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REAL-TIME FRET VISUALIZATION OF FERRIC-PYOVERDINEUPTAKE IN PSEUDOMONAS AERUGINOSA: A ROLE FOR FERROUS

IRONJason GREENWALDa# , Françoise HOEGYa#, Mirella NADERa, Laure JOURNETa,

Gaëtan L. A. MISLINa, Peter L. GRAUMANb and Isabelle J. SCHALKa*

aMétaux et Microorganismes : Chimie, Biologie et Applications. UMR 7175-LC1 InstitutGilbert-Laustriat, CNRS-Université Louis Pasteur, ESBS, Blvd Sébastien Brant, F-67413

Illkirch, Strasbourg, France.b Institüt für Mikrobiologie, Stefan Meier Strasse 19, Albert-Ludwigs Universität Freiburg,

79104 Freiburg, Germany.Running title: The fate of ferric-pyoverdine

*Address correspondence to: Isabelle J. Schalk, Métaux et Microorganismes : Chimie, Biologie etApplications. UMR 7175-LC1 Institut Gilbert-Laustriat, ESBS, Blvd Sébastien Brant, BP 10412, F-67413Illkirch, Strasbourg, France. Tel: 33 3 90 24 47 19; Fax: 33 3 90 24 48 29; E-mail: schalk@esbs.u-strasbg.fr.# These authors contributed equally to this work.

To acquire iron, P s e u d o m o n a saeruginosa secretes a major fluorescentsiderophore, pyoverdine (PvdI) that chelatesiron and shuttles it into the cells via the specificouter membrane transporter, FpvAI. We tookadvantage of the fluorescence properties ofPvdI and its metal chelates, as well as theefficient FRET between donor tryptophans inFpvAI and PvdI, to follow the siderophore’sfate during iron uptake. Our findings withPvdI-Ga and PvdI-Cr uptake indicate that ironreduction is required for the dissociation ofPvdI-Fe, that a ligand exchange for iron occurs,and that this dissociation occurs in theperiplasm. We also observed a delay betweenPvdI-Fe dissociation and the rebinding of PvdIto FpvAI, underlining the kinetic independenceof metal release and siderophore recycling.Meanwhile, PvdI is not modified but recycled tothe media, still competent for iron chelation andtransport. Finally, in vivo fluorescencemicroscopy revealed patches of PvdI,suggesting that uptake occurs viamacromolecular assemblies on the cell surface.

Iron is an essential element for the growthof the vast majority of microorganisms. Underaerobic conditions, the abundance of free iron islimited by the very low solubility of ferrichydroxide. Thus, to maintain the required

intracellular levels of iron, bacteria and fungi havedeveloped efficient ferric ion-chelating agents,called siderophores (1), to scavenge iron from theextracellular environment and import it. A majorsiderophore produced by fluorescent Pseudomonasstrains is pyoverdine (PvdI), which has a stabilityconstant for iron of about 1032 M-1 (2). More than100 different PvdIs have been identified (Dr J-MMeyer, personal communication), forming a largeclass of mixed catecholate-hydroxamatesiderophores characterized by a conserveddihydroxyquinoline-derived chromophore to whicha peptide chain of variable length and compositionis attached (3,4).

In general, the uptake of ferric-siderophores into Gram-negative bacteria involvesa specific outer membrane transporter (OMT) andan inner membrane ABC transporter (5-7). Theenergy required for transport across the innermembrane is provided by ATP hydrolysis. Theproton motive force of the inner membrane drivesOMT-mediated transport across the outermembrane by means of an inner membranecomplex comprising TonB, ExbB and ExbD (8,9).The PvdI OMT (FpvAI) of P. aeruginosa wascloned by Poole et al. in 1993 (10) and its structurewas recently solved (11). FpvAI (11), like FptA(12), FhuA (13,14), FepA (15) and FecA (16,17),— the outer membrane transporters of pyochelin inP. aeruginosa and of ferrichrome, ferric-

http://www.jbc.org/cgi/doi/10.1074/jbc.M609238200The latest version is at JBC Papers in Press. Published on December 5, 2006 as Manuscript M609238200

Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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enterobactin and ferric-citrate in E. coli,respectively — consists of a C-terminal β-barreldomain and an N-terminal plug domain filling thebarrel. The binding site for the ferric siderophore islocated above the plug, well outside themembrane, and is composed of residues of theplug and β-barrel domains. The binding site ofFpvAI consists mostly of aromatic residues,including six Tyr residues and two Trp residues,and only three hydrophilic residues (11).

The P. aeruginosa FpvAI is the best-characterized pyoverdine OMT, while, only threeother transporters of pyoverdines have beencloned: FpvAII and FpvAIII, involved in theuptake of PvdII and PvdIII (18); and FpvB, asecond OMT for PvdI in P. aeruginosa (19). Ofthese known Pvd transporters, only the structure ofFpvAI has been solved, and its interactions with itssiderophore PvdI have been studied using thefluorescent properties of this siderophore. Iron-freePvdI has spectral properties suitable for FRET(Fluorescent Resonance Energy Transfer) with theTrp residues of FpvAI (20). Previous studies usingthis technique have shown that in iron-starved P.aeruginosa cells, FpvAI is initially loaded withPvdI and that, in the first round of iron uptake, thisbound PvdI dissociates from FpvAI and PvdI-Fefrom the extracellular medium takes its place onthe transporter (21,22). The exchange of PvdI-Fefor metal-free PvdI on FpvAI and the translocationof ferric-siderophore are induced by TonB-mediated activation of the transporter (9,23,24). InEscherichia coli, the ferric-siderophore complexesare then transported into the cytoplasm by an ABCtransporter (7). Little is known about this step ofiron acquisition in Pseudomonads. No ABCtransporter has been identified for the PvdI/FpvAIpathway, and the cellular localization of the PvdI-Fe dissociation is unknown. However, we havepreviously utilized the efficient FRET betweenFpvAI and PvdI to show that after iron is releasedfrom the siderophore, the iron-free siderophore isrecycled via an unknown mechanism to the cellsurface, where it again binds to FpvAI (22).

