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Characterization of a membrane-bound NADH-dependent Fe3+ reductase from the dissimilatory Fe3+-reducing bacterium Geobacter sulfurreducens
FEMS Microbiology Letters 185 (2000) 205^211
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Characterization of a membrane-bound NADH-dependent Fe3 reductase from the dissimilatory Fe3 -reducing bacterium Geobacter sulfurreducens Timothy S. Magnuson, Allison L. Hodges-Myerson, Derek R. Lovley * Department of Microbiology, Morrill Science Center, University of Massachusetts, Amherst, MA 01003, USA Received 4 January 2000; received in revised form 15 February 2000; accepted 15 February 2000
Abstract Geobacter sulfurreducens produces a single, membrane-associated Fe3 reductase activity when grown on fumarate or Fe3 . The activity was initially isolated by solubilization of membranes with the non-ionic detergent dodecyl-L-D-maltoside, and partially purified by a combination of ion exchange chromatography and preparative non-denaturing gel electrophoresis. Molecular mass of the reductase, as determined by gel filtration chromatography, was approximately 300 kDa. Cofactor analysis of the purified reductase demonstrates that it contains a hemoprotein and flavin adenine dinucleotide. Kinetic and inhibitor studies show that the reductase is specific for NADH as electron donor, and confirm that the reductase enzymatically reduces Fe3 . The cytochrome associated with the complex undergoes a reoxidation upon addition of Fe3 compounds, indicating an ability to pass reducing equivalents to Fe3 . This is the first description of a purified NADH-dependent Fe3 reductase from a microorganism capable of coupling Fe3 reduction to growth. ß 2000 Published by Elsevier Science B.V. All rights reserved. Keywords : Fe3 reductase ; Cytochrome ; Dissimilatory Fe3 reduction
1. Introduction Dissimilatory iron-reducing bacteria (DIRB) are being increasingly recognized as an ecologically and environmentally important group of microorganisms. Progress is being made toward the understanding of their biochemistry and physiology, particularly with the genera Shewanella and Geobacter, both of which are being studied intensively in terms of the biochemistry and physiology of anaerobic respiration [1^9]. Based on biochemical studies of Shewanella spp. and Geobacter spp., it is currently hypothesized that c-type cytochromes play an important role in electron transport and dissimilatory metal reduction by metal respiring microorganisms [1^3,5^9]. Although some of these proteins have been characterized, there is only one instance where an intact Fe3 reductase enzyme activity has been identi¢ed from an Fe3 respiring organism, and this protein was not puri¢ed in quantities su¤cient for detailed analysis [5].
Members of the genus Geobacter are typically found in environments such as freshwater sediments and aquifers, in which Fe3 reduction plays an important role in the degradation of naturally occurring organic matter or organic contaminants [10^12]. Initial studies on the biochemistry of Fe3 reduction by Geobacter metallireducens and Geobacter sulfurreducens show that the Fe3 reductase activity is localized in the membrane fraction and requires NADH as an electron donor [13,14]. These ¢ndings support the theory that dissimilatory Fe3 reductases are necessarily membrane bound to allow proton gradient generation and to enable contact with insoluble Fe3 oxides as terminal electron acceptors. The purpose of the studies presented here is to isolate and characterize the membrane-bound NADH-dependent Fe3 reductase of G. sulfurreducens. 2. Materials and methods 2.1. Organism and culture conditions
* Corresponding author. Present address. Tel. : +1 (413) 545-9651; Fax: +1 (413) 545-1578; E-mail: [email protected]
G. sulfurreducens strain PCA (ATCC 51573) was ob-
0378-1097 / 00 / $20.00 ß 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 0 8 1 - 1
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tained from our laboratory culture collection. It was cultured under strict anaerobic conditions on modi¢ed freshwater media, containing 20 mM Na^acetate as the electron donor and carbon source, and either 50 mM Fe3 ^citrate or 40 mM fumarate as the electron acceptor [16]. G. sulfurreducens was mass cultured in 10 l glass carboys under N2 ^CO2 (80:20). Late log phase cells were harvested via centrifugation and the pellet resuspended to a cell density of 3 g (wet weight) cells per ml bu¡er in 20 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris^HCl) pH 7.5/10% glycerol, and frozen at 380³C. 2.2. Enzyme assays Fe3 reductase activity was determined by monitoring the reduction of Fe3 to Fe2 with the colorimetric Fe(II) capture reagent ferrozine (3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine). Reaction mixtures (1.0 ml) contained 0.1 M N-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES)^NaOH pH 7.0, 1.0 mM ferrozine, 40 mM MgCl2 , 0.2 mM NADH, 10% glycerol, 0.5 mM Fe3 ^nitrilotriacetic acid, and 10^100 Wg enzyme preparation. Reactions were initiated by addition of Fe3 , and formation of Fe(II)^ferrozine complex (A = 27 900 M31 cm31 ) [17] was measured spectrophotometrically at 562 nm. All activity measurements were made on a Shimadzu (Baltimore, MD, USA) UV2401-PC dual beam spectrophotometer equipped with a temperature-controlled cuvette holder. One milliunit (mU) of activity is de¢ned as the amount of enzyme that catalyzes the formation of 1 nmol Fe2 per min at 40³C. NADH:menaquinone oxidoreductase activity was measured as described previously [18]. Hydrogenase was assayed with hydrogen and benzyl viologen [19], fumarate reductase was measured with fumarate and benzyl viologen [20] and ATPase was measured according to [21]. Total protein was determined by the bicinchoninic acid method [22] with bovine serum albumin as standard. 2.3. Localization and puri¢cation of the Fe3 + reductase activity All manipulations were carried out aerobically at 4³C. Cell suspensions (containing about 10 g wet weight of cells) of G. sulfurreducens were thawed and deoxyribonuclease I (40 U ml31 ), ribonuclease A (10 Wg ml31 ), and lysozyme (0.1 mg ml31 ) were added. The cell suspension was incubated at 37³C for 30 min, and then disrupted with a French pressure cell at 40 000 kPa. After two passes through the cell, the crude cell extract was clari¢ed by centrifugation at 7000Ug for 20 min. The supernatant was removed and centrifuged at 105 000Ug for 1 h to pellet the membrane fraction. The supernatant (soluble fraction) was removed and the pellet (membrane fraction) was resuspended in 20 mM Tris^HCl pH 7.5, 10% glycerol. Washed membrane suspension (1 ml, 10.0 mg protein)
