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Live Flow Cytometry Analysis of c-di-GMP Levels in Single Cell Populations

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c-di-GMP Signaling

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

Abstract

Second-generation RNA-based fluorescent biosensors have been developed that enable flow cytometry experiments to monitor the population dynamics of c-di-GMP signaling in live bacteria. These experiments are high-throughput, provide information at the single-cell level, and can be performed on cells grown in complex media and/or under anaerobic conditions. Here, we describe flow cytometry methods for three applications: (1) high-throughput screening for diguanylate cyclase activity, (2) analyzing c-di-GMP levels under anaerobic conditions, and (3) monitoring cell population dynamics of c-di-GMP levels upon environmental changes. These methods showcase RNA-based fluorescent biosensors as versatile tools for studying c-di-GMP signaling in bacteria.

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References

  1. 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 

  2. Tischler AD, Camilli A (2004) Cyclic diguanylate (c-di-GMP) regulates vibrio cholerae biofilm formation. Mol Microbiol 53(3):857–869. doi:10.1111/j.1365-2958.2004.04155.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Spangler C, Bohm A, Jenal U, Seifert R, Kaever V (2010) A liquid chromatography-coupled tandem mass spectrometry method for quantitation of cyclic di-guanosine monophosphate. J Microbiol Methods 81(3):226–231. doi:10.1016/j.mimet.2010.03.020

    Article  CAS  PubMed  Google Scholar 

  4. Stelitano V, Brandt A, Fernicola S, Franceschini S, Giardina G, Pica A, Rinaldo S, Sica F, Cutruzzola F (2013) Probing the activity of diguanylate cyclases and c-di-GMP phosphodiesterases in real-time by CD spectroscopy. Nucleic Acids Res 41(7):e79. doi:10.1093/nar/gkt028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kellenberger CA, Sales-Lee J, Pan Y, Gassaway MM, Herr AE, Hammond MC (2015) A minimalist biosensor : quantitation of cyclic di-GMP using the conformational change of a riboswitch aptamer. RNA Biol 12(11):1189–1197. doi:10.1080/15476286.2015.1062970

  6. Roelofs KG, Wang J, Sintim HO, Lee VT (2011) Differential radial capillary action of ligand assay for high-throughput detection of protein-metabolite interactions. Proc Natl Acad Sci U S A 108(37):15528–15533. doi:10.1073/pnas.1018949108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wolfe AJ, Berg HC (1989) Migration of bacteria in semisolid agar. Proc Natl Acad Sci U S A 86(18):6973–6977

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. O'Toole GA, Pratt LA, Watnick PI, Newman DK, Weaver VB, Kolter R (1999) Genetic approaches to study of biofilms. Methods Enzymol 310:91–109

    Article  PubMed  Google Scholar 

  9. Koestler BJ, Waters CM (2014) Bile acids and bicarbonate inversely regulate intracellular cyclic di-GMP in vibrio cholerae. Infect Immun 82(7):3002–3014. doi:10.1128/IAI.01664-14

    Article  PubMed  PubMed Central  Google Scholar 

  10. Ho CL, Chong KS, Oppong JA, Chuah ML, Tan SM, Liang ZX (2013) Visualizing the perturbation of cellular cyclic di-GMP levels in bacterial cells. J Am Chem Soc 135(2):566–569. doi:10.1021/ja310497x

    Article  CAS  PubMed  Google Scholar 

  11. Christen M, Kulasekara HD, Christen B, Kulasekara BR, Hoffman LR, Miller SI (2010) Asymmetrical distribution of the second messenger c-di-GMP upon bacterial cell division. Science 328(5983):1295–1297. doi:10.1126/science.1188658

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kellenberger CA, Wilson SC, Sales-Lee J, Hammond MC (2013) RNA-based fluorescent biosensors for live cell imaging of second messengers cyclic di-GMP and cyclic AMP-GMP. J Am Chem Soc 135(13):4906–4909. doi:10.1021/ja311960g

