Detection of 2-Alkyl-4-Quinolones Using Biosensors

  • Matthew P. Fletcher
  • Stephen P. Diggle
  • Miguel Cámara
  • Paul Williams
Part of the Methods in Molecular Biology book series (MIMB, volume 1673)


2-Alkyl-4-quinolones (AQs) such as 2-heptyl-3-hydroxy-4-quinolone (PQS) and 2-heptyl-4-hydroxyquinoline (HHQ) are quorum-sensing signal molecules. Here we describe two methods for AQ detection and quantification that employ thin-layer chromatography (TLC) and microtiter plate assays in combination with a lux-based Pseudomonas aeruginosa AQ biosensor strain. For TLC detection, organic solvent extracts of bacterial cells or spent culture supernatants are chromatographed on TLC plates, which are then dried and overlaid with the AQ biosensor. After detection by the bioreporter, AQs appear as both luminescent and green (from pyocyanin) spots. For the microtiter assay, either spent bacterial culture supernatants or extracts are added to a growth medium containing the AQ biosensor. Light output by the bioreporter correlates with the AQ content of the sample. The assays described are simple to perform, do not require sophisticated instrumentation, and are highly amenable to screening large numbers of bacterial samples.

Key words

Pseudomonas aeruginosa Biosensor 2-Alkyl-4-quinolones Pseudomonas quinolone signal (PQS) 2-Heptyl-4-hydroxyquinoline (HHQ) pqsA 



We gratefully acknowledge the Royal Society (SPD) and Wellcome Trust (MPF) for funding.


  1. 1.
    Williams P, Cámara M (2009) Quorum sensing and environmental adaptation in Pseudomonas aeruginosa: a tale of regulatory networks and multifunctional signal molecules. Curr Opin Microbiol 12:182–191CrossRefPubMedGoogle Scholar
  2. 2.
    Pesci EC, Milbank JB, Pearson JP, McKnight S, Kende AS, Greenberg EP et al (1999) Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 96:11229–11234CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Heeb S, Fletcher MP, Chhabra SR, Diggle SP, Williams P, Cámara M (2011) Quinolones: from antibiotics to autoinducers. FEMS Microbiol Rev 35(2):247–274CrossRefPubMedGoogle Scholar
  4. 4.
    Wade DS, Calfee MW, Rocha ER, Ling EA, Engstrom E, Coleman JP et al (2005) Regulation of Pseudomonas quinolone signal synthesis in Pseudomonas aeruginosa. J Bacteriol 187:4372–4380CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Xiao G, Déziel E, He J, Lépine F, Lesic B, Castonguay MH et al (2006) MvfR, a key Pseudomonas aeruginosa pathogenicity LTTR-class regulatory protein, has dual ligands. Mol Microbiol 62:1689–1699CrossRefPubMedGoogle Scholar
  6. 6.
    Diggle SP, Winzer K, Chhabra SR, Worrall KE, Cámara M, Williams P (2003) The Pseudomonas aeruginosa quinolone signal molecule overcomes the cell density-dependency of the quorum sensing hierarchy, regulates rhl-dependent genes at the onset of stationary phase and can be produced in the absence of LasR. Mol Microbiol 50:29–43CrossRefPubMedGoogle Scholar
  7. 7.
    Allesen-Holm M, Barken KB, Yang L, Klausen M, Webb JS, Kjelleberg S et al (2006) A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms. Mol Microbiol 59:1114–1128CrossRefPubMedGoogle Scholar
  8. 8.
    Diggle SP, Lumjiaktase P, Dipilato F, Winzer K, Kunakorn M, Barrett DA et al (2006) Functional genetic analysis reveals a 2-alkyl-4-quinolone signaling system in the human pathogen Burkholderia pseudomallei and related bacteria. Chem Biol 13:701–710CrossRefPubMedGoogle Scholar
  9. 9.
    Lépine F, Déziel E, Milot S, Rahme LG (2003) A stable isotope dilution assay for the quantification of the Pseudomonas quinolone signal in Pseudomonas aeruginosa cultures. Biochim Biophys Acta 1622:36–41CrossRefPubMedGoogle Scholar
  10. 10.
    Lépine F, Milot S, Déziel E, He J, Rahme LG (2004) Electrospray/mass spectrometric identification and analysis of 4-hydroxy-2-alkylquinolines (HAQs) produced by Pseudomonas aeruginosa. J Am Soc Mass Spectrom 15:862–869CrossRefPubMedGoogle Scholar
  11. 11.
    Ortori CA, Dubern JF, Chhabra SR, Cámara M, Hardie K, Williams P et al (2014) Simultaneous quantitative profiling of N-acyl-L-homoserine lactone and 2-alkyl-4(1H)-quinolone families of quorum-sensing signaling molecules using LC-MS/MS. Anal Bioanal Chem 2:839–850Google Scholar
  12. 12.
    Calfee MW, Coleman JP, Pesci EC (2001) Interference with Pseudomonas quinolone signal synthesis inhibits virulence factor expression by Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 98:11633–11637CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Gallagher LA, McKnight SL, Kuznetsova MS, Pesci EC, Manoil C (2002) Functions required for extracellular quinolone signaling by Pseudomonas aeruginosa. J Bacteriol 184:6472–6480CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Fletcher MP, Diggle SP, Crusz SA, Chhabra SR, Cámara M, Williams P (2007) A dual biosensor for 2-alkyl-4-quinolone quorum-sensing signal molecules. Environ Microbiol 9:2683–2693CrossRefPubMedGoogle Scholar
  15. 15.
    Fletcher MP, Diggle SP, Cámara M, Williams P (2007) Biosensor-based assays for PQS, HHQ and related 2-alkyl-4-quinolone quorum sensing signal molecules. Nat Protoc 2:1254–1262CrossRefPubMedGoogle Scholar
  16. 16.
    Diggle SP, Matthijs S, Wright VJ, Fletcher MP, Chhabra SR, Lamont IL et al (2007) The Pseudomonas aeruginosa 4-quinolone signal molecules HHQ and PQS play multifunctional roles in quorum sensing and iron entrapment. Chem Biol 14:87–96CrossRefPubMedGoogle Scholar
  17. 17.
    Becher A, Schweizer HP (2000) Integration-proficient Pseudomonas aeruginosa vectors for isolation of single-copy chromosomal lacZ and lux gene fusions. Biotechniques 5:948–952Google Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Matthew P. Fletcher
    • 1
  • Stephen P. Diggle
    • 1
  • Miguel Cámara
    • 1
  • Paul Williams
    • 1
  1. 1.Centre for Biomolecular Sciences, School of Life SciencesUniversity of NottinghamNottinghamUK

Personalised recommendations