Analytical and Bioanalytical Chemistry

, Volume 406, Issue 25, pp 6373–6383 | Cite as

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

  • Katharina Buddrus-Schiemann
  • Martin Rieger
  • Marlene Mühlbauer
  • Maria Vittoria Barbarossa
  • Christina Kuttler
  • Burkhard A. Hense
  • Michael Rothballer
  • Jenny Uhl
  • Juliano R. Fonseca
  • Philippe Schmitt-Kopplin
  • Michael Schmid
  • Anton Hartmann
Research Paper


In this interdisciplinary approach, the dynamics of production and degradation of the quorum sensing signal 3-oxo-decanoylhomoserine lactone were studied for continuous cultures of Pseudomonas putida IsoF. The signal concentrations were quantified over time by use of monoclonal antibodies and ELISA. The results were verified by use of ultra-high-performance liquid chromatography. By use of a mathematical model we derived quantitative values for non-induced and induced signal production rate per cell. It is worthy of note that we found rather constant values for different rates of dilution in the chemostat, and the values seemed close to those reported for batch cultures. Thus, the quorum-sensing system in P. putida IsoF is remarkably stable under different environmental conditions. In all chemostat experiments, the signal concentration decreased strongly after a peak, because emerging lactonase activity led to a lower concentration under steady-state conditions. This lactonase activity probably is quorum sensing-regulated. The potential ecological implication of such unique regulation is discussed.


Pseudomonas putida IsoF Continuous culture N-acylhomoserine lactones Mathematical modelling ELISA Quorum sensing 



We enormously appreciated the great dedication to immunochemical analysis of AHL compounds of Dr Petra Krämer, who died unexpectedly in the early phase of these experiments. Furthermore we want to thank Dr Elisabeth Kremmer, Institute of Molecular Immunology, Helmholtz Zentrum München, for providing the monoclonal antibodies. Maria Vittoria Barbarossa was supported by ERC starting grant no. 259559.

Supplementary material

216_2014_8063_MOESM1_ESM.pdf (54 kb)
ESM 1 (PDF 53 kb)


