Bacterial Chemosensing: Methods and Protocols

Part I: Introduction

1. The Diversity of Bacterial Chemosensing

Michael D. Manson

2. Transmembrane Signal Transduction in Bacterial Chemosensing

Michael D. Manson

Part II: Methods for Studying Bacterial Chemotaxis at the Population and Whole-Cell Levels

3. Two Spatial Chemotaxis Assays: The Nutrient-Depleted Chemotaxis Assay and the Agarose-Plug-Bridge Assay

Victoria Korolik and Karen M. Ottemann

4. Quantification of Bacterial Chemotaxis Responses at the Mouths of Hydrogel Capillaries

Benjamin A. Webb, Timofey Arapov, and Birgit Scharf

5. A Static Microfluidic Device for Investigating the Chemotaxis Response to Stable, Non-Linear Gradients

Nitesh Sule, Daniel Penarete-Acosta, Derek L. Englert, and Arul Jayaraman

6. Visualizing Chemoattraction of Planktonic Cells to a Biofilm

Sneha Jani

7. Labeling Bacterial Flagella with Fluorescent Dyes

Part III: In Vivo Analysis of Receptor Function

8. All-Codon Mutagenesis for Structure-Function Studies of Chemotaxis Signaling Proteins

Peter Ames and John S. Parkinson

9. Mutational Analysis of Binding Protein/Chemoreceptor Interactions

Andrew L. Seely

10. In Vitro Assay for Measuring Receptor-Kinase Activity in the Bacillus subtilis Chemotaxis Pathway

Hanna E. Walukiewicz, George W. Ordal, and Christopher V. Rao

11. FRET Analysis of the Chemotaxis Pathway Response

Anja Paulick and Victor Sourjik

12. Monitoring Two-Component Sensor Kinases with a Chemotaxis Signal Readout

Run-Zhi Lai and John S. Parkinson

13. Analyzing Protein Domain Interactions in Chemoreceptors by In Vivo PEGylation

Kylie J. Watts and Mark S. Johnson

14. Tuning Chemoreceptor Signaling by Positioning Aromatic Residues at the Lipid-Aqueous Interface

15. Analyzing Chemoreceptor Interactions In Vivo with the Tri-Functional Crosslinker TMEA

Claudia A. Studdert and Diego A. Massazza

Part IV: Cryo-EM Methods for Studying Chemoreceptor Structure

16. Use of Cryo-EM to Study the Structure of Chemoreceptor Arrays In Vivo

Wen Yang and Ariane Briegel

17. Visualizing Chemoreceptor Arrays in Bacterial Minicells by Cryo-Electron Tomography and Subtomogram Analysis

Zhuan Qin, Bo Hu, and Jun Liu

Part V: Monitoring the Intracellular Movement of Chemosensory Proteins

18. Bacterial Chemoreceptor Imaging at High Spatio-Temporal Resolution Using Photoconvertible Fluorescent Proteins

Jacopo Solari, Francois Anquez, Katharina Scherer, and Thomas S. Shimizu

19. Imaging of Single Dye-Labeled Chemotaxis Proteins in Live Bacteria Using Electroporation

Diana Di Paolo and Richard M. Berry

20. Fluorescence Anisotropy to Detect In Vivo Stimulus-Induced Changes in Chemoreceptor Packing

Part VI: High-Throughput Methods for Screening Novel Chemoeffectors

21. Chemotaxis to Atypical Chemoattractants by Soil Bacteria

Rebecca E. Parales and Jayna L. Ditty

22. Screening Chemoreceptor-Ligand Interactions by High-Throughput Thermal-Shift Assays

Maximilian K.G. Ehrhardt, Suzanne L. Warring, and Monica L. Gerth

23. High-Throughput Screening to Identify Chemoreceptor Ligands

24. Identification of Specific Ligands for Sensory Receptors by Small-Molecule Ligand Arrays and Surface Plasmon Resonance

Christopher J. Day and Victoria Korolik

Part VII: Creating Chemical Tools for Studying Chemosensory Signal Transduction

25. Fluorescence Measurement of Kinetics of CheY Autophosphorylation with Small Molecule Phosphodonors

Ruth E. Silversmith and Robert B. Bourret

26. Synthesis of a Stable Analog of the Phosphorylated Form of CheY: Phosphono-CheY

Daniel B. Lookadoo, Matthew S. Beyersdorf, and Christopher J. Halkides

Part VIII: Computer Simulation of Chemotaxis

27. Quantitative Modeling of Flagellar Motor-Mediated Adaptation

Pushkar P. Lele

28. Molecular Modeling of Chemoreceptor:Ligand Interactions

Asuka A. Orr, Arul Jayaraman, and Phanourios Tamamis

29. Phylogenetic and Protein Sequence Analysis of Bacterial Chemoreceptors

Davi R. Ortega and Igor B. Zhulin