Effect of Fe(II) and Fe(III) Transformation Kinetics on Iron Acquisition

The short-term Fe uptake rate of M. aeruginosa was measured by incubating ...... Kropat , J.; Merchant , S. Copper-dependent iron assimilation pathway...
0 downloads 0 Views 291KB Size
Download PDF
Environ. Sci. Technol. 2010, 44, 1980–1986

Effect of Fe(II) and Fe(III) Transformation Kinetics on Iron Acquisition by a Toxic Strain of Microcystis aeruginosa M A N A B U F U J I I , †,‡ A N D R E W L . R O S E , † TATSUO OMURA,‡ AND T . D A V I D W A I T E * ,† School of Civil and Environmental Engineering, The University of New South Wales, Sydney, NSW 2052, Australia, and Department of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 6-6-06, Sendai, 980-8579, Japan

Received May 3, 2009. Revised manuscript received January 29, 2010. Accepted February 4, 2010.

We have investigated the mechanism of Fe uptake by a toxic strain of the freshwater cyanobacterium Microcystis aeruginosa (PCC7806) with particular attention given to the effect of Fe(II) and Fe(III) transformation kinetics on Fe uptake. Chemiluminescence analysis revealed that M. aeruginosa produces extracellular superoxide (a moderate Fe reducing agent) at rates of 0.4-1.2 amol cell-1 h-1 depending on initial Fe concentration in the culture medium. Short-term assimilation assays using 55Fe showed that reduction of Fe(III) in both organic and inorganic forms by cell-generated superoxide or ascorbate facilitated Fe uptake via formation of unchelated Fe(II), when Fe availability was low because of the use of the strong Fe chelator ethylenediaminetetraacetate (EDTA) as a ligand. In contrast, Fe reduction was unimportant for Fe uptake in the presence of low concentrations (e100 µM) of the weak Febinding ligand citrate because of a high concentration of unchelated Fe(III), indicating that the contribution of reduction to Fe uptake depends on the nature of Fe binding and availability of unchelated Fe(III) in the external medium. A kinetic model incorporating uptake of both unchelated Fe(II) and Fe(III) and based on similar models developed for marine microalgae successfully described Fe uptake rates by M. aeruginosa PCC7806.

Introduction Iron is one of the most important micronutrients for almost all microorganisms because it participates in vital processes, such as biosynthesis of chlorophyll, nitrogen fixation, respiration, and radical scavenging (1). Although Fe is the fourth most abundant element in the Earth’s crust, concentrations of unchelated inorganic Fe(II) and Fe(III) (denoted by Fe(II)′ and Fe(III)′), the chemical species typically available for direct uptake by microorganisms (2), are low in air-saturated surface waters because of the rapid oxidation of Fe(II)′ (3) and extremely low solubility of Fe(III)′ at circumneutral pH (4). Thus, there is now strong evidence * Corresponding author telephone: +61 2 9385 5059; fax: +61 2 9313 8341; e-mail: [email protected]. † The University of New South Wales. ‡ Tohoku University. 1980

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 6, 2010

that Fe may be a limiting micronutrient for the growth of phytoplankton in the open ocean and some coastal waters where Fe availability is very low (5). In freshwater systems Fe is not generally considered a limiting nutrient for the growth of phytoplankton, although Fe limitation has been reported when the concentration of unchelated Fe is unusually low due to low dissolved Fe (12 days), the pH change was small (less than 0.3 units). Experiments were performed using cells harvested during the day in late exponential growth phase at densities between 3-20 × 106 cell mL-1. Prior to both the superoxide generation and short-term iron uptake experiments, the cells were rinsed at 1 mL min-1 with a solution containing 50 mM Na2EDTA and 100 mM Na2oxalate (hereafter referred to as “EDTA/ oxalate”) (18) and subsequently with 2 mM NaHCO3 solution, to eliminate the possible involvement of cell-surface adsorbents in superoxide generation and Fe uptake. Superoxide Determination. The MCLA chemiluminescence method (19) was used to determine superoxide concentrations with an FeLume chemiluminescence system (Waterville Analytical) operating at -1200 V. Approximately 8 mL of culture was filtered onto a 25 mm diameter, 0.22 µm syringe-driven membrane filter unit (MillexGP, Millipore) and rinsed with 10 mL of EDTA/oxalate and 5 mL of NaHCO3. The filter was then positioned upstream of the FeLume flow cell and flushed at 2 mL min-1 with a solution of 15 µM DTPA buffered with 2 mM NaHCO3 at pH 8 (hereafter referred to as “DTPA/NaHCO3”) using a peristaltic pump. The effluent containing cell-generated superoxide was then mixed with MCLA reagent, also delivered by peristaltic pump, in the flow cell and the FeLume response monitored for 240 s. The FeLume was calibrated using the xanthine/xanthine oxidase method (19). The rate of superoxide production by M. aeruginosa (kprod in mol cell-1 h-1) was calculated as kprod )

