Catechol Siderophores Control Tungsten Uptake ... - ACS Publications

The catechol siderophores released by the soil diazotroph Azotobacter vinelandii are used to modulate the uptake of essential (Fe, Mo) and toxic (W) m...
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Environ. Sci. Technol. 2008, 42, 2408–2413

Catechol Siderophores Control Tungsten Uptake and Toxicity in the Nitrogen-Fixing Bacterium Azotobacter vinelandii THOMAS WICHARD,† JEAN-PHILIPPE BELLENGER,† AURÉLIE LOISON,‡ AND A N N E M . L . K R A E P I E L * ,§ Department of Geosciences and Chemistry Department, Princeton Environmental Institute, Guyot Hall, Princeton University, Princeton, New Jersey 08544, and UMR 7512 (CNRS-ULP), ECPM, 25 Rue Becquerel, 67087 Strasbourg Cedex 02, France

Received October 19, 2007. Revised manuscript received December 27, 2007. Accepted January 7, 2008.

Molybdenum (Mo) and tungsten (W), which have similar chemistry, are present at roughly the same concentration in the earth’s continental crust, and both are present in oxic systems as oxoanions, molybdate and tungstate. Molybdenum is a cofactor in the molybdenum-nitrogenase enzyme and is thus an important micronutrient for N2-fixing bacteria such as Azotobacter vinelandii (A. vinelandii). Tungsten is known to be toxic to N2-fixing bacteria, partly by substituting for Mo in nitrogenase. We show that the catechol siderophores produced by A. vinelandii, in addition to being essential for iron acquisition, modulate the relative uptake of Mo and W. These catechol siderophores (particularly protochelin), whose concentrations in the growth medium increase sharply at high W, complex all the tungstate along with molybdate and some of the iron. The molybdenum-catechol complex is taken up much more rapidly than the W complex, allowing A. vinelandii to satisfy its Mo requirement and avoid W toxicity. Mutants deficient in the production of catechol siderophores are more sensitive to tungstate and have higher cellular W quotas than the wild type. The binding of metals by excreted catechol siderophores allows A. vinelandii to discriminate in its uptake of essential metals, such as Fe and Mo, over that of toxic metals, such as W, and to sustain high growth rates under adverse environmental conditions.

Introduction The metal tungsten (W) has a chemistry very similar to that of molybdenum (Mo). In oxic environments, both are present as oxoanions, tungstate (WO42-) and molybdate (MoO42-), which are close structural and electronic analogues and have approximately the same size (1). While molydenum-enzymes are ubiquitous in biology, tungsten-enzymes have been found in only a few prokaryotes (2). In most organisms, W is toxic, in part by replacing Mo in enzymes and making * Corresponding author phone: 1-609-258-7415; fax: 1-609-2585242; e-mail: [email protected]. † Department of Geosciences, Princeton University. ‡ UMR 7512 (CNRS-ULP-ECPM). § Chemistry Department, Princeton University. 2408

