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Arsenic metabolism by microbes in nature and the impacton arsenic remediationShen-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.

Addresses1 Department of Chemical and Environmental Engineering,

University of California, Riverside, CA 92521, United States2 Cell Molecular and Developmental Biology Program,

University of California, Riverside, CA 92521, United States3 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

IntroductionArsenic (As) is a natural and ubiquitous element that

presents in many environmental compartments and is

released through various natural processes or by anthro-

pogenic 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

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 sub-

sequent 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 devel-

opments that are highly selective and economically com-

petitive.

In nature, microbes respond to arsenic in a variety of

different ways. Depending on the species of different

microorganisms, the responses could be chelation, com-

partmentalization, 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 differ-

ent microbes and their impact on environmental arsenic

contamination. In addition, the potential utility of these

natural metabolisms for arsenic remediation will be

discussed.

www.sciencedirect.com Current Opinion in Biotechnology 2009, 20:659–667


Arsenic in the environmentThe 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 mono-

methyl, 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 machi-

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 prokaryotesArsenic 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)

660 Chemical biotechnology

Figure 1

Geo cycling of arsenic.

Current Opinion in Biotechnology 2009, 20:659–667 www.sciencedirect.com


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 mem-

brane in these species.

Basic detoxification mechanisms

Many Gram-negative and Gram-positive bacteria employ

a similar arsenic resistance mechanism based on the ars

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 prokar-

yotes; while E. coli employs GSH and glutaredoxin [28],

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.



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 act-

ing as a transcription repressor and regulating the upper

limit for operon activity, respectively [31]. These regu-

latory 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]. Interest-

ingly, 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 Hydrogeno-phaga 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 resist-

ance mechanism converting the more toxic As(III) to the

less toxic As(V). However, some chemolithotropic bac-

teria 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 methyl-

ated 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 cobala-

mine in many bacterial systems [41]. Methylated forms of

arsenic are volatile and readily released into the environ-

ment where oxidation might convert them back to the

oxidized form As(V). Very little is known about the

demethylation pathways; however, demethylation of mo-

no-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 affi-

nity 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 eukaryotesThe 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 mech-

anism 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




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. cerevisiaeeliminated arsenate resistance [59]. Therefore, the resist-

ance 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 chelat-

ing 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-glutamylcys-

teine 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 Fucusvesiculosus [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

arsenite by engineered S. cerevisiae expressing the Ara-bidopsis 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 eukar-

yotes, incorporation of sulfide to form a more stable, high-

molecular-weight PC–metal–sulfide complex in the

vacuole has been demonstrated [77–79]. In addition,

the formation of metal sulfide particles in Schizosacchar-omyces 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 extru-

sion 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 path-

ways function in a synergistic fashion as the hypersensi-

tivity 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 remediationThe 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 pep-

tides 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 manipu-

lated microorganism.

One of the earliest examples of engineering arsenic

accumulation was demonstrated in plants. The bacterial

enzymes ArsC (arsenate reductase) and g-ECS (GSH




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. thalianawas expressed in E. coli [90�]. This engineered strain

produced PC when exposed to different forms of arsenic,

leading to moderate levels of arsenic accumulation. How-

ever, 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 g-

glutamylcysteine synthetase (GshI*), resulting in higher

PC levels and As accumulation. The significantly

increased PC levels were exploited further by coexpres-

sing 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. colistrain to achieve the highest reported arsenic accumu-

lation 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 com-

plex 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 coexpres-

sing AtPCS and cysteine desulfhydrase, an aminotrans-

ferase 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 PC-

producing cells for biosorption necessitates the use of

zinc for PC induction, making it difficult to implement

in practice. On the other hand, specific arsenic accumu-

lation 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 arsenic-

contaminated site. When the arsenite-specific transporter

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 demon-

strated by the modification of an arsenic resistance operon

using DNA shuffling [96��]. Cells expressing the opti-

mized 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 high-

light the possibility to combine both natural and unna-

tural pathways for hyperarsenic accumulation.

ConclusionArsenic contamination is a major global problem and local

geochemical cycles have been intensified by irresponsi-

ble industrial and mining activities. Fortunately, many

microorganisms have already evolved mechanisms to

cope with this environmental challenge. The fundamen-

tal 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 improve-

ments 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.