[O•2 ] × flowrate number of cells on filter

(1)

using the average superoxide concentration measured in the filter effluent over 240 s. Superoxide decay during the ∼10 s required for the filter effluent to reach the flow cell was negligible for kprod determination because of relatively low superoxide concentrations in the effluent and the presence of DTPA to inhibit reactions of superoxide with metals (20). Short-Term Fe Uptake Experiments. The short-term Fe uptake rate of M. aeruginosa was measured by incubating cells in Fraquil* containing 55Fe complexed by EDTA or citrate. Five milliliters of culture grown in 0.1 µM Fe was filtered on to a 0.22 µm PVDF membrane and rinsed with 5 mL of EDTA/

oxalate then 5 mL of NaHCO3 solutions. Washed cells were resuspended in 2 mL of Fraquil* containing no added Fe and either 1 µM EDTA or 5 µM citrate (to complex other trace metals in the medium). Subsequently appropriate volumes of stock solutions of 55Fe(III)-EDTA or 55Fe(III)-citrate, and additional ligand solutions in the experiment where Fe:ligand ratios were varied, were added (see SI for details). Fe(II) uptake rates were examined at various 55Fe and ligand concentrations by conducting similar incubations in the presence of 1 mM ascorbate. Cells were incubated at 27 °C for 4 h in the EDTA system and 3 h in the citrate system, based on the linearity of Fe uptake over the duration of the experiment (Figure S-1, SI). Fe uptake experiments were conducted in the dark to avoid any photochemical effects. After incubation, samples were vacuum filtered on to 0.22 µm membrane filters, rinsed with 3 mL of EDTA/oxalate then 2 mL of 2 mM NaHCO3, and placed in glass scintillation vials with 5 mL of scintillation cocktail (Beckman ReadyScint). The activity was measured in a Packard TriCarb Liquid Scintillation Counter, with scintillation counts (disintegrations per minute) of the samples converted to moles of Fe using concurrent counts of 5-50 µL of 55Fe-ligand stock in 5 mL scintillation cocktail. Process blanks were determined by performing the procedure in the absence of cells. To determine whether cellular exudates (e.g., siderophores) might be released during the short-term Fe uptake experiments, thereby altering the mode of Fe uptake, an additional experiment was conducted in which cells were initially incubated in Fe-starved Fraquil* ([Fe]T ) 0 µM, [EDTA]T ) 26 µM) for 1 day at cell densities of 4-9 × 106 cells mL-1. Cells were then removed from the media via filtration using a 0.65 µm PVDF membrane and the pH of the filtrate adjusted to pH 8.00 ( 0.05 if needed. The cells were then resuspended in either the filtrate or Fraquil* containing no Fe and [EDTA]T ) 26 µM followed by addition of 55Fe-EDTA stock solution at final [55Fe] ) 0.7 µM and [EDTA] ) 52 µM. 55 Fe uptake experiments were then performed as described above. To examine the effects of superoxide and Fe(II) generation on Fe uptake rate, cells were incubated in the additional presence of either 60 U mL-1 superoxide dismutase (SOD), 60 U mL-1 denatured SOD (d-SOD), 1 mM ferrozine (FZ), 1 mM ascorbate or 1 mM FZ + 1 mM ascorbate; 55Fe uptake experiments were then performed as described above. SOD was used to eliminate superoxide in the system and d-SOD as a control for SOD. FZ is commonly used to inhibit Fe(II) uptake (21) by forming a biologically unavailable Fe(II)-FZ3 complex. There is a concern that high concentrations of FZ (g400 µM) might actively reduce Fe(III)′ and result in inhibition of Fe uptake by simply lowering [Fe(III)′] (21). However, examination of Fe uptake in the citrate system suggested that FZ at 100-1000 µM had a negligible effect on Fe(III)′ uptake in the Fraquil* medium (see SI for details). Ascorbate was used as a Fe reducing agent (14, 15). Some cells were also incubated in 10 mM HgCl2 for 10 min before adding complexed 55Fe as a “killed control”. Calculations of Concentration and Uptake Rate of Unchelated Fe. Fe uptake was modeled assuming that M. aeruginosa can directly internalize either Fe(III)′ or Fe(II)′, but not organically complexed Fe, and that the chemical speciation of Fe is determined by reactions in the medium (Figure 1). The speciation of Fe under the various conditions used during Fe uptake incubations was calculated using a kinetic model based on the chemical reactions shown in Table 1. This model is similar to that developed by Garg et al. (16) to describe Fe uptake by Chattonella marina, except that rate constants for some reactions (including MichaelisMenten parameters) differed as different media and organVOL. 44, NO. 6, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

1981

FIGURE 1. Conceptual model showing potential pathways for Fe acquisition by M. aeruginosa, assuming the organism can acquire both unchelated Fe(III) and unchelated Fe(II) produced via superoxide-mediated reactions in the external medium. Fe(III)L, Fe(II)L, Fe(III)′, and Fe(II)′ represent organically complexed ferric and ferrous iron and unchelated ferric and ferrous iron, respectively. The rate constants defined in Table 1 and parameters related to biological Fe uptake (Ks and GS) are also shown. isms were used. The rate constants used in this study are justified as described in SI.

