INFECTION AND IMMUNITY, Apr. 1985, p. 130-1380019-9567/85/040130-09$02.00/0Copyright ©D 1985, American Society for Microbiology

Siderophore Activity of Pyoverdin for Pseudomonas aeruginosaCHARLES D. COX* AND PATRICIA ADAMS

Department of Microbiology, University of Iowa, Iowa City, Iowa 52242

Received 16 August 1984/Accepted 14 December 1984

Pseudomonas aeruginosa produces an extracellular compound with yellowish green fluorescence, calledpyoverdin, which functions as a siderophore. The production of pyoverdin, formerly called fluorescein, isconcomitant with the production of another siderophore, pyochelin. Pyoverdin is produced by P. aeruginosa inseveral forms, some of which were separated on gel filtration columns and on reverse-phase, high-pressureliquid chromatography columns. An active form of iron-free pyoverdin was purified to homogeneity. Theelution of pyoverdin from the columns was monitored for absorbance, fluorescence, and siderophore activities.These activities, iron binding, and the stimulation of bacterial iron transport indicated that pyoverdin canfunction as a siderophore for P. aeruginosa. The siderophore function of pyoverdin may be related to thepathogenicity of this bacterium because pyoverdin stimulated growth not only in iron-deficient culture medium,but also in defined medium containing transferrin and in human serum or plasma.

Siderophores are bacterial products which bind iron andincrease the rate of bacterial iron transport. The results ofsiderophore function are best observed in low-iron media asa more rapid onset of growth and possibly an increase ingrowth rate. Some iron-binding compounds are also knownto function in medium containing animal or human serum.Since an extension of this phenomenon is the stimulation ofbacterial growth during infections, siderophore synthesis hasbeen linked to bacterial virulence (2).

Liu and Shokrani (21) showed that several compoundsfrom culture media of Pseudomonas aeruginosa were capa-ble of stimulating bacterial growth in rabbit serum. Thesecompounds were separated into fractions based upon theirrelative solubilities in ethyl acetate. A growth-promotingactivity which was extractable from culture media into ethylacetate layers was termed pyochelin A, and several otheractivities which remained in the aqueous phase were termedpyochelins B. Pyochelin A appears to be identical to acompound isolated in our laboratory, which was termedpyochelin (6) and was structurally characterized (7). Thiscompound was shown to promote growth during in vitroculture and also during infections in mice (5). This was theonly growth-promoting factor which we found in the ethylacetate-extractable fraction of spent culture media.

In this report we describe an investigation of the sid-erophore activities which we found in the aqueous phase ofa culture medium extracted with ethyl acetate, the fractioncontaining pyochelins B (21). Other iron-binding compoundswhich could fulfill this function have been described. Shimanand Neilands (32) described iron binding by a Pseudomonasproduct, pyrimine. Teintze et al. (35) described the produc-tion of pseudobactin by a rhizosphere pseudomonad. Re-cently, Philson and Llinas (30) described two iron-bindingcompounds in culture medium filtrates of Pseudomonasfluorescens. These authors found that pyoverdin was themost important compound in iron metabolism and was

structurally similar to pseudobactin. Pyoverdin is producedby a variety of fluorescent pseudomonads (10, 15, 18, 30).Most recently, the structure of pyoverdin from P. aerugi-nosa has been reported (39) to be very similar to thestructure of pseudobactin (35), but detailed analyses of thepotential siderophore activity of this compound have not

* Corresponding author.

been conducted. The other compound described by Philsonand Llinas (30) was ferribactin, which was originally isolatedfrom the same species by Maurer et al. (23). In our study wedefined the siderophores of P. aeruginosa according to theirphysiological activities in growth stimulation and in bacterialiron transport, and we report the presence of a group ofcompounds whose properties correspond to the descriptionof pyoverdin. These pyoverdins promote bacterial growth inhuman plasma.

