Microbial oxidation of arsenite in a subarctic environment: diversity of arsenite oxidase genes and identification of a psychrotolerant arsenite oxidiser
© Osborne et al; licensee BioMed Central Ltd. 2010
Received: 17 May 2010
Accepted: 30 July 2010
Published: 30 July 2010
Arsenic is toxic to most living cells. The two soluble inorganic forms of arsenic are arsenite (+3) and arsenate (+5), with arsenite the more toxic. Prokaryotic metabolism of arsenic has been reported in both thermal and moderate environments and has been shown to be involved in the redox cycling of arsenic. No arsenic metabolism (either dissimilatory arsenate reduction or arsenite oxidation) has ever been reported in cold environments (i.e. < 10°C).
Our study site is located 512 kilometres south of the Arctic Circle in the Northwest Territories, Canada in an inactive gold mine which contains mine waste water in excess of 50 mM arsenic. Several thousand tonnes of arsenic trioxide dust are stored in underground chambers and microbial biofilms grow on the chamber walls below seepage points rich in arsenite-containing solutions. We compared the arsenite oxidisers in two subsamples (which differed in arsenite concentration) collected from one biofilm. 'Species' (sequence) richness did not differ between subsamples, but the relative importance of the three identifiable clades did. An arsenite-oxidising bacterium (designated GM1) was isolated, and was shown to oxidise arsenite in the early exponential growth phase and to grow at a broad range of temperatures (4-25°C). Its arsenite oxidase was constitutively expressed and functioned over a broad temperature range.
The diversity of arsenite oxidisers does not significantly differ from two subsamples of a microbial biofilm that vary in arsenite concentrations. GM1 is the first psychrotolerant arsenite oxidiser to be isolated with the ability to grow below 10°C. This ability to grow at low temperatures could be harnessed for arsenic bioremediation in moderate to cold climates.
Arsenic's toxic and medicinal properties have been appreciated for more than two millennia . Its two soluble inorganic forms, arsenite (+3) and arsenate (+5), entering drinking water from natural sources, have caused poisoning in Taiwan, Chile, Argentina, Bangladesh and West Bengal, and most recently arsenicosis (arsenic poisoning) has been detected in people from Cambodia, Vietnam, Nepal, China, Inner Mongolia, Bolivia and Mexico [2, 3]. In addition, arsenic contamination due to anthropogenic activity (e.g. mining) is increasing in importance in parts of the USA, Canada, Australia, Argentina and Mexico . Although arsenic is toxic to most organisms, some prokaryotes have evolved mechanisms to gain energy by either oxidising or reducing it [5, 6].
Prokaryotic arsenic metabolism has been detected in hydrothermal and temperate environments and has been shown to be involved in the redox cycling of arsenic [7–10]. The arsenite-oxidising bacteria isolated so far are phylogenetically diverse. The oxidation of arsenite may yield useable energy or may merely form part of a detoxification process . To date, all aerobic arsenite oxidation involves the arsenite oxidase that contains two heterologous subunits: AroA (also known as AoxB) and AroB (also known as AoxA) . AroA is the large catalytic subunit that contains the molybdenum cofactor and a 3Fe-4S cluster and AroB contains a Rieske 2Fe-2S cluster .
Although arsenic metabolism has been detected in both moderate and high-temperature environments, and mesophilic and thermophilic arsenite oxidisers have been isolated, no arsenic metabolism (either dissimilatory arsenate reduction or arsenite oxidation) has ever been detected in cold environments (i.e. < 10°C). One such environment with high concentrations of arsenic is the Giant Mine, one of Canada's oldest and largest gold mines. It is located a few kilometres north of Yellowknife, Northwest Territories, 62° north of the equator and 512 kilometres south of the Arctic Circle. Gold was produced from 1948 to 1999 by roasting arsenopyrite (FeAsS)-bearing ore. The mine now contains approximately 300,000 tonnes of arsenic trioxide, stored in underground chambers . Temperatures in the underground stopes range from 4°C to 10°C .
Here we report the detection, isolation and characterisation of an aerobic psychrotolerant arsenite-oxidising bacterium from a subterranean biofilm in the Giant Mine. Unlike other characterised arsenite oxidisers, this organism is capable of growing below 10°C and is the first heterotrophic organism to oxidise arsenite in the early exponential phase of growth. We also compare the diversity of arsenite oxidisers in two subsamples of the biofilm that vary in arsenite concentrations.
