JouRNAL oF BACTERIOLOGY, Jan. 1979, 0021-9193/79/01-0357/08$02.00/0
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Vol. 137, No. 1
Isolation of an Iron-Binding Compound from Pseudomonas aeruginosa CHARLES D. COX* AND REX GRAHAM Department of Microbiology, University of Iowa, Iowa City, Iowa 52242 Received for publication 17 July 1978
An iron-binding compound was isolated from ethyl acetate extracts of culture supernatant fluids of Pseudomonas aeruginosa and was purified by successive paper and thin-layer chromatographic procedures. The purified compound was characterized by UV, visible, infrared, and fluorescence spectroscopy. The compound possesses phenolic characteristics, with little or no similarity to dihydroxybenzoates and no indication of a hydroxamate group. P. aeruginosa synthesized the compound during active growth in culture media containing less than 5 x 10-6 M added FeCl3. When added to iron-poor cultures of P. aeruginosa, the compound promoted the growth of the bacterium and also reversed growth inhibition by the iron chelator ethylenediamine-di-(o-hydroxyphenylacetic acid).
Iron exists predominantly as insoluble com- terial in an ethyl acetate extract of culture media plexes of Fe(III) in aerobic environments. Pseu- of P. aeruginosa would reverse the inhibitory domonas aeruginosa, a microorganism which activity of an iron chelator, ethylenediamine-diinhabits these environments, must have means (o-hydroxyphenylacetic acid) (EDDA), on the of making the iron soluble for transport into the growth of P. aeruginosa and other bacterial cell for metabolic purposes. The terms sidero- species. This report describes the isolation of an chrome (12) and siderophore (8) have been pro- iron-binding compound with growth-promoting posed to describe microbial metabolites pro- activity from ethyl acetate extracts of culture duced for the purpose of iron chelation and iron media of P. aeruginosa. This compound has transport. This functional role has been estab- been given the trivial name pyochelin as a conlished on the basis of promotion of bacterial venient working designation. growth by specific bacterial iron-chelating compounds. Examples of bacterial siderochromes MATERIALS AND METHODS include enterobactin (14), also called enteroBacterial strains and culture conditions. chelin (13), which is produced by several enteric Strains of P. aeruginosa, designated PAO-1 (ATCC bacteria, mycobactins (15), which are produced 15692) and 10145 (ATCC 10145), were obtained from by Mycobacterium species, and schizokinen, the American Type Culture Collection. Clinical isowhich is produced by species of Bacillus (2). lates of P. aeruginosa were obtained from the Burn The functional groups of these molecules Unit of the University of Iowa Hospital. All isolates thought to be involved in the binding of iron are were maintained in the laboratory on slants of brain infusion agar (Difco) with monthly transfers. phenolic groups in enterobactin, hydroxamate heart strains were routinely grown for producgroups in schizokinen, and both phenolic and tionBacterial of the iron-binding compound in 0.25% Casamino hydroxamate groups possessed by mycobactins. Acids with 0.2 mM MgCl2 at pH 7.5 (CAA). Minimal There have been two reports concerning the media used were glucose minimal medium (GMM), isolation from pseudomonads of compounds gluconate minimal medium (GtMM), citrate minimal with iron-binding properties. A decapeptide con- medium (CMM), and succinate minimal medium taining hydroxamate groups was isolated from (SMM). These were composed of 10 mM concentracultures of P. fluorescens and was given the tions of substrate, 4 mM NH4Cl, 0.1 mM K2S04, 0.2 name ferribactin (10). Garibaldi extracted and mM MgCl2, and 1 mM potassium phosphate buffer at NH4Cl, and K2S04 were automeasured the synthesis of a hydroxamate com- pH 7.5. The substrate, Potassium claved phosphate buffer, MgC12, together. the growth and FeCl3 solutions were sterilized pound which appeared to stimulate by passage through of P. fluorescens (6). membrane filters (0.45-,m pore size; Millipore Corp.) P. aeruginosa has been reported to have a and were added to cooled media just prior to inoculadistinct requirement for iron (16), but no sider- tion. All media used in these experiments contained ochrome has been isolated from this bacterium. sufficient iron for limited growth of P. aeruginosa, Miles and Khimji (11) demonstrated that ma- and no attempt was made to remove the traces of iron 357
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from the media. A supplement of 10 to 50 ,uM FeCl3, depending upon the strain of P. aeruginosa, had to be added to these media to obtain maximal growth rates and yields (dry weight per milliliter of medium) of bacteria. All chemicals for culture media were reagent grade and were obtained from Fisher Scientific Co. Purification of pyochelin. Strain PAO-1 was grown in 1-liter volumes of CAA medium in 2.8-liter Fernbach flasks at 300C for 24 h with vigorous shaking. The bacteria were removed by centrifugation (7,500 rpm for 15 min at 230C), and the supernatant fluid was brought to pH 1 to 2 with HCl. Ethyl acetate was added in a 1:5 ratio, and after vigorous shaking in separatory funnels the ethyl acetate layers were collected and concentrated by rotary evaporation. The residue remaining in the flask after evaporation was dissolved in methanol. The methanol solutions were applied to Whatman no. 1 paper in bands by use of microapplicators (Applied Science Laboratories), and the chromatograms were developed ascending in water-acetic acid-acetone (90:10:1) or in water-acetic acid (90:1), with no saturation of the chromatography jar. After development and drying, the paper chromatogram was viewed under UV light, and the fluorescent bands were marked. A test strip of the chromatogram was sprayed with an iron reagent (0.1 M FeCl3 in 0.1 N HCl), and another test strip was treated with a phenolate spray reagent (1 volume of iron spray reagent added to 1 volume of 0.1 M potassium ferricyanide). Pyochelin appeared as a yellow-green fluorescent band when viewed under UV light, turned red with the iron spray reagent, and turned deep blue with the phenolate spray reagent. The fluorescent band on the unsprayed portion of the paper chromatograms was cut out and eluted in two 100-ml volumes of methanol; the methanol solution was concentrated by rotary evaporation. The residue was dissolved in methanol and applied as a band with a microapplicator to a thin-layer chromatography plate spread with silicic acid (Adsorbosil 5, Applied Science Laboratories). The thin-layer plate was developed in a chloroform-methanol (90:1) solvent (tank saturated). After development of the chromatogram, the plate was observed under UV light, and a test strip was sprayed with the iron spray reagent. The band showing yellow-green fluorescence and yielding a red reaction with the spray reagent was scraped from the plate into methanol and clarified by low-speed centrifugation; the methanol solution was decanted into a weighed crucible. The methanol was removed under vacuum in a desiccator jar, and the weight of the residue was determined. The resulting compound was dissolved in a minimal amount of methanol and was checked for purity on thin-layer plates of silicic acid in four solvent systems: (A) chloroform-acetic acid (90:1), (B) chloroformethanol (4:1), (C) chloroform-acetic acid-ethanol (90: 5:2.5), (D) isopropanol-25% ammonium hydroxide-water (100:10:10). The compound was also chromatographed on thin-layer plates of cellulose (Eastman) with water-acetone-acetic acid (90:5:1) as a solvent system. Any contamination revealed by any of these chromatograms necessitated a second preparative
chromatographic step on a thin layer of silicic acid in solvent A. Analysis of pyochei fQr hydroxamate groups followed the methods for hydroxylamine determination (7) and also the method for determination of the cisnitrosoalkane dimer after periodate oxidation (5). The presence of two or more hydroxyl groups on the aromatic ring was determined by the method of Arnow (1).
