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Spectroscopic Observations of Ferric Enterobactin Transport*

Received for publication, October 9, 2002, and in revised form, October 24, 2002Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M210360200

Zhenghua Cao‡, Paul Warfel, Salete M. C. Newton§, and Phillip E. Klebba‡¶

From the Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019

We characterized the uptake of ferric enterobactin(FeEnt), the native Escherichia coli ferric siderophore,through its cognate outer membrane receptor protein,FepA, using a site-directed fluorescence methodology.The experiments first defined locations in FepA thatwere accessible to covalent modification with fluores-cein maleimide (FM) in vivo; among 10 sites that wetested by substituting single Cys residues, FM labeledW101C, S271C, F329C, and S397C, and all these existwithin surface-exposed loops of the outer membraneprotein. FeEnt normally adsorbed to the fluoresceinatedS271C and S397C mutant FepA proteins in vivo, whichwe observed as quenching of fluorescence intensity, butthe ferric siderophore did not bind to the FM-modifiedderivatives of W101C or F329C. These in vivo fluores-cence determinations showed, for the first time, consis-tency with radioisotopic measurements of the affinity ofthe FeEnt-FepA interaction; Kd was 0.2 nM by both meth-ods. Analysis of the FepA mutants with AlexaFluor680, afluorescein derivative with red-shifted absorption andemission spectra that do not overlap the absorbancespectrum of FeEnt, refuted the possibility that the fluo-rescence quenching resulted from resonance energytransfer. These and other data instead indicated thatthe quenching originated from changes in the environ-ment of the fluor as a result of loop conformationalchanges during ligand binding and transport. We usedthe fluorescence system to monitor FeEnt uptake by livebacteria and determined its dependence on ligand con-centration, temperature, pH, and carbon sources and itssusceptibility to inhibition by the metabolic poisons.Unlike cyanocobalamin transport through the outermembrane, FeEnt uptake was sensitive to inhibitors ofelectron transport and phosphorylation, in addition toits sensitivity to proton motive force depletion.

Gram-negative bacteria recognize and transport a variety offerric siderophores (1, 2) through outer membrane (OM)1 re-ceptor proteins that function as ligand-gated porins (LGP) (3).Some of the hallmarks of these transport processes are the

specificity with which LGP select their iron-containing ligands(4–7), the high affinity of the receptor-ligand interactions (8, 9),conformational changes in the receptors during ligand binding(10–13) and transport (14),2 the requirement for the accessoryproteins TonB (16), ExbB, and ExbD (17, 18), and the need forcellular energy to accomplish active transport of the metal-containing ligands through the OM (19–22). This latter energyrequirement is atypical in a bilayer that contains open chan-nels (23–26) and therefore cannot sustain an ion gradient. Atpresent the pathways through which both energy and TonBparticipate in metal transport are unresolved (27–29). Thecrystal structures of FepA (30), FhuA (31, 32), and FecA (13)revealed that their N-terminal 150 amino acids fold into a4-stranded �-sheet domain that lodges within otherwise typicaltransmembrane �-barrels, creating a third mechanistic para-dox: how do ligands pass through such closed pores?

Site-directed biophysical methodologies defined many of thebiochemical properties of FepA (10, 11, 14, 33) by derivatizationof the genetically engineered mutant FepAE280C with fluores-cent or paramagnetic labels. Residue E280C exists on the ex-ternal surface of the third surface loop (L3) (30, 33) of thereceptor, and nitroxide spin labels attached to it reflected struc-tural changes in FepA when FeEnt binds and different motionwhen it passes through the OM protein (14). Similarly, thebinding of either FeEnt or ColB quenched fluorescein labelsattached to purified FepAE280C, which allowed determinationof the thermodynamic and kinetic properties of the bindingreaction (10, 11). One of the conspicuous findings of thoseexperiments was that purification reduced the affinity of FepAfor FeEnt (9, 10) and the affinity of FhuA for its ligand, fer-richrome, (35, 7) at least 100-fold. In this report we appliedfluorescence methodologies to FeEnt transport in vivo; thesensitivity and specificity of the technique provided not only anexplanation for the discrepancies in affinity that were observedin vivo and in vitro but also more detailed information on theligand internalization reaction through the OM, including itstemperature, pH, and energy dependence.

MATERIALS AND METHODS

Bacterial Strains, Plasmids, and Chemicals—Escherichia coli K12strains KDF541 (F�, thi, entA, pro, trp, rpsL, recA, fepA, fhuA, cir) (3)and KDF571 (KDF541, but tonB) (3) were hosts for fepA� pUC19derivatives pITS449 (36) and pT944 (11). Both plasmids encode wildtype FepA, but pT944 contains a series of genetically engineered re-striction sites that facilitate cloning procedures and restriction frag-ment exchange. pMF19 (provided by M. A. Valvano) (37) is a pEXT21derivative that carries wbbL, the structural gene for a rhamnosyltrans-ferase involved in O16 LPS biosynthesis. Fluorescent reagents werepurchased from Molecular Probes.

