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This volume discusses various basic and advanced methods and protocols that have been proven to be successful among certain bacterial species, or a family of species, in type III secretion systems (T3S system). The chapters in this book cover topics such as: site-directed mutagenesis and its application in studying the interactions of T3S components; use of transcriptional control to increase expression and secretion of heterologous proteins in T3S systems; fractionation techniques to examine effector translocation; detecting immune responses to T3S systems; mouse immunization with purified needle proteins from T3S systems and the characterization of the immune responses to these proteins; and detection of protein interactions in T3S systems using yeast 2-hybrid analysis. 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.

Practical and comprehensive, Type 3 Secretion Systems: Methods and Protocols is a valuable resource for anyone interested in learning about the fascinating and ever-changing T3S systems.

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Preface
Foreword
Table of Contents
Contributing Authors

1. Introduction to Type III Secretion Systems
Danielle Condry and Matthew L. Nilles

2. Site-Directed Mutagenesis and its Application in Studying the Interactions of T3S Components
Matthew S. Francis, Ayad A.A. Amer, Debra L. Milton, and Tiago R.D. Costa

3. Blue Native Protein Electrophoresis to Study the T3S System using Yersinia pestis as a Model
Thomas A. Henderson and Matthew L. Nilles

4. In Vivo Photo-Cross-Linking to Study T3S Interactions Demonstrated using the Yersinia pestis T3S System
Thomas A. Henderson and Matthew L. Nilles

5. Isolation of Type III Secretion System Needle Complexes by Shearing
Matthew L. Nilles, Danielle L. Jessen Condry, and Patrick Osei-Owusu

6. Use of Transcriptional Control to Increase Expression and Secretion of Heterologous Proteins in T3S Systems
Kevin J. Metcalf and Danielle Tullman-Ercek

7. Characterization of Type Three Secretion System Translocator Interactions with Phospholipid Membranes
Philip R. Adam, Michael L. Barta, and Nicholas E. Dickenson

8. Analysis of Type III Secretion System Secreted Proteins
Danielle L. Jessen Condry and Matthew L. Nilles

9. Fractionation Techniques to Examine Effector Translocation
Rachel M. Olson and Deborah M. Anderson

10. Measurement of Effector Protein Translocation using Phosphorylatable Epitope Tags and Phospho-Specific Antibodies
Sara Schesser Bartra and Gregory V. Plano

11. A TAL-Based Reporter Assay for Monitoring Type III-Dependent Protein Translocation in Xanthomonas
Sabine Drehkopf, Jens Hausner, Michael Jordan, Felix Scheibner, Ulla Bonas, and Daniela Büttner

12. Subcellular Localization of Pseudomonas syringae pv. tomato Effector Proteins in Plants
Kyaw Aung, Xiufang Xin, Christy Mecey, and Sheng Yang He

13. A Method for Characterizing the Type III Secretion System´s Contribution to Pathogenesis: Homologous Recombination to Generate Yersinia pestis Type III Secretion System Mutants
Patrick Osei-Owusu, Matthew L. Nilles, David S. Bradley, and Travis D. Alvine

14. Detecting Immune Responses to Type III Secretion Systems
Peter L. Knopick and David S. Bradley

15. Recombinant Expression and Purification of the Shigella Translocator IpaB
Michael L. Barta, Philip R. Adam, and Nicholas E. Dickenson

16. Expression and Purification of N-Terminally His-Tagged Recombinant Type III Secretion Proteins
Travis D. Alvine, atrick Osei-Owusu, Danielle L. Jessen Condry, and Matthew L. Nilles

17. Mouse Immunization with Purified Needle Proteins from Type III Secretion Systems and the Characterization of the Immune Response to these Proteins
Travis D. Alvine, David S. Bradley, and Matthew L. Nilles

18. Identification of the Targets of Type III Secretion System Inhibitors
Danielle L. Jessen Condry and Matthew L. Nilles

19. Detection of Protein Interactions in T3S Systems using Yeast 2-Hybrid Analysis
Matthew L. Nilles

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