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them inactive. Mo and W are present at about the same concentrations in soils (ca. 0.6–2.3 ppm (3)), raising the possibility that W might be toxic to Mo-requiring organisms. One of the most important uses of Mo is in the enzyme nitrogenase, which reduces atmospheric nitrogen N2(g) into ammonium and supplies new nitrogen to Earth’s ecosystems. A number of N2-fixing bacteria, including the gram-negative soil bacterium Azotobacter vinelandii (A. vinelandii), use a high-affinity transport system for molybdenum uptake (4, 5). This ATP-binding cassette (ABC) transporter is encoded by the mod operon and consists of a periplasmic protein that binds molybdate (ModA), and two transmembrane proteins (ModB and ModC) that transport Mo into the cytoplasm. Although it does not bind other oxoanions such as sulfate and phosphate, ModA binds molybdate and tungstate with similar affinities (6–8). The transport system encoded by the mod operon thus does not differentiate between MoO42- and WO42-. If taken up by N2-fixing bacteria, W can be incorporated into nitrogenase to yield an inactive form of the enzyme (9). The toxicity of tungstate to N2-fixing bacteria is indeed well-documented (10). It is dependent on the Mo concentration of the medium and appears only when W is in large excess over Mo (10, 11). The uptake of Mo in A. vinelandii is partly controlled by complexation with catechol siderophores (12), which are also known to be involved in Fe uptake (13–15). The wild type strain of A. vinelandii produces at least five different types of siderophores: azotobactin, a pyoverdine-like siderophore with one catechol, one hydroxamate, and one R-hydroxycarboxylic acid group; the tris(catechol) protochelin; the bis(catechol) azotochelin; and the mono(catechols) aminochelin and 2,3-dihydroxybenzoic acid (2,3-DHBA) (15). In this bacterium, the presence of W and Mo affects the nature and the concentration of catechol siderophores which form strong complexes with tungstate in addition to Fe and Mo (16–18). Here, we investigate how the catechol siderophores released by A. vinelandii modulate W uptake and toxicity.

Experimental Section Reagents. HPLC grade acetonitrile and methanol were purchased from Sigma or Fisher Scientific. 2,3-DHBA and 3,4-dihydroxybenzaldehyde (3,4-DHB) were obtained from Sigma and used without further purification. Bacterial Strains and Growth Conditions. The capsulenegative A. vinelandii strain OP (wild type), and F196 and P100, two mutant strains derived from strain OP and defective in siderophore production, graciously provided by the group of Paul E. Bishop (North Carolina State University), were used. The properties of the mutant strains were verified by HPLC analysis. Diazotrophic cultures of A. vinelandii were grown aerobically at 30 °C and pH 6.7 in liquid minimal medium with 10-4 M ethylenediaminetetraacetic acid (EDTA), as previously described (19). Unless otherwise indicated, the bacteria were grown under Fe-sufficient conditions ([FeCl3] ) 5 × 10-6 M (12)). The Mo and W concentrations were adjusted by additions of sodium molybdate and sodium tungstate solutions, respectively. The Fe contamination of the Mo and W stocks, as measured by inductively coupled plasma-mass spectrometry (ICP-MS), was less than 0.05%. Bacterial growth was monitored by measuring the optical density (OD) at 620 nm on a UV–vis spectrophotometer (HP8453E, HewlettPackard). The conversion factor from OD to cell density ()(1.16 ( 0.16) × 108 cells mL-1 OD-1) was determined by counting DAPI stained culture aliquots using epifluorescence 10.1021/es702651f CCC: $40.75

