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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Available online at www.sciencedirect.com Arsenic metabolism by microbes in nature and the impact on arsenic remediation Shen-Long Tsai1,4, Shailendra Singh1,2,3,4 and Wilfred Chen1 In nature, both prokaryotes and eukaryotes have evolved a wide spectrum of pathways such as oxidation/reduction, compartmentalization, exclusion, and immobilization [16] as the main natural defense mechanisms to arsenic. This review highlights our current understanding of the biochemistry and molecular biology involved in these natural arsenic metabolisms, and some successful examples of engineered microbes by harnessing these natural mechanisms for effective remediation. Addresses 1 Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521, United States 2 Cell Molecular and Developmental Biology Program, University of California, Riverside, CA 92521, United States 3 Current address: One MedImmune Way, Gaithersburg, MD 20878, United States. 4 These authors contributed equally to this work. Corresponding author: Chen, Wilfred ([email protected]) Current Opinion in Biotechnology 2009, 20:659–667 This review comes from a themed issue on Chemical biotechnology Edited by Kazuya Watanabe and George Bennett Available online 31st October 2009 0958-1669/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2009.09.013 Introduction Arsenic (As) is a natural and ubiquitous element that presents in many environmental compartments and is released through various natural processes or by anthropogenic inputs. It is recognized as carcinogenic [1] and chronic exposure to arsenic results in a wide range of adverse health effects [2,3]. Depending on the physical– chemical conditions of the environment, some arsenic compounds can be easily solubilized in water [4] and taken up by microorganisms, resulting in high levels of bioavailability [5]. The most notable case was observed in India and Bangladesh where over 50 million people were exposed to highly contaminated water or food [6]. There have been reports of up to 2 mg/kg of arsenic accumulated in grains [7] and up to 92 mg/kg of arsenic in straws [8]. Arsenic occurs in several oxidation states including arsenate As(V), arsenite As(III), elemental As(0) and arsenide As( III). In natural waters, arsenic is mostly www.sciencedirect.com found in its inorganic forms as trivalent arsenite [As(III)] or pentavalent arsenate [As(V)] [9]. Among them, As(III) is generally considered to be more mobile and more toxic than As(V) [10]. Substitutions for phosphate and subsequent inhibition of oxidative phosphorylation is the major toxicity of pentavalent As(V) [11]. On the other hand, the affinity of trivalent As(III) for protein thiols or vicinal sulfhydryl groups makes them highly toxic. As(III) also acts as an endocrine disruptor by binding to hormone receptors and interferes with normal cell signaling [12]. Arsenite-stimulated generation of Reactive Oxygen Species is known to damage proteins, lipids, and DNA and is probably the direct cause of the carcinogenicity of arsenite [10]. Owing to its extreme toxicity, arsenic is ranked number one on the Environmental Protection Agency’s (EPA) priority list of drinking water contaminants and effective from 2006 the maximum contaminant level for arsenic in drinking water was reduced by the US Environmental Protection Agency from 50 ppb to 10 ppb. According to the Natural Resources Defense Council, over 56 million Americans in the 25 reporting states consume water containing arsenic at levels presenting a potential fatal cancer risk. Several treatment technologies have been applied in laboratory-scale and/or field-scale testing for the removal of arsenic from waters, such as coagulation, filtration, ion exchange, adsorption, and reverse osmosis [13–15]. However, these technologies are either too expensive or ineffective for low arsenic concentration treatment. To comply with the current regulatory limit of 10 ppb would require extensive technological developments that are highly selective and economically competitive. In nature, microbes respond to arsenic in a variety of different ways. Depending on the species of different microorganisms, the responses could be chelation, compartmentalization, exclusion, and immobilization [16]. Understanding