Ferric Enterobactin—Enterobactin was purified from E. coli strainAN102 (39), and FeEnt was prepared and purified as previously de-scribed (40). Its concentration was determined from absorbance at 495nm (�495 nm

mM � 5.6) (41).Anti-iodoacetamide Fluorescein (IAF) Sera—Rabbits were immu-

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

‡ Present address: Oklahoma Medical Research Foundation, 825 N.E.13th St., Oklahoma City, OK 73104.

§ Present address: Faculte de Medecine, Necker Enfants Malades,INSERM U570 156, rue de Vaugirard, Paris, 75730 France.

‡ To whom correspondence should be addressed. Tel.: 405-325-4969;Fax: 405-325-6111; E-mail: [email protected].

1 The abbreviations used are: OM, outer membrane; FeEnt, ferricenterobactin; FM, fluorescein maleimide; LPS, lipopolysaccharide; IAF,iodoacetamide fluorescein; LB, Luria Bertani; AM, AlexaFluor680 ma-leimide; L3, third surface loop; MOPS, 4-morpholinepropanesulfonicacid; CCCP, carbonyl cyanide p-chlorophenylhydrazone; DNP, 2,4-dini-trophenol; PMF, proton motive force; BSA, bovine serum albumin. 2 Z. Cao, S. M. Newton, and P. E. Klebba, submitted for publication.

balt3/bc-bc/bc-bc/bc0303/bc2846-03a stafford S�5 6/12/02 4:21 4/Color Figure(s) 1, 5–6 ARTNO: M210360200

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. ??, Issue of ???? ??, pp. 1–xxx, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 1

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nized weekly for one month with 5-IAF (Molecular Probes) conjugatedto BSA and subsequently bled through the ear. Sera were titered byenzyme-linked immunosorbent assay (� 10,000) and Western immuno-blot against ovalbumin-IAF conjugates.

Site-directed Mutagenesis—We used QuikChange (Stratagene) forsite-directed mutagenesis (7, 12) on pITS449 or pT944 and designatedthe mutant FepA proteins FepAXnY, where X represents the one-letterabbreviation for the wild type residue at position n and Y represents theone-letter abbreviation for the substituted amino acid.

We tested the functionality of each FepA mutant by evaluating itsexpression (42), susceptibility to colicins B and D (40), 59FeEnt binding(9), and FeEnt transport in qualitative siderophore nutrition assays (5)and quantitative determinations of 59FeEnt uptake (9).

Fluorescence Labeling of Live Cells—For fluorescein labeling of livebacteria, we grew the cells to stationary phase in LB broth (43) andsubcultured at 1% into MOPS medium (44) containing appropriatenutritional supplements at 37 °C with shaking to mid-log phase. Forlabeling in energy-deficient conditions, after growth in LB and subcul-ture in MOPS the bacteria were collected by centrifugation and resus-pended in the same volume of MOPS media but without glucose orcasamino acids, and with half the usual supplementation of requiredamino acids. The culture was then incubated with shaking for 10 h at37 °C.

Bacteria were collected by centrifugation (5000 � g, 20 min), washedwith and resuspended in Tris-buffered saline (TBS), pH 7.4, to a finalconcentration of 5 � 108 cells/ml. Fluorescein maleimide (FM) or Alex-aFluor680 maleimide (AM) in dimethyl formamide (less than 0.1% offinal volume) was added to a final concentration of 5 �M and incubatedat room temperature for 30 min in the dark with shaking. The labeledcells were centrifuged, washed three times with 25 ml of ice-cold TBSplus 0.05% Tween-20, washed once with and resuspended in ice-coldTBS, and stored on ice for immediate use.

Labeling Specificity—We evaluated labeling specificity with anti-FepA and anti-IAF immunoblots of cell lysates and with fluorescenceemission scans of labeled experimental and control bacterial cultures.In the former case, lysates from 108 cells were subjected to 10% SDS-PAGE and Western immunoblot with anti-BSA-IAF sera; the reactionswere quantified on a Storm Scanner (Molecular Dynamics) after devel-opment with 125I-Protein A (42). The immunoblots showed that expres-sion of FepA did not significantly vary under the culture conditions weemployed. The anti-BSA-IAF serum was equally effective against pro-teins and cells modified by FM, demonstrating its recognition of thefluorescein moiety.

The characteristic orange color of fluorescein facilitated qualitativeevaluation of labeling specificity by visual inspection of treated cellpellets. Strains expressing wild type FepA, which was not labeled byeither IAF or FM, were not colored after treatment with the reagents,whereas bacteria expressing mutant FepA proteins containing single,genetically engineered cysteines in accessible locations were identifiedby the orange color of their cell pellets. To quantitatively determinefluorescein labeling specificity, we performed emission scans at 20 °Cwith an excitation wavelength of 490 nm and a 5 s integration time. Wecompared scans for fluorescein-labeled strains expressing wild type andmutant FepA proteins and used scans of untreated bacteria to establishthe background fluorescence.

