Environ. Sci. Technol. 2010, 44, 834–840
Hydrogen Enhances Nickel Tolerance in the Purple Sulfur Bacterium Thiocapsa roseopersicina OLEG A. ZADVORNYY,† MARK ALLEN,† SUSAN K. BRUMFIELD,‡ ZACK VARPNESS,† ERIC S. BOYD,† NIKOLAY A. ZORIN,§ LARISA SEREBRIAKOVA,§ TREVOR DOUGLAS,† AND J O H N W . P E T E R S * ,† Department of Chemistry and Biochemistry and Plant Sciences and Plant Pathology Department, Montana State University, Bozeman, Montana 59717, and Laboratory of Biochemistry and Biotechnology of Phototrophic Microorganisms, Institute of Basic Biological Problems of the Russian Academy of Sciences, Pushchino, Russia
Received May 29, 2009. Revised manuscript received October 28, 2009. Accepted November 2, 2009.
A common microbial strategy for detoxifying metals involves redox transformation which often results in metal precipitation and/ or immobilization. In the present study, the influence of ionic nickel [Ni(II)] on growth of the purple sulfur bacterium Thiocapsa roseopersicina was investigated. The results suggest that Ni(II) in the bulk medium at micromolar concentrations results in growth inhibition, specifically an increase in the lag phase of growth, a decrease in the specific growth rate, and a decrease in total protein concentration when compared to growth controls containing no added Ni(II). The inhibitory effects of Ni(II) on the growth of T. roseopersicina could be partially overcome by the addition of hydrogen (H2) gas. However, the inhibitory effects of Ni(II) on the growth of T. roseopersicina were not alleviated by H2 in a strain containing deletions in all hydrogenase-encoding genes. Transmission electron micrographs of wild-type T. roseopersicina grown in the presence of Ni(II) and H2 revealed a significantly greater number of dense nanoparticulates associated with the cells when compared to wild-type cells grown in the absence of H2 and hydrogenase mutant strains grown in the presence of H2. X-ray diffraction and vibrating sample magnetometry of the dense nanoparticles indicated the presence of zerovalent Ni, suggesting Ni(II) reduction. Purified T. roseopersicina hyn-encoded hydrogenase catalyzed the formation of zerovalent Ni particles in vitro, suggesting a role for this hydrogenase in Ni(II) reduction in vivo. Collectively, these results suggest a link among H2 metabolism, Ni(II) tolerance, and Ni(II) reduction in T. roseopersicina. * Corresponding author phone: (406) 994-7211; fax: (406) 9945407; e-mail:
[email protected]. † Department of Chemistry and Biochemistry, Montana State University. ‡ Plant Sciences and Plant Pathology Department, Montana State University. § Institute of Basic Biological Problems of the Russian Academy of Sciences. 834
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Introduction Nickel [Ni(II)] has a significant role in biology, not only as a required microelement for growth but also as a toxin (1). As a micronutrient, Ni(II) is utilized in the active sites of metalloenzymes such as [NiFe]-hydrogenase, urease, and carbon monoxide dehydrogenase where it participates in electron transfer reactions (2). In addition, Ni(II) is often present in cofactors, such as the F430 cofactor present in all methanogens (1). Despite the fundamental role of Ni(II) in biology, high concentrations of Ni(II) are toxic. A variety of processes, including alloy production, electroplating baths, steel manufacturing, and the production and disposal of batteries, contribute to elevated concentrations of Ni(II) in the environment (3). Like other heavy metals, Ni(II) inhibits a variety of physiological processes including photosynthesis (4), fermentation (5), and nitrogen fixation (6). Tolerance to Ni(II) has been observed in a number of different species of microorganisms, including Ralstonia metallidurans, Arthrobacter aurescens, Pseudomonas denitrificans, Heliobacter pylori, and Esherichia coli (7, 8). High levels of Ni(II) tolerance, such as that observed in Cupriavidus metallidurans, often depend on the presence of genetic determinants such as the ncc, cnr, and nre operons in the genome or extrachromosomal DNA of the organism in question (9). These genes encode cation efflux systems that function to expel metals from the interior of cells, thereby decreasing the local concentration of Ni(II). For example, strains of R. metallidurans carrying the ncc operon are capable of growth in the presence of bulk medium concentrations of Ni(II) up to 50 mM (10). Similarly, organisms harboring the cnr or nre operon also are capable of growth at Ni(II) concentrations approaching 2.5 mM (9, 11). Among phototrophic bacteria, the nre operon has been shown to be involved in nickel resistance in Synechocystis sp. (12) and Nostoc sp. (8). Importantly, algae and cyanobacteria are reportedly more sensitive to Ni(II) than purple non-sulfur bacteria (13); however, the basis for the enhanced tolerance to Ni(II) ions in purple sulfur bacteria is poorly understood. In addition to efflux detoxification mechanisms, microorganisms absorb heavy metal ions, a characteristic which has been exploited for the bioremediation and detoxification of a variety of metals (14-16). In the case of Ni(II), it has been proposed that absorption involves surface binding and precipitation, resulting in insoluble Ni-containing precipitates. Evidence for precipitation includes the formation of sulfide-containing Ni precipitates on the outer membrane in certain algae (17), while other microbes, such as Citrobacter sp., accumulate nickel in the form of Ni(II)-containing polycrystalline hydrogen uranyl phosphate (18). Direct links between metal reduction and hydrogen (H2) metabolism have been demonstrated previously in organisms suchasDesulfovibriodesulfuricans(19),Lamprobactermodestogalofilus (20), and Desulfomicrobium norvegicum (21). The purple sulfur bacterium Thiocapsa roseopersicina has been shown to reductively precipitate a variety of metal ions including Ni(II), Pd(II), and Pt(II) when grown in the presence of H2 (20). The genome of T. roseopersicina contains three [NiFe]-hydrogenases, each of which have distinct physiological functions in the cell. The hup-encoded and the hynencoded hydrogenases are membrane-associated and are generally involved in H2 uptake in vivo (22). The hyn-encoded hydrogenase has been previously characterized as a supramolecular ring-shaped assembly of ∼576 kDa, containing six hydrogenase molecules (23). The hox-encoded bidirectional hydrogenase is involved in dark fermentative H2 10.1021/es901580n
2010 American Chemical Society
Published on Web 11/23/2009
evolution and light-dependent reactions including H2 uptake when grown in the presence of CO2 and H2 production when grown in the presence of thiosulfate (24). Here, we apply a combination of physiological, genetic, biochemical, and spectroscopic studies to investigate the role of hydrogenases and H2 metabolism in Ni(II) tolerance and precipitation in the purple sulfur bacterium T. roseopersicina.
Materials and Methods Culture Conditions. T. roseopersicina strain BBS and hydrogenase mutant strain GB112131, generously provided by Dr. Gabor Rakhely and Dr. Kornel Kovacs, were grown under anaerobic and phototrophic conditions on modified Pfennig medium (25) containing 2.5 g/L sodium acetate and 2.5 mg/L sodium thiosulfate and without sodium sulfide. Cultures were illuminated with continuous saturating light (80 µE m-2 s-1) at 30 °C. Individual growth experiments were conducted in 120 mL serum bottles containing 70 mL of culture (8 µg of protein/mL). The serum bottles were sealed with a black butyl rubber septum (Geo-Microbial Technologies, Inc.), and the headspace was immediately flushed with anaerobic N2 gas passed over heated (210 °C) copper catalysts. Copper catalysts were routinely regenerated (reduced) by passing 100% H2 gas over the columns. After 12 h of growth and at 2 day intervals thereafter, the gas phase was exchanged with 100% nitrogen (N2), 10% H2 (10% H2/90% N2), or 100% H2. The 10% H2 headspace was included since previous studies indicate that this concentration is sufficient for the stimulation of hydrogenase activity (26). After 24 h of cell growth, Ni(II), in the form of NiCl2 · 6H2O (Fisher Scientific), was added to the culture medium to achieve final