was loaded onto 30^50% sucrose gradients (38 ml total volume) and centrifuged for 16 h at 30 000Ug in a Beckman SW28.1 rotor as described previously [6]. The fractionated membranes, which were separated into inner and outer membrane fractions, were collected and centrifuged at 105 000Ug for 1 h to pellet each membrane fraction. The pellets were resuspended in 1.0 ml 20 mM Tris^HCl pH 7.5 and subsequently assayed for Fe3 reductase activity and total protein as described above. For puri¢cation of the Fe3 reductase activity, the protein concentration of the membrane suspension was determined, and adjusted to 10.0 mg ml31 with bu¡er A (20 mM Tris^HCl pH 7.5, 10% glycerol, 0.1 mg ml31 dodecyl-L-D-maltoside). The membrane suspension was subsequently extracted with dodecyl-L-D-maltoside at a ratio of 1 mg detergent per mg protein. Detergent was added from a 100 mg ml31 aqueous stock solution, and extraction was carried out for 1 h at 4³C with stirring. Insoluble material was pelleted by ultracentrifugation for 1 h at 105 000Ug. The supernatant (detergent extract) was reserved, and the pellet discarded. Detergent extract (400 mg) was loaded onto a column of Q-Sepharose fast £ow ion exchange media (column dimensions 2.5 cmU40 cm) equilibrated with bu¡er A. Bound proteins were eluted with a linear gradient of 0 to 0.6 M NaCl (300 ml total volume). Fractions (5.0 ml) were collected and assayed for Fe3 reductase activity. Fe3 reductase activity eluted at about 0.3 M NaCl. Fractions containing activity were pooled and concentrated with a tangential £ow ultra¢ltration device (Filtron, Northborough, MA, USA) equipped with a 30 kDa cuto¡ membrane. The concentrate from the ion exchange chromatography step was desalted against 1Ustacking gel bu¡er (0.125 M Tris^HCl pH 6.8, 10% glycerol, 0.5% N-lauroylsarcosine) on a Sephadex G-25 column (2.5 cmU40 cm), and then resolved from other membrane proteins on a PrepCell preparative electrophoresis apparatus (Bio-Rad, Hercules, CA, USA). A modi¢ed Tris-bu¡ered system [23,24] was used, in which stacking and resolving gels contained 0.5% N-lauroylsarcosine and 10% glycerol. A 5% acrylamide stacking gel and 6% acrylamide resolving gel were used. Electrophoresis conditions were 20 W constant power for about 15 h with cooling. Six ml fractions were collected by elution with bu¡er A at a £ow rate of 0.7 ml min31 . Fractions were analyzed for Fe3 reductase activity by an in-gel assay (see below), and fractions containing Fe3 reductase were pooled and concentrated using a Filtron Ultrasette with a 30 kDa cuto¡ membrane. Puri¢ed enzyme was stored at 380³C. 2.4. Biochemical characterization Proteins were routinely analyzed by non-denaturing and denaturing polyacrylamide gel electrophoresis (ND^ PAGE and SDS^PAGE) [23,24]. ND^PAGE gels contained 0.5% N-lauroylsarcosine and 10% glycerol. Samples
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were prepared in 0.125 M Tris^HCl pH 6.8, 10% glycerol, 0.5% N-lauroylsarcosine, and 0.01% (w/v) bromphenol blue. Fe3 reductase zymograms were performed by soaking ND^PAGE gels in reaction mixtures containing 0.1 M HEPES^NaOH pH 7.0, 10% glycerol, 1.0 mM MgCl2 , 1.0 mM ferrozine, 0.5 mM Fe3 ^NTA, and 0.2 mM NADH [25]. Positive reactions appeared as purple bands on a pink background. Proteins containing covalently bound heme were detected by staining with N,N,NP,NPtetramethylbenzidine [26]. Native molecular mass was determined by Superdex 200 HR 10/30 gel ¢ltration chromatography in bu¡er A containing 0.05 M NaCl. The column was calibrated with the following molecular mass standards: thyroglobulin (669 000 Da), apoferritin (449 000 Da), L-amylase (200 000 Da), alcohol dehydrogenase (150 000 Da), bovine serum albumin (66 000 Da), carbonic anhydrase (29 000 Da), and horse heart cytochrome c (12 400 Da). UV-visible absorbance spectra of the puri¢ed enzyme were recorded on a Shimadzu (Baltimore, MD, USA) UV2401-PC dual beam spectrophotometer in the wavelength range of 300 to 700 nm, with enzyme diluted to 0.1 mg ml31 in bu¡er A. The enzyme was oxidized by addition of 3 Wl 0.1 M potassium ferricyanide solution. The spectrum was recorded, and then the enzyme was reduced by the addition of sodium dithionite from a 0.1 M stock solution prepared in 0.1 M sodium bicarbonate bu¡er pH 7.0. To measure direct transfer of electrons from the reduced Fe3 reductase to Fe3 , the complex was diluted to 0.1 mg ml31 total protein in bu¡er A (1.0 ml total reaction volume) and titrated with small aliquots (2^5 Wl) of 0.1 M sodium dithionite until reduction of the c-type cytochrome was observed (15^30 Wl were typically required for reduction). Aliquots of 0.1 M Fe3 ^NTA or 0.1 M ferrihydrite (Fe3 ^oxyhydroxide) were then added (5^10 Wl stock solution) and the spectrum re-recorded. Cyclic voltammetry was performed on the puri¢ed Fe3 reductase using a Bioanalytical Systems Inc. (W. Lafayette, IN, USA) Model CS50V potentiostat and a glassy carbon electrode. A 200 WM sample (in a thin-¢lm volume of 1 Wl) was analyzed using an electrolyte solution of 0.1 M Tris^HCl pH 7.6 [27]. Voltammograms were recorded in the range of 0 to 3800 mV versus a saturated calomel reference electrode. Potential values versus the standard hydrogen electrode (SHE) were obtained by adding 210 mV to experimental values.
