Detection of the Bacterial Quorum-Sensing Signaling Molecules N-Acyl-Homoserine Lactones (HSL) and N-Acyl-Homoserine (HS) with an Enzyme-Linked Immunosorbent Assay (ELISA) and via Ultrahigh-Performance Liquid Chromatography Coupled to Mass Spectrometry (UHPLC-MS)

  • Michael Rothballer
  • Jenny Uhl
  • Josie Kunze
  • Philippe Schmitt-Kopplin
  • Anton Hartmann
Part of the Methods in Molecular Biology book series (MIMB, volume 1673)


Quick and reliable quantitative methods requiring low amounts of sample volume are needed for the detection of N-acyl-homoserine lactones (HSL) and their degradation products N-acyl-homoserines (HS) in order to elucidate the occurrence and dynamics of these prevalent quorum-sensing molecules of Gram-negative bacteria in natural samples and laboratory model experiments. A combination of ELISA and UHPLC-MS is presented here which has proven to meet these requirements. Both methods can not only precisely detect and quantify HSLs but also their degradation products HS and thereby enable studying signaling dynamics in quorum sensing, which have been identified to play an essential role in bacterial communication.

Key words

N-acyl-homoserine lactone Quorum sensing Bacterial signaling ELISA UHPLC-MS 


  1. 1.
    Chen X, Kremmer E, Gouzy MF, Clausen E, Starke M, Wollner K et al (2010) Development and characterization of rat monoclonal antibodies for N-acylated homoserine lactones. Anal Bioanal Chem 398:2655–2667CrossRefPubMedGoogle Scholar
  2. 2.
    Shaw PD, Ping G, Daly SL, Cha C, Cronan JEJ, Rinehart KL et al (1997) Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography. Proc Natl Acad Sci U S A 94:6036–6041CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Cataldi TRI, Bianco G, Frommberger M, Schmitt-Kopplin P (2004) Direct analysis of selected N-acyl-L-homoserine lactones by gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom 18:1341–1344CrossRefPubMedGoogle Scholar
  4. 4.
    Rani S, Kumar A, Malik AK (2011) Occurrence of N-acyl homoserine lactones in extracts of bacterial strain of Pseudomonas aeruginosa and in sputum sample evaluated by gas chromatography-mass spectrometry. Am J Anal Chem 2:294CrossRefGoogle Scholar
  5. 5.
    Li X, Fekete A, Englmann M, Götz C, Rothballer M, Frommberger M et al (2006) Development and application of a method for the analysis of N-acylhomoserine lactones by solid-phase extraction and ultra high pressure liquid chromatography. J Chromatogr A 1134:186–193CrossRefPubMedGoogle Scholar
  6. 6.
    Fekete A, Frommberger M, Rothballer M, Li X, Englmann M, Fekete J et al (2007) Identification of bacterial N-acylhomoserine lactones (AHLs) with a combination of ultra-performance liquid chromatography (UPLC), ultra-high-resolution mass spectrometry, and in-situ biosensors. Anal Bioanal Chem 387:455–467CrossRefPubMedGoogle Scholar
  7. 7.
    Buddrus-Schiemann K, Rieger M, Mühlbauer M, Barbarossa MV, Kuttler C, Hense BA et al (2014) Analysis of N-acylhomoserine lactone dynamics in continuous cultures of Pseudomonas putida IsoF by use of ELISA and UHPLC/qTOF-MS-derived measurements and mathematical models. Anal Bioanal Chem 406:6373–6383CrossRefPubMedGoogle Scholar
  8. 8.
    Hense BA, Kuttler C, Müller J, Rothballer M, Hartmann A, Kreft J-U (2007) Does efficiency sensing unify diffusion and quorum sensing? Nat Rev Microbiol 5:230–239CrossRefPubMedGoogle Scholar
  9. 9.
    Fekete A, Kuttler C, Rothballer M, Hense BA, Fischer D, Buddrus-Schiemann K et al (2010) Dynamic regulation of N-acyl-homoserine lactone production and degradation in Pseudomonas putida IsoF. FEMS Microbiol Ecol 72:22–34CrossRefPubMedGoogle Scholar
  10. 10.
    Chen X, Buddrus-Schiemann K, Rothballer M, Krämer PM, Hartmann A (2010) Detection of quorum sensing molecules in Burkholderia cepacia culture supernatants with enzyme-linked immunosorbent assays. Anal Bioanal Chem 398:2669–2676CrossRefPubMedGoogle Scholar
  11. 11.
    Mashburn LM, Whiteley M (2005) Membrane vesicles traffic signals and facilitate group activities in a prokaryote. Nature 437:422–425CrossRefPubMedGoogle Scholar
  12. 12.
    Kadurugamuwa JL, Beveridge TJ (1995) Virulence factors are released from Pseudomonas aeruginosa in association with membrane vesicles during normal growth and exposure to gentamicin: a novel mechanism of enzyme secretion. J Bacteriol 177:3998–4008CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Michael Rothballer
    • 1
  • Jenny Uhl
    • 2
  • Josie Kunze
    • 1
  • Philippe Schmitt-Kopplin
    • 2
  • Anton Hartmann
    • 1
  1. 1.Research Unit Microbe-Plant Interactions, Department Environmental SciencesHelmholtz Zentrum München, German Research Center for Environmental Health (GmbH)NeuherbergGermany
  2. 2.Research Unit Analytical BioGeoChemistry, Department Environmental SciencesHelmholtz Zentrum München, German Research Center for Environmental Health (GmbH)NeuherbergGermany

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