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

Part of the Methods in Molecular Biology book series (MIMB, volume 1673)


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 have 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 to 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. Ligand-insensitive LuxP mutant FRET protein sensors were also developed for use as control biosensors. The FRET-based BAI-2 biosensor responds selectively to both synthetic and biologically derived BAI-2 compounds. 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 AI-2 BAI-2 DPD FRET Biosensor GFP CFP YFP Dissociation constant Quantification Fluorescence 


  1. 1.
    Lilley BN, Bassler BL (2000) Regulation of quorum sensing in Vibrio harveyi by LuxO and sigma-54. Mol Microbiol 36:940–954CrossRefPubMedGoogle Scholar
  2. 2.
    Bassler BL, Wright M, Showalter RE, Silverman MR (1993) Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Mol Microbiol 9:773–786CrossRefPubMedGoogle Scholar
  3. 3.
    Henke JM, Bassler BL (2004) Quorum sensing regulates type III secretion in Vibrio harveyi and Vibrio parahaemolyticus. J Bacteriol 186:3794–3805CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Mok KC, Wingreen NS, Bassler BL (2003) Vibrio harveyi quorum sensing: a coincidence detector for two autoinducers controls gene expression. EMBO J 22:870–881CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    DeKeersmaecker SCJ, Vanderleyden J (2003) Constraints on detection of autoinducer-2 (AI-2) signalling molecules using Vibrio harveyi as a reporter. Microbiology 149:1953–1956CrossRefPubMedGoogle Scholar
  6. 6.
    Turovskiy Y, Chikindas ML (2006) Autoinducer-2 bioassay is a qualitative, not quantitative method influenced by glucose. J Microbiol Methods 66:497–503CrossRefPubMedGoogle Scholar
  7. 7.
    de Lorimier RM, Smith JJ, Dwyer MA, Looger LL, Sali KM, Paavola CD et al (2002) Construction of a fluorescent biosensor family. Protein Sci 11:2655–2675CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Felder CB, Graul RC, Lee AY, Merkle HP, Sadee W (1999) The Venus flytrap of periplasmic binding proteins: an ancient protein module present in multiple drug receptors. AAPS Pharm Sci 1:E2CrossRefGoogle Scholar
  9. 9.
    Rajamani S, Zhu J, Pei D, Sayre R (2007) A LuxP-FRET-based reporter for the detection and quantification of AI-2 bacterial quorum-sensing signal compounds. Biochemistry 46:3990–3997CrossRefPubMedGoogle Scholar
  10. 10.
    Fehr M, Frommer WB, Lalonde S (2002) Visualization of maltose uptake in living yeast cells by fluorescent nanosensors. Proc Natl Acad Sci U S A 99:9846–9851CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Fehr M, Lalonde S, Lager I, Wolff MW, Frommer WB (2003) In vivo imaging of the dynamics of glucose uptake in the cytosol of COS-7 cells by fluorescent nanosensors. J Biol Chem 278:19127–19133CrossRefPubMedGoogle Scholar
  12. 12.
    Shilton BH, Flocco MM, Nilsson M, Mowbray SL (1996) Conformational changes of three periplasmic receptors for bacterial chemotaxis and transport: the maltose-, glucose/galactose- and ribose-binding proteins. J Mol Biol 264:350–363CrossRefPubMedGoogle Scholar
  13. 13.
    Zukin RS, Hartig PR, Koshland DE Jr (1979) Effect of an induced conformational change on the physical properties of two chemotactic receptor molecules. Biochemistry 18:5599–5605CrossRefPubMedGoogle Scholar
  14. 14.
    Zukin RS, Hartig PR, Koshland DE Jr (1977) Use of a distant reporter group as evidence for a conformational change in a sensory receptor. Proc Natl Acad Sci U S A 74:1932–1936CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Bassler BL, Wright M, Silverman MR (1994) Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway. Mol Microbiol 13:273–286CrossRefPubMedGoogle Scholar
  16. 16.
    Semmelhack MF, Campagna SR, Federle MJ, Bassler BL (2005) An expeditious synthesis of DPD and boron binding studies. Org Lett 7:569–572CrossRefPubMedGoogle Scholar
  17. 17.
    Bennett A, Rowe RI, Soch N, Eckhert CD (1999) Boron stimulates yeast (Saccharomyces cerevisiae) growth. J Nutr 129:2236–2238PubMedGoogle Scholar
  18. 18.
    Marques JC, Lamosa P, Russel C, Ventura R, Maycock C, Semmelhack M et al (2011) Processing the interspecies quorum-sensing signal autoinducer-2 (AI-2). J Biol Chem 286:18331–18343CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20:87–90CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.New Mexico ConsortiumLos AlamosUSA

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