207
Flavin cofactor content was determined by trichloroacetic acid extraction and high pressure liquid chromatography (HPLC) [28]. Flavin nucleotides were extracted with 20% trichloroacetic acid, neutralized, and separated on a Hypersil C-18 reversed phase HPLC column (2.5 mm by 25 mm). Standards were glutathione reductase (contains only £avin adenine dinucleotide (FAD)) and glyoxalate oxidase (contains only £avin mononucleotide (FMN)). For donor speci¢city studies, NADH was replaced by NADPH or H2 in the reaction mixture. Inhibition studies were carried out by adding inhibitors (dissolved in appropriate solvents) to standard reaction mixtures, and then monitoring the reaction. Quinacrine, KCN, and p-mercuribenzenesulfonate were dissolved in water ; rotenone, myxathiazole, hydroxyquinolone-N-oxide (HOQNO), and antimycin were dissolved in acetone. 2.5. Materials and chemicals Gel ¢ltration molecular size standards, ferrozine, enzyme inhibitors, glyoxalate oxidase, and glutathione reductase were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Electrophoresis standards were from Bio-Rad Laboratories (Hercules, CA, USA). Q-Sepharose fast £ow chromatography media were obtained from Pharmacia Amersham Biotech (Piscataway, NJ, USA). All other reagents were of analytical grade. 3. Results 3.1. Isolation, puri¢cation and physical characterization of the membrane-bound Fe3 + reductase Cultures of G. sulfurreducens produce a single Fe3 reductase activity, as determined by non-denaturing PAGE (Fig. 1A). Fe3 reductase activity was found to reside primarily in the membrane fraction, with about 67% of the membrane-associated activity distributed in the outer membrane (Table 1). To isolate Fe3 reductase, crude membranes were solubilized with dodecyl-L-D-maltoside, a non-ionic detergent. The crude solubilized material was then fractionated with ion exchange chromatography, which served to remove lipids and other contaminating material from the preparation. Further puri¢cation was achieved with preparative non-denaturing polyacrylamide
Table 1 Distribution of Fe3 reductase activity in soluble and membrane fractions of G. sulfurreducens Fraction
Total protein recovered (mg)
Total activity recovered (mU)
% Total activity recovereda
Soluble Membranes Membrane fractions Cytoplasmic membrane Outer membrane
131.6 98.1
68.3 392.3
14.8 85.2
2.7 4.3
5.1 10.3
32.9 67.1
a
Activity recovered as a percentage of the total activity recovered in all fractions.
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Fig. 1. Analysis of crude and puri¢ed Fe3 reductase by non-denaturing gel electrophoresis. A: Zymogram analysis of crude membrane protein samples resolved by non-denaturing PAGE and visualized by Fe3 reductase staining. Samples were prepared from cultures grown on either Fe3 (Fe) or fumarate (Fum). 100 Wg crude protein loaded per lane. B: PAGE analysis of puri¢ed Fe3 reductase. The puri¢ed protein was visualized by Coomassie Blue (lane 1), Fe3 reductase activity staining (lane 2), and hemoprotein staining (lane 3). 10 Wg protein loaded per lane. C: Denaturing PAGE of partially puri¢ed Fe3 reductase. Molecular mass marker positions are shown at left. 1.0 Wg protein was resolved and detected with silver (lane 1) or with a heme stain (lane 2). The 90 kDa hemoprotein (HP) is indicated. The ¢gure was processed using Adobe Photoshop 5.0 for PC.
gel electrophoresis. Typical yields were about 5%, with about 1 mg enzyme isolated per 10 g wet weight cells. The highly enriched Fe3 reductase migrated as a single band on ND^PAGE gels (Fig. 1B) and stained for Fe3 reductase activity (Fig. 1B). SDS^PAGE analysis of the puri¢ed activity revealed a heterooligomeric polypeptide composition of at least ¢ve major components, with minor constituents as well (Fig. 1C). The native molecular mass was about 300 kDa as determined by gel ¢ltration under non-dissociating conditions. 3.2. Biochemical properties of the Fe3 + reductase Heme staining of the ND^PAGE gels (Fig. 1B) and SDS^PAGE gels (Fig. 1C) indicates that the Fe3 reductase complex contains a hemoprotein, and spectrophoto-
metric analysis of the puri¢ed enzyme con¢rms the presence of a c-type cytochrome (Fig. 2A). The absorbance maxima of the reduced protein lie at 550, 520, and 413 nm, and absorbance maxima of the oxidized protein are at 409 and 535 nm (Fig. 2A). HPLC analysis of the acid-extracted £avins from the complex demonstrates that the Fe3 reductase contains FAD. Electrochemical analysis shows an oxidation/reduction potential of 3100 mV (vs. standard hydrogen electrode), indicating that the Fe3 reductase complex has a redox potential low enough to reduce all Fe3 forms that G. sulfurreducens can use as an electron acceptor, including synthetic ferrihydrite (E³P = 0 to +200 mV). The puri¢ed activity was speci¢c for NADH as an electron donor ; NADPH and H2 did not serve as electron donors for Fe3 reduction (data not shown). The Complex
Fig. 2. UV-visible spectrophotometric characterization of Fe3 reductase. A: Oxidized (dashed line) and reduced (solid line) spectra of puri¢ed Fe3 reductase at 0.05 mg ml31 . B: UV-visible spectra of Fe3 reductase (0.1 mg ml31 ) reduced with dithionite (solid line), and reoxidized with Fe3 ^NTA (dashed line). C: UV-visible spectra of Fe3 reductase (0.1 mg ml31 ) reduced with dithionite (solid line), and reoxidized with Fe3 ^oxyhydroxide (dashed line).
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Table 2 Sensitivity of puri¢ed Fe3 reductase complex to inhibitors
Fig. 3. Kinetic data for Fe3 reductase with NADH (A) and Fe3 (B). Insets show the resultant double reciprocal plots for each.
I inhibitors, rotenone and myxathiazole, inhibit Fe3 reduction (Table 2), providing evidence for an NADH:menaquinone oxidoreductase component in the Fe3 reductase. Other respiratory inhibitors that act at Complex III (ubiquinone :cytochrome c oxidoreductase) and Complex IV (cytochrome oxidase) did not signi¢cantly reduce rates of Fe3 reduction (Table 2). Quinacrine (a £avin homolog) and p-chloromercuribenzoate (a thiol modifying reagent) inhibit Fe3 reduction by the complex, indicating the participation of £avins and thiol groups in the reaction. Results of kinetic studies are shown in Fig. 3. The plots of V vs. [S] show saturation kinetics in both cases, and double reciprocal plots show linearity, consistent with an enzymatic reaction. The kinetic parameters for the enzyme are KNADH = 31 WM and Vm = 0.25 nmol min31 with rem spect to NADH, and KFe3 = 590 WM and Vm = 0.47 nmol m min31 with respect to Fe3 . The enzyme has a temperature optimum of 45³C and a pH optimum of 7.0, with the activity showing thermal inactivation above 50³C (Fig. 4). The Fe3 reductase activity also has signi¢cant NADH menaquinone oxidoreductase activity, but does not show hydrogenase, ATPase, or fumarate reductase activities (Table 3). In order to determine whether the 90 kDa cytochrome c subunit of the reductase was capable of donating electrons to Fe3 , dithionite was added to the Fe3 reductase in an amount just su¤cient to reduce the cytochrome and yield a typical reduced spectrum. The addition of either Fe3 ^NTA (Fig. 2B) or synthetic Fe3 ^oxyhydroxide (Fig. 2C) resulted in oxidation of the cytochrome, demonstrating that the 90 kDa cytochrome can transfer electrons to either form of Fe3 . Studies with
Inhibitor
Site of action
Concentration (mM)
% Inhibitiona
pCMB Quinacrine Rotenone Myxathiazole KCN HOQNO Antimycin
thiols £avins Complex Complex Complex Complex Complex
5.0 2.0 0.05 0.05 10.0 0.04 0.1
70 59 28 9 2 0 0
I I IV III III
a % Inhibition calculated as the decrease in rate compared to a control reaction with no inhibitor present.
puri¢ed preparations of the 90 kDa cytochrome yield similar results [29]. 4. Discussion 4.1. Fe3 + reductase composition and mechanism for Fe3 + reduction Work by Gaspard et al. [5] demonstrated that the Fe3 reductase activity in G. sulfurreducens was found in the membrane fraction, and distributed primarily in the outer membrane. This is consistent with results presented in the present work, which also demonstrates that Fe3 reductase is membrane associated. There is only one detectable Fe3 reductase activity produced by G. sulfurreducens, based on gel electrophoresis experiments on crude protein preparations. In previous work [5], this activity was gel puri¢ed and subjected to SDS^PAGE analysis. The ¢ndings suggested that the Fe3 reductase was composed of at least eight polypeptides, with three of these proteins being cytochromes. In contrast, the results of our work demonstrate that Fe3 reductase puri¢ed by more stringent methods contains only one cytochrome, and at least ¢ve major polypeptides. Additional experimental evidence suggests that the membrane-bound Fe3 reductase complex is an NADH dehydrogenase coupled to a c-type cytochrome that serves as a terminal reductase. This conclusion is supported by the following lines of evidence : (1) the activity is membrane associated and is a heterooligomer of di¡erent peptides, as is commonly seen for other memTable 3 Enzyme activities associated with the puri¢ed Fe3 reductase activity
Fig. 4. Temperature and pH optima of the puri¢ed Fe3 reductase.
Enzyme
Speci¢c activity (mU min31 mg31 )
Relative activitya
NADH:menadione oxidoreductase Fe3 reductase Hydrogenase Fumarate reductase ATPase
20.7 17.1 none detected none detected none detected
1.2 1.0 0 0 0
a
Represents the ratio of activity to Fe3 reductase activity.
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brane NADH dehydrogenases; (2) the activity is inhibited by Complex I inhibitors and contains FAD; (3) the cytochrome associated with the complex undergoes a reoxidation upon addition of Fe3 compounds, indicating an ability to pass reducing equivalents to Fe3 ; (4) the complex has a redox potential low enough to act as an electron donor to Fe3 compounds. 4.2. Potential physiological role of the Fe complex in G. sulfurreducens
3+
[5]
[6]
[7]
reductase [8]
It is generally considered that microorganisms that couple Fe3 reduction to growth must have membrane-bound Fe3 reductases in order to allow direct contact with extracellular insoluble Fe3 oxides, and to provide a mechanism for generation of a proton-motive force via NADH oxidation [15,30^32]. The Fe3 reductase from G. sulfurreducens appears to be situated in the membrane so that both of these functional requirements are met. It is conceivable, due to its large size and membrane distribution, that this reductase can span both inner and outer membranes, and allow for electron transfer from NADH to Fe^oxyhydroxides. Recent work with Shewanella putrefaciens outer membrane proteins suggests, however, that other Fe3 binding proteins may play a role in dissimilatory Fe3 reduction by Fe3 -reducing organisms [4,33]. The Fe3 reductase activity puri¢ed from G. sulfurreducens could function as a respiratory electron transport complex with or without other Fe3 binding components being present. The Fe3 reductase of G. sulfurreducens described in this paper represents the ¢rst active membrane-bound Fe3 -reducing system puri¢ed from a microorganism which can respire on Fe3 . Acknowledgements
[9]
[10]
[11]
[12]
[13] [14] [15]
[16]
[17]
We thank B. Blunt-Harris for excellent technical assistance, and M. Coppi and J. Lloyd for critical review of the manuscript. This research was funded by NSF Grant DEB 9523932 and DOE Grant DE-FG02-97ER62475.