Fluorescence Measurements—Using an SLM-Aminco 8000 fluorime-ter (Rochester, NY) upgraded to 8100 functionality, we recorded thefluorescence intensities of bacteria (5 � 107 cells) suspended in 2 ml ofeither TBS or MOPS media and equilibrated at temperature. For fluo-rescence determinations of FeEnt binding affinity, we added variousconcentrations of FeEnt to the cell suspension and recorded fluores-cence intensity with excitation and emission wavelengths of 490 nmand 518 nm, respectively, for fluorescein and 679 nm and 702 nm forAlexaFluor680. We accounted for background fluorescence and volumechanges and analyzed the data with the bound (1-F/F0) versus totalfunction of GraFit 4 (Erithacus Software Ltd., Middlesex, UK).

Effects of Energy Inhibitors on FeEnt Transport—To study the effectof inhibitors on FeEnt uptake, 5 � 107 FM-labeled, energy-starved cellswere first incubated for 60 min at 37 °C with shaking in TBS buffer plusglucose (0.4%) and the corresponding inhibitors. The cells were trans-ferred to the sample cuvette, equilibrated at the measurement temper-ature, and the uptake time course was recorded.

RESULTS

Site-directed Covalent Modification with Fluorescein Male-imide in Vivo: Effect of Lipopolysaccharide—OM proteins con-tain few unpaired cysteines, and we tested the possibility that

genetically engineered single Cys residues at sites of interestmay be covalently modified in live bacteria with -SH-specificfluorescent probes. Although we previously modified residueE280C in FepA L3 with nitroxide compounds (14, 33, 34), wecould not label it with fluorescent probes (45), probably becauseof the inaccessibility of sites close to the hydrophilic OM sur-face to hydrophobic molecules like fluorescein (Fig. 1A). There-fore, from the FepA crystal structure we designed and intro-duced 10 more individual, unpaired Cys residues at positionseither on the cell surface or within the periplasm: W101C,S150C, S211C, Y260C, S271C, F329C, S397C, S423C, S575C,and S595C. Among these 10 target Cys residues, FM specifi-cally labeled W101C, S271C, F329C, and S397C, which allreside in cell surface-exposed loops of FepA (Fig. 1A) above thelevel of the LPS core sugars; W101 lies in the second loop of theN-domain, whereas S271, F329, and S397 exist in loops 3, 4,and 5, respectively, of the C-domain. FM also specifically mod-ified, at lower levels, sites S63C and S150C, which reside inNL1 and on the periplasmic rim of the FepA �-barrel,respectively.

Further study showed that two circumstances affected thespecificity and efficiency of the fluorescence labeling procedure:the nature of the LPS O-antigen and the bacterial cultureconditions. In our initial experiments the bacteria adsorbedfluorescence probes, but we saw little difference in the fluores-cence intensity of FM-treated KDF541/pFepAS271C andKDF541/pITS449 (fepA�). The use of two different reagents,IAEDANS and coumarin maleimide, did not remedy this lack ofspecificity (data not shown). KDF541, the E. coli strain thatwas the usual host for plasmids, produces rough LPS withoutany O-antigen, and we considered the possibility that LPS wasa determining factor in the specificity of the fluorescence label-ing reactions. The use of deep rough mutant strains did not

FIG. 1. Accessibility of cysteine substitution mutants to fluo-rescein labeling in vivo. A, we engineered 10 cysteine substitutionsin pITS449 (fepA� on pUC18), expressed them in KDF541/pMF19 inMOPS minimal media, and labeled them with FM. The structuralmodel (30) shows the Cys substitutions relative to the OM bilayer andto the position of LPS (32) associated with the exterior of the FepA�-barrel. The N-domain backbone is red, the �-barrel backbone is green.The sites of mutation and LPS, in space-filling format, are shown inCPK (Corey-Pauling-Kolun) colors. The positions of residues Ser271 andPhe329, which were not defined in the crystal structure, are only esti-mated as delimited by the last solved residues of L3 (orange, Gly323,Gln335) and L4 (red, Ala383, Asp400), respectively. B, after fluorescentlabeling, 108-labeled cells were lysed and subjected to Western immu-noblot with anti-BSA-IAF, developed with 125I-Protein A.

Fluorescence Spectroscopy in Vivo2

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improve the labeling specificity of surface-exposed Cys residues(data not shown). Although Western blots revealed specificcovalent modification of some of the Cys mutants, FM nonspe-cifically adsorbed to deep rough strains, which was apparent inthe yellow color of the cell pellets that was not diminished byrepeated washing. These results suggested that in deep roughstrains, and to a lesser extent in rough strains, the fluorescentreagents penetrated into the OM bilayer and resisted washingprocedures. The converse experiment confirmed this inference;the use of bacterial cells synthesizing full-length LPS mini-mized nonspecific adsorption of fluorescent dyes. We achievedthis result by introducing pMF19, which carries a rhamnosyltransferase that allows production of the LPS O-chain (wbbL�)(37). Strains harboring pMF19 and one of the four accessibleCys substitution mutants of FepA were specifically labeled byFM, as seen by fluorescence intensity measurements of livebacteria and in Western immunoblots (Fig. 1B).