MATERIALS AND METHODS

Bacteria and culture conditions. P. aeruginosa strainsPAO1 (= ATCC 15692) and 10145 were obtained from theAmerican Type Culture Collection. A total of 40 clinicalisolates were obtained from human infections in the SurgeryIntensive Care Unit, University of Iowa Hospitals, and were0-serotyped (Difco Laboratories) and pyocin typed (17) inorder to assure a sampling of different strains. These bacteriawere maintained in the laboratory on meat infusion agarslants. Bacteria were grown for inocula in Casamino Acidsthrough three successive transfers in order to obtain uniformiron concentrations in the bacteria. The Casamino Acidsmedium used contained 0.5% Casamino Acids and 0.4 mMMgCl2. Bacteria were grown for siderophore production in a

glucose-succinate minimal medium (G-S medium) which wasbased on the medium developed by Liu and Shokrani (21);this medium contained 10 mM glucose, 10 mM succinate, 40mM NH4CI, 0.5 mM K2SO4, and 0.4 mM MgSO4 and wasbuffered with 5 mM potassium phosphate buffer (pH 7.4),which was added after autoclaving. Cultures were incubatedwith shaking at 37°C.Growth promotion assays were conducted in glucose

minimal medium, which contained the same ingredients asG-S medium except that 10 mM glucose was added in placeof succinate and 10 mM sodium bicarbonate was added.Transferrin (100 ,ug/ml; Sigma Chemical Co.) was added andhad an approximate iron saturation of 40% in this medium.Outdated human plasma was obtained from the Blood DonorCenter, University of Iowa Hospitals, heat inactivated, andadded to this medium at a final concentration of 20%. Noattempt was made to remove trace iron from any of themedia used. Siderophore production was measured in suc-cinate minimal medium (SMM) because glucose appears tohave an inhibitory effect on pyochelin synthesis (4). SMM is

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similar to G-S medium, except that 10 mM sodium succinatereplaces the glucose. Bacteria (strain PAO1) were grown forinoculation in Casamino Acids medium containing 200 ,uMethylenediamine-di-(o-hydroxyphenylacetic acid) for 20 h at37°C, harvested by centrifugation, and washed three times insterile, distilled water. The bacteria were inoculated at aninitial density of 103 bacteria per ml, and growth was assayedeither by absorbance at 600 nm or by measuring viablebacteria. Viable bacteria were measured in dilutions ofmedia as colony-forming units appearing on tryptic soy agaror peptone agar (Difco) after 24 h of incubation at 37°C.Chromatography and purification techniques. Bacteria were

removed from the cultures by centrifugation in two steps,once at 7,000 rpm and once at 10,000 rpm. This was followedby nitrogen-pressurized filtration through membranes (poresize, 0.45 ,um). The filtered culture medium was extractedwith ethyl acetate (1:5, vol/vol), and the aqueous phase wasconcentrated to dryness by rotary evaporation. The driedmaterial was dissolved in sterile, distilled water to yield atleast a 50-fold concentration. This concentrate was appliedto a Bio-Gel P-2 polyacrylamide column (1.8 by 85 cm)equilibrated with a water-methanol (10:1) solvent. In somechromatography experiments, the solvent was a solutioncontaining 5 mM Tris-hydrochloride buffer (pH 7.4), 0.1 MKCl, and 10% methanol. After we verified that the activityeluted from the column in water-methanol, this solvent wasused to prepare active fractions, and the column was thenwashed in the Tris-KCl-methanol solvent between uses. Thefractions were measured for absorbance at 254 nm and forfluorescence emission at 460 nm when the samples wereexcited at 400 nm. These wavelengths were chosen forsensitive detection of pyoverdin, but also detected pyochelin(6).The next step in purification was the concentration of the

active fractions from the Bio-Gel P-2 polyacrylamide columnby rotary evaporation and application in 20- to 50-,ul quan-tities to a C8 reverse-phase column (4 by 250 mm; Ultra-sphere; Beckman). The column had been equilibrated with 1mM ammonium phosphate buffer (pH 7.0), and injection wasfollowed by a linear gradient to 100% methanol over 20 min.Solvent was delivered by Beckman type 110 pumps, andelution was monitored at 254 nm. An alternative chromatog-raphy system relied on a C18 column equilibrated with 100%acetonitrile. At 5 min after sample application, a lineargradient to 50% water was conducted over 15 min. Chroma-tography of crude culture concentrates necessitated makingmethanol extracts of dried culture filtrates so as to avoidprecipitation of medium components on the column in themethanol or acetonitrile gradients. A majority of the fluores-cence due to pyoverdin was soluble in methanol. Beforeinitial use, high-pressure liquid chromatography (HPLC)columns had to be cleaned of iron by repeated injections ofsalicylate in a 10% acetic acid solvent, and the periodic useof 0.5 mM ethylene glycoltetraacetic acid (EGTA) in themobile phase maintained the columns relatively free fromiron contamination.Chromatography of the iron-saturated compounds pro-

ceeded according to the description of Philson and Llinas(30). Iron (1 g of FeCl3 per liter) was added to the originalmedium, and the brown supernatant after centrifugation at2,000 rpm was loaded onto a carboxymethyl cellulose Se-phadex column (2.0 by 30 cm). The column had beenequilibrated with 0.05 M sodium acetate buffer (pH 5.0),which was continued after loading until 300 ml had elutedfrom the column. The elution profile corresponded to com-pounds A and B which have been described previously (30).

