Effects of Siderophores on the Growth of Pseudomonas Aeruginosa in Human Serum and Transferrin ROBERT ANKENBAUER, SOMPORN SRIYOSACHATI, and CHARLES D

INFECTION AND IMMUNITY, JUlY 1985, p. 132-140 Vol. 49, No. 1 0019-9567/85/070132-09$02.00/0 Copyright C 1985, American Society for Microbiology Effects of Siderophores on the Growth of Pseudomonas aeruginosa in Human Serum and Transferrin ROBERT ANKENBAUER, SOMPORN SRIYOSACHATI, AND CHARLES D. COX* Department of Microbiology, University of Iowa, Iowa City, Iowa 52242 Received 15 October 1984/Accepted 27 March 1985 A combination of the siderophores produced by Pseudomonas aeruginosa, pyochelin and pyoverdin, dramatically stimulates the growth of this bacterium in medium containing human transferrin. The amount of growth stimulation observed when each siderophore was added alone was only slighly less than the amount observed with the combination. Siderophore-defective mutants of strain PAO1 were isolated to test the effects of siderophore production on growth in transferrin and human serum. The pyoverdin-proficient (Pvd+), pyochelin-deficient (Pch-) strain (IA5) grows just as well as the parent (PAO1), which produces both siderophores. On the other hand, the Pvd- Pch+ strain (211-5) has severely retarded growth, similar to that demonstrated by a mutant lacking production of both siderophores (IA1), but has an accelerated log phase compared with strain IAl at the later stages of the growth curve. However, the Pvd- Pch+ strain (211-5) had no observable advantage over the Pvd- Pch- strain, IAl, during incubation in human serum. The inability of P. aeruginosa strains to produce pyochelin in glucose-minimal medium may explain the poor growth of 211-5 in this medium and in human serum. The 211-5 strain grows much better than the IAl strain in the medium that allows pyochelin synthesis, but it still does not grow as well as the Pvd+ Pch- strain (IA5). Therefore, pyoverdin appears to be the most important siderophore for growth in human serum.

Pseudomonas aeruginosa must be able to grow in mam- not known to exist for these siderophores or for enterochelin malian tissue to infect humans. Growth, in turn, is depend- and aerobactin. In all of these examples it is believed that ent upon the bacterial acquisition of iron from mammalian multiple siderophores are produced, because one of the iron-binding proteins (3). One of the most important iron- siderophores is critical for growth under severe iron depriva- binding proteins in host defense is transferrin (1). The most tion. In the case of E. coli, the siderophore that allows common mechanism by which bacteria compete with invasiveness is not the one possessing the highest binding transferrin for iron is through the activity of siderophores, coefficient. Therefore, siderophore activities in the presence bacterial products which bind iron and function in high-af- of ferritransferrin must be determined experimentally. finity iron transport (19). In this report we describe the The value of two siderophores to P. aeruginosa is not relative abilities of two siderophores, pyochelin (9, 10, 16) obvious. Pyochelin contains a salicyl ring bonded to a and pyoverdin (8, 17, 22, 31), to stimulate growth and iron thiazoline ring, which is, in turn, bonded to a terminal accumulation by P. aeruginosa during incubation with hu- N-methylthiazolidine ring (10). The structure of pyoverdinpa man transferrin or human serum. Two bacteria that produce from P. aeruginosa, reported by Wendenbaum et al. (31), is more than one siderophore, Escherichia coli and Azotobac- very similar to pseudobactin (29) and pyoverdinpf (23). It ter vinelandii, have been studied to some length. Certain contains a dihydroxyquinoline moiety (the chromophore), invasive strains of E. coli have been found to produce both and two N-hydroxyornithine residues, components which enterochelin (20) (also called enterobactin [24]) and are common to both pseudobactin and pyoverdinpf (8). Six aerobactin (2, 13, 28, 30). The expression of aerobactin has additional amino acids complete the structure. To under- been correlated with increased growth of E. coli in serum stand the effects of these two siderophores, mutants were (13, 27) and with the presence of a ColV plasmid (2, 28, 30). isolated with various synthetic capabilities for siderophores. The other bacterium, A. vinelandii, produces dihydroxyben- The growth capabilities of these mutants revealed that zoic acid, azotochelin, and azotobactin. Azotochelin is N,N'- pyoverdin synthesis is most important for growth