Results and Discussion Growth Kinetics. Growth of M. aeruginosa PCC7806 in Fraquil* with different Fe concentrations exhibited three phases (Figure S-2, SI): (i) early exponential growth phase (9 d) where the growth rate declined for all cultures. Thus, while 0.1 µM Fe was sufficient to support optimal cell growth initially, growth in the 0.1 µM Fe culture was inhibited by depletion of Fe available for uptake in the

later period. The intracellular Fe quota at late exponential growth phase (measured as described in SI) also declined with decreasing Fe concentration in the cultures (7.2 ( 2.1, 32 ( 1.9, and 89 ( 2.2 amol cell-1 for 0.1, 1, and 10 µM Fe cultures, respectively). The growth rate of cyanobacteria may not be an appropriate indicator of iron stress in some cases (22). However, RT-PCR analysis of the cultures described here indicated down-regulated expression of FurA (a gene that encodes a protein to regulate expression of Fe homeostasis polypeptides) in M. aeruginosa PCC7806 grown in 0.1 µM Fe compared to 1 µM Fe under otherwise identical culturing conditions (B. A. Neilan and R. Alexova, personal communication), indicating that the cells were indeed stressed at 0.1 µM Fe. In addition, it has been shown that Synechococcus PCC7942 exhibits stress at total [Fe] < 45 nM in Fraquil with [EDTA] ) 5 µM (23), conditions that are reasonably consistent with iron limited growth at 100 nM Fe and 26 µM EDTA used in this work. Superoxide Production. When DTPA/NaHCO3 was passed through M. aeruginosa cells on an in-line filter, the filter effluent elicited chemiluminescence from reaction with MCLA that was eliminated by addition of 30 unit mL-1 SOD (Figure S-3, SI) but not by the same concentration of d-SOD, indicating that M. aeruginosa releases superoxide into the external medium. Production rates of extracellular superoxide increased under Fe stress (1.2 ( 0.20, 0.62 ( 0.16 and 0.36 ( 0.01 amol cell-1 h-1 for 0.1, 1, and 10 µM cultures, respectively). In addition, there was an inverse relationship between superoxide production and cellular Fe quota (R2 ) 0.82, n ) 3). These rates were substantially less than published values for several marine phytoplankton of 840 amol cell-1 hr-1 for the marine diatom Thalassiosira weissflogii (24) and 0.29-4.3 pmol cell-1 h-1 for the toxic marine phytoplankton C. marina, Fibrocapsus japonica, Heterosigma akashiwo, and Olisthodiscus luteus (16, 25). Production rates normalized to cell surface area (30-110 zmol cell-1 h-1) were also considerably lower than those of the marine microorganisms mentioned above (1.8-420 amol cell-1 hr-1) (Table S-1, SI). The estimated steady-state concentration of superoxide in the growth medium (∼10-9 M, Tables S-2 and S-3, SI) was far less than observed in cultures of C. marina (∼10-7 M). Influence of Cellular Exudates and Chemical Treatments on Fe Uptake. Fe uptake rates were a significantly less by a

TABLE 1. Kinetic Model and Rate Constants Used in This Study reaction inorganic system

EDTA system

citrate system

Fe(II)′ + O2 f Fe(III)′ + O2•Fe(II)′ + O2•- + 2H+ f Fe(III)′ + H2O2 Fe(III)′ + O2•- f Fe(II)′ + O2 cell f O2•O2•- + O2•- + 2H+ f H2O2 + O2 Fe(III)′ + L f Fe(III)L Fe(III)L f Fe(III)′ + L Fe(II)′ + L f Fe(II)L Fe(II)L f Fe(II)′ + L Fe(II)L + O2 f Fe(III)L + O2•Fe(III)L + O2•- f Fe(II)L + O2 Fe(III)′ + L f Fe(III)L Fe(III)L f Fe(III)′ + L Fe(II)′ + L f Fe(II)L Fe(II)L f Fe(II)′ + L Fe(II)L + O2 f Fe(III)L + O2•Fe(III)L + O2•- f Fe(II)L + O2

rate constant kox1 kox2 kred1 kprod kdisp kf1 * kd1 * kf2 * kd2 * kox1 * kred1 * kf1 * kd1 * kf2 * kd2 * kox1 * kred1 *

8.8 1.0 × 107 1.5 × 108 1.2 × 10-18 5.0 × 104 3.5 × 105 1.0 × 10-5 1.4 × 103 2.0 × 10-4 31 1.3 × 105 2.1 × 105 (kd-FeCit1)/(1 + KFeCit2[Cit])b 5.0 × 102 2.0 × 10-3 2.9 8.0 × 102

ref M-1 M-1 M-1 mol M-1 M-1 s-1 M-1 s-1 M-1 M-1 M-1 s-1 M-1 s-1 M-1 M-1

s-1 s-1 s-1 cell-1 h-1 s-1 s-1 s-1 s-1 s-1 s-1 s-1 s-1 s-1

35 36 36 this study 37 38 this studya 16 16 this studya 39 38 40 41 41 42 39

a Detailed methods for determination of rate constants are provided in SI. b k* d1 for citrate depends on the concentration of citrate because of the formation of FeCit and FeCit2 complexes. Values of kd-FeCit1 ) 3.7 × 10-3 s-1 and KFeCit2 ) 1.3 × 104 -4 M-1 were used (40). Values of k* for 2.6 mM Cit to 2.7 × 10-3 s-1 for 26 µM Cit. d1 range from 1.1 × 10

1982

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 6, 2010

FIGURE 2. Effect of chemical treatments on Fe uptake rates by Fe-limited M. aeruginosa. In the control, iron-limited cells were incubated in Fraquil* containing 0.7 µM 55Fe complexed by either EDTA or citrate in the dark. In the chemical treatments, cells were incubated in the additional presence of either 10 mM-1 HgCl2, 60 unit mL-1 SOD, 60 unit mL-1 denatured SOD, 1 mM FZ, 1 mM ascorbate or 1 mM FZ + 1 mM ascorbate. As iron-binding ligands, (A) 26 µM EDTA, (B) 100 µM citrate and (C) 1000 µM citrate were used. The incubation period was 4 h in the EDTA system and 3 h in the citrate system. Error bars represent ( standard deviation from triplicate experiments. One and two asterisks indicate that chemical treatments were significantly different from the control at p < 0.01 and p < 0.05 level, respectively, using a single-tailed heteroscedastic t-test. factor of 1.7 (p < 0.01) when cells were incubated in spent medium amended with 55Fe compared to fresh medium with the same composition, indicating that extracellular compounds did not facilitate Fe uptake by M. aeruginosa during short-term incubation experiments (Figure S-4, SI). To determine if superoxide-mediated reduction was involved in Fe uptake by Fe-limited M. aeruginosa, Fe uptake rates were measured in the presence of various chemical treatments (Figure 2 and Table S-2, SI). When EDTA was used as a ligand, Fe uptake declined significantly in the presence of SOD and FZ. In contrast, addition of d-SOD had no significant effect on Fe uptake. In the presence of Fe-reducing ascorbate and ascorbate plus FZ, 25 and 7-fold increases in the Fe uptake rate occurred, respectively. This series of results suggests that a superoxide-mediated reductive step was involved in Fe uptake in the EDTA-buffered experimental system. When citrate was used as a ligand, effects of chemical treatments depended on the ligand concentration. With 100

µM citrate, additions of SOD, d-SOD, FZ and ascorbate individually had no significant effect on Fe uptake, but the uptake rate decreased significantly upon addition of FZ and ascorbate together. With 1 mM citrate, while decreases in rates of Fe uptake in the presence of SOD and FZ were statistically insignificant, uptake rates in the presence of ascorbate increased significantly (as found in the EDTA system), indicating that the reductant ascorbate increases Fe availability at higher citrate concentrations where Fe(III)′ concentration is low. Inhibition of Fe uptake in the presence of HgCl2 confirmed that Fe is taken up only by living cells (Figure 2). Affinity of Uptake Machinery for Unchelated Fe. The rate of Fe uptake was determined over a range of unchelated Fe concentrations, which were varied using different ligand and Fe concentrations (Tables S-3 and S-4, SI). Assuming Michaelis-Menten saturation kinetics for Fe(II)′ and Fe(III)′ uptake (1), the Fe uptake rate (FS) can be described as VOL. 44, NO. 6, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

1983

FS )

FSmax[S] KS + [S]

(2)

where [S] indicates concentration of bioavailable Fe, FSmax is the maximum uptake rate and KS is the half saturation constant. The total rate of Fe uptake is given by FFe′ ) FFe(III)′ + FFe(II)′. Eadie-Hofstee plots at citrate concentrations e100 µM, where any reduction was found to be unimportant and thus uptake of predominantly Fe(III)′ was assumed, yielded max ) 3.3 ( 0.16 amol cell-1 hr-1 KFe(III)′ ) 33 ( 3.4 pM and FFe(III)′ (Figure S-5A, SI). Applying a similar procedure to Fe uptake rates in the presence of ascorbate, where it was assumed that all iron is reduced and thus uptake of predominantly max ) 2.0 Fe(II)′ occurs, yielded KFe(II)′ ) 970 ( 380 pM and FFe(II)′ -1 -1 ( 0.28 amol cell hr (Figure S-5B, SI). The determined KFe(III)′ was close to that for another freshwater cyanobacterium Synechococcus (43 pM) (23) but much lower than some marine eukaryotic algae (KFe(III)′ ) 0.5-1 nM) (12, 26). The higher affinity for Fe(III)′ than Fe(II)′ is, however, consistent with the Fe uptake behavior of T. weissflogii (12), where values of KFe(III)′ ) 1 nM and KFe(II)′ ) 11 nM are calculated by applying inorganic side reaction coefficients of RFe3+ ) 10-10.0 (27) and RFe2+ ) 0.76 (28) to the reported values of KFe3+ ) 100 zM and KFe2+ ) 8.4 nM. The lower affinity for Fe(II)′ may be because of a slower rate of reaction between Fe(II)′ and a Fe transporter on the cell surface than is the case for Fe(III)′ (1). When normalized to cell surface area, maximum Fe uptake rates in four eukaryotic algae were estimated to be 0.4-1.3 µmol m-2 d-1 (26, 29). max in this work (0.7 This is comparable to the determined FFe(III)′ -2 -1 µmol m d ). Although the surface-normalized maximum Fe uptake rates for M. aeruginosa and a variety of coastal and oceanic phytoplankton are similar, a much higher value (62 µmol m-2 d-1) has been estimated for Synechococcus (23). Plotting all Fe uptake data in the absence of ascorbate against steady-state [Fe(III)′] calculated from the kinetic model shows that Michaelis-Menten kinetics with respect to Fe(III)′ describes the experimental data for the low citrate system well, but Fe uptake rates in incubations with EDTA (at all concentrations) and g1000 µM citrate were on average greater than those predicted by this model by a factor of ∼2 (Figure 3A). At [EDTA] ) 26 µM, the calculated Fe(III)′ uptake rate accounts for 59% of the experimentally measured uptake rate, consistent with the observation that Fe uptake is inhibited by ∼50% by addition of SOD or FZ, that is, that ∼50% of Fe is taken up as Fe(II)′ in the EDTA system. A similar observation has been reported for Synechococcus (23), in which the uptake flux of Fe was found to be about 1 order of magnitude greater than the calculated diffusive supply of Fe(III)′ in an EDTA-buffered system. When Fe uptake rates were calculated assuming that both Fe(III)′ and Fe(II)′ are taken up according to the Michaelis-Menten relationships determined above, much better agreement between predicted and measured rates is obtained (Figure 3B). These findings support the idea that Fe uptake by M. aeruginosa was facilitated by increased [Fe(II)′]. Fe(II) Formation Pathway. Fe(II)′ is formed via either (i) a nondissociative reduction pathway (NDR), where Fe(III)L is directly reduced to Fe(II)L followed by subsequent dissociation of the Fe(II) complex, or (ii) a dissociative reduction pathway (DR) where Fe(III)L initially dissociates to form Fe(III)′ which is then reduced to Fe(II)′ (30). Although the dominant process shifts with nature and concentration of Fe-binding ligand, both processes concurrently occur to some extent. Garg et al. (30) have suggested that the superoxidemediated formation of Fe(II)′ from Fe(III)-EDTA in a bicarbonate solution (pH 8) principally occurs via NDR as a result of the low [Fe(III)′] available for reduction due to slow dissociation and rapid reformation of Fe(III)-EDTA. In the presence of high concentrations of Ca2+ however, DR 1984

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 6, 2010

FIGURE 3. Measured and predicted Fe uptake rates at various unchelated Fe concentrations in Fraquil* containing 55Fe complexed by citrate or EDTA in the absence of chemical treatments. (A) Measured Fe uptake rates as a function of calculated [Fe(III)′] in the presence of citrate (open symbols) or EDTA (closed symbols). The line represents Michaelis-Menten max kinetics with KFe(III)′ ) 33 pM and GFe(III)′ ) 3.3 amol cell-1 h-1. Symbols and error bars are mean and ( standard deviation from triplicate experiments in both panels. (B) Measured Fe uptake rates compared with modeled uptake rates assuming uptake of only Fe(III)′ (closed symbols) or Fe(III)′ + Fe(II)′ (open symbols). The line indicates a slope of 1:1. The standard deviation between measured and calculated rates was reduced by 36% (n ) 16) when superoxide-mediated Fe(II)′ uptake was also considered. Symbols represented by diamonds and squares indicate data for the EDTA and citrate systems, respectively. contributes significantly to Fe(II)′ formation (∼70%). In this work, the steady-state concentration of Fe(II)′ in the EDTAbuffered freshwater medium Fraquil* (which contains a low concentration of Ca2+) occurs predominantly via NDR (93-96%), but DR is relatively minor (4-7%, see SI). The formation rate constant of the Fe(III)-EDTA complex is significantly lower in seawater than in freshwater due to the substantially higher concentrations of alkaline earth metals in seawater and the affinity of EDTA for these major cations (27). The resultant higher Fe(III)′ concentration in seawater than in freshwater results in the shift from NDR to DR in seawater. Only in the presence of a very strong Fe-chelator such as the fungal siderophore desferrioxamine B does NDR become the dominant pathway of Fe uptake in seawater (13, 14). Implications of Findings. Fe reduction was important in the process of Fe uptake by M. aeruginosa in Fraquil* medium when Fe availability was low. Reductive uptake of Fe, possibly mediated by ferric reductase enzymes in the cell membrane, has been suggested to occur in a range of eukaryotes including vascular plants (31), unicellular chlorophytes (32) and diatoms (13, 14), while superoxide-mediated Fe reduction has been shown to be involved in Fe uptake by the marine microalgae C. marina (16) and L. majuscula (15). The freshwater cyanobacterium Synechococcus is also able to access FeEDTA even under conditions where Fe(III)′ is a minor source of bioavailable Fe (23), suggesting the possibility of a reductive Fe uptake strategy similar to that described here for M. aeruginosa under low [Fe(III)′] conditions.

However, reduction was not necessary for Fe uptake by M. aeruginosa when sufficient Fe(III)′ was present to satisfy requirements. Consequently, the contribution of reduction to Fe uptake appears to depend on the nature of Fe(III)′ chelators present in the external medium and the associated availability of Fe(III)′. Although the mechanism of extracellular production of superoxide remains unclear, the increased production under iron deplete conditions might arise through accumulation of hydroquinones located within the cytoplasmic membrane which autoxidize to produce superoxide in the periplasm as suggested in studies of extracellular superoxide generation by Escherichia coli (33). In addition, hydroquinone-mediated superoxide accumulation in the periplasm may also result in diffusion of superoxide to the external medium where it may potentially influence Fe bioavailability through Fe(III) reduction. Although M. aeruginosa can excrete hydroxamate compounds into its extracellular environment (8), the fact that Fe uptake rates were accurately predicted from our kinetic model based solely on a Fe′ acquisition mechanism suggests that siderophore-mediated Fe uptake is unlikely to be important under the conditions employed here. This was also supported by the slight decrease in Fe uptake observed when cells were incubated in spent medium that could potentially contain such compounds compared with freshly prepared medium. A similar phenomenon was considered likely for Synechococcus where Fe uptake during short-term incubations appeared to be associated with a high affinity Fe-transport system that did not involve a siderophore (23). Given that M. aeruginosa is capable of acquiring Fe that is initially present primarily in organically complexed form by taking up Fe′, it appears feasible that Fe′ could be a major substrate for uptake in many natural waters where Fe is typically unlikely to be limiting. However it is important to stress that this does not exclude the possible significance of siderophore-mediated Fe uptake under some conditions, particularly during long-term Fe limitation. While the results presented here provide quantification of the uptake dynamics of Fe(II)′ and Fe(III)′ by M. aeruginosa and insight into the conditions under which reducing agents such as superoxide may influence iron uptake, the conditions used are far removed from those which will be experienced by Microcystis in natural waters. Although EDTA and citrate represent useful ligands for well-defined studies of iron uptake, natural ligands (such as humic substances) are considerably more complex. Hassler and Twiss (23) showed that an increase in concentration of Suwannee River fulvic acid from 1 to 15 mg L-1 decreases Fe bioavailability of Synechococcus 100-fold, consistent with what might be expected for model Fe-binding ligands. However, unlike the simple carboxylate ligands used here, humic substances contain quinone-type moieties that may induce redox transformations of both oxygen and iron thermally (34) and via light-induced reduction of complexed Fe(III). Such transformations are likely to significantly modify iron bioavailability. Indeed, results of preliminary experiments indicate that Fe uptake by M. aeruginosa is significantly higher in the light than dark (but full discussion of these findings will be presented elsewhere). While the impact of such factors on Fe uptake will undoubtedly be the subject of numerous future investigations, we believe that the insights into uptake dynamics of Fe(II)′ and Fe(III)′ under carefully controlled conditions reported here lays the foundation for future rigorous studies of iron uptake kinetics and of the factors controlling iron uptake, cyanobacterial growth, and possibly toxicity, under more complex natural conditions.

Acknowledgments This work was partially funded by Research Fellowships of the Japan Society for the Promotion of Science (JSPS) for Young Scientists. Support provided by the Australian Research Council through projects DP0987188 and LP0883561 is gratefully acknowledged.