A linear gradient from 0.05 to 1 M sodium acetate buffer (pH5.0) allowed the elution of an additional brown-coloredcompound and left the reddish orange color of what has beentermed ferribactin at the top of the column. Washing thecolumn in 2 M sodium acetate buffer allowed the elution ofthis compound.Chromatography to determine the approximate molecular

weight of pyoverdin was conducted by thin-layer gel filtra-tion in a type TLG apparatus (Pharmacia Fine Chemicals) ona glass plate (20 by 40 cm) spread with a 0.8-mm layer of400-mesh Bio-Gel P-2 polyacrylamide gel equilibrated with0.01 M sodium acetate buffer (pH 5.0). The standards usedwere bacitracin, ferrozine, chlorogenic acid, flavin adeninedinucleotide, riboflavin, and lumichrome, and these com-pounds were detected by fluorometric means as describedpreviously (6).

Analytical procedures. The iron solubilization assay wasconducted with precipitated iron made by adding 8.2 ,uCi of55FeC13 to 13 ,uM FeCl3 in 0.1 KOH. This precipitated ironsource was added to column fractions (10 ul/1-ml fraction),and the mixtures were poured over filters (pore size, 0.2 ,um)after 10 min of incubation at 37°C. The filters were washedwith 10 ml of water, dried, and then assayed for radioactivityby scintillation counting. The values were corrected for thetotal amount of iron trapped on the filters.

Siderophore production was measured by inoculatingstrain PAO1, grown for inoculation in Casamino Acidsmedium, into 1 liter of SMM at a density of 103 CFU/ml.Bacterial growth was measured by absorbance at 600 nm,and 60 ml of culture was removed for quantitation ofsiderophores. Pyoverdin was measured by diluting the me-dium into 50 mM Tris-hydrochloride buffer (p11 7.4) andmeasuring the fluorescence emission at 460 nm during exci-tation at 400 nm. For pyochelin measurements, 50 ml ofculture medium was made acid with 5 ml of glacial aceticacid and was extracted with 25 ml of dichloromethane. Thedichloromethane extract was concentrated by rotary evapo-ration and applied to a silica thin layer for chromatographyin chloroform-acetic acid-ethanol (90:5:2.5). Spots corre-sponding to the Rf of a pyochelin standard were scrapedfrom the plate, eluted in ethanol, and assayed fluorometri-cally (emission at 440 nm and excitation at 350 nm) forpyochelin (6).

Bacterial transport of iron was studied by using strainPAO1 cells that were grown in SMM, washed three times insterile, distilled water, and suspended in the reaction bufferat an absorbance at 600 nm of 0.2. This value correspondedto approximately 108 CFU/ml. Samples of the column frac-tions to be assayed were placed under a vacuum to removethe methanol and then added to transport buffer to makefinal 1-ml volumes containing 4 mM sodium-potassium (mo-lar ratio, 3:1) phosphate buffer (pH 7.4), 10 mM EGTA, 1mM MgCl2, and 5 mM sodium succinate. The EGTA wasincorporated because it constitutes an unusable iron chelatefor P. aeruginosa in a manner similar to the use of nitrilo-triacetic acid for Escherichia coli (11). The addition ofEGTA allowed very low amounts of "low-affinity" irontransport, and only those column fractions which containedan iron chelator capable of binding the iron from EGTA andmaking it available to the bacteria resulted in positive resultsfor iron transport. The reactions were initiated by makingthe reaction mixtures 0.1 puM 55Fe in complex with 10 mMEGTA. Bacterial transport was assayed by pouring thereaction mixtures through filters (pore size, 0.45 pum) afterincubation at 37°C for 15 min and determining the radioac-tivity trapped on the dried filters by scintillation counting.

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132 COX AND ADAMS

Control values from reaction mixtures containing no columnfraction were subtracted from the values for the columnfractions. Other reaction mixtures containing no bacteriaand mixtures containing bacteria which had been poisonedwith 5 mM KCN were included to ascertain that activebacterial transport was taking place.