of P. bis-(2,3-dihydroxybenzoyl)-L-lysine (6), and azotobactin is a aeruginosa in human transferrin or serum. Although dihydroxyquinoline bound to an octapeptide (12). pyochelin has impressive effects when added to these media, Siderophore synthesis is controlled in a sequential manner, it was synthesized sparingly in glucose-minimal medium progressing from dihydroxybenzoic acid to azotochelin and (GMM) and in human serum. Therefore, bacteria demon- then to azotobactin (21) and is governed by the increasing strated little growth advantage from pyochelin in these severity of iron limitation. Therefore, the organism appears media. to conserve energy in siderophore synthesis, expending it for the most complex, but most effective, siderophore, azotobactin, when it is essential. There is another example MATERIALS AND METHODS from a recent investigation in which Pseudomonas fluorescens produces both pyoverdinpf and ferribactin (17, Culture media. The medium for siderophore production, 22, 23). Pyoverdinpf is thought to be the most effective of the CAA, contained 0.5% Casamino Acids and 0.4 mM MgSO4. two, but the ferribactin is produced late in culture. There- Other minimal media contained 5 mM potassium phosphate fore, sequential synthesis dependent upon iron demand is buffer (pH 7.4), 5 mM K2SO4, 40 mM NH4Cl, and 0.5 mM MgSO4. Oxidizable substrates, arginine (AMM for arginine minimal medium) or glucose, were added to 20 mM. When * Corresponding author. arginine was used, the NH4Cl was omitted. These media 132 VOL. 49, 1985 EFFECTS OF SIDEROPHORES ON GROWTH OF P. AERUGINOSA 133 were also made into solid media by the addition of 1.5% agar. Chemical Co.) and was mixed with excess 55Fe in 40 mM To institute more stringent iron-limiting conditions, 0.9% Tris-hydrochloride and 20 mM sodium bicarbonate (Tris-bi- Gelrite (Kelco, San Diego, Calif.) was added to broth with 5 carbonate buffer, pH 7.4) to make 100% saturated mM MgSO4 to make solid medium. [55Fe]transferrin by the methods of Simonson et al. (26). This Isolation of mutants. P. aeruginosa PAO1 (ATCC 15692) mixture was dialyzed exhaustively against the same buffer was obtained from the American Type Culture Collection, until no 55Fe appeared in the dialysate. This iron substrate and mutants were derived from this parent strain. Mutagen- was added to culture medium at 6.8 p.g/ml and contained 0.21 esis was carried out with ethyl methanesulfonate by the ,uCi of 55Fe per ml. Bacterial accumulation of iron from procedure of Lin et al. (15). Strain PAO1 was grown in 1% culture medium was measured by passing 1-ml samples of tryptic soy broth (TSB) to an absorbance of 0.7 (600 nm). culture medium through 0.45-p.m pore size filters. The filters The cells were centrifuged from suspension, washed, and were washed with water and dried, and the amounts of iron suspended in 5 ml of the same medium. After 30 min of were determined by scintillation counting. Purified incubation at 37°C, 0.2 ml of ethyl methanesulfonate was siderophores were added to this medium at 10 p.g/ml, ap- added for 60 min. This procedure resulted in a 105-fold proximately the concentrations that could be found in CAA reduction in the number of viable cells. The surviving cells medium culture filtrates (7). Bacteria, prepared for inoculum were washed in sterile, distilled water and suspended in 1% by three passes through CAA culture medium, were washed TSB for overnight incubation in the dark. In some cases three times in water and added to medium at approximately selection was carried out in 0.5% CAA medium with sequen- 5 x 103 CFU/ml. Growth was assayed by measuring absorb- tial cycles of selection with D-cycloserine followed by ance at 600 nm or by diluting culture medium and determin- growth in 1% TSB (4). Siderophore-deficient mutants were ing viable bacteria by plate counts. Viable bacteria in sus- selected against on plating media containing 100 ,uM pensions were counted on agar surfaces (1% tryptic soy agar ethylenediamine-di-(o-hydroxyphenylacetic acid) and on or 1% peptone agar) after 24 h of incubation at 37°C. media containing 200 pLM ethylene glycol-bis(P-aminoethyl Siderophore purification and analysis. Pyochelin was ex- ether)-N,N-tetraacetic acid. Colonies that failed to grow in tracted from media into dichloromethane containing 10% the presence of selective agents, but grew on nonselective acetic acid (mixed 1:2 with medium). Pyochelin was purified media, were plated on 0.5% CAA agar medium and were on consecutive, preparative thin-layer plates as described observed