Download This article appeared in a journal published by Elsevier. The... copy is furnished to the author for internal non-commercial research

This article appeared in a journal published by Elsevier. The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. 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. Am J
Epidemiol 2005, 161:S30-S130.
3.
Tapio S, Grosche B: Arsenic in the aetiology of cancer. Mutat
Res Rev Mutat Res 2006, 612:215-246.
4.
Oremland RS, Kulp TR, Blum JS, Hoeft SE, Baesman S, Miller LG,
Stolz JF: A microbial arsenic cycle in a salt-saturated, extreme
environment. Science 2005, 308:1305-1308.
www.sciencedirect.com
Author's personal copy
Arsenic metabolism by microbes in nature Tsai, Singh and Chen 665
5.
Bryan CG, Marchal M, Battaglia-Brunet F, Kugler V, LemaitreGuillier C, Lievremont D, Bertin PN, Arsene-Ploetze F: Carbon and
arsenic metabolism in Thiomonas strains: differences
revealed diverse adaptation processes. BMC Microbiol 2009,
9:127S.
6.
Hossain MF: Arsenic contamination in Bangladesh — an
overview. Agric Ecosyst Environ 2006, 113:1-16.
7.
Islam FS, Gault AG, Boothman C, Polya DA, Charnock JM,
Chatterjee D, Lloyd JR: Role of metal-reducing bacteria in
arsenic release from Bengal delta sediments. Nature 2004,
430:68-71.
8.
Abedin MJ, Cresser MS, Meharg AA, Feldmann J, CotterHowells J: Arsenic accumulation and metabolism in rice (Oryza
sativa L.). Environ Sci Technol 2002, 36:962-968.
9.
Cullen WR, Reimer KJ: Arsenic speciation in the environment.
Chem Rev 1989, 89:713-764.
10. Liu SX, Athar M, Lippai I, Waldren C, Hei TK: Induction of
oxyradicals by arsenic: implication for mechanism of
genotoxicity. Proc Natl Acad Sci U S A 2001, 98:1643-1648.
11. Goyer RA, Clarkson TW: Toxic effects of metals. Casarett &
Doull’s Toxicology: The basic of poisons. 6th ed. New York:
McGraw Hill; 2001.
12. Kaltreider RC, Davis AM, Lariviere JP, Hamilton JW: Arsenic
alters the function of the glucocorticoid receptor as a
transcription factor. Environ Health Perspect 2001, 109:245-251.
13. Kartinen EO, Martin CJ: An overview of arsenic removal
processes. Desalination 1995, 103:79-88.
14. Zouboulis AI, Katsoyiannis IA: Arsenic removal using iron oxide
loaded alginate beads. Ind Eng Chem Res 2002, 41:6149-6155.
15. DeMarco MJ, SenGupta AK, Greenleaf JE: Arsenic removal
using a polymeric/inorganic hybrid sorbent. Water Res 2003,
37:164-176.
16. Di Toppi LS, Gabbrielli R: Response to cadmium in higher
plants. Environ Exp Bot 1999, 41:105-130.
17. Nordstrom DK: Public health — worldwide occurrences of
arsenic in ground water. Science 2002, 296:2143-2145.
18. Smedley PL, Kinniburgh DG: A review of the source, behavior
and distribution of arsenic in natural waters. Appl Geochem
2002, 17:517-568.
19. Senn DB, Hemond HF: Nitrate controls on iron and arsenic in an
urban lake. Science 2002, 296:2373-2376.
20. Bentley R, Chasteen TG: Microbial methylation of metalloids:
arsenic, antimony, and bismuth. Microbiol Mol Biol Rev 2002,
66:250-271.
21. Qin J, Rosen BP, Zhang Y, Wang GJ, Franke S, Rensing C: Arsenic
detoxification and evolution of trimethylarsine gas by a
microbial arsenite S-adenosylmethionine methyltransferase.
Proc Natl Acad Sci U S A 2006, 103:2075-2080.
22. Stolz JE, Basu P, Santini JM, Oremland RS: Arsenic and selenium
in microbial metabolism. Annu Rev Microbiol 2006, 60:107-130.
23. Rosen BR, Liu ZJ: Transport pathways for arsenic and
selenium: a minireview. Environ Int 2009, 35:512-515.
A concise review regarding the transporter systems of E. coli, S. cerevisiae, and mammals.
24. Sanders OI, Rensing C, Kuroda M, Mitra B, Rosen BP: Antimonite
is accumulated by the glycerol facilitator GlpFin Escherichia
coli. J Bacteriol 1997, 179:3365-3367.
25. Gourbal B, Sonuc N, Bhattacharjee H, Legare D, Sundar S,
Ouellette M, Rosen BP, Mukhopadhyay R: Drug uptake and
modulation of drug resistance in Leishmania by an
aquaglyceroporin. J Biol Chem 2004, 279:31010-31017.
26. Xu C, Zhou TQ, Kuroda M, Rosen BP: Metalloid resistance
mechanisms in prokaryotes. J Biochem 1998, 123:16-23.
27. Butcher BG, Deane SM, Rawlings DE: The chromosomal arsenic
resistance genes of Thiobacillus ferrooxidans have an unusual
arrangement and confer increased arsenic and antimony
www.sciencedirect.com
resistance to Escherichia coli. Appl Environ Microbiol 2000,
66:1826-1833.
28. Shi J, Vlamis-Gardikas V, Aslund F, Holmgren A, Rosen BP:
Reactivity of glutaredoxins 1, 2, and 3 from Escherichia coli
shows that glutaredoxin 2 is the primary hydrogen donor to
ArsC-catalyzed arsenate reduction. J Biol Chem 1999,
274:36039-36042.
29. Ji GY, Silver S: Reduction of arsenate to arsenite by the Arsc
protein of the arsenic resistance operon of Staphylococcus
aureus plasmid-Pi258. Proc Natl Acad Sci U S A 1992,
89:9474-9478.
An important paper describing arsenate reductase and showing its
importance for arsenic resistance.
30. Silver S, Phung LT: Genes and enzymes involved in bacterial
oxidation and reduction of inorganic arsenic. Appl Environ
Microbiol 2005, 71:599-608.
31. Rosen BP: Biochemistry of arsenic detoxification. FEBS Lett
2002, 529:86-92.
32. Rosen BP: Families of arsenic transporters. Trends Microbiol
1999, 7:207-212.
33. Tisa LS, Rosen BP: Molecular characterization of an anion
pump — the Arsb protein is the membrane anchor for the Arsa
protein. 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 the
100 kDa arsenite oxidase from Alcaligenes faecalis in two
crystal forms at 1.64 angstrom and 2.03 angstrom. Structure
2001, 9:125-132.
36. Santini JM, Sly LI, Schnagl RD, Macy JM: A new
chemolithoautotrophic arsenite-oxidizing bacterium isolated
from a gold mine: phylogenetic, physiological, and preliminary
biochemical studies. Appl Environ Microbiol 2000, 66:92-97.
37. Krafft T, Macy JM: Purification and characterization of the
respiratory arsenate reductase of Chrysiogenes arsenatis. Eur
J Biochem 1998, 255:647-653.
38. Saltikov CW, Newman DK: Genetic identification of a
respiratory arsenate reductase. Proc Natl Acad Sci U S A 2003,
100:10983-10988.
39. Saltikov CW, Cifuentes A, Venkateswaran K, Newman DK: The ars
detoxification system is advantageous but not required for
As(V) respiration by the genetically tractable Shewanella
species strain ANA-3. Appl Environ Microbiol 2003,
69:2800-2809.
40. Dombrowski PM, Long W, Farley KJ, Mahony JD, Capitani JF, Di
Toro DM: Thermodynamic analysis of arsenic methylation.
Environ Sci Technol 2005, 39:2169-2176.
41. Gadd GM, White C: Microbial treatment of metal pollution — a
working biotechnology. Trends Biotechnol 1993, 11:353-359.
42. Maki T, Hasegawa H, Watarai H, Ueda K: Classification for
dimethylarsenate-decomposing bacteria using a restrict
fragment length polymorphism analysis of 16S rRNA genes.
Anal Sci 2004, 20:61-68.