FeEnt Binding to Fluorescein-labeled Live Bacteria—Wemeasured the ability of the four surface-localized, Cys substi-tution mutants of FepA to bind 59FeEnt before and after fluo-resceination. Adsorption of the ferric siderophore was weakand barely detectable in W101C- and F329C-FM; attachment offluorescein at these sites sterically hindered the adsorption ofthe siderophore. These data concurred with the prior conclu-sion that residue Phe329 exists in close proximity to, or is acomponent of, an initial FeEnt binding site (B1) (11); the crys-tal structure shows W101C in the middle of the FepA vestibule(Fig. 1A). On the other hand, FM modification of S271C orS397C did not interfere with FeEnt recognition and binding;bacteria expressing either of these mutant proteins manifestednormal (subnanomolar) binding affinities (Fig. 2) even afterfluoresceination. These experiments showed, for the first time,correspondence between the binding affinities measured invivo by radioisotopic and fluorescence methodologies (Kd � 0.3nM). In subsequent experiments we exclusively studied theS271C site, located at the extremity of L3.

FeEnt Binding to Alexa Fluor680-labeled Bacteria—Ligandbinding diminished the fluorescence of probes attached to FepAat E280C, and our modifications and analyses of S271C andS397C showed the same quenching phenomenon in vivo. Pre-

vious determinations of binding kinetics found a biphasic as-sociation reaction between purified FepAE280C-FM and FeEntand ColB (10, 11). We interpreted these data as initial ligandadsorption to an external site (B1), followed by movement to asecond site deeper in the vestibule (B2). The ability ofE280C-FM to reflect these two binding phases presumablyarose from changes in the local environment of the fluor thatoccurred as the receptor protein underwent conformationaldynamics during ligand adsorption. However, because its ab-sorption spectrum overlaps the emission spectrum of fluores-cein, the possibility existed that during its binding FeEntquenched fluorescence by energy transfer between the excitedfluor and the ferric siderophore. Studies with a red-shiftedfluorescein derivative that does not overlap the absorptionspectrum of FeEnt (AM: 679 nm and 702 nm, respectively),refuted this explanation (Fig. 2); during FeEnt binding, FepAproteins modified with AM exhibited equivalent reductions inintensity to those modified with FM. The comparable quench-ing of AM, without the possibility of energy transfer to FeEnt,indicated that the reductions in intensity did not derive fromclose proximity of the ferric siderophore to the fluor. Rather,the data favored the notion that binding triggers conforma-tional dynamics that alter the environment of the reportermolecules.

Temperature-dependent Changes in Polarization—At physi-ological temperatures we expected a decrease in the polariza-tion of fluorescent labels attached to the surface loops of FepAbecause of increased Brownian motion (46, 47). The polariza-tion of FepAS397C-FM (in L5) decreased as the temperature ofthe system was raised, but that of FepAS271C-FM (in L3)increased (Fig. 3). These data confirmed that the temperatureaffected the motion of the attached fluor; L3 became less mobileand L5 became more mobile in response to increased temper-ature. These states were reversible as the temperature of thesystem decreased again.

Fluorescence Measurements of FeEnt Uptake—When exposedto FeEnt at 20 °C, KDF541/pMF19/pS271C-FM bound andtransported it, and we spectroscopically monitored these reac-tions. Subsequent to its binding, we observed FeEnt transportas a recovery of fluorescence intensity, but only when theextracellular free ligand was depleted by its uptake into thecells. At that time, when the population of receptor proteinswas vacated, its original fluorescence intensity returned. Theconsumption of FeEnt was necessary to observe fluorescencechanges because, when present, it bound and quenched again.Thus in the presence of sufficient excess of the ferric sid-erophore, we saw no renewal of fluorescence.2 When it oc-

FIG. 2. Ligand binding by fluorescein-labeled FepA. KDF541expressing S271C (A) or S397C (�) and labeled with FM was diluted to2.5 � 106/ml in TBS buffer, and fluorescence intensity was measured inthe presence of varying amounts of FeEnt. KDF541 expressing wildtype FepA was subjected to the same procedure and used as a control fornonspecific adsorption of FM. After background and dilution correction,1-F/F0 was fit against the FeEnt concentration, using GraFit 4 (Eritha-cus Software Ltd.) “Bound versus Total” equation. The Kd values forFepAS271C and FepAS397C were 0.263 nM (S.E., 0.032 nM) and 0.292nM (0.021 nM), respectively. The inset shows the ability of FeEnt (addedat 200 s) to quench the fluorescence of cells labeled with AM in acomparable manner to its quenching of cells labeled with FM.

