments
3.1.7 Immunoprecipitation of Membrane Protein Complexes Using 3x FLAG Epitope Tags
3.2 Blue Native PAGE
3.2.1 One-Dimensional BN PAGE Using Precast Mini Gels
3.2.2 Two-Dimensional BN/SDS PAGE
3.3 Protein Detection and Analysis
3.3.1 Colloidal Coomassie Staining
3.3.2 Silver Staining (MS Compatible)
3.3.3 Immunoblotting Using Dual-Color Detection
3.3.4 Preparation of Blue Native PAGE-Separated Complexes for Analysis by Mass Spectrometry
4 Notes
References
Chapter 23: Surface Plasmon Resonance: A Sensitive Tool to Study Protein-Protein Interactions
1 Introduction
2 Materials
3 Methods
3.1 Which Protein Should Be Immobilized?
3.2 The Choice of the Immobilization Type and the Sensor Surface
3.3 How Much Protein Should I Immobilize?
3.4 Ligand and Analyte Preparation
3.5 Prepare the Material and the Buffers
3.6 pH Scouting
3.7 Immobilization of the Ligand Using Amine Coupling
3.7.1 Wizard Template Method
3.7.2 Manual Method
3.8 Immobilization of a Control Ligand
3.9 Analyte-Binding Analysis
3.10 Regeneration Optimization
3.11 Affinity and Kinetic Measurements
3.11.1 Affinity: Theory
3.11.2 Affinity: The Experiment
3.11.3 Affinity: Data Analysis
3.11.4 Kinetics: Theory
3.11.5 Kinetics: The Experiment
3.11.6 Kinetics: Data Analysis
4 Notes
References
Chapter 24: Defining Assembly Pathways by Fluorescence Microscopy
1 Introduction
2 Materials
2.1 Strains
2.2 Sample Preparation
2.3 Microscope Slide Preparation
2.4 Image Acquisition
2.5 Software for Image Processing
3 Methods
3.1 Preparation of Bacteria and Setup of Microscopy Equipment
3.2 Microscopy
3.3 Image Processing
3.4 Determination of the Assembly Pathway
4 Notes
References
Chapter 25: Large Complexes: Cloning Strategy, Production, and Purification
1 Introduction
1.1 Cloning, Expression, and Purification of the E. coli Bcs Macrocomplex
1.2 Cloning Expression, Purification, and Stabilization of Multimeric BcsB
2 Materials
2.1 Cloning, Expression, and Purification of the Bcs Macrocomplex
2.1.1 Cloning of the BcsRQABEF Macrocomplex
2.1.2 Expression and Purification of the Bcs Macrocomplex
2.2 Cloning, Expression, Purification, and Stabilization of the Co-polymerase BcsB
2.2.1 Cloning of Multimeric BcsBFL
2.2.2 Expression and Purification of Multimeric BcsBFL
2.2.3 SEC-Coupled On-Column Cross-Linking
3 Methods
3.1 Cloning, Expression, and Purification of the BcsRQABEF Macrocomplex
3.1.1 Cloning of the BcsRQABEF Macrocomplex
3.1.2 Expression and Purification of the Bcs Macrocomplex (See Fig. 2)
3.2 Cloning, Expression, Purification, and Stabilization of the Multimeric BcsBFL
3.2.1 Cloning of Multimeric BcsBFL
3.2.2 Expression and Purification of Multimeric BcsBFL
3.2.3 SEC-Coupled On-Column Cross-Linking
4 Notes
References
Chapter 26: Starting with an Integral Membrane Protein Project for Structural Biology: Production, Purification, Detergent Qua...