Absorption spectra were obtained by using a Perkin-Elmer model 124 or Varian model DMS90 spectrophotome-ter. Fluorescence spectra were obtained by using an Amincocorrected-spectrum type SPF spectrofluorometer which hadbeen calibrated with quinine sulfate. Hydroxamate-contain-ing compounds were measured by the method of Csaky (8),except that the assay was conducted at a variety of iron(FeCl3) concentrations. Pyoverdin was negative in the Csakyassay unless iron was present at the correct concentration.The concentration had to be sufficient to saturate the pyo-verdin, but not much in excess because iron also inhibitedthe assay, probably by impeding the reducing ability ofsodium arsenite. Usually, 0.5 ,umol of FeCl3 per assay wasthe optimum concentration. Hydroxylamine and sali-cylhydroxamate were used as standards. The Arnow test (1)was used to determine dihydroxyphenol content. The assaysused for iron were modifications of methods described byDoeg and Ziegler (9). Increasing iron concentrations wereadded to duplicate pyoverdin samples, and the samples wereassayed by twQ methods, one using thioglycolate reductionand the other using reduction by ascorbic acid in 0.1 M HCI.Both methods relied upon a ferrous-ferrozine chelate (meas-ured at 562 nm) and gave comparable results with FeCl3standards, but only the ascorbate-HCI reduction removedthe iron from pyoverdin for assay. Use of thioglycolatereduction yielded the iron concentration which saturated thepyoverdin in the assay tubes.

RESULTSFractionation of siderophores from spent culture medium.

The aqueous phase of filtered G-S medium from a stationaryphase culture of strain PAO1 after extraction with ethylacetate (ratio of ethyl acetate to medium, 1:5) was concen-trated by rotary evaporation. A 0.5-ml portion of the con-centrate was applied to a Bio-Gel P-2 column equilibratedwith a water-methanol (10:1) solvent. In the elution profilefrom this column, the major peaks of absorbance at 254 nmmatched the peaks of fluorescence (Fig. 1A) and containedthe iron-solubilizing activities (Fig. 1B). All of these peaksappeared to contain activity to stimulate bacterial growth in20% human plasma (Fig. 1B), and they were numberedaccording to their sequence of elution from the column. TheUV and fluorescence spectra of material in peak 4 wereidentical to the spectra of pyochelin (data not shown). Theactive material from peak 4 could be extracted into ethylacetate, and it proved to be identical to pyochelin when itwas subjected to thin-layer chromatography (6). The appear-ance of pyochelin in medium extracted with ethyl acetatecan be explained by the poor extraction efficiency of thiscompound from medium that is not acidified. On the otherhand, peaks 1, 2, and 3 were not similar to pyochelin, butappeared to have absorption spectra identical to each other.The compound in peak 2 appeared to give rise to fluorescentcompounds in the other two peaks because application ofpeak 2 material to another Bio-Gel P-2 column resulted inthe appearance of peaks 1, 2, and 3 (Fig. 2). Application ofpeak 1 or 3 to another Bio-Gel P-2 column resulted in elutionof peak 1 alone or peak 3 alone (data not shown). Assays foractivity in the fractions from this second column showedthat peak 2 contained the maximum ability to enhance

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erophore activities produced by strain PAO1 in G-S medium.Fractions of effluent were collected every 15 min and analyzed forabsorbance at 254 nm (0) apd for fluorescence emission at 460 nmwhen excited at 400 nm (O). Spent G-S medium of strain PAO1 wasfiltered and concentrated 50-fold after extraction with ethyl acetate,and 0.5 ml was applied to a column (1.8 by 85 cm) with awater-methanol (10:1) solvent flowing against gravity at a rate of 0.2ml/min. The numbers at the top represent the peaks of activity. (B)Iron solubilization (0) and growth promotion (0) activities in thefractions from the Bio-Gel P-2 column. Iron solubilization wasmeasured as the iron trapped on filters (pore size, 0.2 ,um) after 10min of incubation at 37°C from mixtures containing 0.082 ,uCi of"FeCl3, 0.13 ,uM FeCl3, 0.001 M KOH, and 1-ml portions of columnfractions. The values represent the total amounts of iron trapped.Growth promotion was measured as the ability of 0.1-ml portions ofeach fraction to stimulate the growth of strain PAO1 cells inoculatedat a density of 103 CFU/ml into glucose minimal medium containing10 mM sodium bicarbonate and 20% human plasma. Growth wasassayed by determining absorbance at 600 nm (A6Wi nm) comparedwith a control plasma after incubation at 37°C for 24 h.

