Manuscript published as: Recent Research Developments in Microbiology 7:103-118 (2003)

Microbial transformations of arsenite and arsenate in natural environments

Colin R. Jackson, Evelyn F. Jackson, Sandra L. Dugas, Karyn Gamble, and Shawn E. Williams.

Department of Biological Sciences, SLU 10736, Southeastern Louisiana University, Hammond, LA 70402 USA

Email: [email protected] Tel: (985) 549-3444 Fax: (985) 549-3851

Running Title: Microbial transformations of arsenic

ABSTRACT Inorganic arsenic primarily exists as oxidized arsenate or reduced arsenite. A number of microorganisms have been identified that show arsenic redox activity and are capable of transforming arsenic between these two states. Dissimilatory arsenate reducers use arsenate as the terminal electron acceptor in anaerobic respiration, while chemoautotrophic arsenite oxidizers use arsenite as the electron donor for autotrophic growth. Both processes appear to be confined to relatively small groups of bacteria. Many more microorganisms are capable of reducing arsenate to arsenite via transcription of the arsC arsenate reductase gene, a component of a fairly widespread arsenic detoxification system that is observed in Bacteria, Archaea, and Eukarya. The various microbial arsenic redox processes can significantly affect the distribution and mobility of arsenic within natural environments, and might influence the toxicity of arsenic to other organisms.

INTRODUCTION Arsenic is a toxic element that can be present in both natural and human-impacted environments. In its inorganic form, arsenic primarily exists in two redox states: the reduced form, arsenite (AsIII), and the oxidized form, arsenate (AsV). Both states are toxic to most 3- organisms. Arsenite (specifically the arsenite ion, AsO3 ) interferes with sulfhydryl groups in 3- amino acids and can disrupt protein structure. Arsenate (specifically the arsenate ion, AsO4 ) is a 3- phosphate (PO4 ) analog and can interfere with both phosphate uptake and a variety of cellular processes that involve phosphate (1). In most environments, arsenite is generally thought to be the more soluble and mobile form, which increase its potential toxicity. However, arsenate is the thermodynamically favorable form in most aerobic systems (2,3). Because of the severe toxicity of both forms of inorganic arsenic, issues of arsenic mobility and toxicity are important on both regional and global scales. Increased arsenic mobility in natural environments is a major concern in the creation of new wells and water supply systems in areas that are rich in arsenic: for example the arsenic crises in West Bengal, India (4) and Bangladesh (5) have resulted in the chronic exposure of millions of people to potentially toxic levels of arsenic. Hot springs of Yellowstone National Park, USA, are rich in arsenic either of magmatic origin or leached from subsurface rocks (6), and runoff from these areas can contain levels of arsenic that make them unsuitable for human consumption or recreation (7). Other geothermal areas in the USA (8), Japan (9), Canada (10), and New Zealand (11) have also been shown to naturally contain high levels of arsenic compounds. In industrialized areas, arsenic can be a waste product from a number of processes including mining and ore refining (12,13,14), fossil fuel combustion (14), and wood treatment (15,16). Elevated levels of arsenic can be severely detrimental to both human and ecological communities bordering on such industries. Microorganisms have evolved a number of mechanisms to cope with arsenic toxicity, and some organisms even benefit from the presence of arsenic. In some cases arsenic toxicity is avoided by minimizing the amount of arsenic that enters the cell. For example, Escherichia coli possesses two different phosphate transport systems: the Pit system, which has low phosphate specificity and will also transport arsenate, and the Pst system, which shows high phosphate specificity (17). It is thought that organisms lacking the Pit system (and thus using the more specific Pst system) have greater resistance to arsenate (18). In certain plant species, arsenate (but not arsenite) uptake and toxicity can be ameliorated by increasing the amount of phosphate in the environment (19,20), and similar patterns might be seen when bacterial cultures are incubated at different concentrations of arsenate and phosphate. However, a number of specific metabolisms also exist in microorganisms to cope with, or benefit from, elevated levels of arsenic, and these metabolisms can have major impacts on the transformation of arsenic between arsenate and arsenite. In this paper we review some of the ways in which microorganisms reduce arsenate and oxidize arsenite, whether as a means of detoxification or as a method of obtaining energy. We conclude by describing some of the potential origins of these metabolic pathways and suggest how these redox transformations might affect the mobility and toxicity of arsenic in the environment.

