The Metal Dependence of Pyoverdine Interactions with Its Outer Membrane Receptor FpvA

Reagents.Carbenicillin disodium salt was a generous gift from SmithKline Beecham (Welwyn Garden City, Herts, United Kingdom). Pvd, Pvd-Fe, and Pvd-Ga were supplied by Isabelle Schalk and prepared as described previously (1, 9, 11). Sodium N-lauroyl sarcosine was purchased from Sigma and n-octylpolyoxyethylene (oPOE) from Bachem. Chemicals for succinate medium (SM) had less than 5 ppm Fe (Merck, Darmstadt), except for the succinic acid, which was ultrapure grade from Sigma. Water with a resistivity of 18.2 MΩcm was obtained directly from a Milli-Q Plus (Millipore) ultrapure water system.

Bacterial strains and growth media.Two derivatives of the PAO1 strain were used: CDC5(pPVR2), a Pvd-deficient mutant which overproduces FpvA (3), and K691(pPVR2), which overproduces FpvA and produces Pvd (22). The strains were grown in an SM [35 mM K₂HPO₄, 22 mM KH₂PO₄, 1 mM MgSO₄, 35 mM succinic acid, 7.5 mM (NH₄)₂SO₄, 75 mM NaOH] (9) plus trace salts in the presence of 150 μg/ml carbenicillin. A low-iron-content trace salts solution (PTM4-lo) was prepared as a 1:100 mixture of PTM4 (28) and PTM4(Fe⁻) (PTM4 contains, per liter, 2 g CuSO₄·5H₂O, 80 mg NaI, 3 g MnSO₄·H₂O, 200 mg NaMoO₄·2H₂O, 20 mg boric acid, 500 mg CoCl₂, 7 g ZnCl₂, 22 g FeSO₄·7H₂O, 200 mg biotin, and 1 ml sulfuric acid). Cultures were routinely grown overnight at 30°C in LB medium to an optical density at 600 nm (OD₆₀₀) of 4 to 5 and then pelleted, washed, and resuspended with SM at a 100-fold dilution. After 7 h at 30°C, the cultures were near an OD₆₀₀ of 1 and overproducing FpvA. The PTM4-lo salts were prepared fresh for each culture from PTM4 and PTM4(Fe⁻) stocks and then diluted 5,000-fold into the medium for a final iron concentration of 160 nM. All metal-deficient cultures were grown in plastic flasks and 50-ml tubes. As precaution against metal contaminants, plastic flasks from the dishwasher were treated with 1 mM EDTA overnight, followed by abundant rinsing with 18.2-MΩcm water before culture growth.

For the preparation of metal-depleted medium, Pvd (2 mg) from strain ATCC 27853 (a gift from Valerie Geoffroy) was coupled to 1 ml of N-hydroxysuccinimide-activated Sepharose (GE Healthcare, Uppsala) via its lysine side chain according to the manufacturer's recommended protocol. An immobilized Pvd column (IPC) was prepared and used to extract the trace metals from 50 ml SM. The medium was passed over the column with a 1-min contact time. IMAC-extracted medium (SM_imac) was prepared by passing 1 liter of SM over a 5-ml HiTrap IMAC Sepharose HP column (GE Healthcare) at 5 ml/min, followed by the addition of 10 ml of the same chelating resin to the flowthrough and mixing with gentle inversion on a rotator for 48 h at 4°C. The resin was allowed to settle and the medium stored at −20°C.

FpvA expression levels in the different medium preparations were monitored by Western blotting of boiled whole-cell lysates. Even loading of the sodium dodecyl sulfate (SDS) gel was ensured by using the equivalent of 200 μl of each culture at an OD₆₀₀ of 1.0 and then boiling these cells for 5 min in 100 μl SDS loading buffer. The gel was transferred to a nitrocellulose membrane in 10 mM CAPS (N-cyclohexyl-3-aminopropanesulfonic acid)-10% methanol (pH 11) for 1 h at 200 mA. A polyclonal rabbit anti-FpvA antibody was used as a primary antibody (1:2,000 dilution), and the blotting was performed using the Pierce ECL reagents according to the manufacturer's recommendations.

Purification of FpvA.K691(pPVR2) or CDC5(pPVR2) cultures were grown 24 h at 30°C in SM with PTM4-lo salts and the cells harvested and stored frozen at −80°C. The frozen pellets were resuspended in 50 mM Tris (pH 8.0) (20 ml/g of cells) and lysed by sonication followed by a spin at 3,000 × g for 5 min. The supernatant was mixed with 1% N-lauroyl sarcosine, followed by centrifugation at 100,000 × g. The pellet was solubilized in 5% oPOE, followed by another centrifugation at 100,000 × g. The supernatant was loaded onto a 10-ml SourceQ (GE Healthcare) column and eluted with a 30-ml gradient from 0 to 1 M NaCl. The fractions with FpvA also contain FptA in an approximately equal concentration. Lipids were removed by use of a sucrose gradient with equal volumes of 15%, 10%, and 5% sucrose in 20 mM Tris-1% oPOE and the SourceQ elutions layered on top. The samples were centrifuged at 39,000 rpm for 16 h at 12°C in an SW41 rotor (Beckman), and the FpvA-FptA fraction visualized under UV illumination was collected with a syringe needle. The FpvA-FptA mixture was separated by chromatofocusing on a MonoP column (GE Healthcare) equilibrated in 25 mM Na cacodylate (pH 6.3) with elution in PBE74 (pH 3.8). The fractions were adjusted to pH 8 with Tris and then injected on a Superdex 200 gel filtration column preequilibrated in 20 mM Tris (pH 8.0)-100 mM NaCl-0.5% oPOE. The eluted sample was concentrated and exchanged into the desired buffer using Millipore Amicon ultracentrifugal filtration devices with a molecular weight cutoff of 50,000.