Three mechanisms have been proposedthat could explain how bacteria overcome thethermodynamic barrier of iron removal fromsiderophore ligands: ligand exchange, reduction ofthe iron, and hydrolysis of the siderophore. Areduction mechanism can be differentiated fromligand exchange by investigating the dissociation

while substituting iron with a metal that has asimilar ionic radius, the same charge, and the samecoordination geometry (25,26). Cr(III) formscomplexes with siderophores that are structurallysimilar to ferric siderophores but are kineticallyinert to ligand substitution while Ga(III) formscomplexes with siderophores that are kineticallylabile and exchange with other ligands occursreadily (25,26). However, Ga has no stable (II)oxidation state so the metal cannot be removedfrom the ligand by reduction.

To investigate the fate of PvdI-Fe in P.aeruginosa after transport across the outermembrane by FpvAI, we combined the FRETtechnique, fluorescence microscopy and the use ofPvdI-Ga and PvdI-Cr complexes. We found thatneither PvdI-Cr nor Pvd-Ga could be dissociatedby iron starved P. aeruginosa, despite the fact thatPvd-Ga is efficiently transported across that outermembrane where it accumulates in the periplasm.No recycling of PvdI to the extracellular mediumwas detected for either Pvd-Cr or Pvd-Ga. Thissuggests that reduction of Fe(III) to Fe(II) and aligand exchange are necessary for the completerelease of the metal from PvdI. We have alsoshown that iron release does not involve apermanent chemical modification of PvdI: thesiderophore is recycled to the extracellularmedium unmodified and fully functional. Finally,fluorescence microscopy showed that there is noaccumulation of PvdI-Ga in the cytoplasm, furtherevidence that the PvdI-metal complex dissociatesin the periplasm immediately after its uptake.

EXPERIMANTAL PROCEDURES

Chemicals. Carbenicillin disodium salt was agenerous gift from SmithKline Beecham (WelwynGarden City, Herts, U.K.). FCCP (carbonylcyanide p -(trifluoromethoxy)phenylhydrazone)and sodium N-lauroyl-sarcosine were purchasedfrom Sigma and oPOE (n-octylpolyoxyethylene)from Bachem. PvdIs were prepared as describedpreviously, (2,27) for PvdI and PvdI-Fe, (28) forPvdI-Ga and (22) for PvdI-Cr.

Baterial Strains and Growth Media. Three mutantswere used: CDC5(pPVR2), which overproducesFpvAI and is PvdI-deficient (29), K691(pPVR2),which overproduces FpvAI and produces PvdI

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(30) and the PvdI- and Pch-deficient strain PAD07(31). The strains were grown overnight in asuccinate medium (27) in the presence of 150µg/mL carbenicillin for CDC5(pPVR2) andK691(pPVR2), and 100 µg/mL streptomycin and50 µg/mL tetracycline for PAD07.

Image acquisition. CDC5(pPVR2) cells growingin succinate medium were incubated with orwithout siderophore, washed once withsiderophore-free buffer, and then mounted ontoS750-agarose coated slides, as describedpreviously in (32). Images were acquired on anOlympus AX70 microscope with a MicroMaxCCD camera using 100 ms exposures. Imageswere captured using MetaMorph 6.0 (UniversalImaging).

Fluorescence Spectroscopy. Fluorescenceexperiments were performed on K691(pPVR2) orCDC5(pPVR2) cells with a PTI (PhotonTechnology International TimeMaster, Bioritech)spectrofluorometer. For all experiments, thesample was stirred at 29°C in a 1 mL cuvette, theexcitation wavelength (λexc) was set at 290 nm (forthe FRET experiments) or 400 nm (for directexcitation), and the emission of fluorescence (λem)was measured at 447 nm. The cells were washedwith 2 volumes of 50 mM Tris-HCl (pH 8.0) andresuspended in the same buffer to a final OD600 of2. PvdI-Fe, PvdI-Ga, PvdI-Cr or PvdI was addedand the fluorescence at 447 nm was measuredevery 300 ms for the duration of the experiment.As a control, the same experiments were repeatedin the absence of the siderophore and an analysisby SDS-PAGE gels showed the same amount ofFpvAI in K691(pPVR2) cells as in CDC5(pPVR2)cells (22).

Preparation of periplasmic, cytoplasmic, innermembrane, and outer membrane fractions.CDC5(pPVR2) cells were grown in 50 mL ofsuccinate medium to an OD600 of 1, at which time600 nM PvdI-Ga or PvdI-Fe was added. After 30min the transport was complete based on aliquotsthat were removed for fluorescence measurements,The cells were pelleted at 6000 g, the pellet gentlyrinsed with water and then resuspended in 3 mL20% sucrose, 1 mM EDTA, 0.2M Tris pH 8.0.After 2 min at ambient temperature, with theintegrity of the bacterial outer membrane

compromised, osmotic shock was achieved by therapid addition of 4.5 mL ice-cold water followedby gentle mixing by inversion. After 2 min on ice,the periplasmic fraction was separated from thecells by centrifugation at 6000 g for 10 min. Thepellet, which was firm and showing no signs oflysis, was gently rinsed with water and thenresuspended in 20 mL of 20 mM Tris pH 8.0. Thecells were lysed by sonication and the cytoplasmicfraction separated from the insoluble material at100,000 g for 30 min. The inner membrane wasextracted from the pellet with 20 mL of 1%sodium N-lauroyl sarcosine in 20 mM Tris pH 8.0followed by a second 100,000 g spin. The outermembrane was extracted from the resulting pelletwith 2% oPOE in 20 mM Tris pH 8.0, followed bya final high speed centrifugation. The fluorescenceintensity (λexc = 400 nm, λem = 450 nm) wasmeasured for each fraction and the appropriatedilution factors applied to the measurements sothat all reported intensities represent the totalfluorescence from the same volume of the initialcultures.