Supporting Information Available Additional details as noted in the text. This information is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) Sunda, W. G., Bioavailability and bioaccumulation of iron in the sea. In the Biogeochemistry of Iron in Seawater; Turner, D. R., Hunter, K. A., Eds.; J. Wiley: Chichester; New York, 2001; pp 41-84. (2) Morel, F. M. M.; Kustka, A. B.; Shaked, Y. The role of unchelated Fe in the iron nutrition of phytoplankton. Limnol. Oceanogr. 2008, 53 (1), 400–404. (3) Rose, A. L.; Waite, T. D. Effect of dissolved natural organic matter on the kinetics of ferrous iron oxygenation in seawater. Environ. Sci. Technol. 2003, 37 (21), 4877–4886. (4) Liu, X. W.; Millero, F. J. The solubility of iron in seawater. Mar. Chem. 2002, 77 (1), 43–54. (5) Boyd, P. W.; Jickells, T.; Law, C. S.; Blain, S.; Boyle, E. A.; Buesseler, K. O.; Coale, K. H.; Cullen, J. J.; de Baar, H. J. W.; Follows, M.; et al. Mesoscale iron enrichment experiments 1993-2005: Synthesis and future directions. Science 2007, 315 (5812), 612– 617. (6) Twiss, M. R.; Auclair, J. C.; Charlton, M. N. An investigation into iron-stimulated phytoplankton productivity in epipelagic Lake Erie during thermal stratification using trace metal clean techniques. Can. J. Fish. Aquat. Sci. 2000, 57 (1), 86–95. (7) Utkilen, H.; Gjolme, N. Iron-stimulated toxin production in Microcystis aeruginosa. Appl. Environ. Microbiol. 1995, 61 (2), 797–800. (8) Imai, A.; Fukushima, T.; Matsushige, K. Effects of iron limitation and aquatic humic substances on the growth of Microcystis aeruginosa. Can. J. Fish. Aquat. Sci. 1999, 56 (10), 1929–1937. (9) Wilhelm, S. W. Ecology of iron-limited cyanobacteria: A review of physiological responses and implications for aquatic systems. Aquat. Microb. Ecol. 1995, 9 (3), 295–303. (10) Winkelmann, G.; Van der Helm, D.; Neilands, J. B. Iron Transport in Microbes, Plants, And Animals. VCH: Weinheim, Germany, 1987; p xxiv, 533 p. (11) Nicolaisen, K.; Moslavac, S.; Samborski, A.; Valdebenito, M.; Hantke, K.; Maldener, I.; Muro-Pastor, A. M.; Flores, E.; Schleiff, E. Alr0397 is an outer membrane transporter for the siderophore schizokinen in Anabaena sp strain PCC 7120. J. Bacteriol. 2008, 190 (22), 7500–7507. (12) Anderson, M. A.; Morel, F. M. M. The influence of aqueous iron chemistry on the uptake of iron by the coastal diatom Thalassiosira-Weissflogii. Limnol. Oceanogr. 1982, 27 (5), 789–813. (13) Shaked, Y.; Kustka, A. B.; Morel, F. M. M. A general kinetic model for iron acquisition by eukaryotic phytoplankton. Limnol. Oceanogr. 2005, 50 (3), 872–882. (14) Maldonado, M. T.; Price, N. M. Reduction and transport of organically bound iron by Thalassiosira oceanica (Bacillariophyceae). J. Phycol. 2001, 37 (2), 298–309. (15) Rose, A. L.; Salmon, T. P.; Lukondeh, T.; Neilan, B. A.; Waite, T. D. Use of superoxide as an electron shuttle for iron acquisition by the marine cyanobacterium Lyngbya majuscula. Environ. Sci. Technol. 2005, 39 (10), 3708–3715. (16) Garg, S.; Rose, A. L.; Godrant, A.; Waite, T. D. Iron uptake by the ichthyotoxic Chattonella marina (Raphidophyceae): Impact of superoxide generation. J. Phycol. 2007, 43 (5), 978–991. (17) Andersen, R. A. Algal Culturing Techniques; Elsevier/Academic Press: Burlington, MA, 2005; p x, 578 p. (18) Tang, D. G.; Morel, F. M. M. Distinguishing between cellular and Fe-oxide-associated trace elements in phytoplankton. Mar. Chem. 2006, 98 (1)), 18–30. (19) Rose, A. L.; Moffett, J. W.; Waite, T. D. Determination of superoxide in seawater using 2-methyl-6-(4-methoxyphenyl)3,7-dihydroimidazo[1,2-a]pyrazin-3(7H)-one chemiluminescence. Anal. Chem. 2008, 80 (4), 1215–1227. (20) Goldstone, J. V.; Voelker, B. M. Chemistry of superoxide radical in seawater: CDOM associated sink of superoxide in coastal waters. Environ. Sci. Technol. 2000, 34 (6), 1043–1048. VOL. 44, NO. 6, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