AcknowledgementsThe financial support from NSF and U.S. EPA are gratefully acknowledged.

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29.�

Ji GY, Silver S: Reduction of arsenate to arsenite by the Arscprotein of the arsenic resistance operon of Staphylococcusaureus plasmid-Pi258. Proc Natl Acad Sci U S A 1992,89:9474-9478.

An important paper describing arsenate reductase and showing itsimportance for arsenic resistance.

30. Silver S, Phung LT: Genes and enzymes involved in bacterialoxidation and reduction of inorganic arsenic. Appl EnvironMicrobiol 2005, 71:599-608.

31. Rosen BP: Biochemistry of arsenic detoxification. FEBS Lett2002, 529:86-92.

32. Rosen BP: Families of arsenic transporters. Trends Microbiol1999, 7:207-212.

33. Tisa LS, Rosen BP: Molecular characterization of an anionpump — the Arsb protein is the membrane anchor for the Arsaprotein. J Biol Chem 1990, 265:190-194.

34. Leist M, Casey RJ, Caridi D: The management of arsenic wastes:problems and prospects. J Hazard Mater 2000, 76:125-138.

35. Ellis PJ, Conrads T, Hille R, Kuhn P: Crystal structure of the100 kDa arsenite oxidase from Alcaligenes faecalis in twocrystal forms at 1.64 angstrom and 2.03 angstrom. Structure2001, 9:125-132.

36. Santini JM, Sly LI, Schnagl RD, Macy JM: A newchemolithoautotrophic arsenite-oxidizing bacterium isolatedfrom a gold mine: phylogenetic, physiological, and preliminarybiochemical studies. Appl Environ Microbiol 2000, 66:92-97.

37. Krafft T, Macy JM: Purification and characterization of therespiratory arsenate reductase of Chrysiogenes arsenatis. EurJ Biochem 1998, 255:647-653.

38. Saltikov CW, Newman DK: Genetic identification of arespiratory arsenate reductase. Proc Natl Acad Sci U S A 2003,100:10983-10988.

39. Saltikov CW, Cifuentes A, Venkateswaran K, Newman DK: The arsdetoxification system is advantageous but not required forAs(V) respiration by the genetically tractable Shewanellaspecies strain ANA-3. Appl Environ Microbiol 2003,69:2800-2809.

40. Dombrowski PM, Long W, Farley KJ, Mahony JD, Capitani JF, DiToro DM: Thermodynamic analysis of arsenic methylation.Environ Sci Technol 2005, 39:2169-2176.

41. Gadd GM, White C: Microbial treatment of metal pollution — aworking biotechnology. Trends Biotechnol 1993, 11:353-359.

42. Maki T, Hasegawa H, Watarai H, Ueda K: Classification fordimethylarsenate-decomposing bacteria using a restrictfragment length polymorphism analysis of 16S rRNA genes.Anal Sci 2004, 20:61-68.

43. Dey S, Ouellette M, Lightbody J, Papadopoulou B, Rosen BP: AnATP-dependent As(III)-glutathione transport system inmembrane vesicles of Leishmania tarentolae. Proc Natl AcadSci U S A 1996, 93:2192-2197.

44. Silver S, Phung LT: Bacterial heavy metal resistance: newsurprises. Annu Rev Microbiol 1996, 50:753-789.

45. Zhao FJ, Ma JF, Meharg AA, McGrath SP: Arsenic uptake andmetabolism in plants. New Phytol 2009, 181:777-794.

46.��

Zhu YG, Rosen BP: Perspectives for genetic engineering for thephytoremediation of arsenic-contaminated environments:from imagination to reality? Curr Opin Biotechnol 2009,20:220-224.

A concise review about arsenic metabolism in plants and how geneticengineering can improve arsenic phytoremediation. This part is notdetailed in our review.




47. Tripathi RD, Srivastava S, Mishra S, Singh N, Tuli R, Gupta DK,Maathuis FJM: Arsenic hazards: strategies for tolerance andremediation by plants. Trends Biotechnol 2007, 25:158-165.

48. Nidhubhghaill OM, Sadler PJ: The structure and reactivity ofarsenic compounds — biological-activity and drug design.Struct Bond 1991, 78:129-190.