concentrations of 6, 20, 30, 40, 60, 140 200, 300, 400, 500, 550, and 600 µM. These concentrations of Ni(II) were used in preliminary characterizations. The concentrations of Ni(II) used for each specific analysis are indicated in the methods and results presented below. Three replicate cultures were grown for each treatment,andgrowthwasmonitoredbytotalproteinaccumulation. Total Cell Protein and Nickel Quantification. At 12 h intervals, 1.2 mL of T. roseopersicina culture was removed and frozen immediately for use in determining the total protein concentration. Total protein was determined in triplicate by combining 0.2 mL of cell suspension with 0.2 mL of 1 N sodium hydroxide and heating at 90 °C for 15 min, followed by neutralization at room temperature (20-21 °C) with 0.04 mL of 6 N HCl. The concentration of protein in these samples was determined using the Bradford assay (27) with bovine serum albumin as the standard. To investigate the fate of Ni(II) during the growth of T. roseopersicina, the concentration of Ni(II) in the bulk medium was determined in cultures grown in the presence of 90 µM Ni(II). This concentration of Ni(II) was chosen since this concentration corresponded to the IC50 for T. roseopersicina grown in the presence of N2 (see below). EPA method 200.8 was used to quantify Ni(II) using an Agilent 7500ce inductively coupled plasma mass spectrometer (28). Triplicate 1.0 mL samples of cell culture were concentrated by centrifugation (13 000 rpm, 25 min), and 0.2 mL of supernatant was prepared for analysis by diluting to a final volume of 6 mL by addition of water containing 1% HNO3 and 0.5% HCl. Growth Parameters. Growth curves were generated by plotting the protein concentration as a function of the incubation time. Protein concentrations determined at different times during growth were expressed as ln(Nt/N0), where Nt is the protein concentration at time t and N0 is the protein concentration at time 0 (29). The lag phase, halftime (as described below), specific growth rate, and maximum protein concentration were used to compare the growth of T. roseopersicina under the various cultivation conditions utilized in this study. The lag phase was extrapolated from polynomial equations describing growth curves by calculating
a third derivative and plotting this value as a function of the incubation time as previously described (29). The maximum protein concentration is defined here as the maximum concentration measured at the onset of the stationary growth phase, and the half-time (t1/2) is the time at which the concentration of protein is half of the maximum value. The derivation dx/dt was extrapolated from polynomial growth curve equations, and this was then used to calculate the specific growth rates using the equation µ ) (1/x)(dx/dt) (30). Three to four replicate growth parameter measurements were made at each sampling interval, and replicate measurements were never found to vary by greater than 5% (data not shown). Curve fitting, as described by Sani et al. (30), yielded r2 > 0.95 in all analyzed data. Data comparisons with p values below 0.05 as determined using a two-tailed Student t test were considered significant. The effect of Ni(II) on the growth of T. roseopersicina was determined by plotting the maximum cell protein in cultures incubated in the presence of different concentrations of Ni(II) in the growth medium. A least-squares fit was used to estimate the Ni(II) concentration, resulting in a 50% inhibition in total cell protein (IC50). Hydrogenase Purification. Cells of T. roseopersicina strain BBS were collected via centrifugation (14000g, 10 min, 4 °C) during logarithmic growth, and the hyn-encoded hydrogenase was purified according to previously published protocols (31) (see the Supporting Information). The purity was confirmed by SDS-PAGE. The activity of the hydrogenase was determined colorometrically as previously described (20) (see the Supporting Information). Nickel Nanoparticle Formation by T. roseopersicina Hydrogenase. Reactions (50 µL) containing 50 mM Tris-HCl (pH 9.0) and 15 mg/mL hyn-encoded hydrogenase were incubated at 8 °C under