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Microbial Ecology

Selenate-Dependent Anaerobic Arsenite Oxidation by a Bacterium from Mono Lake, California

Jenny C. Fisher, James T. Hollibaugh
Jenny C. Fisher
Department of Marine Sciences, University of Georgia, Athens, Georgia 30602-3636
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James T. Hollibaugh
Department of Marine Sciences, University of Georgia, Athens, Georgia 30602-3636
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  • For correspondence: aquadoc@uga.edu
DOI: 10.1128/AEM.01995-07
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ABSTRACT

Arsenate was produced when anoxic Mono Lake water samples were amended with arsenite and either selenate or nitrate. Arsenite oxidation did not occur in killed control samples or live samples with no added terminal electron acceptor. Potential rates of anaerobic arsenite oxidation with selenate were comparable to those with nitrate (∼12 to 15 μmol·liter−1 h−1). A pure culture capable of selenate-dependent anaerobic arsenite oxidation (strain ML-SRAO) was isolated from Mono Lake water into a defined salts medium with selenate, arsenite, and yeast extract. This strain does not grow chemoautotrophically, but it catalyzes the oxidation of arsenite during growth on an organic carbon source with selenate. No arsenate was produced in pure cultures amended with arsenite and nitrate or oxygen, indicating that the process is selenate dependent. Experiments with washed cells in mineral medium demonstrated that the oxidation of arsenite is tightly coupled to the reduction of selenate. Strain ML-SRAO grows optimally on lactate with selenate or arsenate as the electron acceptor. The amino acid sequences deduced from the respiratory arsenate reductase gene (arrA) from strain ML-SRAO are highly similar (89 to 94%) to those from two previously isolated Mono Lake arsenate reducers. The 16S rRNA gene sequence of strain ML-SRAO places it within the Bacillus RNA group 6 of gram-positive bacteria having low G+C content.

Arsenic (As) and selenium (Se) are naturally occurring metalloids, typically present in aqueous environments as the dissolved oxyanions arsenite [As(III)] or arsenate [As(V)] (4) and selenite [Se(IV)] or selenate [Se(VI)] (45), respectively. Global geochemical cycling of arsenic and selenium occurs by tectonic uplift, sedimentation (10), volcanic activity (6, 30), rock weathering (6), and geothermal hydrologic inputs (44). However, local cycling and transformations occur primarily by biological oxidation/reduction, assimilation, and volatilization (38, 40).

Many Bacteria and some Archaea can reduce selenate and/or selenite either as a detoxification strategy or for energy generation via a dissimilatory pathway (40). The ability to respire arsenate is also widely distributed among prokaryotes and is found in gram-positive bacteria having low G+C content, several divisions of the Proteobacteria, and even Archaea (28). Many prokaryotes can also reduce arsenate as a detoxification mechanism using the well-characterized ars system of arsenate reductase genes (24, 39). Other organisms can obtain energy for chemolithoautotrophic growth by oxidizing arsenite (27, 34).

Several organisms capable of transforming selenium and arsenic have been isolated from Mono Lake, a hypersaline, alkaline lake in eastern California (12, 27, 41). One of these organisms, Alkalilimnicola ehrlichii strain MLHE-1, oxidizes arsenite anaerobically using nitrate as an electron acceptor (27). This metabolism was proposed to be the source of the spikes of arsenate observed in anaerobic, sulfidic water in which arsenite and thioarsenic compounds were abundant (14, 26). Nitrate, present in Mono Lake at low concentrations (22), could potentially serve as an oxidant for an equivalent or greater amount of arsenite, depending on whether it was reduced to nitrite or N2. However, another compound may also serve to oxidize arsenite. A previous Mono Lake study found that arsenate reduction in anoxic waters was inhibited by the addition of selenate (13), which is also present in the lake at low concentrations (<1 μM) (J. T. Hollibaugh, unpublished data). The observed inhibition could be due to preferential use of selenate over arsenate or to the oxidation of arsenite (produced from arsenate reduction) by selenate. The E0′ of the selenate/selenite couple (+440 mV) (5) is higher than that of arsenate/arsenite (+60 mV) (20), which suggests that oxidation of arsenite by selenate is thermodynamically favorable. In this study we examined the potential for anaerobic arsenite oxidation using selenate as a terminal electron acceptor. Experiments showed that this pathway was active in enriched lake water samples and could contribute to the observed arsenate disequilibrium in anoxic lake waters. A bacterium that could oxidize arsenite using selenate as the electron acceptor was isolated from these experiments as well.