Results and Discussion
The arsenite-oxidising ability of GM1 was further confirmed by testing for arsenite oxidase (Aro) activity in cells grown in the MSM with 4 mM arsenite and 0.04% (w/v) yeast extract. Aro activity was measured at room temperature (i.e. 24°C) in its optimal buffer, 50 mM 2-(N-Morpholino)ethanesulfonic acid (MES) (pH 5.5) (data not shown). Aro activity was higher when GM1 was grown at 10°C (0.334 U/mg) compared with growth at 4°C (0.247 U/mg) and 20°C (0.219 U/mg) which were comparable. In growth experiments although all the arsenite is oxidised to arsenate in the early exponential growth phase the highest Aro activity was observed in the stationary phase of growth (i.e. 0.334 U/mg compared with 0.236 U/mg at early exponential phase).
The partial aroA gene sequence of GM1 was found to be identical to that of the partial aroA of the putative arsenite oxidiser Limnobacter sp. 83, another member of the Betaproteobacteria  but in a different family. No homologues of aroA were found in the genome sequences of GM1's closest relatives, Polaromonas naphthalenivorans CJ2 and Polaromonas sp. JS666; GM1 is thus clearly distinct from the other Polaromonas spp.
The number of clones from TOP and BOT that clustered within clades A, B and C
In this report we provide the first evidence for bacterial arsenite oxidation below 10°C. The sample site, the Giant Mine, is an extreme environment with arsenic concentrations in excess of 50 mM in the underground waters . In this study we have compared the diversity of arsenite oxidisers in two different subsamples and found that although the composition of arsenite-oxidising communities differs, the diversity does not. The isolated arsenite-oxidising bacterium GM1 was able to grow at low temperatures (< 10°C); its arsenite oxidase was constitutively expressed and displayed broad thermolability.
Sample collection and analyses
Samples were collected from Giant Mine, north of Yellowknife, Northwest Territories, Canada. The microbial biofilm was located growing on a wall in an abandoned stope below the arsenic trioxide storage chambers where liquid was seeping from a diamond drill hole. The first sampling of the biofilm was done in July 2006 and involved collecting some of the biofilm itself, coexisting seepage water, and mineral precipitates from near the top of the biofilm. The biofilm was re-sampled in May 2007 using the same sampling method as in 2006 but this time two samples were collected: one at the top near the seepage point and another near the bottom. All samples were kept at 4°C at all times until microbial or chemical analyses could be performed.
The 2006 biofilm sample was used for mineral characterisation. Mineral precipitates were characterised using beamline X26A at the National Synchrotron Light Source. MicroXANES (at the arsenic K edge) and microXRD followed methods similar to those described previously . The XANES spectra collected on thin layers on sample powder provided clear indication of the presence of both arsenite and arsenate, and a linear combination fit, using scorodite (AsV) and schneiderhohnite (AsIII) as model compounds, estimated the relative proportions at 57% arsenate and 43% arsenite. Synchotron-based microXRD of the biofilm showed clear evidence of microcrystalline yukonite, a Ca-Fe arsenate [Ca7Fe(AsO4)9O10·24.3H2O]  (see reddish-brown colouration in Figure 1a), gypsum and an arsenite mineral [either claudetite (As2O3) or manganarsite (Mn3As2O4(OH)4)].
In 2006 the liquid from the biofilm was extracted 18 days after collection whereas in 2007 the liquid was extracted immediately after collection. The liquid was extracted using a syringe with a 0.22-μm filter. Concentrations of total arsenic and arsenite were determined by hydride generation atomic-absorption spectrometry (HG-AAS) using a Perkin Elmer - Analyst 300.
Scanning electron microscopy
Samples from the top and bottom of the 2007 microbial biofilm were examined using a Jeol JSM-6480LV high-performance, variable pressure analytical scanning electron microscope (SEM) operating in low-vacuum mode using 7-11 kV accelerating voltage and a spot size of 29 nm. Prior to examination, samples were mounted on 12.5-mm pin stubs with sticky carbon discs, freeze-dried in liquid nitrogen using a MODULO 4 k instrument for 30 minutes, and gold coated using a Polaron E5000 instrument.
Enrichment and isolation
In 2006 samples of the microbial biofilm (0.5 g) were inoculated into the MSM  containing 4 mM arsenite and incubated at 4°C, 10°C and 20°C. The enrichments were incubated until all the arsenite was oxidised. The biofilm enrichments took two days to oxidise the 4 mM arsenite irrespective of temperature (data not shown). The enrichments were subcultured three times in the MSM containing 4 mM arsenite before they were serially diluted and plated onto MSM containing 4 mM arsenite and 1.5% (w/v) purified agar (Oxoid). Individual colonies were purified and tested for both chemolithoautotrophic [containing 0.05% (w/v) NaHCO3 as carbon source] and heterotrophic (containing 0.04% (w/v) yeast extract) growth with arsenite .