[18]
[19]
[20]
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membrane associated 90 kDa Fe3 reducing cytochrome c from Geobacter sulfurreducens. Submitted. Lovley, D.R. (1991) Dissimilatory Fe3 and Mn(IV) reduction. Microbiol. Rev. 55, 259^287. Lovley, D.R., Coates, J.D., Sa¡arini, D.A. and Lonergan, D.J. (1997) Dissimilatory iron reduction. In: Iron and Related Transition Metals in Microbial Metabolism (Winkelman, G. and Carrano, C.J., Eds.), pp. 187^215. Harwood Academic Publishers, Switzerland. Nealson, K.H. and Sa¡arini, D. (1994) Iron and manganese in anaerobic respiration: Environmental signi¢cance, physiology, and regulation. Annu. Rev. Biochem. 48, 311^343. Caccavo, F.J. (1999) Protein-mediated adhesion of the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY to hydrous ferric oxide. Appl. Environ. Microbiol. 65, 5017^5022.
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Characterization of a membrane-bound NADH-dependent Fe3 reductase from the dissimilatory Fe3 -reducing bacterium Geobacter sulfurreducens Timothy S. Magnuson, Allison L. Hodges-Myerson, Derek R. Lovley * Department of Microbiology, Morrill Science Center, University of Massachusetts, Amherst, MA 01003, USA Received 4 January 2000; received in revised form 15 February 2000; accepted 15 February 2000
Abstract Geobacter sulfurreducens produces a single, membrane-associated Fe3 reductase activity when grown on fumarate or Fe3 . The activity was initially isolated by solubilization of membranes with the non-ionic detergent dodecyl-L-D-maltoside, and partially purified by a combination of ion exchange chromatography and preparative non-denaturing gel electrophoresis. Molecular mass of the reductase, as determined by gel filtration chromatography, was approximately 300 kDa. Cofactor analysis of the purified reductase demonstrates that it contains a hemoprotein and flavin adenine dinucleotide. Kinetic and inhibitor studies show that the reductase is specific for NADH as electron donor, and confirm that the reductase enzymatically reduces Fe3 . The cytochrome associated with the complex undergoes a reoxidation upon addition of Fe3 compounds, indicating an ability to pass reducing equivalents to Fe3 . This is the first description of a purified NADH-dependent Fe3 reductase from a microorganism capable of coupling Fe3 reduction to growth. ß 2000 Published by Elsevier Science B.V. All rights reserved. Keywords : Fe3 reductase ; Cytochrome ; Dissimilatory Fe3 reduction
1. Introduction Dissimilatory iron-reducing bacteria (DIRB) are being increasingly recognized as an ecologically and environmentally important group of microorganisms. Progress is being made toward the understanding of their biochemistry and physiology, particularly with the genera Shewanella and Geobacter, both of which are being studied intensively in terms of the biochemistry and physiology of anaerobic respiration [1^9]. Based on biochemical studies of Shewanella spp. and Geobacter spp., it is currently hypothesized that c-type cytochromes play an important role in electron transport and dissimilatory metal reduction by metal respiring microorganisms [1^3,5^9]. Although some of these proteins have been characterized, there is only one instance where an intact Fe3 reductase enzyme activity has been identi¢ed from an Fe3 respiring organism, and this protein was not puri¢ed in quantities su¤cient for detailed analysis [5].
Members of the genus Geobacter are typically found in environments such as freshwater sediments and aquifers, in which Fe3 reduction plays an important role in the degradation of naturally occurring organic matter or organic contaminants [10^12]. Initial studies on the biochemistry of Fe3 reduction by Geobacter metallireducens and Geobacter sulfurreducens show that the Fe3 reductase activity is localized in the membrane fraction and requires NADH as an electron donor [13,14]. These ¢ndings support the theory that dissimilatory Fe3 reductases are necessarily membrane bound to allow proton gradient generation and to enable contact with insoluble Fe3 oxides as terminal electron acceptors. The purpose of the studies presented here is to isolate and characterize the membrane-bound NADH-dependent Fe3 reductase of G. sulfurreducens. 2. Materials and methods 2.1. Organism and culture conditions
* Corresponding author. Present address. Tel. : +1 (413) 545-9651; Fax: +1 (413) 545-1578; E-mail: [email protected]
G. sulfurreducens strain PCA (ATCC 51573) was ob-
0378-1097 / 00 / $20.00 ß 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 0 8 1 - 1
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tained from our laboratory culture collection. It was cultured under strict anaerobic conditions on modi¢ed freshwater media, containing 20 mM Na^acetate as the electron donor and carbon source, and either 50 mM Fe3 ^citrate or 40 mM fumarate as the electron acceptor [16]. G. sulfurreducens was mass cultured in 10 l glass carboys under N2 ^CO2 (80:20). Late log phase cells were harvested via centrifugation and the pellet resuspended to a cell density of 3 g (wet weight) cells per ml bu¡er in 20 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris^HCl) pH 7.5/10% glycerol, and frozen at 380³C. 2.2. Enzyme assays Fe3 reductase activity was determined by monitoring the reduction of Fe3 to Fe2 with the colorimetric Fe(II) capture reagent ferrozine (3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine). Reaction mixtures (1.0 ml) contained 0.1 M N-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES)^NaOH pH 7.0, 1.0 mM ferrozine, 40 mM MgCl2 , 0.2 mM NADH, 10% glycerol, 0.5 mM Fe3 ^nitrilotriacetic acid, and 10^100 Wg enzyme preparation. Reactions were initiated by addition of Fe3 , and formation of Fe(II)^ferrozine complex (A = 27 900 M31 cm31 ) [17] was measured spectrophotometrically at 562 nm. All activity measurements were made on a Shimadzu (Baltimore, MD, USA) UV2401-PC dual beam spectrophotometer equipped with a temperature-controlled cuvette holder. One milliunit (mU) of activity is de¢ned as the amount of enzyme that catalyzes the formation of 1 nmol Fe2 per min at 40³C. NADH:menaquinone oxidoreductase activity was measured as described previously [18]. Hydrogenase was assayed with hydrogen and benzyl viologen [19], fumarate reductase was measured with fumarate and benzyl viologen [20] and ATPase was measured according to [21]. Total protein was determined by the bicinchoninic acid method [22] with bovine serum albumin as standard. 