bacterial growth in human plasma and bacterial uptake of55Fe in the presence of EGTA (Fig. 2). Assays for ninhydrin-positive compounds and for carbohydrates indicated thatthese compounds eluted from the columns at elution vol-umes of 120 ml and beyond. The same patterns of elutionwere obtained with both the water-methanol and the Tris-KCI-methanol elution systems. However, some fluorescentmaterial remained on the column after each run in metha-nol-water and had to be eluted in the Tris-KCI-methanolbuffer between runs. Since this material had negligibleactivity compared with peak 2, the Bio-Gel P-2 column wasused with a water-methanol solvent to remove salt, carbo-

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SIDEROPHORE ACTIVITY OF PYOVERDIN 133

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FIG. 2. Fractionation of peak 2 material on a Bio-Gel P-2 polyacrylamide column. Peak 2 material from the experiment shown in Fig. 1was concentrated and applied to the same Bio-Gel P-2 column, and fractions were assayed for fluorescence emission (0), for growthpromotion in human plasma (0), and for stimulation of bacterial iron uptake (*). Chromatography, fluorescence, and growth promotion weremeasured as described in the legend to Fig. 1. Bacterial iron uptake was measured as the amount of 55Fe trapped with bacteria on filters (poresize, 0.45 pLm) after 10 min of incubation of a suspension containing 108 bacteria per ml with a solution containing 4 mM sodium-potassium(3:1) phosphate buffer, 1 mM MgCI,, 10 mM EGTA, 5 mM sodium succinate, and 0.1 ,uM 55Fe added with 10 mM EGTA. Filters were washedwith water and dried, and the radioactivity was determined by scintillation counting. A6w, Absorbance at 600 nm.

hydrates, and amino acids from the concentrated spentculture medium and to yield what appeared to be the nativeform of the active compound in peak 2.

Purification of activity from peak 2. Peak 2 fractions fromthe initial Bio-Gel P-2 column were concentrated by rotaryevaporation and were applied to a C8 reverse-phase HPLCcolumn which had been equilibrated with 1 mM ammoniumphosphate buffer (pH 5.0) containing 1 mM EGTA. Anascending gradient of methanol to 100% yielded the elutionprofile shown in Fig. 3A. Fractions (1 ml) were collectedeach minute and assayed for growth-promoting activity inhuman plasma. The major activity was found in fraction 13(Fig. 3B) and corresponded to a major absorbance peak at12.5 min (Fig. 3A). A 0.6-min delay between the monitor andthe fraction collector accounted for the appearance of the12.5-min peak in fraction 13, the appearance of activity at 11min, and the positive slope of the 12.5-min peak in fractions11 and 12 (Fig. 3B). Application of a concentrate of fraction13 onto the C8 column again yielded a single peak at 12.5 min(data not shown). A comparison of such a chromatogramwith one from a column injected with a methanol-solublefraction of crude culture filtrate (Fig. 4) revealed the removalthrough chromatography of fluorescent peaks at 9, 10, 11,21, and 22 min. Material from fraction 13 demonstratedhomogeneity when it was injected into a C18 reverse-phasecolumn equilibrated with 100% acetonitrile. A linear, de-scending gradient to 50% acetontrile in water revealed asingle, fluorescent peak at 24 min. Chromatography oncellulose, carboxymethyl cellulose, and silica in ethanol-am-monium phosphate buffer revealed single spots at Rf values0.8, 0.6, and 0.2, respectively. The single, fluorescent spotwas phenolate positive and ninhydrin negative. In addition,only when the fluorescent material was overloaded to producestreaming were there positive reactions with a sulfuric acidspray reagent or an iodine vapor to reveal the homogeneityof the compound. The final step in purification was theremoval of salt from fraction 13 by passage through a

Bio-Gel P-2 column in water-methanol or a C18 column inthe acetonitrile-water solvent system.

Proof of iron-binding character. The iron reactivity of thecompound from fraction 13 was verified by using thin-layerchromatography. Although "5Fe alone does not migrate oncarboxymethvl cellulose in an ethanol-ammonium phosphatebuffer solvent system, mixing 55Fe with a concentrate offraction 13 yielded mobile 55Fe on the thin layers (Fig. 5).The binding of iron resulted in some quenching of thefluorescence, and there was a small spot of unchelatedfluorescent compound trailing the ferric chelate. This wasnot an oxidative alteration of the compound by iron becauseno Fe(II) was detected with the ferrozine reagent and Fe(II)did not migrate on cellulose thin layers as the 55Fe complexdid. We also found that the iron bound to the active materialcould not be removed with thioglycolate, but could beremoved through reduction with ascorbate-HCI (9). There-fore, the total amount of iron in a preparation could beassayed by the ferrozine reagent after ascorbate-HCI reduc-tion, and the amount of iron necessary to saturate the activecompound could be assayed with ferrozine after thioglyco-late reduction. The molecular weight of this compound wasestimated to be 1,500 based upon thin-layer molecular siev-ing and comparison of the migration of this fluorescentcompound with the migration of compounds of knownmolecular weights (4). Based upon this molecular weight,the amount of iron necessary to show saturation indicated a1:1 molecular ratio of iron to compound in chelate.