Characterization of the recycled PvdI.CDC5(pPVR2) cells at an OD600 of 2 wereincubated in the presence of 300 nM PvdI-Fe at 30°C in 10 mL of 50 mM TrisHCl pH 8.0 buffer.Iron uptake was followed in parallel in 1 mL of themixture by excitation at 290 nm and monitoringthe emission of fluorescence at 447 nM asdescribed above. After 30 min incubation, the cellswere pelleted and the supernatant containing therecycled PvdI used for the following experiments.The pH of 1 mL of the supernatant (containing therecycled PvdI) was adjusted to 4 with AcOH. Tenµ L of 3 µM FeCl3 was added, the sampleincubated for 5 min, and then the fluorescenceemission monitored (λexc = 400 nm). Additions of10 µL of 3 µM FeCl3 were repeated until there wasno more fluorescence at 447 nm.For the transport assay, a 1 mL aliquot of thissupernatant was incubated with 30 nM 55FeCl3 for10 min. The mixture was then added to PAD07cells prepared at an OD600 of 1 in 1 mL of 50 mMTrisHCl pH 8.0 buffer. After 1, 15 and 30 minincubation at 37°C, 100 µL aliquots were removedand filtered. The filters were washed and counted.This experiment was repeated with the incubationsat 0°C.

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RESULTS

Dissociation of iron from PvdI and PvdIrecycling on FpvAI are kinetically independentsteps. The typical fluorescence signal for ironuptake via PvdI in P. aeruginosa, when excited at290 nm and monitored at 447 nm, displays twosteps: a decrease of fluorescence corresponding tothe formation of FpvAI-PvdI-Fe upon the additionof PvdI-Fe; and a subsequent increasecorresponding to the recycling of PvdI on FpvAI(formation of FpvAI-PvdIrecy) (Figure 1). Duringthe second step, the fluorescence always returnedto exactly the same level as that before the additionof PvdI-Fe, indicating that all the FpvAI receptorswere loaded again with iron-free recycled PvdI.This could be the result of efficient recycling ofPvdI to the media and rebinding to FpvAI or amechanism in which the release of the metal fromthe siderophore occurs on FpvAI. The secondpossibility implies that the PvdI would neverdetach from the FpvAI receptor to enter the cells.If this were case, the increase of fluorescence inFigure 1 (recycling on FpvA) would correspond tothe dissociation of iron from the siderophore whilestill bound to FpvAI.

We tested the possibility of a PvdI-Fedissociation on FpvAI. PvdI-deficientCDC5(pPVR2) cells were incubated with PvdI-Feand the fluorescence recorded at 447 nm withexcitation wavelengths of 290 nm (FRET) and 400nm (direct excitation) (Figure 2A). When PvdI-deficient CDC5(pPVR2) cells are excited at 290nm or 400 nm, no FRET occurs, and nofluorescence is observed at 447 nm because noPvdI is produced (22). Moreover, after addition ofPvdI-Fe, the binding of PvdI-Fe to empty FpvAIdoes not produce any FRET signal. Therefore,with CDC5(pPVR2), only the dissociation of PvdI-Fe (formation of fluorescent PvdI) will produce afluorescence signal while the recycling of PvdI onFpvAI will be the only source of FRET. In thismanner, the dissociation of iron from PvdI wasfollowed with the λexc= 400 nm while the recyclingof PvdI on FpvA was simultaneously followedwith λexc = 290 nm. If PvdI releases the iron whilebound to FpvAI, then the fluorescence signalcorresponding to PvdI-Fe dissociation shouldsuperimpose with that of the PvdI recycling on

FpvAI. There was a lag of about 3 min betweenthe signals, suggesting that PvdI-Fe dissociatesinside the cells, before recycling of PvdI on FpvAI(formation of FpvAI-PvdIrecy). CDC5(pPVR2)cells were also incubated in the presence of metal-free PvdI, and in this case the formation of FpvAI-PvdI started immediately after addition of thesiderophore, with no lag (Figure 2A) (23).

CDC5(pPVR2) cells were incubated in thepresence of various concentrations of PvdI-Fe andthe formation of free PvdI (Figure 2B: λexc = 400nm) and of FpvAI-PvdIrecy complex (Figure 2C:λexc = 290 nm) was followed. The kinetics of PvdI-Fe dissociation and of FpvAI-PvdIrecy formationwere independent of the initital PvdI-Feconcentration. Yet, as expected, there was a directrelationship beween the PvdI-Fe concentration andthe yield of PvdI and of FpvAI-PvdIrecy. Also ofinterest was that the delay before formation ofFpvAI-PvdIrecy was proportional to the initialconcentration of PvdI-Fe: the higher theconcentration of PvdI-Fe, the longer the delaybefore the onset of formation of FpvAI-PvdIrecy

(Figure 2C insert).

PvdI-Cr is an antagonist of the FpvAI/PvdIsystem. Cr forms complexes with siderophores thatare structurally similar to siderophore-Fe but areinert to ligand exchange (33). Therefore, if thePvdI-Fe dissociation mechanism involves ligandexchange with another molecule or protein, PvdI-Cr will not be able to act as metal donor in a ligandexchange mechanism, and the transport will beinhibited.

We have previously shown that the PvdI-Cr complex binds to FpvAI with a 10-fold loweraffinity than that of PvdI-Fe and since PvdI-Cr is anon-fluorescent complex, no FRET occurs uponbinding of PvdI-Cr to FpvAI (22). WhenK691(pPVR2) (FpvAI+, PvdI+) cells wereincubated with various concentrations of PvdI-Cr,a TonB-dependent decrease of fluorescence wasobserved corresponding to displacement of PvdIduring the formation of FpvAI-PvdI-Cr, howeverthere was no subsequent recycling of PvdI onFpvAI (Figure 3A and 3B). Also, no PvdI-Crdissociation (formation of metal-free PvdI) wasdetected with λexc = 400 nm (Figure 3C). Thus,PvdI-Cr acts as an antagonist of the PvdI ironuptake pathway in P. aeruginosa, implicating

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ligand exchange for the iron ion in the PvdI-Fedissociation mechanism.

