Bacterial Protein Secretion Systems, Softcover reprint of the original 1st ed. 2017
Methods and Protocols
Methods in Molecular Biology Series, Vol. 1615

Language: English
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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.

1. Identification of Protein Secretion Systems in Bacterial Genomes using MacSyFinder

Sophie S Abby and Eduardo PC Rocha

 

2. Protein Sorting Prediction 

Henrik Nielsen 

 

3. Cell Fractionation

Melissa Petiti, Laetitia Houot, and Denis Duché

 

4. Defining Lipoprotein Localisation by Fluorescence Microscopy

Maria Guillermina Casabona, Mylène Robert-Genthon, Didier Grunwald, and Ina Attrée

 

5. Components Sub-cellular Localization: Identification of Lipoproteins using Globomycin and Radioactive Palmitate

Nienke Buddelmeijer

 

6. Defining Membrane Protein Localization by Isopycnic Density Gradients

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

 

7. Components Sub-cellular Localisation: Cell Surface Exposure

Anna Konovalova

 

8. Probing Inner Membrane Protein Topology by Proteolysis

Maxence S. Vincent and Eric Cascales

 

9. Mapping of Membrane Protein Topology by Substituted Cysteine Accessibility Method (SCAMTM)

Mikhail Bogdanov

 

10. Defining Membrane Protein Topology using pho-lac Reporter Fusions

Gouzel Karimova and Daniel Ladant

 

11. In vivo and in vitro Protein-peptidoglycan Interactions

Gang Li and S. Peter Howard

 

12. Measure of Peptidoglycan Hydrolase Activity

Yoann G. Santin and Eric Cascales

 

13. Protein-protein Interaction: Bacterial Two-Hybrid

Gouzel Karimova, Emilie Gauliard, Marilyne Davi, Scot P. Ouellette, and Daniel Ladant

 

14. Protein-protein Interactions: Yeast two-hybrid

Jer-Sheng Linand Erh-Min Lai

 

15. Protein-protein Interactions: Cytology Two-hybrid

Krishnamohan Atmakuri

 

16. Fusion Reporter Approaches to Monitor Transmembrane Helix Interactions in Bacterial Membranes

Laureen Logger, Abdelrahim Zoued, and Eric Cascales

17. Protein-protein Interactions: Co-immunoprecipitation

Jer-Sheng Linand Erh-Min Lai

 

18. Protein-protein Interaction: Tandem Affinity Purification in Bacteria

Julie P.M. Viala and Emmanuelle Bouveret

 

19. Site-directed and Time-resolved Photocrosslinking in Cells Metabolically Labeled With Radioisotopes

Raffaele Ieva

 

20. Protein-protein Interactions: Pull-down Assays

Arthur Louche, Suzana P. Salcedo, and Sarah Bigot

 

21. Protein-protein interactions: Surface Plasmon Resonance

Badreddine Douzi

 

22. Assessing Energy-dependent Protein Conformational Changes in the TonB System

Ray A. Larsen

 

23. Defining Assembly Pathways by Fluorescence Microscopy

Abdelrahim Zouedand Andreas Diepold

 

24. Large Complexes: Cloning Strategy, Production, and Purification

Eric Durand and Roland Lloubes

 

25. Shearing and Enrichment of Extracellular Type IV Pili

Alba Katiria, Gonzalez Rivera, and Katrina T. Forest

 

26. Blue Native PAGE Analysis of Bacterial Secretion Complexes

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

 

27. In situ Imaging of Bacterial Secretion Systems by Electron Cryo-tomography

Gregor L. Weiss, João M. Medeiros, and Martin Pilhofer

 

28. Structural Analysis of Protein Complexes by Cryo Electron Microscopy

Tiago R. D. Costa, Athanasios Ignatiou, and Elena V. Orlova

 

29. Bacterial Filamentous Appendages Investigated by Solid-state NMR Spectroscopy

Birgit Habenstein and Antoine Loquet

 

30. Identification of Effectors:  Precipitation of Supernatant Material

Nicolas Flaugnatti and Laure Journet

 

31. Energy Requirements for Protein Secretion via the Flagellar type-III Secretion System

Marc Erhardt

 

32. Screening for Secretion of the Type VI Secretion Protein Hcp by Enzyme-Linked Immunosorbent Assay and Colony Blot

Brent S. Weber, Pek Man Ly, and Mario F. Feldman

33. Effector Translocation: Cya Reporter Assay

Suma Chakravarthy, Bethany Huot, and Brian H. Kvitko

 

34. Monitoring Effector Translocation with the TEM-1 Beta-lactamase Reporter System

Julie Allombert, Anne Vianney, and Xavier Charpentier

 

35. Effector Translocation Assay: Differential Solubilization

Irina S. Franco, Sara V. Pais, Nuno Charro, and Luís Jaime Mota

 

36. Quantitative Determination of Anti-bacterial Activity during Bacterial Co-culture

Juliana Alcoforado Diniz, Birte Hollmann, and Sarah Coulthurst

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts