Role of the Siderophore Azotobactin in the Bacterial Acquisition of

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Environ. Sci. Technol. 2009, 43, 7218–7224

Role of the Siderophore Azotobactin in the Bacterial Acquisition of Nitrogenase Metal Cofactors T H O M A S W I C H A R D , * ,†,‡ JEAN-PHILIPPE BELLENGER,‡ FRANC ¸ OIS M. M. MOREL,‡ AND ANNE M. L. KRAEPIEL§ Institute for Inorganic and Analytical Chemistry, Friedrich Schiller University Jena, Lessingstr. 8, 07743 Jena, Germany, Department of Geosciences, Princeton Environmental Institute, Guyot Hall, Princeton University, Princeton, New Jersey 08544, and Chemistry Department, Princeton Environmental Institute, Guyot Hall, Princeton University, Princeton, New Jersey 08544

Received December 31, 2008. Revised manuscript received March 22, 2009. Accepted March 30, 2009.

Fixation of dinitrogen by soil bacteria is catalyzed by the enzyme nitrogenase which requires iron, molybdenum, and/or vanadium as metal cofactors. Under conditions of iron deficiency, the ubiquitous N2-fixing bacterium Azotobacter vinelandii produces azotobactin, a fluorescent pyoverdine-like compound which serves as a siderophore. Azotobatin’s hydroxamate, catechol, and R-hydroxy-acid moieties endow it with a very high affinity for FeIII, and the Fe complex is taken up by the bacterium. Here we show that azotobactin also serves for the uptake of Mo and V. Azotobactin forms strong complexes with molybdate and vanadate and the complexes are taken up by regulated transport systems. The kinetics of complexation of molybdate and vanadate by azotobactin are faster than the complexation of FeIII, which is either precipitated or bound to strong complexing agents. As a result of this kinetic advantage, the Mo and V complexes of azotobactin form despite the higher affinity of the compound for Fe, which is present in large excess in the environment. The results obtained here for azotobactin and previous data for the bisand tris-catechols produced by A. vinelandii show that those “siderophores” are really “metallophores” that promote the bacterial acquisition of Mo and V in addition to Fe.

Introduction Many microbial enzymatic processes that catalyze key transformations in the cycles of carbon and nitrogen in soils require trace metals as cofactors. For example, nitrogenase, the enzyme responsible for N2-fixation and thus for the input of new nitrogen to ecosystems, exists in three different forms that use three alternative types of metal cofactors: Fe + Mo, Fe + V, and Fe only (1, 2). A modest amount of work has been done on the question of trace metal acquisition by soil microbes (3–5), with the notable exception of Fe which has been extensively studied (6–8). In soils, as in aquatic systems * Corresponding author phone: +49 3641 948169; fax: +49 3641 948172; e-mail: [email protected]. † Friedrich Schiller University Jena. ‡ Department of Geosciences, Princeton Environmental Institute. § Chemistry Department, Princeton Environmental Institute. 7218

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(9) and in the guts of higher organisms (10), bacteria release strong FeIII chelating agents known as siderophores (11), which bind the metal extracellularly; the Fe-siderophore complex is then taken up through various mechanisms (12, 13). Recently we have shown that the ubiquitous freeliving N2-fixing bacterium Azotobacter vinelandii uses catechol siderophores for the uptake of Mo and V, in addition to Fe (5). Under diazotrophic growth (i.e., while fixing N2), A. vinelandii releases compounds such as the tris-catechol protochelin and the bis-catechol azotochelin which bind MoO42- and VO43- in strong complexes. These complexes are then taken up through regulated transport systems (5). The complexation of molybdate and vanadate by the catechol compounds releases them from organic matter and oxide surfaces to which they are bound in soils and makes them available for uptake. At high concentrations, where the free concentrations of MoO42- and VO43-, along with that of WO42-, are toxic (14, 15), catechol complexation and down regulation of the uptake systems serve as an effective detoxification mechanism. Catecholate siderophores, which may more appropriately be called “metallophores”, thus serve multiple functions and help control the acquisition of several metals by A. vinelandii (16). In addition to catechol siderophores, A. vinelandii releases the pyoverdine-like siderophore azotobactin, which is also produced by other Azotobacter isolates (8, 17–19). Catechol siderophores, particularly protochelin, are released by A. vinelandii (wildtype) under a variety of conditions but azotobactin seems to be released only under severe Fe limitation (3, 7). Therefore azotobactin is sometimes considered the “true” siderophore of this bacterium (8). While only one study looked directly at the uptake of Fe-azotobactin in A. vinelandii (8), pyoverdine-type siderophores and their role in iron acquisition, in particular in pseudomonas species, have been extensively studied (20–23). Azotobactin possesses three different types of coordinating moieties, a hydroxamate, an R-hydroxy acid, and a catechol, making it a representative of several classes of siderophores simultaneously. The known interference of V with the pyoverdine-mediated uptake of FeIII (24) provides indirect evidence that azotobactin binds vanadium in addition to FeIII. Azotobactin is thus a good model compound to test whether the results obtained on the uptake of Mo and V complexes with bis- and tris-catechols can be extended to other siderophores and whether these compounds may have other functions in addition to previously described Fe acquisition (23). In this study we examine whether the pyoverdine-like siderophore azotobactin is involved in the acquisition of Mo and V by A. vinelandii grown under diazotrophic conditions.