43. Dey S, Ouellette M, Lightbody J, Papadopoulou B, Rosen BP: An
ATP-dependent As(III)-glutathione transport system in
membrane vesicles of Leishmania tarentolae. Proc Natl Acad
Sci U S A 1996, 93:2192-2197.
44. Silver S, Phung LT: Bacterial heavy metal resistance: new
surprises. Annu Rev Microbiol 1996, 50:753-789.
45. Zhao FJ, Ma JF, Meharg AA, McGrath SP: Arsenic uptake and
metabolism in plants. New Phytol 2009, 181:777-794.
46. Zhu YG, Rosen BP: Perspectives for genetic engineering for the
phytoremediation 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 genetic
engineering can improve arsenic phytoremediation. This part is not
detailed in our review.
Current Opinion in Biotechnology 2009, 20:659–667
Author's personal copy
666 Chemical biotechnology
47. Tripathi RD, Srivastava S, Mishra S, Singh N, Tuli R, Gupta DK,
Maathuis FJM: Arsenic hazards: strategies for tolerance and
remediation by plants. Trends Biotechnol 2007, 25:158-165.
66. Foyer CH, Noctor G: Oxidant and antioxidant signalling in
plants: a re-evaluation of the concept of oxidative stress in a
physiological context. Plant Cell Environ 2005, 28:1056-1071.
48. Nidhubhghaill OM, Sadler PJ: The structure and reactivity of
arsenic compounds — biological-activity and drug design.
Struct Bond 1991, 78:129-190.
67. Guo JB, Dai XJ, Xu WZ, Ma M: Overexpressing GSH1 and
AsPCS1 simultaneously increases the tolerance and
accumulation of cadmium and arsenic in Arabidopsis thaliana.
Chemosphere 2008, 72:1020-1026.
49. Persson BL, Petersson J, Fristedt U, Weinander R, Berhe A,
Pattison J: Phosphate permeases of Saccharomyces
cerevisiae: structure, function and regulation. Biochim Biophys
Acta Rev Biomembr 1999, 1422:255-272.
68. Morris CA, Nicolaus B, Sampson V, Harwood JL, Kille P:
Identification and characterization of a recombinant
metallothionein protein from a marine alga, Fucus
vesiculosus. Biochem J 1999, 338:553-560.
50. Wysocki R, Bobrowicz P, Ulaszewski S: The Saccharomyces
cerevisiae ACR3 gene encodes a putative membrane
protein involved in arsenite transport. J Biol Chem 1997,
272:30061-30066.
69. Merrifield ME, Ngu T, Stillman MJ: Arsenic binding to Fucus
vesiculosus metallothionein. Biochem Biophys Res Commun
2004, 324:127-132.
51. Wysocki R, Chery CC, Wawrzycka D, Van Hulle M, Cornelis R,
Thevelein JM, Tamas MJ: The glycerol channel Fps1p mediates
the uptake of arsenite and antimonite in Saccharomyces
cerevisiae. Mol Microbiol 2001, 40:1391-1401.
70. Maitani T, Kubota H, Sato K, Yamada T: The composition of
metals bound to class III metallothionein (phytochelatin and
its desglycyl peptide) induced by various metals in root
cultures of Rubia tinctorum. Plant Physiol 1996, 110:1145-1150.
52. Ghosh AS, Kar AK, Kundu M: Impaired imipenem uptake
associated with alterations in outer membrane proteins and
lipopolysaccharides in imipenem-resistant Shigella
dysenteriae. J Antimicrob Chemother 1999, 43:195-201.
71. Schmoger MEV, Oven M, Grill E: Detoxification of arsenic by
phytochelatins in plants. Plant Physiol 2000, 122:793-801.
53. Liu J, Liu YP, Powell DA, Waalkes MP, Klaassen CD: Multidrugresistance mdr1a/1b double knockout mice are more
sensitive than wild type mice to acute arsenic toxicity, with
higher 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 AQP7
and AQP9. Proc Natl Acad Sci U S A 2002, 99:6053-6058.
55. Liu ZJ, Boles E, Rosen BP: Arsenic trioxide uptake by hexose
permeases in Saccharomyces cerevisiae. J Biol Chem 2004,
279:17312-17318.