Growth of GM1
Growth experiments of GM1 were conducted in MSM containing 0.04% (w/v) yeast extract in the presence and absence of 4 mM arsenite at 4°C, 10°C and 20°C with shaking at 130 rpm in batch cultures. Experiments were commenced with a 5% (v/v) inoculum of late exponential phase cells grown in the same medium at the same temperature. At regular time intervals samples were taken to measure optical density and pH, and for arsenic analyses. Samples for arsenic analyses were centrifuged in a bench-top centrifuge and the supernatant stored at -20°C until required. All growth experiments were performed on at least two separate occasions with two to three replicates.
Arsenite oxidase assays
GM1 cultures were harvested and crude cell extracts produced by passing them through a French pressure cell at 14 kPSI and arsenite oxidase activity determined by measuring the reduction of the artificial electron acceptor 2,6-dichlorophenolindophenol . All assays were performed in the optimum buffer for the enzyme, 50 mM MES buffer (pH 5.5). Reactions were incubated at the specific temperature with a Cary Dual Cell Peltier for 5 mins prior to the addition of arsenite.
16S rRNA gene sequence determination and phylogenetic analyses
Genomic DNA was extracted using the Wizard® Genomic DNA purification kit (Promega). 16S rDNA was amplified by PCR using the 27f and 1525r primers described previously , with Phusion high fidelity DNA polymerase (New England Biolabs) under the following conditions: 98°C for 30 s, followed by 40 cycles of 98°C for 30 s, 55°C for 30 s and 72°C for 90 s with a final extension at 72°C for 10 min. Both strands of the PCR product were sequenced at the Wolfson Institute for Biomedical Research (WIBR) (UCL) using the primers 27f, 342r, 357f, 518r, 530f, 1100r, 1114f, 1392r, 1406f, 1492r and 1525r . [GM1 16S rRNA gene sequence GenBank accession number: EU106605].
Amplification of aroA, library construction and sequencing
Genomic DNA was extracted from GM1 using the Wizard® Genomic DNA purification kit (Promega) and from the top and bottom biofilm samples using the PowerSoil DNA isolation kit (MoBio Laboratories). The degenerate oligonucleotides used to amplify a portion of the aroA gene were primer set #2 as described previously  using Phusion high fidelity DNA polymerase (New England Biolabs). The aroA PCR products from GM1 and the two biofilm samples were cloned into pBluescript II KS+ (Stratagene). Both strands of the cloned GM1 aroA gene and 50 individual aroA clones from each library were sequenced using the T7 and T3 promoter primers at the WIBR (UCL). Database searches were performed using BLASTP . [GM1 partial aroA sequence GenBank accession number: EU106602. The TOP and BOT aroA library sequences GenBank accession numbers: FJ151018-FJ151051].
Sequences were aligned with CLUSTALX 2.0  using default settings and were manually edited. Phylogenetic analyses were performed with PHYLIP 3.67  and trees constructed and edited with TREEVIEW . Nucleotide and protein distance analyses were performed with the F84 and Jones-Taylor-Thornton computations, respectively and the trees constructed using the neighbour-joining method using a boostrap value of 100.
Accession numbers of reference sequences used in AroA phylogenetic analysis are given in parentheses following the organism name: Achromobacter sp. str. SY8 (ABP63660), Aeropynum pernix (NP_148692), Agrobacterium tumefaciens str. 5A (ABB51928), 'Alcaligenes faecalis' (AAQ19838), Burkholderia multivorans (YP_001585661), Chlorobium limicola (ZP_00512468), Chlorobium phaeobacteroides (ZP_00530522), Chloroflexus aurantiacus (YP_001634827), Herminiimonas arsenicoxydans (YP_001098817), Nitrobacter hamburgensis (YP_571843), NT-26 (AAR05656), Ochrobacterum tritici (ACK38267), Pseudomonas sp. str. TS44 (ACB05943), Pyrobaculum calidifontis (YP_001056256), Rhodoferax ferrireducens (YP_524325), Roseovarius sp. 217 (ZP_01034989), Thermus thermophilus str. HB8 (YP_145366), Thiomonas sp. 3As (CAM58792), Sulfolobus tokodaii str. 7 (NP_378391) and Xanthobacter autotrophicus Py2 (YP_001418831).