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microscopy (20). The phosphorus content of the cells ranged from 1.7 × 10-15 to 2.5 × 10-15 mol cell-1. Measurements of Metal Quotas. Cellular metal quotas in bacterial cultures (OD ) 0.6 ( 0.1) were measured as described before (19). Preparation of Azotochelin and Protochelin. Several grams of the (bis)catechol azotochelin were synthesized in its metal-free form according to published procedures (18, 21). The metal-free (tris)catechol protochelin was purified following a procedure modified from Taraz et al. (22), making possible a few experiments with a relatively low concentration of this compound. Briefly, A. vinelandii OP was grown at high tungstate concentration ([W] ) 10-5 M, [Mo] ) 10-8 M). A 500 mL aliquot of the culture (OD ) 1.5) was centrifuged (16000g, 1 h at 10 °C), and the supernatant passed through an OASIS-HLB cartridge (Waters) preconditioned with 6 mL of methanol and equilibrated with 6 mL of water. After loading, the cartridge was rinsed with 4 mL of water and the compounds were eluted with 4 mL of methanol. The eluate was concentrated under an argon stream down to 0.2–0.4 mL and further purified on a Sephadex LH20 (GE Healthcare) column in an isocratic gradient (80% methanol (v/v)). The elution was monitored by UV absorption at 254 nm, and catechols in collected fractions (V ) 1 mL) were identified by UV–vis spectroscopy. Negative ion ESI/MS of the collected fractions was performed by direct infusion (Thermo Scientific MSQ Plus). Metal-free protochelin and the tungsten-protochelin complex were identified by the pseudomolecular ion m/z 623 [M - H+]- and the doubly charged complex corresponding to m/z 418 [M - 4H+ + WO22-]2-, respectively. In a typical separation, 500 mL of culture yielded about 0.4 mg of metal-free protochelin. Iron- and tungsten-protochelin complexes (1:1) were prepared with purified protochelin (1.4 × 10-4 M) in 80% methanol (18) for use as standards during HPLC analysis. HPLC Determination of Catechol Compounds in the Growth Medium. A 20 mL aliquot of the bacterial culture was centrifuged (16000g, 20 min at 10 °C). The supernatant was spiked with the internal standard (20 µL of 3,4-DHB (200 µg mL-1 in MeOH)) and concentrated on an OASIS-HLB cartridge (see above). A 20 µL aliquot of the concentrated methanolic eluate was mixed with 30 µL of 0.07% aqueous TFA and subsequently analyzed by reversed-phase HPLC at pH 2.2 for the determination of catechol compounds (for further details see Supporting Information). Quantification of Metal Complexes in Growth Medium. For determination of metal complexes, the methanolic eluate (see above) was analyzed by HPLC at pH 6.6 using a solvent system of water/acetonitrile containing 10 mM ammonium acetate, followed by ICP-MS measurements of collected fractions (0.5–1.0 mL; see Supporting Information). Calibration curves were linear (r2 > 0.99) for W and Fe in the measurement range obtained by ICP-MS. Metal complexes were identified by co-injections of the corresponding (1:1) metal complexes and UV–vis spectroscopy of fractions containing high [Fe] or [W]. Short-Term Uptake of Mo and W. A. vinelandii OP was grown in a culture medium with [98Mo] ) 2 × 10-8 M and [W] ) 10-7 M. When the OD was close to 0.5, 50 mL of the bacterial suspension was aseptically centrifuged (16,000g, 10 min at 20 °C) washed with 10 mL of fresh medium and split into two subsamples. Each subsample was resuspended into 50 mL of fresh medium containing 2 × 10-8 M of sodium molybdate (Na295MoO4), 10-7 M of sodium tungstate (Na2WO4), and 10-4 M of either protochelin or azotochelin. The resuspension media, which contained no EDTA, were left to equilibrate for at least 20 min before addition of the bacteria to ensure complete complexation of the oxoanions (18). The short-term uptake of Mo or W was monitored by collecting aliquots of the cell suspension

FIGURE 1. (A) Growth rates of wildtype A. vinelandii (strain OP) as a function of [W] at various Mo concentrations. (B) Molar cellular quotas of W, Mo, and Fe normalized to phosphorus (P) in strain OP as a function of [W] ([Mo] ) 10-8 M, [Fe] ) 5 × 10-6 M, and OD ) 0.6 ( 0.1). Values represent results from three independent experiments (means ( std dev). Error bars that are not visible are within the symbols. over time and measuring cellular 95Mo or W quotas by ICP-MS (12). A similar experiment was repeated with cells preconditioned and resuspended at [Mo] ) 2 × 10-8 M (no W added, OD ) 0.5). Monitoring of Catechol Production and Metal Quotas in Response to High [W]. Duplicate 2 L cultures of A. vinelandii OP were grown with [Mo] ) 10-7 M and [Fe] ) 5 × 10-6 M. At OD ) 0.38 ( 0.01 the medium was spiked with tungstate to achieve a final concentration of [W] ) 5 × 10-6 M. Aliquots of the culture (10–50 mL depending on the OD) were harvested over time for measurement of catechol siderophores in the medium and intracellular Fe, W, and Mo quotas.