Experimental Section Reagents. HPLC grade methanol and all other chemicals were purchased from Sigma or Fisher Scientific. Azotochelin was synthesized as previously described (25). Bacterial Strain, Culture Medium, and Growth Conditions. The mutant strains F196 (Tn5luxAB, a catechol negative mutant, which produces only the pyoverdin-like siderophore azotobactin (26)) and CA11.70 (∆nifHDK ∆anfHD70::kan, which expresses only the V-nitrogenase (27)) of Azotobacter vinelandii were used in this work. The cultures were grown under diazotrophic conditions in minimal liquid medium at pH 6.7 (25). The Fe, Mo and V concentrations were adjusted by additions of FeCl3, Na2MoO4, or NaVO3 solutions, respectively. Cultures of A. vinelandii (F196, CA11.70) were grown in 250 mL flasks containing 25-50 mL of medium 10.1021/es8037214 CCC: $40.75

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and were continuously agitated at 180 rpm at 22 °C. Bacterial growth was monitored by measuring the optical density (OD) at 620 nm in a 1 cm polypropylene cell on a Cary-UV 100 spectrophotometer (Varian, USA). The conversion from OD to cell density (1.16 ( ( 0.16) × 108 cell mL-1 OD-1) was obtained by counting DAPI stained culture aliquots using epifluorescence microscopy (28). Azotobactin Isolation. Azotobacter vinelandii (mutant strain F196) was grown under iron-limited but Mo-replete conditions ([Fe] ) 5 × 10-7 M, [Mo] ) 2 × 10-8 M). After centrifugation of 250 mL of medium (OD ) 0.2) the yellow-green supernatant was pumped (6 mL min-1) through a Sep-Pak RP-C18 cartridge (Waters, USA), conditioned with 4 mL of methanol and subsequently equilibrated with water. The fluorescent material retained on the column was directly eluted with 2 mL of cold methanol. The methanol fraction was slightly concentrated under an argon stream and then stored at -18 °C for precipitation of azotobactin overnight (18). After centrifugation, the yellow precipitate was stored at -80 °C. For further purification, azotobactin was dissolved in water and applied on a Bio-Gel P2 column (Biorad, USA) and eluted isocratically with water. Free azotobactin was recovered from the column. The purity of the collected azotobactin was verified by HPLC on a C18 reverse phase column (JASCO, Japan) at pH 6.6 using a fluorescent and UV detectors as previously described (15) and by UPLC/MS analysis (Figure S1). Azotobactin was quantified by UV-vis spectroscopy (λmax ) 380 nm at pH 4.0 and  ) 23500 M-1 cm-1) (8, 17). The standard stock solution of azotobactin (1.7 × 10-4 M) contained both azotobactin D and azotobactin δ (lacton-form), whose structures are closely related and which have equivalent siderophore activity (8). The stock contained a small amount of Fe ([Fe] ) 9 × 10-9 M, measured by inductively coupled mass spectrometry (ICP-MS, Element2, Thermo-Finnigan) as previously described (15). The molecular masses of azotobactin D and δ were determined by direct inlet electrospray time-of-flight mass spectroscopy (ESI-TOF-MS, Qmicro, Waters-Micromass, UK) (see also Figure S1). For analysis of Mo-azotobactin, the source capillary and cone energy were set on 2.7 kV and 5 V. Scans were acquired in negative-ion mode from m/z 200 to 3200. Fluorescence Measurements. Fluorescence was measured on a spectrofluorophotometer (PerkinElmer Instruments, USA) equipped with a xenon lamp as the light source. The emission of fluorescence was generally measured at λ ) 487 nm upon excitation at λ ) 380 nm using bandwidths of 8 and 2 nm, respectively. The fluorescence signal was calibrated with purified azotobactin in the concentration range from 0.1 to 4.0 × 10-6 M. To measure free azotobactin concentrations, aliquots of a culture of A. vinelandii were collected over time, centrifuged, and azotobactin concentrations were measured in the supernatant by fluorescence. ThebindingratioandstabilityconstantfortheMo-azotobactin complex were determined by measuring the absorbance at λ ) 415 nm (Cary-100 UV-vis spectrometer, Varian, USA) of a 2.2 × 10-5 M solution of azotobactin in 10 mM sodium acetate (pH ) 4) titrated with sodium molybdate (1.1 × 10-4 M). Standard solutions were prepared by weighing known amounts of molybdate (Na2MoO4 · 2H2O) and diluting the stock solution of azotobactin. For vanadate, complex formation was quantified by measuring the quenching of fluorescence of a 4 × 10-6 M solution of azotobactin (pH ) 6.6) upon titration with sodium vanadate (1.0 × 10-4 M). The concentrations of Mo and V in stock solutions were subsequently verified by ICP-MS. The concentration of azotobactin was verified by absorbance based on its extinction coefficient. The titration data were analyzed with the Hyperquad2006 software (Protonic Software, USA) (25). Short-Term Uptake of Iron, Vanadium, and Molybdenum. The uptake rates of vanadate and FeIII were studied