FIG. 3. Effects of temperature on the polarization ofFepAS271C-FM and FepAS397C-FM. Bacteria were grown and fluo-rescently labeled and washed; 5 � 107 bacteria were deposited into a2-ml cuvette for polarization measurements. Polarization was deter-mined at 4 °C, and at 500 s the temperature of the sample cuvettejacket was raised to 37 °C.

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curred, the resurgence of fluorescence paralleled the kinetics ofFeEnt uptake, independently measured in 59Fe uptake exper-iments (Fig. 4). In those studies, the increase in fluorescenceintensity occurred between 1200 and 1400 s. The correspondingradioisotope uptake experiment at 20 °C showed that accumu-lation of 59FeEnt stopped at the same time as a result ofdepletion of the substrate from the media. So the recovery offluorescence intensity precisely mirrored the depletion of theligand from the culture.

Consistent with prior observations of the energy-dependentuptake reaction (9, 48–51), the spectroscopic measurement ofFeEnt transport was affected by media composition, FeEntconcentration, and temperature. It occurred more slowly inbacteria deprived of nutritional requirements, either in mini-mal media lacking auxotrophic amino acid supplements orsuspended in TBS (Fig. 5A). As discussed above, at 20 °C thetransport of FeEnt was visible as a return of fluorescenceintensity, and the concentration-dependence of the phenome-non was striking. For bacteria exposed to 2, 5, 10, and 20 nM

FeEnt, the lag time preceding transport was 250, 550, 1000,and 2050 s, respectively (Fig. 5B). Furthermore, AM-labeledcells showed the same spectroscopic response as FM-labeledbacteria. These data indicated that the intensity changes thatoccurred during uptake also derived from conformational mo-tion in the surface loops, in this case associated with ligandinternalization. At 37 °C, in the presence of equimolar or sub-saturating FeEnt, the reaction occurred most rapidly (Fig. 5B,inset), too rapidly to permit a detailed analysis. However, thefluorescence intensity changes were fully TonB-dependent,whether the experiments were conducted at 20 °C (Fig. 5B) or37 °C. A complete description of the fluorescence changes as-sociated with the 37 °C transport reaction, including its ener-gy- and TonB-dependence, is given elsewhere.2

The uptake reaction was pH-dependent. We attempted tostudy the process at pH 10 but found that FeEnt does notadsorb to FepA at high pH. This result concurred with the factthat basic residues (Arg286, Arg316, Ref. 42; Lys483, Ref. 12) play

FIG. 4. Transport of FeEnt by KDF541/pS271C-FM. The timecourses of spectroscopic intensity change (A) and 59FeEnt uptake (B)were compared in fluorescently labeled bacteria. The cells were sus-pended to 2.5 � 107 cells/ml in MOPS minimal media with glucose andcasamino acids at 20 °C. For the fluorescence measurements, at t � 0FeEnt was added to 10 nM, and intensity changes were measured in thefluorescence spectrometer. In the radioactivity determinations, at t � 059FeEnt was added to 10 nM, and aliquots were removed at the indi-cated times, filtered and the filters were counted (o-o). The data monitorthe accumulation of 59FeEnt by the bacteria.

FIG. 5. Effects of environmental conditions on the fluores-cence time course. KDF541 (tonB�)/pMF19/S271C was cultured incomplete MOPS media (containing 0.4% glucose, 0.2% casamino acids,and supplementary auxotrophic amino acids), labeled with FM, anddiluted to 2.5 � 107 cells/ml in physiological buffers and differentconditions. A, media: The fluorescence response of the cells to 2 nM

FeEnt (added at 300 s) was observed at 20 °C in complete MOPS media(green) or MOPS media without amino acids (teal) or TBS (blue); allmedia contained 0.4% glucose. The response of bacteria to which fluo-rescein was nonspecifically adsorbed is also shown (black). KDF541/pMF19/pITS449 was grown in LB for 14 h and treated with FM asusual; the fluorescence intensity of 5 � 107 cells in 2 ml of MOPS mediawas monitored at 20 °C. FeEnt was added at 425 s. B, concentration,TonB-deficiency, and pH: Bacteria were diluted into complete MOPSmedia at 20 °C and pH 7.4, FeEnt was added (at 300 s) to 2 nM (green),5 nM (teal), 10 nM (blue), or 20 nM (black), and fluorescence intensitychanges were recorded. KDF541/pMF19/S271C-AM was also tested inthe same media with 2 nM FeEnt (magenta). The red curve derives fromKDF571 (tonB)/pMF19/S271C-FM, subjected to the same regimen inthe same media; the gray curve derives from KDF541/pMF19/S271C-FM tested in complete MOPS media that was adjusted to pH 4.C, temperature: Bacteria were diluted into complete MOPS media andequilibrated at 20 °C (green), 15 °C (teal), 10 °C (blue), or 5 °C (black).FeEnt was added to 10 nM, and the changes in fluorescence wererecorded. The experiment was also performed with 2 nM FeEnt (inset) at37 °C (magenta) or 20 °C (green).


