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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 andpractical guide to new researchers and experts looking to expand their knowledge.
List of contents
Identification of protein secretion systems in bacterial genomes using MacSyFinder version 2.- Protein sorting prediction.- Cell fractionation.- Components sub-cellular localization: identification of lipoproteins using globomycin and radioactive palmitate.- Components sub-cellular localization: identification of lipoproteins using alkyne fatty acids and click-chemistry.- Defining membrane protein localization by isopycnic density gradients.- Components sub-cellular localization: Cell surface exposure.- Probing protein topology and conformation by limited proteolysis.-Exploring uniform, dual and dynamic topologies of membrane proteins by substituted cysteine accessibility method (SCAMTM).- Preparation of uniformly oriented inverted inner (cytoplasmic) membrane vesicles from Gram-negative bacterial cells.- Defining membrane protein topology using pho-lac reporter fusions.- Measure of peptidoglycan degradation activity.- Protein-protein interaction: Bacterial Two-Hybrid.- Protein-protein interactions: oxidative Bacterial Two-Hybrid.- Protein-protein interactions: Yeast two-hybrid.- Protein-protein interactions: Bimolecular Fluorescence Complementation and Cytology two-hybrid.- Bacterial one- and two-hybrid assays to monitor transmembrane helix interactions.- Protein-protein interactions: Co-immunoprecipitation.- Protein-protein interaction: Tandem Affinity Purification in bacteria.- In vivo site-directed and time-resolved photocrosslinking of envelope proteins.- Identification of protein partners by APEX2 proximity labeling.- Blue native PAGE analysis of bacterial secretion complexes.- Surface Plasmon Resonance: a sensitive tool to study protein-protein interactions.- Defining assembly pathways by fluorescence microscopy.- Large complexes: cloning strategy, production and purification.- Starting with an integral membrane protein project for structural biology: production, purification, detergent quantification and buffer optimization - case study of the exporter CntI from Pseudomonas aeruginosa.- Structural analysis of protein complexes by cryo electron microscopy.- CryoEM data analysis of membrane proteins. Practical considerations on amphipathic belts, ligands and variability analysis.- Structural analyses of bacterial effectors by X-ray crystallography.- Structural analysis of proteins from bacterial secretion systems and their assemblies by NMR spectroscopy.- Quantifying substrate protein secretion via the type-III secretion system of the bacterial flagellum.- Use of Bastion for the identification of secreted substrates.- Identification of effectors: Precipitation of supernatant material.- Metabolic labelling: snapshot of the effect oftoxins on the key cellular processes.- Effector translocation assay: differential solubilization.- Monitoring effector translocation with the TEM-1 beta-lactamase reporter system: from endpoint to time course analysis.- Quantitative determination of anti-bacterial activity during bacterial co-culture.- Investigating secretion systems and effectors on Galleria mellonella.
