Skip to main content
SpringerLink
Log in

Analysing traces of autoinducer-2 requires standardization of the Vibrio harveyi bioassay

  • Original Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Autoinducer-2 (furanosyl borate diester) is a biologically active compound whose role as a universal bacterial signalling molecule is currently under intense investigation. Because of its instability and the low concentrations of it found in biological samples, its detection relies at present on a bioassay that measures the difference in the timing of the luminescence of the Vibrio harveyi BB170 sensor strain with and without externally added AI-2. Here we systematically investigated which parameters affected the fold induction values of luminescence obtained in the bioassay and developed a modified protocol. Our experiments showed that growth and luminescence of V. harveyi BB170 are strongly influenced by trace elements. In particular, addition of Fe3+ within a certain concentration range to the growth medium of the preinoculum culture improved the reproducibility and reduced the variance of the bioassay. In contrast, trace elements and vitamins introduced directly into the bioassay caused inhibitory effects. The initial density and luminescence of the sensor strain are very important and the values required for these parameters were defined. Borate interferes with the detection of AI-2 by giving false positive results. The response of V. harveyi BB170 to chemically synthesized AI-2 in the bioassay is nonlinear except over a very small concentration range; it is maximum over three orders of magnitude and shows inhibition above 35 μM. Based on the modified protocol, we were able to detect AI-2 in the absence of inhibitors with maximum fold induction values for the positive control (chemically synthesized AI-2) of >120 with a standard deviation of ~30% in a reliable and reproducible way.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5a–d
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Nealson KH, Platt T, Hastings JW (1970) J Bacteriol 104:313–322

    CAS  Google Scholar 

  2. Fuqua WC, Winans SC, Greenberg EP (1994) J Bacteriol 176:269–275

    CAS  Google Scholar 

  3. Bassler BL, Greenberg EP, Stevens AM (1997) J Bacteriol 179:4043–4045

    CAS  Google Scholar 

  4. Bassler BL, Wright M, Showalter RE, Silverman MR (1993) Mol Microbiol 9:773–786

    Article  CAS  Google Scholar 

  5. Henke JM, Bassler BL (2004) J Bacteriol 186:3794–3805

    Article  CAS  Google Scholar 

  6. Winzer K, Hardie KR, Williams P (2003) Adv Appl Microbiol 53:291–396

    Article  CAS  Google Scholar 

  7. Vendeville A, Winzer K, Heurlier K, Tang CM, Hardie KR (2005) Nat Rev Microbiol 3:383–396

    Article  CAS  Google Scholar 

  8. DeKeersmaecker SC, Vanderleyden J (2003) Microbiology 149:1953–1956

    Google Scholar 

  9. Miller ST, Xavier KB, Campagna SR, Taga ME, Semmelhack MF, Bassler BL, Hughson FM (2004) Mol Cell 15:677–687

    Article  CAS  Google Scholar 

  10. Coulthurst SJ, Whitehead NA, Welch M, Salmond GP (2002) Trends Biochem Sci 27:217–219

    Article  CAS  Google Scholar 

  11. Turovskiy Y, Chikindas ML (2006) J Microbiol Methods 66:497–503

    Article  CAS  Google Scholar 

  12. Greenberg EP, Hastings JW, Ulitzur S (1979) Arch Microbiol 120:87–91

    Article  CAS  Google Scholar 

  13. De Keersmaecker SC, Varszegi C, van Boxel N, Habel LW, Metzger K, Daniels R, Marchal K, De Vos D, Vanderleyden J (2005) J Biol Chem 280:19563–19568

    Article  CAS  Google Scholar 

  14. Stevens RV, Beaulieu N, Chan WH, Daniewski AR, Takeda T, Waldner A, Williard PG, Zutter U (1986) J Am Chem Soc 108:1039–1049

    Article  CAS  Google Scholar 

  15. Chen X, Schauder S, Potier N, Van Dorsselaer A, Pelczer I, Bassler BL, Hughson FM (2002) Nature 415:545–549

    Article  CAS  Google Scholar 

  16. Semmelhack MF, Campagna SR, Hwa C, Federle MJ, Bassler BL (2004) Org Lett 6:2635–2637

    Article  CAS  Google Scholar 

  17. Mariscal A, Peinado MT, Carnero-Varo M, Fernandez-Crehuet J (2003) Chemosphere 50:349–354

    Article  Google Scholar 

  18. Rodicheva EK, Trubachev IN, Medvedeva SE, Egorova OI, Shitova LY (1993) J Biolumin Chemilumin 8:293–299

    Article  CAS  Google Scholar 

  19. Crosa JH (1997) Microbiol Mol Biol Rev 61:319–336

    CAS  Google Scholar 

  20. Panina EM, Mironov AA, Gelfand MS (2001) Nucleic Acids Res 29:5195–5206

    Article  CAS  Google Scholar 

  21. Juhas M, Wiehlmann L, Huber B, Jordan D, Lauber J, Salunkhe P, Limpert AS, von Gotz F, Steinmetz I, Eberl L, Tummler B (2004) Microbiology 150:831–841

    Article  CAS  Google Scholar 

  22. Cornelis P, Aendekerk S (2004) Microbiology 150:752–756

    Article  CAS  Google Scholar 

  23. Kim EJ, Wang W, Deckwer W-D, Zeng A-P (2005) Microbiology 151:1127–1138

    Article  CAS  Google Scholar 

  24. Makemson JC, Hastings JW (1982) Curr Microbiol 7:181–186

    Article  CAS  Google Scholar 

  25. Dunlap PV (1992) Arch Microbiol 157:235–241

    Article  CAS  Google Scholar 

  26. Dunlap PV (1992) J Biolumin Chemilumin 7:203–214

    Article  CAS  Google Scholar 

  27. Rajan R, Zhu J, Hu X, Pei D, Bell CE (2005) Biochemistry 44:3745–3753

    Article  CAS  Google Scholar 

Download references

Acknowledgements

R. Vilchez thanks the Environmental Microbiology Research Group (RNM-270, Universidad de Granada, Spain) for supporting him with a postdoctoral grant.

Remark

The maximum fold induction values that we obtained with newer instruments (e.g. Victor Light 1420 Luminescence Counter) were above 800 up to 1300; however, the effects described in this paper were the same.

Author information

Authors and Affiliations

  1. “Microbial Communication” Group, Department of Cell Biology and Immunology, Helmholtz-Center for Infection Research, Inhoffenstr. 7, 38124, Braunschweig, Germany

    Ramiro Vilchez, André Lemme, Helena Sztajer & Irene Wagner-Döbler

  2. Institute of Organic Chemistry, Technical University of Braunschweig, Hagenring 30, 38106, Braunschweig, Germany

    Verena Thiel & Stefan Schulz

Authors
  1. Ramiro Vilchez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. André Lemme
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Verena Thiel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stefan Schulz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Helena Sztajer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Irene Wagner-Döbler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ramiro Vilchez.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Vilchez, R., Lemme, A., Thiel, V. et al. Analysing traces of autoinducer-2 requires standardization of the Vibrio harveyi bioassay. Anal Bioanal Chem 387, 489–496 (2007). https://doi.org/10.1007/s00216-006-0824-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-006-0824-4

Keywords

Search

Navigation