The iron-free form of the compound was saturated with iron by adding 0.1 M FeCl3 in 0.1 M HCI to make 0.05 M FeCl3 concentrations in methanol solutions of the compound, and then conducting ethyl acetate extractions of the aqueous layer. The combined ethyl acetate extracts containing the red compound were taken to dryness by rotary evaporation, and the dried material was dissolved in ethyl acetate and extracted with 0.1 M HCI. Successive changes of the HCl layer removed any free iron from the ethyl acetate layer. This solution was then evaporated to dryness under vacuum, weighed, and stored in the cold. Iron in the pyochelin complex was measured by the method for nonheme iron deterniination of Doeg and Ziegler (4). Spectrocopic procedures. Absorption spectra in the visible and UV ranges were obtained with a model 124 Perkin-Elmer double-beam spectrophotometer. Infrared spectra were obtained with a Perkin-Elmer Infracord. Baker instra-analyzed KBr (J. T. Baker Chemical Co.) was used to make the pellets. Fluorometry was conducted with an Aminco-Bowman corrected spectra (SPF) spectrofluorometer. The instrument was standardized before each experiment with a reference solution of quinine sulfate (AmincoBowman). The concentration of pyochelin was determined by exciting methanol solutions at 350 am, measunng the fluorescence at 440 nm, and converting the relative fluorescence to weight (micrograms) of pyochelin by fitting the values to a standard curve. Molecular weight and binding coefficient de. terndation. Estimations of molecular size were determined by thin-layer gel filtration in a TLG apparatus (Pharmacia) us*g a 20 by 40 cm glass plate spread with *0.8-mm layer of G-25 superfine Sephadex gel (Pharmacia). The g6l was swollen and washed in 0.01 M sodium acetate buffer, pH 4.6. Iron was removed from the buffer by adding bathophenanthroline and coaducting multiple extractions with isoamyl alcoh6l to remove the iron complex. Samples of 5 to 10 1 were applied to the layer nd chromatographed in the sodium acetate buffer for 24 h with the plate inclined at a 150 angle. Distances of migration of most samples and standards were measured directly on the plate by detecting fluorescent com,pounds with an UV lamp. Bacitracin was detected by spraying the plate with fluorescamine. Ferrozine, chlorogenic acid, and iron-free pyochelin were also detected with the iron spray reagent. Measurement of the decrease in fluorescence after small additions of iron to pyochelin was used to determine the concentration of pyochelin-iron complex and from this value the binding coefficient (3). The relative fluorescence yields of iron-free, iron-saturated, and partially iron-bound compounds were determined in
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IRON-BINDING COMPOUND FROM P. AERUGINOSA
relation to quinine sulfate prepared at a concentration to equal the absorbance of pyochelin at 350 nm. The concentration required for this technique necessitated the use of ethanol as the solvent. Measurement of pyochelin in supernatant fluids. Bacteria from slants of meat infusion agar were inoculated into 50 ml of CAA medium and grown for 24 h at 300C with vigorous shaking. Bacteria were centrifuged from 40 ml of the culture, and the supernatant fluid was extracted with 20 ml of ethyl acetate in a manner identical to the extraction for purification. Pyochelin concentrations were determined fluorometrically in methanol solutions of those extracts. The extracts were then concentrated and chromatographed on Adsorbosil-5 in solvent C to verify the identity of pyochelin. Production of pyochelin during growth was measured by growing strain PAO-1 for inoculum in CAA medium, harvesting the cells from 40 ml of medium, washing the cells in in three 40-ml volumes of distilled water, and resuspending the cells in 10 ml of distilled water. This inoculum grown with limiting iron was diluted to yield approximately 102 colony-forming units (CFU)/ml in 1 liter of SMM medium. The culture was incubated at 300C with vigorous shaking, and 40-ml quantities were removed at intervals for measurement of optical density at 600 nm and pH, after which the sample was extracted with a 20-ml volume of ethyl acetate as described before and the methanol solution was analyzed fluorometrically for pyochelin. Growth studies. Experiments on the promotion of growth by pyochelin were constructed along the theoretical basis used by Lankford et al. (9). Strains PAO1 and 10145 were grown in CAA medium and prepared for inocula as described before. The cells were diluted into 10.0 ml of media in tubes (20 by 150 mm) to yield approximately 102 CFU/ml. Studies using inocula containing high bacterial numbers were conducted by making the final dilution to yield approximately 10' CFU/ml. Other experiments measured the growth of strain PAO-1 incubated with the iron chelator EDDA. EDDA has been used as an inhibitor of the growth of a variety of bacteria on the basis of its iron-chelating behavior (11). EDDA was incorporated into CAA medium at a final concentration of 1.25 mg/ml, and the pH of the medium was adjusted to 7.5. This concentration of EDDA retarded the onset of growth of P. aeruginosa, but allowed final growth comparable to that in control medium. Purified iron-free and iron-bound pyochelin were added as methanol solutions in volumes up to 0.1 ml and in water solutions which had been adjusted to a pH of 7.0 and sterilized by filtration through a filter with a 0.45-pm pore size (Millipore Corp.). Aqueous solutions of citrate, nitrilotriacetic acid, and FeCl3 were adjusted to pH 7.5, made bacteria-free by filtration, and added to the appropriate tubes. Control cultures were also constructed by adding methanol and water extracts of silicic acid prepared from areas of the thin-layer chromatograms containing compounds other than pyochelin and also areas not used for chromatography. The inoculated tubes were shaken vigorously in a
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water bath at 37°C, and the turbidities of the tube contents were measured at various sampling times in a Spectronic 20 colorimeter (Bausch & Lomb) at 600 nm. Growth was also measured by plating dilutions of the cultures on tryptic soy agar (Difco) and recording the CFU per milliliter.
RESULTS Thin-layer chromatography of ethyl acetate extracts of P. aeruginosa culture media revealed as many as three compounds which turned red when sprayed with 0.1 M FeCl3 in 0.1 N HCI. One of these compounds was produced consistently in low-iron-containing media, possessed a dull yellow-green fluorescence, and yielded the most intense reaction with the iron spray reagent. This compound was purified (Materials and Methods), yielding a light-yellow solid when dried. The material is hygroscopic and was stored at room temperature in a desiccator. The iron-free compound is very soluble in methanol, ethanol, chloroform, ethyl acetate, and benzene, giving colorless to light-yellow solutions in these solvents. The iron-bound compound is more soluble in water than the iron-free form and can also be stored dry for months with no deterioration. Thin-layer chromatography of purified pyochelin yielded single spots of the yellow-green fluorescent compound with the following Rf values: solvent A, 0.19; solvent B, 0.75; solvent C, 0.69; and solvent D, 0.6. The spots gave red reactions with the iron spray reagent and intense blue reactions with the phenolate spray reagent. Material allowed to remain on the thin-layer plates turns an intense yellow color with time and changes from ninhydrin negative to ninhydrin positive. Analysis of as much as 1.0 mg of purified pyochelin for hydroxamate groups generated no hydroxylamine and no absorbance maximum at 267 nm after periodate oxidation. Analysis of similar amounts of the iron-free compound by the Arnow test yielded no red color with the nitrite-molybdate reagent and, therefore, no evidence for an aromatic ring with two or more hydroxyls. A methanol solution containing 50 ,ug of ironfree pyochelin/ml displayed an absorption spectrum with maxima at 218, 248, and 310 nm (curve a, Fig. 1). Pyochelin in the iron-free state is extremely labile and deteriorates to a compound that absorbs at 290 nm and a compound that imparts an intense yellow color to the solution. Iron was then added to the sample cuvette in increasing amounts until there was no further increase in absorption at 520 nm. At pH 2.5, the absorption spectrum of the iron-saturated com-
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form by adding iron and then back'to the free form by dithionite addition, and each form could be recognizd by thin-layer chromatography. While conducting these reversible reactions, only the iron-bound form could be detected by spraying the plates with 1.0% potassum ferrocyanide and oberving the blue reaction with iron. Colorimetric assay for iron indicated no detectable iron in the free form ofthe compound, but 0.104 jg of iron per pg of the iron-bound form. The migration of compounds with known molecular weights on G-25 superfine Sephadex yielded the linear relationship shown in Fig. 4. Fitting the distances of migration of iron-free and iron-bound pyochelin to this curve implies
1.8
1.6 1.4 LU
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04 0.2 -..
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WAVELENGTH (nm)
FIG. 1. UV and visibk absorption pectra of pu-
rified pyochdlin. (a) Spectrum of iron-free pyochelin (50 pg/mi) in methanol. (b) Spectrum offerripyochelin
(50 ps/lmi in methanol at pH 2.5. The reference solution was methanol. 0I
pound displayed maxima at 237, 2i5, 325, 425, and 520 nm (curve b, Fig. 1). This wine-red solution changed to orange and displayed a shift in absorption maximum from 520 to 488 nm when the pH was adjusted to values between 4.0 and 7.0. The iron-free forn of pyochelin is fluorescent; its corrected excitation spectrum has mama at 235, 272, ad32 nm and prominent troughs at 252 an 0 nm when the emission is measured at 442/mm (curve a, Fig. 2). The corrected emission setrum has a single maximum at 442 nm when excited at 352 nm (curve b, Fig. 2). Excitng the sample at 235 or 272 nm also yields a single emision maximum at 442 nm. Addition of iron yields a nonfluorescent compound. The infrared spectra of iron-free pyochelin (panel A) and ferripyochelin (panel B) are shown in Fig. 3. Iron binding by pyochelin was demonstrated by two-dimensional chromatography on silicic acid in chloroform-acetic acid-ethanol (90:5:5). The iron-free compound yielded one fluorescent spot at R1 0.80 when chromatographed in the first dimension, and yielded one red spot at Rf 0.24 when iron was added to the edge of the chromatogram containing the compound and the plate was turned and developed in the same solvent in the second dimension. Pyochelin could be changed from the free to the bound
Wavelength (nanometes)
fluoresence spectra of pyochein (a) Excitation spectum of a methanol solution ofpyochelin (10 pg/mi); emision was measred at 42 nm. (b) Emission spectnm of the same pyochelin solution, with the sampk excited at 352 nm. FIG. 2. Corrcted
Wavenumber (cm ')
3.000 100 -1 A so
2.000
1.500
I
I
800
1.000 I
I
I
I
I
I
-
60
c
0
2 S
40
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20
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a
a
100
so
Wavelength (microns)
FIG. 3. Infrared spectra of pyochelin. (A) Absorption spectrum of iron-free pyochelin. (B) Absorption spectrum of ferpyochelin. Sampls of I mg of each preparation were pressed into KBr di"sk, with the ue of 2 mg of KBr.