Fluorescence spectroscopy.Fluorescence experiments were performed on a QuantaMaster UV/visible spectrofluorometer (Photon Technology International, Birmingham, NJ) with a four-cuvette carousel. For in vivo experiments, the sample was stirred at 29°C in a 1-ml cuvette. The excitation wavelength (λ_exc) was set at 290 nm (for the fluorescent resonance energy transfer [FRET] experiments) or at 400 nm (for direct excitation), and for kinetics experiments, the emission of fluorescence (λ_em) was monitored at 450 nm. Recycling of Pvd on FpvA and Pvd-Fe dissociation were monitored simultaneously in the various growth media by using a four-sample carousel and alternating λ_exc between 290 nm and 370 nm for each time point. Excitation at 370 nm was used instead of excitation at Pvd's maximum of absorbance of 400 nm in order to minimize the monochromator travel distance between time points.

Preparation of periplasmic, cytoplasmic, inner membrane, and outer membrane fractions.CDC5(pPVR2) cells were grown in 50 ml of SM or SM_imac to an OD₆₀₀ of 1, at which time 600 nM Pvd was added. After 30 min, the cells were pelleted at 6,000 × g and the cellular fractions separated as previously described (12). Briefly, the pellet was resuspended in 3 ml 20% sucrose-1 mM EDTA-0.2 M Tris (pH 8.0), and after 2 min at ambient temperature osmotic shock was applied by the rapid addition of 4.5 ml ice-cold water followed by gentle sample inversion. After 2 min on ice, the periplasmic fraction was separated from the cells by centrifugation at 6,000 × g for 10 min and the pellet rinsed with water and resuspended in 20 ml of 20 mM Tris (pH 8.0). The cells were lysed by sonication and the cytoplasmic fraction separated from the insoluble material at 100,000 × g for 30 min. The inner membrane was extracted from the pellet with 20 ml of 1% sodium N-lauroyl sarcosine in 20 mM Tris (pH 8.0), followed by a second spin at 100,000 × g. The outer membrane was extracted from the resulting pellet with 2% oPOE in 20 mM Tris (pH 8.0), followed by a final high-speed centrifugation. The fluorescence intensity (λ_exc = 400 nm and λ_em = 450 nm) was measured for each fraction and the appropriate dilution factors applied to the measurements so that all reported intensities represent the total fluorescence from the same volume of the initial cultures. Minimal cross-contamination of the fractions was verified with Coomassie blue- and silver-stained SDS-polyacrylamide gels (12). Although we consistently found a small amount of FpvA in the periplasmic fraction, it was not significant compared to the amount of Pvd that was recovered there.

Elemental analyses.FpvA-Pvd was purified from K691(pPVR2) as described above, with the addition of 1 mM EDTA in all buffers until the final gel filtration into analysis buffer (10 mM Tris [pH 8.0], 100 mM NaCl, 0.5% oPOE). The elution from the penultimate column (MonoP) was split in two, and half was treated with a 20-fold excess of Pvd-Fe for 1 week before both samples were further purified on a Superdex 200 gel filtration column. The eluted samples (16.7 μM for FpvA-Pvd and 14.5 μM for FpvA-Pvd-Fe) were assayed for Fe and Al contents.

ICP-AES (inductively coupled plasma-atomic emission spectroscopy) analyses were performed to determine Fe concentrations in nondiluted samples. Samples were injected via a peristaltic pump (Gilson) equipped with Tygon tubing at a 1-ml/min flow rate. Nebulization of samples was performed by means of a concentric nebulizer (Meinhardt). A JY 38 ICP-AES (Jobin Yvon) was used as the detector. ICP conditions were the following: nebulization gas flow rate, 0.35 liter/min; outer gas flow rate, 12.0 liter/min; and auxiliary gas flow rate, 0.2 liter/min. Detection was performed at 238.204 nm.

ICP-mass spectrometry (ICP-MS) analyses were performed to determine Al and Mn concentrations in samples diluted 10-fold. Samples were injected via a peristaltic pump (Gilson) equipped with Tygon tubing at a 1-ml/min flow rate. Nebulization of samples was performed by means of a concentric nebulizer (Meinhardt type C; flow rate, 1 ml/min). A PlasmaQuad 3 ICP-MS (thermo-elemental) was used as the elemental detector. ICP conditions were the following: nebulization gas flow rate, 0.75 liter/min; outer gas flow rate, 13.5 liter/min; auxiliary gas flow rate, 1.8 liter/min. The plasma power was set to 1,350 W, and ion lens voltages were adjusted to maximize the signals detected at m/z = 27 and m/z = 55 for Al and Mn, respectively.

