JOURNAL OF BACTERIOLOGY, May 1980, p. 581-5870021-9193/80/05-0581/07$02.00/0

Vol. 142, No. 2

Iron Uptake with Ferripyochelin and Ferric Citrate byPseudomonas aeruginosa

CHARLES D. COXDepartment ofMicrobiology, University ofIowa, Iowa City, Iowa 52242

Pyochelin is an iron-binding compound produced by Pseudomonas aeruginosaand demonstrates siderophore activity by its involvement in iron transport.During the transport process, an energy-independent association of [5Fe]ferri-pyochelin with bacteria occurred within the initial 30 s of reaction, followed by anenergy-dependent accumulation of iron. The energy-independent association withiron appeared to be at the surface of the bacteria because the iron could bewashed from the cells with thioglycolate, whereas accumulated iron was notwashed from the bacteria. Energy-independent association of iron with bacteriaand energy-dependent accumulation of iron in the presence of ferripyochelinvaried concomitantly in cells grown under various conditions, but pyochelinsynthesis appeared to be controlled separately. 'Fe complexed with citrate wasalso taken up by P. aeruginosa with a lower level of initial cell association.Bacterial mechanisms for iron uptake from ferric citrate were present in cellsgrown in a variety of media and were in lowest levels in cells grown in citrate.The synthesis of bacterial components for iron uptake from ferric citrate and fromferripyochelin was inhibited by high concentrations of iron supplied in growthmedia.

Siderophores are microbial iron-binding com-pounds which function in iron solubilization andmembrane transport of iron. Siderophores areoften classified according to the phenolic or hy-droxamic acid composition of the compounds.The phenolic siderophore studied most inten-sively is enterobactin, isolated from Salmonellatyphimurium (14), which is the same compoundas enterochelin isolated from Escherichia coli(12). Iron uptake with ferrienterochelin (6, 13)had an apparent Km of 0.1 yIM (4) and wasinhibited by respiratory poisons (5, 16). How-ever, there was an energy-independent compo-nent of iron accumulation by E. coli revealedduring the inhibition of uptake by dinitrophenol(15). It was also noted that accumulation of ironfrom ferrienterochelin in the presence of dinitro-phenol was fractionally present in cells grown in100 ,uM FeCl3. It was hypothesized that theenergy-independent accumulation of iron wasdue to both nonspecific association of iron-sid-erophore with cells and to specific binding pro-teins for ferrienterochelin.

In addition to iron uptake from this sidero-phore, there is a separate mechanism in somestrains of E. coli which is induced by citrate andrecognizes ferric citrate for iron transport (4).There are other mechanisms described in E. coliand S. typhimurium (9) for iron transport fromcompounds containing hydroxamate groups, butthis paper will not deal with this class of sider-ophore.

Pyochelin is a phenolic iron-binding com-pound which can be extracted into ethyl acetatefrom culture media ofPseudomonas aeruginosaand promotes the growth of the bacterium whenadded in the pure form to iron-deficient media(1). This compound appears to be similar topyochelin A described by Liu and Shokrani (8),although a detailed comparison is not possibleat this time. On the other hand, pyochelin shouldnot be confused with the water-soluble pigment,pyoverdinepf, which has been described as asiderophore for Pseudomonas fluorescens (10).This paper verifies the function of pyochelin

as a siderophore for P. aeruginosa by describinga role for pyochelin in iron transport. A mecha-nism for iron uptake from ferric citrate wasstudied in comparison with the mechanism withferripyochelin. These two mechanisms were ex-amined in bacteria grown under a variety ofconditions.

MATERIALS AND METHODSBacteria and culture conditions. P. aeruginosa

strain PAO-1 was obtained from the American TypeCulture Collection (ATCC 15692) and was maintainedin the laboratory on brain heart infusion agar. Bacteriafor uptake assays were grown in minimal mediumcomposed of 20 mM sodium succinate, 4 mM NH4Cl,0.4mM MgCl2, 1 mM K2S04, 1 ,uM ZnCl2, 1 1LM MnCl2,and 4 mM potassium phosphate buffer at pH 7.4. Insome experiments, minimal media were also madewith 20 mM concentrations of glucose, citrate, orasparagine as oxidizable substrates in place of succi-