PvdI-Ga is efficiently transported andaccumulates in the periplasm. A ligand exchangemechanism for PvdI-Fe dissociation does notexclude a requirement for ferric iron reduction toits lower affinity ferrous species. PvdI-Ga wasused to investigate this possibility since Ga(III) hasno stable (II) oxidation state, and therefore cannotbe removed from the siderophore by reduction.Siderophore-Ga complexes are kinetically labileand exchange with other ligands occurs readily(25,26) and Ga(III) is the closest ion to the ferricion in size and ligand coordination. Therefore,PvdI-Ga should act as an antagonist of the ironuptake via PvdI only if reduction is involved in thePvdI-metal dissociation process.

We have shown previously that PvdI-Gahas a Ki of 16 nM for FpvAI and is transportedinto P. aeruginosa (28). Since PvdI-Ga is alsofluorescent but with about a two-fold higherquantum yield than PvdI, the removal of Ga fromPvdI-Ga would result in a reduction influorescence intensity (28). Likewise, there is alarge FRET signal when Pvd-Ga binds to FpvAI.We incubated PvdI-deficient CDC5(pPVR2) cellsin the presence of 75 nM PvdI-Ga or PvdI-Fe anddissociation of the metal from the siderophore wasmonitored by the emission of fluorescence at 447nm (λexc = 400 nm) (Figure 4). The addition of thefluorescent PvdI-Ga complex causes an immediatejump in the fluorescence, followed by a decreaseof fluorescence that could correspond to PvdI-Gadissociation (28) in P. aeruginosa cells. Whencompared to the kinetic of PvdI-Fe dissociation(Figure 4), the plateau for PvdI-Ga fluorescencewas reached after 4 min (10 min for PvdI-Fe) andwas of a higher fluorescence. This means thateither differing amounts of metal-free PvdI wereliberated in both experiments or that somethingelse, such as an incomplete dissociation of PvdI-Ga, modulated the Pvd-Ga fluorescence.

To further investigate whether PvdI-Gacan dissociate in P. aeruginosa cells, wemonitored the recycling of PvdI into theextracellular medium during PvdI-Ga uptake.PvdI-deficient CDC5(pPVR2) cells wereincubated in the presence of 600 nM Pvd-Fe, PvdI-Ga or PvdI-Cr (Figure 5A). Aliquots wereremoved at various times, the cells pelleted, and

the quantity of PvdI or PvdI-Ga measured in theextracellular medium (λexc =400 nm and λem = 447nm). Efficient recycling of the siderophore to theextracellular medium was observed for PvdI-Fe, asdescribed previously (22), and as expected norecycling of PvdI-Cr was detected. However,PvdI-Ga was nearly depleted from the media atabout the same rate as the recycling of PvdI fromPvdI-Fe (Figure 5A). If PvdI-Fe and PvdI-Ga weretransported by P. aeruginosa with the samekinetics and mechanism, then the same amount offluorescent PvdI would be recycled from bothcomplexes and a plateau of equivalentfluorescence reached. However, PvdI from PvdI-Ga was not recycled as it is from PvdI-Fe so itmust therefore remain associated with the bacteria(Figure 5A).

After the uptake of the PvdI-Fe and PvdI-Ga complexes, we measured the amount offluorescent PvdI or PvdI-Ga in the periplasm,cytoplasm, and inner and outer membranes todetermine where the PvdI-Ga was localized(Figure 5B). The majority of PvdI-Ga remained inthe periplasm, apparently trapped and unable to berecycled to the extracellular media. After PvdI-Feuptake, there was less PvdI in the periplasm thanin the case of PvdI-Ga due to the efficientrecycling of PvdI. The remaining fluorescence inthe media and outer membrane (Figure 5B) mayreflect an equilibrium at these unnaturally highlevels of Pvd-metal complexes or similarly anoverloading of the PvdI transport pathway aswould be consistent with the previously observedsaturation of FpvAI at 450nM PvdI-Fe (22). Inorder to confirm that the observed partioning ofPvd-Ga into the periplasmic fraction was not dueto an inefficient fractionation procedure, samplesfrom all four cell fractions were analyzed ontricine-SDS-PAGE and visualized by both silverand coomassie staining (Figure 5C). The tricinebuffer system allowed the clear visualization ofpeptides down to 10 kD, revealing a uniquestaining pattern for each fraction with no visiblecontamination of small peptides from thecytoplasm in the periplasm. Furthermore, the largedifference in Pvd-Ga concentration observed ineach fraction is not likely to originate from a minorcontamination during the cell fractionation.

P. aeruginosa imported PvdI-Ga with asimilar kinetic to PvdI-Fe (Figure 5A) however,the PvdI-Ga accumulated in the periplasm (Figure

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5B) instead being recycled to the media. Ourfindings are consistent with an incompletedissociation of PvdI-Ga (kinetic in Figure 4), suchthat the siderophore is unable to be recycled in theextracellular medium. A reduction of Fe(III),thereby lowering PvdI affinity for iron, appears tobe required for a complete and efficient release ofthe metal from PvdI in vivo.

Recycled PvdI is able to chelate iron and to starta new iron uptake cycle. Siderophore-irondissociation in Gram-negative bacteria could alsoinvolve hydrolysis of the siderophore to overcomethe high affinity for iron. Recycled PvdI has thesame spectral properties as PvdI extracted from P.aeruginosa cultures (22), suggesting that thechromophore is not modified by the bacteria. Totest whether that the peptide moiety is modified,particularly the two formylhydroxy-ornithinesinvolved in the metal coordination, the ability ofPvdIrecy to chelate iron was investigated. PvdIrecy

was incubated with stoichiometric amounts ofFeCl3 and formation of PvdIrecy-Fe was observedby the decrease in fluorescence (λexc = 400 nm, λem

= 447 nm). After confirming that PvdIrecy was ableto chelate iron efficiently (Figure 6), we thenloaded PvdIrecy with 55Fe and demonstrated that itwas able to transport iron into PvdI- and Pch-deficient P. aeruginosa cells (Figure 7). Thus, thePvdI recycled after iron uptake and release, is afully functional siderophore, able to chelate ironand to start a new iron uptake cycle.

