Arsenic accumulation by Pseudomonas stutzeri and its response to some thiol chelators
Environ Health Prev Med
Arsenic accumulation by Pseudomonas stutzeri and its response to some thiol chelators
D. N. Joshi 0 1
J. S. Patel 0 1
S. J. S. Flora 0 1
K. Kalia 0 1
0 S. J. S. Flora Division of Pharmacology and Toxicology, Defence Research and Development Establishment , Jhansi Road, Gwalior 474 002, Madhya Pradesh , India
1 D. N. Joshi J. S. Patel K. Kalia (&) Laboratory of Biochemistry, B.R.D. School of Biosciences, Sardar Patel University , Vallabh Vidyanagar 388 120, Gujarat , India
Objective The aim of this study is to examine arsenic accumulation by Pseudomonas stutzeri and its response to some thiol chelators, DMPS and MiADMSA. Methods Determination of arsenic accumulation by Pseudomonas sp. was carried out using an atomic absorption spectrophotometer, a TEM and an EDAX. Arsenate reductase enzyme assay was carried out from a cell-free extract of Pseudomonas sp. The effect of chelating agents on arsenite accumulation was analyzed. Total cellular proteins were analyzed using 1-D SDS-PAGE. Results Pseudomonas sp. exhibited a maximum accumulation of 4 mg As g-1 (dry weight). TEM and EDAX analysis showed the presence of As-containing electrondense particles inside the cells. Data on arsenate reductase enzyme kinetics yielded a Km of 0.40 mM for arsenate and a Vmax of 5,952 lmol arsenate reduced per minute per milligram of protein. The chelating agents MiADMSA and DMPS were found to reduce the arsenic accumulation by 60 and 35%, respectively, whereas the presence of both chelating agents in medium containing cells pretreated with arsenite reduced it by up to 90%. The total protein profile of the cellular extract, obtained by 1-D SDS-PAGE, indicated five upregulated proteins, and three of these proteins exhibited differential expression when the cells were grown with MiADMSA and DMPS. Conclusion This study shows a new approach towards arsenic detoxification. A combination treatment with MiADMSA and DMPS may be useful for removing intracellular arsenic. The proteins that were found to be induced in this study may play an important role in the extrusion of arsenic from the cells, and this requires further characterization.
Arsenic bioaccumulation; Arsenate reductase; Chelating agent; Arsenic-induced protein/s
The metalloid arsenic (As) is a member of group V of the
periodic table and is thus classified as a heavy metal [
Although arsenic is generally toxic to life, it has been
demonstrated that microorganisms can use arsenic
compounds as electron donors or electron acceptors, and that
they can possess arsenic detoxification mechanisms [
Arsenic occurs in nature in four oxidation states (?5, ?3, 0
and -3), with pentavalent arsenate [?5, As(V)] and
trivalent arsenite [?3, As(III)] being the most common forms.
Both forms are toxic: arsenite disrupts sulfhydryl groups of
proteins and interferes with enzyme function, whereas
arsenate acts as a phosphate analog and can interfere with
phosphate uptake and transport. Arsenic, like other heavy
metals, cannot be destroyed once it has entered the
environment . Microorganisms have evolved a variety of
mechanisms for coping with arsenic toxicity, including
minimizing the amount of arsenic that enters the cell (e.g.,
through increased specificity of phosphate uptake),
oxidizing the arsenite (through the activity of arsenite
oxidase), or arsenite peroxidation with membrane lipids.
Resistance to arsenic species in both Gram-positive and
Gram-negative organisms results from energy-dependent
efflux of either arsenate or arsenite from the cell, mediated
by the ars operon [
]. Our earlier studies confirmed the
existence of a bacterium with an arsC gene that is
responsible for the conversion of As(V) to As(III) [
], which may
be either extruded from the cells or sequestered in the
intracellular compartment in its free form and/or in
conjugation with glutathione (GSH) or other thiols. AQP7 and
AQP9 conduct the transmembrane movement of the likely
substrate, the neutral species As(OH)3, which may be
considered an inorganic equivalent of glycerol [
]. On the other
hand, microorganisms take up As(III) through the
glyceroporin membrane protein [
]. It can be assumed that the
aquaglyceroporin transport system found in mammalian
cells may be similar to the transport system that facilitates
arsenite uptake by bacterial cells.