Rarefaction curves and Chi-squared
Rarefaction calculations were performed to compare the DNA sequence diversity of the TOP and BOT libraries, and to assess whether full coverage of sequence diversity was obtained. This was performed with the program ANALYTICAL RAREFACTION 1.3 http://www.uga.edu/~strata/software/index.html which uses the rarefaction calculations given by Hulbert  and Tipper . Sequences were clustered with BLASTclust http://toolkit.tuebingen.mpg.de/blastclust# based on a 99% identity threshold over 100% of the sequence length to create operating taxonomic units.
JMS would like to acknowledge support from the University of London Central Research fund (Grant AR/CRF/B). THO is supported by a Natural Environment Research Council studentship (14404A). HEJ and SRW acknowledge support from Natural Sciences and Engineering Research Council and Indian and Northern Affairs Canada, and from A. Lanzirotti at the National Synchrotron Light Source. DKN acknowledges support from the National Research Program of the US Geological Survey. We would like to thank R. Blaine McCleskey with technical help for biofilm arsenic analyses, James Davy for technical help with the SEM, Anthony Osborn for ICP-OES analysis of culture solutions, and S. Simpson for the underground photograph of the biofilm. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the USGS.
- Cullen WR: Is Arsenic an Aphrodisiac? The Sociochemistry of an Element. 2008, UK: Royal Society of Chemistry PublishingGoogle Scholar
- Nordstrom DK: Worldwide occurrences of arsenic in ground water. Science. 2002, 296: 2143-2145. 10.1126/science.1072375.View ArticlePubMedGoogle Scholar
- Ravenscroft P, Brammer H, Richards K: Arsenic Pollution: a Global Synthesis. 2009, UK: Wiley-BlackwellView ArticleGoogle Scholar
- Smedley PL, Kinniburgh DG: A review of the source, behaviour and distribution of arsenic in natural waters. Appl Geochem. 2002, 17: 517-568. 10.1016/S0883-2927(02)00018-5.View ArticleGoogle Scholar
- Oremland RS, Stolz JF: The ecology of arsenic. Science. 2003, 300: 939-944. 10.1126/science.1081903.View ArticlePubMedGoogle Scholar
- Stolz JF, Basu P, Santini JM, Oremland RS: Arsenic and selenium in microbial metabolism. Annu Rev Microbiol. 2006, 60: 107-130. 10.1146/annurev.micro.60.080805.142053.View ArticlePubMedGoogle Scholar
- Inskeep WP, Macur RE, Hamamura N, Warelow TP, Ward SA, Santini JM: Detection, diversity and expression of aerobic bacterial arsenite oxidase genes. Environ Microbiol. 2007, 9: 934-943. 10.1111/j.1462-2920.2006.01215.x.View ArticlePubMedGoogle Scholar
- Quéméneur M, Heinrich-Salmeron A, Muller D, Lièvremont D, Jauzein M, Bertin PN, Garrido F, Joulian C: Diversity surveys and evolutionary relationships of aoxB genes in aerobic arsenite-oxidising bacteria. Appl Environ Microbiol. 2008, 74: 4567-4573. 10.1128/AEM.02851-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Quéméneur M, Cébron A, Billard P, Battaglia-Brunet F, Garrido F, Leyval C, Joulian C: Population structure and abundance of arsenite-oxidising bacteria along an arsenic pollution gradient in waters of the Upper Isle River Basin, France. Appl Environ Microbiol. 2010, 76: 4566-4570. 10.1128/AEM.03104-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Rhine ED, Garcia-Dominguez E, Phelps CD, Young LY: Environmental microbes can speciate and cycle arsenic. Environ Sci Technol. 2005, 39: 9569-9573. 10.1021/es051047t.View ArticlePubMedGoogle Scholar
- Clark ID, Raven KG: Sources and circulation of water and arsenic in the Giant Mine, Yellowknife, NWT, Canada. Isotopes Environ Health Stud. 2004, 40: 1-14. 10.1080/10256010410001671014.View ArticleGoogle Scholar
- Coleman NV, Mattes TE, Gossett JM, Spain JC: Biodegradation of cis-dichloroethene as the sole carbon source by a β-Proteobacterium. Appl Environ Microbiol. 2002, 68: 2726-2730. 10.1128/AEM.68.6.2726-2730.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Jeon CO, Park W, Padmanabhan , DeRito C, Snape JR, Madsen EL: Discovery of a bacterium, with distinctive dioxygenase, that is responsible for in situ biodegradation in contaminated sediment. Proc Natl Acad Sci USA. 2003, 100: 13591-13596. 10.1073/pnas.1735529100.PubMed CentralView ArticlePubMedGoogle Scholar