using the radiotracers 49V (Los Alamos National Laboratory) or 55Fe (Perkin-Elmer, USA), respectively. A. vinelandii cells (F196 or CA11.70 mutant strain) were grown diazotrophically in minimal medium under Fe-replete conditions and low [V] and low [Mo]. To measure V uptake rates, 25 mL of culture of exponentially growing cells (OD ) 0.4) was aseptically centrifuged, and the pellet was rinsed with 50 mL of V-free and Fe-free medium and subsequently resuspended in 25 mL of medium containing an excess of azotobactin or azotochelin and spiked with 49V. To measure Fe uptake rates, 6 mL of the cell suspension (OD ) 0.15) was aseptically centrifuged, and the pellet was rinsed with 15 mL of V-free and Fe-free medium and resuspended in 6 mL of medium. The cell suspension was then spiked with a solution of 55Feazotobactin prepared by adding 55FeCl3 to a solution of azotobactin at pH 6-7 and let to equilibrate for 5 days at 8 °C. At regular time intervals, cells were harvested and rinsed twice with 30 mL of V-free and Fe-free medium and subsequently with 30 mL of an oxalate-EDTA-KCl solution for 2 min to remove metals adsorbed at the cell surface (29). Intracellular 49V and 55Fe in whole cells were measured by liquid scintillation counting (Beckman-Coulter LSC6500). Molybdate uptake was measured using the stable isotopes 95 Mo and 98Mo, supplied as 95MoO3 and 98MoO3 by Cambridge Isotope Laboratories, Inc. A. vinelandii (strain F196) was grown diazotrophically in liquid medium containing 95 MoO42-. When the culture reached OD ) 0.4, a 15 mL cell suspension was aseptically centrifuged (16,000g, 10 min at 22 °C) and rinsed with 10 mL of Mo-free medium. The cells were then resuspended in 70 mL of medium containing an excess of azotobactin and 98Mo. Aliquots were collected at regular time intervals. The cells were filtered (polycarbonate filter, 0.6 µm pore size, 45 mm diameter) and rinsed as described above. Finally, the cells were digested in HNO3 optima in a microwave digester (CEM, Mars) and the digest was analyzed by ICP-MS to determine the intracellular concentration of 95Mo and 98Mo. Before each uptake experiment, the resuspension medium was left to equilibrate with the ligand (azotobactin or azotochelin) and the metal (49VO3-, 98MoO42-, or 55FeCl3, ligand:metal ratio 20:1). This is important for the free molybdate and vanadate are rapidly taken up by A. vinelandii (5, 14). Kinetics of Complex Formation. The kinetics of Mo complex formation with azotobactin were measured and compared with the kinetics of Fe complexation. An azotobactin solution (1 × 10-6 M azotobactin in 10 mM ammonium acetate, pH 6.6) was spiked with Mo or Fe-EDTA (see Figure 5). The evolution of the fluorescence intensity at λ ) 487 nm was measured (λexc ) 380 nm) over time. Dissolution of Ferrihydrite. Ferrihydrite (Fe5HO8 · 4H2O) was prepared according to established procedures, dried in the oven at 90 °C, and used within one week (30). A 60 mg portion of ferrihydrite was added to 1 mL of a 9.8 × 10-5 M solution of azotobactin in 10 mM ammonium acetate (pH ) 6.6). The suspension was kept in the dark at room temperature and continuously agitated to prevent sedimentation of the iron oxides. After centrifugation, aliquots (10 µL) of the supernatant were collected over time and diluted in 1 mL of a 10 mM ammonium acetate solution (pH 6.6) before analysis by fluorescence spectroscopy to measure the quench of fluorescence corresponding to complexation of Fe by azotobactin (31). The lack of azotobactin degradation was verified at the end of the experiment by collecting a 10 µL aliquot of the supernatant and adding it to 1 mL of an EDTA/oxalic acid solution (1.0 × 10-4 M, (29)). This resulted in the dissociation of the FeIII-azotobactin complex, and restoration of the expected fluorescence corresponding to free azotobactin (Figure S2). The fluorescence over time of VOL. 43, NO. 19, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. (A) Azotobactin δ (lacton-form). (B) Growth and azotobactin production by A. vinelandii (mutant strain F196). Optical density (OD, open diamonds) and azotobactin concentration (black circles) in the bacterial culture as a function of time after inoculation. The bacteria were grown under very low [Fe] and Mo-replete conditions ([Fe] ) 1 × 10-7 M and [Mo] ) 1 × 10-6 M). a solution of azotobactin incubated without ferrihydrite was also measured in a control experiment.