ARSENIC BREATHING BACTERIA: DISSIMILATORY REDUCTION OF ARSENATE While a number of bacteria are capable of reducing arsenate as a form of detoxification, it was only recently that organisms capable of using arsenate as the terminal electron acceptor in anaerobic respiration were discovered. Although thermodynamic considerations suggest that dissimilatory reduction of arsenate could provide enough energy for microbial growth (21), it is possible that the overall toxicity of arsenic has limited the distribution of this process amongst bacteria. In 1994, bacterial strain MIT-13 was isolated from arsenic-contaminated river sediments and this organism is capable of obtaining energy from arsenate respiration, when coupled to growth on lactate (22). A second bacterial strain, SES-3, isolated from a selenate- contaminated freshwater marsh (and capable of anaerobic respiration through selenate reduction; (23) is also able to obtain energy from arsenate respiration (21). Both MIT-13 and SES-3 were originally assigned to a new genus, Geospirillum (24,25), but have more recently been classified as two species of Sulfurospirillum (a clade within the epsilon Proteobacteria) based on 16S rRNA sequence analysis and DNA-DNA hybridizations. Strain MIT-13 is designated S. arsenophilum and strain SES-3 is S. barnesii (26). Thus, the organisms are closely related (16S rRNA sequences are 97% identical), but differ slightly in metabolism. Both are capable of using arsenate, nitrate, thiosulfate, or sulfur as the terminal electron acceptor in anaerobic respiration, and can also grow microaerobically with low levels of oxygen or fermentatively on fumarate (26). S. barnesii is also capable of respiratory iron or selenate reduction (23,26), while S. arsenophilum can reduce sulfate (22). In terms of arsenate reduction, S. arsenophilum shows optimal growth at around 10 mM AsV (22), whereas S. barnesii shows optimum growth at 5 mM AsV. Growth at higher arsenate concentrations may be inhibited by accumulating levels of arsenite (21). A third dissimilatory arsenate reducing bacterium was isolated from lake sediments that had low levels of arsenic contamination (27). This organism (originally referred to as strain OREX-4) is capable of respiring lactate when using either arsenate or sulfate as the terminal electron acceptor, with arsenate being the acceptor of choice. Unlike the other two isolates, this organism was phylogenetically affiliated with the low G+C Gram-positive bacteria, and has been designated Desulfotomaculum auripigmentum (27). The specific epithet auripigmentum refers to its ability to form the yellow colored arsenic trisulfide (As2S3) through the joint reduction of arsenate and sulfate (28). The arsenic trisulfide precipitates both intra- and extra-cellularly, being found near the cell membrane and on the surface of the cell wall (28). As well as growing on sodium arsenate in laboratory cultures, D. auripigmentum was capable of respiring arsenate released from the mineral scorodite, suggesting that naturally occurring rocks might serve as a source of AsV for dissimilatory arsenate reducers (28). Two other dissimilatory AsV reducing bacteria that are phylogenetically affiliated with the low G+C Gram-positive bacteria were isolated from an alkaline, arsenic-rich, saline lake, and have been designated Bacillus arsenicoselenatis and Bacillus selenitireducens (29). Both isolates are alkaliphiles that are also capable of growth through the respiratory reduction of selenium compounds. An organism that represents a novel phylogenetic group of bacteria was isolated from a reed bed exposed to goldmine wastewater (30). This bacterium is strictly anaerobic, using arsenate, nitrate, or nitrite as the terminal electron acceptor in respiration. 16S rRNA sequence analysis for this organism suggests that it is the first representative of a deeply branching lineage of Domain Bacteria, and the organism has been named Chrysiogenes arsenatis (30). Unlike the other arsenate reducers, C. arsenatis can use acetate, which combined with its unique phylogeny, suggests a novel mechanism of arsenate reduction. The respiratory arsenate reductase from this organism has been characterized and is inducible by arsenate, does not recognize alternative electron acceptors (i.e. it is arsenate specific), and is located in the periplasm (31). This is in contrast to the respiratory arsenate reductase of S. barnesii, which is thought to span the cell membrane, with its active site facing the cytoplasm (32). Among the Archaea, a strictly anaerobic hyperthermophile was isolated from a hot spring in Italy, and this organism (Pyrobaculum arsenaticum) is capable of respiratory growth using arsenate, sulfur, or selenate (33). The isolate can also grow chemoautotrophically using carbon dioxide, hydrogen as an electron donor, and arsenate or sulfur as the electron acceptor. 16S rRNA gene sequence analysis showed that this organism belongs to the Thermoproteales order of Archaea, and when tested, another species of this order (Pyrobaculum aerophilum) was also able to grow anaerobically in the presence of arsenate or selenate (33). Other recently discovered arsenate respiring organisms include a species (strain GBFH) of Desulfitobacterium (belonging to the low G+C Gram positive bacteria) isolated from arsenic-contaminated lake sediments (34), and a new strain of Thermus (strain HR13; Deinococcus/Thermus phylum of Bacteria) isolated from the drainage channel of an arsenic-rich hot spring (35). For a more in depth discussion of dissimilatory arsenate reduction see reviews by Newman et al. (32), and Stolz and Oremland (36).