PvdI bound to FpvAI is located in patches at thecell surface. Because of its fluorescent properties,Pvd is suitable for imaging in living cells byfluorescence microscopy. We immobilizedgrowing Pvd- and Pch-deficient P. aeruginosa(PAD07) cells on agarose, and as expected, onlylow levels of background fluorescence weredetectable in the CFP filter set (Figure 8A).However, when 300 nM of PvdI was added to thecells, there was strong fluorescence at theperiphery of washed cells (Figure 8B).Interestingly, most cells (85%, 180 cells counted)contained one region of higher fluorescenceintensity, most often located close to one cell pole,but also occurring at the lateral edges (seeenlargements in Fig. 8B). These experimentsclearly demonstrate that PvdI binding sites arenon-uniformly distributed in the cell outer

membrane. However, the significance of this non-uniform staining pattern is not immediatelyevident. This result also supports the findings thatPvdI binds to the FpvAI OMT in the absence ofiron, and is retained at the receptor for an extendedperiod of time (at least 10 min). In agreement within vitro data, addition of PvdI-Fe to the cells doesnot give rise to a fluorescent signal (Figure 8C),even after incubation for 30 min. Thus, there wasno change in intracellular fluorescence that mighthave indicated the dissociation of PvdI-Fe. Wepreviously estimated that only 10% of thereceptors are activated by TonB and able totransport PvdI-Fe (23) so the amount of PvdI-Fetransported and dissociated inside the cells maynot be sufficient for detection of PvdIrecy byfluorescence microscopy. Further experiments arenecessary before conclusions can be drawn.Addition of PvdI-Ga resulted in a fluorescentpattern that was indistinguishable from that of freePvdI (Figure 8D, compared with 8A). Even after20 min, PvdI-Ga fluorescence was only observedat the periphery of cells, and not inside the cell,further evidence that PvdI-Ga does not accumulatein the cytoplasm.

DISCUSSION

Two mechanisms for the release of ironfrom a siderophore-iron(III) complex have beenreported in the literature. One involves hydrolysisof the siderophore-Fe complex, as in the case offerric enterobactin in E. coli, in which thecyclictriester is hydrolyzed by esterases (34). Thesecond mechanism involves the reduction ofFe(III) to Fe(II), the ferrous ion having a muchlower affinity for the siderophore (35). Fe(II) isthen easily released to a ligand with a higheraffinity for Fe(II) than that of the siderophore (36).We demonstrated that PvdI, after having releasediron inside the bacteria, is recycled to theextracellular medium in a form that is still able tobind and transport iron (Figures 6 and 7). Clearly,the PvdI-Fe dissociation does not involvehydrolysis or chemical modifications of thesiderophore that permanently lower its affinity forFe(III). Despite the huge amount of PvdI producedby the cells in iron limited planktonic cultures (200

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mg/L), PvdI is not consumed during the ironuptake process but is efficiently recycled to theextracellular medium. This efficient use of PvdImight be because the synthesis of PvdI is complex(37), involving many enzymes and consumingsubstantial energy and substrates.

Fluorescence measurements during PvdI-Cr uptake showed that there was no dissociation ofthe kinetically stable metal-siderophore complex(Figure 3C), evidence that there is a ligandexchange for iron during its uptake. Although nocandidates have been identified, this exchangemechanism might involve a periplasmic bindingprotein of an ABC transporter since thedissociation appears to occur in the periplasm.

Unlike the PvdI-Cr complex, which issubstitution inert, the PvdI-Ga complex iskinetically labile, and exchange with other ligandsoccurs readily. S ince Ga has no stable (II)oxidation state, the bacteria cannot remove themetal from the ligand by reduction. Based on thedecrease in fluorescence during its uptake, PvdI-Ga seemed to dissociate in P. aeruginosa cells(Figure 4). However, the rate of decrease wasmore than 2x faster than PvdI-Fe dissociation,suggesting that we might actually be observing arapid step that occurs just before the completedissociation of PvdI-Ga. Subsequently we foundthat PvdI from PvdI-Ga was not recycled to theextracellular medium but accumulates in theperiplasm (Figure 5), as if the siderophore was stillassociated to the metal in this cellularcompartment. Presumably, Ga does not completelydissociate from PvdI despite the decrease influorescence observed during uptake (Figure 4).Formation of a ternary complex between PvdI-Gaand another protein, like a periplasmic bindingprotein or a reductase could be an explanation forthe decrease of fluorescence observed in Figure 4.Such a mechanism would be similar to the onedescribed for Fe(III) release from ferrioxamine Bin vitro (36) in which, a ternary complex formsbetween ferrioxamine B and another iron-chelatingmolecule, sulfonated bathophenanthroline, in arapid step followed by a rate-limiting reduction ofthe ternary complex by glutathione or ascorbate.Another explanation for the decrease offluorescence observed in Figure 4 could be aprotonation of the catecholate coordination in thePvdI-Ga complex. It would be expected to lowerthe fluorescence (28) while at the same time

