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

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c-di-GMP Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1657))

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.

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Acknowledgments

DC was supported by a Federation of European Microbiological Societies (FEMS) fellowship. TER was supported by a Queen Elizabeth II Scholarship. HA was supported by an Eyes High Postdoctoral Fellowship. JJH has been supported by a Canada Research Chair from the Canadian Institutes for Health Research (CIHR), and a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC). EB was supported by the Israel Science Foundation Grant 1124/12 and the Dyna and Fala Weinstock Foundation.

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Author notes

  1. Dorit Eli and Trevor E. Randall contributed equally to this work.

Authors and Affiliations

  1. The Mina and Everard Goodman Faculty of Life Sciences, Center for Advanced Materials and Nanotechnology, Bar-Ilan University, Ramat-Gan, 5290002, Israel

    Dorit Eli & Ehud Banin

  2. Department of Biological Sciences, University of Calgary, Calgary, AB, Canada, T2N 1N4

    Trevor E. Randall, Henrik Almblad & Joe J. Harrison

Authors
  1. Dorit Eli
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  2. Trevor E. Randall
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  3. Henrik Almblad
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  4. Joe J. Harrison
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  5. Ehud Banin
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Corresponding authors

Correspondence to Joe J. Harrison or Ehud Banin .

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Editors and Affiliations

  1. Department of Biological Sciences, Binghamton University Department of Biological Sciences, Binghamton, New York, USA

    Karin Sauer

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Eli, D., Randall, T.E., Almblad, H., Harrison, J.J., Banin, E. (2017). Measuring Cyclic Diguanylate (c-di-GMP)-Specific Phosphodiesterase Activity Using the MANT-c-di-GMP Assay. In: Sauer, K. (eds) c-di-GMP Signaling. Methods in Molecular Biology, vol 1657. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7240-1_20

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  • DOI: https://doi.org/10.1007/978-1-4939-7240-1_20

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7239-5

  • Online ISBN: 978-1-4939-7240-1

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