  13. Kellenberger CA, Hallberg ZF, Hammond MC (2015) Live cell imagin g using Riboswitch-Spinach tRNA fusions as metabolite-sensing fluorescent biosenso r s. Methods Mol Biol 1316:87–103. doi:10.1007/978-1-4939-2730-2_8

  14. Kellenberger CA, Hammond MC (2015) In vitro analysis of riboswitch-Spinach aptamer fusions as metabolite-sensing fluorescent biosensors . Methods Enzymol 550:147–172. doi:10.1016/bs.mie.2014.10.045

  15. Paige JS, Wu KY, Jaffrey SR (2011) RNA mimics of green fluorescent protein. Science 333(6042):642–646. doi:10.1126/science.1207339

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Paige JS, Nguyen-Duc T, Song W, Jaffrey SR (2012) Fluorescence imaging of cellular metabolites with RNA. Science 335(6073):1194. doi:10.1126/science.1218298

  17. Wang XC, Wilson SC, Hammond MC (2016) Next-generation RNA-based fluorescent biosensors enable anaerobic detection of cyclic di-GMP. Nucleic Acids Res 44(17):e139. doi:10.1093/nar/gkw580

  18. Song W, Strack RL, Svensen N, Jaffrey SR (2014) Plug-and-play fluorophores extend the spectral properties of Spinach. J Am Chem Soc 136(4):1198–1201. doi:10.1021/ja410819x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hallberg ZF, Wang XC, Wright TA, Nan B, Ad O, Yeo J, Hammond MC (2016) Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3′, 3′-cGAMP). Proc Natl Acad Sci U S A 113(7):1790–1795. doi:10.1073/pnas.1515287113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Tuckerman JR, Gonzalez G, Sousa EH, Wan X, Saito JA, Alam M, Gilles-Gonzalez MA (2009) An oxygen-sensing diguanylate cyclase and phosphodiesterase couple for c-di-GMP control. Biochemistry 48(41):9764–9774. doi:10.1021/bi901409g

    Article  CAS  PubMed  Google Scholar 

  21. Lacey MM, Partridge JD, Green J (2010) Escherichia coli K-12 YfgF is an anaerobic cyclic di-GMP phosphodiesterase with roles in cell surface remodelling and the oxidative stress response. Microbiology 156(Pt 9):2873–2886. doi:10.1099/mic.0.037887-0

    Article  CAS  PubMed  Google Scholar 

  22. An S, Wu J, Zhang LH (2010) Modulation of Pseudomonas Aeruginosa biofilm dispersal by a cyclic-di-GMP phosphodiesterase with a putative hypoxia-sensing domain. Appl Environ Microbiol 76(24):8160–8173. doi:10.1128/AEM.01233-10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Reid BG, Flynn GC (1997) Chromophore formation in green fluorescent protein. Biochemistry 36(22):6786–6791. doi:10.1021/bi970281w

    Article  CAS  PubMed  Google Scholar 

  24. Craggs TD (2009) Green fluorescent protein: structure, folding and chromophore maturation. Chem Soc Rev 38(10):2865–2875. doi:10.1039/b903641p

    Article  CAS  PubMed  Google Scholar 

  25. Yeo J, Dippel, AB, Wang, XC, Hammond, MC Manuscript in preparation

    Google Scholar 

  26. Zahringer F, Lacanna E, Jenal U, Schirmer T, Boehm A (2013) Structure and signaling mechanism of a zinc-sensory diguanylate cyclase. Structure 21(7):1149–1157. doi:10.1016/j.str.2013.04.026

    Article  CAS  PubMed  Google Scholar 

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Acknowledgment

The work on which this chapter is based was supported by NIH grant DP2 OD008677 (to M.C.H.). The authors thank Zachary Hallberg for assistance with figures.

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Correspondence to Ming C. Hammond .

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Yeo, J., Wang, X.C., Hammond, M.C. (2017). Live Flow Cytometry Analysis of c-di-GMP Levels in Single Cell Populations. 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_10

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  • DOI: https://doi.org/10.1007/978-1-4939-7240-1_10

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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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