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A Novel Fluorescent Protein-Based Biosensor for Gram-Negative Bacteria

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A Novel Fluorescent Protein-Based Biosensor for Gram-Negative Bacteria
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   A  PPLIED AND  E NVIRONMENTAL   M ICROBIOLOGY , Dec. 2002, p. 6343–6352 Vol. 68, No. 120099-2240/02/$04.00  0 DOI: 10.1128/AEM.68.12.6343–6352.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.  A Novel Fluorescent Protein-Based Biosensor forGram-Negative Bacteria Yan Y. Goh, 1 † Bow Ho, 2 and Jeak L. Ding 1 *  Department of Biological Sciences 1  and Department of Microbiology, 2  National University of Singapore, Singapore 117543 Received 22 April 2002/Accepted 5 September 2002 Site-directed mutagenesis of enhanced green fluorescent protein (EGFP) based on rational computationaldesign was performed to create a fluorescence-based biosensor for endotoxin and gram-negative bacteria.EGFP mutants (EGFP i ) bearing one (G10) or two (G12) strands of endotoxin binding motifs were constructedand expressed in an  Escherichia coli  host. The EGFP i  proteins were purified and tested for their efficacy as anovel fluorescent biosensor. After efficient removal of lipopolysaccharide from the  E. coli  lysates, the bindingaffinities of the EGFP i  G10 and G12 to lipid A were established. The  K   D  values of 7.16  10  7 M for G10 and8.15  10  8 M for G12 were achieved. With high affinity being maintained over a wide range of pH and ionicstrength, the binding of lipid A/lipopolysaccharide to the EGFP i  biosensors could be measured as a concen-tration-dependent fluorescence quenching of the EGFP mutants. The EGFP i  specifically tagged gram-negativebacteria like  E. coli  and  Pseudomonas aeruginosa , as well as other gram-negative bacteria in contaminated watersampled from the environment. This dual function of the EGFP i  in detecting both free endotoxin and livegram-negative bacteria forms the basis of the development of a novel fluorescent biosensor. Increasing concern regarding the microbiological safety of food, water, dairy products, industrial waste, and pharmaceu-tical preparations has provided an urgency for detection meth-ods that are fast, sensitive, specific, reliable, and quantitativefor quality assurance in order to prevent infections and epi-demics (10). There are a large number of detection methodsfor microorganisms, including immunomagnetic separationand flow cytometry (18), flexural plate wave (16), quartz crystal(8), and surface acoustic wave (9). In addition, due to theubiquity and lethality of endotoxin (or lipopolysaccharide)from the outer cell-wall of gram-negative bacteria, many phar-maceutical products are rigorously tested for the presence of contaminating lipopolysaccharide or gram-negative bacteriabefore the products are sold for human use. Both the UnitedStates Pharmacopoeia and the European Pharmacopoeia spec-ify the rabbit pyrogen test and the  Limulus  amoebocyte lysatetest as the quality control tests for the presence of endotoxin ininjectables and medical devices. A number of new approaches to pyrogen testing have beenreported. These are mainly based on an in vitro pyrogen testinvolving the use of human cells such as leukocyte cell lines,isolated primary blood, and whole blood (4, 20). Recently,genetic engineering of an endotoxin-sensitive  Limulus  amoe-bocyte lysate protein, recombinant factor C expressed in abaculovirus system, produced an enzyme with remarkable sen-sitivity to endotoxin, at 0.001 endotoxin unit (EU)/ml (2).The green fluorescent protein (GFP) has been a popularchoice for development of reporter-biosensors to detect var-ious environmentally hazardous compounds (7, 11, 19).Through computer-aided simulation and rational design, wehave recently developed a fluorescent biosensor for lipopoly-saccharide and lipid A (the bioactive moiety of lipopolysaccha-ride) with enhanced green fluorescent protein (EGFP) as ascaffold protein (6). Previously, we have shown (5) that lipo-polysaccharide or lipid A can interact strongly with short cat-ionic amphipathic sequences of five alternating basic (B) andhydrophobic (H) residues (BHBHB). Thus, such sequence