Results Growth Rates and Cellular Quotas of W and Mo in A. vinelandii. The growth rate of A. vinelandii OP (wild type) depends on the tungstate concentration [W] in the growth medium. At a total molybdate concentration, [Mo], of 10-8 M, the growth rate of strain OP remains at its maximum for tungstate concentrations below 1 µM; higher tungstate concentrations result in decreasing growth rates (Figure 1A). As observed in earlier studies (10, 11), tungstate is more toxic at low molybdate concentration. For a Mo concentration of 10-8 M, the decrease in growth rates at high [W] corresponds to an increase in the cellular W quotas (measured at OD ) 0.6, ≈6.9 × 1010 cells L-1), while the cellular quotas of Mo and Fe remain approximately constant (Figure 1B). It thus appears that it is the accumulation of W, not a change in the cellular concentration of Mo or Fe due to W interference, that causes the decrease in growth rates (although it should be noted that the Mo quotas correspond to the uptake of practically all Mo in the medium). We know that the uptake of Mo and Fe depends on their complexation by catechol siderophores and that these compounds also complex W (12, 18, 23). We thus examined the release of catechols by A. vinelandii in the presence of tungsten and their effect on W speciation and uptake. Catechol Production. In cultures of A. vinelandii (wild type strain OP) growing in an EDTA-containing medium VOL. 42, NO. 7, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Quantification of total concentrations of protochelin (hatched), azotochelin (gray), and DHBA (black) released into the growth medium of Fe-sufficient cultures of strain OP (wild type) as a function of [W] ([Mo] ) 10-8 M, [Fe] ) 5 × 10-6 M, and OD ) 0.42 ( 0.05). Values represent results from three independent experiments (means ( std dev).

FIGURE 3. Complexation of W and Fe by catechol siderophores in the growth medium of strain OP (wild type, [Mo] ) 10-8 M, [Fe] ) 5 × 10-6 M, [W] ) 8 × 10-6, and OD ) 0.5). HPLC chromatograms of extracts from the growth medium and metal analysis of collected fractions by ICP-MS. Circles and triangles indicate concentrations of metal complexes in the growth medium calculated on the basis of metal concentrations in HPLC fractions. (1, DHBA; 2, iron-protochelin; 3, tungsten-protochelin; 4, protochelin; IS, internal standard). under Fe-sufficient conditions (12), the mono(catechol) DHBA and the tris(catechol) protochelin are the most abundant catechols in the growth medium at all W concentrations tested, while azotochelin is only a minor component (Figure 2). In the concentration range [W] ) 10-8 to 10-6 M, where the bacteria grow at their maximum rate, DHBA and protochelin concentrations remain approximately constant. At [W] ) 10-5 M, which is toxic, the protochelin concentration, but not that of DHBA, increases by a factor of 5. Complexation of W in the Growth Medium. In our medium, EDTA complexes Fe but not Mo or W (24). To determine which metals in the growth medium are bound by the catechols released by the bacteria, we used an HPLC separation coupled to an ICP-MS analysis of collected fractions (Figure 3). In diazotrophic cultures of the wild type grown at high [W] (8 × 10-6 M) under Fe-sufficient conditions, a major fraction of protochelin is in the form of tungsten-protochelin, with smaller fractions as iron-protochelin and free protochelin. While only a minor fraction (16%) of the iron is bound to protochelin, we accounted for 82% of the W 2410