IRON-BINDING COMPOUND FROM P. AERUGINOSA
VOL. 137, 1979
BaciIracnr)
300
/
5.2 g (wet weight) of cells did not yield detectable amounts of it. In media of high iron content, P. aeruginosa produces less pyochelin. Strain PAO-1 was
Flavin Adenine Dinuicleotide
c
'0
200 o
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A
-
E /
Riboflavin 1
Ferrozine
05 .
Iron-free Pyochelin ..fron-bound Pyochelin
Chiorogenic
Acid
Lumichrome
100
1000 500 Molecular Weight
2000
c 01 0
FIG. 4. Thin-layergel filtration. Samples of5 to l10
0005 -
pl of known compounds and purified pyochelin were
applied to a 0.8-mm layer of G-25 superfilne Sephadex which had been equilibrated to the flow of sodium acetate (pH 4.6) buffer for 24 h at an inclined angle of 15°. The plate was developed for 24 h, and the fluorescent compounds were marked directly on the layer. Bacitracin was detected with fluorescamine, and iron chelators were detected with the iron spray reagent. Distances were measured from the origin to the center of the spots.
that the iron-bound form is a molecule of smaller size than the iron-free compound. Hydrophobic behavior of the pyochelin molecules might have retarded its migration somewhat in the aqueous solvent, but it was concluded that the molecular weight of pyochelin probably lies between 400 and 600. These values were used to obtain an estimate of the affinity of pyochelin for iron by using fluorescence methods (3). The association constants obtained were 1.37 x 105 for a compound with a molecular weight of 400 and 2.04 x 105 for a molecule with a molecular weight of
0.,1
10 0
B
5
-. - 0 os )
'O1 E
0
005
600.
Synthesis of pyochelin. Pyochelin produc0 001 tion attained levels of 3 to 5 ng/ml very early in the culture (Fig. 5A) and remained constant 0005 0 until the culture assumed a reduced rate of growth (Fig. 5B), at which time there was a 3 3 2 25 1 1 . massive production of pyochelin. The identity of 0 001 1 0 35 15 20 30 25 the pyochelin produced early in culture was T -e HOOrs verified by thin-layer chromatography. When FIG. 5. Synthesis of pyochelin by strain PAO-1I the bacteria ceased to grow exponentially, pyochelin synthesis also slowed, and the concen- during growth in SMM. Bacteria were inoculated attF tration did not decrease over the next 20 h of 102 CFU/ml in I liter ofSMM, and 50-ml amounts of for mea-I times subsequent removed incubation. The compound was not altered chro- medium were the amount and of pyochelim (A) at growth xtendd inuba-surement matogaphiaRy dringthe incuba- extracted of during the extended matographically into ethyl acetate (B). Pyochelin concentra tion. Experiments with GMM and CAA medium tions were determined fluorometrically in methanolI yielded similar results. Pyochelin is an extracel- solutions and presented as micrograms per milliliterr lular product, and ethyl acetate extracts from of culture medium.
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grown from inocula of 10' CFU/ml in 50-ml quantities of SMM in 250-ml flasks with the following iron concentrations: 0, 0.1, 0.5, 1.0, 5.0, and 10.0 IM FeCh3. The quantities of pyochelin in methanol solutions of ethyl acetate extracts from the flasks containing the increasing concentrations of iron were 5.6, 7.0, 4.5, 0.3, and 0 ug/ml, respectively. All of the 43 clinical isolates from the Burn Unit, University of Iowa Hospital, produced large quantities of pyochelin. Inoculation of CAA medium with large numbers of cells from rich media often resulted in low pyochelin production, however.
Growth-promoting effects of pyochelin. To determine the effect of the iron-binding compound on the growth of P. aeruginosa, iron-free or iron-bound pyochelin was added to 10.0-ml quantities of various culture media in tubes (50 by 120 mm) inoculated with strain 10145 at 102 CFU/ml. Both the iron-free and iron-bound forms of the compound decreased the lag phase and increased the growth rate in GMM, SMM, GtMM, and CAA media, with the observed effects decreasing in the order the media are listed. The most notable stimulation was found in GMM, evidenced by a decrease in the lag phase of 15 h with 6.0 ,ug of iron-bound pyochelin/ml added (curve c, Fig. 6), in comparison with the same culture lacking pyochelin (curve a, Fig. 6). The effects of pyochelin on the lag phase of growth were dependent upon inoculum size; in experiments using 106 CFU/ml, the lag phase was very short and there was little or no effect of pyochelin. Iron in concentrations of 1.0 or 10.0 ,uM yielded some growth stimulation (curve b, Fig. 6). In CMM there was always rapid onset of growth with little or no stimulation of growth by pyochelin addition, and citrate added to GMM enhanced the growth of the bacteria (curve d, Fig. 6) to the same extent as pyochelin. Miles and Khimji (11) showed that a component of the ethyl acetate extract of P. aeruginosa cultures can reverse the inhibition of the iron chelator EDDA, EDDA was incorporated into CAA medium at a concentration of 1.25 mg/ml, and strain PAO-1 was inoculated at 102 CFU/ml, with and without pyochelin. This concentration of EDDA resulted in a long lag (curve b, Fig. 7) compared to growth in CAA rmedium without EDDA (curve a, Fig. 7). Addition of either iron-bound or iron-free pyochelin to CAA medium without EDDA gave the slight stimulation of growth characteristic for strain PAO-1 in CAA medium (curve c, Fig. 7). Addition of both iron-free pyochelin (curve d, Fig. 7) and iron-bound pyochelin (curve e, Fig. 7) to CAA
10
0.5
0.
0.10
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o~ . 10
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50
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FIG. 6. Growth-promoting effects offerripyochelin for strain 10145 grown in GMM. Bacteria weregrown for inoculum in SMM, washed three times, and inoculated at approximately 102 CFU/ml in 10-ml quantities of GMM. Growth was measured by absorbance at 600 nm in tubes (20 by 150 mm) incubated with shaking at 37°C. (a) Growth in GMM; (b) growth in GMM with 1.0 isM FeCl3; (c) growth in GMM with 6 pg of ferripyochelin/ml; (d) growth in GMM supplemented with 0.5 mM sodium citrate.
medium containing EDDA reversed this compound's growth inhibition. Citrate and nitrilotriacetic acid were also added to cultures containing EDDA at 0.05 and 0.05 mM. Neither compound reversed EDDA inhibition. Extracts of the silicic acid thin-layer chromatograms outside the pyochelin band did not reverse EDDA inhibition. Addition of a 10 AM concentration of FeCl3 to the CAA medium containing EDDA allowed more rapid onset of growth than with EDDA alone, but did not reverse the inhibition as well as iron-free pyochelin.
DISCUSSION The iron-binding compound pyochelin can be prepared in 20-mg amounts from 6 liters of culture media within a 2-day period by use of preparative paper and thin-layer chromatography. Strain PAO-1 in CAA medium typically
VOL. 137, 1979
IRON-BINDING COMPOUND FROM P. AERUGINOSA
01 (0.