2.3. Localization and puri¢cation of the Fe3 + reductase activity All manipulations were carried out aerobically at 4³C. Cell suspensions (containing about 10 g wet weight of cells) of G. sulfurreducens were thawed and deoxyribonuclease I (40 U ml31 ), ribonuclease A (10 Wg ml31 ), and lysozyme (0.1 mg ml31 ) were added. The cell suspension was incubated at 37³C for 30 min, and then disrupted with a French pressure cell at 40 000 kPa. After two passes through the cell, the crude cell extract was clari¢ed by centrifugation at 7000Ug for 20 min. The supernatant was removed and centrifuged at 105 000Ug for 1 h to pellet the membrane fraction. The supernatant (soluble fraction) was removed and the pellet (membrane fraction) was resuspended in 20 mM Tris^HCl pH 7.5, 10% glycerol. Washed membrane suspension (1 ml, 10.0 mg protein)
was loaded onto 30^50% sucrose gradients (38 ml total volume) and centrifuged for 16 h at 30 000Ug in a Beckman SW28.1 rotor as described previously [6]. The fractionated membranes, which were separated into inner and outer membrane fractions, were collected and centrifuged at 105 000Ug for 1 h to pellet each membrane fraction. The pellets were resuspended in 1.0 ml 20 mM Tris^HCl pH 7.5 and subsequently assayed for Fe3 reductase activity and total protein as described above. For puri¢cation of the Fe3 reductase activity, the protein concentration of the membrane suspension was determined, and adjusted to 10.0 mg ml31 with bu¡er A (20 mM Tris^HCl pH 7.5, 10% glycerol, 0.1 mg ml31 dodecyl-L-D-maltoside). The membrane suspension was subsequently extracted with dodecyl-L-D-maltoside at a ratio of 1 mg detergent per mg protein. Detergent was added from a 100 mg ml31 aqueous stock solution, and extraction was carried out for 1 h at 4³C with stirring. Insoluble material was pelleted by ultracentrifugation for 1 h at 105 000Ug. The supernatant (detergent extract) was reserved, and the pellet discarded. Detergent extract (400 mg) was loaded onto a column of Q-Sepharose fast £ow ion exchange media (column dimensions 2.5 cmU40 cm) equilibrated with bu¡er A. Bound proteins were eluted with a linear gradient of 0 to 0.6 M NaCl (300 ml total volume). Fractions (5.0 ml) were collected and assayed for Fe3 reductase activity. Fe3 reductase activity eluted at about 0.3 M NaCl. Fractions containing activity were pooled and concentrated with a tangential £ow ultra¢ltration device (Filtron, Northborough, MA, USA) equipped with a 30 kDa cuto¡ membrane. The concentrate from the ion exchange chromatography step was desalted against 1Ustacking gel bu¡er (0.125 M Tris^HCl pH 6.8, 10% glycerol, 0.5% N-lauroylsarcosine) on a Sephadex G-25 column (2.5 cmU40 cm), and then resolved from other membrane proteins on a PrepCell preparative electrophoresis apparatus (Bio-Rad, Hercules, CA, USA). A modi¢ed Tris-bu¡ered system [23,24] was used, in which stacking and resolving gels contained 0.5% N-lauroylsarcosine and 10% glycerol. A 5% acrylamide stacking gel and 6% acrylamide resolving gel were used. Electrophoresis conditions were 20 W constant power for about 15 h with cooling. Six ml fractions were collected by elution with bu¡er A at a £ow rate of 0.7 ml min31 . Fractions were analyzed for Fe3 reductase activity by an in-gel assay (see below), and fractions containing Fe3 reductase were pooled and concentrated using a Filtron Ultrasette with a 30 kDa cuto¡ membrane. Puri¢ed enzyme was stored at 380³C. 2.4. Biochemical characterization Proteins were routinely analyzed by non-denaturing and denaturing polyacrylamide gel electrophoresis (ND^ PAGE and SDS^PAGE) [23,24]. ND^PAGE gels contained 0.5% N-lauroylsarcosine and 10% glycerol. Samples
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were prepared in 0.125 M Tris^HCl pH 6.8, 10% glycerol, 0.5% N-lauroylsarcosine, and 0.01% (w/v) bromphenol blue. Fe3 reductase zymograms were performed by soaking ND^PAGE gels in reaction mixtures containing 0.1 M HEPES^NaOH pH 7.0, 10% glycerol, 1.0 mM MgCl2 , 1.0 mM ferrozine, 0.5 mM Fe3 ^NTA, and 0.2 mM NADH [25]. Positive reactions appeared as purple bands on a pink background. Proteins containing covalently bound heme were detected by staining with N,N,NP,NPtetramethylbenzidine [26]. Native molecular mass was determined by Superdex 200 HR 10/30 gel ¢ltration chromatography in bu¡er A containing 0.05 M NaCl. The column was calibrated with the following molecular mass standards: thyroglobulin (669 000 Da), apoferritin (449 000 Da), L-amylase (200 000 Da), alcohol dehydrogenase (150 000 Da), bovine serum albumin (66 000 Da), carbonic anhydrase (29 000 Da), and horse heart cytochrome c (12 400 Da). UV-visible absorbance spectra of the puri¢ed enzyme were recorded on a Shimadzu (Baltimore, MD, USA) UV2401-PC dual beam spectrophotometer in the wavelength range of 300 to 700 nm, with enzyme diluted to 0.1 mg ml31 in bu¡er A. The enzyme was oxidized by addition of 3 Wl 0.1 M potassium ferricyanide solution. The spectrum was recorded, and then the enzyme was reduced by the addition of sodium dithionite from a 0.1 M stock solution prepared in 0.1 M sodium bicarbonate bu¡er pH 7.0. To measure direct transfer of electrons from the reduced Fe3 reductase to Fe3 , the complex was diluted to 0.1 mg ml31 total protein in bu¡er A (1.0 ml total reaction volume) and titrated with small aliquots (2^5 Wl) of 0.1 M sodium dithionite until reduction of the c-type cytochrome was observed (15^30 Wl were typically required for reduction). Aliquots of 0.1 M Fe3 ^NTA or 0.1 M ferrihydrite (Fe3 ^oxyhydroxide) were then added (5^10 Wl stock solution) and the spectrum re-recorded. Cyclic voltammetry was performed on the puri¢ed Fe3 reductase using a Bioanalytical Systems Inc. (W. Lafayette, IN, USA) Model CS50V potentiostat and a glassy carbon electrode. A 200 WM sample (in a thin-¢lm volume of 1 Wl) was analyzed using an electrolyte solution of 0.1 M Tris^HCl pH 7.6 [27]. Voltammograms were recorded in the range of 0 to 3800 mV versus a saturated calomel reference electrode. Potential values versus the standard hydrogen electrode (SHE) were obtained by adding 210 mV to experimental values.