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FIGURE 4. Short-term uptake of tungsten (open symbols) and molybdenum (closed symbols) by strain OP (wild type). Open symbols and downward triangles: bacterial cells preconditioned at [98Mo] ) 2 × 10-8 M and [W] ) 10-7 M, harvested and resuspended into fresh medium for measurement of Mo and W bacterial uptake. The resuspension medium contains [95Mo] ) 2 × 10-8 M, [W] ) 10-7 M and an excess ()10-6 M) of ligand. Ligand ) azotochelin (Az) or protochelin (Pro). Upward triangles: same as downward triangles but no W was added to the preconditioning and resuspension media. One representative experiment is shown. originally present in the medium in the form of the tungsten-protochelin complex (Figure 3). Such a high recovery rate for W after the concentration and separation steps demonstrates that the bulk of W is complexed by catechols in the medium. The other catechol siderophores (DHBA, aminochelin, azotochelin, and azotobactin) are not measurably involved in the complexation of W or Fe under our culture conditions (Figure 3). Because of the low concentration of Mo (10-8 M) compared to W in the medium, it was not possible to measure directly molybdenum-catechol complexes by HPLC in this experiment. But in all experiments, except at the highest W concentration (10-5 M), protochelin was in large excess of Mo and W. Under these conditions, the formation of molybdenum-protochelin in the growth medium of A. vinelandii was documented in a previous study (12). Short-Term Uptake of Tungstate and Molybdate. To understand how the binding of W to protochelin in the medium affects its bioavailability, we measured the uptake of W complexed to protochelin by A. vinelandii in shortterm experiments and compared it to that of the molybdenum-protochelin complex (Figure 4). (We have previously shown that the uptake of Mo in the presence of excess protochelin is proportional to the concentration of the molybdenum-protochelin complex (12) and refer to it as molybdenum-protochelin uptake with no implication regarding the actual mechanism of transport.) A. vinelandii cells (strain OP) grown at low [Mo] and high [W] were collected and resuspended into fresh medium with an excess of protochelin (10-6 M) to complex all Mo (2 × 10-8 M) and W (10-7 M). Even though Mo is present at a concentration five times lower than W, it is taken up at least 10 time faster, demonstrating the preferential uptake of molybdenum-protochelin over tungsten-protochelin. In a similar experiment with azotochelin, we found that the uptake rate of tungsten-azotochelin (like that of tungsten-protochelin) is close to, or below, our detection limit (∼3 × 10-22 mol cell-1 min-1, Figure 4). Further, at high [W] and in the presence of an excess of protochelin, the uptake rate of Mo by high W cells is reduced by only about 30% compared to its uptake rate by low W cells in the absence of W. This high uptake rate is consistent with the Mo quotas measured in high W cells (Figure 1B). The uptake system(s) for molybdenum-protochelin and molybdenum-azotochelin is apparently able to discriminate against the W complexes. Thus, the excretion of catechol siderophores by the bacteria results in the

FIGURE 5. Growth, catechol production, and metal accumulation in strain OP (wild type) grown at [Fe] ) 5 × 10-6 M and [Mo] ) 10-7 M in response to W addition (A). Cell density of the culture (diamonds) and release of protochelin (circles) and azotochelin (squares) by the bacteria into the medium before and after W addition ([W] ) 5 × 10-6 M, 25.5 h after inoculation, dotted line). Cross-hatched areas indicate protochelin concentrations higher than [W] )5 × 10-6 M. (B) Production rate of protochelin based on its concentration in the growth medium. (C) Accumulation of Fe (closed triangles), Mo (open circles), and W (open triangles) in the bacteria: Values represent means of two independent experiments. Error bars indicate minimum/maximum values. complexation of Mo and W in the growth medium and allows the preferential uptake of Mo (which is needed for growth) over W (which is toxic). Catechol Release after W Addition. The large increase in protochelin concentration observed at high [W] (Figure 2) suggests that protochelin excretion may be a direct or indirect response to W toxicity. To better understand the relation between W and the production of catechol siderophores, we followed the concentration of these compounds after the addition of a toxic concentration of W (5 × 10-6 M) to a culture of A. vinelandii that had been growing exponentially in the absence of tungsten (Figure 5). After W addition, the growth rate of the culture decreased abruptly and, for about 2 h, protochelin excretion stopped completely. Over the same time interval, W was taken up very rapidly: within 2 h the molar W quota in the cells reached W:P ) 4 × 10-3, five times higher than the Mo quota, Mo:P ) 7 × 10-4 (Table S4 of the Supplementary information). This corresponds to a rate of uptake of W (present initially as WO42-) that is much larger than that measured for the tungsten-protochelin complex. During that time we saw no accumulation of Fe or Mo in the cells; this is evidence for a decrease in the Fe uptake rate but not in Mo uptake since, at the time W was added to the