ARSENIC EATING BACTERIA: CHEMOAUTOTROPHIC OXIDATION OF ARSENITE In contrast to the dissimilatory arsenate reducers, which use arsenate as the terminal electron acceptor in anaerobic respiration, some bacteria are capable of using arsenite as the electron donor for chemoautotrophic growth. The energetics of the oxidation of arsenite to arsenate suggest that enough energy for growth can be produced through this reaction (37), although very few organisms appear to grow in this way. A bacterium isolated from mine waters draining a gold-arsenic deposit in the USSR was described as Pseudomonas arsenitoxidans and grew aerobically through the chemoautotrophic oxidation of AsIII (38). The isolate was also capable of growth on arsenopyrite when this mineral was added to the growth medium. Unfortunately this isolate seems to have been lost, preventing further characterization of its mechanisms for AsIII oxidation. A second chemoautotrophic arsenite oxidizer (NT-26) was isolated from arsenopyrite samples taken from a gold mine in Australia (39). This isolate appears to represent a previously unknown clade in the Agrobacterium-Rhizobium branch of the alpha Proteobacteria, and as with P. arsenitoxidans uses arsenite as an electron donor and oxygen as an electron acceptor. However, NT-26 appears to grow much more efficiently on AsIII than P. arsenitoxidans, with doubling times of approximately 8 hours, versus the 2-3 days reported for P. arsenitoxidans (39). NT-26 can also grow heterotrophically, although arsenite is oxidized throughout growth, even in the presence of organic substrates. In fact, the addition of AsIII to NT-26 cultures growing on organic substrates results in increased yields, suggesting that energy is gained from arsenite oxidation (39). Thus, while just two chemoautotrophic arsenite oxidizers have been identified to date, both theoretically and practically the reaction yields sufficient energy for microbial growth.