This paper clearly demonstrated that hexose permeases catalyze the
majority 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 of
arsenic trioxide and methylarsenous acid. Biochem Biophys
Res Commun 2006, 351:424-430.
72. Wunschmann J, Beck A, Meyer L, Letzel T, Grill E, Lendzian KJ:
Phytochelatins are synthesized by two vacuolar serine
carboxypeptidases in Saccharomyces cerevisiae. FEBS Lett
2007, 581:1681-1687.
This study showed that the vacuolar serine carboxypeptidases CPY and
CPC 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 encoding
phytochelatin synthase promotes tolerance to and
accumulation of Cd and As in Saccharomyces cerevisiae. J
Plant Biol 2005, 48:440-447.
74. Singh S, Lee W, DaSilva NA, Mulchandani A, Chen W: Enhanced
arsenic accumulation by engineered yeast cells expressing
Arabidopsis thaliana phytochelatin synthase. Biotechnol
Bioeng 2008, 99:333-340.
75. Thomas D, SurdinKerjan Y: Metabolism of sulfur amino acids
in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 1997,
61:503-512.
57. Ghosh M, Shen J, Rosen BP: Pathways of As(III) detoxification
in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 1999,
96:5001-5006.
This paper reported the two major pathways for arsenic detoxification in
S. cerevisiae. These results clearly demonstrated that Arr3p and Ycf1p
represent separated pathways for the detoxification of arsenite in yeast.
76. Rochette EA, Bostick BC, Li GC, Fendorf S: Kinetics of arsenate
reduction by dissolved sulfide. Environ Sci Technol 2000,
34:4714-4720.
58. Mukhopadhyay R, Shi J, Rosen BP: Purification and
characterization of Acr2p, the Saccharomyces cerevisiae
arsenate reductase. J Biol Chem 2000, 275:21149-21157.
78. Mendoza-Cozatl DG, Moreno-Sanchez R: Cd2+ transport and
storage in the chloroplast of Euglena gracilis. Biochim Biophys
Acta Bioenerget 2005, 1706:88-97.
59. Mukhopadhyay R, Rosen BP: Saccharomyces cerevisiae ACR2
gene encodes an arsenate reductase. FEMS Microbiol Lett
1998, 168:127-136.
60. Knowles FC, Benson AA: The biochemistry of arsenic. Trends
Biochem Sci 1983, 8:178-180.
61. Cobbett C, Goldsbrough P: Phytochelatins and
metallothioneins: roles in heavy metal detoxification and
homeostasis. Annu Rev Plant Biol 2002, 53:159-182.
62. Singhal RK, Anderson ME, Meister A: Glutathione, a 1st line of
defense against cadmium toxicity. FEBS J 1987, 1:220-223.
63. Ngu TT, Easton A, Stillman MJ: Kinetic analysis of arsenicmetalation of human metallothionein: significance of the twodomain structure. J Am Chem Soc 2008, 130:17016-17028.
64. Noctor G: Metabolic signalling in defence and stress: the
central roles of soluble redox couples. Plant Cell Environ 2006,
29:409-425.
65. Wysocki R, Clemens S, Augustyniak D, Golik P, Maciaszczyk E,
Tamás MJ, Dziadkowiec D: Metalloid tolerance based on
phytochelatins is not functionally equivalent to the arsenite
transporter Acr3p. Biochem Biophys Res Commun 2003,
304:293-300.
Current Opinion in Biotechnology 2009, 20:659–667
77. Kneer R, Zenk MH: The formation of Cd–phytochelatin
complexes in plant cell cultures. Phytochemistry 1997,
44:69-74.
79. Mendoza-Cozatl DG, Rodriguez-Zavala JS, RodriguezEnriquez S, Mendoza-Hernandez G, Briones-Gallardo R,
Moreno-Sanchez R: Phytochelatin–cadmium–sulfide highmolecular-mass complexes of Euglena gracilis. FEBS J 2006,
273:5703-5713.