under fluorescent lamps to screen for nonfluores- previously (9). The first silica thin-layer plate was developed cent colonies. One such colony, 211-5, has been determined in chloroform-acetic acid-ethanol (90:5:2.5), and the second to be pyoverdin defective (Pvd-), but proficient in pyochelin plate was developed in the same solvent in a 90:5:5 ratio. synthesis (Pch+). Strain IAl, Pvd- Pch-, was isolated after Pyochelin was eluted from the silica in dichloromethane, and ethyl methanesulfonate mutagenesis of strain 211-5 as a this solution was filtered and taken to dryness for determina- minute colony appearing on AMM agar containing 200 ,uM tion of the dry weight of the product. Screening mutants ethylene glycol-bis(3-aminoethyl ether)-N,N-tetraacetic involved the extraction of 5 ml of spent culture medium and acid after incubation at 37°C for 72 h. Each colony suspected chromatography of concentrated extracts on silica thin-layer of being a mutant was inoculated into 5 ml of CAA medium plates (Eastman Kodak Co.) in chloroform-acetic acid to be tested for siderophore synthesis. (90:5). Pyochelin production was scored by fluorescence No mutants with a phenotype Pvd+ Pch- have been found appearing at the same Rf as a pyochelin standard. Measure- during the screening of thousands of slow-growing colonies. ment of pyochelin in culture medium was conducted by Alternative time intervals used in mutagenesis and different high-pressure liquid chromatography of concentrated methods of selection yielded no improvements. Ultimately, dichloromethane extracts on a C18 (4 by 250-mm a Pvd+ Pch- strain (IA5) was constructed through transduc- Ultrasphere; Beckman Instruments, Inc.) column equili- tion of IAl with phage F116L lysates of strain PA01. The brated with 50% methanol-1% acetic acid-0.5 mM ethylene transduction procedure was essentially that of Krishnapillai glycol-bis(3-aminoethyl ether)-N,N-tetraacetic acid flowing (14), except that the adsorption mixture was plated directly at 1 ml/min. A linear gradient to 100% methanol, starting 5 onto CAA agar containing 100 ,uM ethylene diamine-di-(o- min after injection and proceeding for 20 min, was formed by hydroxyphenylacetic acid). The Pvd+ Pch- transductant a Beckman 322 MP chromatograph and allowed pure appeared as a fluorescent colony on a barely perceptible pyochelin, eluting at approximately 20 min, to be quantified lawn ofIAl. The IA5 transductant fed the surrounding IAl by peak height (monitored at 254 nm). cells, resulting in increased growth of surrounding colonies. Pyoverdin was purified by molecular sieving and high- Growth assays. GMM was supplemented with 20 mM pressure liquid chromatography as described previously (8). sodium bicarbonate for growth experiments involving A concentrate of filter-sterilized spent culture medium was transferrin or serum. Transferrin was added to this medium applied to a P-2 Bio-Gel column (1.8 by 85 cm; Bio-Rad in final concentrations of 6.8 to 500 p.g/ml. Heat-inactivated Laboratories) in a water-methanol (10:1) solvent flowing (56°C for 30 min) human serum was added from a pool against gravity at 0.2 ml/min. The major fluorescent peak obtained from 10 normal donors. In some experiments it was eluting from the column, at approximately 70 ml, was important to reduce the iron contamination of the medium by concentrated and applied to a C8 (Ultrasil, Beckman) pre- mixing the medium constituents with MgCO3 and removing parative column (10 by 250 mm) equilibrated with 10% the MgCO3 by centrifugation and filtration. This treatment acetonitrile in water. The solvent was pumped at 1 ml/min by lowered the iron concentration in the medium from 2.3 to 0.4 an LDC Constametric pump. The second fluorescent peak to puM. Iron (5) and the iron saturation of transferrin (32) were emerge from the column at 28 min was collected and measured by using ferrozine. In some experiments, minerals concentrated. This was pure pyoverdin by subsequent analy- were added to make sure that no other mineral limitations ses on high-pressure liquid chromatography columns and were affecting growth. These minerals were added to make a thin-layer chromatography (8). Screening mutants and final concentration of 5,uM (NH4)6Mo7024 * 4H20 and 1 ,uM quantitation of pyoverdin involved diluting culture media in CaSO4, ZnSO4, Na2SeO4, MnC12, and CaCl2. 50 mM Tris-hydrochloride buffer (pH 7.4) and measuring Apotransferrin was prepared from transferrin (Sigma fluorescence at 460 nm while exciting the sample at 400 nm. 134 ANKENBAUER ET AL. INFECT. IMMUN.