0~0
0
01
5
10
15 20 Time (Hours)
25
30
35
FIG. 7. Inhibition of growth by ElDDA and reversal of inhibition by pyochelin. Stra,in PAO-I was inoculated (102 CFU/ml) into tubes (20 by 150 mm) containing 10 ml of CAA medium. The tubes were shaken vigorously at 37°C, and growtih was measured at various times by ubsorbance at 6(X) nm in (a) medium alone, (b) medium with 1.25 nmg ofEDDA/mi, (c) medium with either 7.2 pg ofpyoci Wieln/ml or 6 g of ferripyochelin/ml, (d) medium cor plus 7.2 pg of pyochelin/ml, and (e')medium with EDDA plus 6.0 pg of ferripyochelinlnaXl.
produced 6 mg of pyochelin per iter. The bacteria produced the compound in media having less than 5.0 ,uM added FeCl3. Thin-layer chromatography was used to demonst rate the widespread occurrence of this compouLnd in the culture media of over 40 different strains of P. aeruginosa. These aspects of th e synthesis of pyochelin are similar to the produ ction of enterochelin by Escherichia coli. Althoiugh pyochelin resembles enterochelin in solubiliity and a positive reaction with the phenolate.spray reagent, absorption and fluorescence sp5 ectra indicate that the compounds are different. The fluorescence of the molecule indicates the presence of an aromatic ring. Akn absorption maximum at 350 nm matches th e fluorescence excitation maximum at 350 nm aLt alkaline pH, but the other maxima of the absorption spectra do not match the fluorescence exciLtation spectra. There is indication of the absorpttion of an aromatic hydroxyl at 310 nm; howevrer, there is no matching peak in the fluoresceince excitation spectrum. The bathochromic shilft of this peak in more alkaline conditions woulid also suggest the ionization of an aromatic hydr*oxyl. The negative Arnow test suggests that the phenolic char-
363
acter of this compound is expressed by one hydroxyl on the aromatic ring rather than the two hydroxyls as in the case of enterochelin. A positive ninhydrin reaction was not observed with the intact molecule, but became apparent after deterioration of the compound on the thin-layer chromatograms. The infrared spe6trum was included to demonstrate further the- lack of similarity between pyochelin and enterochelin. Notable is the absence of absorption at 1,760 cm-', where enterochelin and 2,3-dihydroxybenzoyl-serine absorb strongly as a result of strained ester group carbonyl stretch (12, 14). Gel permeation on thin layers of G-25 Sephadex suggests a molecular weight in the range of 400 to 600. However, this may be an underestimate due to the use of aqueous solvents. Hydrophobic molecules such as 8-hydroxyquinoline chromatographed more slowly than their molecular weights would suggest. Ferripyochelin is more water soluble than iron-free pyochelin and should be less susceptible to hydrophobic retardation. These data suggest that iron-free pyochelin may be an open molecule which closes around iron during binding and assumes a smaller size. In the absence of added iron, the iron-free compound will enhance the growth of both strains 10145 and PAO-1. Theoretically, pyochelin may aid the bacteria by chelating and making available the trace amounts of iron in the medium. Adding 6 ,tg of iron-bound compound/mnl is equivalent to adding a 10 ,LM concentration of FeCl3, and there was dramatic stimulation of growth, greater than from adding that concentration of iron alone. Ferric chloride added at 1.0 or 10.0 ,uM concentrations caused only a sLight decrease in the lag phase. It was expected that strain 10145 would be more sensitive to the effects of iron chelators since the growth of this strain exhibits a longer lag phase and is slower than that of strain PAO-1 in all of the media used. Two rates of growth were observed in Fig. 5A and in media containing only trace amounts of iron. In these media there is rapid logarithmic growth followed by a slower rate beginning at an absorbance of around 0.2. This may be noticed in Fig. 6, but was negligible in Fig. 7, for in CAA medium the bacteria typically display shorter lag phases and higher final absorbance values. This phenomenon is probably due to iron deprivation at high bacterial densities, and this deprivation elicits a second phase of pyochelin synthesis (Fig. 5B). The slower rate of growth occurred at bacterial densities of around 2 x 108 CFU/ml in GMM and SMM and was less ap-
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parent with the addition of iron or ferripyochelin (Fig. 6). Pyocheln is the only compound detected in ethyl acetate extracts of culture media capable of reversing the inhibition of growth by EDDA. On the basis of this activity, it appears to be the compound extracted by Miles and Khimji (11) which was capable of reversing the EDDA in-
port Grant RR 06372 from the Biomedical Research Support Branch, Division of Research Facilities and Resources, Na-
tional Institutes of Health
LlrrFTUE
1.
1937. Colorimetric determination of the components of 3,4-dihydroxyphenylalanine-tyrosine
mixur. J. Bio. Chem. 118:531-537. 2. Boyers, B.R., AL V. Powell, and C. E. aord 1967.
active in (schizokinen) Iron-chelating J. of cellhydroxamic division inacidBacillus initiation megaterium.
hibition of a number of bacterial genera. The
Bacteriol. 93:286-294.
nhibition of growth by EDDA was relieved by adding 10.0 pM FeCla; other iron chelators which
CrriD
Arnow, L E.
3.
Davenport, D. 1971. Use of fluoresence in binding studies, p. 203-240. In A. Peace, C.-G. Rose, and T. L. Pasby
enhanced growth, citrate and nitrilotriacetic (ed.), Fluorescence spectroscopy. M. Dekker, Inc., New acid, did not completely relieve the inhibition. York. The finding that no other metals or metal che- 4. Doeg, K. A., and D.ML Ziegler. 1962. Simplified methods for the estimation of iron in mitochondria and lates of pyochelin stimulate the growth of P. submitochondrial fractions. Arch. Biochem. Biophys. aeruginosa in the media used (data not shown) 97:37-40. 'indlicates that the growth-promoting effects of 5.s. Emery, T. 1962. Further observations concerning the periodic acid oxidation of hydroxylamine derivatives. J. pyochelin are on the basis of iron uptake. TheOrg. Chem. 27:1075. oretically, the iron in the medium exists in the EDDA complex, and initiation of growth de- 6. Garibaldi, J. A. 1971. Influence of temperature on the ism of a fluorescent peudomonad. J. Baciron m pends upon accumulation of sufficient pyochelin 1":103-103. the pseuform for frtepe-ii sbefn 7. Gibson, F., and D. I. McGrath. 1969. The isolation and irniin a usable tondsequpone to sequester iron characteization of a hydroxamic acid (arobactin) domonads. The estimate of the binding coeffiformed by Aerobacter aerogenes. Biochim Biophys. cient describes the iron-binding by pyochelin in Acta 192:175-181. ethaol. The binding coefficient in aqueous meBacterial assimilation of iron. Crit. C. K 1973. 8Laor be higher than this value (2.4 x 1W) dia mamay Microbiol. 2:273-331. ~~~~~~~~~~~~~~Rev. 9. J. R. of to inhipyochelin the reverse Walker, J. B. Reeves, N. EL Lankfod, C. the F., and ability Nabbut B. R. Beyers, and R. J. Jons 1965. Inocbition of EDDA (binding coefficient, 10'") sugpyochelin~ tulum-dependent division lag of Bacilus culture and binding coefficient If this. gests If the ofiin offpoh th.idi tb. gesU~~~~~~~. for iron in aqueous media is found to be near 10 reversal of EDDA inhibition may indicate some specificity in the interaction of P. aerugosa
its reaion to an ononous factor(s)
lo.
( uiolnn).J.
Biactsriol. 91:1070t1079. Maure, B., A. Miiller, W. Keller4Schierlein, and H. zibner. 1968Stoffechaelproducktevonmikroorgan-
n. Frribactin, sin siderochrom aus Peudomonaa with feripyochelin. However, it is also possible Mikrobiol 60:326-339. iron for that pyochelin acts to solubieweMAies, A. A., Migula. and P. Arch. L Khlmi. 1975. Enterob l transport without having a receptor siteon-te chatora of iron: their oc n, d on, and rdat to pathognicity. J. Med. Microbiol. 8:477-490. bacterial surface for specific transport processes. J. B. 1973. Microbial iron traportcompounds Current studies on the structure of pyochelin 12. N In G. Eichorn (ed.), Inor(siderochromes), aer.ginosa by P.. aerugua of iron by transport ofIron andthe the active active transport a and bioch p.. 167-202. Elsevier, srdm pganic will resolve the status of this compound. Even if 13. OYBrien, L G, and F. Gibson. 1970. The structure of enterochlin and related 2,3-dihydrozy-N-benzoyl erno specific transport system is found for pyine conuates from Escherichia coi. Biochim. Bioochelin, the evidence suggests that the molecule A,ca1dJ9 binds iron and prsents iron to the bacteria in a 14. PohaysJ. N3-402 B.1.70.. usable form. from Salmoeua tpan iron tanport compo munw. Biocem Biophys Res. Commun. 3:88-992. iron-chelating growth A. Mycobactin 15. Snow, ACKNOWLEDGMENTS factosB.from 1970. Bacteriol. Rev. 34:99-125. Mycobacteria. This investigation was supported by Public Health Service 16. WarIn, W. 8, and C H. Werkman. 1942. Iron requiegrant Al 13120 from the National Institute of Allorgy and Infectious Diseases and in part by Biomedica Research Sup-
ments of hetrotrophic bactria. Arch. Biochem. 1: 425-433.
p.