Synthesis of NP-Pvd.An amine-containing derivative of Pvd was synthesized by coupling a diamino-polyethylene glycol (PEG) [2,2′-(ethylenedioxy)bis(ethylamine)] (Aldrich) to the succinate moiety on Pvd using standard carbodiimide chemistry. Pvd-Fe (0.78 mg, 0.56 μmol) was dissolved in 0.7 ml dimethyl sulfoxide-H₂O (7:1, vol/vol), to which was added 3 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (ABCR Karlsruhe). After 1.5 h at room temperature, 2 μl of the diamino-PEG was added to give approximately 20 mM, or a 25-fold excess over Pvd-Fe. After 3 h, the reaction mixture was diluted 20-fold into 0.5% acetic acid (AcOH)-1 mM EDTA and injected on a 6-ml ResourceS column equilibrated in the same buffer. Elution was carried out at 3 ml/min with a 30-ml gradient from 0 to 0.5 M NaCl. The first major peak was the metal-free starting material, and the second was the desired product based on matrix-assisted laser desorption ionization-time-of-flight MS. The product was diluted threefold in 0.5% AcOH, reinjected on the same column, and eluted with a 30 ml gradient to 1.25 M ammonium acetate in 1% AcOH. The eluted peak (1 ml) was frozen at −80°C, lyophilized, and resuspended in 50 μl H₂O, giving a concentration of 1 mM amino-PEG-Pvd (NP-Pvd) based on the extinction coefficient of Pvd at pH 5 of 16,500 M⁻¹cm⁻¹. The NP-Pvd was reloaded with iron by the addition of 20 mM sodium citrate (pH 5) with 1 mM FeCl₃ to give a twofold excess of iron to Pvd.

SPR.Surface plasmon resonance (SPR) measurements were performed at 25°C using a Biacore 2000 instrument. The response measured in Biacore experiments is related to the accumulation of mass at the sensor surface and is recorded as a function of time using arbitrary resonance units (RU), where a signal of 1 RU corresponds to the binding of approximately 1 pg of material per mm². The NP-Pvd-Fe was immobilized (48 RU) on a CM5 sensor surface from a 10 μM solution in 0.5 mM sodium citrate (pH 5.0) via standard amine-coupling chemistry (16). A surface treated with the same chemistry but omitting the NP-Pvd-Fe injection was used as reference surface. The running buffer for the NP-Pvd-Fe immobilization was 10 mM HEPES-150 mM NaCl (pH 7.5). For the FpvA interaction and siderophore competition analysis, the running buffer was 10 mM Tris-150 mM NaCl-0.8% oPOE (pH 8.0) (TNO). EDTA (0.4 mM) was added to this running buffer for experiments involving iron stripping from immobilized Pvd surfaces and reloading. To remove iron from the immobilized NP-Pvd-Fe surface, the surface was treated by three pulses (2 min) of formic acid 1%-8 mM EDTA. The NP-Pvd surface was reloaded with iron by the injection (twice for 2 min each) of 10 mM citrate-0.5 mM FeCl₃ (pH 5.0).

Interaction assays and data processing and analysis.Solubilized and purified FpvA (7.5 nM to 2 μM) was injected for 120 s on the NP-Pvd-Fe surfaces, followed by a 600-s buffer injection. All sensorgrams were processed by double referencing, and the binding profiles were analyzed by global fitting using the simple 1:1 Langmuir model from the BIAevaluation 4.1 software (21).

For competition experiments, increasing amounts of Pvd-Fe or Pvd were incubated (>60 min, room temperature) with a constant concentration of FpvA (250 nM or 1,000 nM, respectively). The FpvA-siderophore mixture was then allowed to flow over the NP-Pvd-Fe surface to measure the free FpvA that can still bind to the surface. The free protein concentration was deduced from the initial slope recorded between 12 and 20 s after the injection start, using a calibration curve established by injecting known FpvA concentrations on the same surface. The “solution affinity” model from the BIAevaluation 4.1 software was use to evaluate the equilibrium constant from the plot of free concentrations of FpvA against the total concentration of siderophore.