An Aminco Bowman corrected spectra spectrofluorometer A 1 .00 - o No Addition was calibrated with quinine sulfate for pyoverdin measure- - * Pch ments. o Pvd RESULTS 0.50 * Pch+Pvd Effects of added siderophores on growth and iron accumula- tion by PAO1 in the presence of transferrin. Siderophores can have appreciable effects on the logarithmic phase of bacterial growth. In strain PA01 inoculated at 5 x 103 CFU/ml in GMM, 10 h was required before turbidity could be detected (data not shown). When 100 ,ug of apotransferrin and 6.8 ,g of [55Fe]transferrin were added per ml of GMM, the appear- Ec ance of turbidity was delayed until after 30 h of incubation 0.10 (Fig. 1A), even though there was sufficient iron contamina- tion to yield 89% saturation of the transferrin. The iron C) contamination came predominantly from the sodium bi- a) 0.05 carbonate, and no attempt was made to remove the iron in 0 this experiment. The combination of siderophores, U_cJ pyochelin and pyoverdin, stimulated growth to the greatest .0D0 extent; this effect was followed by pyoverdin added alone, 4 which was followed by pyochelin alone (Fig. 1A). The levels of stimulation of iron accumulation from the [55Fe]transfer- rin by the siderophores were similar to the levels of growth stimulation (Fig. 1B). 0.01 I I I Effects of siderophore production on growth and iron ac- cumulation in the presence of transferrin. Additions of siderophores affected the early phases of growth of strain B 50.0 PA01. Since this strain is capable of producing both siderophores, later growth characteristics may have been similar regardless of the initial siderophore added. There- 0- fore, an investigation of the relative effects of siderophore La production in the presence of transferrin is relevant to the LO 10.0 , effects of individual siderophores on both early and late X stages of growth. To determine these effects, mutants defec- o) /, .-/ tive in siderophore production were isolated as described E 5.0 above. These strains were inoculated into GMM containing .0 20 mM sodium bicarbonate and 200 ,ug transferrin carrying 0.21 ,uCi of 55Fe per ml. Approximately 0.4 ,uM iron existed as a trace contaminant, representing 8.0% saturation of transferrin. Pvd+ Pch- cells (IA5) grew as well as the E 1.0 wild-type, Pvd+ Pch+ (PA01) cells (Fig. 2A). The effect of pyochelin synthesis without pyoverdin (Fig. 2A) was only observed later during incubation when strain 211-5 cells had 4SC. 0.5 .1 reached 0.05 absorbance unit or approximately 107 CFU/ml. The Pvd- Pch- mutant (IA1) continued at a very slow rate of , growth, finally reaching approximately 0.4 absorbance unit (5 x 108 cfu/ml) at 270 h of incubation, 90 h after the 211-5 culture (Fig. 2A). The relative abilities of these strains to accumulate iron from the [55Fe]transferrin (Fig. 2B) were 5 1 0 1 5 20 25 30 35 similar to their growth capabilities. Time (h) The growth capabilities of these strains were dependent FIG. 1. Effect added siderophores on the growth (A), measured upon the concentration of the iron saturation of transferrin. by absorbance at 600 nm, of strain PA01 inoculated into GMM The logarithmic rates of the same strains were more rapid containing 100 ,ug of transferrin per ml (no addition), with added than in Fig. 2 when they were inoculated into GMM contain- pyoverdin and pyochelin each at 10 ,ug/ml (Pch+Pvd), with ing 500 ,ug of transferrin per ml. There was 45% saturation of pyochelin added at 10 ,ug/ml (Pch), and with pyoverdin added at 10 transferrin in this experiment. Measurements of the growth p.g/ml (Pvd). Iron accumulation (B) was measured as 51Fe trapped of viable cells, instead of measurement by absorbance, with