207
Flavin cofactor content was determined by trichloroacetic acid extraction and high pressure liquid chromatography (HPLC) [28]. Flavin nucleotides were extracted with 20% trichloroacetic acid, neutralized, and separated on a Hypersil C-18 reversed phase HPLC column (2.5 mm by 25 mm). Standards were glutathione reductase (contains only £avin adenine dinucleotide (FAD)) and glyoxalate oxidase (contains only £avin mononucleotide (FMN)). For donor speci¢city studies, NADH was replaced by NADPH or H2 in the reaction mixture. Inhibition studies were carried out by adding inhibitors (dissolved in appropriate solvents) to standard reaction mixtures, and then monitoring the reaction. Quinacrine, KCN, and p-mercuribenzenesulfonate were dissolved in water ; rotenone, myxathiazole, hydroxyquinolone-N-oxide (HOQNO), and antimycin were dissolved in acetone. 2.5. Materials and chemicals Gel ¢ltration molecular size standards, ferrozine, enzyme inhibitors, glyoxalate oxidase, and glutathione reductase were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Electrophoresis standards were from Bio-Rad Laboratories (Hercules, CA, USA). Q-Sepharose fast £ow chromatography media were obtained from Pharmacia Amersham Biotech (Piscataway, NJ, USA). All other reagents were of analytical grade. 3. Results 3.1. Isolation, puri¢cation and physical characterization of the membrane-bound Fe3 + reductase Cultures of G. sulfurreducens produce a single Fe3 reductase activity, as determined by non-denaturing PAGE (Fig. 1A). Fe3 reductase activity was found to reside primarily in the membrane fraction, with about 67% of the membrane-associated activity distributed in the outer membrane (Table 1). To isolate Fe3 reductase, crude membranes were solubilized with dodecyl-L-D-maltoside, a non-ionic detergent. The crude solubilized material was then fractionated with ion exchange chromatography, which served to remove lipids and other contaminating material from the preparation. Further puri¢cation was achieved with preparative non-denaturing polyacrylamide
Table 1 Distribution of Fe3 reductase activity in soluble and membrane fractions of G. sulfurreducens Fraction
Total protein recovered (mg)
Total activity recovered (mU)
% Total activity recovereda
Soluble Membranes Membrane fractions Cytoplasmic membrane Outer membrane
131.6 98.1
68.3 392.3
14.8 85.2
2.7 4.3
5.1 10.3
32.9 67.1
a
Activity recovered as a percentage of the total activity recovered in all fractions.
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Fig. 1. Analysis of crude and puri¢ed Fe3 reductase by non-denaturing gel electrophoresis. A: Zymogram analysis of crude membrane protein samples resolved by non-denaturing PAGE and visualized by Fe3 reductase staining. Samples were prepared from cultures grown on either Fe3 (Fe) or fumarate (Fum). 100 Wg crude protein loaded per lane. B: PAGE analysis of puri¢ed Fe3 reductase. The puri¢ed protein was visualized by Coomassie Blue (lane 1), Fe3 reductase activity staining (lane 2), and hemoprotein staining (lane 3). 10 Wg protein loaded per lane. C: Denaturing PAGE of partially puri¢ed Fe3 reductase. Molecular mass marker positions are shown at left. 1.0 Wg protein was resolved and detected with silver (lane 1) or with a heme stain (lane 2). The 90 kDa hemoprotein (HP) is indicated. The ¢gure was processed using Adobe Photoshop 5.0 for PC.
gel electrophoresis. Typical yields were about 5%, with about 1 mg enzyme isolated per 10 g wet weight cells. The highly enriched Fe3 reductase migrated as a single band on ND^PAGE gels (Fig. 1B) and stained for Fe3 reductase activity (Fig. 1B). SDS^PAGE analysis of the puri¢ed activity revealed a heterooligomeric polypeptide composition of at least ¢ve major components, with minor constituents as well (Fig. 1C). The native molecular mass was about 300 kDa as determined by gel ¢ltration under non-dissociating conditions. 3.2. Biochemical properties of the Fe3 + reductase Heme staining of the ND^PAGE gels (Fig. 1B) and SDS^PAGE gels (Fig. 1C) indicates that the Fe3 reductase complex contains a hemoprotein, and spectrophoto-
metric analysis of the puri¢ed enzyme con¢rms the presence of a c-type cytochrome (Fig. 2A). The absorbance maxima of the reduced protein lie at 550, 520, and 413 nm, and absorbance maxima of the oxidized protein are at 409 and 535 nm (Fig. 2A). HPLC analysis of the acid-extracted £avins from the complex demonstrates that the Fe3 reductase contains FAD. Electrochemical analysis shows an oxidation/reduction potential of 3100 mV (vs. standard hydrogen electrode), indicating that the Fe3 reductase complex has a redox potential low enough to reduce all Fe3 forms that G. sulfurreducens can use as an electron acceptor, including synthetic ferrihydrite (E³P = 0 to +200 mV). The puri¢ed activity was speci¢c for NADH as an electron donor ; NADPH and H2 did not serve as electron donors for Fe3 reduction (data not shown). The Complex
Fig. 2. UV-visible spectrophotometric characterization of Fe3 reductase. A: Oxidized (dashed line) and reduced (solid line) spectra of puri¢ed Fe3 reductase at 0.05 mg ml31 . B: UV-visible spectra of Fe3 reductase (0.1 mg ml31 ) reduced with dithionite (solid line), and reoxidized with Fe3 ^NTA (dashed line). C: UV-visible spectra of Fe3 reductase (0.1 mg ml31 ) reduced with dithionite (solid line), and reoxidized with Fe3 ^oxyhydroxide (dashed line).