medium, the bacteria had already taken up all Mo initially present in the medium. Two hours after W addition, the production of protochelin by the bacteria increased dramatically up to 34.0 amol cell-1 h-1 (compared to 10.9 amol cell-1 h-1 before W addition). The concentration of protochelin reached 6 × 10-6 M within 5 h, enough to complex all the WO42- added to the growth medium. While azotochelin in the medium increased also, it remained a minor fraction of total catechols and its increase in concentration lagged behind the increase in protochelin. When there was enough protochelin in the medium to bind all the tungstate, the accumulation of W in the cells stopped almost completely, even though 90% of the W originally added was still in solution (Figure 5C, hatched range). At the same time, cellular Fe uptake resumed, but not Mo uptake because the medium was already depleted in Mo (see above). Metal quotas of Co, Cu, Zn, and Mn were not affected by the addition of W to the culture (data not shown). W Detoxification by Siderophores. Taken at face value, our data indicate that the excretion of protochelin into the growth medium allows A. vinelandii to take up preferentially the metals Fe and Mo, which it needs, over W, which is toxic. This suggests that the sensitivity to W toxicity should depend on the organism’s ability to synthesize and release catechol siderophores. This prediction can be tested by examining the effect of [W] on the growth of mutant strains P100 and F196, which are deficient in the production of catechol siderophores: F196 produces only the pyoverdin-like siderophore azotobactin, while P100 produces only a low-affinity siderophore that has yet to be identified (25). We found that both mutants are indeed more sensitive to W toxicity than the wild type (OP) and that P100 is more sensitive than F196 at all [W] (Figure 6A, Table S1 of the Supporting Information). The intracellular W quotas increase with [W] in all three strains, and the W quotas of the mutant strains are markedly higher than that of strain OP for [W] > 1 × 10-7 M (Figure 6B). These data are consistent with the observation that OP (unlike F196 and P100) releases large amounts of protochelin (Figure 2) which forms an unavailable complex with W, thereby reducing its uptake (Figure 4). As shown earlier for the wild type (Figure 1), the measured Fe quotas (as well as those of Mo which is all taken up) did not show a decrease with increasing [W]; in fact those of the mutants increased slightly at high [W] (Table S2 of the Supporting Information). The protection afforded by catechols against W toxicity was further demonstrated by growing the wild type and the mutants with an excess of azotochelin, which was used as a model for the catechol compounds of A. vinelandii. With azotochelin added, the toxicity of tungstate was greatly reduced and the growth rate of strains P100, F196, and OP remained essentially constant at all [W] (Figure 6C, Table S1). As shown by HPLC analysis of cultures of the wild type, the release of protochelin by the bacteria was inhibited by the large excess of azotochelin (Figure S1 of the Supporting Information). Tungsten-azotochelin was the dominant catechol complexes in the medium, as opposed to tungstenprotochelin (compare Figure 3 and Figure S1). Again, the concentration of W recovered in the tungsten-azotochelin complex accounted for nearly all the W added ([tungstenazotochelin] ) (8.0 ( 0.1) × 10-6 M, when [W] ) 10-5 M was added). In the presence of azotochelin, the Fe quotas of the mutants but not of the wild type increased markedly (Table S2), presumably reflecting the role of the catechol siderophore in iron uptake. In contrast, the Mo quotas of the wild type and the mutants were not affected by azotochelin addition; they represent an essentially complete uptake of Mo from the medium, indicating efficient Mo uptake in the presence of siderophores (OP and F 196) or in their absence (P100). VOL. 42, NO. 7, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. Effect of W concentrations ([Mo] ) 10-8 M) on growth and W quotas in A. vinelandii (wild type strain OP and mutant strains F196 and P100). (A) Relative growth rates (means ( std dev; n ) 3); (B) cellular W:P quotas (OD ) 0.6 ( 0.1). Values represent means of two independent experiments. Error bars indicate minimum/maximum values (n ) 2); (C) relative growth rates as in A with 10-4 M azotochelin (Az) added to the growth medium.