Properties of the growth-promoting compound. Figure 6shows the absorption spectrum of the compound as itappeared in fraction 13 from the C8 column and the spec-trum of the compound in the presence of 100 ,uM FeCl3. Thefluorescence excitation spectrum, which was obtained bymeasuring emission at the maximum value of 460 nm, wassimilar to the absorption spectrum, having characteristicpeaks in acidic solutions at 360 and 380 nm (data not shown).In solutions at neutral pH, the excitation peak at 400 nm,

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134 COX AND ADAMS

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column on a C8 reverse-phase, HPLC column. The profile ofabsorbance at 254 nm was monitored in a linear gradient of 0 to100% methanol in ammonium phosphate buffer (1 mM, pH 5.0)flowing at a rate of 1 ml/min. (B) Activities in HPLC fractions tostimulate bacterial iron (155Fe) uptake (A\) and to promote bacterialgrowth in human plasma (O). The 1-ml fractions were taken todryness under a vacuum and were included in uptake and growthpromotion assays as described in the legends to Fig. 2 and 1,respectively. Fluorescence assays (data not shown) indicated aminor peak in fraction 11 and a major peak in fraction 13. A6w0,Absorbance at 600 nm.

5 10 15 20 25Elution time (min)

FIG. 4. Crude, methanol extract of G-S culture medium fromstrain PAO1 fractionated on a C8 HPLC column. The chromatog-raphy conditions were identical to those described in the legend toFig. 3. The fluorescence assay indicated peaks at 9, 10, 11, 12.5, 21,and 22 min.

with emission measured at 460 nm, was more characteristicof this compound in culture media and during chromatogra-phy in neutral solvents. These spectral characteristics areidentical to those which have been reported for pyoverdin(10, 30, 37, 38) isolated from both P. aeruginosa and P.fluorescens. The fluorescence could be quenched to a limitedextent by the addition of FeCl3. Pyoverdin has been reportedto be an hydroxamate (24, 30), and the shoulder at 450 to 480nm in the absorption spectrum of the ferric chelate issuggestive of this (Fig. 6). The Csaky assay was positive forthe purified product, but only when the compound was in theferripyoverdin form. The Arnow test for dihydroxyl deriva-tives was negative.

Activity of purified pyoverdin. Presumably, pyoverdin wasactive in promoting growth in human plasma by providingiron to the bacteria. To test this presumption, strain PAO1was inoculated into glucose minimal medium containing 100,ug of transferrin per ml (Fig. 7). Purified pyoverdin fromfraction 13 dramatically stimulated the growth rate anddecreased the lag phase (Fig. 7) when it was added at aconcentration of 10 jig/ml, a level which was below thevisible detection of yellow color, but was detectable byfluorescence when excited by a hand-held UV lamp.

Production of pyoverdin during growth. Pyoverdin produc-tion was measured with reference to the growth of strain

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Thin-Layer Chromatogram

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The migration on a carboxymethyl cellulose thin layer of a mixtureof 0.05 ,uCi of "FeCl3 and pyoverdin (55Fe+Pv) is compared withthe migration of 0.05 ,uCi of "Fe alone in an ethanol-ammoniumphosphate buffer (1 mM, pH 5.0) (1:1) solvent. After chromatogra-phy, the dried chromatogram was cut into 0.5-cm sections to assayfor "Fe by scintillation counting.

PA01 and pyochelin production in SMM. This medium wasused in place of G-S medium because glucose appears toinhibit pyochelin synthesis (4). Although both siderophoreswere detectable after 13 h of incubation, the maximumsynthesis of both pyoverdin and pyochelin was noted duringentry of the culture into stationary phase (Fig. 8). However,unlike pyochelin synthesis, pyoverdin was not produced byall of the P. aeruginosa strains examined. Of 40 strains fromhuman infections, 2 were pyoverdin negative.