MATERIALS AND METHODS

Sample collection.Samples were collected from station 6 of Mono Lake, CA (37°57.822′N, 119°01.305′W) in April and August of 2004. Water samples (from a depth of 28 to 35 m; anoxic) were collected and stored as previously described (14).

Mono Lake water incubations.All enrichments were set up in an anaerobic chamber (<2% H2 and 98% N2 atmosphere). Anaerobic conditions were maintained throughout the experiments. Whole (live) lake water samples were distributed into serum vials, capped with butyl rubber stoppers, and crimp sealed. Filter-sterilized (0.22-μm pore size) As(III) as NaAsO2, and Se(VI) as Na2SeO4, or nitrate as NaNO3 were added to final concentrations of 2 mM or 5 mM. Controls were 0.22-μm-pore-size filtered lake water with buffered formalin (1% [vol/vol] final concentration). Whole lake water was also diluted 1:25 with artificial Mono Lake water (AMLW) with no organic carbon source. These treatments were amended with 1 mM arsenite and selenate.

Isolation and growth of strain ML-SRAO.Enrichment cultures from lake water were transferred to an alkaline (150 mM carbonate; pH ∼9) and saline (∼80 g/liter) AMLW medium that was modified from the medium used previously for the isolation of another Mono Lake bacterium (12). The AMLW medium contained (in g/liter) the following: NaCl (60), (NH4)2SO4 (0.5), MgSO4·7H2O (0.25), KCl (1.7), Na2BO4 (2), Na2SO4 (16.5), KH2PO4 (0.25), and KH2PO4 (0.5). The medium also contained vitamins (25) and trace metals (46). The medium was bubbled with nitrogen and transferred to an anaerobic chamber. Aliquots were dispensed into serum bottles, amended with 3 to 5 mM arsenite and selenate, and sealed with butyl rubber stoppers and crimp seals. Enrichments were subcultured into fresh medium every 4 to 8 weeks and were monitored periodically for the production of arsenate. The medium initially contained no added organic carbon, but later a small amount of yeast extract (0.01%, wt/vol) was added to increase biomass and growth rates. Serial dilutions were made after >6 months of transfers. Attempts to grow the isolate on solid medium were not initially successful, but serial dilution yielded a pure culture. The purity of the culture was confirmed by its uniform cell morphology under an epifluorescence microscope, denaturing gradient gel electrophoresis analysis of 16S rRNA genes in the culture, and an unambiguous, nearly full-length 16S rRNA gene sequence obtained directly from DNA extracted from the culture. Growth curves for ML-SRAO were measured with 10 mM lactate as an electron donor and carbon source and 10 mM arsenate or selenate as a terminal electron acceptor. Cells were enumerated by direct counts using epifluorescence microscopy after staining with DAPI (4′,6′-diamidino-2-phenylindole) (29).

Coupled arsenite oxidation/selenate reduction experiment.Anaerobic arsenite oxidation and selenate reduction rates were determined for cultures amended with 5 mM selenate as the electron acceptor and 5 mM arsenite, 5 mM lactate, or 2.5 mM arsenite and lactate as the electron donors. An inoculum of selenate- and lactate-grown cells from a pure culture of strain ML-SRAO was added to a final cell concentration of ∼107 cells per ml.

Washed-cell experiments.Strain ML-SRAO was grown to a density of ∼108 cells per ml on lactate and selenate. Cells were centrifuged (15 min at 5,000 × g at 4°C) and resuspended twice with fresh mineral medium to remove any residual organic carbon. Selenate and arsenite (each, 3 mM) were added to three replicate treatments to initiate the reaction. The control treatment had only selenate (3 mM). The final cell densities for the treatments and controls were ∼107 cells per ml.