80. Dameron CT, Winge DR: Peptide-mediated formation of
quantum 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-batch
bioprocess. 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 yeast
Saccharomyces cerevisiae. Yeast 1997, 13:819-828.
83. Ortiz DF, Ruscitti T, McCue KF, Ow DW: Transport of metalbinding peptide by HMT1, a fission yeast ABC-type vacuolar
membrane-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 in
Escherichia coli and its effect on heavy-metal uptake. Appl
Microbiol Biotechnol 1995, 43:1112-1117.
www.sciencedirect.com
Author's personal copy
Arsenic metabolism by microbes in nature Tsai, Singh and Chen 667
85. Li Y, Cockburn W, Kilpatrick J, Whitelam GC: Cytoplasmic
expression of a soluble synthetic mammalian metallothioneinalpha domain in Escherichia coli — enhanced tolerance and
accumulation of cadmium. Mol Biotechnol 2000, 16:211-219.
86. Bae W, Chen W, Mulchandani A, Mehra RK: Enhanced
bioaccumulation of heavy metals by bacterial cells displaying
synthetic phytochelatins. Biotechnol Bioeng 2000, 70:518-524.
87. Bae W, Mehra RK, Mulchandani A, Chen W: Genetic engineering
of Escherichia coli for enhanced uptake and bioaccumulation
of 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 and
hyperaccumulation of arsenic in plants by combining arsenate
reductase and gamma-glutamylcysteine synthetase
expression. Nat Biotechnol 2002, 20:1140-1145.
An excellent paper showing how the arsenic defense mechanism in
microbes 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 and
accumulation of lead and cadmium in transgenic plants. Nat
Biotechnol 2003, 21:914-919.
90. Sauge-Merle S, Cuine S, Carrier P, Lecomte-Pradines C, Luu DT,
Peltier G: Enhanced toxic metal accumulation in engineered
bacterial cells expressing Arabidopsis thaliana phytochelatin
synthase. Appl Environ Microbiol 2003, 69:490-494.
This paper reported the use of phytochelatin producing E. coli as a
potential arsenic accumulating biosorbent.
91. Rittle KA, Drever JI, Colberg PJS: Precipitation of arsenic during
bacterial 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
www.sciencedirect.com
pathway and its application to precipitation of cadmium on the
cell surface. Appl Environ Microbiol 2000, 66:4497-4502.
93. Kostal J, Yang R, Wu CH, Mulchandani A, Chen W: Enhanced
arsenic accumulation in engineered bacterial cells expressing
ArsR. Appl Environ Microbiol 2004, 70:4582-4587.
94. Singh S, Mulchandani A, Chen W: Highly selective and rapid
arsenic removal by metabolically engineered Escherichia coli
cells expressing Fucus vesiculosus metallothionein. Appl
Environ Microbiol 2008, 74:2924-2927.
For the first time, a metallothionein able to bind to arsenic was utilized
along with an arsenic transporter for the selective removal of arsenic.
Resting cells can be used to completely remove 35 ppb of As(III) in
20 min, making this a low-cost option for arsenic removal from water.
95. Dai MH, Copley SD: Genome shuffling improves degradation of
the anthropogenic pesticide pentachlorophenol by
Sphingobium chlorophenolicum ATCC 39723. Appl Environ
Microbiol 2004, 70:2391-2397.
96. Crameri A, Dawes G, Rodriguez E, Silver S, Stemmer WPC:
Molecular evolution of an arsenate detoxification pathway
DNA shuffling. Nat Biotechnol 1997, 15:436-438.
One of the few reports to show application of irrational approaches for
functional evolution of the arsenic resistance operon.
97. Chauhan NS, Ranjan R, Purohit HJ, Kalia VC, Sharma R:
Identification of genes conferring arsenic resistance
to Escherichia coli from an effluent treatment plant
sludge metagenomic library. FEMS Microbiol Ecol 2009,
67:130-139.
98. Singh S, Kang SH, Lee W, Mulchandani A, Chen W: Systematic
Engineering of Phytochelatin Synthesis and Arsenic Transport
for Enhanced Arsenic Accumulation in E. coli. Biotechnol.
Bioeng., in press.
Current Opinion in Biotechnology 2009, 20:659–667