1985

(21) Shaked, Y.; Kustka, A. B.; Morel, F. M. M.; Erel, Y. Simultaneous determination of iron reduction and uptake by phytoplankton. Limnol. Oceanogr.: Methods 2004, 2, 137–145. (22) Henley, W. J.; Yin, Y. Growth and photosynthesis of marine Synechococcus (Cyanophyceae) under iron stress. J. Phycol. 1998, 34 (1), 94–103. (23) Hassler, C. S.; Twiss, M. R. Bioavailability of iron sensed by a phytoplanktonic Fe-bioreporter. Environ. Sci. Technol. 2006, 40 (8), 2544–51. (24) Kustka, A. B.; Shaked, Y.; Milligan, A. J.; King, D. W.; Morel, F. M. M. Extracellular production of superoxide by marine diatoms: Contrasting effects on iron redox chemistry and bioavailability. Limnol. Oceanogr. 2005, 50 (4), 1172–1180. (25) Oda, T.; Nakamura, A.; Shikayama, M.; Kawano, I.; Ishimatsu, A.; Muramatsu, T. Generation of reactive oxygen species by raphidophycean phytoplankton. Biosci., Biotechnol., Biochem. 1997, 61 (10), 1658–1662. (26) Sunda, W. G.; Huntsman, S. A. Interrelated influence of iron, light and cell size on marine phytoplankton growth. Nature 1997, 390 (6658), 389–392. (27) Hudson, R. J. M.; Covault, D. T.; Morel, F. M. M. Investigations of iron coordination and redox reactions in seawater using Fe59 radiometry and ion-pair solvent-extraction of amphiphilic iron complexes. Mar. Chem. 1992, 38 (3-4), 209–235. (28) Millero, F. J.; Yao, W. S.; Aicher, J. The speciation of Fe(II) and Fe(III) in natural-waters. Mar. Chem. 1995, 50 (1-4), 21–39. (29) Sunda, W. G.; Huntsman, S. A. Iron uptake and growth limitation in oceanic and coastal phytoplankton. Mar. Chem. 1995, 50 (1-4), 189–206. (30) Garg, S.; Rose, A. L.; Waite, T. D. Superoxide-mediated reduction of organically complexed iron(III): Impact of pH and competing cations (Ca2+). Geochim. Cosmochim. Acta 2007, 71 (23), 5620– 5634. (31) Gonzalez-Vallejo, E. B.; Gonzalez-Reyes, J. A.; Abadia, A.; LopezMillan, A. F.; Yunta, F.; Lucena, J. J.; Abadia, J. Reduction of ferric chelates by leaf plasma membrane preparations from Fedeficient and Fe-sufficient sugar beet. Aust. J. Plant Physiol. 1999, 26 (6), 601–611. (32) La Fontaine, S.; Quinn, J. M.; Nakamoto, S. S.; Page, M. D.; Gohre, V.; Mosely, J. L.; Kropat, J.; Merchant, S. Copper-

1986

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 6, 2010

(33) (34) (35)

(36)

(37) (38)

(39) (40)

(41) (42)

dependent iron assimilation pathway in the model photosynthetic eukaryote Chlamydomonas reinhardtii. Eukaryotic Cell 2002, 1 (5), 736–757. Korshunov, S.; Imlay, J. A. Detection and quantification of superoxide formed within the periplasm of Escherichia coli. J. Bacteriol. 2006, 188 (17), 6326–6334. Garg, S.; Rose, A. L.; Waite, T. D. Production of reactive oxygen species on photolysis of dilute aqueous quinone solutions. Photochem. Photobiol. 2007, 83 (4), 904–913. Pham, A. N.; Waite, T. D. Oxygenation of Fe(II) in natural waters revisited: Kinetic modeling approaches, rate constant estimation and the importance of various reaction pathways. Geochim. Cosmochim. Acta 2008, 72 (15), 3616–3630. Rush, J. D.; Bielski, B. H. J. Pulse radiolytic studies of the reactions of HO2/O2- with Fe(II)/Fe(III) ionssThe reactivity of HO2/O2with ferric ions and its implication on the occurrence of the Haber-Weiss reaction. J. Phys. Chem. 1985, 89, 5062–5066. Bielski, B. H. J.; Cabelli, D. E.; Arudi, R. L.; Ross, A. B. Reactivity of H2O2/O2- radicals in aqueous-solution. J. Phys. Chem. Ref. Data 1985, 14 (4), 1041–1100. Fujii, M.; Rose, A. L.; Waite, T. D.; Omura, T. Effect of divalent cations on the kinetics of Fe(III) complexation by organic ligands in natural waters. Geochim. Cosmochim. Acta 2008, 72 (5), 1335– 1349. Rose, A. L.; Waite, T. D. Reduction of organically complexed ferric iron by superoxide in a simulated natural water. Environ. Sci. Technol. 2005, 39 (8), 2645–2650. Fujii, M.; Ito, H.; Rose, A. L.; Waite, T. D.; Omura, T. Superoxidemediated Fe(II) formation from organically complexed Fe(III) in coastal waters. Geochim. Cosmochim. Acta 2008, 72 (24), 6079– 6089. Rose, A. L.; Waite, T. D. Kinetics of iron complexation by dissolved natural organic matter in coastal waters. Mar. Chem. 2003, 84 (1-2), 85–103. Pham, A. N.; Waite, T. D. Modeling the kinetics of Fe(II) oxidation in the presence of citrate and salicylate in aqueous solutions at pH 6.0-8.0 and 25 degrees C. J. Phys. Chem. A 2008, 112 (24), 5395–5405.

ES901315A