decreasing the affinity of the siderophore for themetal. The kinetic parameters for the formationand the dissociation of PvdI-Fe in acidicconditions have been reported (2). The in vitrodissociation mechanism occurs by an unfolding ofPvdI via successive protonations of thecoordination sites starting with the catecholatecoordination site (2). Thus it is reasonable tospeculate that an in vivo protonation would occuron the catechol, explaining the decrease offluorescence observed in Figure 4. Following sucha protonation, even though the chromophore wouldno longer be involved in the chelation of the metal,there would be significant residual affinity fromthe two remaining hydroxamates. However, for acomplete dissociation of the metal from thesiderophore, a reduction step and capture of theiron by another ligand must be necessary. Furtherinvestigations are needed to identify the source ofthe protons for the putative first step of the PvdI-Fe dissociation, the source of electrons for thereduction, and the periplasmic Fe bindingmolecule for the ligand exchange. Ferrisiderophorereductase activities have been reported in P .aeruginosa, so the reduction of iron in PvdI-Fe isplausible, however the corresponding enzymeshave not been identified and the activities havebeen located in the cytoplasm (38,39). Also it hasbeen shown in vitro that ferric-siderophore fromGram-negative bacteria could be reduced by asimple chemical process rather than by anenzymatic process (40). In this case, flavins in theoxidized form (FMN, FAD, riboflavin) areenzymatically reduced by electron transfer fromNADH or NADPH and the reduced flavins in turntransfer their electrons to the ferric-siderophores.This reduction reaction is also greatly stimulatedby Fe(II) acceptors such as ferrozine.

After uptake through the outer membraneby FpvAI, dissociation of PvdI-Fe is rapid (Figure2A) and there is no intracellular accumulation ofthe metal-form of this siderophore. Cellsosmotically shocked after incubating with[55Fe]ferri[14C]Pvd (41) and Mössbauerspectroscopy studies (42) showed no accumulationof PvdI-Fe inside the cells. Furthermore, we foundthat after overloading the PvdI uptake pathwaywith PvdI-Fe, less than 0.5% of the totalfluorescence was in the cytoplasm (Figure 5B), alevel that is barely above the background level inuntreated cells (data not shown). In the case of

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PvdI-Ga overloading, ~2% of the fluorescence wasin the cytoplasmic fraction and ~70% in theperiplasm. The small amount in the cytoplasmicfraction is likely a contamination from incompleteextraction of the periplasm during osmotic shock.In contrast to PvdI-Fe uptake, cytoplasmicaccumulation of ferric-enterobactin in E. coli andferricoprogens in Neurospara crassa (43) beforethe ligand is released has been observed byMössbauer spectrocopy.

To further confirm the cellular location ofPvdI-Ga during transport and the likely location ofPvdI-Fe dissociation, we used fluorescencemicroscopy. PvdI-deficient CDC5(pPVR2) cellswere incubated in the presence of PvdI-Ga, afterwhich the complex was not detectable in thecytoplasm (Figure 8D). The absence of PvdI-Ga inthe cytoplasm points to the periplasm as the likelylocation of PvdI-Fe dissociation. Moreover, thegenome of P. aeruginosa contains the genes ofonly a few ABC transporters, whereas there arenumerous outer membrane transporter genes(44,45). Therefore, it would be logical that theferric-siderophore complexes used by P .aeruginosa dissociate in the periplasm, and thatonly the iron is transported into the cytoplasm byan ABC transporter. In addition, we have recentlyreported that at least one enzyme involved in thesynthesis of Pvd is localized in the periplasm andthat the final Pvd is probably only produced in thiscellular compartment (46). It is therefore possiblethat no mechanism exists for the export of Pvdfrom the cytosol in P. aeruginosa, al thoughperhaps one exists for a synthetic precursor of Pvd.The reason that the disociation of Pvd-Fe and thefinal step of Pvd synthesis occur in the periplasmmay in fact be that it obviates the risk ofaccumulating such a potent iron chelator in thecytoplasm where it would compete with thecytoplasmic iron dependent enzymes.

The novel use of fluorescence microscopyto investigate the PvdI iron-uptake pathway inliving P. aeruginosa cells allowed us to visualizePvdI and PvdI-Ga bound to their OMT, FpvAI, atthe cell surface. While the data confirmed thatunder iron-limited conditions the FpvAI receptorsat the cell surface are loaded with iron-free PvdI(Figure 6B) (6,20,21), we also found that about85% of the cells showed a region of higherfluorescence intensity, usually located close to one

cell pole. This observation of high density PvdIbinding sites merits further investigation.

The delay in PvdI recycling after ironrelease (Figure 2) is sufficiently long for the Pvd-Fe complex to be transported into the periplasm,dissociated and be re-exported to the outermembrane. However, the FRET after the full ironuptake cycle returns to the same level as beforeaddition of PvdI-Fe (Figure 1) which is alsoconsistent with a mechanism in which PvdI bringsthe iron to the cells without dissociating from theOMT. The delay between PvdI-Fe dissociation andPvdI recycling on FpvAI can also be explained bya mechanism in which the plug domain of FpvA ispulled into the periplasm. In this scenario, PvdI-Fecould remain bound to this partially or totallyunfolded plug domain where iron release occurs bya reduction and ligand exchange, possiblyinvolving an apo periplasmic protein. With theplug domain dislodged from the barrel, none of theTrp residues in FpvAI would be close to the metal-free fluorescent siderophore and no FRET wouldoccur. FpvAI contains 17 Trps, but they are alllocated in the β-barrel domain. After release ofiron by the plug-bound siderophore, the plug withfluorescent PvdI refolds into the β-barrel domainsuch that FRET is restored. Regardless of theactual mechanism of recycling, the delay beforethe observed onset of PvdI recycling in Figure 2can be due to another phenomenon. Themacroscopic FRET only begins to appear after thePvdI-Fe uptake process has reduced theconcentration of PvdI-Fe in the media to athreshold that allows PvdI to begin to accumulateon FpvAI. Before this threshold is reached, thethermodynamics favor a displacement of PvdI bythe surplus of Pvd-Fe in the media.