an atmosphere of H2 in 3 mL vials to investigate nickel nanoparticle formation by purified hydrogenase. The vials were sealed, and 6.3 µL of 200 mM NiCl2 prepared in 50 mM Tris-HCl (pH 9.0) was added incrementally (1 µL at each step, with a final addition of 1.3 µL). Each incremental addition of Ni was allowed to react for 4 days before the next addition with a final incubation of 4 days following the final addition. Dynamic Light Scattering (DLS). DLS measurements were performed using a Brookhaven 90-PALS (Brookhaven Instrument Corp., Holtsville, NY) using a 661 nm diode laser with the instrument set to 90°. The correlation functions were fit using a non-negatively constrained least-squares analysis. Magnetic Characterization of Nickel Nanoparticles in T. roseopersicina Hydrogenase. Vibrating sample magnetometry (VSM) measurements were performed on a Physical Property Measurement System (Quantum Design, San Diego, CA). Protein/mineral composites were isolated from incubations using a centrifugal ultrafiltration device with a 100 000 nominal molecular weight limit cutoff membrane (Microcon, Billerica, MA). Samples were washed with dH2O (18.2 MΩ resistivity) followed by lyophilization, yielding a solid pellet. Approximately 0.3 mg (weighed to an accuracy of 0.01 mg) of lyophilized product was immobilized on a quartz paddle using Duco cement, and this was placed in the VSM instrument. Measurements were made at 5 K, and the field was swept between +6.4 × 106 and -6.4 × 106 A/m while vibrating at a frequency of 40 Hz. The data were analyzed using IgorPro (Wavemetrics, Lake Oswego, OR), and the diamagnetic signal from the large amount of protein was subtracted. Transmission electron microscopy (TEM). Thin cell sections for TEM were prepared according to previously described protocols (32) (see the Supporting Information). Cellular thin sections and purified hydrogenase samples, both unstained and stained with 2% uranyl acetate and Reynold’s VOL. 44, NO. 2, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Influence of the Ni(II) Concentration on the Lag Phase, Maximum Specific Growth Rate, and Total Protein Concentration of T. roseopersicina Cultivated under N2, 10% H2 (10% H2/90% N2), and 100% H2 Atmospheres 10% H2 + 90% N2
N2 Ni(II) concn
lag phase, h
control 6 µM 60 µM
12.4 ( 0.8 14.3 ( 0.7 22.6 ( 1.2
a
µ, h
-1
0.044 ( 0.003 0.039 ( 0.003 0.025 ( 0.001
total cell protein, mg/mL
lag phase, h
1.41 ( 0.04 1.34 ( 0.03 0.95 ( 0.01
10.1 ( 0.6 12.2 ( 0.8 14.3 ( 0.9
a
µ, h
-1
0.053 ( 0.003 0.046 ( 0.002 0.033 ( 0.002
100% H2 total cell protein, mg/mL
lag phase, h
µ,a h-1
total cell protein, mg/mL
1.44 ( 0.06 1.36 ( 0.02 1.15 ( 0.02
7.2 ( 0.5 7.2 ( 0.4 7.1 ( 0.6
0.055 ( 0.004 0.050 ( 0.003 0.041 ( 0.004
1.48 ( 0.03 1.45 ( 0.02 1.19 ( 0.01
a µ ) maximum specific growth rate calculated using total protein concentrations (see the Materials and Methods for a description of the calculation).
lead citrate (33), were viewed with a LEO 912AB transmission electron microscope. X-ray diffraction (XRD) patterns were collected on thin sections with the LEO 912AB TEM instrument, and the d spacings were calculated and compared with those from the powder diffraction file for Ni after calibration of the diffraction camera with a Au standard.
Results Effect of Ni(II) on the Growth of T. roseopersicina in the Absence of H2. The influence of Ni(II) on the growth of T. roseopersicina was evaluated in cultures grown under a N2 headspace to establish baseline growth parameters. The lag phase of T. roseopersicina cultures increased with increasing Ni(II) concentrations in the bulk medium when incubated in the presence of N2 (Table 1). The presence of 6 µM Ni(II) resulted in a slight increase in the lag phase of growth when compared to no Ni(II) controls. In contrast, the presence of 60 µM Ni(II) in the bulk medium increased the lag phase by 10 h and decreased the maximum specific growth rate by 43%, when compared to no Ni(II) controls, indicating that Ni(II) at a concentration of 60 µM inhibits growth in cultures grown under a N2 atmosphere. Significant reductions in growth were observed at Ni(II) concentrations >60 µM (data not shown). Effect of H2 on the Growth of T. roseopersicina in the Absence of Ni(II). Prior to comparing growth in cultures of T. roseopersicina cultivated in the presence of H2 and Ni(II) with those cultivated in the presence of N2 and Ni(II), it was first necessary to compare the effect of H2 on growth in the absence of Ni(II). Growth of T. roseopersicina in the presence of H2 resulted in a 2-5 h shorter lag phase and 20-25% increased specific growth rate when compared to cultures grown in an atmosphere of N2, indicating that T. roseopersicina couples H2 oxidation to growth (Table 1). Importantly, the maximum cell protein in cultures grown in the absence of Ni(II) during the stationary phase was not statistically different (p g 0.05) under the three different cultivation conditions (N2, 10% H2, and 100% H2) (Table 1), indicating that while H2 increases the growth rate, the cells still reach a similar maximum protein concentration. Thus, any difference observed in maximal protein concentration in cultures grown in the presence of Ni(II) and H2 when compared to cultures grown in the presence of Ni(II) and N2 can be attributed to the effect of the headspace gas composition on Ni(II) tolerance. Importantly, the lag phase was significantly decreased in cultures grown in the presence of 10% H2 and 100% H2 when compared to N2. Thus, the lag phase is not a useful parameter in describing the effect of Ni(II) on the growth of T. roseopersicina in the presence or absence of H2. Effect of Ni(II) on the Growth of T. roseopersicina in the Presence of Hydrogen. The effect of Ni(II) on the growth of T. roseopersicina was evaluated in cultures grown in the presence and absence of H2. Growth in the presence of 60 µM Ni(II) and 10% H2 resulted in a 25% increase in the specific growth rate when compared to growth in the presence of 60 836
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µM Ni(II) but in the absence of H2 (Table 1). Importantly, the maximum cell protein in cultures grown in the presence of 60 µM Ni(II) and 10% H2 and 100% H2 was 21% and 26% higher than that in cultures grown in the presence of 60 µM Ni(II) and in the absence of H2, respectively. These observations suggest that the inhibitory effects of Ni(II) on growth of T. roseopersicina can be partially overcome by the presence of H2 in the headspace. IC50 is a useful parameter for comparing the effects of a substance on the growth of a particular organism cultivated under multiple conditions. Thus, the IC50 for Ni(II) in cultures of T. roseopersicina was evaluated in the presence and absence of H2 gas. The IC50 was determined to be 90 ( 3, 115 ( 3, and 122 ( 2 µM Ni(II) under N2, 10% H2, and 100% H2, respectively. The IC50 for Ni(II) in cultures grown in the presence of N2 was significantly lower than that for growth in the presence of 10% H2 (p < 0.01). Similarly, the IC50 for Ni(II) in cultures grown in the presence of 10% H2 was significantly lower than that for growth in the presence of 100% H2 (p ) 0.03). Thus, the IC50 appears to be dependent on the presence of H2 in the gas phase. These observations indicate that while 10% H2 is likely sufficient for the H2dependent mitigation of the inhibitory effects of Ni(II), it may become limiting during the incubations. Effect of Ni(II) on the Growth of T. roseopersicina (∆hynSL ∆hupSL ∆hoxH). Growth of the hydrogenase deletion mutant T. roseopersicina strain GB112132 (∆hynSL ∆hupSL ∆hoxH), which contains deletions in all three hydrogenases, did not exhibit elevated levels of Ni(II) tolerance when cultivated in the presence of H2 when compared to growth of the wild-type cultivated under the same conditions. Addition of 60 µM Ni(II) to the GB112132 mutant cultures grown in the presence of both N2 and 100% H2 resulted in a 75% decrease in the maximum protein concentration (both conditions) when compared to growth of GB112132 mutant cultures grown under the same conditions but in the absence of Ni(II) (Table S1, Supporting Information). Moreover, the growth rates were not significantly different in the mutant strain cultivated under a headspace of