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nate. CAA medium was composed of 0.5% CasaminoAcids adjusted to pH 7.5 with 1 N KOH and made 0.4mM with MgC12 before inoculation. Trace iron inmedia was reduced in concentration by adding 20 g ofMgCO3 per liter and after 2 h removing the MgCO3 bycentrifugation. The medium was then adjusted to pH7.4 and made bacteria-free by passage through mem-brane ifiters of 0.45-pm pore size (Millipore). All min-erals were filtered through identical membrane filtersand added to media at the time of inoculation. Allglassware was cleaned with concentrated nitnc acid.Bacterial growth was determined by measuring in-creases in absorbance at 600 nm in 1-cm cuvettes in aGilford model 240 spectrophotometer. Growth curvesresulted from inoculations of washed bacteria fromlogarithmic-phase cultures in CAA medium into 500-ml quantities of various media which were shaken in1-liter flasks at 370C.

Puriflcation and assay of pyochelin. Pyochelinwas extracted into ethyl acetate from spent culturemedia which had been adjusted to pH 2.5. Purificationwas by paper chromatography in water-acetic acid-acetone (90:10:.1), chromatography on a first thin layerof silicic acid in a 90:.5:2.5 ratio of chloroform-aceticacid-ethanol, and chromatography on a second thin-layer plate developed in a 90.5:5 ratio of the samesolvents (1). Pyochelin resulting from this procedurewas determined to be pure by thin-layer chromatog-raphy on silicic acid in several solvents (1) and byreversed-phase high-pressure liquid chromatography.Pyochelin was measured by dry weight after solventwas removed by vacuum. The pure compound wasstored at 4°C in the dark and dissolved in ethanolimmediately before use. Pyochelin in the pure form isvery labile, and degradation products may be detected24 h after ethanol addition.

Pyochelin in culture media was measured by ex-traction of the compound into ethyl acetate (20 mladded to 50 ml of acidified medium), conducting thefirst thin-layer purification step on silicic acid, andfluorometrically comparing ethanol eluents of silicicacid from spots contsining unknown pyochelin sam-ples with varying concentrations ofpure pyochelin runon the same thin-layer plate. Pyochelin was measuredin a concentration range from 10 to 250 ng/ml byexciting samples at 350nm and measuring fluorescenceat 440 nm (1).The molecular weight of pyochelin was estimated

to be 650 on the basis of titration with FeCI6 andmeasurement of the iron content (3) of ferripyochelimThis value has been verified by mas spectroscopy(data not shown).Assay ofiron uptake. Bacteria were harvested by

centrfugation at 5,900 x g for 10 min at 24°C fromcultures in logarithmic phase (absorbance values of0.2 to 0.5 at 600 nm). The cells were washed threetimes in 40-ml volumes of 1 mM morpholinopropanesulfonate, pH 7.4, with 1 mM MgCl2 (MOPS-Mgbuffer), and suspended in MOPS-Mg buffer to yield0.1 absorbance at 60 nm for assay of iron uptake.Bacteria at this density corresponded to approxi-mately 3 x 10' bacteria per ml or 0.05 mg (dry weight)of cells per ml. Oxidizable substrate, the same as usedin the growth medium, and inhibitors were added andincubated with the cells at 24°C for 10 min in a shaking

water bath before addition of the iron-containing sub-strate.

Stock solutions of ferripyochelin were made with 5pM TMFeCl3 and 6.5 pM pyochelin, and solutions offerric citrate were made with 5pM 'FeCl3 and 100 uMsodium citrate. The solutions were passed throughcellulose acetate filters of 0.45-pm pore size (GA-6,Gelman) before use. Uptake reactions were initiatedby additions of varying amounts of the stock solutions,and 1-ml samples of the reaction mixtures were re-moved at intervals to be placed on 0.45-pm-pore-sizefilters, with a vacuum making filtration complete in 1a. The bacteria trapped on the filters were washedwith 10-ml volumes of distilled water or 0.5% thiogly-colate prepared immediately before use. Washing withthioglycolate removed 96% of 'Fe intentionally pre-cipitated on filters with 0.1 M KOH. It was shown bycolorimetric assay (2) that 94% ofa 5pM concentrationof iron with citrate, pyochelin, or precipitated withKOH was reduced to Fe(ll) within 5s by thioglycolate.No cell death was detectable during the washing pro-cedure.The dried filters were placed in scintillation fluid