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Vol. 137, No. 1
Isolation of an Iron-Binding Compound from Pseudomonas aeruginosa CHARLES D. COX* AND REX GRAHAM Department of Microbiology, University of Iowa, Iowa City, Iowa 52242 Received for publication 17 July 1978
An iron-binding compound was isolated from ethyl acetate extracts of culture supernatant fluids of Pseudomonas aeruginosa and was purified by successive paper and thin-layer chromatographic procedures. The purified compound was characterized by UV, visible, infrared, and fluorescence spectroscopy. The compound possesses phenolic characteristics, with little or no similarity to dihydroxybenzoates and no indication of a hydroxamate group. P. aeruginosa synthesized the compound during active growth in culture media containing less than 5 x 10-6 M added FeCl3. When added to iron-poor cultures of P. aeruginosa, the compound promoted the growth of the bacterium and also reversed growth inhibition by the iron chelator ethylenediamine-di-(o-hydroxyphenylacetic acid).
Iron exists predominantly as insoluble com- terial in an ethyl acetate extract of culture media plexes of Fe(III) in aerobic environments. Pseu- of P. aeruginosa would reverse the inhibitory domonas aeruginosa, a microorganism which activity of an iron chelator, ethylenediamine-diinhabits these environments, must have means (o-hydroxyphenylacetic acid) (EDDA), on the of making the iron soluble for transport into the growth of P. aeruginosa and other bacterial cell for metabolic purposes. The terms sidero- species. This report describes the isolation of an chrome (12) and siderophore (8) have been pro- iron-binding compound with growth-promoting posed to describe microbial metabolites pro- activity from ethyl acetate extracts of culture duced for the purpose of iron chelation and iron media of P. aeruginosa. This compound has transport. This functional role has been estab- been given the trivial name pyochelin as a conlished on the basis of promotion of bacterial venient working designation. growth by specific bacterial iron-chelating compounds. Examples of bacterial siderochromes MATERIALS AND METHODS include enterobactin (14), also called enteroBacterial strains and culture conditions. chelin (13), which is produced by several enteric Strains of P. aeruginosa, designated PAO-1 (ATCC bacteria, mycobactins (15), which are produced 15692) and 10145 (ATCC 10145), were obtained from by Mycobacterium species, and schizokinen, the American Type Culture Collection. Clinical isowhich is produced by species of Bacillus (2). lates of P. aeruginosa were obtained from the Burn The functional groups of these molecules Unit of the University of Iowa Hospital. All isolates thought to be involved in the binding of iron are were maintained in the laboratory on slants of brain infusion agar (Difco) with monthly transfers. phenolic groups in enterobactin, hydroxamate heart strains were routinely grown for producgroups in schizokinen, and both phenolic and tionBacterial of the iron-binding compound in 0.25% Casamino hydroxamate groups possessed by mycobactins. Acids with 0.2 mM MgCl2 at pH 7.5 (CAA). Minimal There have been two reports concerning the media used were glucose minimal medium (GMM), isolation from pseudomonads of compounds gluconate minimal medium (GtMM), citrate minimal with iron-binding properties. A decapeptide con- medium (CMM), and succinate minimal medium taining hydroxamate groups was isolated from (SMM). These were composed of 10 mM concentracultures of P. fluorescens and was given the tions of substrate, 4 mM NH4Cl, 0.1 mM K2S04, 0.2 name ferribactin (10). Garibaldi extracted and mM MgCl2, and 1 mM potassium phosphate buffer at NH4Cl, and K2S04 were automeasured the synthesis of a hydroxamate com- pH 7.5. The substrate, Potassium claved phosphate buffer, MgC12, together. the growth and FeCl3 solutions were sterilized pound which appeared to stimulate by passage through of P. fluorescens (6). membrane filters (0.45-,m pore size; Millipore Corp.) P. aeruginosa has been reported to have a and were added to cooled media just prior to inoculadistinct requirement for iron (16), but no sider- tion. All media used in these experiments contained ochrome has been isolated from this bacterium. sufficient iron for limited growth of P. aeruginosa, Miles and Khimji (11) demonstrated that ma- and no attempt was made to remove the traces of iron 357
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from the media. A supplement of 10 to 50 ,uM FeCl3, depending upon the strain of P. aeruginosa, had to be added to these media to obtain maximal growth rates and yields (dry weight per milliliter of medium) of bacteria. All chemicals for culture media were reagent grade and were obtained from Fisher Scientific Co. Purification of pyochelin. Strain PAO-1 was grown in 1-liter volumes of CAA medium in 2.8-liter Fernbach flasks at 300C for 24 h with vigorous shaking. The bacteria were removed by centrifugation (7,500 rpm for 15 min at 230C), and the supernatant fluid was brought to pH 1 to 2 with HCl. Ethyl acetate was added in a 1:5 ratio, and after vigorous shaking in separatory funnels the ethyl acetate layers were collected and concentrated by rotary evaporation. The residue remaining in the flask after evaporation was dissolved in methanol. The methanol solutions were applied to Whatman no. 1 paper in bands by use of microapplicators (Applied Science Laboratories), and the chromatograms were developed ascending in water-acetic acid-acetone (90:10:1) or in water-acetic acid (90:1), with no saturation of the chromatography jar. After development and drying, the paper chromatogram was viewed under UV light, and the fluorescent bands were marked. A test strip of the chromatogram was sprayed with an iron reagent (0.1 M FeCl3 in 0.1 N HCl), and another test strip was treated with a phenolate spray reagent (1 volume of iron spray reagent added to 1 volume of 0.1 M potassium ferricyanide). Pyochelin appeared as a yellow-green fluorescent band when viewed under UV light, turned red with the iron spray reagent, and turned deep blue with the phenolate spray reagent. The fluorescent band on the unsprayed portion of the paper chromatograms was cut out and eluted in two 100-ml volumes of methanol; the methanol solution was concentrated by rotary evaporation. The residue was dissolved in methanol and applied as a band with a microapplicator to a thin-layer chromatography plate spread with silicic acid (Adsorbosil 5, Applied Science Laboratories). The thin-layer plate was developed in a chloroform-methanol (90:1) solvent (tank saturated). After development of the chromatogram, the plate was observed under UV light, and a test strip was sprayed with the iron spray reagent. The band showing yellow-green fluorescence and yielding a red reaction with the spray reagent was scraped from the plate into methanol and clarified by low-speed centrifugation; the methanol solution was decanted into a weighed crucible. The methanol was removed under vacuum in a desiccator jar, and the weight of the residue was determined. The resulting compound was dissolved in a minimal amount of methanol and was checked for purity on thin-layer plates of silicic acid in four solvent systems: (A) chloroform-acetic acid (90:1), (B) chloroformethanol (4:1), (C) chloroform-acetic acid-ethanol (90: 5:2.5), (D) isopropanol-25% ammonium hydroxide-water (100:10:10). The compound was also chromatographed on thin-layer plates of cellulose (Eastman) with water-acetone-acetic acid (90:5:1) as a solvent system. Any contamination revealed by any of these chromatograms necessitated a second preparative
chromatographic step on a thin layer of silicic acid in solvent A. Analysis of pyochei fQr hydroxamate groups followed the methods for hydroxylamine determination (7) and also the method for determination of the cisnitrosoalkane dimer after periodate oxidation (5). The presence of two or more hydroxyl groups on the aromatic ring was determined by the method of Arnow (1).