49. Persson BL, Petersson J, Fristedt U, Weinander R, Berhe A,Pattison J: Phosphate permeases of Saccharomycescerevisiae: structure, function and regulation. Biochim BiophysActa Rev Biomembr 1999, 1422:255-272.

50. Wysocki R, Bobrowicz P, Ulaszewski S: The Saccharomycescerevisiae ACR3 gene encodes a putative membraneprotein involved in arsenite transport. J Biol Chem 1997,272:30061-30066.

51. Wysocki R, Chery CC, Wawrzycka D, Van Hulle M, Cornelis R,Thevelein JM, Tamas MJ: The glycerol channel Fps1p mediatesthe uptake of arsenite and antimonite in Saccharomycescerevisiae. Mol Microbiol 2001, 40:1391-1401.

52. Ghosh AS, Kar AK, Kundu M: Impaired imipenem uptakeassociated with alterations in outer membrane proteins andlipopolysaccharides in imipenem-resistant Shigelladysenteriae. J Antimicrob Chemother 1999, 43:195-201.

53. Liu J, Liu YP, Powell DA, Waalkes MP, Klaassen CD: Multidrug-resistance mdr1a/1b double knockout mice are moresensitive than wild type mice to acute arsenic toxicity, withhigher arsenic accumulation in tissues. Toxicology 2002,170:55-62.

54. Liu ZJ, Shen J, Carbrey JM, Mukhopadhyay R, Agre P, Rosen BP:Arsenite transport by mammalian aquaglyceroporins AQP7and AQP9. Proc Natl Acad Sci U S A 2002, 99:6053-6058.

55.��

Liu ZJ, Boles E, Rosen BP: Arsenic trioxide uptake by hexosepermeases in Saccharomyces cerevisiae. J Biol Chem 2004,279:17312-17318.

This paper clearly demonstrated that hexose permeases catalyze themajority of the transport of arsenite in S. cerevisiae.

56. Liu ZJ, Sanchez MA, Jiang X, Boles E, Landfear SM, Rosen BP:Mammalian glucose permease GLUT1 facilitates transport ofarsenic trioxide and methylarsenous acid. Biochem BiophysRes Commun 2006, 351:424-430.

57.�

Ghosh M, Shen J, Rosen BP: Pathways of As(III) detoxificationin Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 1999,96:5001-5006.

This paper reported the two major pathways for arsenic detoxification inS. cerevisiae. These results clearly demonstrated that Arr3p and Ycf1prepresent separated pathways for the detoxification of arsenite in yeast.

58. Mukhopadhyay R, Shi J, Rosen BP: Purification andcharacterization of Acr2p, the Saccharomyces cerevisiaearsenate reductase. J Biol Chem 2000, 275:21149-21157.

59. Mukhopadhyay R, Rosen BP: Saccharomyces cerevisiae ACR2gene encodes an arsenate reductase. FEMS Microbiol Lett1998, 168:127-136.

60. Knowles FC, Benson AA: The biochemistry of arsenic. TrendsBiochem Sci 1983, 8:178-180.

61. Cobbett C, Goldsbrough P: Phytochelatins andmetallothioneins: roles in heavy metal detoxification andhomeostasis. Annu Rev Plant Biol 2002, 53:159-182.

62. Singhal RK, Anderson ME, Meister A: Glutathione, a 1st line ofdefense against cadmium toxicity. FEBS J 1987, 1:220-223.

63. Ngu TT, Easton A, Stillman MJ: Kinetic analysis of arsenic-metalation of human metallothionein: significance of the two-domain structure. J Am Chem Soc 2008, 130:17016-17028.

64. Noctor G: Metabolic signalling in defence and stress: thecentral roles of soluble redox couples. Plant Cell Environ 2006,29:409-425.

65. Wysocki R, Clemens S, Augustyniak D, Golik P, Maciaszczyk E,Tamas MJ, Dziadkowiec D: Metalloid tolerance based onphytochelatins is not functionally equivalent to the arsenitetransporter Acr3p. Biochem Biophys Res Commun 2003,304:293-300.

66. Foyer CH, Noctor G: Oxidant and antioxidant signalling inplants: a re-evaluation of the concept of oxidative stress in aphysiological context. Plant Cell Environ 2005, 28:1056-1071.

