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.
Key words
- bis-(3′,5′)-cyclic dimeric guanosine monophosphate
- c-di-GMP
- Cyclic dinucleotides
- Reversed phase
- Ion exchange
- High-performance liquid chromatography
- Biofilm
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References
Römling U, Simm R (2009) Prevailing concepts of c-di-GMP signaling. Contrib Microbiol 16:161–181
Sondermann H, Shikuma NJ, Yildiz FH (2012) You’ve come a long way: C-di-GMP signaling. Curr Opin Microbiol 15:140–146
Ross P, Aloni Y, Weinhouse C et al (1985) An unusual guanyl oligonucleotide regulates cellulose synthesis in Acetobacter xylinum. FEBS Lett 186:191–196
Ross P, Weinhouse H, Aloni Y et al (1987) Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature 325:279–281
Schirmer T (2016) C-di-GMP synthesis: structural aspects of evolution, catalysis and regulation. J Mol Biol 428:3683–3701
Hengge R (2009) Principles of c-di-GMP signalling in bacteria. Nat Rev Microbiol 7:263–273
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
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
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
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
Hickman JW, Harwood CS (2008) Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Mol Microbiol 69:376–389
Burhenne H, Kaever V (2013) Quantification of cyclic dinucleotides by reversed-phase LC-MS/MS. Methods Mol Biol 1016:27–37
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
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
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
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
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
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
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
Van Lookeren Campagne MM, Van Haastert PJM (1983) A sensitive cyclic nucleotide phosphodiesterase assay for transient enzyme kinetics. Anal Biochem 135:146–150
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
Vogel H, Bonner D (1956) Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem 218:97–106
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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
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DOI: https://doi.org/10.1007/978-1-4939-7240-1_4
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