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a role in the binding of the acidic ferric siderophore. At pH 4,FeEnt bound and quenched the fluorescence intensity ofKDF541/pMF19/pS271C-FM; however, the magnitude of thequenching was slightly less, and the recovery of fluorescencethat normally occurred as the temperature approached 37 °Cdid not take place at pH 4 (Fig. 5B), suggesting that uptake wasimpaired under these conditions.

We investigated the range of permissive temperatures forFeEnt uptake (Fig. 5C). As temperature decreased, so also didthe slope of the fluorescence recovery. Conversely, the bacteriaexperienced a lag prior to the rebound of fluorescence increase,the magnitude of which was inversely proportional to temper-ature. These data supported the conclusion that the recovery offluorescence derived from the transport reaction. Somewhatunexpectedly, the bacteria transported FeEnt, although veryslowly, even at 5 °C (Fig. 5C).

Energy-dependence and Susceptibility to Poisons—The pro-ton ionophore CCCP, and the electron transport inhibitorscyanide and azide completely and indefinitely abrogated up-take of the ferric siderophore (Fig. 6A); glucose starvation pro-duced comparable effects (Fig. 6B). On the other hand, theproton ionophore DNP and the phosphate analog arsenate in-hibited uptake but not to the same extent. Transport occurredin their presence, but the slope of the fluorescence increase wassignificantly reduced in both cases, such that the bacteria uti-lized the quantity of extrinsic FeEnt (2 nM) in 1 or 2 h, respec-tively. In the absence of inhibitors the cells exhausted theexogenous ligand within 10 min. The effects of arsenate specif-ically related to its inhibition of phosphate metabolism becausethe presence of 30 mM phosphate effectively suppressed arse-nate inhibition (Fig. 6C). Finally, in the absence of glucose,ascorbic acid and succinate effectively restored transport activ-ity, although in the latter they supported transport only at amuch slower rate.

DISCUSSION

Variations in the fluorescence intensity of bacteria labeledwith fluorescein or AlexaFluor680 at FepA residue S271C re-flected the active transport of FeEnt through the OM layer ofthe cell envelope. The spectroscopic phenomena recapitulatedthe characteristics of the energy-dependent active transportreaction: high affinity, occurring at concentrations less than 2nM; a direct relationship between lag time and initial ligandconcentration; temperature-dependence of the rate of intensitychange; consistency with uptake measurements by radioiso-topic determinations; susceptibility to energy poisons. Thesedata unambiguously demonstrate that the recovery of fluores-cence after FeEnt binding originates from its uptake reactionthrough the OM. The spectroscopic system offers an alternativeto existing methods for observation of OM transport that issensitive, precise, continuous, and amenable to quantitativeanalysis.

The evidence that the fluorescence measurements describeconformational motion during the uptake reaction stems fromseveral independent observations. (i) In prior work utilizingFepAE280C-FM, we concluded that conformational change oc-curs in L3 in vitro because the modified side chain of E280Cresides on the outside of the surface vestibule where it cannotcontact FeEnt and similar quenching was engendered by bind-ing of colicin B, which contains no metal complex. These datamade collisional quenching unlikely for either ligand and ex-cluded energy transfer as a mechanism to explain the effects ofColB, which has no chromophores. (ii) In the current experi-ments, the FepA crystal structure suggests that fluoresceinattached to S271C at the extremity of L3 likely projects towardthe interior of the vestibule. The observation that FeEnt bind-ing quenches S271C-AM in the same way that it quenches

S271C-FM eliminates energy transfer as an explanation of theinhibition. (iii) These data do not rule out collisional quenchingof fluors attached to S271C, but this possibility seems unlikely.Modification of S271C with fluorescein had no effects on theaffinity, capacity, or transport of 59FeEnt, indicating that thisresidue does not likely contact FeEnt during binding or uptake.Fluoresceination of Phe329, on the other hand, abrogated FeEntadsorption and transport (45), reinforcing this inference. (iv) Inaddition, given the extensive spectral overlap of FeEnt and

FIG. 6. Effects of metabolic poisons. In all panels, KDF541/pMF19/S271C was grown in complete MOPS media, labeled with FM,washed, and resuspended at 2.5 � 107/ml. A, energy poisons: Thelabeled cells were suspended in MOPS media containing 0.4% glucoseand shaken for 40 min at 37 °C in the absence of any poison (green) orthe presence of CCCP (0.1 mM, red), cyanide (20 mM, magenta), azide(10 mM, black), DNP (2 mM, blue), or arsenate (10 mM, teal). The cellswere equilibrated at 20 °C, FeEnt was added to 2 nM (at 800 s), andfluorescence changes were monitored. B, carbon sources: The cells wereresuspended in MOPS media without glucose, shaken for 10 h at 37 °C,and equilibrated at 20 °C in the presence of 0.4% glucose (green), suc-cinate (teal), ascorbate (blue), or the absence of an extraneous carbonsource (black). FeEnt was added to 2 nM (at 1000 s), and fluorescencechanges were monitored. C, arsenate and phosphate: Labeled cells wereresuspended in MOPS media plus glucose and shaken for 1 h at 37 °Cin the absence of test compounds (green) or the presence of 30 mM