Meso-2,3-dimercaptosuccinic acid (MiADMSA) and
2,3-dimercaptopropane-1-sulfonate (DMPS) have been
considered promising antidotes to acute or chronic arsenic
] due to the ability of their vicinal thiol
groups to react with trivalent arsenicals, forming a
saturated five-member heterocyclic ring . Therefore, we
made an attempt to study the response of thiol chelating
agents on arsenic accumulation using Pseudomonas sp. as a
model system, which may help to improve understanding
of the role of chelating agents in the arsenic detoxification
Materials and methods
Pseudomonas stutzeri that had been isolated and
characterized in our lab, and which has the ability to grow in the
presence of arsenic [
], was used for the present study. This
bacterium has shown maximum tolerance levels of 50 mM
for arsenate and 0.2 mM for arsenite, respectively. Na
arsenate, Na meta-arsenite, NADPH, and DTT were
procured from Sigma (St. Louis, MO, USA); DMPS and
MiADMSA were a gift from Dr S.J.S. Flora, DRDE,
Growth kinetics for Pseudomonas sp.
Culture slants were made and kept at 4 C. The bacteria
were grown at 37 C in nutrient broth medium with
continuous shaking at 110 rpm in the orbital shaker for all of
the given conditions (control, arsenic stress). Cells were
harvested by centrifugation (5,0009g for 10 min) at
different time intervals during the lag, log and the stationary
phases. Optical density was measured after different time
intervals at 600 nm using a Cary (Varian, Palo Alto, CA,
USA) 50 UV-visible spectrophotometer. The growth rate
constant (k) for the log phase of growth was determined by
plotting the log10 of the optical density against time [
Experiments were performed in triplicate and repeated
Determination of arsenic accumulation
by Pseudomonas sp.
Cells were harvested by centrifugation (5,0009g for
10 min) and the pH of the supernatant was measured. The
cell pellets were washed 2–3 times with normal saline,
dried, and then used in the measurements of arsenic
accumulation. One-milliliter samples were taken at various
time intervals for cell mass determination and for arsenic
Arsenate reductase enzyme assay
Pseudomonas sp. bacteria were grown to mid-log phase
in 200 ml of NB medium supplemented with 50 mM of
arsenate, harvested by centrifugation for 10 min at
5,000 rpm, and washed twice in 25 ml reaction buffer
(10 mM Tris, pH 7.5, with 1 mM Na2EDTA and 1 mM
MgCl2). The cells were resuspended in 5 ml of reaction
buffer, disrupted by sonication and cell-free extract was
prepared by centrifugation at 5,0009g for 10 min at 4 C.
Arsenate reductase activity was measured using a method
based on NADPH oxidation [
]. The reaction was
initiated at 37 C by mixing 50 ll of cell-free crude extract in
820 ll of reaction buffer, 20 ll of 10 mM DTT (final
concentration 300 lM), and 50 ll of 3 mM NADPH (final
concentration 0.15 mM). Arsenate concentrations of 200,
500 and 1 mM were assayed along with ‘‘no arsenic’’ for
controls. Absorbance decreases at 340 nm were recorded
as NADPH oxidization coupled to the reduction of arsenate
to arsenite. Enzyme activity was calculated using a molar
extinction coefficient of 6.2 9 103 for NADP?. The
endogenous NADPH oxidation rate was subtracted from
the arsenate-induced NADPH oxidation.
TEM and EDAX analysis
Pseudomonas sp. bacteria grown without (control) and
with (experimental) 50 mM sodium arsenate were
harvested and fixed for 2 h at room temperature in 4%
glutaraldehyde and then washed four times at the stationary
phase in 0.1 M phosphate buffer pH (7.2). Pre-embedding
of bacterial cells was done in 4% agar, and small pieces
(1–2 mm2) were cut from solidified agar blocks. These
pieces were fixed overnight at 4 C in 2% osmium
tetroxide (OsO4) in phosphate buffer before being
dehydrated with acetone and embedded in polyepoxy resin.