mo-tifs were introduced into the  -sheets located on the surface of the EGFP barrel in the vicinity of the chromophore (6). TheEGFP mutants (EGFP i ) showed a range of lipid A bindingaffinities (26.12 to 0.13  M), resulting in concentration-depen-dent fluorescence quenching (6).The high level of endogenous lipopolysaccharide, which isthe ligand that binds EGFP i , and a serious host incompatibilityproblem that may result in its growth inhibition or even celldeath during the expression of recombinant EGFP i  in  Esche- richia coli  are the major challenges facing the expression of EGFP i  in a gram-negative bacterial host. However, this prob-lem did not arise in the present study. Furthermore, Schnait-man (17) has demonstrated that treatment of   E. coli  with thecombination of Triton X-114, EDTA, and lysozyme resulted insolubilization of all lipopolysaccharide from the cell wall.Hence, our strategy to overcome lipopolysaccharide contami-nation of EGFP i  proteins was to target recombinant EGFP i either into insoluble inclusion bodies or into the periplasmicspace or to efficiently remove lipopolysaccharide from solublecytoplasmic EGFP i  after lysing the cells.We report here the construction of EGFP i , expression in  E. coli , purification, and lipopolysaccharide removal, followed bycharacterization and the use of EGFP i  as a novel biosensor fordetection of endotoxin and live gram-negative bacteria. Wetested the binding affinity of EGFP i  to lipid A over a widerange of pHs and ionic strengths. In addition, we demonstratedfluorescence quenching of EGFP i  upon interaction with lipo- * Corresponding author. Mailing address: Department of BiologicalSciences, National University of Singapore, 14, Science Drive 4, Sin-gapore 117543. Phone: (65) 6874 2776. Fax: (65) 6779 2486. E-mail:dbsdjl@nus.edu.sg.† Present address: Temasek Applied Science School, Temasek Poly-technic, Singapore 529757.6343  polysaccharide and lipid A. The fluorescent EGFP i  was used todetect gram-negative bacteria directly in laboratory cultures as well as in environmental water samples. MATERIALS AND METHODSConstruction of EGFP i  plasmid.  After insertion of EGFP into the pBluescriptSK II vector (Stratagene, La Jolla, Calif.) to yield pBS-EGFP, a lipid A bindingmotif was introduced into the   -sheet(s) of the EGFP scaffold via site-directedmutagenesis (6). The full-length EGFP i  mutants were synthesized by PCR withforward primer 5  -CCGCCCATATGGTGAGCAAGGGCG-3  , containing an  Nde I site, and reverse primer 5  -GGGGATCCCGCGGGCCCTCTAGACT-3  ,containing a  Bam HI site. For directional cloning, the PCR products were cleavedat the  Nde I and  Bam HI sites and cloned into the pET3b (Novagen, Madison,Wis.) vector linearized with compatible restriction sites. With this strategy, thesingle- and double-motif mutants, collectively referred to as pET3b-EGFP i , weretransformed into  E. coli  TOP10 competent cells (Invitrogen, Carlsbad, Calif.),from which plasmid DNAs were extracted for verifying the DNA sequencesbefore their transformation into the expression host,  E. coli  BL21(DE3) (Nova-gen, Madison, Wis.) for protein production. Expression of EGFP i .  The EGFP i  proteins were expressed optimally in 5 ml of Luria-Bertani (LB) medium containing 80  g of ampicillin per ml and incubatedat 37°C without isopropylthiogalactopyranoside (IPTG) induction for 16 h withconstant shaking at 230 rpm. The culture was subsequently scaled up to 200-ml volume for the production of EGFP i  under the same conditions. The cultures were pelleted at 5,000   g   for 10 min at 4°C and resuspended with 40 ml of lysisbuffer containing 10 mM Tris-Cl, pH 7.5. The bacterial cells in the suspension were passed through a French press (Basic Z model; Constant System, Warwick,United Kingdom) at 100 MPa of pressure for four rounds in order to generate  90% cell disruption. Purification of EGFP i  proteins.  