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Table 2 Sensitivity of puri¢ed Fe3 reductase complex to inhibitors
Fig. 3. Kinetic data for Fe3 reductase with NADH (A) and Fe3 (B). Insets show the resultant double reciprocal plots for each.
I inhibitors, rotenone and myxathiazole, inhibit Fe3 reduction (Table 2), providing evidence for an NADH:menaquinone oxidoreductase component in the Fe3 reductase. Other respiratory inhibitors that act at Complex III (ubiquinone :cytochrome c oxidoreductase) and Complex IV (cytochrome oxidase) did not signi¢cantly reduce rates of Fe3 reduction (Table 2). Quinacrine (a £avin homolog) and p-chloromercuribenzoate (a thiol modifying reagent) inhibit Fe3 reduction by the complex, indicating the participation of £avins and thiol groups in the reaction. Results of kinetic studies are shown in Fig. 3. The plots of V vs. [S] show saturation kinetics in both cases, and double reciprocal plots show linearity, consistent with an enzymatic reaction. The kinetic parameters for the enzyme are KNADH = 31 WM and Vm = 0.25 nmol min31 with rem spect to NADH, and KFe3 = 590 WM and Vm = 0.47 nmol m min31 with respect to Fe3 . The enzyme has a temperature optimum of 45³C and a pH optimum of 7.0, with the activity showing thermal inactivation above 50³C (Fig. 4). The Fe3 reductase activity also has signi¢cant NADH menaquinone oxidoreductase activity, but does not show hydrogenase, ATPase, or fumarate reductase activities (Table 3). In order to determine whether the 90 kDa cytochrome c subunit of the reductase was capable of donating electrons to Fe3 , dithionite was added to the Fe3 reductase in an amount just su¤cient to reduce the cytochrome and yield a typical reduced spectrum. The addition of either Fe3 ^NTA (Fig. 2B) or synthetic Fe3 ^oxyhydroxide (Fig. 2C) resulted in oxidation of the cytochrome, demonstrating that the 90 kDa cytochrome can transfer electrons to either form of Fe3 . Studies with
Inhibitor
Site of action
Concentration (mM)
% Inhibitiona
pCMB Quinacrine Rotenone Myxathiazole KCN HOQNO Antimycin
thiols £avins Complex Complex Complex Complex Complex
5.0 2.0 0.05 0.05 10.0 0.04 0.1
70 59 28 9 2 0 0
I I IV III III
a % Inhibition calculated as the decrease in rate compared to a control reaction with no inhibitor present.
puri¢ed preparations of the 90 kDa cytochrome yield similar results [29]. 4. Discussion 4.1. Fe3 + reductase composition and mechanism for Fe3 + reduction Work by Gaspard et al. [5] demonstrated that the Fe3 reductase activity in G. sulfurreducens was found in the membrane fraction, and distributed primarily in the outer membrane. This is consistent with results presented in the present work, which also demonstrates that Fe3 reductase is membrane associated. There is only one detectable Fe3 reductase activity produced by G. sulfurreducens, based on gel electrophoresis experiments on crude protein preparations. In previous work [5], this activity was gel puri¢ed and subjected to SDS^PAGE analysis. The ¢ndings suggested that the Fe3 reductase was composed of at least eight polypeptides, with three of these proteins being cytochromes. In contrast, the results of our work demonstrate that Fe3 reductase puri¢ed by more stringent methods contains only one cytochrome, and at least ¢ve major polypeptides. Additional experimental evidence suggests that the membrane-bound Fe3 reductase complex is an NADH dehydrogenase coupled to a c-type cytochrome that serves as a terminal reductase. This conclusion is supported by the following lines of evidence : (1) the activity is membrane associated and is a heterooligomer of di¡erent peptides, as is commonly seen for other memTable 3 Enzyme activities associated with the puri¢ed Fe3 reductase activity
Fig. 4. Temperature and pH optima of the puri¢ed Fe3 reductase.
Enzyme
Speci¢c activity (mU min31 mg31 )
Relative activitya
NADH:menadione oxidoreductase Fe3 reductase Hydrogenase Fumarate reductase ATPase
20.7 17.1 none detected none detected none detected
1.2 1.0 0 0 0
a
Represents the ratio of activity to Fe3 reductase activity.
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brane NADH dehydrogenases; (2) the activity is inhibited by Complex I inhibitors and contains FAD; (3) the cytochrome associated with the complex undergoes a reoxidation upon addition of Fe3 compounds, indicating an ability to pass reducing equivalents to Fe3 ; (4) the complex has a redox potential low enough to act as an electron donor to Fe3 compounds. 4.2. Potential physiological role of the Fe complex in G. sulfurreducens
3+
[5]
[6]
[7]
reductase [8]
It is generally considered that microorganisms that couple Fe3 reduction to growth must have membrane-bound Fe3 reductases in order to allow direct contact with extracellular insoluble Fe3 oxides, and to provide a mechanism for generation of a proton-motive force via NADH oxidation [15,30^32]. The Fe3 reductase from G. sulfurreducens appears to be situated in the membrane so that both of these functional requirements are met. It is conceivable, due to its large size and membrane distribution, that this reductase can span both inner and outer membranes, and allow for electron transfer from NADH to Fe^oxyhydroxides. Recent work with Shewanella putrefaciens outer membrane proteins suggests, however, that other Fe3 binding proteins may play a role in dissimilatory Fe3 reduction by Fe3 -reducing organisms [4,33]. The Fe3 reductase activity puri¢ed from G. sulfurreducens could function as a respiratory electron transport complex with or without other Fe3 binding components being present. The Fe3 reductase of G. sulfurreducens described in this paper represents the ¢rst active membrane-bound Fe3 -reducing system puri¢ed from a microorganism which can respire on Fe3 . Acknowledgements
[9]
[10]
[11]
[12]
[13] [14] [15]
[16]
[17]
We thank B. Blunt-Harris for excellent technical assistance, and M. Coppi and J. Lloyd for critical review of the manuscript. This research was funded by NSF Grant DEB 9523932 and DOE Grant DE-FG02-97ER62475.
[18]
[19]
[20]
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