(Budgetsolve, Reearch Products Inc.) and countedwith the tritium channel with 33% efficiency in aPackard model 2420 counter. 56FeCI3 and ['4C]succi-nate were obtained from Amersham Searle. Quenchwas determined by extemal channels ratio. Weights ofbacteria were determined from duplicate samples ofseveral concentrations ofbacteria washed in water anddried at 110°C. Control reactions were run withoutcells to determine the association of iron and ironchelates with the filters, and these values were sub-tracted from the quantities of iron trapped on filtersfrom reactions run with bacteria to yield values ofbacterial accumulation of 'MFe.

RESULTSIron uptake with ferripyochelin. Iron sup-

plied to bacteria as ferripyochelin was taken upby P. aerugmnosa (Fig. la) with a remarkablylarge amount of 'Fe accumulated by cells at 5s. There was a rapid initial rate ofuptake duringthe first 30 s of reaction, followed by a slowerrate of uptake. Filtrations of reaction mixtureslacking bacteria were conducted to ensure thatthe trapping of 'Fe was an association of ironwith bacteria and not with the filters. Both theinitial association with iron and the second ratewere reduced by over 90% in assays containingunlabeled ferripyochelin in a 20:1 ratio with['Fe]ferripyochelin. Addition of pyochelin(without iron) in a 20:1 ratio with [MFe]ferripy-ochelin did not affect either of the mechanismsof iron accumulation (data not shown). CelLsincubated with ['Fe]ferripyochelin and withoutoxidizable substrate (succinate) had a compara-ble level of initial association with 'Fe (Fig. lb)to the rate in the presence of succinate (Fig. la),but the second rate of uptake was much slower.The initial accumulation of 'Fe appeared to beindependent of the generation of energy by the

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FIG. 1. 'Fe uptake with ferripyochelin. Reactionmixtures contained 50 nM [5Felferripyochelin with2mM succinate (a), without succinate (b), with 2mMsuccinate and 2 mM DNP (c), and with 2 mM succi-nate and 2mMKCN (d). Reactions were initiated byaddition of[5Fe]ferripyochelin, and iron uptake wasmeasured as 'Fe which was trapped on filters withbacteria from 1-mi samples of reaction mixtures andwhich remained with the bacteria during waterwashes of the filters. Reaction mixtures containedbacteria which had been grown in succinate minimalmedium containing 0.01 pM added FeCl3, and sus-pended in MOPS-Mg buffer at 50 pg (dry weight) ofcells per ml. Succinate and inhibitors were preincu-bated with bacteria for 10min at24°C before additionofferripyochelin. Thepoints represent average valuesfrom assays run in triplicate.

bacteria, occurring in cells incubated with 2 mMDNP (Fig. lc) or with 2 mM KCN (Fig. ld).Incubation of bacteria at 40C also inhibited thesecond rate of uptake, leaving the initial associ-ation of bacteria with [5Fe]ferripyochelin unaf-fected (data not shown).Iron uptake with ferric citrate. Only one

rate of uptake was observed when cells weremixed with [5Fe]ferric citrate instead of ferri-pyochelin (Fig. 2a), but the initial concentrationof 'Fe associated with cells at 5 s was elevated.Increasing the sodium citrate concentration inthe substrate solution from 0.1 mM to 1 or 10mM decreased the initial level ofiron associationwith bacteria and also the rate of iron uptake.The level of initial association with iron re-mained approximately the same, whereas only

the rate of uptake was inhibited by exclusion ofsuccinate (Fig. 2b), the inclusion of 2 mM DNP(Fig. 2c), or the inclusion of 2 mM KCN (Fig.2d) in reaction mixtures.Determination of cell association and up-

take of "5Fe. Additional information regardingthe initial events of iron accumulation was ob-tained by washing the filters and trapped bac-teria from reaction mixtures with 10-ml volumesof 0.5% thioglycolate. Thioglycolate was used inconjunction with a colorimetric assay for Fe(II)(3) to demonstrate the reduction and release ofFe(II) from ferric citrate and ferripyochelin intime intervals similar to those for the washingprocedure for filters. Washing bacteria on filterswith thioglycolate did not disturb the uptake of["4C]succinate by P. aeruginosa and was notlethal to the bacteria (data not shown). How-ever, thioglycolate washes of filters from assayswith [5Fe]ferripyochelin removed much of theinitial association of 'Fe with the trapped bac-teria and left a rate of uptake (Fig. 3a) nearlyidentical to the second rate of iron uptake inreactions with water-washed filters (Fig. 3b).Nearly total inhibition of 'Fe accumulation with