Soluble EGFP i  proteins were subjected toorganic extraction (22). Briefly, insoluble material in the disrupted cell suspen-sion which did not display green fluorescence was first removed by centrifugationat 20,000    g   for 30 min at 4°C. Triethanolamine base (Sigma, St. Louis, Mo.)and ammonium sulfate were added to the fluorescent green supernatant to finalconcentrations of 100 mM and 1.6 M, respectively. After incubation on ice for1 h, the precipitated proteins were removed by centrifugation at 5,000   g   for 20min at 4°C. Ammonium sulfate was added to the supernatant at room temper-ature to a final concentration of 2.8 M to achieve 70% saturation. The entiresuspension was extracted twice by vigorous shaking for 1 min each with 1/4(vol/vol) followed by 1/16 (vol/vol) ethanol. The aqueous and ethanol phases were separated by centrifugation at 3,000   g   for 5 min at room temperature. A 1/4 (vol/vol) concentration of   n -butanol was added to the combined ethanolextract. After vigorous shaking for 30 s, the phases formed were separated bycentrifugation as before. At this step, EGFP i  moved almost completely into thelower aqueous phase. The upper organic phase was discarded, and an equal volume of chloroform was added to the aqueous phase. After extraction for 30 s,the phases were separated by centrifugation as before. The upper aqueous phasecontaining EGFP i  was collected and dialyzed twice with 200 ml each of 10 mMTris-HCl, pH 7.5, for 4 h with snakeskin (Pierce, Rockford, Ill.) dialysis tubing with a 3,500-Da cutoff. Lipopolysaccharide removal with Triton X-114 and affinity chromatography. The dialyzed EGFP i  extracts were subjected to various methods of lipopolysac-charide removal before functional studies. Triton X-114 was added to the proteinpreparation to a final concentration of 1%. The mixture was incubated at 4°C for30 min with constant stirring to ensure a homogenous solution (12). The sample was then incubated at 37°C for 10 min and centrifuged at 20,000   g   for 10 minat 25°C. The upper aqueous phase containing the protein was carefully removedand subjected to Triton X-114 phase separation for three more cycles.To further remove endotoxin, 6 to 8 ml of EGFP i  extracts was passed through1 ml of Detoxi-Gel endotoxin-removing resin, prepacked in a 5-ml disposablecolumn (Pierce, Rockford, Ill.) by gravity. The column was washed once with 5ml of 1% sodium deoxycholic acid (Sigma), followed by 5 ml of 2 M NaCl, andthrice with 5 ml each of pyrogen-free water before and after each lipopolysac-charide removal. Under pyrogen-free conditions, the EGFP i  extracts were fur-ther chromatographed through a column of 1 ml of S3  peptide affinity gel (3)to further remove traces of endotoxin, following the same steps as for theDetoxi-Gel endotoxin-removing column. The resulting EGFP i  was assayed forendotoxin by the  Limulus  amoebocyte lysate chromogenic assay with Kinetic-QCL (BioWhittaker Inc., Walkersville, Md.). Protein quantification and determination of expression level of EGFP i .  Thetotal protein of all cell lysates and extracts obtained from various steps of  FIG. 1. (A) Coomassie blue-stained SDS-PAGE gel of EGFP i  in whole-cell (W), soluble lysate (L), insoluble fractions (I), dialyzed organicextract (D), and after S3  column lipopolysaccharide removal (S3  ). G12 showed the lowest expression level at 3% of total protein, while nativeEGFP (G0) and G10 represented 7 to 8% of total protein. The majority of G12 was insoluble, and hence it was not purified by the organicextraction method. (B) Western analysis of EGFP i  in whole-cell, soluble lysate, and insoluble fractions. Most of the expressed native EGFP (G0)and G10 were in the soluble lysate, while G12 was mostly in the insoluble pellet.6344 GOH ET AL. A  PPL  . E NVIRON . M ICROBIOL  .  purification was quantified by the method of Bradford (1). To determine theexpression level of the EGFP i  mutant proteins in the lysates and insolublefractions, the proteins were electrophoresed by sodium dodecyl sulfate-polyac-rylamide gel electrophoresis (SDS-PAGE), and the relative amount of EGFP i  inthe bands was quantified densitometrically with Image Master VDS software(Amersham Biosciences, Buckinghamshire, United Kingdom). Measurement of binding affinity of EGFP i  with lipid A under different con-ditions.  