N2 or 100% H2 (Table S1). Collectively, these results suggest a role for hydrogenases in H2-mediated Ni(II) tolerance in T. roseopersicina. Ni(II) Removal and Nickel Nanoparticle Formation in T. roseopersicina Cells. Nickel nanoparticle formation was examined in cultures grown in the presence of Ni(II) at concentrations corresponding to their IC50. A qualitative comparison of Ni(II) concentrations in the bulk medium with the growth state of cultures when cultivated under N2, 10% H2, and 100% H2 atmospheres indicates that the removal of Ni(II) from the aqueous phase corresponds with the onset of the lag phase. Following 150 h of incubation in the presence of 90 µM Ni(II) under a N2 headspace, less than 40% of the nickel present at the start of the experiment remained in the bulk medium (Figure 1B). Importantly, the concentration of Ni(II) in the bulk medium of cultures of T. roseopersicina grown in the presence of 10% H2 and 100% H2 was
FIGURE 1. Effect of 90 µM Ni(II) on the growth of T. roseopersicina cultivated under N2, 10% H2, or 100% H2 as determined by the total protein concentration as a function of time (A). Ni(II) concentration in the bulk medium in cultures of T. roseopersicina cultivated under N2, 10% H2, or 100% H2 in the presence of 90 µM Ni(II) (B). The arrow represents the time at which Ni(II) was added to the cultivation medium. significantly lower (p e 0.01 for both conditions) than that in the bulk medium of cultures grown in the presence of N2. For example, 66% and 80% reductions of aqueous-phase Ni(II) were observed in T. roseopersicina cultures grown in the presence of 10% H2 and 100% H2 atmospheres when compared to growth under a N2 atmosphere after ∼60 h of growth (Figure 1B). TEM was used to investigate the fate of Ni(II) in cultures of T. roseopersicina grown in the presence and the absence of H2. TEM images of unstained thin sections of T. roseopersicina grown for 200 h in the presence of 120 µM Ni(II) indicated a significantly greater (p < 0.01) number of dense nanoparticles in cells grown in the presence of 10% H2 (32 ( 4 particles/500 nm2) when compared to cells grown in the absence of H2 (11 ( 3 particles/500 nm2) (Figure 2). In some thin sections, the particles appeared to accumulate within and/or on the membranes of the cells (Figure 2B,D). The observed decrease in the Ni(II) in the medium (Figure 1B) coupled with the observation of the precipitate formation in the presence of H2 (Figure 2) suggested that the enhanced Ni(II) tolerance in T. roseopersicina could be a result of Ni(II) reduction and precipitation by hydrogenase. Moreover, precipitate formation was not observed in the T. roseopersicina mutant strain lacking hydrogenase activity. To investigate the possibility of Ni(II) reduction, X-ray diffraction was performed to determine the molecular structure and composition of the particles. The X-ray diffraction patterns (inset in Figure 2C) of the particles indicated a d spacing within 5% of the theoretical values of Ni metal [Ni(0)] (ICSD no. 64989), suggesting that the particles contained Ni(0) metal formed by the reduction of Ni(II). In Vitro Ni Nanoparticle Formation by T. roseopersicina hyn-Encoded Hydrogenase. To investigate the potential role of hyn-encoded hydrogenase in Ni(II) reductive precipitation in T. roseopersicina, we purified HynSL and investigated nickel nanoparticle formation by the isolated enzyme. Unstained
FIGURE 2. Transmission electron micrographs of T. roseopersicina cells grown for 200 h in the presence of 120 µM Ni(II) under a N2 (A, B) or 10% H2 (C, D) atmosphere. High-resolution transmission electron microscopic images showing black precipitates and cellular localization of black particles (B, D). Inset in (C): electron diffraction ring of black particles corresponding to Ni(0) in the metal form. Scale bar: (A, C) 1000 nm, (B, D) 500 nm.
FIGURE 3. TEM analysis of Ni nanoparticles. Unstained TEM image of Ni nanoparticles (A). Inset: electron diffraction results, average diameter of 2.6 ( 0.6 nm. Stained TEM image of Ni nanoparticles stained with 2% uranyl acetate (B). The scale bar for both images is 50 nm.
