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Use of Nonradiochemical DNAse Footprinting to Analyze c-di-GMP Modulation of DNA-Binding Proteins

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c-di-GMP Signaling

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

Abstract

The transition of bacteria from a planktonic lifestyle to a collaborative, sessile biofilm lifestyle is a regulated process orchestrated by the intracellular second-messenger c-di-GMP (bis-(3′–5′)-cyclic dimeric guanosine monophosphate). To modulate this transition, c-di-GMP acts at the transcriptional, posttranscriptional, and posttranslational levels. In this chapter, we describe a method to study of how a transcriptional regulator modulates gene expression in response to c-di-GMP binding. DNase I footprinting is a valuable tool for use in analyzing how regulatory proteins bind to DNA, the location of their binding sites or how c-di-GMP affects their binding to DNA. This chapter describes a protocol for nonradiochemical DNase I footprinting experiments using a capillary electrophoresis method based on the interaction of the Pseudomonas aeruginosa FleQ protein with the promoter regions of biofilm-related genes.

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Acknowledgments

We gratefully thank Hidetada Hirakawa, Keiji Murakami, and Gael Panis for useful discussions and technical advice. The National Institute of General Medical Sciences (NIGMS) provided funding to Caroline S. Harwood, under grant number GM56665.

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Authors and Affiliations

  1. Department of Microbiology, University of Washington, Seattle, WA, 98195-7242, USA

    Claudine Baraquet & Caroline S. Harwood

  2. Université de Toulon, MAPIEM, EA4323, 83957, La Garde, France

    Claudine Baraquet

Authors
  1. Claudine Baraquet
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  2. Caroline S. Harwood
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Corresponding author

Correspondence to Caroline S. Harwood .

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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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Baraquet, C., Harwood, C.S. (2017). Use of Nonradiochemical DNAse Footprinting to Analyze c-di-GMP Modulation of DNA-Binding Proteins. 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_24

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  • DOI: https://doi.org/10.1007/978-1-4939-7240-1_24

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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

  • eBook Packages: Springer Protocols

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