the molecular and genetic level of arsenic metabolism will be, therefore, an important knowledge base for developing efficient and selective arsenic bioremediation approaches, which has so far been considered as a cost-effective and environmental friendly way for heavy-metal removal. In this review, we will highlight the natural arsenic metabolism in different microbes and their impact on environmental arsenic contamination. In addition, the potential utility of these natural metabolisms for arsenic remediation will be discussed. Current Opinion in Biotechnology 2009, 20:659–667 Author's personal copy 660 Chemical biotechnology Figure 1 Geo cycling of arsenic. Arsenic in the environment The major source of As contamination is from naturally existing minerals; however, anthropogenic activities have also contributed extensively [17] (Figure 1). As exists in several oxidation states ( 3, 0, +3, and +5), enabling it to mobilize under various environmental conditions and hinders many remediation technologies from efficiently removing it from water. Under oxidizing conditions, As(V) is the dominant form at lower pH while As(III) becomes dominant at higher pH (Figure 1). However, the uncharged form of As(III) [As(OH)3] becomes dominant under reducing environments, which is more toxic and difficult to remove [18]. Nitrate can greatly influence As cycling by oxidizing ferrous iron to produce As-sorbing particles [19]. Elemental arsenic is not common and organic arsines are only found in extremely reducing environments [20]. A number of microorganisms have been shown to methylate arsenic giving rise to monomethyl, dimethyl, and/or tri-methyl derivatives [21]. These methylated arsines are volatile and are rapidly released to the atmosphere. The widespread presence of arsenic has forced different microorganisms to develop arsenic detoxification machiCurrent Opinion in Biotechnology 2009, 20:659–667 neries. Microorganisms have developed various strategies to counter-act arsenic toxicity: firstly, active extrusion of arsenic; secondly, intracellular chelation (in eukaryotes) by various metal-binding peptides including glutathione (GSH), phytochelatins (PCs), and metallothioneins (MTs); thirdly, arsenic transformation to various organic forms which could be potentially less toxic (Figure 2). In the forthcoming sections we will discuss in detail about these mechanisms in prokaryotes and eukaryotes. Arsenic metabolism by prokaryotes Arsenic uptake pathways Arsenic could potentially act as an electron donor or acceptor and be part of the electron transport chain in some bacteria. However, specific uptake transporters have not evolved because of the extreme toxicity [22]. As(III) and As(V) are typically taken up using the glycerol and phosphate transporter, respectively, because of their structure chemical similarities to As(III) and As(V). In E. coli, for example, two phosphate transporters (Pit and Pst) are used for As(V) uptake, with Pst being the dominant uptake pathway [23]. The uncharged As(III) is taken up by the glycerol transporter GlpF [24], a member of glycerol channels of the major intrinsic protein (MIP) www.sciencedirect.com Author's personal copy Arsenic metabolism by microbes in nature Tsai, Singh and Chen 661 Figure 2 Schematic representations of (a) prokaryotes’ and (b) eukaryotes’ processes involved in arsenic metabolism in the environment. In both cases, arsenic enters the cells through transporters. Arsenate is reduced to arsenite by a reductase, which further extrudes out of the cell by a specific membrane pump. In eukaryotes, arsenite can also be detoxified by complexation with Cys-rich peptides such as phytochelatins and storage in the vacuole. In addition, arsenite can serve as an electron donor by oxidation to arsenate. Arsenate can be used as the ultimate electron acceptor during respiration and inorganic arsenic can also be transformed into organic species in a methylation cascade. family. Mutation in GlpF resulted in As(III)-tolerant E. coli strains [24]. GlpF homologs have been identified in Leishmania major [25] or Pseudomonas putida, and are likely to facilitate As(III) transport across the cell membrane in these species. Basic detoxification mechanisms Many Gram-negative and Gram-positive bacteria employ a similar arsenic resistance mechanism based on the ars www.sciencedirect.com