TEM images of purified hyn-encoded hydrogenase incubated in the presence of Ni(II) and H2 revealed the presence of nanoparticles that resembled those produced by intact T. roseopersicina cells. The nanoparticles produced by HynSL contained electron-dense cores with an average diameter of 2.6 ( 0.6 nm (Figure 3A). A comparison of X-ray diffraction d spacings for the electron-dense cores with d spacings for Ni(0) metal standards suggested that the particles contained Ni(0) (Figure 3A, inset; Table S1, Supporting Information). Interestingly, halos with diameters of ∼6, ∼8, and ∼11 nm were observed surrounding the Ni nanoparticle. Recent cryoEM reconstructions of HynSL from T. roseopersicina indicate that the protein complex is on the order of 8-10 nm in diameter (23), suggesting that the 8 and 11 nm halos may represent HynSL protein complexes with a Ni nanoparticle in the core. DLS indicated that the size of the hydrogenase ring structure was concentration-dependent (Figure S1, Supporting Information). At high protein concentrations (>15 mg/mL), the hydrogenase is in the 11 nm ring conformation, whereas, at lower concentrations (1 mg/mL), the ∼6 nm protein complex is preferentially formed. In the VOL. 44, NO. 2, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. VSM characterization of Ni nanoparticles. Raw VSM measurement of Ni nanoparticles (A). VSM measurement of Ni nanoparticles after subtraction of the diamagnetic signal at high field due to the presence of protein (B). uranyl acetate-stained TEM analysis of Ni nanoparticles (Figure 3B), all of the sizes of the protein complexes are present. Hydrogenase-synthesized Ni nanoparticles were further characterized by VSM. The results indicate that nickel nanoparticles have a typical superparamagnetic signal, similar to Ni nanoparticles prepared using dendrimer templates (34). The saturation magnetic moment (Ms) of 1 emu/g of the studied material was determined at 5 K (Figure 4). High protein to Ni ratios may also account for the high diamagnetic signal that had to be subtracted (Figure 4A). In the corrected loops, there was no apparent coercive field with any measurable hysteresis (Figure 4B). Thus, the presented results confirm the formation of metallic Ni nanoparticles by stable T. roseopersicina hydrogenase in the presence of H2.
Discussion The data presented here indicate that Ni(II) ions at concentrations as low as 6 µM inhibit growth of T. roseopersicina cells when grown in the presence of N2. These results are consistent with previous studies which have documented the inhibitory effects of Ni(II) in a variety of phototrophic bacteria, including the purple non-sulfur bacteria Rhodobacter photometricum (35) and Rhodobacter sphaeroides (36), the cyanobacterium Anacystis nidulans (37), and the green alga Scenedesmus acutus f. alternans strain B4 (38). Each of these strains exhibited markedly different tolerances to Ni(II). For example, whereas R. photometricum and R. sphaeroides grow in the presence of >400 µM Ni(II), growth in the cyanobacterium A. nidulans was completely inhibited in the presence of 170 µM Ni(II). While some of the observed differences in Ni(II) tolerance in the aforementioned organisms were likely due to differences in Ni(II) bioavailability 838
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due to the composition of the various growth media and/or the mechanism by which growth was quantified, it is unlikely that the differences in Ni(II) tolerance can be attributed solely to these variables, especially considering the numerous mechanisms which organisms utilize to detoxify their local environment of metals. In the present study, H2 was shown to significantly reduce the inhibitory effects of Ni(II) in cultures of T. roseopersicina. The removal of Ni(II) from the bulk medium was dependent on the presence of cells, suggesting that abiotic precipitation reactions were negligible in conferring tolerance to Ni(II). Moreover, the rate of Ni(II) removal from the bulk medium increased with increasing concentrations of H2 in the headspace, suggesting that metals were actively being removed from solution by a H2-dependent physiological process. Previous studies indicate that D. desulfuricans cells containing hydrogenase are capable of reductively precipitating Pd(II) to Pd(0) using H2 as the electron donor (19). TEM examination indicated that cells of T. roseopersicina accumulated a significantly greater number of black nanoparticles when grown in the presence of H2 and Ni(II) when compared to N2 and Ni(II). These nanoparticles were shown to contain Ni metal using XRD, suggesting Ni(II) reduction and precipitation. Although the presence of H2 resulted in an increase in Ni(II) tolerance and precipitate formation, some level of tolerance and precipitate formation are observed even when the cultures are grown under a N2 gas phase (Figure 2). It has been previously shown that, in addition to H2, T. roseopersicina can utilize sulfur compounds as donors of electrons for metabolic processes (39), and these, along with other undefined exogenous electron donors, may also be able to reduce Ni(II) ions. The enhanced tolerance to Ni(II) in wild-type cultures of T. roseopersicina grown in the presence of H2 was not observed in a mutant strain of T. roseopersicina lacking functional hydrogenase when grown in the presence of H2, suggesting a role for hydrogenases in the enhanced tolerance to Ni(II) during growth in the presence of H2. Previously, it was reported that the stable hydrogenases of T. roseopersicina and L. modestohalophilus are involved in Ni(II), Pd(II), and Pt(II) reduction (20) and that the cellular localization of dark precipitates generally corresponds to the location of hydrogenase (40). Importantly, the Ni(II) nanoprecipitates observed in the present study appeared to be localized near the membrane of the cells, which corresponds to the cellular location of the hup- and hyn-encoded hydrogenases that function in H2 uptake in vivo. The stable hyn-encoded hydrogenase is capable of directly reducing Ni(II) in vitro, resulting in nanoparticulate Ni(0), suggesting this as a potential mechanism by which Ni(II) is reductively precipitated in vivo. Investigation of the magnetic properties of nanoparticles synthesized by hyn-encoded hydrogenase indicates that the magnetic signal is similar to the nickel nanoparticles formed in vivo and to nickel nanoparticles obtained through reaction with dendrimer polymers (34). Numerous studies have shown that biological polymers can serve as templates for the synthesis of inorganic nanomaterials (41, 42). For example, alginate has been used to produce various sizes and structures of Co, Ni, and CoNi nanoparticles with different magnetic properties (43). Moreover, nickel nanoparticles have been shown to be formed by apoferritin protein cages (44), and DNA-templated nanofabrication has been used to form nickel nanowires on the surface of DNA and protein assemblies (45). The evidence presented here indicates that catalytic proteins, such as HynSL, can also serve as templates for nanoparticle synthesis. Presumably, the concentrations of Ni(II) and H2 are also important variables in controlling the synthesis rate and structure of nanoparticles in vitro.
In conclusion, T. roseopersicina cells grown in the presence of H2 demonstrate a significant increase in Ni(II) tolerance indicated by a H2-dependent increase in the specific growth rate, an increase in the maximum protein concentration, and an increase in IC50 for Ni(II) when Ni(II) is present in the growth media at inhibitory concentrations. A hydrogenase-deficient deletion strain of T. roseopersicina did not exhibit enhanced tolerance to Ni(II) when grown in the presence of H2 when compared to N2, implicating a role for hydrogenases and H2 metabolism in Ni(II) tolerance. The accumulation of Ni(0) nanoparticles is greater in cells cultivated in the presence of H2 when compared to N2, suggesting a link between H2 metabolism and Ni(II) reduction. In the presence of H2, purified hyn-encoded hydrogenase can reduce Ni(II) to Ni(0) in vitro, resulting in the formation of Ni(0) nanoparticles. Collectively these results provide a direct link between H2 oxidation and Ni(II) tolerance in the purple sulfur bacterium T. roseopersicina and implicate a role for hydrogenases in Ni(II) tolerance and Ni(II) reductive precipitation in a model bacterium. Further, these results point to the efficacy of the hyn-encoded hydrogenase as a unique template for Ni nanoparticle synthesis.
Acknowledgments This work is supported by a grant from the Department of Energy (DE-FG36-06GO86060) to J.W.P. and T.D. E.S.B. was supported by a NASA Astrobiology Institute Postdoctoral Fellowship. We acknowledge Dr. Robin Gerlach and Howard Christiansen for help with the ICP-MS analysis and Dr. Gabor Rakhely and Dr. Kornel Kovacs for T. roseopersicina hydrogenase mutant strain GB112131. We gratefully acknowledge Dr. Brent Peyton, Dr. Shane Ruebush, David Mulder, and John Heilman, M.A., for helpful discussion and the critical reading of the manuscript.
Supporting Information Available Supplemental methods used to purify and quantify the activity of the hyn-encoded hydrogenase and TEM analysis, supplemental Table 1 presenting growth characteristics of the T. roseopersicina hydrogenase mutant strain when grown in the presence and absence of H2, supplemental Table 2 indicating the measured d spacing values of the Ni nanoparticles, and supplemental Figure 1 exhibiting the size distribution of hydrogenase supermolecular ring structures as analyzed by DLS. This material is available free of charge via the Internet at http://pubs.acs.org.
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