The surface plasmon resonance sensorgrams were recorded to deter-mine the association and dissociation kinetics of EGFP i  to the immobilized lipid A with a BIACORE 2000 instrument (Biacore, Uppsala, Sweden). Briefly, lipid A (f583,  E. coli ; Sigma) at 1 mg/ml (0.5 mM) in water was immobilized on theHPA (hydrophobic) chip according to the manufacturer’s specifications. Stocksolutions of EGFP i  purified by affinity chromatography through an S3  column were diluted with different buffers and injected at five different concentrations(0.5 to 6   M) over the monolayer of immobilized lipid A at a flow rate of 20  l/min, with the diluent as the running buffer. Purified recombinant GFP (Clon-tech, Palo Alto, Calif.) was injected as the negative control. The dissociationconstant of each lipid A-EGFP i  complex was calculated with the BiaEvaluationsoftware, version 3.0 (Biacore). To regenerate the HPA chip, 100 mM NaOH wasinjected until the response unit (RU) returned to baseline. S3   (NH 2 -HAEHKVKIKVKQKYGQFPQGTEVTYTCSGNYFLM-COOH), which binds lipid A at high affinity (21), was used as a control. Fluorescence measurements of complexes of lipopolysaccharide-EGFP i  andlipid A-EGFP i.  To assess changes in the fluorescence of the EGFP i  after inter-action with lipopolysaccharide and lipid A, fluorimetric assays were carried out.Ten picomoles (270 ng) of G10 (or G12) sampled from three stages of lipopoly-saccharide removal (dialysis, Triton X-114 treatment, and S3   affinity chroma-tography) was diluted in 100  l of pyrogen-free buffer containing 50 mM Tris-Cl(pH 7.3) and mixed with 2  l of various amounts of lipid A at 2.5 to 80 ng (1.25to 40 pmol) or lipopolysaccharide at 7.5 to 240 ng (1.25 to 40 pmol). Lipid A andlipopolysaccharide were warmed at 37°C, sonicated for 30 min, and vortexedagain immediately before addition to the EGFP i  extracts.The fluorescence spectra were measured with an LS-50B spectrofluorimeter(Perkin Elmer, Beaconsfield, United Kingdom). In the recorded emission scans,emission intensities were monitored from 370 to 600 nm, while the excitation wavelength was fixed at 488 nm. The scanning speed was fixed at 1,500 nm/min,and the emission wavelength window was set at 10 nm. Changes in the emittedfluorescent light intensity at 508 nm of the lipid A-EGFP i  and lipopolysaccha-ride-EGFP i  complexes due to increasing amounts of lipid A or lipopolysaccha-ride were recorded. Native EGFP extract was used as a control to normalize thechanges of fluorescence to G10 and G12. Fluorescent tagging of live bacteria.  Laboratory cultures of   Pseudomonas aeruginosa  ATCC 27853 and  E. coli  TOP10 were challenged with EGFP i . Briefly,1 ml of overnight cultures containing 10 10 CFU/ml was pelleted and resuspendedin 1 ml of 0.9% saline. Aliquots (20  l) of the bacterial suspension were mixed with 5   l of EGFP i  in a final concentration of 2.0   M. The reactions werestopped at 3, 5, and 10 min by pelleting the bacteria, washing the pellets threetimes with 200   l of saline, and finally resuspending the cells in 20   l of 0.9%saline. Bacteria tagged with EGFP i  were viewed with a FluoView 300 confocallaser scanning microscope with an IX70 inverted microscope (Olympus). To testfor the specificity of EGFP i  for gram-negative bacteria, other microorganismssuch as the gram-positive bacterium  Staphylococcus aureus  ATCC 25923 and the TABLE 1. Purification and LPS removal of the expression of EGFP i Purification step  a Protein EGFP i LipopolysaccharideremovalVol(ml)Protein concn  b (mg/ml)Totalprotein(mg)Protein/proteinratio  c (  g/   g of total protein) Amt(mg)Recovery(%)Purification(fold)Concn  d (EU/ml)Efficiency(fold) EGFP i  G0Crude lysate 67 24.29 1,627.43 0.035 56.96 100 1 —  e — After centrifugation(30,000   g  )65 17.05 1,108.25 0.021 23.27 40.85 0.6   5,000 1Organic extraction 19.5 2.14 41.73 0.53 22.12 38.83 15.14 NA   f  NA Dialysis 22 1.67 36.74 0.54 19.84 34.83 15.43 400   12.5Triton X-114 15 1.84 27.6 0.67 18.49 32.34 19.14 0.6   8,000Detoxi-Gel chromatography 12 2.4 25.68 0.71 18.23 32 20.29 0.04   125,000S3  chromatography 12 1.5 18 0.74 13.32 23.38 21.14 0.02   250,000EGFP i  G10Crude lysate 68 20.16 1,370.88 0.033 45.24 100 1 —  e — After centrifugation(30,000   g  )66 9.86 650.76 0.019 12.36 27.32 0.58   5,000 1Organic extraction 10.5 2.13 22.365 0.52 11.63 25.71 15.76 NA NA Dialysis 18 1.03 18.54 0.54 10.01 22.13 16.36 1,000   5Triton X-114 7 1.09 7.63 0.62 4.73 10.46 18.79 5   1,000Detoxi-Gel chromatography 6.5 1.02 6.63 