operon (typically arsRDABC) encoded either on the chromosome or on plasmids [26]. In both cases, there are two necessary components: a reductase enzyme (ArsC) for the reduction of As(V) to As(III), which is subsequently extruded using an As(III) expulsion pump (ArsB). Additional ars genes have recently been found suggesting parallel evolution and complex regulations [27]. The source of reducing power varies among prokaryotes; while E. coli employs GSH and glutaredoxin [28], Current Opinion in Biotechnology 2009, 20:659–667 Author's personal copy 662 Chemical biotechnology Staphylococcus aureus utilizes thioredoxin [29]. During the reduction step, arsenate binds to a recognition domain comprising of Arg Residues, resulting in a disulfide bond between the cysteine residues on ArsC and the reducing equivalents. Reduction of the disulfide bond via electron transfer results in As(V) reduction into As(III) [30]. ArsR and ArsD are regulatory components primarily acting as a transcription repressor and regulating the upper limit for operon activity, respectively [31]. These regulatory proteins have extremely high affinity for As(III) and bind via their cysteine residues, resulting in altered DNA binding for transcriptional activation [32]. ArsA is an ATPase that assists ArsB in As(III) efflux by providing the necessary energy via ATP hydrolysis [33]. Interestingly, the relatively less toxic As(V) is converted to the more toxic As(III) before efflux; it is possible that the As(III) efflux system was first evolved under reducing environments, which was subsequently coupled with As(V) reduction to accommodate As(V) toxicity once the earth atmosphere became more oxidized [31]. Arsenite oxidation/reduction Oxidization of As(III) can be important for arsenic removal since As(V) is less soluble and is much more effectively removed by physico-chemical methods [34]. In nature, microorganisms carry out As(III) oxidation using the enzyme As(III) oxidase, which is classified as a member of the DMSO reductase family and was only recently identified and sequenced [35]. Most arsenite oxidases, like the one (AoxAB) isolated from Hydrogenophaga sp. strain NT-14, work as a heterodimer (from the gene aoxAB) and contain Fe and molybdenum as part of the catalytic unit [35]. Phylogenetic lineages suggested that the enzyme had an early origin primarily as a resistance mechanism converting the more toxic As(III) to the less toxic As(V). However, some chemolithotropic bacteria do extract energy from oxidizing arsenite [36]. In addition to the intracellular reduction of As(V) using the arsenate reductase, arsenate reduction can also be part of the anaerobic arsenate respiration in some bacteria (e.g. Shewanella sp. strain ANA-3) [37], where arsenate acts as a terminal electron acceptor. This respiratory arsenate reductase (ArrA and ArrB) is membrane-bound like other members of the electron transport chain [38] and contains a molybdopterin center in ArrA and a Fe–S center in ArrB. A Shewanella sp. strain ANA-3 containing a mutation in the arrAB gene cluster is unable to grow on As(V) [39]. Methylation/demethylation Methylation is originally thought as a detoxification step; however, recent literature suggests that not all methylated arsenic products are less toxic [20]. The primary mode of arsines and methyl arsenicals generation is As(V) reduction and subsequent oxidative addition of methyl groups [40] from various sources such as methyl cobalaCurrent Opinion in Biotechnology 2009, 20:659–667 mine in many bacterial systems [41]. Methylated forms of arsenic are volatile and readily released into the environment where oxidation might convert them back to the oxidized form As(V). Very little is known about the demethylation pathways; however, demethylation of mono-methyl and dimethyl arsenic compounds have been demonstrated and even the use of methylated arsenicals as a carbon source is possible [42]. The understanding of these mechanisms will not only shed light on the arsenic mobilization but may also open up new horizons in metabolic pathway engineering to exploit those pathways for arsenic remediation. Arsenic efflux machinery As(III) can either be extruded via an arsenite carrier protein or via an arsenite efflux pump ArsB. The first approach exploits the membrane potential for energy while the latter utilizes