sodium phosphate (magenta), 30 mM sodium phosphate and 10 mM

sodium arsenate (blue), or 10 mM sodium arsenate (teal). The cells, inthe same buffers, were re-equilibrated at 20 °C, FeEnt was added to 2nM (at 800 s), and fluorescence intensity changes were recorded.


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fluorescein, it seems unlikely that significant collisionalquenching can occur without significant energy transfer, whichwas not found. (v) From a logistics perspective, FeEnt binds tothe outside of a closed channel, so some sort of conformationalchange is necessary to promote its internalization. (vi) Finally,the loops of FepA (L7) (12) and FecA (L7 and L8) (13) areconformationally active during FeEnt binding, and loop motionwas previously seen in vivo (14). In summary, a body of evi-dence argues against either collisional or energy transfer mech-anisms of quenching, leaving conformational dynamics thatrelocalize the fluor to a quenching environment as a singleviable explanation for the quenching phenomena. This conclu-sion concurs with data acquired by a variety of methodologies(7, 10, 12–14, 32–34).

Because L3, L5, and L7 in FepA change in conformationduring the interaction with ligand, it appears likely that suchmotion is a general property of ligand-gated porin surfaceloops. We envision a model of FepA conformation in which thereceptor exists in an extended, open form in the absence ofligand but closes around ligands when they bind. At either 4 °Cor 20 °C, FeEnt adsorption creates the closed state, in whichthe fluor undergoes maximal quenching from increased colli-sions with water or other polar or charged molecules in theenvironment created by the aggregation of the loops around theligand. This mechanism agrees with the observation of biphasicbinding kinetics for ligand adsorption to FepA and with con-tractions from open to closed states of FecA loops 7 and 8 andFepA L7. As the temperature warms and FepA transportsFeEnt, fluorescence rebounds because the now empty receptorproteins revert from the closed state to the open state. At 37 °C,further motion occurs that results in additional quenching (18).

One perplexing finding about FepA and FhuA was that pu-rification from the OM quite drastically decreased their abilityto bind ligands. This decrease questioned the relevance of thecrystallographic data to the structures of the siderophore re-ceptors in vivo. However, two different techniques, 59FeEntbinding in vivo (9) and fluorescence spectroscopy in vitro (10),defined the discrepancy, raising the possibility of methodolog-ical bias. The results we report eliminate this contingency bydetermining with a fluorescence methodology that the affinityof FepA for FeEnt in live bacteria is in fact subnanomolar: Kd

� 0.1–0.2 nM, as now established by two independent methods.Thus these data confirm that extraction from the OM reducesthe affinity of FepA for FeEnt about 100-fold (Kd � 10 nM; Ref.10). Furthermore, the affinity of native FepA for FeEnt in vivois 20,000-fold greater than that of the isolated FepA N-domainin vitro (Kd � 5 �M; Ref. 52). The result of these considerationsis that at the Kd measured in vivo, purified FepA is less than1% saturated with FeEnt, but why? The crystal structures ofboth ligand-free and ligand-bound FepA and FhuA revealed aclosed form of the proteins with loops condensed together abovethe membrane channel, whereas later crystallographic andcross-linking studies in vivo showed that the loops may extendinto an open conformation. Thus, the likely explanation for thediscrepancy in binding affinities between the native and puri-fied receptors is that in the living cell, in the absence of ligands,ligand-gated porins adopt an open conformation but when com-plexed with a ferric siderophore, or removed from the OM, theirloops close.

Besides its prior application to the binding reactions betweenFepA and its ligands, site-directed fluorescence spectroscopydefined conformational changes in FhuA L5 during ferrichromebinding (53). Furthermore, the intrinsic fluorescence of pyover-din facilitated characterization of its adsorption to the pseudo-monad OM protein, FpvA (54–56). Our experiments are thefirst to use site-directed fluorescence to directly observe protein

motion associated with uptake of a ligand through a mem-brane. It is important to note that the methodology does notnecessarily reflect exclusively OM transport. Although the in-tensity changes in fluors attached to FepA derive from fluctu-ations of its loops between open and closed states, we onlyobserved the recovery of fluorescence when the exogenous li-gand was completely exhausted. In our experiments the bacte-ria had an 59FeEnt binding capacity of 80 pmol/109 cells (datanot shown); when we provided 10 nM FeEnt in the medium, the2.5 � 107 cells/ml underwent five rounds of transport to fullydeplete the external ferric siderophore. This quantity of FeEntexceeds the concentration of FepB in the periplasm (15), so itscomplete utilization by the bacteria requires transport throughthe inner membrane to the cytoplasm.