67. Guo JB, Dai XJ, Xu WZ, Ma M: Overexpressing GSH1 andAsPCS1 simultaneously increases the tolerance andaccumulation of cadmium and arsenic in Arabidopsis thaliana.Chemosphere 2008, 72:1020-1026.

68. Morris CA, Nicolaus B, Sampson V, Harwood JL, Kille P:Identification and characterization of a recombinantmetallothionein protein from a marine alga, Fucusvesiculosus. Biochem J 1999, 338:553-560.

69. Merrifield ME, Ngu T, Stillman MJ: Arsenic binding to Fucusvesiculosus metallothionein. Biochem Biophys Res Commun2004, 324:127-132.

70. Maitani T, Kubota H, Sato K, Yamada T: The composition ofmetals bound to class III metallothionein (phytochelatin andits desglycyl peptide) induced by various metals in rootcultures of Rubia tinctorum. Plant Physiol 1996, 110:1145-1150.

71. Schmoger MEV, Oven M, Grill E: Detoxification of arsenic byphytochelatins in plants. Plant Physiol 2000, 122:793-801.

72.�

Wunschmann J, Beck A, Meyer L, Letzel T, Grill E, Lendzian KJ:Phytochelatins are synthesized by two vacuolar serinecarboxypeptidases in Saccharomyces cerevisiae. FEBS Lett2007, 581:1681-1687.

This study showed that the vacuolar serine carboxypeptidases CPY andCPC are responsible for PC synthesis in S. cerevisiae.

73. Kim YJ, Chang KS, Lee MR, Kim JH, Lee CE, Jeon YJ, Choi JS,Shin HS, Hwang SB: Expression of tobacco cDNA encodingphytochelatin synthase promotes tolerance to andaccumulation of Cd and As in Saccharomyces cerevisiae. JPlant Biol 2005, 48:440-447.

74. Singh S, Lee W, DaSilva NA, Mulchandani A, Chen W: Enhancedarsenic accumulation by engineered yeast cells expressingArabidopsis thaliana phytochelatin synthase. BiotechnolBioeng 2008, 99:333-340.

75. Thomas D, SurdinKerjan Y: Metabolism of sulfur amino acidsin Saccharomyces cerevisiae. Microbiol Mol Biol Rev 1997,61:503-512.

76. Rochette EA, Bostick BC, Li GC, Fendorf S: Kinetics of arsenatereduction by dissolved sulfide. Environ Sci Technol 2000,34:4714-4720.

77. Kneer R, Zenk MH: The formation of Cd–phytochelatincomplexes in plant cell cultures. Phytochemistry 1997,44:69-74.

78. Mendoza-Cozatl DG, Moreno-Sanchez R: Cd2+ transport andstorage in the chloroplast of Euglena gracilis. Biochim BiophysActa Bioenerget 2005, 1706:88-97.

79. Mendoza-Cozatl DG, Rodriguez-Zavala JS, Rodriguez-Enriquez S, Mendoza-Hernandez G, Briones-Gallardo R,Moreno-Sanchez R: Phytochelatin–cadmium–sulfide high-molecular-mass complexes of Euglena gracilis. FEBS J 2006,273:5703-5713.

80. Dameron CT, Winge DR: Peptide-mediated formation ofquantum semiconductors. Trends Biotechnol 1990, 8:3-6.

81. Krumov N, Oder S, Perner-Nochta I, Angelov A, Posten C:Accumulation of CdS nanoparticles by yeasts in a fed-batchbioprocess. J Biotechnol 2007, 132:481-486.

82. Bobrowicz P, Wysocki R, Owsianik G, Goffeau A, Ulaszewski S:Isolation of three contiguous genes, ACR1, ACR2 and ACR3,involved in resistance to arsenic compounds in the yeastSaccharomyces cerevisiae. Yeast 1997, 13:819-828.

83. Ortiz DF, Ruscitti T, McCue KF, Ow DW: Transport of metal-binding peptide by HMT1, a fission yeast ABC-type vacuolarmembrane-protein. J Biol Chem 1995, 270:4721-4728.

