FRET-Based Biosensors for the Detection and Quantification of AI-2 Class of Quorum Sensing Compounds

  • Sathish Rajamani
  • Richard SayreEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 692)


Intercellular small molecular weight signaling molecules modulate a variety of biological functions in bacteria. One of the more complex behaviors mediated by intercellular signaling molecules is the suite of activities regulated by quorum sensing molecules. These molecules mediate a variety of population-dependent responses, including the expression of genes that regulate bioluminescence, type III secretion, siderophore production, colony morphology, biofilm formation, and metalloprotease production. Given their central role in regulating these responses, the detection and quantification of QS molecules has important practical implications. Until recently, the detection of QS molecules from Gram-negative bacteria has relied primarily on bacterial reporter systems. These bioassays though immensely useful are subject to interference by compounds that affect bacterial growth and metabolism. In addition, the reporter response is highly dependent on culture age and cell population density. To overcome such limitations, we developed an in vitro protein-based assay system for the rapid detection and quantification of the furanosyl borate diester (BAI-2) subclass of autoinducer-2 (AI-2) QS molecules. The biosensor is based on the interaction of BAI-2 with the Vibrio harveyi QS receptor LuxP. Conformation changes associated with BAI-2 binding to the LuxP receptor change the orientation of cyan and yellow variants of GFP (CFP and YFP) fused the N- and C-termini, respectively, of the LuxP receptor. LuxP-BAI2 binding induces changes in fluorescence resonance energy transfer (FRET) between CFP and YFP, whose magnitude of change is ligand concentration dependent. A set of ligand-insensitive LuxP-mutant FRET protein sensor was also developed for use as control biosensors. The FRET-based BAI-2 biosensor responds selectively to both synthetic and biologically derived BAI-2compounds. This report describes the use of the LuxP-FRET biosensor for the detection and quantification of BAI-2.

Key words

Autoinducer Quorum sensing LuxP Ligand BAI-2 DPD FRET Biosensor GFP CFP YFP Dissociation constant Quantification Fluorescence 


  1. 1.
    Lilley, B. N., and Bassler, B. L. (2000) Regulation of quorum sensing in Vibrio harveyi by LuxO and sigma-54, Mol Microbiol 36, 940.PubMedCrossRefGoogle Scholar
  2. 2.
    Bassler, B. L., Wright, M., Showalter, R. E., and Silverman, M. R. (1993) Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence, Mol Microbiol 9, 773–786.PubMedCrossRefGoogle Scholar
  3. 3.
    Henke, J. M., and Bassler, B. L. (2004) Quorum sensing regulates type III secretion in Vibrio harveyi and Vibrio parahaemolyticus, J Bacteriol 186, 3794.PubMedCrossRefGoogle Scholar
  4. 4.
    Mok, K. C., Wingreen, N. S., and Bassler, B. L. (2003) Vibrio harveyi quorum sensing: a coincidence detector for two autoinducers controls gene expression, EMBO J 22, 870–881.PubMedCrossRefGoogle Scholar
  5. 5.
    DeKeersmaecker, S. C. J., and Vanderleyden, J. (2003) Constraints on detection of autoinducer-2 (AI-2) signalling molecules using Vibrio harveyi as a reporter, Microbiology 149, 1953–1956.PubMedCrossRefGoogle Scholar
  6. 6.
    Turovskiy, Y., and Chikindas, M. L. (2006) Autoinducer-2 bioassay is a qualitative, not quantitative method influenced by glucose, J Microbiol Methods 66, 407–503.CrossRefGoogle Scholar
  7. 7.
    de Lorimier, R. M., Smith, J. J., Dwyer, M. A., Looger, L. L., Sali, K. M., Paavola, C. D., Rizk, S. S., Sadigov, S., Conrad, D. W., Loew, L., and Hellinga, H. W. (2002) Construction of a fluorescent biosensor family, Protein Sci 11, 2655–2675.PubMedCrossRefGoogle Scholar
  8. 8.
    Felder, C. B., Graul, R. C., Lee, A. Y., Merkle, H. P., and Sadee, W. (1999) The Venus flytrap of periplasmic binding proteins: an ancient protein module present in multiple drug receptors, AAPS PharmSci 1, E2.PubMedCrossRefGoogle Scholar
  9. 9.
    Rajamani, S., Zhu, J., Pei, D., and Sayre, R. (2007) A LuxP-FRET-based reporter for the detection and quantification of AI-2 bacterial quorum-sensing signal compounds, Biochemistry 46, 3990–3997.PubMedCrossRefGoogle Scholar
  10. 10.
    Fehr, M., Frommer, W. B., and Lalonde, S. (2002) Visualization of maltose uptake in living yeast cells by fluorescent nanosensors, Proc Natl Acad Sci USA 99, 9846–9851.PubMedCrossRefGoogle Scholar
  11. 11.
    Fehr, M., Lalonde, S., Lager, I., Wolff, M. W., and Frommer, W. B. (2003) In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors, J Biol Chem 278, 19127–19133.PubMedCrossRefGoogle Scholar
  12. 12.
    Shilton, B. H., Flocco, M. M., Nilsson, M., and Mowbray, S. L. (1996) Conformational changes of three periplasmic receptors for bacterial chemotaxis and transport: the maltose-, glucose/galactose- and ribose-binding proteins, J Mol Biol 264, 350–363.PubMedCrossRefGoogle Scholar
  13. 13.
    Zukin, R. S., Hartig, P. R., and Koshland, D. E., Jr. (1979) Effect of an induced conformational change on the physical properties of two chemotactic receptor molecules, Biochemistry 18, 5599–5605.PubMedCrossRefGoogle Scholar
  14. 14.
    Zukin, R. S., Hartig, P. R., and Koshland, D. E., Jr. (1977) Use of a distant reporter group as evidence for a conformational change in a sensory receptor, Proc Natl Acad Sci USA 74, 1932–1936.PubMedCrossRefGoogle Scholar
  15. 15.
    Bassler, B. L., Wright, M., and Silverman, M. R. (1994) Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway, Mol Microbiol 13, 273–286.PubMedCrossRefGoogle Scholar
  16. 16.
    Semmelhack, M. F., Campagna, S. R., Federle, M. J., and Bassler, B. L. (2005) An expeditious synthesis of DPD and boron binding studies, Org Lett 7, 569–572.PubMedCrossRefGoogle Scholar
  17. 17.
    Bennett, A., Rowe, R. I., Soch, N., and Eckhert, C. D. (1999) Boron stimulates yeast (Saccharomyces cerevisiae) growth, J Nutr 129, 2236–2238.PubMedGoogle Scholar
  18. 18.
    Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., and Miyawaki, A. (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications, Nat Biotechnol 20, 87–90.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Microbiology and ImmunologyDartmouth Medical SchoolHanoverUSA
  2. 2.Life Sciences InstituteAnn ArborUSA
  3. 3.Donald Danforth Plant Science CenterSt. LouisUSA

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