  1. 1.
    Fuqua WC, Winans SC, Greenberg EP (1994) Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol 176(2):269–275Google Scholar
  2. 2.
    Hartmann A, Schikora A (2012) Quorum sensing of bacteria and trans-kingdom interactions of N-acyl homoserine lactones with eukaryotes. J Chem Ecol 38(6):704–713. doi: 10.1007/s10886-012-0141-7 CrossRefGoogle Scholar
  3. 3.
    Williams P, Winzer K, Chan WC, Cámara M (2007) Look who's talking: communication and quorum sensing in the bacterial world. Phil Trans R Soc B Biol Sci 362(1483):1119–1134. doi: 10.1098/rstb.2007.2039 CrossRefGoogle Scholar
  4. 4.
    Huber B, Riedel K, Hentzer M, Heydorn A, Gotschlich A, Givskov M, Molin S, Eberl L (2001) The cep quorum-sensing system of Burkholderia cepacia H111 controls biofilm formation and swarming motility. Microbiology 147(Pt 9):2517–2528Google Scholar
  5. 5.
    Koiv V, Mae A (2001) Quorum sensing controls the synthesis of virulence factors by modulating rsmA gene expression in Erwinia carotovora subsp. carotovora. Mol Gen Genomics 265(2):287–292. doi: 10.1007/s004380000413 CrossRefGoogle Scholar
  6. 6.
    Shih P-C, Huang C-T (2002) Effects of quorum-sensing deficiency on Pseudomonas aeruginosa biofilm formation and antibiotic resistance. J Antimicrob Chemother 49(2):309–314. doi: 10.1093/jac/49.2.309 CrossRefGoogle Scholar
  7. 7.
    Yazgan A, Özcengiz G, Marahiel MA (2001) Tn10 insertional mutations of Bacillus subtilis that block the biosynthesis of bacilysin. Biochim Biophys (BBA) Gene Struct Expr 1518(1–2):87–94. doi: 10.1016/S0167-4781(01)00182-8 CrossRefGoogle Scholar
  8. 8.
    Sokol PA, Malott RJ, Riedel K, Eberl L (2007) Communication systems in the genus Burkholderia: global regulators and targets for novel antipathogenic drugs. Future Microbiol 2(5):555–563. doi: 10.2217/17460913.2.5.555 CrossRefGoogle Scholar
  9. 9.
    Cooley M, Chhabra SR, Williams P (2008) N-acylhomoserine lactone-mediated quorum sensing: a twist in the tail and a blow for host immunity. Chem Biol 15(11):1141–1147. doi: 10.1016/j.chembiol.2008.10.010 CrossRefGoogle Scholar
  10. 10.
    Steidle A, Allesen-Holm M, Riedel K, Berg G, Givskov M, Molin S, Eberl L (2002) Identification and characterization of an N-acylhomoserine lactone-dependent quorum-sensing system in Pseudomonas putida strain IsoF. Appl Environ Microbiol 68(12):6371–6382. doi: 10.1128/aem.68.12.6371-6382.2002 CrossRefGoogle Scholar
  11. 11.
    Steidle A, Sigl K, Schuhegger R, Ihring A, Schmid M, Gantner S, Stoffels M, Riedel K, Givskov M, Hartmann A, Langebartels C, Eberl L (2001) Visualization of N-acylhomoserine lactone-mediated cell-cell communication between bacteria colonizing the tomato rhizosphere. Appl Environ Microbiol 67(12):5761–5770CrossRefGoogle Scholar
  12. 12.
    Fekete A, Kuttler C, Rothballer M, Hense BA, Fischer D, Buddrus-Schiemann K, Lucio M, Müller J, Schmitt-Kopplin P, Hartmann A (2010) Dynamic regulation of N-acyl-homoserine lactone production and degradation in Pseudomonas putida IsoF. FEMS Microbiol Ecol 72(1):22–34. doi: 10.1111/j.1574-6941.2009.00828.x CrossRefGoogle Scholar
  13. 13.
    Krysciak D, Schmeisser C, Preuß S, Riethausen J, Quitschau M, Grond S, Streit WR (2011) Involvement of multiple loci in quorum quenching of Autoinducer I molecules in the nitrogen-fixing symbiont Rhizobium (Sinorhizobium) sp. strain NGR234. Appl Environ Microbiol 77(15):5089–5099. doi: 10.1128/aem.00112-11 CrossRefGoogle Scholar
  14. 14.
    Uroz S, Oger PM, Chapelle E, Adeline M-T, Faure D, Dessaux Y (2008) A Rhodococcus qsdA-encoded enzyme defines a novel class of large-spectrum quorum-quenching lactonases. Appl Environ Microbiol 74(5):1357–1366. doi: 10.1128/aem.02014-07 CrossRefGoogle Scholar
  15. 15.
    Wahjudi M, Papaioannou E, Hendrawati O, van Assen AHG, van Merkerk R, Cool RH, Poelarends GJ, Quax WJ (2011) PA0305 of Pseudomonas aeruginosa is a quorum quenching acylhomoserine lactone acylase belonging to the Ntn hydrolase superfamily. Microbiology 157(7):2042–2055. doi: 10.1099/mic.0.043935-0 CrossRefGoogle Scholar