90

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200 d

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1 2 3 4Time (min)

FIG. 2. 55Fe uptake with ferric citrate. Reactionmixtures contained 50 nM [5Fe]ferric citrate with 2mM succinate (a), without succinate (b), with 2 mMsuccinate and 2 mM DNP (c), and with 2 mM succi-nate and 2mMKCN (d). Reactions were initiated byaddition of [fFe]ferric citrate, and iron uptake wasmeasured in a manner identical to those in Fig. 1.

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FIG. 3. Cell association and uptake of [FeJferri-pyochelin. Uptake assays containing 20 nM [5Fel-ferripyocheim and 2 mM succinate were terminatedby washing filters with 0.5% thioglycolate (a) andterminated by washing filters with water (b), and anassay containing 20 nM [mFe]ferripyochelin, 2 mMsuccinate, and 2 mMKCN was terminated by wash-ing filters with 0.5% thioglycolate (c). Reactions wereconducted in a manner identical to those in Fig. 1.

ferripyochelin was achieved by conducting thi-oglycolate washes of bacteria from reaction mix-tures containing 2mM KCN (Fig. 3c). The sameinhibition was noticed with DNP, but KCN wasused to avoid the quench of scintillation count-ing caused by the small amounts of DNP in thefilters. A lower concentration of [MFe]ferripy-ochelin (20 nM) was used in these assays becauseof the prominence of the initial rate of ironaccumulation at this concentration. The smeeffect of thioglycolate washes of filters was ob-served at higher concentrations of [MFe]ferri-pyochelin. Therefore, iron associated with bac-teria in the initial process of accumulation wasvery likely near the bacterial surface to be ac-cessible to reduction with thioglycolate. It wasalso apparent that when [MFe]ferric citrate wasused as substrate thioglycolate washes of filtersyielded a lower quantity of cell-associated 'Fe(Fig. 4a), but a comparable uptake rate to reac-tions with water-washed filters (Fig. 4b). Com-bination of thioglycolate washes of filters withthe use ofKCN in the reaction mixtures yieldeda reduction of both components of [5Fe]ferriccitrate accumulation (Fig. 4c).

It appeared that rates of iron accumulation by

bacteria consisted of cell association and uptakeof iron and that thioglycolate washes merelyreversed the cell association of iron complexesat the terminations of reactions. Although thepresence of at least two reactions made theprocess of uptake by whole cells difficult toanalyze, measurements with thioglycolatewashes of triplicate assays yielded apparent Kmvalues of 78 nM for [5Fe]ferripyochelin uptakeand 100 nM for [5Fe]ferric citrate uptake. Cellassociation with iron complexes was also esti-mated in later studies by the difference betweenvalues obtained at 5 s of reaction with thiogly-colate-washed filters and water-washed filtersfrom assays containing 2 mM KCN.

Effecta of iron concentration on the for-mation ofmechanisms for iron uptake. Suc-cinate minimal medium was reduced in traceiron content by treatment with MgCO3 and thenwas made with increasing concentrations ofeither FeCl3 or ferric citrate. Both pyochelinsynthesis and cell association of 'Fe complexesappeared to correlate with the rates of uptakeand with maximum capabilities appearing incells grown in 0.01 to 0.1 WM FeCk6 and 0.028 to0.28 uM ferric citrate (Table 1).Formation of uptake mechanisms in dif-

ferent growth media. Earlier studies demon-

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FIG. 4. CeU assciation and uptake of['6FeJferriccitrate. Uptake assays containing 50 nM ["6Felferriccitrate and2 mMsuccinate were terminated by wash-ing filters with 0.5% thioglycolate (a) and terminatedby washing filters with water (b), and an assay con-taining 50 nM [FeJferric citrate, 2 mM succinate,and 2 mM KCN was terminated by washing filerswith 0.5% thioglycolate (c). Reactions were conductedin a manner identical to those in Fig. 1.