Discussion Like previous authors, we have found that tungstate is toxic to diazotrophic cultures of A. vinelandii only when in large excess over molybdate and that W toxicity depends on the Mo concentration in the medium (10, 11, 26). Our growth data (Figure 1, Table S3 of the Supporting Information) are in quantitative agreement with the results of Benneman and co-workers (11) who found that for a Mo concentration of 10-7 M, a [W]:[Mo] ratio of 200 is required to achieve a 50% decrease in growth rate. Further, in agreement with the findings of Cornish and Page (14), we have found that when exposed to toxic levels of W, the bacteria respond quickly by producing large amounts of protochelin (Figure 5). A more detailed comparison with other studies of the nature and concentrations of the siderophores excreted by A. vinelandii is precluded by the different chemical compositions of the growth media, most of which do not contain EDTA. By direct HPLC-ICP-MS measurements, we have been able to show that essentially all W in the growth medium becomes complexed to protochelin (Figure 2), which is consistent with the high equilibrium constant measured for W ) 9.0 (18)). As shown tungsten-catechol complexes (log K AZ in earlier studies, other metals such as Mo and V are also bound to protochelin, as is a significant part of the iron (12). Bis- and tris(catechols) are among the few classes of ligands that can bind strongly to oxoanions such as molybdate, 2412

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vanadate, and tungstate. The only structure that has been completelyresolvedisthatofthe1:1molybdenum-azotochelin complex, where Mo is bound to two oxo groups in a cis position and to two catechol groups (13, 18). It is likely that azotochelin binds W in a similar geometry. According to ESIMS measurements, protochelin also binds Mo and W in 1:1 complexes (12). The affinity of these ligands for Fe is higher than for the oxoanions, and the reason that these are actually bound in the growth medium is because of the slow kinetics of Fe complexation (27). In our system the slow kinetics result from the exchange with iron-EDTA, but the same result should be obtained when Fe is precipitated as an oxyhydroxide, which must serve as the ultimate source of Fe in the environment. The reaction with molybdate and tungstate, which are not bound to EDTA, is fast (16, 28). The effect of siderophores on metal speciation thus depends as much on kinetics as on thermodynamics. Once Mo, W, and Fe are complexed with catechols, their uptake is tightly controlled by the transport system. Earlier studies have shown that the protochelin and azotochelin complexes of Fe and Mo (as well as V) can be taken up (12, 14, 19, 23). We have found that the uptake of tungsten-protochelin and tungsten-azotochelin is very slow or nil. The production of catechol siderophores thus provides the bacteria with a means to take up preferentially the metals it needs, Fe and Mo (and V), and not W which is toxic. This was confirmed by demonstrating the high sensitivity to W toxicity of catechol-deficient mutants and the detoxification brought about by the addition of azotochelin. Unlike the tungsten-catechol complexes, free tungstate appears to be taken up very rapidly by A. vinelandii cells and to be highly toxic. Although the exact biochemical mechanism of toxicity is not known, the data show that iron uptake stops immediately after tungstate addition. It is known that tungstate can inhibit the activity of the enzyme ferric reductase, which is responsible for an essential step in iron uptake (14). The abundant release of protochelin that follows W addition may thus result from a decrease in Fe uptake since the regulation of protochelin production by Fe is welldocumented (13). Part of the protochelin release might also result from Mo depletion, since 2 µM protochelin has been measured in low Mo-media (12), or, more directly, from the high cellular W concentration. Regardless of the actual trigger, the accelerated production of protochelin ceases when all tungstate in the medium is complexed. An insight into the reason why WO42- is taken up rapidly by A. vinelandii but not the tungsten-catechol complexes is provided by what is known of the Mo uptake system. Like most ions, tungstate can presumably diffuse inside the periplasmic space where it should bind to ModA. ModA does not differentiate between tungstate and molybdate, and both oxoanions are taken up when present in the medium (4). The discrimination against a tungsten-catechol complex might be effected at the outer membrane (12, 29), but even if the complex enters the periplasm, discrimination will be achieved by ModA. Compared to azotochelin, the periplasmic ModA protein has a slightly higher affinity for molybdate Mo Mo ) 7.3), and a much lower one for ) 7.7 vs logK AZ (logK ModA W W ) 9.0 8, 18). In view of = 7.7 vs logK AZ tungstate (logK ModA the similar functionalities and stoichiometries of the azotochelin and protochelin complexes, ModA should presumably outcompete protochelin for molybdate, but not for tungstate, in the periplasm. Further, the kinetics of dissociation of the tungsten-catechol complexes are slow (t1/2 > a few hours), and W in catechol complexes may be kineticallyunavailable.Incontrast,themolybdenum-catechol complexes dissociate readily, making molybdate available for complexation by ModA and uptake (18). Complexation of W by catechols decreases W toxicity but does not alleviate it completely. This may be due to an