Chemical analyses.Oxygen was measured in lake water with a YSI oxygen meter equipped with a Clark-type electrode. Arsenate was measured by the molybdate blue method on a spectrophotometer for the experiment with whole lake water (18). Samples from the lake water dilution enrichment experiment in AMLW were frozen for analysis of arsenic and selenium species by a modified method using ion chromatography-inductively coupled plasma mass spectrometry (Dionex IC and Perkin Elmer Elan 6000 ICP-MS) (17). Samples were diluted with anoxic Milli-Q water and were filtered (pore size, 0.22 μm) prior to analysis. Selenium and arsenic species were separated with a Dionex AS16 anion exchange column and eluted by a step gradient (20 to 50 mM tetramethylammonium hydroxide) at a flow rate of 1 to 1.5 ml min−1. Arsenate, arsenite, and selenite in ML-SRAO culture experiments were analyzed using high-performance liquid chromatography as previously described (8, 13). Samples were analyzed for total dissolved Se at the University of Georgia Chemical Analysis Lab in Athens, GA, and selenate concentrations were calculated as Se(total) minus selenite.

Molecular analysis.Environmental water samples, enrichment cultures, and isolate cultures were filtered onto Sterivex or Millipore (pore size, 0.22 μm) nitrocellulose filters. DNA was extracted from cells collected on the filters using either a phenol-chloroform method (7) or a MoBio soil extraction kit according to the manufacturer's instructions (MoBio Laboratories, Inc., Carlsbad, CA). PCR was performed on the extracted DNA to amplify the 16S rRNA gene as previously described using universal primers 27F and 1492R (3). PCR products of the correct size (∼1,500 bp) were excised from a 1.5% agarose gel and purified with a Qiaquik gel extraction kit (Qiagen, Valencia, CA). The amplicon was sequenced directly using a BigDye Terminator sequencing reaction kit (Applied Biosystems, Foster City, CA). All nucleotide sequences were determined at the Molecular Genetics Instrumentation Facility at the University of Georgia (Athens, GA). The 16S rRNA gene sequence was compared to others in the GenBank database using BLAST (2) and was aligned with similar sequences using Clustal W (43). A phylogenetic tree and bootstrap values (1,000 replicates) based on 16S rRNA gene sequences were generated using Jukes-Cantor evolutionary distances and the neighbor-joining method in the MEGA4 program (42).

A portion of the arrA (respiratory arsenate reductase) gene of strain ML-SRAO was also amplified (15) using published primers (23). The amplicon (∼112 bp) was cloned using a TOPO 2.1 kit, purified with a Qiagen plasmid prep kit, prepared for sequencing as previously described (21), and sequenced at the Molecular Genetics Instrumentation Facility. Primer walking was performed to obtain a larger portion (∼600 bp) of the gene. The full-length arrA sequences of Bacillus arseniciselenatis and Bacillus selenitireducens were aligned in BioEdit (9), and primers were designed from conserved regions in these two sequences to be used as the forward primer (arrAf235, 5′-ATACGATTGCCGATAA-3′). An internal sequence from the arrA gene of ML-SRAO was used as the reverse primer (arrArw2, 5′-TATCTTTTAGCGCTAAATTC-3′). PCR was performed in 25-μl reaction mixtures with puRe Taq Ready-To-GO PCR Beads (Amersham Biosciences, Buckinghamshire, United Kingdom) and a 0.4 μM concentration of each primer. Samples were denatured at 95°C for 10 min, followed by 40 cycles of denaturing at 95°C for 15 s, annealing at 50°C for 45 s, and extension at 72°C for 1 min. The cycles were followed by a final extension at 72°C for 15 min. Bands of the expected size were excised from a 1% agarose gel, purified, cloned, and sequenced as described above. A neighbor-joining tree and bootstrap values (1,000 replicates) based on inferred amino acid sequences were computed using the maximum composite likelihood method in MEGA4 (42).