On the other hand, 2 nM FeEnt, the amount that we em-ployed to study the susceptibility of the fluorescence phenom-ena to energy inhibitors, is a concentration that precisely sat-urates the bacteria. Therefore, its depletion from the mediainvolves only a single round of transport by the FepA proteinsof the OM, without requirement for uptake through the innermembrane. The fact that we saw an immediate recovery offluorescence, without a lag period, when we provided 2 nM

FeEnt supports this inference. At this stoichiometric concen-tration, the assay exclusively measured the activity of the OMcomponent. The ability to monitor OM transport without theneed for null mutants in the subsequent inner membrane per-mease components (i.e., fepC, fepD, or fepG) is a significantadvantage of the spectroscopic approach. Our characterizationof the energetics of the OM transport stage diverged from thoseobtained for cyanocobalamin uptake (21, 22) in that inhibitorsof electron transport (cyanide and azide), phosphorylation (ar-senate), and proton motive force (CCCP and DNP) all impairedFeEnt uptake through FepA, in some cases abrogating trans-port. In the vitamin B12 transport system, DNP-mediated de-pletion of PMF inhibited OM transport, but the process wasinsensitive to cyanide. Bradbeer (22, 38) conducted those ex-periments by radioisotopic methods in an atp genetic back-ground, whereas our strains were atp�. Most other consider-ations were similar between his experiments and ours, exceptfor the preincubation times of the bacteria with the poisons,which were 10 min (22) and 40 min (this report). Our dataalmost exactly reprise, nevertheless, a prior characterization ofthe energy dependence of FeEnt uptake by E. coli (50) that wasperformed by radioisotopic methods. The explanation for thesedifferences and similarities awaits further experimentation.

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JOBNAME: AUTHOR QUERIES PAGE: 1 SESS: 1 OUTPUT: Mon Nov 18 09:42:53 2002/balt3/bc�bc/bc�bc/bc0303/bc2846�03a

A—Au/In general, Journal requires abbreviations to be used three times in the abstract and fivetimes in the text, or else be spelled out.

B—Au/Standard query: The Journal guidelines state that the summary should be no longer than200 words. At this stage, we only require that the summary fit into the left-hand column. Ifyour summary is long enough to run over into the right-hand column, please cut text.

C—Au/Journal uses medium for one type of media. Please confirm or correct here and elsewherein text.

D—Au/In the sentence beginning ‘For labeling in energy-deficient‘ is without glucose or casaminoacids correct? Or should it be and?

E—Au/Please provide company name for fluorimeter.

F—Au/‘equilibrated at temperature.‘ Should it be ‘for room temperature?‘

G—Au/Please confirm Erithacus Software is what is meant.

H—Au/Please confirm change of ‘engineering‘ to ‘engineered‘ in first sentence of paragraph.

I—Au/The � in fepA� was made superscript to agree with remainder of text. Please confirm orcorrect.

J—Au/Journal uses three-letter codes when position numbers are used.

K—Au/Editor added ‘of‘ to ‘utilized the quantity of extrinsic. . . ‘ Please confirm or correct.

L—Au/Sentence beginning ‘Finally, in the absence‘ has been slightly rearranged.

M—Au/Please confirm or correct definition for IM.

N—Au/For future submissions to the Journal, please use expanded page numbers in referencesection.

O—Au/For Ref. 43, please supply editors, if applicable, and page range if whole book was notused.

P—Au/For Ref. 45, please supply title of Ph.D. thesis.

Q—Au/Please confirm abbreviation for journal in Ref. 46. It is not found in our standard sources.

AUTHOR QUERIES

AUTHOR PLEASE ANSWER ALL QUERIES 1

JOBNAME: AUTHOR QUERIES PAGE: 2 SESS: 1 OUTPUT: Mon Nov 18 09:42:53 2002/balt3/bc�bc/bc�bc/bc0303/bc2846�03a

R—Au/Please confirm journal in Ref. 47. Is that the complete name?

S—Au Ref. 58 is a duplicate of Ref. 50. Therefore, Ref.58, which was the last reference, has beendeleted and the appropriate changes have been made in the text. Please check text carefullyand DO NOT renumber references.

T—Au/Please check the colors on the computer screen against what appears in the legend andamend the legend as necessary. Are the color figures acceptable for publication?

U—Au/Is it okay to include telephone and fax numbers in the correspondence footnote?

V—Au Please confirm definitions in abbreviations footnote.

W—The Journal does not include unpublished information in the reference list. Therefore Ref. 15has become footnote 2. Ref. 57, the last reference, has been moved up to take the place ofRef.15, and the appropriate changes have been made in the text. Please check text carefully.DO NOT renumber references. You may update the information in its footnote location atpage proofs.

AUTHOR QUERIES

AUTHOR PLEASE ANSWER ALL QUERIES 2

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