DISCUSSIONThe results of this investigation verified the original de-

scription of siderophore activity remaining in the aqueousphase of spent G-S medium after extraction with ethylacetate (21). The assays used to search for this activity wereinclusive for all compounds which were capable of solubi-lizing precipitated iron, stimulating iron transport in thepresence of the nontransportable [55Fe]EGTA substrate, andstimulating bacterial growth in human plasma. Using thesecriteria for activity, we detected one peak of residual pyo-chelin and three peaks of fluorescent siderophore activity inculture media which had been extracted with ethyl acetate.The three peaks of activity corresponded to the originalfinding of numerous pyochelins B (21); however, the spectralcharacteristics of these compounds corresponded to theprevious descriptions of pyoverdin (10, 15, 24, 37, 38). Sincethere has been a considerable quantity of literature pub-lished about pyoverdin and since there has already been onechange in terminology, from bacterial fluorescein to pyover-din (A. Turfreijer, Ph.D. thesis, University of Amsterdam,Amsterdam, The Netherlands, 1941), we suggest that theterm pyoverdin be maintained for these compounds. Onereason for the early interest in this pigment is the dramatic

green color which it imparts to wounds (green pus orpyoverdin) and to culture media. Many of the early publica-tions described the nutritional requirements for pyoverdinproduction (10, 14, 18, 29, 33, 36). Although many mediumconstituents were found to support pyoverdin production, itwas notable that iron inhibited production (10, 13, 14, 19, 20,22, 36).An early insight into the structure and function of pyo-

verdin came when Garibaldi noticed the correlation betweeniron requirement by a fluorescent pseudomonad and theproduction of an hydroxamate-containing fluorescent com-pound (13). There have been numerous attempts to deter-mine the structure of pyoverdin. One compound producedby a fluorescent pseudomonad was reported to contain apterin ring system (3). Another research group presentedevidence that a cyclic peptide is the fluorescent compoundfrom Pseudomonas mildenbergii (16, 27). Most recently, the

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FIG. 6. Absorption spectra of purified growth-promoting activi-ties from fraction 13 in the iron-free form (Pv) and in the ferricchelate (Pv+Fe) obtained by adding 100 nmol of FeCl3 to thecuvette.

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136 COX AND ADAMS

structures of a group of green fluorescent compounds havebeen found to be similar. Teintze et al. have reported thatthe structure of pseudobactin, which is produced by arhizophere pseudomonad strain (B10), contains a dihy-droxyquinoline (the fluorescent moiety) connected to ahexapeptide containing a terminal N-hydroxyornithine (34,35). In addition, Philson and Llinas described a similarstructure produced by P. fluorescens (30, 31). This structurewas composed of dihydroxyquinoline connected to a hepta-peptide containing amino acids different from those found inpseudobactin, but containing a terminal N-hydroxyornith-ine. Although the iron-binding or siderophore properties ofthe compound from P. flluorescens were not determined,presumably this compound is the same pyoverdin that wasfound by Meyer et al. (24, 25) to be an hydroxamate-con-taining siderophore for this species. Most important to thisreport is the structure of pyoverdin from P. aeruginosa,which has been revealed by Wendenbaum et al. (39) tocontain a dihydroxyquinoline connected to two N-hydroxy-ornithines via a peptide chain. Again, the amino acidspresent in the peptide chain were different from those foundin the pseudobactin and pyoverdin produced by P. flutores-cens. In addition to Pseudomonas species, Azotobactervinelandii produces azotobactin, a green fluorescent com-pound with structural similarities to pyoverdins and pseudo-bactin (12).The pyoverdin molecule purified in this study conforms to

the description previously published by Wendenbaum et al.(39). We found a fluorescent, 1,500-dalton, hydroxamate-containing compound which has a binding ratio with iron of1:1. The absorption spectrum of this compound is identicalto the spectra described previously for pyoverdin or fluores-cein (11, 14, 15, 33, 36-38; Turfreijer, Ph.D. thesis). Thereason for the lability of the iron-free compound in theCsaky assay is not known. Other investigators probablyhave not confronted this problem because they have purifiedthe iron chelates of pyoverdins. The presence of the N-hydroxypiperidone ring in pyoverdin, instead of the N-ace-tyl-N-hydroxyl derivatives usually found in hydroxamatesiderophores (26), may have importance in this regard. Thefinding of Garibaldi (13) of hydroxamate-positive com-pounds produced by pseudomonads in low-iron media isconsistent with our data. We found other compounds pro-duced by P. aeruginosa which gave media and columnfractions hydroxamate-positive characteristics in the ab-sence of iron, but these compounds had negligible activity iniron transport compared with pyoverdin. Although theyeluted with pyoverdin from the Bio-Gel P-2 column, thehydroxamate-positive compounds could be separated frompyoverdin during HPLC. These compounds were found ingreater concentrations in older cultures and may representdegradation products of pyoverdin.