Electron microscopy.Samples were fixed with 2% glutaraldehyde in 0.1 M phosphate-buffered saline and 1% osmium tetraoxide (OsO4) in 0.1 M phosphate-buffered saline. After dehydration with ethanol, samples were dried with liquid CO2 in a critical point drier and sputter coated with gold. Electron micrographs were taken with a LEO model 982 field emission scanning electron microscope.

Nucleotide sequence accession numbers.The 16S rRNA gene and arrA sequences of strain ML-SRAO were deposited in GenBank under the accession numbers EU186648 and EU188649.

RESULTS

Lake water enrichment experiment.Arsenate was produced after an initial lag period in live lake water treatments amended with arsenite and either selenate or nitrate (Fig. 1). Little or no arsenate was produced in killed control samples with either electron acceptor or in live controls with no added electron acceptor. Potential arsenite oxidation rates were 12.3 ± 1.3 μmol liter−1 h−1 with selenate as the terminal electron acceptor and 14.5 ± 2.8 μmol liter−1 h−1 for nitrate. Final arsenate concentrations (2.1 ± 0.2 mM) in treatments with selenate were equal to the amount of arsenite added. Arsenite oxidation occurred after a slightly longer lag time (∼96 h) in the nitrate treatment. Most of the added arsenite (5 mM) was oxidized to arsenate (3.8 ± 0.9 mM).

FIG. 1.
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FIG. 1.

Arsenate production in water collected from Mono Lake amended with 2 mM arsenite and selenate ○, 5 mM arsenite and nitrate ▾, and filter-sterilized controls •.

Lake water dilution experiment.A lag time of ∼3 weeks occurred when lake water samples were diluted (1:25) into mineral medium (AMLW) to assess the potential for autotrophic arsenite oxidation with selenate (Fig. 2). Decreases in selenate and arsenite were mirrored by increases in arsenate and selenite (Fig. 2), suggesting tight coupling between arsenite oxidation and selenate reduction in this medium. Arsenite oxidation rates were much slower in AMLW medium than in lake water samples.

FIG. 2.
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FIG. 2.

Arsenic and selenium speciation in Mono Lake water diluted 1:25 with mineral medium and amended with 1 mM arsenite and selenate. Symbols: •, arsenite; ○, arsenate; ▾, selenite; ▵, selenate.

Isolation and growth of strain ML-SRAO.A selenate-reducing, arsenite-oxidizing culture was established after several months of repeated transfers in artificial lake water. Doubling times for cells in the enrichment culture were extremely long (≥1 week), and cell densities were low (optical density at 600 nm of <0.01) when grown in mineral medium with selenate, arsenite, and trace amounts of yeast extract (0.01%, wt/vol). Growth and arsenite oxidation rates were more rapid when cells were grown with arsenite, excess selenate, and a low concentration (<1 mM) of sucrose. Serial dilution was performed, and strain ML-SRAO was isolated from the highest dilution exhibiting selenate-driven anaerobic arsenite oxidation (10−7 cells per ml). The purity of the culture was confirmed by sequencing the 16S rRNA gene directly from DNA extracted from the culture, by denaturing gradient gel electrophoresis analysis, and by observation of uniform cell morphology by epifluorescence and phase-contrast microscopy.

Strain ML-SRAO is a gram-positive rod (∼0.5 μm wide and ∼4 to 6 μm in length) (Fig. 3) that catalyzes a novel, selenate-dependent anaerobic arsenite oxidation. This organism cannot grow autotrophically on these substrates; it requires an organic carbon source. ML-SRAO can also grow using lactate or sucrose as a carbon source and electron donor with selenate or arsenate as the terminal electron acceptor. Cells grown on lactate and selenate or arsenate reached densities of >108 cells per ml (optical density at 600 nm of >0.100) (Fig. 4). Cells grew to the highest densities (∼109 cells per ml) and had shorter doubling times for growth (∼7.2 h) on selenate and lactate (Fig. 4A) than on arsenate and lactate (∼12 h) (Fig. 4B).

FIG. 3.
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FIG. 3.

Scanning electron micrograph of strain ML-SRAO. Scale bar, 5 μm.