0.68 4.51 9.97 20.61 0.07   70,000S3  chromatography 6.5 0.48 3.12 0.71 2.22 4.91 21.52 0.01   500,000EGFP i  G12Crude lysate 78 40.05 3,123.9 0.029 90.5931 100 1 —  e — After centrifugation(30,000   g  )76 8.65 657.4 0.005 3.287 7.27 0.17   5,000 1Organic extraction 18 1.65 29.7 0.045 1.3365 2.95 1.55 NA NA Dialysis 28 0.84 23.52 0.054 1.27008 2.81 1.86 200   25Triton X-114 14 1.22 17.08 0.074 1.26392 2.79 2.55 4   1,250Detoxi-Gel chromatography 14 1.04 14.56 0.076 1.10656 2.44 2.62 0.04   125,000S3  chromatography 13.5 1.01 13.635 0.08 1.0908 2.41 2.76 0.025   200,000  a Native EGFP (G0) and mutant EGFP i  (G10 and G12) were expressed in  E. coli.  G10 and G12 bear single and double strands of lipopolysaccharide/lipid A bindingmotif of the type BHBHB, respectively (5, 6), on the  -sheet in the vicinity of the EGFP chromophore.  b Total protein concentration was determined by the Bradford method (1).  c EGFP i  concentration was determined by loading a fixed amount of total protein in SDS-PAGE gels, and bands were densitometrically analyzed with Image MasterVDS software version 2.0 (Amersham Pharmacia Biotech).  d LAL-QCL kinetic endotoxin test (BioWhittaker), the most effective and sensitive kit for detecting minute quantities (lowest limit, 0.005 EU/ml) of endotoxin insolutions, was used to quantify the efficiency of lipopolysaccharide removal.  e —, not detected in crude lysates because they contained soluble as well as insoluble lipopolysaccharide on the cell wall.  f  NA, not applicable. Levels in organic extracts were not determined because they contain high salt concentrations, which may interfere with the enzymatic reaction. V OL  . 68, 2002 FLUORESCENT BIOSENSOR FOR GRAM-NEGATIVE BACTERIA 6345   yeast  Pichia pastoris  were treated the same way. In principle, lack of lipopoly-saccharide on the gram-positive bacterium and yeast strains would not result inEGFP i  tagging on these microorganisms. Testing of contaminated environmental water samples.  Water samples col-lected from a fish aquarium, drain water, and a stagnant pool were centrifugedat 10,000    g   for 5 min at room temperature. From each milliliter of samplecollected, the pellet was resuspended in 20   l of saline, except for the fishaquarium water, where 5 ml of sample was pelleted and resuspended into 20  ldue to anticipated low cell density. EGFP i  at a 2   M final concentration wasadded to the mixture for 10 min, and the sample was pelleted as before, washedthree times with 200  l of saline, and finally resuspended in 20  l of 0.9% salinefor observation under the confocal microscope. In parallel, these water samples were plated on Mueller-Hinton II agar (Becton Dickinson, Cockeysville, Md.)plates and incubated at 37°C overnight. Gram staining was carried out on boththe water samples and the cultures. RESULTSExpression of EGFP i  mutant proteins in  E. coli .  EGFP i  G10and G12, bearing one and two strands, respectively, of theendotoxin binding motif(s), containing symmetrical sequencesof alternating basic and hydrophobic (BHBHB) residues onone and two adjacent  -sheets were constructed and expressedin a bacterial host. The expression used the advantageousproperties of EGFP: fluorescent, highly soluble, and of lowtoxicity. Furthermore, it requires no posttranslational modifi-cations, which is highly in contrast to the potentially toxic effectof lipopolysaccharide-binding cationic peptides. Clones har-boring the pET3b-EGFP (native) and pET3b-EGFP i  mutants(G10 or G12) constitutively expressed native EGFP and mu-tant EGFP i  intracellularly at 3 to 8% of total cellular protein(Fig. 1A). Consistent with expression profiles in COS-1 cells(6), the native EGFP and mutant G10 were expressed mainlyin the  E. coli  soluble lysates, while most of G12 was located inthe insoluble inclusion bodies (Fig. 1B). Other constructs weregenerated to target the EGFP i  to the periplasm or to carry aHis tag. However, the yield was low, and most of the mutantproteins were probably misfolded and hence showed low or nofluorescence (data not shown). EGFP i  protein extraction and lipopolysaccharide removal.  