c-di-GMP Signaling, Softcover reprint of the original 1st ed. 2017 Methods and Protocols Methods in Molecular Biology Series, Vol. 1657

This volume provides a collection of protocols for the common experimental approaches used in the in the burgeoning field of c-di-GMP-dependent signaling. The chapters, divided into eight major parts, guide readers through methods on synthesis, detection, quantitation, modulation of the levels of c-di-GMP present in cells, procedures to detect and evaluate the interaction of c-di-GMP, and up and coming approaches focusing on the inhibition of c-di-GMP signaling.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, c-di-GMP Signaling: Methods and Protocols aims to inspire researchers to try new approaches.

1. Discovery of the Intracellular Signaling Molecule Cyclic di-GMP

Ute Römling and Michael Y. Galperin

PARt I: Synthesis of c-di-GMP

2. Enzymatic Production of C-di-GMP Using a Thermophilic Diguanylate Cyclase

3. Synthesis of [³²P]-c-di-GMP for Diguanylate Cyclase and Phosphodiesterase Activity Determinations

Barbara I. Kazmierczak

Part II: Detection and Quantitation of c-di-GMP

4. High-performance Liquid Chromatography (HPLC)-based Detection and Quantitation of Cellular c-di-GMP

Heike Bähre and Volkhard Kaever

6. Detection of Cyclic Dinucleotides by STING

Xiao-Xia Du and Xiao-Dong Su

Geoffrey B. Severin and Christopher M. Waters

Part III: Visualizing  c-di-GMP levels using biosensors

8. Gauging and Visualizing c-di-GMP Levels in Pseudomonas aeruginosa using Fluorescence-based Biosensors

Morten Rybtke, Song Lin Chua, Joey Kuok Hoong Yam, Michael Givskov, Liang Yang, and Tim Tolker-Nielsen

9. Cyclic di-GMP-responsive Transcriptional Reporter Bioassays in Pseudomonas aeruginosa

Bradley R. Borlee, Grace I. Borlee, Kevin H. Martin, and Yasuhiko Irie

10. Live Flow Cytometry Analysis of c-di-GMP levels in Single Cell Populations

Jongchan Yeo, Xin C. Wang, and Ming C. Hammond

Part IV: Indirect detection of c-di-GMP levels

11. Experimental Detection and Visualization of the Extracellular Matrix in Macrocolony Biofilms

Diego O. Serra and Regine Hengge

12. Congo Red Stain Identifies Matrix Overproduction and is an Indirect Measurement for c-di-GMP in many Species of Bacteria

Christopher J. Jones and Daniel J. Wozniak

13. Type IV Pili-dependent Motility as a Tool to Determine the Activity of c-di-GMP Modulating Enzymes in Myxococcus xanthus

Dorota Skotnicka and Lotte Søgaard-Andersen

PART V: Modulation of c-di-GMP levels And bacterial responses

14. Using Light-Activated Enzymes for Modulating Intracellular c‑di-GMP Levels in Bacteria

Min-Hyung Ryu, Anastasia Fomicheva, Lindsey O’Neal, Gladys Alexandre,and Mark Gomelsky

15. Analysis of c-di-GMP Levels Synthesized by a Photoreceptor Protein in Response to Different Light Qualities using an in vitro Enzymatic Assay

Veronika Angerer, Lars-Oliver Essen, and Annegret Wilde

16. Probing the Role of Cyclic di-GMP Signalling Systems in Disease using Chinese Radish

Shi-Qi An, Ji-Liang Tang, and J. Maxwell Dow

Ronan R McCarthy, Martina Valentini, and Alain Filloux

18. Semi-quantitative Analysis of the Red, Dry, and Rough Colony Morphology of Salmonella enterica serovar Typhimuriumand Escherichia coli using Congo Red

Annika Cimdins and Roger Simm

Part VI: Measuring c-di-GMP modulating activities

19. Fluorescent 2-aminopurine c-di-GMP and GpG analogs as PDE Probes

Jie Zhou, Clement Opoku-Temeng, and Herman O. Sintim

Dorit Eli, Trevor E. Randall, Henrik Almblad, Joe J. Harrison, and Ehud Banin

21. Determining Phosphodiesterase Activity (Radioactive Assay)

22. Determining Diguanylate Cyclase Activity (Radioactive Assay)

Barbara I. Kazmierczak

23. Detection of c-di-GMP-responsive DNA Binding

Jacob R. Chambers and Karin Sauer

24. Use of Nonradiochemical DNAse Footprinting to Analyze c-di-GMP Modulation of DNA-binding Proteins

Claudine Baraquet and Caroline S. Harwood

25. Detection of Cyclic di-GMP Binding Proteins Utilizing a Biotinylated Cyclic di-GMP Pulldown Assay

Jacob R. Chambers and Karin Sauer

26. Probing Protein-protein Interactions with Genetically Encoded Photo-activatable Crosslinkers

Richard B. Cooley and Holger Sondermann

27. Identification of c-di-AMP-Binding Proteins Using Magnetic Beads

Jan Kampf, Jan Gundlach, Christina Herzberg, Katrin Treffon, and Jörg Stülke

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28. Pulldown with a c-di-GMP-specific Capture Compound Coupled to Mass Spectrometry as a Powerful Tool to Identify Novel Effector Proteins

Benoît-Joseph Laventie, Timo Glatter,andUrs Jenal

29. Identification of c-di-GMP-responsive Riboswitches

Johann Peltier and Olga Soutourina

30. Isothermal Titration Calorimetry to Determine Apparent Dissociation Constants (K_d) and Stoichiometry of Interaction (n) of c-di-GMP Binding Proteins

Bruno Y. Matsuyama, Petya V. Krasteva, and Marcos V. A. S. Navarro

PART VIII: Targeting c-di-GMP signaling

31. Targeting c-di-GMP Signaling, Biofilm Formation and Bacterial Motility With Small Molecules

Clement Opoku-Temeng and Herman O. Sintim

32. Discovering Selective Diguanylatecyclases Inhibitors: From PleD to Discrimination of the Active Site of cyclic-di-GMP Phosphodiesterases

S. Rinaldo, G. Giardina, F. Mantoni, A. Paiardini, and A. Paone, F. Cutruzzolà

33. High throughput Screening for Compounds that Modulate the Cellular c-di-GMP Level in Bacteria

34. Genetic Tools to Study c-di-GMP-dependent Signaling in Pseudomonas aeruginosa

Livia Leoni, Sarika Vishnu Pawar, and Giordano Rampioni

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts

Includes supplementary material: sn.pub/extras