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IRON UPTAKE BY P. AERUGINOSA 585

TABLE 1. 'Fe uptake in relation to iron supplied in 8uCccinate minimal medium'Fe accumulation (pmol/min/mg of dry cell weight)b

Form ofiroConcn Pyochelina Ferripyochelin Ferric citratesupplid Conc (uM) formed (mg/supplied ~~~~~~~~liter)Upae Cellc associa- Cell associa-

hte)Uptake tion Uptake tionFeCl3 0 5.1 15.0 4.1 11.0 1.0

0.01 6.0 28.1 14.2 12.7 2.10.10 7.0 22.3 15.1 14.3 2.51.00 4.5 19.4 11.0 5.1 1.1

10.00 NDd 2.3 2.8 1.7 0.4Ferric citrate 0.028 6.8 10.5 4.1 15.2 3.1

0.28 7.5 7.7 3.5 13.1 2.72.80 5.6 2.3 1.7 4.0 1.0

28.00 0.4 0.7 0.5 2.4 0.6a Pyochelin was measured fluorometrically in ethanol extracts of areas on chromatograms at the Rf of

pyochelin.b Uptake measured as in Fig. 1.'Cell association of 5Fe estimated by subtracting values determined from thioglycolate-washed filters from

values determined with water-washed filters. All reactions were poisoned with 2 mM KCN.d ND, Not detected.

strated that the lag phase and growth rate were 10prolonged in glucose minimal medium, but thatthe onset of growth could be accelerated byincluding pyochelin in the growth medium (1).On the other hand, stimulatory effects of py-ochelin were less prominent in CAA, succinate,and asparagine minimal media and were negli- Citgible in citrate minimal medium. Figure 5 dis- cplays typical patterns of growth of strain PAO-1 in the various media used to grow celLs for E 1.0-

CAA

assay of iron uptake. CelLs grown in succinate, c CAA Aspasparagine, or CAA media demonstrated mod- da derate rates of growth, relatively high levels of (0pyochelin produced, and relatively high rates of e[5Fe]ferripyochelin uptake (Table 2). On the cother hand, growth in citrate miniimal medium l Dwas comparable to that in succinate, but py- Succ Gluochelin was produced in very low concentra- eS GIutions, and there was very little iron uptake with 9 0.1 b[5Fe]ferripyochelin. In comparison, bacteriagrew slowly in glucose minimal medium with along lag phase and did not produce detectablepyochelin, but had a very active uptake mecha-nism for iron from [5Fe]ferripyochelin. There-fore, separate control mechanisms appear tocontrol pyochelin synthesis and the formation ofthe uptake mechanism for ferripyochelin. The 1 r I Imechanisms for iron uptake from [5Fe]ferric 001 2 3citrate was lower in citrate-grown cells, but was 0 1e0 20 30relatively uniform in all other cells. It is also Time (hours)clear that ferric citrate uptake did not correlate FIG. 5. Growth of P. aeruginosa strain PAO-1 inwith pyochelin synthesis and that the uptake CAA medium (a), succinate minimal medium (b),mechanism for ferric citrate was not induced by citrate minimal medium (c), asparagine minimal me-citrate. dium (d), and glucose minimal medium (e). Growth

DISCUSSION was measured by absorbance at 600nm after inocula-tion of 10' bacteria per ml at zero time during incu-bation at 37°C with shaking.Pyochelin was previously isolated as an iron-

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TABLE 2. Pyochelin production and 5Fe uptake by P. aeruginosa grown in various media55Fe accumulationb (pmol/min/mg of dry cell weight)

Generation Pyochein(/ Ferripyochelin Ferric citrateGrowthsbstratetime (mini) litmer)(g/litei(m)ter) Uptake Cell associa- Uptake Cell associa-Uptke

tionUpae

tionc

Casamino Acids 30 8.3 15.8 9.2 9.3 2.5Succinate 29 6.2 16.7 10.0 10.2 2.5Citrate 32 0.4 2.7 1.2 5.3 1.8Asparagine 40 5.4 20.9 14.0 11.0 2.2Glucose 58 NDd 21.4 15.5 16.5 3.1

a Pyochelin was measured fluorometrically in ethanol extracts of areas on chromatograms at the Rf ofpyochelin.