  16. 16.
    Yates EA, Philipp B, Buckley C, Atkinson S, Chhabra SR, Sockett RE, Goldner M, Dessaux Y, Cámara M, Smith H, Williams P (2002) N-acylhomoserine lactones undergo lactonolysis in a pH-, temperature-, and acyl chain length-dependent manner during growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa. Infect Immun 70(10):5635–5646. doi: 10.1128/iai.70.10.5635-5646.2002 CrossRefGoogle Scholar
  17. 17.
    Dunlap PV (1999) Quorum regulation of luminescence in Vibrio fischeri. J Mol Microbiol Biotechnol 1(1):5–12Google Scholar
  18. 18.
    De Lay N, Gottesman S (2009) The Crp-activated small noncoding regulatory RNA CyaR (RyeE) links nutritional status to group behavior. J Bacteriol 191(2):461–476. doi: 10.1128/jb.01157-08 CrossRefGoogle Scholar
  19. 19.
    Mellbye B, Schuster M (2014) Physiological framework for the regulation of quorum sensing-dependent public goods in pseudomonas aeruginosa. J Bacteriol 196(6):1155–1164CrossRefGoogle Scholar
  20. 20.
    Fekete A, Frommberger M, Rothballer M, Li X, Englmann M, Fekete J, Hartmann A, Eberl L, Schmitt-Kopplin P (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(2):455–467. doi: 10.1007/s00216-006-0970-8 CrossRefGoogle Scholar
  21. 21.
    Li X, Fekete A, Englmann M, Götz C, Rothballer M, Frommberger M, Buddrus K, Fekete J, Cai C, Schröder P, Hartmann A, Chen G, Schmitt-Kopplin P (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(1–2):186–193. doi: 10.1016/j.chroma.2006.09.047 CrossRefGoogle Scholar
  22. 22.
    Chen X, Buddrus-Schiemann K, Rothballer M, Krämer P, Hartmann A (2010) Detection of quorum sensing molecules in Burkholderia cepacia culture supernatants with enzyme-linked immunosorbent assays. Anal Bioanal Chem 398(6):2669–2676. doi: 10.1007/s00216-010-4045-5 CrossRefGoogle Scholar
  23. 23.
    Chen X, Kremmer E, Gouzy M-F, Clausen E, Starke M, Wöllner K, Pfister G, Hartmann A, Krämer P (2010) Development and characterization of rat monoclonal antibodies for N-acylated homoserine lactones. Anal Bioanal Chem 398(6):2655–2667. doi: 10.1007/s00216-010-4017-9 CrossRefGoogle Scholar
  24. 24.
    Barbarossa MV, Kuttler C, Fekete A, Rothballer M (2010) A delay model for quorum sensing of Pseudomonas putida. Biosystems 102(2–3):148–156CrossRefGoogle Scholar
  25. 25.
    Clark DJ, Maaløe O (1967) DNA replication and the division cycle in Escherichia coli. J Mol Biol 23:99–112CrossRefGoogle Scholar
  26. 26.
    Englmann M, Fekete A, Kuttler C, Frommberger M, Li X, Gebefügi I, Fekete J, Schmitt-Kopplin P (2007) The hydrolysis of unsubstituted N-acylhomoserine lactones to their homoserine metabolites: analytical approaches using ultra performance liquid chromatography. J Chromatogr A 1160(1–2):184–193CrossRefGoogle Scholar
  27. 27.
    Buddrus-Schiemann K, Schmid M, Schreiner K, Welzl G, Hartmann A (2010) Root colonization by Pseudomonas sp. DSMZ 13134 and impact on the indigenous rhizosphere bacterial community of barley. Microb Ecol 60(2):381–393CrossRefGoogle Scholar
  28. 28.
    Wöllner K, Chen X, Kremmer E, Krämer PM (2010) Comparative surface plasmon resonance and enzyme-linked immunosorbent assay characterisation of a monoclonal antibody with N-acyl homoserine lactones. Anal Chim Acta 683(1):113–118CrossRefGoogle Scholar
  29. 29.
    Sha Y, Deng C, Liu B (2008) Development of C-18-functionalized magnetic silica nanoparticles as sample preparation technique for the determination of ergosterol in cigarettes by microwave-assisted derivatization and gas chromatography/mass spectrometry. J Chromatogr A 1198:27–33. doi: 10.1016/j.chroma.2008.05.049 CrossRefGoogle Scholar
  30. 30.
    Aguilar-Arteaga K, Rodriguez JA, Barrado E (2010) Magnetic solids in analytical chemistry: a review. Anal Chim Acta 674(2):157–165. doi: 10.1016/j.aca.2010.06.043 CrossRefGoogle Scholar
  31. 31.
    Deng H, Li XL, Peng Q, Wang X, Chen JP, Li YD (2005) Monodisperse magnetic single-crystal ferrite microspheres. Angew Chem Int Ed 44(18):2782–2785. doi: 10.1002/anie.200462551 CrossRefGoogle Scholar