84. Pazirandeh M, Chrisey LA, Mauro JM, Campbell JR, Gaber BP:Expression of the Neurospora-crassa metallothionein gene inEscherichia coli and its effect on heavy-metal uptake. ApplMicrobiol Biotechnol 1995, 43:1112-1117.




85. Li Y, Cockburn W, Kilpatrick J, Whitelam GC: Cytoplasmicexpression of a soluble synthetic mammalian metallothionein-alpha domain in Escherichia coli — enhanced tolerance andaccumulation of cadmium. Mol Biotechnol 2000, 16:211-219.

86. Bae W, Chen W, Mulchandani A, Mehra RK: Enhancedbioaccumulation of heavy metals by bacterial cells displayingsynthetic phytochelatins. Biotechnol Bioeng 2000, 70:518-524.

87. Bae W, Mehra RK, Mulchandani A, Chen W: Genetic engineeringof Escherichia coli for enhanced uptake and bioaccumulationof mercury. Appl Environ Microbiol 2001, 67:5335-5338.

88.��

Dhankher OP, Li YJ, Rosen BP, Shi J, Salt D, Senecoff JF,Sashti NA, Meagher RB: Engineering tolerance andhyperaccumulation of arsenic in plants by combining arsenatereductase and gamma-glutamylcysteine synthetaseexpression. Nat Biotechnol 2002, 20:1140-1145.

An excellent paper showing how the arsenic defense mechanism inmicrobes can be applied to plants.

89. Song WY, Sohn EJ, Martinoia E, Lee YJ, Yang YY, Jasinski M,Forestier C, Hwang I, Lee Y: Engineering tolerance andaccumulation of lead and cadmium in transgenic plants. NatBiotechnol 2003, 21:914-919.

90.�

Sauge-Merle S, Cuine S, Carrier P, Lecomte-Pradines C, Luu DT,Peltier G: Enhanced toxic metal accumulation in engineeredbacterial cells expressing Arabidopsis thaliana phytochelatinsynthase. Appl Environ Microbiol 2003, 69:490-494.

This paper reported the use of phytochelatin producing E. coli as apotential arsenic accumulating biosorbent.

91. Rittle KA, Drever JI, Colberg PJS: Precipitation of arsenic duringbacterial sulfate reduction. Geomicrobiol J 1995, 13:1-11.

92. Wang CL, Maratukulam PD, Lum AM, Clark DS, Keasling JD:Metabolic engineering of an aerobic sulfate reduction

pathway and its application to precipitation of cadmium on thecell surface. Appl Environ Microbiol 2000, 66:4497-4502.

93. Kostal J, Yang R, Wu CH, Mulchandani A, Chen W: Enhancedarsenic accumulation in engineered bacterial cells expressingArsR. Appl Environ Microbiol 2004, 70:4582-4587.

94.��

Singh S, Mulchandani A, Chen W: Highly selective and rapidarsenic removal by metabolically engineered Escherichia colicells expressing Fucus vesiculosus metallothionein. ApplEnviron Microbiol 2008, 74:2924-2927.

For the first time, a metallothionein able to bind to arsenic was utilizedalong with an arsenic transporter for the selective removal of arsenic.Resting cells can be used to completely remove 35 ppb of As(III) in20 min, making this a low-cost option for arsenic removal from water.

95. Dai MH, Copley SD: Genome shuffling improves degradation ofthe anthropogenic pesticide pentachlorophenol bySphingobium chlorophenolicum ATCC 39723. Appl EnvironMicrobiol 2004, 70:2391-2397.

96.��

Crameri A, Dawes G, Rodriguez E, Silver S, Stemmer WPC:Molecular evolution of an arsenate detoxification pathwayDNA shuffling. Nat Biotechnol 1997, 15:436-438.

One of the few reports to show application of irrational approaches forfunctional evolution of the arsenic resistance operon.

97. Chauhan NS, Ranjan R, Purohit HJ, Kalia VC, Sharma R:Identification of genes conferring arsenic resistanceto Escherichia coli from an effluent treatment plantsludge metagenomic library. FEMS Microbiol Ecol 2009,67:130-139.

98. Singh S, Kang SH, Lee W, Mulchandani A, Chen W: SystematicEngineering of Phytochelatin Synthesis and Arsenic Transportfor Enhanced Arsenic Accumulation in E. coli. Biotechnol.Bioeng., in press.



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