Ultrathin sections were cut with an ultramicrotome
(Reichert OMU3, Vienna, Austria) equipped with a diamond
knife and then stained with uranyl acetate and lead citrate
as contrasting agents. The sections were mounted on
copper grids. Micrographs of both control (without arsenate)
and experimental cells (treated with arsenate) were taken
with a 2000FX II transmission electron microscope (TEM)
(JEOL, Eching, Germany), operating at 200 kV.
Energydispersive X-ray analysis (EDAX) of the cell pellets was
performed with a Philips (Eindhoven, The Netherlands)
XL-30 electron microscope equipped with an ESEM-TMP
EDAX microanalysis system (Philips).
Use of chelating agents to remove arsenite
from Pseudomonas sp.
Bacterial cells were grown in a nutrient broth medium
containing 0.2 mM arsenite. The cells were harvested at
different time intervals during the lag, log and stationary
phases by centrifugation at 5,0009g for 10 min at 4 C
and transferred into a medium containing the chelating
agents MiADMSA (50 lg 100 ml-1) and DMPS
(50 lg 100 ml-1) either alone or in combination, and
allowed to grow for 4 h as follows:
Group 1: Arsenic control (bacterial cells grown with
0.2 mM arsenite prepared in nutrient broth medium and
transferred into N saline for 4 h)
Group 2: Cells grown with 0.2 mM arsenite and then
transferred into a medium containing DMPS
(0.5 lg ml-1) for 4 h
Group 3: Cells grown with 0.2 mM arsenite and then
transferred into a medium containing MiADMSA
(0.5 lg ml-1) for 4 h.
Group 4: Cells grown with 0.2 mM arsenite and then
transferred into a medium containing MiADMSA and
DMPS (0.5 lg ml-1) for 4 h.
Group 5: Control (bacterial cells grown in nutrient
One-milliliter samples were taken at various time points
for cell mass determinations and for arsenic analysis, while
Fig. 1A–B Growth of
Pseudomonas sp. in (A) the
presence of 50 mM arsenate and
(B) the presence of 0.2 mM
arsenite. The change in OD600
versus extracellular pH over
48 h is shown. Values are
mean ± SE (n = 9)
5 ml samples were taken after 24 h during the mid-log
phase to evaluate the cellular protein profiles of all of the
groups. SDS gel electrophoresis was performed as per the
method of Laemmli [
The arsenic concentrations in all of the samples were
measured using an atomic absorption spectrophotometer
with an autosampler (AS-72, AAS PerkinElmer, Norwalk,
CT, USA) and a graphite furnace (MHS) (Analyst 100,
AAS PerkinElmer) following wet acid digestion of the
bacterial cells. Pellets were dried at 90–100 C to constant
weight and digested with concentrated nitric acid using a
microwave digestion system (Multiwave 3000, Anton Paar,
Austria, Europe). Samples were brought to a constant
volume before analysis.
Effect of arsenic on the growth kinetics
of Pseudomonas sp.
Growth comparisons of the cells grown in arsenic-free
media and arsenic-containing media revealed an
approximately twofold decrease in growth following arsenic
treatment as compared to the cells grown in arsenic-free
media. The growth rate calculated in the absence of arsenic
was 0.76 h-1 (a doubling time of 1.30 h), in the presence
of arsenate it was 0.43 h-1 (a doubling time of 2.32 h), and
in the presence of arsenite it was 0.33 h-1 (a doubling time
of 3.30 h) at the maximum tolerance limit of the bacteria,
resulting in 43 and 56% reductions in the cellular growth of
the bacterial isolate by arsenate and arsenite, respectively.
The data thus suggests a 1.3-fold decrease in cellular
growth due to the higher toxicity of arsenite compared to
arsenate (Fig. 1A,B). The pH of the extracellular medium
was found to increase gradually from 7.2 to 9.2 from the
lag to the stationary phase due to the arsenate in the
medium (Fig. 1A), while the pH was only slightly
increased, from 7.2 to 7.6, by arsenite (Fig. 1B).
