Methods for Analysis of Bacterial Autoinducer‐2 Production

Michiko E. Taga1, Karina B. Xavier2

1 Department of Plant and Microbial Biology, University of California, Berkeley, California, 2 Instituto Gulbenkian de Ciencia, Bacterial Signaling Laboratory, Oeiras, Portugal
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 1C.1
DOI:  10.1002/9780471729259.mc01c01s23
Online Posting Date:  November, 2011
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Quorum sensing is a cell‐cell signaling process that many bacteria use to regulate gene expression as a function of the density of the population. This phenomenon involves the production, release, and response to small chemical molecules termed autoinducers. Most autoinducers are species‐specific; however, one autoinducer called autoinducer‐2 (AI‐2) is produced and detected by many species of bacteria and thus can foster inter‐species communication. This unit describes two assays to detect and quantify AI‐2 from biological samples. The first uses a bacterial reporter strain, which produces bioluminescence in response to AI‐2. The second is an in vitro assay based on a modified version of an AI‐2 receptor fused to a cyan fluorescent protein and a yellow fluorescent protein. Binding of AI‐2 to this fusion protein induces a dose‐dependent decrease in fluorescence resonance energy transfer (FRET), enabling quantification of the AI‐2 concentration in the samples. Curr. Protoc. Microbiol. 23:1C.1.1‐1C.1.15. © 2011 by John Wiley & Sons, Inc.

Keywords: AI‐2; autoinducer‐2; luminescent proteins luminescent measurements; Vibrio harveyi; bacterial LuxS protein; 4,5‐dihydroxy‐2,3‐pentanedione; quorum sensing; BB170; FRET; CFP‐LuxP‐YFP

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Table of Contents

  • Introduction
  • Basic Protocol 1: Detection of AI‐2 Using the Vibrio harveyi Reporter Strain
  • Alternate Protocol 1: Detection of AI‐2 Using an In Vitro CLPY‐FRET‐Based Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Detection of AI‐2 Using the Vibrio harveyi Reporter Strain

  • Bacterial strain to be tested
  • Growth medium specific for bacterial strain
  • V. harveyi strain in BB152 (luxM::Tn5), frozen glycerol stock
  • V. harveyi strain in BB170 (luxN::Tn5), frozen glycerol stock
  • AB medium (see )
  • Sterile syringe filters, 0.2‐µm pore size
  • 1‐ml syringes
  • Sterile 1.5‐ml microcentrifuge tubes
  • Luminometer, scintillation counter, or equivalent device for measuring light
CAUTION: V. harveyi is a Biosafety Level 1 (BSL‐1) organism. Such organisms are not known to consistently cause disease in healthy adult humans, and are of minimal potential hazard to laboratory personnel and the environment. Standard microbiological practices should be followed when working with these organisms. See unit 1.1 and other pertinent resources ( appendix 1B) for more information.NOTE: If preparing a large number of samples, use a 96‐well microtiter filter plate with a sterile 96‐well plate for collection rather than individual syringes, syringe filters, and microcentrifuge tubes.NOTE: BB170 and BB152 strains (Bassler et al., ) are available at American Type Culture Collection ( with strain ATCC #BAA‐1117 and ATCC #BAA‐1119, respectively. To create frozen stocks these strains should be cultured in AB medium for 14 to 16 hr and frozen with 20% (v/v) glycerol. Loss of bioluminescence has been observed in V. harveyi stocks cultured in different media.

Alternate Protocol 1: Detection of AI‐2 Using an In Vitro CLPY‐FRET‐Based Assay

  • AI‐2 [purchased from OMM Scientific ( or synthesized enzymatically by a simple procedure as described in Schauder et al. ( )]
  • Reaction buffer (see )
  • CLPY‐FRET protein (purified as explained in protocol 3)
  • 96‐well microtiter plates to measure fluorescence (use black opaque plates, such as Optiplate‐96 from Perkin Elmer)
  • Multilabel 96‐well plate reader equipped to measure fluorescence using the following conditions: 440 nm excitation (5 nm slit) and 527 and 485 nm emission filters (5 nm slit)

Support Protocol 1:

  • Escherichia coli BL21(luxS) carrying the plasmid pQE30‐CLPY, frozen stock
  • LB medium, supplemented with ampicillin (see appendix 4A)
  • Isopropyl thiogalactoside (IPTG)
  • Nickel affinity resin (e.g., Sigma P6611)
  • Equilibration buffer (see recipe)
  • Elution buffer (see recipe)
  • Cocktail of protease inhibitors
  • Protein quantification kit (Bradford)
  • 15‐ml tubes (or other tubes of similar volume)
  • Shaking incubator
  • Centrifuge
  • Disposable plastic columns for protein purification (volume 15 ml)
  • French press (or other cell disruption equipment)
CAUTION: E. coli is a Biosafety Level 1 (BSL‐1) organism. Standard microbiological practices should be followed when working with this organism. See unit 1.1 and other pertinent resources ( appendix 1B) for more information.NOTE:Escherichia coli BL21(luxS) carrying the plasmid pQE30‐CLPY (Rajamani et al., ) were constructed by Dr. Satish Rajamani, Darmouth Medical School, and Dr. Richard Sayer, Donald Danforth Plant Science Center.
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  •   FigureFigure 1.C0.1 Proposed pathway for formation of AI‐2 molecules. DPD cyclizes spontaneously to form two stereoisomers, A and B. Spontaneous addition of water to A and B results in formation of C and S. typhimurium AI‐2, respectively. V. harveyi AI‐2 is formed by addition of boric acid to C (modified from Miller et al., ).
  •   FigureFigure 1.C0.2 Light production in the BB170 bioassay. Induction of bioluminescence is calculated at the time point marked by the vertical line.
  •   FigureFigure 1.C0.3 AI‐2 concentration in cell‐free fluids taken from an E. coli culture determined using the CLPY‐FRET assay. (A) Calibration curve of FRET ratio versus AI‐2 concentration in standard samples. (B) Concentration of extracellular AI‐2 in E. coli cultured in LB. These concentrations were determined using the equation obtained in A.


Literature Cited

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