The purified in vivo-formed FpvA-Pvd complex contains aluminum.The purification of an iron-free FpvA-Pvd complex from the Pvd-producing and FpvA-overexpressing strain K691(pPVR2) has been previously reported (26) and has been used to solve the crystal structure of the FpvA-Pvd complex (6). Although those authors noted in their description of the structure that there was a significant density in the Pvd molecule where an iron would normally be bound, it was not attributed to a metal and thus was not modeled. Therefore, we analyzed the purified protein by ICP-AES and ICP-MS in order to see what metals could be associated with Pvd in the complex. As a control, the same protein was incubated in a 20-fold excess of Pvd-Fe for 1 week to completely exchange the bound Pvd for Pvd-Fe. Both protein samples were passed over a Superdex 200 column before being subjected to elemental analysis. The buffer from the gel filtration (10 mM Tris, 100 mM NaCl, 0.5% oPOE) was also analyzed as a blank. The analyses showed that there was more aluminum in the FpvA-Pvd sample than in either the FpvA-Pvd-Fe or blank sample (Table 1) and that it was present in the same concentration as the Pvd in the FpvA-Pvd complex. In our hands there is a batch-to-batch variability in FpvA-Pvd preparations, in that they have different amounts of the copurified contaminants apo-FpvA and FpvA-Pvd-Fe. To assess whether Pvd in the preformed in vivo FpvA-Pvd complex might be picking up iron or aluminum from the buffers during purification, we performed the lysis and membrane extraction steps of the purification in 50 μM Fe(II)SO₄ and 90 μM Fe(III)Cl₃ and found that this excess of iron did not reduce the FRET in the detergent-solubilized outer membrane compared to the control (data not shown). Therefore, aluminum must be already bound to Pvd in the FpvA-Pvd complex in vivo, which explains why iron is not picked up by the complex during or after purification. The small contaminant of FpvA-Pvd-Fe could form from apo-FpvA and metal-free Pvd, which is always present in excess during the cell lysis step of the purification.

Trace metals present in minimal media modulate Pvd fluorescence.We use SM for iron-limited Pseudomonas cultures, and since metal binding modulates Pvd fluorescence, we used this fluorescence as a probe of trace metals in SM. In Fig. 1A the fluorescence of 2 μM Pvd in SM is compared to that in SM plus 100 μM EDTA. The fluorescence intensity of Pvd in SM was nearly twofold higher, and remarkably, it was not immediately affected by the subsequent addition of EDTA. Thus, this modulation in Pvd fluorescence appears to be the result of a competition between EDTA and Pvd for a metal that displays slow kinetics of ligand exchange. A similar fluorescence modulation can be replicated with aluminum and EDTA in 10 mM Tris. As demonstrated in Fig. 1B, EDTA had no direct effect on Pvd fluorescence, but instead it was through the sequestration of a metal that the fluorescence was modulated. When Al(III) was added to 20 mM Tris with Pvd, there was an increase in fluorescence (trace 6), which was not reduced by subsequent addition of EDTA (trace 5); however, when EDTA was added to the Al(III) before the Pvd, then the fluorescence (trace 4) remained the same as Pvd alone (trace 1). Also, in both SM and aluminum-spiked Tris buffer, there was a concomitant red shift of the emission spectra by 5 nm upon a metal-induced fluorescence increase.

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FIG. 1.

Fluorescence of Pvd in SM and Tris buffer with EDTA and aluminum. Emission spectra of Pvd in various solutions (λ_exc = 400 nm) are shown. When EDTA was present, the additions of EDTA and Pvd were always separated by 5 min to allow a more complete metal chelation. (A) Two micromolar Pvd in SM (1), SM plus 100 μM EDTA (2), and SM with addition 5 min later of 100 μM EDTA (3). (B) One micromolar Pvd, 40 μM EDTA, and/or 20 μM AlCl₃ in 20 mM Tris (pH 8.0) listed in the order of addition: Pvd alone (1); Pvd and EDTA; (2) EDTA and Pvd (3); EDTA, Al, and Pvd (4); Al, Pvd, and EDTA (5); and Pvd and Al (6).

Immobilized Pvd and IMAC Sepharose extract a Pvd fluorescence-enhancing metal.In order to study the role of trace metals in the Pvd-Fe uptake pathway, a method was developed for making a defined minimal medium in which the majority of trace multivalent metals that are present in standard medium preparations have been removed. Without a prior knowledge of the metals which might influence ferric Pvd transport, a metal extraction system that mimics the potentially broad spectrum of Pvd-metal affinities was initially chosen. Pvd from P. aeruginosa strain ATCC 27853 was immobilized on N-hydroxysuccinimide-activated Sepharose via its lysine side chain, thus creating a metal affinity resin. SM was passed over the immobilized Pvd to yield SM_ip. Addition of 160 nM Pvd to SM and SM_ip confirmed via a 30% drop in fluorescence that a fluorescence-enhancing species had been removed by the immobilized Pvd column (data not shown).