After organic extraction of the bacterial lysates, the enrichedsoluble EGFP i  yielded green fluorescence except for G12, which contained much less soluble protein. Since G12 wasexpressed mainly as insoluble inclusion bodies, only minimallevels of soluble protein were purified. The majority of G12 would have to be denatured and renatured from the inclusionbodies.Stepwise treatment with Triton X-114 successfully removedthe bulk of lipopolysaccharide, while the Detoxi-Gel endotox-in-removing column further purified the EGFP i . The recalci-trant problematic trace levels of lipopolysaccharide remaining with EGFP i  were removed with an S3  affinity column, result-ing in virtually pyrogen-free EGFP i  containing   0.05 EU of lipopolysaccharide per ml (Table 1).  Affinity of G10 and G12 for lipid A remains high over a widerange of pHs and salt conditions.  The binding affinity of G10and G12 for lipid A was measured by surface plasmon reso-nance under various conditions of pH and ionic strength. Fig-ure 2A shows a series of sensorgrams obtained by injectingincreasing concentrations of G12. The slight increase in theinitial response was partially attributed to the mass transfer of the bulk proteins in EGFP i  and the other minor molecules which may be present in the extracts. Thus, sensorgrams were obtained under various buffer conditions and salt con-centrations, from which the corresponding  K   D  values werecalculated.It is noteworthy that in the absence of lipid A/lipopolysac- FIG. 2. (A) Surface plasmon resonance sensorgrams of G12 protein after purification through the S3   affinity column, injected at differentconcentrations (0.5 to 6  M) into the flow cell, which was coated with lipid A (  E. coli  f583). The buffer was 50 mM Tris-HCl (pH 7.3) containing25 mM NaCl. The surface plasmon resonance curves were used for calculation of   K   D . (B) The effects of pH on binding affinity (  K   D ) of EGFP i  tolipid A. As the pH increased from 6 to 9, the  K   D  increased from 1.07  10  7 M  1 to 4.68  10  6 M  1 for G12 and from 3.94  10  7 M  1 to 2.70   10  5 M  1 for G10. (C) Effects of salt (0 to 200 mM NaCl) in 50 mM Tris-Cl (pH 7.3) on the binding affinity of EGFP i  to lipid A. EGFP i  istolerant to salt. Between 0 and 200 mM NaCl, the binding affinity improved by 0.5 to 1 order of magnitude, with a  K   D  of 3.15  10  6 M  1 to 7.16  10  7 M  1 for G10 and 7.56  10  7 M  1 to 8.18  10  8 M  1 for G12. In contrast, the S3  control peptide showed a slight drop in binding affinityas the salt concentration increased. The binding affinity of native EGFP (G0), which has no specific binding site for lipopolysaccharide, remainedlow at all salt concentrations tested. The  K   D  values are means    standard deviation of three individual experiments with five differentconcentrations.6346 GOH ET AL. A  PPL  . E NVIRON . M ICROBIOL  .  charide, at pH 6.0, the fluorescence of EGFP i  itself was re-duced by 50%, and all extracts showed turbidity at pH 5.0 andprecipitated out at pH 4.0 (data not shown). The fluorescenceintensity of G10 and G12 decreased between pH 7.0 and 4.5but remained stable between pH 7.0 and 11.0, as was alsoobserved for GFP (14). Increase in pH decreases the bindingaffinity of G10 and G12, as reflected by the lowest  K   D  of 1.07  10  7 M at pH 6.0 to the highest  K   D  of 4.68  10  6 M at pH9.0 for G12, and a  K   D  of 3.94  10  7 M at pH 6.0 to the highest  K   D  of 2.70  10  5 M at pH 9.0 for G10 (Fig. 2B). The apparentincrease in  K   D  of G10 over pH 6 to 9 did not, in practice,adversely affect its binding to lipid A, as there is probablycompensation by hydrophobic interaction between the lipid A acyl chains and the hydrophobic residues in the lipopolysac-charide-binding motif, BHBHB .  This shows that the initialdriving force for the biointeraction between EGFP i  and lipid A is via electrostatic interaction of the phosphate head groups of the lipid A and lysine residues on the endotoxin binding site(s)on EGFP i , which presumably protrudes from the immobilizedsurface. The binding affinity increased by 5- to 10-fold whenthe salt concentration was raised from 0 to 200 mM NaCl (Fig.2C), with optimal  K   D  at the physiological range. This may be FIG. 2— Continued .V OL  . 68, 2002 FLUORESCENT BIOSENSOR FOR GRAM-NEGATIVE BACTERIA 6347
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