Bacterial Chemosensing: Methods and Protocols

This volume covers a wide range of up-to-date technologies that have been successfully applied to study the chemosensing behavior of the traditional model species, such as Escherichia coli and Salmonella typhimurium, while being also applicable to a wide spectrum of other species. Beginning with an introduction, the sections of the book explore methods for studying bacterial chemotaxis at the population and whole-cell levels, in vivo analysis of receptor function, cryo-EM methods for studying chemoreceptor structure, as well as intracellular movement of chemosensory proteins, high-throughput methods to screen for novel chemoeffectors, and chemical tools and computer simulations for analyzing chemotaxis. Written for the highly successful Methods in Molecular Biology series, chapters include brief introductions to their topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips for troubleshooting and avoiding known pitfalls.  Authoritative and cutting-edge, Bacterial Chemosensing: Methods and Protocols provides an extensive repertoire of approaches that can be extended to understanding chemotaxis, in particular, and chemosensing, in general, in the context of the enormously varied lifestyles adopted in the larger bacterial world.

Table of Contents:
The Diversity of Bacterial Chemosensing.- Transmembrane Signal Transduction in Bacterial Chemosensing.- Two Spatial Chemotaxis Assays: The Nutrient-Depleted Chemotaxis Assay and the Agarose-Plug-Bridge Assay.- Quantification of Bacterial Chemotaxis Responses at the Mouths of Hydrogel Capillaries.- A Static Microfluidic Device for Investigating the Chemotaxis Response to Stable, Non-Linear Gradients.- Visualizing Chemoattraction of Planktonic Cells to a Biofilm.- Labeling Bacterial Flagella with Fluorescent Dyes.- All-Codon Mutagenesis for Structure-Function Studies of Chemotaxis Signaling Proteins.- Mutational Analysis of Binding Protein/Chemoreceptor Interactions.- In Vitro Assay for Measuring Receptor-Kinase Activity in the Bacillus subtilis Chemotaxis Pathway.- FRET Analysis of the Chemotaxis Pathway Response.- Monitoring Two-Component Sensor Kinases with a Chemotaxis Signal Readout.- Analyzing Protein Domain Interactions in Chemoreceptors by In Vivo PEGylation.- Tuning Chemoreceptor Signaling by Positioning Aromatic Residues at the Lipid-Aqueous Interface.- Analyzing Chemoreceptor Interactions In Vivo with the Tri-Functional Crosslinker TMEA.- Use of Cryo-EM to Study the Structure of Chemoreceptor Arrays In Vivo.- Visualizing Chemoreceptor Arrays in Bacterial Minicells by Cryo-Electron Tomography and Subtomogram Analysis.- Bacterial Chemoreceptor Imaging at High Spatio-Temporal Resolution Using Photoconvertible Fluorescent Proteins.- Imaging of Single Dye-Labeled Chemotaxis Proteins in Live Bacteria Using Electroporation.- Fluorescence Anisotropy to Detect In Vivo Stimulus-Induced Changes in Chemoreceptor Packing.- Chemotaxis to Atypical Chemoattractants by Soil Bacteria.- Screening Chemoreceptor–Ligand Interactions by High-Throughput Thermal-Shift Assays.- High-Throughput Screening to Identify Chemoreceptor Ligands.- Identification of Specific Ligands for Sensory Receptors by Small-Molecule Ligand Arrays and Surface Plasmon Resonance.- Fluorescence Measurement of Kinetics of CheY Autophosphorylation with Small Molecule Phosphodonors.- Synthesis of a Stable Analog of the Phosphorylated Form of CheY: Phosphono-CheY.- Quantitative Modeling of Flagellar Motor-Mediated Adaptation.- Molecular Modeling of Chemoreceptor:Ligand Interactions.- Phylogenetic and Protein Sequence Analysis of Bacterial Chemoreceptors.