The cost of a large-scale immobilized Pvd column, which would be needed to prepare several liters of SM_ip, inspired the development of an alternative approach. Although we found that iminodiacetate-based metal chelating resins did not remove the fluorescence-enhancing metals in SM, the more efficient chelation by IMAC Sepharose (a proprietary metal affinity resin from GE Healthcare) proved to be more effective. To overcome the slow kinetics of ligand exchange for some metals, the metal removal was performed in two steps: on a column (5-min contact time) and then for 48 h in batch with fresh resin. For these manipulations, there was ∼1 mM MgSO₄ in the medium, and thus the only trace metals effectively removed were those that are chelated by IMAC Sepharose with a much higher affinity than Mg(II). The fluorescence emission spectra of 2 μM and 100 nM Pvd in SM and SM_imac showed that the IMAC resin removes a large amount of the fluorescence-enhancing metal (Table 2). However, the smaller difference in fluorescence with the lower Pvd concentration suggests that there are still some metals left in solution. This is supported by the fact that preaddition of EDTA can further decrease the fluorescence of 100 nM Pvd but has little effect on 2 μM Pvd. Once again, the red shift in the emission is correlated with trace metals in solution. Fluorescence measurements at 24 h after sample preparation showed an increased intensity, suggesting that the kinetics of ligand exchange for some metals are slow and that, for the metals concerned, Pvd has a higher affinity than EDTA. Because there is a competition between Pvd and EDTA for metals that can both enhance and quench fluorescence, the observed time-dependent fluorescence modulation is a function of the concentrations and binding kinetics of all the metals in solution. Therefore, the fluorescence modulation is too complex to interpret apart from the conclusions that a trace metal that enhances Pvd fluorescence exists in SM at a concentration on the order of 2 μM and that it is largely removed in SM_ip and SM_imac.

Reduction of in vivo FRET in P. aeruginosa grown in metal-poor media.Previous studies have demonstrated that Pvd is bound to FpvA in vivo when P. aeruginosa is cultured in iron-deficient media by taking advantage of a fortuitous signal of the interaction between FpvA and Pvd: the FRET which occurs between the tryptophans of FpvA and the bound Pvd (25, 26). To investigate the effect of metal depletion on in vivo FRET, SM_ip and SM_imac were compared with SM as media for the growth of P. aeruginosa. However, the metal-depleted media did not support bacterial growth above a cell density (A ₆₀₀) of 0.3 OD unit, compared to 1.2 OD units in SM, likely due to a lack of some essential nutrients such as iron and other trace metals that were extracted by IMAC Sepharose and immobilized Pvd. Therefore, in all subsequent experiments, the media were supplemented with a modified PTM4 trace salts solution which included 160 nM Fe₂SO₄ (see Materials and Methods). When the FpvA-overexpressing strain K691(pPVR2) was grown in the trace salts-supplemented SM, SM_ip, and SM_imac, we observed a large difference in the amount of in vivo FRET despite similar levels of FpvA expression and Pvd production (Fig. 2A). FpvA expression was qualitatively compared by Western blotting of the whole-cell lysates, and the Pvd concentration in the medium was calculated from its extinction coefficient at 405 nm. The Pvd concentrations varied from 50 to 150 μM depending on the age of the culture, and FpvA expression levels were similar in all the media (Fig. 2B). Although both metal removal protocols cause a reduction in the in vivo FRET, the 48-h batch method used in the SM_imac preparation is more efficient in reducing the FRET than the short exposure used in the SM_ip preparation.

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FIG. 2.

FRET and FpvA expression from bacteria cultured in metal-deficient medium. K691(pPVR2) was grown overnight in SM, SM_{ip ,} and SM_imac with PTM4-lo trace salts (see Materials and Methods), and then the cells were centrifuged, washed with 50 mM Tris (pH 8.0), and resuspended at an OD₆₀₀ of 2. (A) FRET (λ_exc = 290 nm), expressed as the emission intensity normalized to the tryptophan fluorescence in order account for small differences in cell density between samples. Dashed line, SM; thin line, SM_ip; thick line, SM_imac. (B) Western blot with polyclonal anti-FpvA antibodies of whole-cell lysates. For each lane, 500 μl of the cells at an OD₆₀₀ of 2 were pelleted, resuspended in 100 μl SDS sample loading buffer, and incubated for 5 min at 95°C, and then 20 μl was run on a 13.5% SDS-acrylamide gel. Lanes: 1, 250 ng purified FpvA; 2, K691(pPVR2) in SM_imac; 3, K691(pPVR2) in SM; 4, K691(pPVR2) in SM_ip; 5, CDC5(pPVR2) in SM; 6, CDC5(pPVR2) in SM_imac.

Loss of Pvd binding in P. aeruginosa grown in SM_imac.The in vivo binding kinetics and affinity of Pvd were reported for CDC5(pPVR2) cells grown in standard SM preparations. In order to test whether this reported binding is metal dependent, we grew CDC5(pPVR2), a Pvd-deficient strain that overexpresses FpvA, in SM and SM_imac. Growing cultures of CDC5(pPVR2) at an OD of 0.8 were put on ice, followed by the addition of 1 μM Pvd or Pvd-Ga. After 10 min, the cells were centrifuged, washed, and resuspended in Tris buffer for FRET measurements. The addition of metal-free Pvd gave rise to FRET only in SM (Fig. 3A). A similar expression level of FpvA in the different media was confirmed by Western blotting (Fig. 2B) as well as by monitoring the FRET between FpvA and the fluorescent complex of Ga(III) with Pvd (Pvd-Ga) (Fig. 3B). The binding assay was performed on ice in order to slow the transport of Pvd to better observe the binding of Pvd without the internalization of Pvd-metal complexes. However, the same result was obtained at room temperature (data not shown). This experiment was repeated for bacteria grown in SM supplemented with 80 μM FeSO₄, in an effort to suppress FpvA expression, as a control for nonspecific or FpvA-independent interactions of Pvd-Ga or Pvd with the bacteria. Neither Pvd nor Pvd-Ga incubation produced FRET for the cells in the iron-supplemented medium, confirming that the FRET induced by Pvd-Ga incubation arises from FpvA-Pvd-Ga complexes in the outer membrane. Thus, despite similar FpvA expression levels, only the cells grown in the standard SM and not those grown in SM_imac were able to bind Pvd.