This study provides the first evidence forthe role of iron reduction in the in vivo dissociationof PvdI-Fe, improving our understanding of themechanism of PvdI-Fe uptake in P. aeruginosaand iron uptake by pyoverdines into fluorescentPseudomonads in general. We conclude that metal-siderophore dissociation via the FpvAI/PvdIpathway occurs without chemical modification ofPvdI, but rather by a reduction of Fe(III) followedby ligand exchange. Our data indicate that PvdIdissociates from Fe in the periplasm, the locationof PvdI-Ga accumulation and that free PvdI is thenrecycled to the extracellular medium, where it canundergo a new cycle of iron uptake. Thus, the

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mechanism reported here for PvdI and relevant formore than 100 currently known pyoverdines isdifferent from that described previously for ferric-enterobactin and ferrichrome. Further studies arenecessary to determine whether the PvdI-Fedissociation occurs on FpvAI, to identify whatcatalyzes the reduction of Fe(III) and the apo

periplasmic binding protein involved in the captureof the iron released from PvdI.

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Moras, D. (1998) Cell 95, 771-77815. Buchanan, S. K. (1999) Curr Opin Struct Biol 9, 455-46116. Ferguson, A. D., Chakraborty, R., Smith, B. S., Esser, L., van der Helm, D., and

Deisenhofer, J. (2002) Science 295, 1715-171917. Yue, W. W., Grizot, S., and Buchanan, S. K. (2003) J Mol Biol 332, 353-36818. de Chial, M., Ghysels, B., Beatson, S. A., Geoffroy, V., Meyer, J. M., Pattery, T., Baysse,

C., Chablain, P., Parsons, Y. N., Winstanley, C., Cordwell, S. J., and Cornelis, P. (2003)Microbiology 149, 821-831

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21. Schalk, I. J., Hennard, C., Dugave, C., Poole, K., Abdallah, M. A., and Pattus, F. (2001)Mol Microbiol 39, 351-360

22. Schalk, I. J., Abdallah, M. A., and Pattus, F. (2002) Biochemistry 41, 1663-167123. Clément, E., Mesini, P. J., Pattus, F., Abdallah, M. A., and Schalk, I. J. (2004)

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(2002) Biochemistry 41, 14591-1460129. Ankenbauer, R., Hanne, L. F., and Cox, C. D. (1986) J Bacteriol 167, 7-1130. Poole, K., Neshat, S., Krebes, K., and Heinrichs, D. E. (1993) J Bacteriol 175, 4597-460431. Takase, H., Nitanai, H., Hoshino, K., and Otani, T. (2000) Infect Immun 68, 1834-183932. Mascarenhas, J., Soppa, J., Strunnikov, A. V., and Graumann, P. L. (2002) EMBO J. 21,

3108-311833. Leong, J., and Neilands, J. B. (1976) J Bacteriol 126, 823-83034. Langman, L., Young, I. G., Frost, G. E., Rosenberg, H., and Gibson, F. (1972) J Bacteriol

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(2004) Biochemistry 43, 1386-139236. Mies, K. A., Wirgau, J. I., and Crumbliss, A. L. (2006) Biometals 19, 115-12637. Ravel, J., and Cornelis, P. (2003) Trends Microbiol 11, 195-20038. Halle, F., and Meyer, J. M. (1992) Eur J Biochem 209, 621-62739. Halle, F., and Meyer, J. M. (1992) Eur J Biochem 209, 613-62040. Coves, J., and Fontecave, M. (1993) Eur J Biochem 211, 635-64141. Royt, P. W. (1990) Biol Met 3, 28-3342. Mielczarek, E. V., Royt, P. W., and Toth-Allen, J. (1990) Biol Met 3, 34-3843. Matzanke, B. F. (1997) Iron tranport in Microbes, Plants and animals. (Winkelmann, G.

v. d. H., D.; Neilnads, J. B., Ed.), VCH Verlagsgesellschaft, Weinheim, Germany44. Poole, K., and McKay, G. A. (2003) Front Biosci 8, d661-68645. Schalk, I. (2006) New insights on the iron metabolism in pathogneic Pseudomonas.

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FOOTNOTES

We thank SmithKline Beecham for generously providing carbenicillin. This work was partly funded bythe Centre National de la Recherche Scientific, the Association Vaincre la Mucoviscidose (FrenchAssociation against Cystic Fibrosis) and a grant from the ANR (Agence Nationale de Recherche, ANR-05-JCJC-0181-01) to I. S. and by the Deutsche Forschungegemeinschaft (P. G.). J. G. and L. J. aresupported by EMBO postdoctoral fellowships.

The abbreviations used are: Pvd, pyoverdine; Pch, pyochelin; OMT, Outer Membrane Transporter; FRET,Fluorescence Resonance Energy Transfer t ; FCCP, carbonyl cyanide p -(trifluoromethoxy)phenylhydrazone; oPOE, n-octylpolyoxyethylene.

LEGENDS

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Figure 1: Iron uptake kinetics monitored by FRET in K691(pPVR2) cells (22). The PvdI-producing,FpvAI-overproducing K691(pPVR2) cells were washed and resuspended at an OD600 of 2 in 50 mM Tris-HCl (pH 8.0) and incubated at 29°C. After the addition of 300 nM PvdI-Fe (arrow), fluorescence at 447nm was measured every 300 ms for 20 min, with the excitation set at 290 nm (black line). The samefluorescence measurements were repeated in the absence of siderophore () or with cells preincubated inthe presence of 200 µM FCCP (), a protonophore that abolishes the proton gradient across the innermembrane.