ARSENIC RESISTANT MICROORGANISMS A relatively large number of microorganisms are capable of resisting the toxic effects of arsenic, using methods such as arsenite oxidation (to produce the less toxic arsenate), extrusion of arsenic from the cell, and minimizing the uptake of arsenic from the environment. Early studies on isolates obtained from arsenical tanks used as cattle dips reported arsenite oxidation in strains identified as species of Bacillus (40), Achromobacter, and Pseudomonas (41), and this oxidation did not release energy for growth (i.e. it was not chemoautotrophic). In an Alcaligenes faecalis isolate obtained from soil, pre-exposure to AsIII induced arsenite oxidation, suggesting the existence of an inducible enzyme system (42). Respiratory inhibitors affected this oxidation, suggesting that some form of cytochrome system was required for arsenite oxidation, as well as the action of the arsenite oxidase enzyme itself (42). Other strains of A. faecalis obtained from raw sewage, were also capable of AsIII oxidation, which begin during the stationary phase of growth in batch cultures (43). As with the other strains, arsenite oxidation appeared to involve an inducible enzyme and components of the electron transport system. A reexamination of the earlier arsenite oxidizing isolates of Bacillus (40) and Achromobacter (41), suggested that these were also strains of A. faecalis (43). Two bacterial isolates, identified as Pseudomonas putida (strain 18) and Alcaligenes eutrophus (strain 280), isolated from gold-arsenic deposits in Kazakhstan were capable of arsenite oxidation, which required the presence of live cells (44). P. putida strain 18 showed increased catalase activity following exposure to AsIII, and this increase in catalase activity correlated with arsenite oxidation. A mechanism for AsIII oxidation in this organism was proposed, that required the presence of peroxide (H2O2) to oxidize AsIII, and the subsequent dissimilation of H2O2 by catalase (45). AsIII has also been shown to suppress that transport and accumulation of glucose and phosphate in arsenite sensitive strains of Pseudomonas, whereas in the arsenite resistant strain of P. putida 18, glucose and phosphate transport is unaffected by low levels of AsIII. Thus, it is possible that arsenite resistance might also involve changes in the cell membrane as well as arsenite oxidation ability (46). As well as an increase in catalase activity concordant with arsenite oxidation, cellular levels of other antioxidants also increase when P. putida 18 is exposed to AsIII, including superoxide dismutase, glutathionine peroxidase, and glutathione reductase (47). Synthetic antioxidants and vitamin E inhibit arsenite oxidation in this organism, suggesting that an oxidative or peroxide-mediated mechanism is involved (48). Hydroperoxides of unsaturated fatty acids were found to accumulate in the growth medium when P. putida 18 was grown in the presence of AsIII, particularly as the cells approached the stationary phase of growth in batch culture. This corresponded to an increase in the amount of unsaturated fatty acids within stationary phase cells exposed to AsIII compared to those grown in AsIII free media (49). Microscopic examination of AsIII exposed cells revealed changes in the outer membrane, which showed increased folding, particularly in the stationary phase. AsIII also extended the stationary phase in P. putida 18, which lasted up to 20-25 days compared to just 5 days when the cells were grown without AsIII (50). The changes in the phospholipid membrane and cellular fatty acids observed when P. putida 18 is exposed to arsenite, along with the increased activity of oxidative stress enzymes, suggests that lipid mediated peroxidation may be involved in arsenite oxidation in this organism. Arsenite resistance might be related to a cells ability to over-synthesize fatty acids to replace those in the membrane that form hydroperoxides during the reaction with AsIII (51). The importance of unsaturated fatty acids for arsenite oxidation can be demonstrated by the addition of linetol (containing polyunsaturated fatty acids) to the growth medium, which increases the arsenite oxidation rate of P. putida 18 cultures by up to 50% (48). The Alcaligenes eutrophus 280 isolate obtained from the same gold-arsenic deposits (44) showed the same peroxide interactions between unsaturated fatty acids and arsenite as P. putida 18 (52), and both organisms could also oxidize iron or manganese via the same lipid peroxide mechanism (53). Thus, for these two arsenite-oxidizers, the method of oxidation seems to involve reactions with lipid components of the cell membrane rather than activity of an arsenite oxidize enzyme. An arsenite oxidase is involved in arsenite oxidation in Alcaligenes faecalis and has been obtained and purified (37) and more recently its crystalline structure identified (54). The enzyme has a molecular weight of 85,000 to 100,000 Daltons, and contains molybdenum, iron, and sulfur residues. During arsenite oxidation, azurin or cytochrome c can serve as the electron acceptor, and the energetics of the reaction suggest that it could be couple to chemoautotrophic growth, although this does not appear to be the case in A. faecalis (37). The crystal structure suggests that arsenite oxidase consists of large (825 amino acids) and small (134 amino acids) subunits. The large subunit is similar to the dimethylsulfoxide family of proteins, and shows the most similarity to the periplasmic nitrate reductase of Desulfovibrio desulfuricans, whereas the small subunit is homologous to parts of some cytochrome complexes (54). Thus in Alcaligenes faecalis, AsIII oxidation is dependent upon activity of an arsenite oxidase and components of the electron transport system. Because the organism appears to gain no energy from AsIII oxidation, the function of the process is probably to detoxify arsenic by converting it to the less toxic form (AsV). Two bacterial isolates (Thermus aquaticus and Thermus thermophilus) obtained from a hot spring are also thought to oxidize arsenite as part of a detoxification process (55). The best characterized, and probably the most widespread, arsenic resistance system in microorganisms is the ars gene system. Early studies observed that plasmid-borne genes mediated arsenite and arsenate resistance in certain bacteria (56), for example in Escherichia coli (57), Staphylococcus aureus (58), and Pseudomonas aeruginosa (59). The underlying genetic system was subsequently characterized in plasmids of Staphylococcus aureus (60) and Staphylococcus xylosus (61) when the ars operon was sequenced. At the most basic level, the ars system consists of a series of three or more genes coding for a transmembrane pump system and an arsenate reductase. Specifically, the operon includes a regulatory gene (arsR), a gene coding for an arsenite-specific transmembrane pump (arsB), and a gene coding for an arsenate reductase (arsC) (62). Arsenite is pumped directly out of the cell by the membrane protein encoded for by arsB, however arsenate must first be reduced to arsenite by the soluble arsenate reductase coded from arsC. arsR codes for a repressor protein that regulates ars gene expression (63). In some bacteria the operon contains other genes: arsA produces an oxyanion-stimulated ATPase (64,65) that couples ATP hydrolysis to the extrusion of arsenicals (and antimonite; 66) through the arsB protein; arsD encodes for a regulatory protein that controls the upper level of ars expression (67); arsH has an uncertain function but is essential for arsenic resistance in some bacteria (68). Thus, resistance to both arsenite and arsenate can be provided for by the series of genes, arsRBC, with increased efficiency at extruding arsenite if arsA is also expressed (Fig. 1). As well as in plasmids of S. aureus and S. xylosus, the ars operon and its role in arsenic resistance has been identified in other bacteria, either in plasmids or as part of the bacterial chromosome. Within the Proteobacteria, ars-mediated arsenic resistance has been observed in Escherichia coli (69,70), Pseudomonas aeruginosa (71), a number of Yersinia species (68,72), Acidiphilum multivorum (73,74), Thiobacillus ferrooxidans (now called Acidithiobacillus ferrooxidans; 75), and Acidithiobacillus caldus (76). Gram-positive bacteria possessing the ars operon system include the Staphylococci and Bacillus subtilis (77). In Saccharomyces cerevisiae, the ACR (or ARR) operon is homologous to ars and also confers arsenic resistance (78), suggesting that the system might be widespread in eukaryotic microorganisms as well as prokaryotes. A search for ars genes in GenBank (79) reveals an even greater number of putative ars genes in Bacteria and Archaea that have not been specifically characterized in terms of arsenic resistance. Thus, the ars system seems to be a relatively common mechanism by which microorganisms can resist the toxic effects of arsenite and arsenate.