the energy provided by the ATPase ArsA via ATP hydrolysis [43]. The majority of prokaryote systems employ the ArsA/B system while some bacteria can suffice only with ArsB. Reduced affinity for As(III) after cysteine residue mutations suggests that ArsA activation by As(III) occurs via metal-thiolate complex formed among three cysteine residues and As(III) [44]. Arsenic metabolism by eukaryotes The arsenic metabolism by plant cells has recently been reviewed elaborately elsewhere [45,46,47]. In this part, we will only highlight the metabolism of yeast, fungi, and algae when exposed to arsenic compounds. These would include the mechanism of arsenic uptake, metabolism, and efflux. Arsenic uptake Arsenic uptake by Saccharomyces cerevisiae occurs through three different transport systems. The pentavalent arsenate, because of the similarity to phosphate [48], is taken up through a phosphate transporter, Pho87p [49]. In addition, two transporter systems for the trivalent arsenite have been identified. Similar to bacterial systems, arsenite is taken up by an aquaglyceroporin Fps1p, a glycerol transporter [50,51]. Disruption of the FPS1 gene resulted in a reduction in arsenite uptake, which confirms the important role of the Fpslp channel for arsenite uptake [52,53]. However, the FPS1 deletion strain was still sensitive to arsenite in the absence of glucose suggesting the existence of an additional transport mechanism related to glucose uptake [54]. In 2004, Liu et al. found that a class of hexose permeases (Hxt1p to Hxt1 plus Gal2p) of S. cerevisiae adventitiously catalyzed the uptake of arsenite [55]. Arsenite uptake was reduced by 80% in the presence of glucose even when FPS1 was deleted, confirming that the hexose transporters are mainly responsible for arsenite uptake. Recently, the same group demonstrated that a mammalian glucose www.sciencedirect.com Author's personal copy Arsenic metabolism by microbes in nature Tsai, Singh and Chen 663 permease GLUT also catalyzed the uptake of arsenite when heterogeneously expressed in yeast [56]. Arsenic metabolism Once arsenic enters the cells, a series of detoxification steps are used to reduce the acute cytotoxic effects. The most comprehensive mechanism of arsenic tolerance in yeast is provided by three contiguous gene clusters: ARR1, ARR2, and ARR3. ARR1 encodes a transcription factor that regulates the transcription of arsenate reductase Arr2p and the arsenite extrusion transporter Arr3p [57]. After arsenate is transported inside the yeast cells, arsenate is reduced to arsenite by an arsenate reductase Arr2p [58]. However, unlike the bacterial arsenate reductase ArsC (a 141-residue monomer), Arr2p is a homodimer of two 130-residue monomers. It has been shown that the yeast gene ARR2 can complement an E. coli strain with a deletion of the chromosomal arsC gene [58]. In addition, the disruption of ARR2 in S. cerevisiae eliminated arsenate resistance [59]. Therefore, the resistance of cultured cells for arsenic toxicity has long been thought to reduce the accumulation of arsenite since no arsenate efflux transporter has been found so far. To date Arr2p is still the sole arsenate reductase in eukaryote and no ARR2 gene has yet been found with the fission yeast S. pombe or other fungi. Intracellular sequestration Questions have been raised as to why cells were designed to reduce arsenate to the more reactive arsenite, which is at least 100 times more toxic [60]. The answer is that by taking advantage of the chemical reactivity, arsenite can bind to many intracellular chelating proteins or peptides containing thiol ligands, such as GSH, PCs, and MTs to form inactive complexes [61–63]. GSH is a major reservoir of nonprotein thiols [64], and the availability of GSH is important in arsenate reduction as well as in arsenite transport into the vacuoles [65]. Guo et al. showed that overexpression of the S. cerevisiae GSH1 gene encoding a g-glutamylcysteine synthetase (g-ECS), the first enzyme in the GSH biosynthesis pathway [66], elevated the tolerance and accumulation of arsenic in Arabidopsis thaliana [67]. MTs belong to a family of cysteine-rich proteins with the unique ability to form stable metal-thiolate clusters with their two metal-binding, cysteine-rich domains [68], and are the major metal-binding ligands in animals. Although As-binding MTs have