Km: 0.40 mM arsenate
Vmax: 5952 U/mg
Arsenic accumulation by cells grown with arsenate was
found to be 1.6 times higher than that for cells grown with
arsenite during the lag phase, whereas cells grown with
arsenate exhibited 2–3 times higher arsenic accumulations
during different growth phases than cells grown with
arsenite (Fig. 2). The maximum arsenic accumulation of
4.08 ± 0.08 mg As g-1 in the dry pellets of Pseudomonas
sp. was found during the mid-log phase; after this, the
organism started to produce an efflux of the intracellular
arsenic into the extracellular environment.
Arsenate reductase activity
The arsenate reductase activity was analyzed during
the mid-log phase of growth, which corresponded to the
maximum accumulation of arsenic by the cells before the
organism started to produce an efflux of intracellular
arsenic. The data on the enzyme kinetics, calculated using
a Lineweaver–Burk plot, showed a Km of 0.40 mM for
arsenate and a Vmax of 5,952 lmol arsenate
reduced min-1 mg-1 protein for the arsenate reductase of
Pseudomonas sp. (Fig. 3). Under these conditions, the
cellular concentration of arsenic was found to 0.01 mM,
whereas the Km value for arsenate of 0.40 mM indicates
that the cellular concentration is too low in cells during the
mid-log phase, and so the arsenate reductase is less active,
leading to insufficient conversion of arsenate to arsenite,
which may result in arsenate accumulation by the cells.
TEM and EDAX analysis
Transmission electron microscopy was used to localize
the arsenic that was accumulated by Pseudomonas sp.
cells. Electron-dense deposits were found in the
cytoplasm of the cells grown in the presence of 50 mM
arsenate. It was also possible to observe that some of the
arsenic accumulated in the periplasm (Fig. 4A,B), which
did not occur in the cells grown without the metalloid
(Fig. 4C,D). The presence of arsenic in the electron-dense
areas was confirmed by EDX analysis (Fig. 4E,F), thus
suggesting cytoplasmic accumulation of the metalloid by
the Pseudomonas sp.
Use of chelating agents to remove arsenite
from bacterial cells
Figure 5 shows the effect of MiADMSA and DMPS used
individually and in combination on arsenic accumulation
by Pseudomonas sp. previously grown in
arsenite-containing medium. The presence of MiADMSA and DMPS
in the medium was found to reduce the arsenic
accumulation by cells pre-grown in arsenite-containing medium
up to different growth stages. The highest reduction was
observed when the cells were transferred into the medium
with both of the chelating agents used in combination.
Arsenic accumulation was significantly reduced (by 15,
75 and 93%) in cells in the lag phase when DMPS,
MiADMSA and a combination of them, respectively,
were applied, as compared to cells grown in
arsenitecontaining media. Cells in the initial log phase reduced
their arsenic accumulation by up to 30 and 37% when
DMPS and MiADMSA were applied, respectively, and a
reduction of 90% was observed in the presence of both of
the chelating agents. The use of MiADMSA and DMPS
individually was found to be effective at reducing arsenic
accumulation by 65 and 61%, respectively, in cells in the
mid-log phase as compared to cells in the initial log
Fig. 4A–F Transmission
electron micrograph and EDAX
analysis of the Pseudomonas sp.
grown in the absence and
presence of arsenate.
A Pseudomonas sp. grown in the
absence of arsenate (915K),
B Pseudomonas sp. grown in the
absence of arsenate (930K).