- Wang Q, Garrity GM, Tiedje JM, Cole JR: Naïve Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol. 2007, 73: 5261-5267. 10.1128/AEM.00062-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Santini JM, Sly LI, Schnagl RD, Macy JM: A new chemolithoautotrophic arsenite-oxidising bacterium isolated from a gold mine: phylogenetic, physiological, and preliminary biochemical studies. Appl Environ Microbiol. 2000, 66: 92-97. 10.1128/AEM.66.1.92-97.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Drewniak L, Matlakowska R, Sklodowska A: Arsenite and arsenate metabolism of Sinorhizobium sp. M14 living in the extreme environment of Zloty Stok gold mine. Geomicrobiol J. 2008, 22: 363-370. 10.1080/01490450802402836.View ArticleGoogle Scholar
- Duquense K, Lieutaud A, Ratouchniak J, Muller D, Lett M, Bonnefoy V: Arsenite oxidation by a chemoautotrophic moderately acidophilic Thiomonas sp.: from the strain to gene study. Environ Microbiol. 2008, 10: 228-237.Google Scholar
- Kashyap DR, Botero LM, Franck WL, Hassett DJ, McDermott TR: Complex regulation of arsenite oxidation by Agrobacterium tumefaciens. J Bacteriol. 2006, 188: 1081-1088. 10.1128/JB.188.3.1081-1088.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Hamamura N, Macur RE, Korf S, Ackerman G, Taylor WP, Kozubal M, Reysenbach A-L, Inskeep WP: Linking microbial oxidation of arsenic with detection and phylogenetic analysis of arsenite oxidase genes in diverse geothermal environments. Environ Microbiol. 2009, 11: 421-431. 10.1111/j.1462-2920.2008.01781.x.View ArticlePubMedGoogle Scholar
- Magurran AE: Ecological diversity and its measurement. 1996, London: ChapmanGoogle Scholar
- Cullen WR, Polishchuk E, Reimer KJ, Sun YM, Wang L, Lai VWM: Arsenic in Yellowknife, North West Territories, Canada. Arsenic exposure and health effects V. Edited by: Chappell WR, Abernathy CO, Calderson RL, Thomas DJ. 2003, USA: Elsevier, 79-88.Google Scholar
- Walker SW, Jamieson HE, Lanzirotti A, Andrade CF: Determining arsenic speciation in iron oxides derived from a gold-roasting operation: application of synchrotron micro-XRD and micro-XANES at the grain scale. Can Mineral. 2005, 43: 1205-1224. 10.2113/gscanmin.43.4.1205.View ArticleGoogle Scholar
- Meng SG, Bang SB, Korfiatis GP: Effects of silicate, sulphate, and carbonate on arsenic removal by ferric chloride. Water Res. 2000, 34: 1255-1261. 10.1016/S0043-1354(99)00272-9.View ArticleGoogle Scholar
- Meng XG, Korfiatis GP, Jing CY, Christodoulatos C: Redox transformations of arsenic in water treatment sludge during aging and TCLP extraction. Environ Sci Technol. 2001, 35: 3476-3481. 10.1021/es010645e.View ArticlePubMedGoogle Scholar
- Tu S, Ma LQ, MacDonald GE, Bondada B: Effects of arsenic species and phosphorus on arsenic absorption, arsenate reduction and thiol formation in excised parts of Pteris vittata L. Environ Exp Bot. 2004, 51: 121-131. 10.1016/j.envexpbot.2003.08.003.View ArticleGoogle Scholar
- Lane DJ: 16S/23S rRNA sequencing. Nucleic Acid Techniques in Bacterial Systematics. Edited by: Stackebrandt E, Goodfellow M. 1991, UK: John Wiley & Sons, 115-163.Google Scholar
- Alschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215: 403-10.View ArticleGoogle Scholar
- Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res. 1997, 25: 4876-4882. 10.1093/nar/25.24.4876.PubMed CentralView ArticlePubMedGoogle Scholar
- Felsenstein J: PHYLIP - Phylogeny Inference Package (Version 3.2). Cladistics. 1989, 5: 164-166.Google Scholar
- Page RDM: TREEVIEW: An application to display phylogenetic trees on personal computers. Comput Appl Biosci. 1996, 12: 357-358.PubMedGoogle Scholar
- Hurlbert SH: The nonconcept of species diversity: a critique and alternative parameters. Ecology. 1971, 52: 577-586. 10.2307/1934145.View ArticleGoogle Scholar
- Tipper JC: Rarefaction and rarefiction - the use and abuse of a method in paleontology. Paleobiol. 1979, 5: 423-434.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.