Results and Discussion Azotobactin Production. To simplify the experiments, we used as our test organism for most experiments the mutant F196 of A. vinelandii, which can express all three nitrogenases (i.e., the V, Mo, and Fe only nitrogenases), but produces only azotobactin as a siderophore (26) (Figure 1A). The production of azotobactin can be conveniently followed by measuring the fluorescence of the compound in the growth medium. Under diazotrophic growth at low Fe ([FeIII]T ) 0.1 µM and [EDTA] ) 100 µM), the bacterium released azotobactin into the medium during the lag phase, reaching concentrations of 0.1-1 µM by the onset of exponential growth, and in excess of 10 µM afterward (Figure 1B). This is similar to the pattern of protochelin production in the wildtype A. vinelandii grown at higher Fe concentration (3, 14). Azotobactin Complexes. Its various coordination groups make azotobactin a versatile chelating agent and pyoverdines which have similar binding groups, are known to complex V and other transition metals in addition to Fe (24, 32, 33). Evidence for the formation of the Fe3+, MoO42-, and VO43complexes with azotobactin can be seen in the UV-vis 7220

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absorption spectra in the presence and absence of the metals (Figure 2A). In the absence of the metal, the UV-vis absorption spectrum of azotobactin is identical to previously published spectra (17, 34) and shows a clear maximum at λ ) 380 nm. Upon addition of the metals, this peak is shifted to markedly higher wavelengths, λ ) 415 nm. Titration of azotobactin with molybdate shows evidence of a strong 1:1 complex with a log Kapp g 1 × 107 (Figure 2B). We performed mass spectrum analysis of the isolated Mo-azotobactin δ complex (see above). As expected for a 1:1 stochiometry, the molecular mass m/z 1519 for the negatively charged Mo-azotobactin δ complex is 126 u higher than the mass of the positively charged free azotobactin m/z 1393. Further, the mass distribution pattern is consistent with that predicted from the distribution of stable Mo isotopes for a doubly charged molecule ion m/z 759 (Figure 2B inset). These data are consistent with the displacement by MoO22+ of 4H+ on azotobactin, as MoO22+ binds to the catechol and R-hydroxy acid groups of azotobactin in an octrahedral structure, along with the additional loss of one H+. This type of coordination geometry was previously observed in the Mo complex with the bis-catechol azotochelin (25) and in a bis-catecholamide siderophore analogue (35). The loss of 4H+ suggests that molybdenum might be bound exclusively to the catechol and R-hydroxy acid groups (31). Evidence of complex formation can also be obtained at lower concentrations by examining the quenching of azotobactin fluorescence upon metal binding (31, 36). This is seen in Figure 2C which shows the fluorescence quenching of azotobactin upon the addition of vanadate, although the fluorescence of the V-azotobactin complex(es) is significant as observed upon addition of vanadate in excess of the azotobactin. These data are consistent with the formation of a strong 1:1 V-azotobactin complex at low metal to ligand ratios, possibly followed by the formation of one or several polynuclear complexes as the metal to ligand ratio increases. Uptake of Azotobactin