(a) arsR arsD arsA arsB arsC

350 bp 350 bp 1700 bp 1200 bp 450 bp (b) arsenate reductase ATP ADP AsIII AsV arsA Cytoplasm

t B i

s P r

a

Environment

AsIII AsV

Figure 1. Conceptual illustration of the genes of the ars operon (a) and the actions of the proteins involved in arsenic resistance (b). Arsenate can be taken up by bacterial cells via none specific phosphate transport, such as the Pit system (b). Arsenate reductase (arsC) converts arsenate to arsenite, which is extruded from the cell via the arsB protein. arsA codes for an ATPase which enhances arsenite extrusion in some organisms.

The arsC gene is particularly interesting in that it codes for an arsenate reductase that catalyzes the transformation of AsV to AsIII. Given the widespread presence of this gene in microorganisms, the activity of this enzyme could be important in the transformation of arsenate to arsenite in natural environments. Arsenate reductase is a soluble protein with approximately 135 amino acid residues (18), and enzymatic reduction of arsenate by the E.coli and Pseudomonas fluorescens arsenate reductases requires the intracellular presence of reduced glutathione and glutaredoxin (80,81). The crystal structure of the E. coli arsC protein shows 138 amino acid residues with cysteine residues being important features of the active site (82). The plasmid-borne arsenate reductase of Staphylococcus aureus is coupled to thioredoxin, thioredoxin reductase, and NADPH to be enzymatically active. As with the arsenate reductases of the Proteobacteria, certain cysteine residues are essential for redox activity (83,84). The same is true for the arsenate reductase of Bacillus subtilis, where three cysteine residues seem to be involved in a "triple cysteine redox relay" mechanism for AsV reduction (85). The ACR2 gene of Saccharomyces cerevisiae is homologous to the arsC of bacteria, and even shows arsenate reductase activity when cloned into a bacterial expression vector and expressed in E. coli (86). Glutaredoxin (but not thioredoxin) is also required and, as with the bacterial enzymes, specific cysteine residues are essential for redox activity (86,87). As well as transforming arsenic between the oxidized and reduced inorganic forms, various microorganisms are capable of converting arsenite of arsenate into organic forms. These methylation reactions can form both volatile and nonvolatile organo-arsenicals such as methylarsines and methylarsonic acids, and can also occur in the tissues of larger organisms. A number of reviews of these processes and the mechanisms underlying them are available (3,88,89).