b Uptake measured as in Fig. 1.c Cell association of 'Fe estimated by subtracting values determined from thioglycolate-washed filters from

values determined with water-washed filters. All reactions were poisoned with 2 mM KCN.d ND, Not detected.

binding compound produced by P. aeruginosa(1). The present paper contains evidence thatpyochelin is a siderophore (7, 11), in that fen-i-pyochelin acts as a substrate for iron transportin P. aeruginosa. Uptake of iron from [5Fe]-ferripyochelin appeared to be composed of tworeactions: an energy-independent associationwith ferripyochelin in the initial 30 s of thereaction followed by a slower rate of energy-dependent transport. Cell association with fer-ripyochelin was measured as the concentrationof 'Fe trapped with bacteria, resisting removalby water washes and the inhibition with respi-ratory poisons, 5 s after substrate addition. Thisphenomenon is similar to one reported in E. coli(15) which was also resistant to the respiratorypoisons DNP and KCN. The rapid rate involvingcell association which occurred in the initial 30s ofreaction appeared to be saturated, or at leastcould not be detected, at a substrate concentra-tion of 60 nM [5Fe]ferripyochelin. A decrease inthe appearance of the initial rate may be ob-served in Fig. 3b in relation to Fig. la, whichcorrelates with an increase in substrate concen-tration. A portion of the cell association with[5Fe]ferripyochelin was reversed with thiogly-colate washes of filters (Fig. 3). The release ofiron from ferripyochelin by reduction of thebound Fe(III) to free Fe(II) (1) can help explainthis reversal. Iron accumulation was a complexof at least two reactions, and even at 5 s therewas considerable iron taken up by bacteriawhich was resistant to the thioglycolate wash.Only when the respiratory poison KCN was usedin conjunction with thioglycolate washes did theinitial values of cell association fall to valuesclose to the 6% residual iron predicted from thecolorimetric assays for the reduction of precipi-tated or complexed iron. Whether this processof cell association is due to specific proteins for

ferripyochelin or is nonspecific, the effect is ahigh concentration of ferripyochelin near thebacterial membrane.Although the amounts of 'Fe uptake ob-

served with P. aeruginosa were approximatelyfivefold less than those values reported with E.coli (5, 15, 16) or with S. typhimurium (9, 13),the concentration of ferripyochelin was also ap-proximately one-fifth that used for ferrientero-chelin. Utilization of this concentration was im-portant in allowing an observation of the tworates in the reaction between P. aeruginosa andferripyochelin. This concentration is also com-parable to the pyochelin concentrations foundin culture media of P. aeruginosa in logarithmicphase (1).

Iron from [5Fe]ferric citrate was also accu-mulated by P. aeruginosa in an energy-depend-ent process. This mechanism is different fromthe one reported in E. coli (4) in that it is notinduced by citrate. There was also an initialenergy-independent cell association of substratewhich was reversed with thioglycolate. The like-lihood of nonspecific mechanisms for cell asso-ciation of ferric citrate was suggested by thecompetition of free citrate with cell-associated[5Fe]ferric citrate, and the small amounts ofvariation in cell association observed in cellsgrown under various conditions (Tables 1 and 2)compared with the large variations in cell asso-ciation of [5Fe]ferripyochelin.

Pyochelin formation, cell association, andmechanisms of iron uptake appeared to be con-trolled by iron concentration in growth media.The large requirement for iron by P. aeruginosa(17) could explain the high concentrations ofiron necessary to produce appreciable effects onformation of iron sequestration mechanisms (10,uM FeCl3 and 28 AM ferric citrate). The greaterability of ferric citrate to inhibit formation of

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IRON UPTAKE BY P. AERUGINOSA 587

iron transport mechanisms and the low levels ofiron sequestration mechanisms in cells grown incitrate minimal medium are probably due to thechelating ability of citrate and the utilization offerric citrate in iron transport. Although no con-clusive explanations for effects of media on ironuptake mechanisms can be given at this time, itis important that growth in glucose minimalmedium allowed no detectable pyochelin pro-duction. This finding indicates that separatecontrol mechanisms govern siderophore synthe-sis and the formation of transport mechanisms.Inability to synthesize pyochelin in glucose min-imal medium also explains the slow growth rateof P. aeruginosa in this medium and the reasonthat this medium is optimal for demonstratingthe growth-promoting effects of pyochelin (1).