FIG. 4.
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FIG. 4.

(A) Growth of strain ML-SRAO on selenate and lactate. Symbols: ○, selenite; • selenate; ▴, cell density. (B) Growth of strain ML-SRAO on arsenate and lactate. Symbols: • arsenate; ○, arsenite; ▴, cell density. Symbols represent the mean of three replicates; error bars represent ±1 standard deviation.

Coupled arsenite oxidation/selenate reduction.We examined the ability of strain ML-SRAO to grow in the presence of arsenite to determine if energy could be conserved during the arsenite oxidation process. Arsenate and selenite both increased linearly in the 5 mM selenate and arsenite treatment (Fig. 5A) as arsenite and selenate concentrations decreased. Most, but not all, of the added arsenite (∼75%) and selenate (∼80%) was transformed during the course of the experiment. Selenite production was compared with treatments amended with 5 mM selenate, 2.5 mM arsenite, and 2.5 mM lactate (Fig. 5B) or 5 mM selenate and 5 mM lactate (Fig. 5B). Selenate was completely converted to selenite only in the treatments that also contained lactate. The selenite production rate was linear in the selenate-arsenite and selenate-arsenite-lactate treatments, in contrast to increasing rates of selenite production observed in the selenate-lactate treatment. Cell counts taken at the end of this experiment showed that the selenate-lactate treatment had the highest cell density (∼108 cells per ml); cultures grown with selenate-arsenite-lactate exhibited some growth (7 × 107 cells per ml); and cultures grown with selenate-arsenite had little or no growth (∼107 cells per ml).

FIG. 5.
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FIG. 5.

(A) Arsenate (•) and selenite (○) production and arsenite (▾) and selenate (▵) consumption by strain ML-SRAO in AMLW amended with 5 mM selenate and arsenite. (B) Selenite produced by strain ML-SRAO in AMLW amended with 5 mM arsenite and selenate (○); 5 mM selenate, 2.5 mM lactate, and 2.5 mM arsenite (▾); or 5 mM selenate and lactate (•). Symbols represent the mean of three replicates; error bars represent ±1 standard deviation.

Several control experiments were performed to determine if this reaction was of a chemical nature or if an intermediate metabolite that formed during selenate reduction could catalyze the oxidation of arsenite. No arsenate was produced in filtered selenate-reducing culture medium amended with arsenite, cell-free medium with selenate and arsenite, or culture medium with heat-killed cells (data not shown). Live treatments produced no arsenate unless selenate was added as well. All control experiments indicated that live, active cells were required for this process to occur.

Washed-cell experiment.Washed cell suspensions of strain ML-SRAO were examined to determine if the coupled arsenite oxidation/selenate reduction process was dependent on the presence of dissolved organic carbon as well. The washed-cell suspensions achieved partial (∼50%) transformation of the arsenite and selenate added. Previous experiments (data not shown) showed that no arsenite was oxidized if selenate was not present or if cells were absent or heat killed, and selenate was not reduced if arsenite was not present. Arsenate and selenite production were closely coupled (Fig. 6) and had close to, but significantly different from, a 1:1 relationship (slope, 0.93; r2 = 0.9998; P < 0.0001) based on model II-type correlation and regression. No growth was observed under these experimental conditions, but cell density did not decrease.

FIG. 6.
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FIG. 6.

Arsenate production versus selenite production (•) in washed-cell experiments. The solid line (slope, 0.93; r2 = 0.9998, n = 18, and P < 0.0001) shows the relationship of these two variables. The dashed line represents a 1:1 slope.

Phylogenetic analysis.Strain ML-SRAO belongs to the Bacillus RNA group 6 of gram-positive bacteria having low G+C content based on BLAST analysis of its 16S rRNA gene. Alignments using Clustal W revealed that it was clearly different from, but closely related to, Mono Lake isolates B. arseniciselenatis and B. selenitireducens (Fig. 7). The 16S rRNA gene sequence of ML-SRAO was ≤95% similar to any of the sequences previously recovered from Mono Lake (15, 16) and only 96% similar to the nearest cultured relative, Bacillus agaradhaerens.