C Pseudomonas sp. grown in
the presence of arsenate
(915K), D Pseudomonas sp.
grown in the presence of
arsenate (930K). E Cytoplasmic
accumulation of arsenic
Pseudomonas sp. when bacterial
growth occurs in metal-free
medium. F Cytoplasmic
accumulation of arsenic in
Pseudomonas sp. when bacterial
growth occurs in medium with
50 mM of arsenate
Fig. 5 Effects of MiADMSA and DMPS on Pseudomonas sp.
preexposed to arsenite. Units: arsenic concentration in the bacterial cells
as milligram per gram of dry weight. Values are mean ± SE (n = 9)
phase, whereas MiADMSA was found to be more
effective (an 84% reduction was observed) in cells in the late
log phase compared to DMPS (for which a 50% reduction
was observed). Similarly, MiADMSA showed a 78%
reduction and was found to be more effective than DMSA
(45% reduction) in cells at the stationary phase. Our
results indicate that DMPS was effective at reducing
arsenic accumulation (reductions of 15–60%) in cells
pregrown with arsenite up to different stages of growth,
while MiADMSA was found to be more effective than
DMPS at reducing arsenic accumulation (reductions of
37–84%), although the presence of both the chelating
agents yielded reductions of 81–95%, indicating the
synergistic effect of MiADMSA and DMPS in the
removal of intracellular arsenic.
Effect of chelating agents on the cellular protein profile
The total protein profile of the cellular extract of cells
grown with arsenic alone exhibited five upregulated
proteins with molecular weights of 90, 52, 35, 25 and 14 kDa,
indicating their roles in metal resistance, accumulation and/
or transport of arsenic, while one protein with a molecular
weight of 12 kDa showed downregulation (Fig. 6; lane 2).
Two of these proteins, those of molecular weights 52 and
25 kDa, were repressed when the cells were transferred to a
medium containing DMPS (Fig. 6; lane 3), and were
shown to be downregulated when the cells were transferred
into a medium containing MiADMSA alone or in
combination with DMPS, indicating the probable role of these
proteins in arsenic uptake from the medium (Fig. 6; lanes
4, 5). The low molecular weight 12 kDa protein which was
repressed by arsenite exposure was significantly expressed
in the cells transferred to the medium containing
MiADMSA alone or in combination with DMPS (Fig. 6; lanes
4, 5), indicating the role of this protein in the intracellular
efflux of arsenic and thus in the reduction of arsenic
The effects of environmental arsenic on human health can
be devastating. This aspect, together with the
environmental ubiquity of arsenic, led to the evolution of arsenic
defense mechanisms in every organism studied, from
Escherichia coli to humans. Organisms take up As(V) via
phosphate transporters and As(III) by glyceroporin
membrane protein [
] or hexose transporters [
]. As(V) is then
reduced to As(III), which may be either extruded from the
cells, sequestered in the intracellular compartment in its
free form and/or in conjugation with glutathione (GSH) or
other thiols. In this study we focused on arsenic uptake by
Pseudomonas sp. and its response to some conventional
thiol chelating agents like DMPS and MiADMSA.
Our isolate could grow in up to 50 mM arsenate and
could maintain its character even after being grown for 3–4
generations in metal-free medium. The decrease in the
growth rate in the presence of a high concentration of
arsenic may be due to the association of this ion with the
membrane fraction, resulting in an expanded membrane,
which may increase the number of binding sites and make
it less effective at transporting materials needed for growth
]. Macy et al. [
] reported that the increase in the
external pH to 9.4 when organisms used acetate as an
electron donor was linked to arsenate reduction. A number
of organisms have been isolated that use arsenic as a
terminal electron acceptor in anaerobic respiration [
is a decrease in growth rate under these conditions and an
increase in the final pH of the medium from 7.2 to 9.2,
suggesting that the reduction in growth caused by arsenate
and the alkalization of the medium might be due to the
reduction of arsenate to arsenite. The pH of the medium
was not found to be altered when cells grown with arsenite.