Present research is concentrated on separat-ing the events of cell association from the eventsof transport so that accurate kinetic measure-ments may be made of the separate processes.This will also be important for the determinationof the nature of the iron which is transportedacross the membrane, Fe(ll), Fe(llI), or Fe(E)in an iron complex.

ACKNOWLEYDIGMENSThis investigation was supported by Public Health Service

grant Al 13120 from the National Institute of Allergy andInfectious Diseases.

LITERATURE CITED1. Cox, C. D., and R. Graham. 1979. Isolation of an iron-

binding compound from Pseudomonas aeruginosa. J.Bacteriol. 137:357-364.

2. Dailey, H. A., and J. Lascelles. 1977. Reduction of ironand synthesis of protoheme by Spirillum itersonii andother organisms. J. Bacteriol. 129:815-820.

3. Doeg, K. A., and D. M. Ziegler. 1962. Simplified meth-ods for the estimation of iron in mitochondria andsubmitochondrial fractions. Arch. Biochem. Biophys.

97:37-40.4. Frost, G., and H. Rosenberg. 1973. The inducible cit-

rate-dependent iron transport system in Escherichiacoli K12. Biochim. Biophys. Acta 330:90-101.

5. Hancock, R. E. W., K. Hantke, and V. Braun. 1977.Iron transport in Escherichia coli K-12: 2,3-dihydrox-ybenzoate-promoted iron uptake. Arch. Microbiol. 114:231-239.

6. Langman, L., I. G. Young, G. E. Frost, H. Rosenberg,and F. Gibson. 1972. Enterochelin system ofiron transport in Escherichia coli: mutations affecting ferric-en-terochelin esterase. J. Bacteriol. 112:1142-1149.

7. Lankford, C. E. 1973. Bacterial aimiltion of iron. Crit.Rev. Microbiol. 2:273-331.

8. Liu, P. V., and F. Shokrani. 1978. Biological activitiesof pyochelins: iron chelating agents of Pseudomonasaeruginosa. Infect. Immun. 22:878-890.

9. Luckey, ML, J. R. Poliack, R. Wayne, B. N. Ames, andJ. B. Neilands. 1972. Iron uptake in Salmonella typhi-murium: utfliztion of exogenous siderochromes as ironcarriers. J. Bacteriol. 111:731-738.

10. Meyer, J. M., and J. M. Hornsperger. 1978. Role ofpyoverdinepr, the iron-binding fluorescent pigment ofPseudomonas fluorescens, in iron transport. J. Gen.Microbiol. 107:329-331.

11. Neilands, J. B. 1973. Microbial iron transport compounds(siderochromes), p. 167-202. In G. Eichorn (ed.), Inor-ganic biochemistry. Elsevier, Amsterdam.

12. O'Brien, I. G., G. B. Cox, and F. Gibson. 1970. Thestructure of enterochelin and related 2,3-dihydroxyben-zoylserine conjugates from Escherichia coli. Biochim.Biophys. Acta 218:393402.

13. Pollack, J. R., B. N. Ames, and J. B. Neilands. 1970.Iron transport in Salnonella typhimurium mutantsblocked in the biosynthesis of enterobactin. J. Bacteriol.104:635-639.

14. Poflack, J. R., and J. B. Neilands. 1970. Enterobactin,an iron bwanport compound from Salmonella typhi-murium. Biochim. Biophys. Res. Commun. 38:989-992.

15. Pugsley, A. P., and P. Reeves. 1976. Iron uptake incolicin resistant mutants of Escherichia coli K-12. J.Bacteriol. 126:1052-1062.

16. Pugsley, A. P., and P. Reeves. 1977. Uptake of ferrien-terochelin by Escherichia coli: energy dependent stageof uptake. J. Bacteriol. 130:26-36.

17. Waring, W. S., and C. H. Werkman. 1942. Iron require-ments of heterotrophic bacteria. Arch. Biochem. 1:425-433.

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