1 Introduction
2 Materials
2.1 Bacterial Culture and CntI Production
2.2 Bacterial Cell Lysis and Membrane Isolation
2.3 Solubilization of CntI with Detergent
2.4 Purification of CntI Solubilized in Detergent
2.4.1 Immobilized Affinity Chromatography (IMAC)
2.4.2 Size Exclusion Chromatography (SEC)
2.5 Thermofluor Assay (or Differential Scanning Fluorescence)
2.6 SEC-MALLS (Size Exclusion Chromatography-Multi Angle Laser Light Scattering)
3 Methods
3.1 Cell Culture and CntI Expression
3.2 Bacterial Cell Lysis and Membrane Preparation
3.3 Extraction and Solubilization of CntI
3.4 Purification of Solubilized CntI
3.4.1 Immobilized Affinity Chromatography (IMAC)
3.4.2 Size Exclusion Chromatography (SEC)
3.5 Stability Condition Screening by Fluorescent Thermal Stability Assay (FTSA)
3.5.1 Determination of Appropriate Concentration of Fluorophore and Protein
3.5.2 Screen of Buffer Solutions and Additives
3.5.3 Data Analysis
3.6 Analysis of Protein Detergent Complexes (PDC) by SEC-MALLS
3.6.1 Preparation of the SEC-MALLS System
3.6.2 Preparation and Analysis of PDC (Protein-Detergent Complex)
3.6.3 Evaluation of Detergent Micelles Accumulated During Protein Concentration
4 Notes
References
Chapter 27: Structural Analysis of Protein Complexes by Cryo-Electron Microscopy
1 Structural Studies by Cryo-EM of Macro-complexes as Illustrated by Studies of Type IV Secretion Systems
2 Sample Preparation in Cryo-EM
3 Data Acquisition
3.1 Direct Electron Detectors
3.2 Micrograph Sub-Frame Alignment
3.3 Radiation Damage
4 Processing of 2D Images
4.1 Contrast Transfer Function
4.2 Defocus Determination and Correction for the CTF Effects
4.3 Particle Selection
4.4 Normalization of Data
4.5 Classification of Particle Images
4.5.1 Principal Component Analysis
4.5.2 Maximum Likelihood Estimation
4.5.3 K-Means
4.5.4 Modification and New Developments in Classification
4.6 Determination of Particle Orientation
4.6.1 Projection Matching
4.6.2 Angular Reconstitution
5 3D Analysis of EM Structures
5.1 3D Reconstruction in Fourier Space
5.2 3D Reconstruction in Real Space
5.3 Structure Refinement
5.4 3D Classification for Analysis of Heterogeneity Molecular Complexes
6 Evaluation of the Structure Quality
6.1 Fourier Shell Correlation and the Gold Standard Approach
6.2 Local Estimation of Resolution
7 Interpretation and Fitting of Atomic Models
8 Application of the 3D Analysis to the T4SS
9 Conclusions
References
Chapter 28: CryoEM Data Analysis of Membrane Proteins. Practical Considerations on Amphipathic Belts, Ligands, and Variability...
1 Introduction
2 Typical Workflow of Membrane Protein Structure Determination
2.1 Typical Workflow
2.2 Membrane Proteins and Special Orientations: Grid-Type
2.3 How to Distinguish Real Membrane Protein Particles from Micelles
2.4 Types of Amphipathic Solvents Available for Membrane Protein Structural Investigations
2.5 The Case of Small Membrane Proteins
2.6 Membrane Protein and Low Resolution: Model Building
3 Visualization of Amphipathic Belts
3.1 Visualization of Amphipathic Belts in cryoEM Maps
3.2 Influence of Averaging and Symmetry on the Visualization of Amphipathic Belts in cryoEM
3.3 Lipids or Detergents in CryoEM Maps
4 Visualization of Ligands
4.1 Ligand Visualization and Resolution
4.2 Effect of Symmetry on Ligand Visualization: Case of Flexible Ligands or Plastic Binding-Sites
5 Variability Analysis of cryoEM Structures
5.1 Membrane Proteins are Flexible Objects
5.2 Interpretation of Membrane Protein Movements
6 Conclusion
References
Chapter 29: Structural Analyses of Bacterial Effectors by X-Ray Crystallography
1 Introduction
2 Materials
2.1 Drop Setup Using Mosquito Robot
2.2 Manual Optimization of Crystallization Conditions
2.3 Crystal Harvesting Freezing
2.4 Data Collection, Processing, Model Building, and Analysis
3 Methods
3.1 Crystal Screening
3.2 Optimization
3.3 Crystal Harvesting and Freezing
3.4 X-Ray Diffraction Screening and Data Collection
3.5 Data Processing and Analysis
3.6 Structure Determination and Model Building
3.7 Refinement/Validation/Deposition
3.8 Structure Analysis
3.8.1 PBDSUM: General Properties of Your Protein
3.8.2 PISA Analysis, Interface and Multimeric Assemblies
3.8.3 Comparing Structures Using DALI
3.8.4 Analysis of Surface Properties
Charge and Hydrophobicity
Metal Ligand Properties
3.8.5 Conserved Surface Residues
4 Notes
References
Chapter 30: Structural Analysis of Proteins from Bacterial Secretion Systems and Their Assemblies by NMR Spectroscopy
1 Introduction
1.1 Overall Folding, Interaction, and Structure/Dynamics Changes Upon Complex Formation
2 Materials
2.1 Sample Preparation
2.2 NMR Experiments
3 Methods
3.1 Sample Preparation
3.2 NMR Experiments
3.2.1 Preliminary Checking for Protein Folding and Stability
3.2.2 Mapping Ligand-Protein Binding and Interfaces
3.2.3 CSP Measurement
4 Notes
References
Chapter 31: Use of Bastion for the Identification of Secreted Substrates
1 Introduction
2 Method
2.1 Known Substrate´s Analysis
2.2 Novel Substrate Prediction
2.3 The Relationship Analysis Between the Known and the Novel Substrates
3 Notes
4 Conclusion
References
Chapter 32: Identification of Effectors: Precipitation of Supernatant Material
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 33: Metabolic Labeling: Snapshot of the Effect of Toxins on the Key Cellular Processes
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 34: Effector Translocation Assay: Differential Solubilization
1 Introduction
2 Materials (See Note 1)
2.1 Cell Culture, Infection, and Preparation of Cell Extracts
2.2 Sodium Dodecyl Sulfate Polyacrylamide Gel (SDS-PAGE)
2.3 Immunoblotting
3 Methods
3.1 Infection of RAW 264.7 Cells by Y. enterocolitica and Preparation of Triton-Soluble and Triton-Insoluble Fractions
3.2 Infection of HeLa Cells by S. typhimurium and Preparation of Triton-Soluble and Triton-Insoluble Fractions
3.3 SDS-PAGE and Immunoblotting
4 Notes
References
Chapter 35: Monitoring Effector Translocation with the TEM-1 Beta-Lactamase Reporter System: From Endpoint to Time Course Anal...