FIG. 7.
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FIG. 7.

Phylogenetic relationships among cultured members of the genus Bacillus, environmental clones from Mono Lake (*), and strain ML-SRAO based on nearly full-length (>1,400 bp) 16S rRNA gene sequences. The neighbor-joining tree is rooted with the sequence from Paenibacillus polymyxa. Bootstrap values (numbers at nodes) were calculated from 1,000 iterations; values less than 50% are not shown. Scale bar indicates 0.01 nucleotide substitutions per position. GenBank accession numbers for the sequences are given in parentheses.

Strain ML-SRAO possesses a respiratory arsenate reductase (arrA), a portion of which (>600 base pairs) was amplified, translated to amino acids, and compared to sequences from the GenBank. The inferred amino acid sequences from the arrA of strain ML-SRAO were highly similar to those of B. arseniciselenatis (94%) and B. selenitireducens (89%) (Fig. 8) based on a BLAST analysis.

FIG. 8.
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FIG. 8.

Neighbor-joining tree based ∼200 deduced amino acid sequences based on the respiratory arsenate reductase gene (arrA) of strain ML-SRAO and other cultured arsenate reducing bacteria. GenBank accession numbers are given in parentheses. Evolutionary distances were computed using the maximum composite likelihood method. The tree is rooted with the putative deduced ArrA sequence from A. ehrlichii MLHE-1 (NC_008340). Bootstrap values (>50% only; based on 1,000 iterations) are shown at the nodes of the tree. The scale bar indicates 0.05 nucleotide substitutions per position.

DISCUSSION

Arsenite oxidation rates in the whole lake water treatments with either selenate or nitrate as the terminal electron acceptor (Fig. 1) were similar to rates observed previously in Mono Lake enrichment cultures, which also had a lag time of ∼100 h before oxidation began (27). The filtered controls produced almost no arsenate (Fig. 1), confirming the biological nature of the arsenite oxidation for both terminal electron acceptors. The much slower arsenite oxidation rates observed in the AMLW experiment inoculated with lake water (Fig. 2) were most likely due to the significant dilution factor.

The ability to oxidize arsenite has been reported for many prokaryotes (40) in recent years. Some of these organisms use oxygen as an electron acceptor either for the detoxification of arsenite (33) or to conserve energy for growth (31, 35). Others can grow with arsenite using nitrate as the terminal electron acceptor, reducing it to nitrite (27) or completely denitrifying to N2 (32). Strain ML-SRAO cannot use oxygen or nitrate to oxidize arsenite but instead couples arsenite oxidation with selenate reduction.

The reaction appears to occur via a two-electron transfer from selenate to arsenite, producing selenite and arsenate: H2AsO3− + HSeO4− → HAsO42− + SeO32− + 2H+ (ΔG0′ = −94.5 kJ/mol).

Arsenite oxidation with selenate is thermodynamically more favorable than oxidation with nitrate (ΔG0′ = −87.2 kJ/mol), which can support autotrophic growth, e.g., A. ehrlichii (27). The As(III)/Se(VI) redox couple could provide enough energy for chemolithoautotrophic growth; however, strain ML-SRAO does not grow without a source of organic carbon. Approximately 0.93 mole of arsenate was produced for every mole of selenite in our experiments with washed cells free of added dissolved organic carbon (Fig. 6). This is very close to the theoretical 1:1 ratio predicted by the equation above yet statistically different.

Strain ML-SRAO is able to oxidize arsenite rapidly in the presence of selenate, with or without the addition of a dissolved organic carbon source (Fig. 5 and 6). However, complete reduction of selenate occurred only when lactate was also present. Even in washed-cell experiments, the production of selenite was greater than that of arsenate, suggesting the simultaneous oxidation of some endogenous carbon substrate (Fig. 6). The benefit of arsenite oxidation and the mechanism by which it occurs are therefore uncertain. The organism does not use the reaction for growth energy, as cell counts were much lower in treatments with arsenite only and slightly lower in treatments with lactate and arsenite compared to growth with selenate and lactate. Additionally, the process is unlikely to be a detoxification pathway, as strain ML-SRAO was able to grow in the presence of high arsenite concentrations produced during arsenate reduction (Fig. 4B).

