Journet Laure, Cascales Eric (eds.) Bacterial Protein Secretion Systems: Methods and Protocols

Humana Press, 2017. — 521 p. — (Methods in Molecular Biology 1615). — ISBN: 978-1-4939-7031-5.This volume details protocols that cover the broad arsenal of techniques used to study a secretion system from A to Z. 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 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 tips on troubleshooting and avoiding known pitfalls.Authoritative and cutting-edge, Bacterial Protein Secretion Systems: Methods and Protocol aims to provide techniques that are not restricted to the study of secretion systems but are also of specific interest for any researcher interested on multi-protein complexes of the bacterial cell envelope.Identification of Protein Secretion Systems in Bacterial Genomes Using MacSyFinder
Protein Sorting Prediction
Cell Fractionation
Defining Lipoprotein Localisation by Fluorescence Microscopy
Identification of Lipoproteins Using Globomycin and Radioactive Palmitate
Defining Membrane Protein Localization by Isopycnic Density Gradients
Cell Surface Exposure
Probing Inner Membrane Protein Topology by Proteolysis
Mapping of Membrane Protein Topology by Substituted Cysteine Accessibility Method (SCAM)
Defining Membrane Protein Topology Using pho-lac Reporter Fusions
In Vivo and In Vitro Protein–Peptidoglycan Interactions
Measure of Peptidoglycan Hydrolase Activity
Protein–Protein Interaction: Bacterial Two-Hybrid
Protein–Protein Interactions: Yeast Two-Hybrid System
Protein–Protein Interactions: Cytology Two-Hybrid
Fusion Reporter Approaches to Monitoring Transmembrane Helix Interactions in Bacterial Membranes
Protein–Protein Interactions: Co-Immunoprecipitation
Protein–Protein Interaction: Tandem Affinity Purification in Bacteria
Site-Directed and Time-Resolved Photocrosslinking in Cells Metabolically Labeled with Radioisotopes
Protein–Protein Interactions: Pull-Down Assays
Protein–Protein Interactions: Surface Plasmon Resonance
Assessing Energy-Dependent Protein Conformational Changes in the TonB System
Defining Assembly Pathways by Fluorescence Microscopy
Large Complexes: Cloning Strategy, Production, and Purification
Shearing and Enrichment of Extracellular Type IV Pili
Blue Native PAGE Analysis of Bacterial Secretion Complexes
In Situ Imaging of Bacterial Secretion Systems by Electron Cryotomography
Structural Analysis of Protein Complexes by Cryo Electron Microscopy
Bacterial Filamentous Appendages Investigated by Solid-State NMR Spectroscopy
Energy Requirements for Protein Secretion via the Flagellar Type III Secretion System
Identification of Effectors: Precipitation of Supernatant Material
Screening for Secretion of the Type VI Secretion System Protein Hcp by Enzyme-Linked Immunosorbent Assay and Colony Blot
Effector Translocation: Cya Reporter Assay
Monitoring Effector Translocation using the TEM-1 Beta-Lactamase Reporter System
Effector Translocation Assay: Differential Solubilization
Quantitative Determination of Anti-bacterial Activity During Bacterial Co-culture