Complexes. We measured the uptake of the Mo-azotobactin and V-azotobactin complexes in short-term experiments with the stable isotope 98Mo or the radioisotope 49V, using diazotrophically growing cells that had been preconditionned at low Mo and V concentrations. The F196 mutant took up rapidly the Mo-azotobactin complex when grown at low Mo concentration, and the V-azotobactin complex when grown at low V concentrations in the absence of Mo (Figure 3A). We verified that the shortterm uptake rate of V in the presence of azotobactin is due to the uptake of the V-azotobactin complex rather than the free vanadate in the medium by showing that the uptake rate is independent of the excess concentration of azotobactin, and thus of the free vanadate ion concentration (Figure 3B). We have previously obtained a similar result with V complexed to catechol siderophores (5). The organisms also took up the 55Fe-azotobactin complex as previously documented (8). The uptake rates we measured were similar to those previously observed for the catechol complexes of Mo and V by the wild type A. vinelandii and they are sufficient to support rapid N2-fixation and growth of the bacterium (5). It is noteworthy that those cultures which were preconditioned under Fe-replete and low Mo conditions took up Mo slightly faster than Fe (Figure 3A). Regulation of Uptake. The uptake of the catechol complexes of Mo and V by A. vinelandii is regulated as a function of the bacterium’s deficiency/sufficiency in Mo or V (14). We verified that the uptake system for the azotobactin complexes in the F196 mutant is regulated in the same way by comparing the uptake of the V-azotobactin complex in cells grown at high and low V concentrations in the absence of Mo (Figure 4A and B). When cells were grown at high V concentration, the uptake of V-azotobactin was indeed only a fraction of that observed for cells grown at low V

FIGURE 2. Metal binding by azotobactin. (A) A. vinelandii (mutant strain F196) was grown under low Fe and Mo conditions ([Fe] ) 5 × 10-7 M and [Mo] ) 2 × 10-8 M). The spent growth medium (pH ) 6.0) was collected and its metal binding properties were investigated by UV-vis absorbance after addition of an excess of metals (10-5 M FeCl3, NaVO3, or Na2MoO4). The absorbance spectrum of purified azotobactin is shown for comparison. (B) Titration curve of purified azotobactin by molybdate at pH ) 4.0, measured by absorbance at λ ) 415 nm and demonstrating the formation of a strong 1:1 Mo-azotobactin complex. Inset: selected part of ESI-MS spectrum of the Mo-azotobactin complex showing the characteristic isotopic pattern of the Mo-containing peak at m/z 759, corresponding to the doubly negatively charged [Mo-azotobactin]2-. (C) Titration curve of purified azotobactin by vanadate at pH ) 6.6, measured by fluorescence emission. Inset: fluorescence emission spectra of azotobactin upon addition of vanadate. The initial solution contains 4 × 10-6 M of purified azotobactin (pH ) 6.6). Each curve corresponds to the addition of 4 µL of a 10-4 M solution of vanadate. To quantify the extent of V binding by azotobactin, the y axis shows the measured fluorescence normalized to the fluorescence in the absence of V (I0), after the fluorescence of the V-azotobactin complex (Imin) has been subtracted from both signals.

