Quorum Sensing pp 207-217 | Cite as

Quantifying Pseudomonas aeruginosa Quinolones and Examining Their Interactions with Lipids

  • Gregory C. Palmer
  • Jeffrey W. Schertzer
  • Lauren Mashburn-Warren
  • Marvin WhiteleyEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 692)


Pseudomonas aeruginosa produces a quorum sensing molecule termed the Pseudomonas Quinolone Signal (2-heptyl-3-hydroxy-4-quinolone; PQS) that regulates an array of genes involved in virulence. This chapter addresses four related techniques useful for detecting and quantifying PQS. First, extraction of PQS from complex mixtures (e.g. cell cultures) is described. Separation of PQS from extracts by Thin-Layer Chromatography (TLC) is used in combination with the natural fluorescence of the molecule for quantification. A second separation technique for the PQS precursor HHQ using High-Performance Liquid Chromatography (HPLC) is also described, and this assay exploits the molecule’s characteristic absorbance for quantification. A third method for quantification of PQS from simple mixtures (e.g. enzyme assays) using fluorescence is outlined. Finally, a protocol for determining PQS interactions with membrane lipids through Fluorescence Resonance Energy Transfer (FRET) is presented. These techniques allow for quantification and characterization of PQS from diverse environments, a prerequisite to understanding the biological functions of QS molecules.

Key words

Pseudomonas quinolone signal Thin-layer chromatography High-pressure liquid chromatography Fluorescence resonance energy transfer 


  1. 1.
    Parsek, M. R., and Greenberg, E. P. (2000) Acyl-homoserine lactone quorum sensing in gram-negative bacteria: a signaling mechanism involved in associations with higher organisms, Proc Natl Acad Sci USA 97, 8789–8793.PubMedCrossRefGoogle Scholar
  2. 2.
    Pesci, E. C., Milbank, J. B., Pearson, J. P., McKnight, S., Kende, A. S., Greenberg, E. P., and Iglewski, B. H. (1999) Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa, Proc Natl Acad Sci USA 96, 11229–11234.PubMedCrossRefGoogle Scholar
  3. 3.
    Deziel, E., Gopalan, S., Tampakaki, A. P., Lepine, F., Padfield, K. E., Saucier, M., Xiao, G., and Rahme, L. G. (2005) The contribution of MvfR to Pseudomonas aeruginosa pathogenesis and quorum sensing circuitry regulation: multiple quorum sensing-regulated genes are modulated without affecting lasRI, rhlRI or the production of N-acyl-l-homoserine lactones, Mol Microbiol 55, 998–1014.PubMedCrossRefGoogle Scholar
  4. 4.
    Mashburn, L. M., and Whiteley, M. (2005) Membrane vesicles traffic signals and facilitate group activities in a prokaryote, Nature 437, 422–425.PubMedCrossRefGoogle Scholar
  5. 5.
    Mashburn-Warren, L., Howe, J., Garidel, P., Richter, W., Steiniger, F., Roessle, M., Brandenburg, K., and Whiteley, M. (2008) Interaction of quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation, Mol Microbiol 69, 491–502.PubMedCrossRefGoogle Scholar
  6. 6.
    Schaefer, A. L., Taylor, T. A., Beatty, J. T., and Greenberg, E. P. (2002) Long-chain acyl-homoserine lactone quorum-sensing regulation of Rhodobacter capsulatus gene transfer agent production, J Bacteriol 184, 6515–6521.PubMedCrossRefGoogle Scholar
  7. 7.
    Marketon, M. M., Gronquist, M. R., Eberhard, A., and Gonzalez, J. E. (2002) Characterization of the Sinorhizobium meliloti sinR/sinI locus and the production of novel N-acyl homoserine lactones, J Bacteriol 184, 5686–5695.PubMedCrossRefGoogle Scholar
  8. 8.
    Deziel, E., Lepine, F., Milot, S., He, J., Mindrinos, M. N., Tompkins, R. G., and Rahme, L. G. (2004) Analysis of Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs) reveals a role for 4-hydroxy-2-heptylquinoline in cell-to-cell communication, Proc Natl Acad Sci USA 101, 1339–1344.PubMedCrossRefGoogle Scholar
  9. 9.
    Gallagher, L. A., McKnight, S. L., Kuznetsova, M. S., Pesci, E. C., and Manoil, C. (2002) Functions required for extracellular quinolone signaling by Pseudomonas aeruginosa, J Bacteriol 184, 6472–6480.PubMedCrossRefGoogle Scholar
  10. 10.
    Palmer, K. L., Aye, L. M., and Whiteley, M. (2007) Nutritional cues control Pseudomonas aeruginosa multicellular behavior in cystic fibrosis sputum, J Bacteriol 189, 8079–8087.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Gregory C. Palmer
    • 1
  • Jeffrey W. Schertzer
    • 1
  • Lauren Mashburn-Warren
    • 1
  • Marvin Whiteley
    • 1
    Email author
  1. 1.Section of Molecular Genetics and MicrobiologyThe University of Texas at AustinAustinUSA

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