Figure 2: PvdI-Fe uptake and dissociation kinetics monitored by FRET and by direct excitation ofPvdI in CDC5(pPVR2) cells. CDC5(pPVR2) (FpvA +, ∆PvdI) cells were washed and resuspended at anOD600 of 2 in 50 mM Tris-HCl (pH 8.0) and incubated at 29°C.A. The transport assay was initiated by the addition of 300 nM PvdI-Fe to the cells (arrow). Fluorescenceat 447 nm was monitored with excitation at 290 nm (black line) and at 400 nm (). CDC5(pPVR2) cellswere also incubated with 300 nM PvdI and fluorescence monitored at 447 nm with excitation at 290 nm().B. The transport assay was initiated by the addition of 50 nM (), 100 nM (), 300 nM () or 600 nM() PvdI-Fe to the cells (arrow). Fluorescence at 447 nm under excitation at 400 nm was monitored.C. The transport assay was initiated by the addition of 300 nM () or 600 nM () PvdI-Fe to the cells(arrow). Fluorescence was monitored at 447 nm with excitation at 290 nm. The experiment was repeatedalso with 50 nM, 100 nM and 1 µM PvdI-Fe (data not shown). Insert: Plot of the time delay for the start offormation of FpvAI-PvdIrecy versus the concentration of PvdI-Fe incubated with CDC5(pPVR2) cells.

Figure 3: Pvd-Cr binding kinetics monitored by fluorescence in K691(pPVR2) cells. K691(pPVR2)(FpvAI+, PvdI+) cells were washed and resuspended at an OD600 of 2 in 50 mM Tris-HCl (pH 8.0) andincubated at 29°C. A. After the addition of 3 µM PvdI-Cr (arrow), fluorescence at 447 nm was monitored with excitation at290 nm (). The experiment was repeated with K691(pPVR2) cells in the absence of siderophore ()and with cells preincubated with 200 µM FCCP ().B. After the addition of 300 nM (), 1.2 µM () or 3 µM PvdI-Cr () (arrow), fluorescence at 447 nmwas monitored with excitation at 290 nm. The experiment was repeated in the absence of siderophore ()and in the presence of 300 nM PvdI-Fe (black line).C. After addition of 3 µM PvdI-Cr, fluorescence was monitored at 447 nm with excitation at 290 nm ()and at 400 nm ().

Figure 4: Pvd-Ga uptake kinetics and dissociation monitored by FRET and by direct excitation offluorescence in CDC5(pPVR2) cells. CDC5(pPVR2) (FpvAI+, ∆PvdI) cells at an OD600 of 2 wereincubated in 50 mM Tris-HCl (pH 8.0) at 29°C. The transport assay was initiated by the addition of 75 nMPvdI-Ga () or PvdI-Fe () to the cells (arrow). Fluorescence was monitored at 447 nm with excitationat 400 nm. The experiment was repeated in the absence of siderophore (black line).

Figure 5: A. Recycling of PvdI to the extracellular medium. The appearance of fluorescence (λexc =400 nm, λem = 447 nm) in the culture media of CDC5(pPVR2) (fpvA+, ∆Pvd) upon incubation in theabsence () or in the presence of 600 nM PvdI-Fe (), PvdI-Ga (), or PvdI-Cr ().B. Cellular distribution of PvdI after PvdI-metal uptake. Growing cultures of CDC5(pPVR2) at anOD600 of 0.8 were incubated for 30 min in the presence of 600 nM Pvd-Fe (black) or Pvd-Ga (white), afterwhich the amount of PvdI and Pvd-Ga was quantified in the media and each cellular location (periplasm,cytoplasm, inner membrane, and outer membrane). Values are expressed as a percentage of the totalfluorescence recovered from each culture.C. Tricine SDS-PAGE of cell fractions. Equal volumes of each cell fraction were run on a 11% tricineSDS-PAGE gel and visulaized by both silver and coomassie staining. The molecular weight standards (M)

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are in lane 1 and lanes 2-5 are P:periplasm, C:cytoplasm, I:inner membrane, O:outermembrane. Theunique band pattern for each fraction demonstrates that there is no significant contamination of theperiplasm by other cell fractions. The protein standard masses, noted on the left, are in kilodaltons.

Figure 6: Iron chelation by PvdIrecy. CDC5(pPVR2) at an OD600 of 2 were incubated with 300 nM PvdI-Fe at 29°C in 10 mL of 50 mM TrisHCl pH 8.0 buffer. After 30 min the cells were pelleted and 1 mL ofthe supernatant containing recycled PvdI was adjusted to pH 4 with AcOH. Aliquots of 10 µL of 3 µMFeCl3 were added and the fluorescence emission spectra recorded (λex = 400 nm). Spectrum of PvdIrecy

before addition of FeCl3 is in bold line.

Figure 7: 55Fe uptake by PvdIrecy in Pvd- and Pch-deficient P. aeruginosa cells (PAD07).CDC5(pPVR2) at an OD600 of 2 were incubated with 300 nM PvdI-Fe at 29°C in 10 mL of 50 mMTrisHCl pH 8.0 buffer. After 30 min, the cells were pelleted and 1 mL of the supernatant containingPvdIrecy was incubated with 30 nM 55FeCl3 for 15 min. This mixture was then incubated with 1 mL ofPAD07 cells at an OD600 of 2 in 50 mM TrisHCl pH 8.0 buffer at 37 °C. After 30 min, the cells werepelleted and aliquots (100 µL) of cell suspension were removed at different times, filtered and theradioactivity retained counted (). The experiment was repeated for the PAD07 cells at 0°C (). Eachpoint is the average of triplicate determinations.

Figure 8: Fluorescence microscopy of PvdI-deficient CDC5(pPVR2) P. aeruginosa cells. A) PvdI-deficient CDC5(pPVR2) cells, B) cells after addition of PvdI and after washing to eliminate unboundPvdI, C) cells after addition of PvdI-Fe and after washing, D) cells after addition of PvdI-Ga and afterwashing. Images were taken 10 min after washing, Filter specification Ex 425-445HQ, Em 460-510HQ,Dc 450. White bar = 2 µm.

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Peter L. Grauman and Isabelle J. SchalkJason Greenwald, Françoise Hoegy, Mirella Nader, Laure Journet, Gaëtan L.A. Milsin,

A role for ferrous ironReal-time fret visualization of ferric-pyoverdine uptake in Pseudomonas aeruginosa:

published online December 5, 2006J. Biol. Chem. 

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