EVOLUTIONARY RELATIONSHIPS A number of different mechanisms exist by which microorganisms transform inorganic arsenic between the two dominant redox states, arsenite and arsenate. Arsenate reduction can occur as a respiratory process (dissimilatory AsV reduction) or as part of arsenic resistance (through the arsC encoded arsenate reductase). Arsenite oxidation can be coupled to chemoautotrophic growth, or occur as a method of detoxification through the action of either cellular lipids or an arsenite oxidase. The latter method also seems to require components of the electron transport system in addition to the AsIII oxidase enzyme (37,43) suggesting that some similarities to chemoautotrophic AsIII oxidation might exist. Given the relatively narrow distribution of arsenite oxidizing ability for both detoxification (shown for Pseudomonas, Alcaligenes, and Thermus isolates, but only confirmed as enzymatic in Alcaligenes faecalis) and chemoautotrophy (shown for just two species) it is tempting to speculate that the two processes are related. Chemoautotrophic oxidation in Pseudomonas arsenitoxidans was poorly characterized (38), but the NT-26 AsIII oxidizer (a member of the Agrobacterium-Rhizobium branch of alpha Proteobacteria) appeared to possess a periplasmic arsenite oxidase (39). In contrast, the arsenite oxidase of A. faecalis (beta Proteobacteria) is located on the outer cell membrane (37), and the specific activity and optimum pH of the two enzymes differ (39). Thus, although the two types of arsenite oxidase catalyze similar reactions, there are some differences between them in terms of activity and cellular location. While it is possible that chemoautotrophic growth through arsenite oxidation evolved from a method of arsenite oxidation for detoxification (perhaps similar to the suggested development of aerobic respiration from a method for detoxifying oxygen), the scarcity of identified arsenite oxidizers precludes any firm conclusions from being drawn. The similarity in amino acid sequence between the A. faecalis arsenite oxidase and the nitrate reductase of Desulfovibrio desulfuricans (54), does suggest a possible starting point from which to explore AsIII oxidase evolution. Although few dissimilatory arsenate reducers have been discovered, it is interesting that they represent a number of distinct phylogenetic lineages within Domain Bacteria, including Gram-positives (Desulfotomaculum auripigmentum, Bacillus arsenicoselenatis and Bacillus selenitireducens), Proteobacteria (Sulfurospirillum spp.), and a previously unrecognized clade (Chrysiogenes arsenatis), as well as one group of Archaea (Pyrobaculum arsenaticum and Pyrobaculum aerophilum), and a Thermus sp. Thus, the respiratory reduction of arsenate seems to be both rare but also widespread phylogenetically, suggesting that is has either independently evolved multiple times or has been spread through lateral gene transfer. To some extent the former is supported by the different forms of respiratory arsenate reductase that are observed in Sulfurospirillum (a trans-membrane reductase; 32) and C. arsenatis (periplasmic; 31). Thus, unlike dissimilatory sulfate reduction, which probably arose a single time and evolved through divergence (90) and lateral gene transfer (91), there may be multiple origins (and hence multiple methods) of dissimilatory arsenate reduction. The relative rarity of arsenate respiration is possibly explained by the production of the more toxic arsenite as a waste product, which inhibits arsenate respiration in the recognized dissimilatory AsV reducers (21,27). The general scarcity of arsenic in the environment (compared to other potential electron acceptors) would also reduce any selection for dissimilatory arsenate reducers. All of the recognized arsenate respiring organisms are also capable of using at least one other terminal electron acceptor, suggesting that for the majority of these organism dissimilatory arsenate reduction may be just one of many metabolic options, and not an essential process. The reduction of arsenate as part of a detoxification system appears to be relatively common in microorganisms. Representatives of all three domains (Bacteria, Archaea, and Eukarya) possess either an arsC encoded AsV reductase or an enzyme encoded for by a homologous gene. When the sequences of known and putative arsC genes are obtained from GenBank (79), aligned, and a phylogenetic tree constructed, there is a clear separation of the three domains (Fig.2). This suggests that arsC is an evolutionarily old gene and a prototypic arsC gene must have existed before the divergence of Bacteria and Archaea/Eukarya. Furthermore, there is no separation of plasmid-borne and chromosomal arsC genes, suggesting multiple incidents of chromosome-plasmid transfer. It has been suggested that the plasmid-borne arsC in Staphylococcus aureus shows sequence similarities to tyrosine phosphatases, and differs from the Escherichia coli and Saccharomyces cerevisiae arsC genes in its evolutionary origins (92). While the S. aureus arsC gene sequence is noticeably different from those of E. Coli and S. cerevisiae, this would be expected from its position (with the other Gram-positive Bacteria) in the phylogenetic tree (Fig.2) and does not imply different evolutionary origins. An earlier phylogenetic tree showing the relationships between arsenate reductase proteins of seven Bacteria supports this argument (75). For a more thorough examination of the phylogenetic relationships between over 40 arsC genes see a study by Jackson and Dugas (93).

