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

descriptionPublicationkeyboard_double_arrow_right Part of book or chapter of book , Article 01 Jan 2017Publisher:Springer New York

Authors: Dorit Eli; Joe J. Harrison; Trevor E. Randall; Henrik Almblad; Ehud Banin;

doi: 10.1007/978-1-4939-7240-1_20

pmid: 28889300

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

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Abstract

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.

Related Organizations

University of Calgary
Canada
Bar-Ilan University
Israel

Keywords

Enzyme Activation, Bacterial Proteins, Phosphoric Diester Hydrolases, ortho-Aminobenzoates, Cyclic GMP, Recombinant Proteins, Enzyme Assays

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