  32. 32.
    Smith HL, Waltman P (1995) The theory of the chemostat: dynamics of microbial competition. Cambridge University Press, New YorkCrossRefGoogle Scholar
  33. 33.
    Brady JF (1995) In: Karu A, Nelson J, Wong R (eds) Immunoanalysis of agrochemicals: emerging technologies. American Chemical Society, Washington, pp 266–287CrossRefGoogle Scholar
  34. 34.
    Meyer A, Megerle JA, Kuttler C, Mueller J, Aguilar C, Eberl L, Hense BA, Raedler JO (2012) Dynamics of AHL mediated quorum sensing under flow and non-flow conditions. Phys Biol 9(2). doi: 10.1088/1478-3975/9/2/026007
  35. 35.
    Adar YY, Simaan M, Ulitzur S (1992) Formation of the LuxR protein in the Vibrio fischeri lux system is controlled by HtpR through the GroESL proteins. J Bacteriol 174(22):7138–7143Google Scholar
  36. 36.
    Ulitzur S (1989) The regulatory control of the bacterial luminescence system–a new view. J Biolumin Chemilumin 4(1):317–325. doi: 10.1002/bio.1170040144 CrossRefGoogle Scholar
  37. 37.
    Rampioni G, Bertani I, Pillai CR, Venturi V, Zennaro E, Leoni L (2012) Functional characterization of the quorum sensing regulator RsaL in the plant-beneficial strain Pseudomonas putida WCS358. Appl Environ Microbiol 78(3):726–734. doi: 10.1128/aem.06442-11 CrossRefGoogle Scholar
  38. 38.
    Gupta R, Schuster M (2013) Negative regulation of bacterial quorum sensing tunes public goods cooperation. ISME J 7(11):2159–2168. doi: 10.1038/ismej.2013.109 CrossRefGoogle Scholar
  39. 39.
    Kuttler C, Hense BA (2010) Finetuning for the mathematical modelling of quorum sensing regulation systems. Int J Biomath Biostat 1:151–168Google Scholar
  40. 40.
    Zhang H-B, Wang L-H, Zhang L-H (2002) Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens. Proc Natl Acad Sci 99(7):4638–4643. doi: 10.1073/pnas.022056699 CrossRefGoogle Scholar
  41. 41.
    Chen CC, Riadi L, Suh SJ, Ohman DE, Ju LK (2005) Degradation and synthesis kinetics of quorum-sensing autoinducer in Pseudomonas aeruginosa cultivation. J Biotechnol 117(1):1–10. doi: 10.1016/j.jbiotec.2005.01.003 CrossRefGoogle Scholar
  42. 42.
    Henkel M, Schmidberger A, Kuehnert C, Beuker J, Bernard T, Schwartz T, Syldatk C, Hausmann R (2013) Kinetic modeling of the time course of N-butyryl-homoserine lactone concentration during batch cultivations of Pseudomonas aeruginosa PAO1. Appl Microbiol Biotechnol 97(17):7607–7616. doi: 10.1007/s00253-013-5024-5 CrossRefGoogle Scholar
  43. 43.
    De Kievit TR, Gillis R, Marx S, Brown C, Iglewski BH (2001) Quorum-sensing genes in Pseudomonas aeruginosa biofilms: their role and expression patterns. Appl Environ Microbiol 67(4):1865–1873. doi: 10.1128/aem.67.4.1865-1873.2001 CrossRefGoogle Scholar
  44. 44.
    Lopez D, Vlamakis H, Kolter R (2009) Generation of multiple cell types in Bacillus subtilis. FEMS Microbiol Rev 33(1):152–163. doi: 10.1111/j.1574-6976.2008.00148.x CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Katharina Buddrus-Schiemann
    • 1
  • Martin Rieger
    • 1
  • Marlene Mühlbauer
    • 1
  • Maria Vittoria Barbarossa
    • 2
  • Christina Kuttler
    • 3
  • Burkhard A. Hense
    • 4
  • Michael Rothballer
    • 1
  • Jenny Uhl
    • 5
  • Juliano R. Fonseca
    • 5
  • Philippe Schmitt-Kopplin
    • 5
    • 6
  • Michael Schmid
    • 1
  • Anton Hartmann
    • 1
  1. 1.Research Unit Microbe-Plant Interactions, Helmholtz Zentrum MünchenGerman Research Centre for Environmental Health (GmbH)NeuherbergGermany
  2. 2.Bolyai InstituteUniversity of SzegedSzegedHungary
  3. 3.Centre for Mathematical SciencesTechnische Universität MünchenGarchingGermany
  4. 4.Institute of Computational Biology, Helmholtz Zentrum MünchenGerman Research Centre for Environmental Health (GmbH)NeuherbergGermany
  5. 5.Research Unit Analytical BioGeoChemistry, Helmholtz Zentrum MünchenGerman Research Centre for Environmental Health (GmbH)NeuherbergGermany
  6. 6.Analytical Food ChemistryTechnische Universität MünchenFreising-WeihenstephanGermany

Personalised recommendations