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FIG. 3.

Pvd binding to FpvA in vivo monitored by FRET. CDC5(pPVR2) was grown to an OD₆₀₀ of 1, then incubated with 1 μM Pvd (A) or 1 μM Pvd-Ga (B) for 5 min on ice, and then washed in and resuspended in 50 mM Tris (pH 8.0). The emission spectrum for each sample was normalized to the tryptophan fluorescence, and then the control without Pvd or Pvd-Ga was subtracted. The three traces are for cells grown in SM (1), SM_imac (2), and SM plus 80 μM FeSO₄ (3).

The recycling of Pvd onto FpvA is diminished in SM_imac.It has been previously shown that after the transport and dissociation of Pvd-Fe by P. aeruginosa, the Pvd is recycled (Pvd_recy) to the extracellular medium as well as onto FpvA (24). In wild-type bacteria, the recycling is observed as a decrease in FRET following the addition of Pvd-Fe, followed by a rise back to the original level. This corresponds to a displacement of Pvd by Pvd-Fe, followed by a rebinding of Pvd to FpvA. Although the mechanism of recycling is unknown, the binding of Pvd_recy to FpvA suggested that the receptor might be involved in the Pvd-Fe dissociation and/or recycling. In order to test the metal dependence of this process, we compared the recycling of Pvd on FpvA in CDC5(pPVR2) cells that were grown in either SM or SM_imac. Since CDC5 cells do not produce Pvd, the recycling is observed as a drop in FRET only upon successive additions of Pvd-Fe. This recycling was much more pronounced in the SM-cultured cells, which is consistent with a metal dependence (Fig. 4, top). The kinetics of Pvd-Fe dissociation were simultaneously followed the by direct excitation of Pvd fluorescence (Fig. 4, middle). The dissociation was faster in the SM_imac-cultured cells, which can be explained by a lack of competition with other metals (see Discussion). The lower overall fluorescence intensity in SM_imac is due to the fluorescence-enhancing Pvd-metal complexes that occur in SM. The ratio between the direct excitation and FRET fluorescence is a sensitive signal for the amount of Pvd that is free in solution versus bound to FpvA. Free Pvd fluorescence at 450 nm with excitation at 290 nm was about 1/12th as much as with excitation at 370 nm. The FRET between Pvd and FpvA makes this ratio close to 1 in purified FpvA-Pvd complexes. Consequently, the bottom panel of Fig. 4, which plots this ratio during Pvd-Fe uptake, depicts the kinetics and extent of FpvA-Pvd complex formation in vivo. For the SM_imac-cultured cells, this ratio remained higher and more stable during the three additions of Pvd-Fe, while for the bacteria grown in SM, the addition of Pvd-Fe displaced Pvd from the receptor (rise in ratio), followed by a rebinding of the Pvd to FpvA (drop in ratio).

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FIG. 4.

Kinetics of Pvd-Fe dissociation and recycling in bacteria cultured in metal-deficient medium. CDC5(pPVR2) was grown to an OD₆₀₀ of 1 and then washed with 20 mM Tris (pH 8.0) and resuspended at an OD₆₀₀ of 2. The kinetics of Pvd recycling (top panel; λ_exc = 290 nm) and Pvd-Fe dissociation (middle panel; λ_exc = 370 nm) were monitored at 450 nm following three additions of 300 nM Pvd-Fe (vertical dashed lines) and plotted as the change in fluorescence intensity from t = 0. The ratio between the two signals (direct excitation and FRET) is plotted in the lower panel.