interference with Mo uptake. For example, in a medium where tungsten-protochelin is 5 times more abundant than molybdenum-protochelin, the uptake rate of molybdenumprotochelin is reduced by a factor of 2 compared to its maximum value without any W (Figure 4). The toxicity of W bound to protochelin may also result from a cellular accumulation of W. Intracellular W quotas increase with [W], implying that either a small fraction of tungsten-protochelin is taken up or the small fraction of free tungstate that is at equilibrium with the complex is taken up. Once inside the cell, W can be toxic in various ways, particularly by substituting for Mo in nitrogenase, making it inactive (9). The binding of metals by catechol siderophores excreted into the medium provides the bacteria with a precise tool to control metal acquisition. The uptake of toxic metals, such as W, is repressed to very low rates compared to that of essential metals, such as Fe and Mo. This result explains why A. vinelandii is sensitive to W toxicity only when W is in large excess of Mo despite similar affinity of the ModA protein for molybdate and tungstate. This is an important attribute for organisms living in soils where W and Mo are present at similar concentrations. In addition, the presence of catechol ligands can help in capturing essential metals from other unavailable forms, such as particles or organic complexes (19). The role of catechol siderophores is thus not confined to iron acquisition; these compounds also play an essential part in fine-tuning the uptake of other essential and toxic metals.

Acknowledgments The authors thank Susan Brantley, Telissa M. Loveless, and Paul E. Bishop for providing the mutant strains F196 and P100. We are also grateful to Florence Lagarde for her help and François M. M. Morel for useful discussions and his support throughout this work. We thank Dorothy Little and Eric Chan for their help with mass spectrometry. This work was supported by grants from the NSF (Grant CHE-0221978, Center for Environmental Bioinorganic Chemistry, and Grant DEB-0614116) and from the French Department of Research, as well as fellowships from the French Department of Education to J.-P.B and from the Camille and Henry Dreyfus Postdoctoral Program in Environmental Chemistry to T.W. T.W. and J.-P.B. contributed equally to this work.

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(9) (10) (11) (12)

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(14) (15) (16) (17)

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Supporting Information Available Additional information on methods, growth rates, and cellular Mo and W quotas of the wild type and mutant strains. This material is available free of charge via the Internet at http://pubs.acs.org.

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