All of the structural and phylogenetic relationships amongthese pigments and different species are not yet known. Inaddition to differences among pigments produced by dif-ferent species, other investigators (24, 30, 34) have men-tioned the multiple forms of pyoverdin found in cultures ofP.fluorescens, just as we found in P. aeruginosa. Therefore,there will be many problems in the purification, analysis, andnomenclature of these pigments. Meyer et al. (24, 25) havemade the reasonable suggestion that the pigment from P.fluorescens be named pyoverdinePf and the pigment from P.aeruginosa be named pyoverdinePa (39). Dropping the ter-minal "e" from pyoverdine is compatible with the terminol-ogy for pigments produced by pseudomonads (pyocyanin,pyorubrin, pseudobactin, pyochelin). The purification pro-

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FIG. 7. Growth promotions of strain PAO1 by purified pyo-verdin in glucose minimal medium containing transferrin. The levelsof viable bacteria were measured in glucose minimal mediumcontaining 10 mM sodium bicarbonate and 100 ,ug of transferrin (Tr)per ml (A) and in the same medium supplemented with 10 p.g ofpyoverdin (Tr+Pv) per ml from fraction 13 of the C8 column (0).Washed bacterial cells grown in Casamino Acids medium containing200 ,uM ethylenediamine-di-(o-hydroxyphenylacetic acid) were in-oculated at a density of 103 CFU/ml.

cedure for pyoverdinPa described here is different from themethods used for pseudobactin or pyoverdinPf because it isbased on the isolation of the iron-free form instead of theferric chelate. The purification scheme for the ferric chelatewas attempted, but the techniques used to remove the ironfrom the chelate were unsatisfactory. Pyoverdin appeared toelute from desalting columns in complex with the ax,ot-dipyridyl or ferrozine which had been added to chelate theFe(II) removed from the pyoverdin complex. 8-Hydroxy-quinoline was not used in the removal of iron from thecomplex because of potential problems in contaminating thefinal product in our structural analyses.

In addition to pyoverdinPa, we found ferribactin in culturemedia as described by Philson and Llinas (30). We alsofound low amounts of this compound and negligible irontransport activity compared with pyoverdin. This compoundis currently under investigation to determine its importanceto P. aeruginosa. Ferribactin contains some of the sameamino acids found in pyoverdinPf (23, 30) and may be adegradation product or a product of enzymatic processing inthe removal of iron from pyoverdins.The structure of pyoverdinPa has been published previ-

ously, but the activity of this compound has not beenverified. In the present study we verified the siderophoreactivity of pyoverdinPa, with special reference to its ability

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SIDEROPHORE ACTIVITY OF PYOVERDIN 137

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to stimulate growth in human plasma and transferrin. SincepyoverdinPa stimulates bacterial growth in these media, itmay compete with transferrin for iron. It will be interestingto determine whether all fluorescent, pyoverdin-like pig-ments have this activity or whether specific amino acidsequences in these compounds allow specific iron-sequester-ing activities. In this work we also demonstrated that puri-fication and separation of iron-free forms of pyoverdin byHPLC is possible. Important questions which are underinvestigation include the differences in the structures of thepyoverdins which have been separated, the reason for theproduction of multiple forms, and the reason for bacterialproduction of both pyochelin and pyoverdin at the same timein culture and under the same conditions. A similar investi-gation with A. vinelandii (28) has shown that there issequential derepression for specific siderophores under var-ious degrees of iron deprivation, with azotobactin beingsynthesized under the most severe iron-limiting conditions.We have not been able to detect sequential synthesis ofsiderophores by P. aeruginosa. It is possible that only one ofthese siderophores is crucial to growth in mnammalian tissue.Although the binding coefficient of pyoverdinPa for iron is1032 (39), which is competitive with the value for transferrin,we have isolated pyoverdin-negative strains from humaninfections. Alternatively, we have never found a pyochelin-negative strain, and this siderophore has dramatic effects onthe virulence of P. aeruginosa (5). To understand andcontrol the growth of P. aeruginosa during infections, wecontinue to study the activities of these siderophores.

ACKNOWLEDGMENTS

We thank Marcia Reeve for preparation of the manuscript.This investigation was supported by Public Health Service grant

Al 13120 from the National Institute of Allergy and InfectiousDiseases.

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