MICROBIAL ARSENIC TRANSFORMATIONS IN NATURAL ENVIRONMENTS In most environments, arsenate is the thermodynamically favorable form under oxygenated conditions, although arsenite is generally more mobile and more toxic (2,3,94). Abiotic transformations can occur between AsV and AsIII, and these may be mediated by changes in redox potential, pH, or sorption processes although such transformations are generally slow (95,96,97,98). Microbial processes can indirectly influence arsenic geochemistry, for example bacterial sulfate reduction can result in AsIII precipitation (99), but the processes describe earlier suggest more direct ways in which microorganisms can transform arsenic in natural environments. Early work on arsenic containing minerals showed that Acidithiobacillus species were capable of releasing arsenic from orpiment, arsenopyrite, and enargite (100,101), through a method of arsenite oxidation. The microbial community in raw sewage, activated sludge and even sewage effluent is also capable of accelerating the oxidation of arsenite under aerobic conditions (102). In lake sediments AsIII oxidation may be largely an abiotic process with microorganisms playing a relatively minor role in some systems (103). Arsenite oxidizing organisms have, however, been isolated from aquatic environments and it is possible that these organisms might be important in the remediation of arsenic contaminated environments (104,105). In hot springs, microbial mat communities are undeniably responsible for rapid rates of AsIII oxidation (106) and molecular studies have shown that changes in the microbial community coincide with changes in arsenite oxidation rates (107). Arsenite oxidizing strains of Thermus have been isolated from such geothermal systems (55), although other microorganisms that have not been tested for arsenite oxidizing ability are also likely to be involved (107). Thus, given the slow rates of abiotic redox transformations, it seems that microbially mediated AsIII oxidation might be an important process in some environments. Staphylococcus aureus pl Staphylococcus xylosus pl Bacillus halodurans* Bacillus subtilis Bacillus geothermoleavorans* Thiobacillus ferrooxidans Mycobacterium tuberculosis* Pseudomonas aeruginosa Pseudomonas putida* Acidiphilum multivorum pl Klebsiella oxytoca pl Escherichia coli Escherichia coli pl Salmonella typhimurium pl Serratia marcescens pl Yersinia enterocolitica pl Archaeoglobus fulgidus* Halobacterium* pl Saccharomyces cerevisiae 0.10

Figure 2. Phylogenetic relationships among arsC genes of various microorganisms. Plasmid borne arsC genes are identified by a pl, and putative arsC genes recognized from genome sequencing projects designated with *. The tree was constructed by nearest neighbor clustering of manually aligned sequences using the ARB software package.

