Bacterial Secretion Systems Methods and Protocols

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. 

1               Identification of protein secretion systems in bacterial genomes using MacSyFinder version 2

Sophie S Abby, Rémi Denise, and Eduardo PC Rocha

 

2               Protein sorting prediction

Henrik Nielsen

 

3               Cell fractionation

Melissa Petiti, Laetitia Houot, and Denis Duché

 

4               Components sub-cellular localization: identification of lipoproteins using globomycin and radioactive palmitate

Nienke Buddelmeijer

 

5               Components sub-cellular localization: identification of lipoproteins using alkyne fatty acids and click-chemistry

Karine Nozeret, and Nienke Buddelmeijer

 

6               Defining membrane protein localization by isopycnic density gradients

Rhys A. Dunstan, Iain D. Hay, and Trevor Lithgow

 

7               Components sub-cellular localization: Cell surface exposure

Anna Konovalova

 

8               Probing protein topology and conformation by limited proteolysis

Maïalène Chabalier, Thierry Doan, and Eric Cascales

 

9               Exploring uniform, dual and dynamic topologies of membrane proteins by substituted cysteine accessibility method (SCAM^TM)

Mikhail Bogdanov

 

10            Preparation of uniformly oriented inverted inner (cytoplasmic) membrane vesicles from Gram-negative bacterial cells

Mikhail Bogdanov

 

11            Defining membrane protein topology using pho-lac reporter fusions

Gouzel Karimova, and Daniel Ladant

 

12            Measure of peptidoglycan degradation activity

Yoann G. Santin, and Eric Cascales

 

13            Protein-protein interaction: Bacterial Two-Hybrid

Gouzel Karimova, Emilie Gauliard, Maryline Davi, Scot P. Oulelette, and Daniel Ladant

 

14            Protein-protein interactions: oxidative Bacterial Two-Hybrid

Callypso Pellegri, Emmanuelle Bouveret, and Laetitia Houot

 

15            Protein-protein interactions: Yeast two-hybrid

Jer-Sheng Lin, and Erh-Min Lai

 

16            Protein-protein interactions: Bimolecular Fluorescence Complementation and Cytology two-hybrid

Dvuti Purkait, Mohd Ilyas, and Krishnamohan Atmakuri

 

17            Bacterial one- and two-hybrid assays to monitor transmembrane helix interactions

Abdelrahim Zoued, Jean-Pierre Duneau, and Eric Cascales

 

18            Protein-protein interactions: Co-immunoprecipitation

Jer-Sheng Lin, Jemal Ali, and Erh-Min Lai

 

19            Protein-protein interaction: Tandem Affinity Purification in bacteria

Julie P. M. Viala, and Emmanuelle Bouveret

 

20            In vivo site-directed and time-resolved photocrosslinking of envelope proteins

Yassin A. Abuta’a, Anne Caumont-Sarcos, Cécile Albenne, and Raffaele Ieva

 

21            Identification of protein partners by APEX2 proximity labeling

Ophélie Remy, and Yoann G. Santin

 

22            Blue native PAGE analysis of bacterial secretion complexes

Susann Zilkenat, Eunjin Kim, Tobias Dietsche, Julia V. Monjaràs Feria, Claudia E. Torres-Vargas, Mehari Tesfazgi Mebrhatu, and Samuel Wagner

 

23            Surface Plasmon Resonance: a sensitive tool to study protein-protein interactions

Badreddine Douzi

 

24            Defining assembly pathways by fluorescence microscopy

Andreas Diepold

 

25            Large complexes: cloning strategy, production and purification

Samira Zouhir, Wiem Abidi, and Petya V. Krasteva

 

26            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.

Maxime Mégret-Cavalier, Alexandre Pozza, Quentin Cece, Françoise Bonneté, Isabelle Broutin, and Gilles Phan

 

27            Structural analysis of protein complexes by cryo electron microscopy

Athanasios Ignatiou, Kévin Macé, Adam Redzej, Tiago R. D. Costa, Gabriel Waksman, and Elena V. Orlova

 

28            CryoEM data analysis of membrane proteins. Practical considerations on amphipathic belts, ligands and variability analysis

Alexia Gobet, Loïck Moissonnier, and Vincent Chaptal

 

29            Structural analyses of bacterial effectors by X-ray crystallography

Chloé Dugelay, Virginie Gueguen-Chaignon, and Laurent Terradot

 

30            Structural analysis of proteins from bacterial secretion systems and their assemblies by NMR spectroscopy

Gisele Cardoso de Amorim, Benjamin Bardiaux, and Nadia Izadi-Pruneyre

 

31            Quantifying substrate protein secretion via the type-III secretion system of the bacterial flagellum