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Gauging and Visualizing c-di-GMP Levels in Pseudomonas aeruginosa Using Fluorescence-Based Biosensors

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c-di-GMP Signaling

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

Abstract

Recent research has shown that the molecule c-di-GMP is an important second messenger regulating various functions in bacteria. In particular, the implication of c-di-GMP as a positive regulator of adhesion and biofilm formation has gained momentum as a highly relevant research topic, as detailed knowledge about the underlying regulatory mechanisms may enable the development of measures to control biofilms in both industrial and medical settings. Accordingly, it is in many cases of interest to measure the c-di-GMP level in bacteria under specific conditions or in specific mutant strains. We have developed a collection of fluorescence-based c-di-GMP biosensors capable of gauging the c-di-GMP level in Pseudomonas aeruginosa and closely related bacteria. Here, we describe protocols for the use of these biosensors in gauging and visualizing cellular c-di-GMP levels of P. aeruginosa both in in vitro setups such as continuous-culture flow-cell biofilms, and in in vivo settings such as a murine corneal infection model.

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References

  1. Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: a common cause of persistent infections. Science 284(5418):1318–1322

    Article  CAS  PubMed  Google Scholar 

  2. Romling U, Galperin MY, Gomelsky M (2013) Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev 77(1):1–52. doi:10.1128/MMBR.00043-12

    Article  PubMed  PubMed Central  Google Scholar 

  3. Irie Y, Parsek MR (2014) LC/MS/MS-based quantitative assay for the secondary messenger molecule, c-di-GMP. Methods Mol Biol 1149:271–279. doi:10.1007/978-1-4939-0473-0_22

    Article  CAS  PubMed  Google Scholar 

  4. Rybtke MT, Borlee BR, Murakami K, Irie Y, Hentzer M, Nielsen TE, Givskov M, Parsek MR, Tolker-Nielsen T (2012) Fluorescence-based reporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa. Appl Environ Microbiol 78(15):5060–5069. doi:10.1128/AEM.00414-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Crusz SA, Popat R, Rybtke MT, Camara M, Givskov M, Tolker-Nielsen T, Diggle SP, Williams P (2012) Bursting the bubble on bacterial biofilms: a flow cell methodology. Biofouling 28(8):835–842. doi:10.1080/08927014.2012.716044

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Choi KH, Kumar A, Schweizer HP (2006) A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 64(3):391–397. doi:10.1016/j.mimet.2005.06.001. S0167-7012(05)00158-2 [pii]

    Article  CAS  PubMed  Google Scholar 

  7. Klausen M, Heydorn A, Ragas P, Lambertsen L, Aaes-Jorgensen A, Molin S, Tolker-Nielsen T (2003) Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol Microbiol 48(6):1511–1524

    Article  CAS  PubMed  Google Scholar 

  8. Saraswathi P, Beuerman RW (2015) Corneal biofilms: from planktonic to Microcolony formation in an experimental keratitis infection with Pseudomonas aeruginosa. Ocul Surf 13 (4): 331–345. doi: 10.1016/j.jtos.2015.07.001 [pii]

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Acknowledgments

This work was supported by grants from and the Danish Council for Independent Research (DFF–1323-00177) to TTN, and from the Danish Strategic Research Council and the Lundbeck Foundation (R198-2015-486) to MG.

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Correspondence to Tim Tolker-Nielsen .

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Rybtke, M., Chua, S.L., Yam, J.K.H., Givskov, M., Yang, L., Tolker-Nielsen, T. (2017). Gauging and Visualizing c-di-GMP Levels in Pseudomonas aeruginosa Using Fluorescence-Based Biosensors. 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_8

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  • DOI: https://doi.org/10.1007/978-1-4939-7240-1_8

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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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