The microbial reduction of AsV to AsIII in natural communities has major implications in that AsIII is much more mobile, and potentially much more toxic (94). Thus, the activity of dissimilatory arsenate reducers or an arsC-encoded arsenate reductase is likely to increase the toxicity of arsenic to other organisms. In the case of arsC-mediated arsenate reduction, this would represent a situation where a single microbial gene might have profound influences on the responses of plant or animal communities to arsenic pollution, potentially shifting the competitive balance to species that are more capable of tolerating high arsenite conditions. Expression of the microbial arsC gene might be a major determinant of community structure in some arsenic contaminated environments, in essence behaving as a keystone gene, analogous in some ways to a keystone species (i.e. it exerts a greater influence on community structure than might be first thought). It is also possible to envision that the expression of microbial arsC genes triggers differential gene expression in other organisms, which must now respond to arsenite rather than arsenate. Genomic cascades might exist, whereby gene expression in one organism triggers differential expression in another organism, which in turn triggers a different response in a third species, and so forth. The interactions between microbial arsenic activity and higher organisms have not been studied at either a physiological or genetic level, but increased interest and research in environmental genomics suggests that bacterial gene expression can have major consequences in some ecosystems. Microbial AsV reduction has been shown to occur in anoxic saltmarsh sediments and inhibition of AsV reduction by respiration blocking chemicals suggested that this was dissimilatory reduction (108). Addition of arsenate to these sediments inhibited dissimilatory sulfate reduction and methanogenesis, presumably because of increased competitiveness of dissimilatory arsenate reducers. In contrast, addition of nitrate inhibited AsV reduction (108). Thus, in arsenic contaminated systems it is likely that dissimilatory AsV reduction is energetically less favorable than denitrification, but more favorable than either sulfate reduction or methanogenesis. This is consistent with theoretical energy yields for the different redox couplets (27) and suggests that, given the common occurrence of the other anaerobic respiratory processes, one of the major factors limiting the prevalence of dissimilatory arsenate respiration is simply the distribution and availability of arsenate. Dissimilatory AsV reduction has also been observed in arsenic contaminated sediments obtained from freshwater lakes (109,110), although dissimilatory sulfate or iron reducers can also be important in mobilizing AsIII from lake sediments (110,111). A recently developed assay for the detection and enumeration of dissimilatory AsV reducers should facilitate further studies of their distribution (112). Arsenate reduction was reported for samples of an agricultural soil that contained naturally high levels of arsenic (113). However, AsV reduction rates for bulk soil were significantly lower than rates observed for an AsV reducing isolate obtained from that soil, suggesting that arsenate reducers represented a small subset of the entire microbial community. The isolate (believed to be a Clostridium species) grew through fermentation, suggesting that arsenate reduction was occurring via a detoxification mechanism. While the Clostridia have not been conclusively characterized in terms of arsenic resistance, at least two species possess genes that are undoubtedly arsC (93), so it is likely that the action of an arsC encoded arsenate reductase was responsible for the observed AsV reduction. Biological reduction of AsV to AsIII was also observed in arsenic contaminated mine tailings and was attributable to arsenic detoxification pathways occurring in various microbial populations within the tailings (114). In both the agricultural soil and the mine tailings, microbial reduction of AsV to AsIII increased arsenic mobility (113,114). It should be noted, however, that in some cases microbial arsenate reduction can reduce arsenic mobility: for example, the dissimilatory AsV reducer, Desulfotomaculum auripigmentum, precipitates As2S3 when respiring on AsV and sulfur, reducing the amount of soluble arsenic (28). Through a variety of detoxification and respiratory mechanisms microorganisms have the ability to greatly influence the speciation of arsenic within the environment. While a number of microorganisms have been identified that possess these mechanisms, these are relatively few compared to the vast number of microorganisms that are still uncharacterized in terms of arsenic biochemistry. A recent phylogenetic analysis of known arsC genes found almost 50 arsC gene sequences to be available in GenBank (93), but this is a minute fraction of the number of bacterial taxa that are thought to exist. Many more species are likely to possess arsC genes, especially given that these genes are not restricted to those organisms that are found solely in arsenic contaminated environments. The numbers of known dissimilatory arsenate reducers or arsenite oxidizers are far lower, but bacteria possessing these abilities might also be much more widespread than is currently thought. Testing for AsV reduction, AsIII oxidation, or even overall arsenic resistance, is not a common procedure when characterizing bacterial isolates, and undoubtedly many of the existing isolates obtained from both clinical and environmental samples will be capable of some form of arsenic activity. Even greater numbers of microbial species remain to be identified. How these organisms transform arsenic in natural environments and influence the distribution and dynamics of larger organisms is currently unknown.

ACKNOWLEDGMENTS Funding for arsenic research in our laboratory has been provided by award LEQSF(2002- 05)-RD-A-25 from the Louisiana Board of Regents Support Fund, Research and Development Program, to Colin R. Jackson.

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