The question remains as to how strain ML-SRAO is able to couple arsenite oxidation and selenate reduction and why it carries out this reaction. ML-SRAO could not oxidize arsenite in the presence of nitrate or oxygen (data not shown), so the process appears to be selenate dependent. We were unable to amplify an arsenite oxidase gene from ML-SRAO using several different primer sets, but we did successfully sequence a portion of the arrA gene (Fig. 8). Many of the characterized anaerobic respiratory enzymes (e.g., selenate reductase, nitrate reductase, and arsenate reductase) appear to be substrate specific (19, 36, 37), while others may serve more than one function in vitro and possibly in vivo (1). The purified arsenate reductase enzyme from B. selenitireducens can reduce selenate, selenite, and arsenite in the presence of an artificial electron donor although the organism does not respire selenate or arsenite in vivo (1). It has been proposed that A. ehrlichii (MLHE-1), which oxidizes arsenite but does not have arsenite oxidase genes in its genome (11), may express one or both of its two arsenate reductase-like genes (arrA) when oxidizing arsenite (11). Strain ML-SRAO may be able to use its ArrA for both the reduction and oxidation of arsenic, but our studies have yet to confirm this.

Strain ML-SRAO is related to members of the Bacillus genus based on its 16S rRNA gene sequence (Fig. 7) and shares several of the metabolic capabilities of Bacillus species from Mono Lake. B. arseniciselenatis can respire arsenate and selenate, and B. selenitireducens respires arsenate and selenite (41). Preliminary experiments with B. arseniciselenatis indicate that it, too, can couple the oxidation of arsenite to the reduction of selenate in the presence of a dissolved organic carbon source. This suggests that this process is not limited to one organism and potentially may be carried out by selenate reducers from other clades as well.

The ecological relevance of selenate-dependent arsenite oxidation is yet to be fully understood, as this is the first account of this process. The selenate/arsenite couple has the thermodynamic potential to support chemoautotrophic growth, although this was not the case for strain ML-SRAO. The discovery of a new mechanism for arsenite oxidation provides a broader context for the biological transformation of toxic metalloids not only in Mono Lake but also in other environments. Similar processes could occur in other soda lakes, hydrothermal vents, or metal-polluted soils and waters. The study of novel pathways for arsenite oxidation (e.g., coupled to selenate reduction) can suggest mechanisms for arsenic mobilization or sequestration in diverse natural or impacted environments and may further contribute to the understanding of potential early earth metabolisms.

ACKNOWLEDGMENTS

This research was funded by an NSF grant (MCB 99-77886) to J.T.H. and a University of Georgia Graduate School Dissertation Completion Fellowship to J.C.F.

We thank Robert Jellison and Kim Rose for assistance with sample collection at Mono Lake and Peter Jackson and Morris Jones for assistance with ion chromatography-inductively coupled plasma mass spectrometry analysis. Rebecca Hale assisted with arrA amplification and sequencing. Erin Biers and two anonymous reviewers provided helpful suggestions for improving the manuscript.

FOOTNOTES

    • Received 31 August 2007.
    • Accepted 27 February 2008.
  • Copyright © 2008 American Society for Microbiology

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Selenate-Dependent Anaerobic Arsenite Oxidation by a Bacterium from Mono Lake, California
Jenny C. Fisher, James T. Hollibaugh
Applied and Environmental Microbiology Apr 2008, 74 (9) 2588-2594; DOI: 10.1128/AEM.01995-07

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Selenate-Dependent Anaerobic Arsenite Oxidation by a Bacterium from Mono Lake, California
Jenny C. Fisher, James T. Hollibaugh
Applied and Environmental Microbiology Apr 2008, 74 (9) 2588-2594; DOI: 10.1128/AEM.01995-07
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KEYWORDS

Arsenites
Bacillus
Fresh Water
Selenium Compounds

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