FIGURE 3. Short-term uptake of Fe, V, and Mo complexes with azotobactin by A. vinelandii. (A) Mutant strain F196. Squares: Fe uptake ([FeIII] ) 2.7 × 10-8 M, [azotobactin] ) 1.0 × 10-6 M); circles: V uptake ([V] ) 4.2 × 10-8 M, [azotobactin] ) 1.0 × 10-6 M); triangles: Mo uptake ([Mo] ) 2.0 × 10-8 M, [azotobactin] ) 1.0 × 10-6 M). The bacteria were preconditioned under Fe-replete conditions ([Fe] ) 5 × 10-6 M) and low [Mo] (1.7 × 10-8 M) or low [V] (2.0 × 10-8 M). (B) Mutant strain CA11.70. V uptake ([V] ) 4.0 × 10-8 M and [azotobactin] ) 8.3 × 10-6 M (circles) or ([V] ) 4.0 × 10-8 M and [azotobactin] ) 9.2 × 10-7 M (triangles). The bacteria were preconditioned under Fe-replete conditions ([Fe] ) 5 × 10-6 M) and low [V] ) (2.0 × 10-8 M). concentration (Figure 4B). Interestingly, the uptake rate of the V-azotobactin complex by the F196 mutant was similar to that observed for the uptake of the V-azotochelin complex by the CA11.70 mutant, which produces only the V-nitrogenase and uses catechol siderophores for V uptake (14). This uptake rate is consistent with the steady-state uptake rates calculated using previously measured growth rates and cellular V quotas in diazotrophic culture of CA11.70 (14). In contrast, the uptake rates of V-azotochelin by F196 and of V-azotobactin by CA11.70 were comparatively much slower (Figure 4C). These differences in uptake kinetics imply that the azotobactin and catechol complexes are taken up by separate uptake systems that can be differentially regulated. Even at very high vanadate concentration (10-4 M), A. vinelandii maintains a high growth rate (Figure 4A). It appears that V is effectively detoxified through complexation by azotobactin and down-regulation of the transport of the V-azotobactin complex. Such detoxification mechanism has previously been demonstrated for the V and Mo complexes of the bis- and tris-catechols produced by A. vinelandii (5, 14). The mechanisms of uptake for the Mo and V azotobactin complexes remain to be investigated. The complexes might be taken up by the Fe-siderophore transporters, as found for VOL. 43, NO. 19, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Vanadium-dependent growth and regulation of vanadium uptake. (A) Growth curves (optical density vs time) of cultures of A. vinelandii (mutant strain F196) grown at increasing vanadium concentrations. (B) Short-term uptake of V-azotobactin by strain F196 ([V] ) 4.0 × 10-8 M, [azotobactin] ) 1.0 × 10-6 M in resuspension medium). Bacteria were conditioned either at low [V] ) 2.0 × 10-8 M or high [V] ) 1.0 × 10-5 M. (C) Comparison of the short-term uptake rates of V-azotobactin and V-azotochelin ([V] ) 4.0 ( 0.2 × 10-8 M, [ligand] ) 1.0 × 10-6 M) by the mutant strains F196 and CA11.79 of A. vinelandii. Bacterial cells were preconditioned at the same V concdentration as used in short-term uptake experiment. Values represent means ( s.d. (n ) 2-4). other metals in Pseudomonas aeruginosa (37, 38), but the tight regulation of their uptake suggests that they might instead be taken up by other specific transporters that remain to be discovered. 7222