Periplasmic accumulation of Pvd is metal dependent.The accumulation of Pvd-Ga complexes in the periplasm of P. aeruginosa was recently demonstrated and presented as evidence for the periplasmic dissociation of Pvd-Fe and involvement of a ferrous intermediate during iron transport by FpvA (12). In these experiments, most of the Pvd fluorescence after transport of Pvd-Fe was recovered in the extracellular medium; however, it also accumulated, although to a much lesser extent than for Pvd-Ga, in the periplasm. The accumulation in the periplasm could be due to inefficient recycling of metal-free Pvd or perhaps due to another Pvd-metal complex that cannot be dissociated by the bacteria. To further investigate these possibilities, the cellular localization of Pvd fluorescence was monitored after Pvd treatment of CDC5(pPVR2) cells grown in SM and SM_imac. The cell fractions were separated as previously described (12) and the Pvd concentration reported as the percentage of recovered fluorescence from an equivalent culture volume (Fig. 5). In order to directly compare the intensities from the two cultures, 1 μM AlCl₃ was added to all the samples prior to fluorescence measurements. In this manner, all of the Pvd is in a metal complex and the differences in quantum yield between Pvd and Pvd-metal complexes are minimized. Figure 5 shows that for cells grown in the metal-poor medium, there was much less Pvd accumulation in the periplasm. Also, there was less Pvd in the outer membrane fraction, in agreement with the findings in Fig. 2 that show reduced in vivo binding to FpvA in the absence of metals. The experiment was also performed with cells grown in SM_imac supplemented with 1 μM AlCl₃. In this case the results were like those for bacteria cultured in SM. Thus, the accumulation of Pvd in the periplasm is metal dependent, and 1 μM Al(III) is sufficient to reproduce this accumulation in metal-poor medium. Consistent with this metal dependence is the fact the addition of Al(III) to the fractionated samples had no effect on the SM or SM-Al samples but caused a dramatic increase in the fluorescence of the SM_imac samples, putting their total fluorescence (sum of individual cell fractions) on par with the fluorescence from the SM samples. It is worth noting that the volume of the periplasm (as well as that of the other cell fractions) is many hundreds if not thousands of times smaller than the culture volume that it occupies, so the concentration of Pvd in the periplasm is significantly higher than that in the extracellular medium despite the lower fluorescence measurement. We also tested the growth of P. aeruginosa in SM with a range of AlCl₃ concentrations (10 to 800 μM) to see if it could be a growth inhibitor. A twofold reduction in doubling time was observed for all samples with aluminum, but with a similar final cell density.

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FIG. 5.

Cellular distribution of fluorescence after exposure to exogenous Pvd. CDC5(pPVR2) was cultured in SM, SM_imac, and SM_imac plus 1 μM AlCl₃ to an OD₆₀₀ of 0.8, at which point it was incubated for 30 min in the presence of 600 nM Pvd. The cell fractions (periplasm, cytoplasm, inner membrane, and outer membrane) were separated (see Materials and Methods), and the fluorescence in the media and in four cellular locations was quantified. Values are expressed as a percentage of the total fluorescence recovered from each culture.

Purified FpvA binding to Pvd is metal dependent in vitro.The reported values for the apparent association kinetics between FpvA and Pvd in vitro were derived from FRET measurements with purified and detergent-solubilized FpvA that had been expressed in the Pvd-deficient strain CDC5 (5). An unusual feature of this association was that its rate was independent of the concentration of Pvd. Although this can be explained by a fast initial binding step that does not give rise to FRET, the possibility that trace metals in the buffers were the cause of the concentration independence needs to be investigated. This is particularly relevant since the measurements were performed only in the concentration range near the reported K_D (equilibrium dissociation constant) (∼10 nM), a range that is below the limits of detection for trace metals such as iron and aluminum. If the binding of Pvd to FpvA in vitro is dependent on the trace metals in the buffers, then when the buffers are treated with the IMAC resin, a loss of binding should be observed. Also, if the trace metal concentration in protein solubilization buffer (20 mM Tris [pH 8], 1% oPOE) is not higher than that in the SM recipe, there will be a loss of Pvd binding when the concentrations of Pvd and FpvA are relatively high if the binding is metal dependent. When 30 μM Pvd was mixed with 20 μM FpvA, a small amount of complex quickly formed, but based on the degree of FRET, there was no further complex formation after 2 days (Fig. 6A). However, following a 200-fold dilution of this mixture (100 nM FpvA and 150 nM Pvd), the complex continued to form, reaching completion within 24 h. Our experience with the purification of FpvA-Pvd complexes from a Pvd-producing strain indicates that upon excitation at 290 nm, the ratio of Trp fluorescence to Pvd fluorescence (FRET) falls in the range of 1:3 to 1:3.5 when all FpvA molecules are bound to a Pvd. By this measure, the FpvA molecules in the dilute samples are 100% bound to Pvd, whereas the higher-concentration samples reach only 10 to 20% binding. This inverse dependence on the concentration could be explained by a self-association of FpvA or Pvd that inhibits their interaction with each other. This possibility can be excluded in a number of ways but most simply by adding Fe(III) or Al(III) at the same concentration as Pvd, which quickly leads to complete complex formation even at 20 μM FpvA (data not shown). Furthermore, the exposure of buffers to IMAC Sepharose or the addition of metal chelators such as EDTA or ferrichrome all led to a reduction of complex formation even at low concentrations of FpvA and Pvd (Fig. 6C and D). Only a trace metal contaminant in the buffers or protein stocks can explain the observed inverse concentration dependence seen in Fig. 6A.

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FIG. 6.