Aquaglyceroporins have been shown to facilitate the
uptake of As(III), including E. coli GlpF [
], S. cerevisiae
], mouse AQP7 , and AQP9 from rat [
and humans [
]. It can be assumed that the
aquaglyceroporin transport system found in the mammalian cells may
be similar to the transport system that facilitates arsenite
uptake by bacterial cells. Pseudomonas sp. exhibited a
maximum arsenic accumulation of 4 mg As g-1 in dry
bacterial pellets after 18 h of growth when supplemented
with 50 mM arsenate, which may be due to the
intracellular sequestration of arsenic. The arsenate reductase
activity was found to be maximum during the mid-log
phase of growth, indicating the conversion of arsenate to
arsenite, which may be the mechanism driving the
intracellular removal of arsenite by these cells after 18 h of
growth. TEM and EDAX analysis showed the presence of
As-containing electron-dense particles inside the cells,
confirming the intracellular accumulation of the metalloid
anion by Pseudomonas sp.
Sodium 2,3-dimercaptopropane sulfonate (DMPS) is
another analog of BAL and is mainly distributed in the
extracellular space. It can enter cells through a specific
transport mechanism. No major adverse effects on humans
or animals have been reported after DMPS administration
]. The monoisoamyl ester of DMSA (MiADMSA; a C5
branched chain alkyl monoester of DMSA) has been found
to be more effective than DMSA at reducing the cadmium
and mercury burden [
]. As(III), which is reduced
form of As(V), may form conjugates with either
glutathione (GSH) or another thiol. Cells of Pseudomonas sp.
pretreated with arsenite have shown to reduce their arsenic
accumulation when in the presence of DMPS and
MiADMSA either individually or in combination, indicating the
role of these chelators in the arsenic uptake mechanism.
These chelating agents probably form complexes with the
arsenic and these complexes can then be extruded from the
cells, suggesting that DMPS and MiADMSA could be
useful for the removal of arsenic. MiADMSA would be
especially advantageous, as it possesses high reactivity
toward arsenite. Chelating agents can also affect the
specific transport of proteins like glyceroporin membrane
protein, leading to a reduction in arsenite uptake by these
cells, or it may act as a competitive inhibitor for arsenite,
thus aiding in the removal of intracellular arsenic by
Psudomonas sp. The ability of these bacteria to remove
arsenic–chelate complexes from solution relies on the
presence of specific As-chelator transporter proteins; this
topic needs further characterization.
It is established that many microorganisms survive in
the presence of toxic metals or metalloids by inducing the
expression of an array of resistance proteins. The highly
specific nature of these resistance mechanisms is the result
of a cleverly designed genetic circuit that is tightly
controlled by specific metalloregulatory proteins. Patel et al.
] have recently reported on multiple physiological
responses induced by arsenate stress in P. stutzeri which
are not exclusively associated with the expression of
classical arsenic resistance operons, indicating the probable
role of these proteins in the arsenic resistance mechanism.
The role of proteins in the mechanism of the resistance of
P. fluorescens to heavy-metal-induced stress, such as from
Cu, Co and Pb, was demonstrated by Sharma et al. [
Our results indicate differential expression when cells
grown with MiADMSA and DMPS either individually or
in combination, indicating the probable role of these
proteins in the intracellular efflux of arsenite, which require
further detailed studies.
The removal of toxic components like arsenic is of great
importance, not only because of the resulting
decontamination but also because this removal is important to human
welfare and for maintaining ecological balance. Its high
accumulation of and tolerance toward arsenic indicates that
P. stutzeri could be a suitable candidate for developing
bioremediation processes. The similarity between the
aquaglyceroporin transport system found in bacterial cells
and mammalian cells facilitating arsenite uptake suggests
that MiADMSA and DMPS may be useful for the removal
of intracellular arsenic, although this subject requires
Acknowledgment The authors thank the Director of the Defence
Research and Development Establishment, Government of India,
Gwalior, India for financial support.
1. Wackett LP , Dodge AG , Ellis LB . Microbial genomics and the periodic table . Appl Environ Microbiol . 2004 ; 70 : 647 - 55 .
2. Ahmann D , Roberts AL , Krumholz LR , Morel FM . Microbe grows by reducing arsenic . Nature . 1994 ; 371 : 750 .
3. Cervantes C , Ji G , Ramirez JL , Silver S. Resistance to arsenic compounds in microorganisms . FEMS Microbiol Rev . 1994 ; 15 : 355 - 67 .