The iron-free form of the compound was saturated with iron by adding 0.1 M FeCl3 in 0.1 M HCI to make 0.05 M FeCl3 concentrations in methanol solutions of the compound, and then conducting ethyl acetate extractions of the aqueous layer. The combined ethyl acetate extracts containing the red compound were taken to dryness by rotary evaporation, and the dried material was dissolved in ethyl acetate and extracted with 0.1 M HCI. Successive changes of the HCl layer removed any free iron from the ethyl acetate layer. This solution was then evaporated to dryness under vacuum, weighed, and stored in the cold. Iron in the pyochelin complex was measured by the method for nonheme iron deterniination of Doeg and Ziegler (4). Spectrocopic procedures. Absorption spectra in the visible and UV ranges were obtained with a model 124 Perkin-Elmer double-beam spectrophotometer. Infrared spectra were obtained with a Perkin-Elmer Infracord. Baker instra-analyzed KBr (J. T. Baker Chemical Co.) was used to make the pellets. Fluorometry was conducted with an Aminco-Bowman corrected spectra (SPF) spectrofluorometer. The instrument was standardized before each experiment with a reference solution of quinine sulfate (AmincoBowman). The concentration of pyochelin was determined by exciting methanol solutions at 350 am, measunng the fluorescence at 440 nm, and converting the relative fluorescence to weight (micrograms) of pyochelin by fitting the values to a standard curve. Molecular weight and binding coefficient de. terndation. Estimations of molecular size were determined by thin-layer gel filtration in a TLG apparatus (Pharmacia) us*g a 20 by 40 cm glass plate spread with *0.8-mm layer of G-25 superfine Sephadex gel (Pharmacia). The g6l was swollen and washed in 0.01 M sodium acetate buffer, pH 4.6. Iron was removed from the buffer by adding bathophenanthroline and coaducting multiple extractions with isoamyl alcoh6l to remove the iron complex. Samples of 5 to 10 1 were applied to the layer nd chromatographed in the sodium acetate buffer for 24 h with the plate inclined at a 150 angle. Distances of migration of most samples and standards were measured directly on the plate by detecting fluorescent com,pounds with an UV lamp. Bacitracin was detected by spraying the plate with fluorescamine. Ferrozine, chlorogenic acid, and iron-free pyochelin were also detected with the iron spray reagent. Measurement of the decrease in fluorescence after small additions of iron to pyochelin was used to determine the concentration of pyochelin-iron complex and from this value the binding coefficient (3). The relative fluorescence yields of iron-free, iron-saturated, and partially iron-bound compounds were determined in
VOL. 137, 1979
IRON-BINDING COMPOUND FROM P. AERUGINOSA
relation to quinine sulfate prepared at a concentration to equal the absorbance of pyochelin at 350 nm. The concentration required for this technique necessitated the use of ethanol as the solvent. Measurement of pyochelin in supernatant fluids. Bacteria from slants of meat infusion agar were inoculated into 50 ml of CAA medium and grown for 24 h at 300C with vigorous shaking. Bacteria were centrifuged from 40 ml of the culture, and the supernatant fluid was extracted with 20 ml of ethyl acetate in a manner identical to the extraction for purification. Pyochelin concentrations were determined fluorometrically in methanol solutions of those extracts. The extracts were then concentrated and chromatographed on Adsorbosil-5 in solvent C to verify the identity of pyochelin. Production of pyochelin during growth was measured by growing strain PAO-1 for inoculum in CAA medium, harvesting the cells from 40 ml of medium, washing the cells in in three 40-ml volumes of distilled water, and resuspending the cells in 10 ml of distilled water. This inoculum grown with limiting iron was diluted to yield approximately 102 colony-forming units (CFU)/ml in 1 liter of SMM medium. The culture was incubated at 300C with vigorous shaking, and 40-ml quantities were removed at intervals for measurement of optical density at 600 nm and pH, after which the sample was extracted with a 20-ml volume of ethyl acetate as described before and the methanol solution was analyzed fluorometrically for pyochelin. Growth studies. Experiments on the promotion of growth by pyochelin were constructed along the theoretical basis used by Lankford et al. (9). Strains PAO1 and 10145 were grown in CAA medium and prepared for inocula as described before. The cells were diluted into 10.0 ml of media in tubes (20 by 150 mm) to yield approximately 102 CFU/ml. Studies using inocula containing high bacterial numbers were conducted by making the final dilution to yield approximately 10' CFU/ml. Other experiments measured the growth of strain PAO-1 incubated with the iron chelator EDDA. EDDA has been used as an inhibitor of the growth of a variety of bacteria on the basis of its iron-chelating behavior (11). EDDA was incorporated into CAA medium at a final concentration of 1.25 mg/ml, and the pH of the medium was adjusted to 7.5. This concentration of EDDA retarded the onset of growth of P. aeruginosa, but allowed final growth comparable to that in control medium. Purified iron-free and iron-bound pyochelin were added as methanol solutions in volumes up to 0.1 ml and in water solutions which had been adjusted to a pH of 7.0 and sterilized by filtration through a filter with a 0.45-pm pore size (Millipore Corp.). Aqueous solutions of citrate, nitrilotriacetic acid, and FeCl3 were adjusted to pH 7.5, made bacteria-free by filtration, and added to the appropriate tubes. Control cultures were also constructed by adding methanol and water extracts of silicic acid prepared from areas of the thin-layer chromatograms containing compounds other than pyochelin and also areas not used for chromatography. The inoculated tubes were shaken vigorously in a
359
water bath at 37°C, and the turbidities of the tube contents were measured at various sampling times in a Spectronic 20 colorimeter (Bausch & Lomb) at 600 nm. Growth was also measured by plating dilutions of the cultures on tryptic soy agar (Difco) and recording the CFU per milliliter.
RESULTS Thin-layer chromatography of ethyl acetate extracts of P. aeruginosa culture media revealed as many as three compounds which turned red when sprayed with 0.1 M FeCl3 in 0.1 N HCI. One of these compounds was produced consistently in low-iron-containing media, possessed a dull yellow-green fluorescence, and yielded the most intense reaction with the iron spray reagent. This compound was purified (Materials and Methods), yielding a light-yellow solid when dried. The material is hygroscopic and was stored at room temperature in a desiccator. The iron-free compound is very soluble in methanol, ethanol, chloroform, ethyl acetate, and benzene, giving colorless to light-yellow solutions in these solvents. The iron-bound compound is more soluble in water than the iron-free form and can also be stored dry for months with no deterioration. Thin-layer chromatography of purified pyochelin yielded single spots of the yellow-green fluorescent compound with the following Rf values: solvent A, 0.19; solvent B, 0.75; solvent C, 0.69; and solvent D, 0.6. The spots gave red reactions with the iron spray reagent and intense blue reactions with the phenolate spray reagent. Material allowed to remain on the thin-layer plates turns an intense yellow color with time and changes from ninhydrin negative to ninhydrin positive. Analysis of as much as 1.0 mg of purified pyochelin for hydroxamate groups generated no hydroxylamine and no absorbance maximum at 267 nm after periodate oxidation. Analysis of similar amounts of the iron-free compound by the Arnow test yielded no red color with the nitrite-molybdate reagent and, therefore, no evidence for an aromatic ring with two or more hydroxyls. A methanol solution containing 50 ,ug of ironfree pyochelin/ml displayed an absorption spectrum with maxima at 218, 248, and 310 nm (curve a, Fig. 1). Pyochelin in the iron-free state is extremely labile and deteriorates to a compound that absorbs at 290 nm and a compound that imparts an intense yellow color to the solution. Iron was then added to the sample cuvette in increasing amounts until there was no further increase in absorption at 520 nm. At pH 2.5, the absorption spectrum of the iron-saturated com-
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COX AND GRAHAM
form by adding iron and then back'to the free form by dithionite addition, and each form could be recognizd by thin-layer chromatography. While conducting these reversible reactions, only the iron-bound form could be detected by spraying the plates with 1.0% potassum ferrocyanide and oberving the blue reaction with iron. Colorimetric assay for iron indicated no detectable iron in the free form ofthe compound, but 0.104 jg of iron per pg of the iron-bound form. The migration of compounds with known molecular weights on G-25 superfine Sephadex yielded the linear relationship shown in Fig. 4. Fitting the distances of migration of iron-free and iron-bound pyochelin to this curve implies
1.8
1.6 1.4 LU
1.2
0
0.6
04 0.2 -..
500
600
WAVELENGTH (nm)
FIG. 1. UV and visibk absorption pectra of pu-
rified pyochdlin. (a) Spectrum of iron-free pyochelin (50 pg/mi) in methanol. (b) Spectrum offerripyochelin
(50 ps/lmi in methanol at pH 2.5. The reference solution was methanol. 0I
pound displayed maxima at 237, 2i5, 325, 425, and 520 nm (curve b, Fig. 1). This wine-red solution changed to orange and displayed a shift in absorption maximum from 520 to 488 nm when the pH was adjusted to values between 4.0 and 7.0. The iron-free forn of pyochelin is fluorescent; its corrected excitation spectrum has mama at 235, 272, ad32 nm and prominent troughs at 252 an 0 nm when the emission is measured at 442/mm (curve a, Fig. 2). The corrected emission setrum has a single maximum at 442 nm when excited at 352 nm (curve b, Fig. 2). Excitng the sample at 235 or 272 nm also yields a single emision maximum at 442 nm. Addition of iron yields a nonfluorescent compound. The infrared spectra of iron-free pyochelin (panel A) and ferripyochelin (panel B) are shown in Fig. 3. Iron binding by pyochelin was demonstrated by two-dimensional chromatography on silicic acid in chloroform-acetic acid-ethanol (90:5:5). The iron-free compound yielded one fluorescent spot at R1 0.80 when chromatographed in the first dimension, and yielded one red spot at Rf 0.24 when iron was added to the edge of the chromatogram containing the compound and the plate was turned and developed in the same solvent in the second dimension. Pyochelin could be changed from the free to the bound
Wavelength (nanometes)
fluoresence spectra of pyochein (a) Excitation spectum of a methanol solution ofpyochelin (10 pg/mi); emision was measred at 42 nm. (b) Emission spectnm of the same pyochelin solution, with the sampk excited at 352 nm. FIG. 2. Corrcted
Wavenumber (cm ')
3.000 100 -1 A so
2.000
1.500
I
I
800
1.000 I
I
I
I
I
I
-
60
c
0
2 S
40
-
20
-
0
a
a
a
100
so
Wavelength (microns)
FIG. 3. Infrared spectra of pyochelin. (A) Absorption spectrum of iron-free pyochelin. (B) Absorption spectrum of ferpyochelin. Sampls of I mg of each preparation were pressed into KBr di"sk, with the ue of 2 mg of KBr.
