Bacterial Protein Secretion Systems

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 arealso of specific interest for any researcher interested on multi-protein complexes of the bacterial cell envelope.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts

Inhalt
Identification of Protein Secretion Systems in Bacterial Genomes using MacSyFinder.- Protein Sorting Prediction .- Cell Fractionation.- Defining Lipoprotein Localisation by Fluorescence Microscopy.- Components Sub-cellular Localization: Identification of Lipoproteins using Globomycin and Radioactive Palmitate.- Defining Membrane Protein Localization by Isopycnic Density Gradients.- Components Sub-cellular Localisation: Cell Surface Exposure.- Probing Inner Membrane Protein Topology by Proteolysis.- Mapping of Membrane Protein Topology by Substituted Cysteine Accessibility Method (SCAM TM ).- 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.- Protein-protein Interactions: Cytology Two-hybrid.- Fusion Reporter Approaches to Monitor 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 Cryo-tomography.- Structural Analysis of Protein Complexes by Cryo Electron Microscopy.- Bacterial Filamentous Appendages Investigated by Solid-state NMR Spectroscopy.- Identification of Effectors: Precipitation of Supernatant Material.- Energy Requirements for Protein Secretion via the Flagellar type-III Secretion System.- Screening for Secretion of the Type VI Secretion Protein Hcp by Enzyme-Linked Immunosorbent Assay and Colony Blot.- Effector Translocation: Cya Reporter Assay.- Monitoring Effector Translocation with the TEM-1 Beta-lactamase Reporter System.- Effector Translocation Assay: Differential Solubilization.- Quantitative Determination of Anti-bacterial Activity during Bacterial Co-culture.