Skip to main content

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

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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

Similar content being viewed by others

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

Authors

Corresponding author

Correspondence to Olga E. Petrova .

Editor information

Editors and Affiliations

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