Skip to main content
SpringerLink
Log in

High-Performance Liquid Chromatography (HPLC)-Based Detection and Quantitation of Cellular c-di-GMP

  • Protocol
  • First Online:
c-di-GMP Signaling

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

Abstract

The modulation of c-di-GMP levels plays a vital role in the regulation of various processes in a wide array of bacterial species. Thus, investigation of c-di-GMP regulation requires reliable methods for the assessment of c-di-GMP levels and turnover. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis has become a commonly used approach to accomplish these goals. The following describes the extraction and HPLC-based detection and quantification of c-di-GMP from Pseudomonas aeruginosa samples, a procedure that is amenable to modifications for the analysis of c-di-GMP in other bacterial species.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Römling U, Simm R (2009) Prevailing concepts of c-di-GMP signaling. Contrib Microbiol 16:161–181

    Article  PubMed  Google Scholar 

  2. Sondermann H, Shikuma NJ, Yildiz FH (2012) You’ve come a long way: C-di-GMP signaling. Curr Opin Microbiol 15:140–146

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ross P, Aloni Y, Weinhouse C et al (1985) An unusual guanyl oligonucleotide regulates cellulose synthesis in Acetobacter xylinum. FEBS Lett 186:191–196

    Article  CAS  PubMed  Google Scholar 

  4. Ross P, Weinhouse H, Aloni Y et al (1987) Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature 325:279–281

    Article  CAS  PubMed  Google Scholar 

  5. Schirmer T (2016) C-di-GMP synthesis: structural aspects of evolution, catalysis and regulation. J Mol Biol 428:3683–3701

    Article  CAS  PubMed  Google Scholar 

  6. Hengge R (2009) Principles of c-di-GMP signalling in bacteria. Nat Rev Microbiol 7:263–273

    Article  CAS  PubMed  Google Scholar 

  7. Ross P, Mayer R, Weinhouse H et al (1990) The cyclic diguanylic acid regulatory system of cellulose synthesis in Acetobacter xylinum. Chemical synthesis and biological activity of cyclic nucleotide dimer, trimer, and phosphothioate derivatives. J Biol Chem 265:18933–18943

    CAS  PubMed  Google Scholar 

  8. Thormann KM, Duttler S, Saville RM et al (2006) Control of formation and cellular detachment from Shewanella oneidensis MR-1 biofilms by cyclic di-GMP. J Bacteriol 188:2681–2691

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ueda A, Wood TK (2009) Connecting quorum sensing, c-di-GMP, pel polysaccharide, and biofilm formation in Pseudomonas aeruginosa through tyrosine phosphatase TpbA (PA3885). PLoS Pathog 5:e1000483

    Article  PubMed  PubMed Central  Google Scholar 

  10. Liu X, Beyhan S, Lim B et al (2010) Identification and characterization of a phosphodiesterase that inversely regulates motility and biofilm formation in Vibrio cholerae. J Bacteriol 192:4541–4552

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hickman JW, Harwood CS (2008) Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Mol Microbiol 69:376–389

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Burhenne H, Kaever V (2013) Quantification of cyclic dinucleotides by reversed-phase LC-MS/MS. Methods Mol Biol 1016:27–37

    Article  CAS  PubMed  Google Scholar 

  13. Spangler C, Böhm A, Jenal U et al (2010) A liquid chromatography-coupled tandem mass spectrometry method for quantitation of cyclic di-guanosine monophosphate. J Microbiol Methods 81:226–231

    Article  CAS  PubMed  Google Scholar 

  14. Simm R, Morr M, Remminghorst U et al (2009) Quantitative determination of cyclic diguanosine monophosphate concentrations in nucleotide extracts of bacteria by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Anal Biochem 386:53–58

    Article  CAS  PubMed  Google Scholar 

  15. Petrova OE, Sauer K (2012) PAS domain residues and prosthetic group involved in BdlA-dependent dispersion response by Pseudomonas aeruginosa biofilms. J Bacteriol 194:5817–5828

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Li Y, Heine S, Entian M et al (2013) NO-induced biofilm dispersion in Pseudomonas aeruginosa is mediated by an MHYT domain-coupled phosphodiesterase. J Bacteriol 195:3531–3542

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ryjenkov DA, Tarutina M, Moskvin OV et al (2005) Cyclic Diguanylate is a ubiquitous signaling molecule in bacteria: insights into biochemistry of the GGDEF protein domain. J Bacteriol 187:1792–1798

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Schmidt AJ, Ryjenkov DA, Gomelsky M (2005) The ubiquitous protein domain EAL is a cyclic Diguanylate-specific Phosphodiesterase: enzymatically active and inactive EAL domains. J Bacteriol 187:4774–4781

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wojcik W, Olianas M, Parenti M et al (1981) A simple fluorometric method for cAMP: application to studies of brain adenylate cyclase activity. J Cyclic Nucleotide Res 7:27–35

    CAS  PubMed  Google Scholar 

  20. Van Lookeren Campagne MM, Van Haastert PJM (1983) A sensitive cyclic nucleotide phosphodiesterase assay for transient enzyme kinetics. Anal Biochem 135:146–150

    Article  PubMed  Google Scholar 

  21. Martinez-Valdez H, Kothari RM, Hershey HV et al (1982) Rapid and reliable method for the analysis of nucleotide pools by reversed-phase high-performance liquid chromatography. J Chromatogr A 247:307–314

    Article  CAS  Google Scholar 

  22. Vogel H, Bonner D (1956) Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem 218:97–106

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biological Sciences, Binghamton Biofilm Research Center (BBRC), Binghamton University, 2401 ITC Building, 85 Murray Hill Road, Binghamton, NY, 13902, USA

    Olga E. Petrova & Karin Sauer

Authors
  1. Olga E. Petrova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Karin Sauer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Olga E. Petrova .

Editor information

Editors and Affiliations

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

    Karin Sauer

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Petrova, O.E., Sauer, K. (2017). High-Performance Liquid Chromatography (HPLC)-Based Detection and Quantitation of Cellular c-di-GMP. 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_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7240-1_4

  • Published:

  • 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

Publish with us

Policies and ethics

Search

Navigation