IRON-BINDING COMPOUND FROM P. AERUGINOSA
VOL. 137, 1979
BaciIracnr)
300
/
5.2 g (wet weight) of cells did not yield detectable amounts of it. In media of high iron content, P. aeruginosa produces less pyochelin. Strain PAO-1 was
Flavin Adenine Dinuicleotide
c
'0
200 o
361
A
-
E /
Riboflavin 1
Ferrozine
05 .
Iron-free Pyochelin ..fron-bound Pyochelin
Chiorogenic
Acid
Lumichrome
100
1000 500 Molecular Weight
2000
c 01 0
FIG. 4. Thin-layergel filtration. Samples of5 to l10
0005 -
pl of known compounds and purified pyochelin were
applied to a 0.8-mm layer of G-25 superfilne Sephadex which had been equilibrated to the flow of sodium acetate (pH 4.6) buffer for 24 h at an inclined angle of 15°. The plate was developed for 24 h, and the fluorescent compounds were marked directly on the layer. Bacitracin was detected with fluorescamine, and iron chelators were detected with the iron spray reagent. Distances were measured from the origin to the center of the spots.
that the iron-bound form is a molecule of smaller size than the iron-free compound. Hydrophobic behavior of the pyochelin molecules might have retarded its migration somewhat in the aqueous solvent, but it was concluded that the molecular weight of pyochelin probably lies between 400 and 600. These values were used to obtain an estimate of the affinity of pyochelin for iron by using fluorescence methods (3). The association constants obtained were 1.37 x 105 for a compound with a molecular weight of 400 and 2.04 x 105 for a molecule with a molecular weight of
0.,1
10 0
B
5
-. - 0 os )
'O1 E
0
005
600.
Synthesis of pyochelin. Pyochelin produc0 001 tion attained levels of 3 to 5 ng/ml very early in the culture (Fig. 5A) and remained constant 0005 0 until the culture assumed a reduced rate of growth (Fig. 5B), at which time there was a 3 3 2 25 1 1 . massive production of pyochelin. The identity of 0 001 1 0 35 15 20 30 25 the pyochelin produced early in culture was T -e HOOrs verified by thin-layer chromatography. When FIG. 5. Synthesis of pyochelin by strain PAO-1I the bacteria ceased to grow exponentially, pyochelin synthesis also slowed, and the concen- during growth in SMM. Bacteria were inoculated attF tration did not decrease over the next 20 h of 102 CFU/ml in I liter ofSMM, and 50-ml amounts of for mea-I times subsequent removed incubation. The compound was not altered chro- medium were the amount and of pyochelim (A) at growth xtendd inuba-surement matogaphiaRy dringthe incuba- extracted of during the extended matographically into ethyl acetate (B). Pyochelin concentra tion. Experiments with GMM and CAA medium tions were determined fluorometrically in methanolI yielded similar results. Pyochelin is an extracel- solutions and presented as micrograms per milliliterr lular product, and ethyl acetate extracts from of culture medium.
362
COX AND GRAHAM
grown from inocula of 10' CFU/ml in 50-ml quantities of SMM in 250-ml flasks with the following iron concentrations: 0, 0.1, 0.5, 1.0, 5.0, and 10.0 IM FeCh3. The quantities of pyochelin in methanol solutions of ethyl acetate extracts from the flasks containing the increasing concentrations of iron were 5.6, 7.0, 4.5, 0.3, and 0 ug/ml, respectively. All of the 43 clinical isolates from the Burn Unit, University of Iowa Hospital, produced large quantities of pyochelin. Inoculation of CAA medium with large numbers of cells from rich media often resulted in low pyochelin production, however.
Growth-promoting effects of pyochelin. To determine the effect of the iron-binding compound on the growth of P. aeruginosa, iron-free or iron-bound pyochelin was added to 10.0-ml quantities of various culture media in tubes (50 by 120 mm) inoculated with strain 10145 at 102 CFU/ml. Both the iron-free and iron-bound forms of the compound decreased the lag phase and increased the growth rate in GMM, SMM, GtMM, and CAA media, with the observed effects decreasing in the order the media are listed. The most notable stimulation was found in GMM, evidenced by a decrease in the lag phase of 15 h with 6.0 ,ug of iron-bound pyochelin/ml added (curve c, Fig. 6), in comparison with the same culture lacking pyochelin (curve a, Fig. 6). The effects of pyochelin on the lag phase of growth were dependent upon inoculum size; in experiments using 106 CFU/ml, the lag phase was very short and there was little or no effect of pyochelin. Iron in concentrations of 1.0 or 10.0 ,uM yielded some growth stimulation (curve b, Fig. 6). In CMM there was always rapid onset of growth with little or no stimulation of growth by pyochelin addition, and citrate added to GMM enhanced the growth of the bacteria (curve d, Fig. 6) to the same extent as pyochelin. Miles and Khimji (11) showed that a component of the ethyl acetate extract of P. aeruginosa cultures can reverse the inhibition of the iron chelator EDDA, EDDA was incorporated into CAA medium at a concentration of 1.25 mg/ml, and strain PAO-1 was inoculated at 102 CFU/ml, with and without pyochelin. This concentration of EDDA resulted in a long lag (curve b, Fig. 7) compared to growth in CAA rmedium without EDDA (curve a, Fig. 7). Addition of either iron-bound or iron-free pyochelin to CAA medium without EDDA gave the slight stimulation of growth characteristic for strain PAO-1 in CAA medium (curve c, Fig. 7). Addition of both iron-free pyochelin (curve d, Fig. 7) and iron-bound pyochelin (curve e, Fig. 7) to CAA
10
0.5
0.
0.10
0 01
o~ . 10
20
J. BACTERIOL.
b1
Eay
40 30 Time (Hours)
50
60
FIG. 6. Growth-promoting effects offerripyochelin for strain 10145 grown in GMM. Bacteria weregrown for inoculum in SMM, washed three times, and inoculated at approximately 102 CFU/ml in 10-ml quantities of GMM. Growth was measured by absorbance at 600 nm in tubes (20 by 150 mm) incubated with shaking at 37°C. (a) Growth in GMM; (b) growth in GMM with 1.0 isM FeCl3; (c) growth in GMM with 6 pg of ferripyochelin/ml; (d) growth in GMM supplemented with 0.5 mM sodium citrate.
medium containing EDDA reversed this compound's growth inhibition. Citrate and nitrilotriacetic acid were also added to cultures containing EDDA at 0.05 and 0.05 mM. Neither compound reversed EDDA inhibition. Extracts of the silicic acid thin-layer chromatograms outside the pyochelin band did not reverse EDDA inhibition. Addition of a 10 AM concentration of FeCl3 to the CAA medium containing EDDA allowed more rapid onset of growth than with EDDA alone, but did not reverse the inhibition as well as iron-free pyochelin.
DISCUSSION The iron-binding compound pyochelin can be prepared in 20-mg amounts from 6 liters of culture media within a 2-day period by use of preparative paper and thin-layer chromatography. Strain PAO-1 in CAA medium typically
VOL. 137, 1979
IRON-BINDING COMPOUND FROM P. AERUGINOSA
01 (0.