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Kinetics of Complexation. In earlier studies, we have observed that MoO42- and VO43- are complexed by catechol siderophores in A. vinelandii growth medium despite the much higher affinity of these siderophores for Fe3+, which is present in excess. We surmised that this is caused by the faster reaction of the siderophores with the free oxoanions than with EDTA- bound FeIII (39). The intense fluorescence of azotobactin and its quenching upon binding to metals provides a convenient model system to study the kinetics of complex formation. Under the same conditions as used in our growth medium, complexation of VO43- by azotobactin was more rapid than could be followed by our fluorescence measurements (data not shown). The complexation of MoO42- was essentially complete within 2 min (Figure 5A); but the complexation of Fe3+, which was bound to an excess of EDTA (100 µM), took about 2 days (Figure 5B). So, despite unfavorable thermodynamics, the much faster kinetics of the complexation reactions with the free oxoanions compared to that with EDTA-bound Fe insures that MoO42- and VO43are complexed by azotobactin released into the growth medium. In soils, the concentration of Fe is many times greater than those of Mo and V: a few percent by weight for Fe compared to 1-2 ppm for Mo and 100 ppm for V (40). But most of the Fe is present as various oxyhydroxide precipitates while Mo and V are either bound to organic matter or adsorbed on oxides and clays (41–43). We have previously shown that the ligand exchange reactions for the oxoanions are relatively fast and argued that the slow dissolution of the Fe precipitates by siderophores would allow complexation of MoO42- and VO43- to occur in nature as it does in our growth medium (16). Taking advantage again of the fluorescence of azotobactin and its quenching by Fe binding, we followed the kinetics of Fe-azotobactin formation at pH 6.6 in the presence of ferrihydrite, the most labile of the Fe oxyhydroxides. A few studies using chemical force microscopy and computational modeling have shown that azotobactin interacts strongly with iron oxides (44, 45). Consistent with these results, we find that azotobactin promotes the dissolution of ferrihydrite, and the decrease in the free azotobactin concentration (which corresponds to the formation of Fe-azotobactin) followed first order kinetics with a constant of l ) 1.3 × 10-2 h-1 (Figure 5C). To verify that the observed decrease in free azotobactin concentration is due to complexation of dissolved Fe rather than adsorption of azotobactin on the ferrihydrite, we filtered the solution at the end of the experiment and recovered the initial free azotobactin concentration after exchanging the metal with an excess of EDTA and oxalate (Figure S2). Under these conditions, the rate of formation of the Fe-azotobactin complex, which is equal to the rates of disappearance of the free ligand and of dissolution of the ferrihydrite, is much slower than the rates of complexation of MoO42- and VO43-. Our data show that azotobactin, a compound that possesses a catechol, an R-hydroxy acid, and a hydroxamate binding group and is produced by the N2-fixing bacterium A. vinelandii can serve the same multiple functions previously demonstrated for the bis- and tris-catechols produced by the same organism. As previously shown for catechol siderophores (5), azotobactin binds molybdate and vanadate in strong complexes and those complexes can form even in the presence of excess of iron, which is either precipitated or complexed with strong ligands. Under conditions of metal deficiency, the Mo and V complexes are rapidly taken up by the bacteria via uptake systems which appear to be both specific and regulated. As complexation of oxoanions is kinetically favored over Fe binding by azotobactin, it is possible that iron uptake may become limited at high Mo:Fe or high V:Fe ratios (24).

produced by A. vinelandii must play a role in the acquisition of the oxoanions of Mo and V in addition to Fe in nature and should really be considered “metallophores.” It remains to be seen if siderophores that do not possess catechol moieties are also used for uptake of multiple metals and if bacteria other than A. vinelandii use a similar strategy for metal acquisition.

Acknowledgments We thank Susan Brantley, Telissa M. Loveless, and Paul E. Bishop for providing the mutant strains F196 and CA11.70. This work was supported by the NSF (CHE-0221978, Center for Environmental Bioinorganic Chemistry) and by a postdoctoral fellowship from the Camille and Henry Dreyfus Program in Environmental Chemistry to T.W.

Supporting Information Available Additional information on methods of azotobactin-purification and dissolution of ferrihydrite. This material is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited

FIGURE 5. Kinetics of metal complexation by azotobactin. (A) Fluorescence quenching of azotobactin as a function of time after addition of molybdate ([azotobactin] ) 1 × 10-6 M; [Mo] ) 1 × 10-5 M). Values represent means ( s.d. (n ) 3). (B) Fluorescence quenching of azotobactin as a function of time after addition of Fe-EDTA. [azotobactin] ) 1 × 10-6 M; [EDTA] ) 1 × 10-4 M, [FeIII] ) 5 × 10-6 M. (C) Kinetics of ferrihydrite dissolution at pH ) 6.6 after addition of 9.8 × 10-5 M of azotobactin (closed symbols). Controls show amount of free azotobactin in solution without ferrihydrite (open symbols). The decrease in the concentration of free azotobactin is due to the formation of Fe-azotobactin as Fe is released from ferrihydrite. The concentration of free azotobactin was calculated based on the fluorescence intensity at λ ) 487 nm. Circles and triangles correspond to two independent experiments. Our new results with azotobactin and previous data with bis- and tris-catechols show that all the “siderophores”

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