bacteria from 1-ml sample of culture medium on 0.45-,um pore revealed the same size membrane filters. GMM contained 20 mM sodium bicarbonate, patterns of growth that were observed in 100 ,ug of transferrin per ml, 6.8 ,ug of [55Fe]transferrin (100% Fig. 2. Pvd+ Pch- cells (IA5) grew as well as cells capable of saturated with 0.21 ,uCi of"Fe per ml) per ml, and a mixture of trace producing both siderophores (PAO1) (Fig. 3). Pvd- Pch+ metals. The overall saturation of transferrin was 89%. bacteria (211-5) demonstrated the effects of pyochelin at approximately 107 CFU/ml, reaching 109 CFU/ml ap- proximately 30 h before Pvd- Pch- bacteria (IA1) (Fig. 3). proximately 60 h. The differences between strains were in Visible turbidity was reached by the IA5 and PAO1 strains at their growth rates and not in their lag phases. approximately 30 h, similar to the growth of PAO1 in Fig. 1. Growth of siderophore-deficient strains in human serum. Strains 211-5 and IAl reached visible turbidity at ap- There was a major difference between the results observed VOL. 49, 1985 EFFECTS OF SIDEROPHORES ON GROWTH OF P. AERUGINOSA 135 A

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1 0 30 60 90 120 150 180 210 240 270 300 330 Time(h) FIG. 2. Growth (A) of the Pvd+ Pch+ (PAO1), Pvd+ Pch- (IA5), Pvd- Pch+ (211-5), and Pvd- Pch- (IA1) strains was measured by absorbance at 600 nm in GMM containing transferrin after inoculation at 103 bacteria per ml. Iron accumulation (B) by the strains was measured as in Fig. 1. GMM contained 20 mM sodium bicarbonate, 200 ,ug of transferrin per ml, 0.21 ,Ci of 55Fe, and no trace metals and was treated to remove contaminating iron. Overall iron saturation of transferrin was 8.0%. in minimal medium containing transferrin (Fig. 3) and results for pyochelin production. Analyses of pyochelin production after inoculation of the same strains into 20% human serum. during incubation of PAO1 cells in GMM or GMM contain- The Pvd+ Pch- strain (IAS) still grew as well as the wild-type ing transferrin (data not shown) substantiated the earlier strain (PA01) (Fig. 4). However, the Pvd- Pch+ cells (211-5) finding (7) that glucose is not conducive to pyochelin produc- did not grow better than the mutant deficient in both tion. When the siderophore-deficient strains were inoculated siderophores (IA1) even at the later stages of the growth into a CAA medium, a medium which supports prolific curves (Fig. 4). This result indicated that pyochelin was pyochelin synthesis (7), containing 200 ,ug of transferrin per either not produced or not effective in human serum. There ml, 0.4 ,uM iron as trace contaminant, and 20 mM sodium was also an indication of longer lag phases as well as slower bicarbonate, the Pvd- Pch+ cells (211-5) grew much better growth rates for strains 211-5 and IAl in human serum (Fig. than the mutant that was deficient in both siderophores (IA1) 4). (Fig. 5). The Pvd- Pch- strain, IAl, did not grow as well in

Growth of siderophore-defcient Istrains in media designed this medium as it did in GMM containing transferrin (Fig. 2). 136 ANKENBAUER ET AL. INFECT. IMMUN.

* PA01 [ o LA5 10': * 211- 5 [ o IAl

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20 40 60 80 100 120 140 160 Time(h) FIG. 3. Growth (A) of Pvd+ Pch+ (PAO1), Pvd+ Pch- (IA5), Pvd- Pch+ (211-5), and Pvd- Pch- (IA1) strains was measured in GMM containing 500 ,ug of transferrin per ml after inoculation of 5 x 103 bacteria per ml. Growth is presented as the mean values of triplicate determinations of viable bacteria appearing on TSB plates after the plating of culture dilutions and overnight incubation at 37°C. The preparation of medium and inoculum was as in Fig. 1.