been described in the alga Fucus vesiculosus [69], none have been isolated in bacteria. On the other hand, PCs are small enzymatically synthesized cysteine-rich peptides widely found in plants and yeasts, and have been shown to bind arsenite efficiently [70,71,72]. Overexpression of a tobacco PC synthase in yeast S. cerevisiae resulted in increased tolerance for Cd and As [73] without any enhancement in accumulation. However, our lab reported enhanced accumulation of www.sciencedirect.com arsenite by engineered S. cerevisiae expressing the Arabidopsis thaliana PC synthase [74]. For some yeasts such as Candida glabrata, extracellular sulfate is metabolized to sulfide [75], which acts as an electron donor for arsenate reduction [76]. In some eukaryotes, incorporation of sulfide to form a more stable, highmolecular-weight PC–metal–sulfide complex in the vacuole has been demonstrated [77–79]. In addition, the formation of metal sulfide particles in Schizosaccharomyces pombe and Candida glabrata is also part of their intracellular detoxification [80,81]. Arsenic resistant via intracellular and extracellular transport S. cerevisiae has two different mechanisms to reduce arsenite cytotoxicity. One is through the arsenite extrusion pump Arr3p, which transports the As(III)–GSH complexes out of the membrane. Overexpression of Arr3p in yeast results in As(III) tolerance [82], while deletion of ARR3 results in sensitivity to both As(V) and As(III) [50– 52]. In addition to the membrane efflux pump, a second mechanism of arsenic resistance is via the transport of GSH-conjugated arsenite into the vacuole [52]. The Ycf1p protein associated with the vacuolar membrane is a member of the ABC transporter superfamily that is responsible for the ATP-dependent transport of a wide range of GSH-conjugated substrates (such as As(GS)3) into the vacuole. Both of these mechanisms are essential for survival at high arsenic concentrations as deletion of the YCF1 gene results in arsenic hypersensitivity. Further genetic analyses support the notion that these two pathways function in a synergistic fashion as the hypersensitivity of yeast cells to arsenic is additive in a mutant lacking both genes. While S. cerevisiae transports the GSH–As complex into the vacuole, S. pombe transports high-molecular PC–Cd–S complexes into the vacuole via the Hmt1 transporter [83]. Engineered microbes for arsenic remediation The use of engineered microbes as selective biosorbents is an attractive green technology for the low-cost and efficient removal of arsenic [74]. Although efforts have been reported in engineering microbes for the removal of cadmium or mercury by expressing metal-binding peptides such as human MTs [84,85] or synthetic peptides [86,87], the relatively low specificity and affinity of these peptides for arsenic make them ineffective for arsenic remediation. Development of an arsenic accumulating microbe should comprise the ability to firstly, modify the naturally existing defense mechanisms and secondly, develop novel or hybrid pathways into one easily manipulated microorganism. One of the earliest examples of engineering arsenic accumulation was demonstrated in plants. The bacterial enzymes ArsC (arsenate reductase) and g-ECS (GSH Current Opinion in Biotechnology 2009, 20:659–667 Author's personal copy 664 Chemical biotechnology synthase) were expressed in A. thaliana, resulting in the accumulation of As(V) as GSH–As complexes [88]. A similar effort was subsequently reported by expressing the yeast YCF1 in A. thaliana for enhanced As storage in the vacuole [89]. These reports open up the possibility of engineering metabolisms and pathways for arsenic sequestration. On the basis of these early examples, similar efforts have been demonstrated with engineered microbes. In one case, the PC synthase from A. thaliana was expressed in E. coli [90]. This engineered strain produced PC when exposed to different forms of arsenic, leading to moderate levels of arsenic accumulation. However, the level of GSH, a key PC precursor, became limiting for higher level of PC production and arsenic accumulation. Our lab has recently expressed the PC synthase from S. pombe (SpPCS) in E. coli, resulting in higher As accumulation [98]. PC production was further increased by coexpressing a feedback desensitized gglutamylcysteine synthetase (GshI*), resulting in higher PC levels and As accumulation. The significantly increased PC levels were exploited further by coexpressing an arsenic transporter GlpF, leading to an additional 1.5-fold higher As accumulation. These engineering steps were finally combined in an arsenic efflux deletion E. coli strain to achieve the highest reported arsenic accumulation in E. coli of 16.8 mmol/g cells. Naturally, sulfur reducing bacteria are used for As(V) precipitation by the formation of insoluble sulfide complex with H2S [91]. Metabolic engineering approaches have been utilized for intracellular production of H2S in bacteria, leading to higher cadmium accumulation [92]. Our lab has recently engineered a yeast strain coexpressing AtPCS and cysteine desulfhydrase, an aminotransferase that converts cysteine into hydrogen sulfide under aerobic condition, to elevate the accumulation of arsenic by the formation of PC–metal–sulfide complexes (Tsai, 2009, unpublished). The use of resting cells as a high-affinity biosorbent for arsenic removal has also been exploited. By expressing AtPCS in S. cerevisiae, which naturally has a higher level of GSH, the engineered yeast strain accumulated high levels of arsenic and was effective in removing arsenic in resting cell cultures [74]. However, the utility of PCproducing cells for biosorption necessitates the use of zinc for PC induction, making it difficult to implement in practice. On the other hand, specific arsenic accumulation was achieved in E. coli cells by overexpressing the arsenic-specific regulatory protein ArsR. Resting cells expressing ArsR were effective in removing 50 ppb of As(III) within one hour [93]. The concept of resting cell sorbents has been extended to the use of a naturally occurring As-binding MT [94]. Singh and coworkers developed an engineered E. coli strain expressing the fMT from F. vesiculosus [69] isolated from an arseniccontaminated site. When the arsenite-specific transporter Current Opinion in Biotechnology 2009, 20:659–667 GlpF was co-overexpressed with fMT, the engineered E. coli accumulated arsenic at high levels even in the presence of 10-fold excess amounts of competing heavy metals [94]. Resting cells were able to completely remove 35 ppb of As(III) within 20 min, making this an attractive low-cost option for arsenic remediation. New irrational approaches such as directed evolution, genome shuffling, and metagenomic studies can be used for developing new arsenic resistant pathways that are suitable for arsenic remediation [95]. This was demonstrated by the modification of an arsenic resistance operon using DNA shuffling [96]. Cells expressing the optimized operon grew in 0.5 M arsenate, a 40-fold increase in resistance. Along the same line, Chauhan and coworkers constructed a metagenomic library from an industrial effluent treatment plant sludge, and identified a novel As(V) resistance gene (arsN) encoding a protein similar to acetyltransferases. Overexpression of ArsN led to higher arsenic resistance in E. coli [97]. These examples highlight the possibility to combine both natural and unnatural pathways for hyperarsenic accumulation. Conclusion Arsenic contamination is a major global problem and local geochemical cycles have been intensified by irresponsible industrial and mining activities. Fortunately, many microorganisms have already evolved mechanisms to cope with this environmental challenge. The fundamental understanding of the biochemistry and metabolic pathways involved in arsenic resistance are now being gradually translated into strategies for engineering microbes for effective arsenic remediation. Although the initial reports are promising, substantial improvements are necessary to move these approaches from the bench to practice. In this respect, new tools in synthetic biology will certainly enable us to increase our efforts toward this end. Acknowledgements The financial support from NSF and U.S. EPA are gratefully acknowledged. References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1. Rosen P: Theoretical significance of arsenic as a carcinogen. J Theor Biol 1971, 32:425. 2. Chen Y, Factor-Litvak P, Parvez F, Graziano J, Howe G, Ahsan H: Association between lower-dose arsenic exposure from drinking water and high blood pressure in Bangladesh. 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