Concentration and metal dependence of the FpvA-Pvd complex in vitro monitored by FRET. FpvA in pure or crude (initial ion-exchange column elution contaminated with FptA, the pyochelin receptor) form was mixed with pure Pvd in 20 mM Tris-1% oPOE at different concentrations and with different metal chelators. The FRET (λ_exc = 290 nm) was measured 48 h after the initial mixture of FpvA and Pvd. (A) Mixtures of 20 μM pure FpvA plus 30 μM Pvd were diluted 200-fold (to 100 nM FpvA) for fluorescence measurements. Black line, 48 h of incubation and then measurement immediately after dilution; gray line, 50-fold dilution (to 400 nM FpvA) for 48 h and then 4-fold dilution for immediate measurement; dotted line, 24 h of incubation, 200-fold dilution, and then measurement 24 h after dilution. (B) Pure FpvA at 150 nM mixed with 300 nM Pvd in standard (dashed line) or IMAC-treated (solid line) buffer. (C) Crude ∼3 μM FpvA mixed with 5 μM Pvd in standard buffer (dashed line) or buffer pretreated for 5 min with 100 μM EDTA (solid line). (D) Pure FpvA at 150 nM in standard buffer (dashed line) or in buffer with 700 nM ferrichrome (solid line) and 24 h later mixed with 300 nM Pvd.

SPR confirms an iron requirement for FpvA-Pvd interactions.We introduced a primary amine to Pvd via a PEG linker on the succinate moiety in order to be able to immobilize Pvd to a Biacore sensor chip by primary amine coupling. The succinate moiety was chosen as the attachment site because it has been previously demonstrated that the attachment of bulky groups to this part of Pvd had no effect on iron chelation and transport (27). Using the immobilized Pvd surface, we were able to compare the affinities of FpvA for Pvd and Pvd-Fe in a controlled and quantitative manner. Figure 7A shows that the binding of FpvA to the immobilized Pvd is iron dependent. A 0.5 μM FpvA sample gave a response of 90 RU for binding to the immobilized Pvd-Fe surface, while a 5 μM sample gave 5 RU on the iron-free surface. Upon subsequent reloading of the surface with iron, it was possible to recover 90% the initial FpvA binding activity.

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FIG. 7.

SPR analysis of FpvA-Pvd interactions with and without iron. The binding of FpvA to immobilized NP-Pvd was monitored by SPR and was shown to be iron dependent. (A) The profiles in black correspond to the binding of 500 nM FpvA to iron-loaded, immobilized NP-Pvd-Fe (solid line) and again after the surface was stripped of iron and subsequently reloaded with iron (dashed line). In red is the binding profile of FpvA (5 μM) for the iron-free NP-Pvd surface. (B) Kinetic analysis of FpvA binding to immobilized NP-Pvd-Fe. The binding curves (red) corresponding to the injection of various concentrations FpvA (from 7.5 to 250 nM) on the NP-Pvd-Fe surface are overlaid with the global fit for a simple one-to-one interaction (black). (C) The binding activities of NP-Pvd-Fe and Pvd-Fe are similar in solution. Iron-free Pvd (empty circles), Pvd-Fe (black circles), and NP-Pvd-Fe (gray circles) were mixed in a molar ratio of 0.1 to 10 with a fixed concentration of FpvA (250 nM for Pvd-Fe and NP-Pvd-Fe or 1,000 nM for Pvd without iron). The solution binding is observed as a decrease in the free FpvA that can bind to the surface and is expressed as a relative response compared to the response for FpvA alone.

The FpvA binding parameters for immobilized Pvd-Fe were evaluated from a global fit of the binding profiles to a simple 1:1 Langmuir model (Fig. 7B). The kinetic association and dissociation constants calculated from three independent experiments were k _on = (7 ± 3) × 10⁴ M⁻¹ s⁻¹ and k _off = (1.2 ± 0.13) × 10⁻² s⁻¹, yielding an equilibrium constant (K _D = k _off/k _on) of (1.9 ± 0.8) ×10⁻⁷ M. The maximum binding (R _max) values were less than 10% of the theoretical R _max in all three experiments, suggesting limited access of the binding site of the immobilized NP-Pvd to the FpvA. The immobilization of ligands can in some cases markedly affect the access and the binding site (18), and solution equilibrium measurements can provide an alternative method for measuring binding activity without these effects. Figure 7C demonstrates FpvA binding to Pvd in solution, observed as the inhibition of FpvA binding to the immobilized Pvd-Fe surface. Pvd-Fe and NP-Pvd-Fe showed similar binding activities, while the metal-free Pvd, even at 10 μM (a 10-fold molar excess over FpvA), did not inhibit FpvA binding to the immobilized Pvd-Fe surface. The range of K _D values that can be evaluated with this method depends on the measurable range of free FpvA. The lowest free FpvA concentration that can be accurately measured using a calibration curve constructed with known concentrations of FpvA is approximately 5 nM (slope, <0.18 RU/s) (data not shown).

Theoretical considerations for equilibrium affinity measurements indicate that a high affinity (K _D in the range of a few nM or less) cannot be determined with good accuracy because the free protein concentrations are too low to be measured under these experimental conditions (33). An equilibrium dissociation constant in the nanomolar range (0.5 ± 3.6 nM) was evaluated for the NP-Pvd-Fe interaction with FpvA, and since Pvd-Fe and the amine derivative have very similar binding profiles (Fig. 7C), it is expected that they have similar affinities in solution. This is in relatively good agreement with the literature values of 0.5 to 1.5 nM for the in vivo Pvd-Fe binding to FpvA (5, 15, 25).