The pyochelin synthesis by 211-5 in CAA medium allowed iron-limiting conditions that were enacted by transferrin. growth that was similar to, but less than, that demonstrated These conditions should be pertinent to those experienced by the Pvd+ Pch- strain (IAS) or the wild-type strain (PA01) during infections of human tissues. Both siderophores dra- (Fig. 5). The growth of the IA5 strain was always terminated matically stimulated bacterial growth in human serum and in in this medium by a lytic phage F116L infection, but suf- medium containing transferrin (Fig. 1). We had expected ficient growth was observed to determine the growth capa- that one siderophore would demonstrate superior activity, bility endowed by pyoverdin production. thus exposing the reason for an alternative siderophore and the one siderophore that would be most crucial for the DISCUSSION infectious process. Theoretically, the binding coefficients of the compounds for iron should allow predictions of their In the present investigation we tested the relative activi- activities. Although the binding coefficient of pyoverdin, 1032 ties of two siderophores produced by P. aeruginosa under (31), is much higher than that of pyochelin, approximately VOL. 49, 1985 EFFECTS OF SIDEROPHORES ON GROWTH OF P. AERUGINOSA 137

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3 I I 0 10 20 30 40 50 60 70 80 90 100 Time (h) FIG. 4. Growth of Pvd+ Pch+ (PAO1), Pvd+ Pch- (IA5), Pvd- Pch+ (211-5), and Pvd- Pch- (IA1); strains in 20% heat-inactivated, normal human serum. Numbers of bacteria are presented as the mean values of triplicate determinations. GMM and the inoculum were prepared as in Fig. 2.

105 (9), pyoverdin was only slightly more stimulatory than but the lack of its synthesis, that may explain the need for an pyochelin (Fig. 1). The measurement of the binding co- alternative siderophore for P. aeruginosa. In GMM contain- efficient for pyochelin was conducted at acid pH and may not ing transferrin, neither the Pvd+ Pch+ strain (PAO1) nor the pertain to physiological conditions, but the 2:1 ratio of Pvd- Pch+ strain (211-5) demonstrated significantly better pyochelin to iron in the chelate suggests that the pyochelin growth than its Pch- counterpart. Only at the later stages of may not function as well as pyoverdin (which has a 1:1 ratio growth, at cell densities above 107 CFU/ml, did the ability to to iron) in binding iron. synthesize pyochelin have an effect on the Pvd- Pch+ 211-5 Since strain PAQ1 can synthesize its own siderophores strain. We had previously found (7) and have repeatedly and feed itself iron, siderophore-defective mutants had to be found during this investigation that all strains of P. used to determine the effects of individual siderophores. The aeruginosa produce low amounts of pyochelin in medium growth capabilities of these mutants (Fig. 2) demonstrated containing glucose as the sole carbon source. Young (33) had the superior nature of pyoverdin production in GMM con- also shown glucose to inhibit the synthesis of pyoverdin and taining transferrin. It is not the lack of activity of pyochelin, pyocyanin. However, in serum that contains 4 to 5 mM 138 ANKENBAUER ET AL. INFECT. IMMUN. 1.0

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0.01 0 Time (hours) FIG. 5. Growth of Pvd+ Pch+ (PAO1), Pvd+ Pch- (AS), Pvd- Pch+ (211-5), and Pvd- Pch- (IA), strains in CAA medium containing 200 ,ug of transferrin per ml was measured by absorbance at 600 nm. The medium and inoculum were prepared as in Fig. 2 with removal of iron from the medium and no addition of trace minerals. glucose and in GMM with transferrin, pyoverdin production to determine why strain IAl grows as well as it does in appears to be responsible for the rapid growth manifested by transferrin and serum. strain PAO1. Enzymatic mechanisms for iron release from ferritransfer- The mutants used in this investigation may have multiple rin might also work in concert with siderophore mecha- lesions which affect growth. However, we found that the nisms. Pyochelin might be active through the delivery of addition of