Measuring Cyclic Diguanylate (c-di-GMP)-Specific Phosphodiesterase Activity Using the MANT-c-di-GMP Assay

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1657; p. 263
• Main Authors: Eli, Dorit, Randall, Trevor E, Almblad, Henrik, Harrison, Joe J, Banin, Ehud
• Format: Journal Article
• Language: English
• Published: United States 2017
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Cyclic GMP - analogs & derivatives; Cyclic GMP - chemistry; Cyclic GMP - metabolism; Enzyme Activation; Enzyme Assays - methods; ortho-Aminobenzoates - chemistry; Phosphoric Diester Hydrolases - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; RocR; pGpG; Cyclic diguanylate; MANT-c-di-GMP; Phosphodiesterase
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-7240-1_20

The second messenger, cyclic diguanylate (c-di-GMP), regulates a variety of bacterial cellular and social behaviors. A key determinant of c-di-GMP levels in cells is its degradation by c-di-GMP-specific phosphodiesterases (PDEs). Here, we describe an assay to determine c-di-GMP degradation rates in vitro using 2'-O-(N'-methylanthraniloyl)-cyclic diguanylate (MANT-c-di-GMP). Additionally, a protocol for the production and purification of recombinant Pseudomonas aeruginosa RocR, a c-di-GMP-specific PDE that may serve as a control in MANT-c-di-GMP assays, is provided. The use of the fluorescent MANT-c-di-GMP analogue can deliver fundamental information about PDE function, and is suitable for identifying and investigating c-di-GMP-specific PDE activators and inhibitors.