1 Introduction
2 Materials
2.1 Bacterial Strains, Beta-Lactamase Constructs, and Host Cells
2.2 Legionella pneumophila Media and Bacterial Growth
2.3 Cell Culture and Differentiation
2.4 Translocation Assays
3 Methods
3.1 Growth of the Infecting Legionella pneumophila Strains
3.2 Maintenance and Differentiation of U937 Target Cells
3.3 Detection of Effector Translocation Using a Fluorescence Plate Reader
3.4 Visualization of Effector Translocation Using a Fluorescence Microscope
4 Notes
References
Chapter 36: Quantifying Substrate Protein Secretion via the Type III Secretion System of the Bacterial Flagellum
1 Introduction
1.1 Investigating f-T3SS Substrate Protein Export in the Presence or Absence of Compounds Interfering with the pmf
1.2 Quantifying Protein Secretion of Individual f-T3SS Using Fluorescent Labeling of Flagellar Filaments
1.3 Quantifying Protein Secretion Via the f-T3SS Using Split NanoLuc Luciferase Fusion Proteins
2 Materials
2.1 Investigating f-T3SS Substrate Protein Export in the Presence or Absence of Compounds Interfering with the pmf
2.1.1 FlgM-Secretion Assay
2.1.2 Protein Fractionation
2.1.3 Protein Extraction by Filtration over a Nitrocellulose Filter
2.1.4 Protein Precipitation Using Trichloroacetic Acid
2.1.5 Immunoblotting
2.1.6 Assays to Inhibit the Proton Motive Force
2.2 Quantifying Protein Secretion of Individual f-T3SS Using Fluorescent Labeling of Flagellar Filaments
2.2.1 Flagellin Multi-Labeling
2.2.2 Preparation of Custom-Made Flow Cell
2.2.3 Microscopy Imaging and Analysis Software
2.3 Protein Secretion via the f-T3SS Using Split NanoLuc Luciferase Fusion Proteins (See Note 11 and Fig. 3)
3 Methods
3.1 Investigating f-T3SS Substrate Protein Export in the Presence or Absence of Compounds Interfering with the pmf
3.1.1 FlgM-Secretion Assay
3.1.2 Protein Fractionation
3.1.3 Protein Extraction by Filtration over a Nitrocellulose Filter (See Note 18)
3.1.4 Protein Precipitation Using Trichloroacetic Acid
3.1.5 Immunoblotting
3.1.6 Assays to Inhibit the Proton Motive Force: Disruption of the pmf Using Carbonyl Cyanide M-Chlorophenylhydrazone (See Not...
3.1.7 Assays to Inhibit the Proton Motive Force: Disruption of the ΔΨ Component of the pmf by K+/Valinomycin (See Note 22 and ...
3.1.8 Assays to Inhibit the Proton Motive Force: Disruption of the ΔpH Component of the pmf by Potassium Acetate (See Note 24 ...
3.2 Quantifying Protein Secretion of Individual f-T3SS Using Fluorescent Labeling of Flagellar Filaments
3.2.1 Fluorescent Labeling of Flagellar Filaments
3.2.2 Preparation of Microscope Flow Cell
3.2.3 Microscopy of Multi-Labeled Flagellar Filaments
3.3 Quantifying Protein Secretion Via the f-T3SS Using Split NanoLuc Luciferase Fusion Proteins
3.3.1 Split NanoLuc Secretion Assay
3.3.2 Detecting Luminescence Using the Nano-Glo HiBiT Extracellular Detection System
3.3.3 Analysis of NanoLuc Secretion Assay
4 Notes
References
Chapter 37: Quantitative Determination of Antibacterial Activity During Bacterial Coculture
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 38: Investigating Secretion Systems and Effectors on Galleria mellonella
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Insects
3.2 Preparation of Bacteria
3.3 Infection
3.4 Death Monitoring
3.5 Assess Bacterial Burden at the Time of Death
4 Notes
References
Index