siderophores, pyoverdin or pyochelin, can re- sufficient contaminating iron (not from transferrin) to allow store the growth capabilities of the 211-5 or IAl strain to the initial growth and exoenzyme synthesis. Limited hydrolysis levels of the wild type or the IA5 strain (Fig. 2). It is also of transferrin by an enzyme might allow bacterial acquisition possible that there are other methods of iron acquisition of iron, either directly or through the action of siderophores. from ferritransferrin functioning in these assays. For ex- However, if this were true, one should observe the Pvd- ample, we had expected the IAl strain to be totally defective Pch+ 211-5 strain growing much better than the Pvd- Pch- at growth in transferrin, but the strain displays a slow, IAl, strain in media that contain large amounts of contami- consistent rate to reach over 109 CFU/ml (Fig. 2) and a more nating iron (Fig. 3). This did not occur. Our present theory, rapid rate in human serum (Fig. 4). Although there could be based on the speed of its effectiveness on strain PA01 (Fig. another siderophore such as ferribactin (9, 22), the lack of a 1) and strains 211-5 and IAl (data not shown), is that similar growth capability in CAA medium (Fig. 5) may be pyochelin must be acting as a siderophore. However, we more suggestive of exoenzyme activity. For example, a have no explanation at this time for the siderophore capabili- proteolytic enzyme could nick transferrin and ruin the ties of pyochelin in the presence of ferritransferrin. affinity of the protein for iron. Investigations are underway It is important that there is no effect of pyochelin produc- VOL. 49, 1985 EFFECTS OF SIDEROPHORES ON GROWTH OF P. AERUGINOSA 139 tion in serum (Fig. 4) even at the later stages of growth. 3. Bullen, J. J. 1981. The significance of iron in infection. Rev. Konopka and Neilands (13) have demonstrated the binding Infect. Dis. 3:1127-1138. of the catechol siderophore, enterobactin, to serum albumin. 4. Carhart, G., and G. Hegeman. 1975. Improved method of Since this may be a general behavior of all phenolate selection for mutants of Pseudomonas putida. Appl. Microbiol. siderophores, pyochelin may be produced and then bound 30:1046-1047. in 5. Carter, P. 1971. Spectrophotometric determination of serum an inactive form in the presence of human serum (Fig. 4). iron at the submicrogram level with a new reagent (ferrozine). Just as the binding of enterobactin to albumin has been Anal. Biochem. 40:450-458. proposed as an explanation for the beneficial activity of 6. Corbin, J. L., and W. A. Bulen. 1969. The isolation and aerobactin during the growth of E. coli in serum, we are identification of 2,3-dihydroxybenzoic acid and 2-N,6-N- currently investigating whether pyoverdin production by P. dihydroxybenzoly-L-lysine formed by iron-deficient Azotobac- aeruginosa is more beneficial than pyochelin production ter vinelandii. Biochemistry 8:757-762. because of the binding of pyochelin to albumin. Additional 7. Cox, C. D. 1980. Iron uptake with ferripyochelin and ferric antibacterial mechanisms that are known to exist in serum citrate by Pseudomonas aeruginosa. J. Bacteriol. 142:581-587. 8. Cox, C. D., and P. Adams. 1985. Siderophore activities of and inhibit siderophore systems in other bacteria may also pyoverdin for Pseudomonas aeruginosa. Infect. Immun. 48: be responsible for the inhibition of pyochelin activity. There 130-138. are antibodies against the lipopolysaccharide carbohydrate, 9. Cox, C. D., and R. Graham. 1979. Isolation of an iron-binding colitose, of E. coli which inhibit the synthesis of enterochelin compound from Pseudomonas aeruginosa. J. Bacteriol. 93: (11, 25). There are also antibodies that react with 144-148. enterochelin (18). Antibodies against pyochelin might ef- 10. Cox, C. D., K. L. Rinehart, M. L. Moore, and J. C. Cook. 1981. fectively remove it from serum and inhibit our attempts to Pyochelin: novel structure of an iron-chelating growth promoter extract and measure this siderophore. We have detected the for Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U.S.A. synthesis of pyochelin late in the stationary phase in GMM 78:42564260. 