0~0
0
01
5
10
15 20 Time (Hours)
25
30
35
FIG. 7. Inhibition of growth by ElDDA and reversal of inhibition by pyochelin. Stra,in PAO-I was inoculated (102 CFU/ml) into tubes (20 by 150 mm) containing 10 ml of CAA medium. The tubes were shaken vigorously at 37°C, and growtih was measured at various times by ubsorbance at 6(X) nm in (a) medium alone, (b) medium with 1.25 nmg ofEDDA/mi, (c) medium with either 7.2 pg ofpyoci Wieln/ml or 6 g of ferripyochelin/ml, (d) medium cor plus 7.2 pg of pyochelin/ml, and (e')medium with EDDA plus 6.0 pg of ferripyochelinlnaXl.
produced 6 mg of pyochelin per iter. The bacteria produced the compound in media having less than 5.0 ,uM added FeCl3. Thin-layer chromatography was used to demonst rate the widespread occurrence of this compouLnd in the culture media of over 40 different strains of P. aeruginosa. These aspects of th e synthesis of pyochelin are similar to the produ ction of enterochelin by Escherichia coli. Althoiugh pyochelin resembles enterochelin in solubiliity and a positive reaction with the phenolate.spray reagent, absorption and fluorescence sp5 ectra indicate that the compounds are different. The fluorescence of the molecule indicates the presence of an aromatic ring. Akn absorption maximum at 350 nm matches th e fluorescence excitation maximum at 350 nm aLt alkaline pH, but the other maxima of the absorption spectra do not match the fluorescence exciLtation spectra. There is indication of the absorpttion of an aromatic hydroxyl at 310 nm; howevrer, there is no matching peak in the fluoresceince excitation spectrum. The bathochromic shilft of this peak in more alkaline conditions woulid also suggest the ionization of an aromatic hydr*oxyl. The negative Arnow test suggests that the phenolic char-
363
acter of this compound is expressed by one hydroxyl on the aromatic ring rather than the two hydroxyls as in the case of enterochelin. A positive ninhydrin reaction was not observed with the intact molecule, but became apparent after deterioration of the compound on the thin-layer chromatograms. The infrared spe6trum was included to demonstrate further the- lack of similarity between pyochelin and enterochelin. Notable is the absence of absorption at 1,760 cm-', where enterochelin and 2,3-dihydroxybenzoyl-serine absorb strongly as a result of strained ester group carbonyl stretch (12, 14). Gel permeation on thin layers of G-25 Sephadex suggests a molecular weight in the range of 400 to 600. However, this may be an underestimate due to the use of aqueous solvents. Hydrophobic molecules such as 8-hydroxyquinoline chromatographed more slowly than their molecular weights would suggest. Ferripyochelin is more water soluble than iron-free pyochelin and should be less susceptible to hydrophobic retardation. These data suggest that iron-free pyochelin may be an open molecule which closes around iron during binding and assumes a smaller size. In the absence of added iron, the iron-free compound will enhance the growth of both strains 10145 and PAO-1. Theoretically, pyochelin may aid the bacteria by chelating and making available the trace amounts of iron in the medium. Adding 6 ,tg of iron-bound compound/mnl is equivalent to adding a 10 ,LM concentration of FeCl3, and there was dramatic stimulation of growth, greater than from adding that concentration of iron alone. Ferric chloride added at 1.0 or 10.0 ,uM concentrations caused only a sLight decrease in the lag phase. It was expected that strain 10145 would be more sensitive to the effects of iron chelators since the growth of this strain exhibits a longer lag phase and is slower than that of strain PAO-1 in all of the media used. Two rates of growth were observed in Fig. 5A and in media containing only trace amounts of iron. In these media there is rapid logarithmic growth followed by a slower rate beginning at an absorbance of around 0.2. This may be noticed in Fig. 6, but was negligible in Fig. 7, for in CAA medium the bacteria typically display shorter lag phases and higher final absorbance values. This phenomenon is probably due to iron deprivation at high bacterial densities, and this deprivation elicits a second phase of pyochelin synthesis (Fig. 5B). The slower rate of growth occurred at bacterial densities of around 2 x 108 CFU/ml in GMM and SMM and was less ap-
364
J. BAcTricira.
COX AND GRAHAM
parent with the addition of iron or ferripyochelin (Fig. 6). Pyocheln is the only compound detected in ethyl acetate extracts of culture media capable of reversing the inhibition of growth by EDDA. On the basis of this activity, it appears to be the compound extracted by Miles and Khimji (11) which was capable of reversing the EDDA in-
port Grant RR 06372 from the Biomedical Research Support Branch, Division of Research Facilities and Resources, Na-
tional Institutes of Health
LlrrFTUE
1.
1937. Colorimetric determination of the components of 3,4-dihydroxyphenylalanine-tyrosine
mixur. J. Bio. Chem. 118:531-537. 2. Boyers, B.R., AL V. Powell, and C. E. aord 1967.
active in (schizokinen) Iron-chelating J. of cellhydroxamic division inacidBacillus initiation megaterium.
hibition of a number of bacterial genera. The
Bacteriol. 93:286-294.
nhibition of growth by EDDA was relieved by adding 10.0 pM FeCla; other iron chelators which
CrriD
Arnow, L E.
3.
Davenport, D. 1971. Use of fluoresence in binding studies, p. 203-240. In A. Peace, C.-G. Rose, and T. L. Pasby
enhanced growth, citrate and nitrilotriacetic (ed.), Fluorescence spectroscopy. M. Dekker, Inc., New acid, did not completely relieve the inhibition. York. The finding that no other metals or metal che- 4. Doeg, K. A., and D.ML Ziegler. 1962. Simplified methods for the estimation of iron in mitochondria and lates of pyochelin stimulate the growth of P. submitochondrial fractions. Arch. Biochem. Biophys. aeruginosa in the media used (data not shown) 97:37-40. 'indlicates that the growth-promoting effects of 5.s. Emery, T. 1962. Further observations concerning the periodic acid oxidation of hydroxylamine derivatives. J. pyochelin are on the basis of iron uptake. TheOrg. Chem. 27:1075. oretically, the iron in the medium exists in the EDDA complex, and initiation of growth de- 6. Garibaldi, J. A. 1971. Influence of temperature on the ism of a fluorescent peudomonad. J. Baciron m pends upon accumulation of sufficient pyochelin 1":103-103. the pseuform for frtepe-ii sbefn 7. Gibson, F., and D. I. McGrath. 1969. The isolation and irniin a usable tondsequpone to sequester iron characteization of a hydroxamic acid (arobactin) domonads. The estimate of the binding coeffiformed by Aerobacter aerogenes. Biochim Biophys. cient describes the iron-binding by pyochelin in Acta 192:175-181. ethaol. The binding coefficient in aqueous meBacterial assimilation of iron. Crit. C. K 1973. 8Laor be higher than this value (2.4 x 1W) dia mamay Microbiol. 2:273-331. ~~~~~~~~~~~~~~Rev. 9. J. R. of to inhipyochelin the reverse Walker, J. B. Reeves, N. EL Lankfod, C. the F., and ability Nabbut B. R. Beyers, and R. J. Jons 1965. Inocbition of EDDA (binding coefficient, 10'") sugpyochelin~ tulum-dependent division lag of Bacilus culture and binding coefficient If this. gests If the ofiin offpoh th.idi tb. gesU~~~~~~~. for iron in aqueous media is found to be near 10 reversal of EDDA inhibition may indicate some specificity in the interaction of P. aerugosa
its reaion to an ononous factor(s)
lo.
( uiolnn).J.
Biactsriol. 91:1070t1079. Maure, B., A. Miiller, W. Keller4Schierlein, and H. zibner. 1968Stoffechaelproducktevonmikroorgan-
n. Frribactin, sin siderochrom aus Peudomonaa with feripyochelin. However, it is also possible Mikrobiol 60:326-339. iron for that pyochelin acts to solubieweMAies, A. A., Migula. and P. Arch. L Khlmi. 1975. Enterob l transport without having a receptor siteon-te chatora of iron: their oc n, d on, and rdat to pathognicity. J. Med. Microbiol. 8:477-490. bacterial surface for specific transport processes. J. B. 1973. Microbial iron traportcompounds Current studies on the structure of pyochelin 12. N In G. Eichorn (ed.), Inor(siderochromes), aer.ginosa by P.. aerugua of iron by transport ofIron andthe the active active transport a and bioch p.. 167-202. Elsevier, srdm pganic will resolve the status of this compound. Even if 13. OYBrien, L G, and F. Gibson. 1970. The structure of enterochlin and related 2,3-dihydrozy-N-benzoyl erno specific transport system is found for pyine conuates from Escherichia coi. Biochim. Bioochelin, the evidence suggests that the molecule A,ca1dJ9 binds iron and prsents iron to the bacteria in a 14. PohaysJ. N3-402 B.1.70.. usable form. from Salmoeua tpan iron tanport compo munw. Biocem Biophys Res. Commun. 3:88-992. iron-chelating growth A. Mycobactin 15. Snow, ACKNOWLEDGMENTS factosB.from 1970. Bacteriol. Rev. 34:99-125. Mycobacteria. This investigation was supported by Public Health Service 16. WarIn, W. 8, and C H. Werkman. 1942. Iron requiegrant Al 13120 from the National Institute of Allorgy and Infectious Diseases and in part by Biomedica Research Sup-
ments of hetrotrophic bactria. Arch. Biochem. 1: 425-433.
