Abstract
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Lilley BN, Bassler BL (2000) Regulation of quorum sensing in Vibrio harveyi by LuxO and sigma-54. Mol Microbiol 36:940–954
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 9:773–786
Henke JM, Bassler BL (2004) Quorum sensing regulates type III secretion in Vibrio harveyi and Vibrio parahaemolyticus. J Bacteriol 186:3794–3805
Mok KC, Wingreen NS, Bassler BL (2003) Vibrio harveyi quorum sensing: a coincidence detector for two autoinducers controls gene expression. EMBO J 22:870–881
DeKeersmaecker SCJ, Vanderleyden J (2003) Constraints on detection of autoinducer-2 (AI-2) signalling molecules using Vibrio harveyi as a reporter. Microbiology 149:1953–1956
Turovskiy Y, Chikindas ML (2006) Autoinducer-2 bioassay is a qualitative, not quantitative method influenced by glucose. J Microbiol Methods 66:497–503
de Lorimier RM, Smith JJ, Dwyer MA, Looger LL, Sali KM, Paavola CD et al (2002) Construction of a fluorescent biosensor family. Protein Sci 11:2655–2675
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:E2
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 46:3990–3997
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 99:9846–9851
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 278:19127–19133
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 264:350–363
Zukin RS, Hartig PR, Koshland DE Jr (1979) Effect of an induced conformational change on the physical properties of two chemotactic receptor molecules. Biochemistry 18:5599–5605
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 74:1932–1936
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 13:273–286
Semmelhack MF, Campagna SR, Federle MJ, Bassler BL (2005) An expeditious synthesis of DPD and boron binding studies. Org Lett 7:569–572
Bennett A, Rowe RI, Soch N, Eckhert CD (1999) Boron stimulates yeast (Saccharomyces cerevisiae) growth. J Nutr 129:2236–2238
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 286:18331–18343
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 20:87–90
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Rajamani, S., Sayre, R. (2018). Biosensors for the Detection and Quantification of AI-2 Class Quorum-Sensing Compounds. In: Leoni, L., Rampioni, G. (eds) Quorum Sensing. Methods in Molecular Biology, vol 1673. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7309-5_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7309-5_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7308-8
Online ISBN: 978-1-4939-7309-5
eBook Packages: Springer Protocols