11. Fitzgerald, S. P., and H. J. Rogers. 1980. Bacteriostatic effect of containing transferrin and in human serum (data not shown). serum: role of antibody to lipopolysaccharide. Infect. Immun. This synthesis might be explained by the saturation of 27:302-308. pyochelin-binding antibodies or by the depletion of glucose 12. Fukosawa, K., M. Goto, K. Sasaki, and Y. Hirata. 1972. Struc- through bacterial utilization. Although glucose may repress ture of the yellow-green fluorescent peptide produced by iron- pyochelin synthesis in minimal medium, there may be a deficient Azotobacter vinelandii strain 0. Tetrahedron 28: variety of inhibitory phenomena present in human serum. 5359-5365. For example, human serum also causes a lag phase (Fig. 4) 13. Konopka, K., and J. B. Neilands. 1984. Effect of serum albumin for Pch- strains which is not appreciable in GMM containing on siderophore-mediated utilization of transferrin iron. Bio- transferrin (Fig. 3). We have not been able to find evidence chemistry 23:2122-2127. 14. Krishnapillai, V. 1971. A novel transducing phage: its role in for sequential derepression of siderophore synthesis, as has recognition of a possible new host-controlled modification sys- been found in A. vinelandii. However, it could still be argued tem in Pseudomonas aeruginosa. Mol. Gen. Genet. 114:134- that GMM containing transferrin exacts sufficient iron de- 143. mand to elicit sequential derepression. According to this 15. Lin, E. E. E., S. A. Lerner, and S. E. Jorgensen. 1962. A method theory, as bacteria approach conditions of severe iron limita- for isolating constitutive mutants for carbohydrate-catabolizing tion, they would synthesize pyochelin and then pyoverdin. enzymes. Biochim. Biophys. Acta 60:422428. However, we have not observed this sequence in GMM. In 16. Liu, P. V., and F. Shokrani. 1978. Biological activities of other media both pyochelin and pyoverdin are produced at pyochelins: iron-chelating agents of Pseudomonas aeruginosa. low iron concentrations, and pyoverdin is produced alone at Infect. Immun. 22:878-890. 17. Meyer, J. M., and M. A. Abdallah. 1978. The fluorescent higher iron concentrations of 2 to 5 xuM. pigment of Pseudomonasfluorescens: biosynthesis, purification Although enzymatic activities can not be ruled out, both and physiochemical properties. J. Gen. Microbiol. 107:319-328. pyoverdin and pyochelin are able to provide iron from 18. Moore, D. G., R. J. Yancey, C. E. Lankford, and C. F. Earhart. ferritransferrin in P. aeruginosa. Pyoverdin appears to be 1980. Bacteriostatic enterochelin-specific immunoglobulin from the active siderophore for rapid growth in serum (Fig. 4). It normal human serum. Infect. Immun. 27:418423. will be most important to determine whether pyoverdin 19. Neilands, J. B. 1981. Microbial iron compounds. Annu. Rev. synthesis is also sufficient for growth during infections in Biochem. 50:715-731. mice. Until we have more information concerning the genet- 20. O'Brien, I. G., and F. Gibson. 1970. The structure of enterochelin and related 2,3-dihydroxy-N-benzoylserine conju- ics of siderophore synthesis and can construct isogenic gates from Escherichia coli. Biochim. Biophys. Acta 215: strains possessing specific expressions of siderophores, we 393402. will continue to use the present strains to delineate the 21. Page, W. J., and M. Huyer. 1984. Derepression of the relative abilities of pyoverdin and pyochelin to deliver iron Azotobacter vinelandii siderophore system, using iron-contain- to P. aeruginosa under different conditions. ing minerals to limit iron repletion. J. Bacteriol. 158:496-502. 22. Philson, S. B., and M. Llinas. 1982. Siderochromes from Pseu- ACKNOWLEDGMENTS domonasfluorescens. I. Isolation and characterization. J. Biol. Chem. 257:8081-8085. We thank Marcia Reeve for preparation of this manuscript. 23. Philson, S. B., and M. Llinas. 1982. Siderochromes from Pseu- This investigation was supported by Public Health Service grant domonas fluorescens. lI. 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