4. Ji GY , Silver S. Regulation and expression of the arsenic resistance operon from Staphylococcus aureus plasmid pI258 . J Bacteriol . 1992 ; 174 : 3684 - 94 .
5. 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 USA . 1992 ; 89 : 9474 - 8 .
6. Valls M , De Lorenzo V. Exploiting the genetic and biochemical capacities of bacteria for the remeidation of heavy metal pollution . FEMS Microbiol Rev . 2002 ; 26 : 327 - 38 .
7. Patel PC , Goulhen F , Botthman C , Gault AG , Charnock JM , Kalia K , et al. Arsenate detoxification in a Pseudomonas hypertolerant to arsenic . Arch Microbiol . 2007 ; 187 : 171 - 83 .
8. Rosen BP . Biochemistry of arsenic detoxification . FEBS Lett . 2002 ; 52 : 986 - 92 .
9. Mukhopadhyay R , Rosen BP , Phung T , Silver S. Microbial arsenic: from geocycles to genes and enzymes . FEMS Microbiol Rev . 2002 ; 26 : 311 - 25 .
10. Anderson O. Principles and recent developments in chelation treatment of metal intoxication . Chem Rev . 1999 ; 99 : 2683 - 710 .
11. Flora SJS , Bhattacharya R , Vijayraghvan R . Combined therapeutic potential of meso-2,3-dimercapto succinic acid and calcium dosodium edentate in the mobilization and distribution of lead in experimental lead intoxication in rats . Fundam Appl Toxicol . 1995 ; 25 : 233 - 40 .
12. Pirt SJ . Principles of microbe and cell cultivation . Oxford: Blackwell; 1975 .
13. Anderson CR , Cook GM . Isolation and characterization of arsenate reducing bacteria from arsenic contaminated sites in New Zealand . Curr Microbiol . 2004 ; 48 : 341 - 7 .
14. Laemmli UK . Cleavage of structural proteins during the assembly of head of the bacteriophage T4 . Nature . 1970 ; 277 : 680 - 5 .
15. Suzuki Y , Matsushita H . Interaction of metal ions and phospholipids monolayers as a biological membrane model . Ind Health . 1968 ; 6 : 128 - 33 .
16. Liu Z , Carbrey JM , Agre P , Rosen BP . Arsenic trioxide uptake by human and rat aquaglyceroporins . Biochem Biophys Res Commun . 2004 ; 316 : 1178 - 85 .
17. Macy JM , Nunan K , Hagen KD , Dixon DR , Harbour PJ , Cahill M , et al. Chrysiogenes arsenatis gen . nov., sp. nov., a new arsenaterespiring bacterium isolated from gold mine wastewater . Int J Syst Bacteriol . 1996 ; 46 : 1153 - 7 .
18. Sanders OI , Rensing C , Kuroda M , Mitra B , Rosen BP . Antimonite is accumulated by the glycerol facilitator GlpF in Escherichia coli . J Bacteriol . 1997 ; 179 : 3365 - 7 .
19. Liu Z , Shen J , Carbrey JM , Mukhopadhyay R , Agre P , Rosen BP . Arsenite transport by mammalian aquaglyceroporins AQP7 and AQP9 . Proc Natl Acad Sci USA . 2002 ; 99 : 6053 - 8 .
20. Wysocki R , Chery CC , Wawrzycka D , Van HM , Cornelis R , Thevelein JM . The glycerol channel Fps1p mediates the uptake of arsenite and antimonite in Saccharomyces cerevisiae . Mol Microbiol . 2001 ; 40 : 1391 - 401 .
21. Hurby K , Donner A . 2 , 3 -Dimercapto-1 -propanesulphonate in heavy metal poisoning . Med Toxicol Adverse Drug Exp . 1987 ; 2 : 317 - 23 .
22. Jones MM , Singh PK , Gale GR , Smith AB , Atkins LM . Cadmium mobilization in vivo by intraperitoneal or oral administration of mono-alkyl esters of meso-2,3-dimercaptosuccinic acid . Pharmacol Toxicol . 1992 ; 70 : 336 - 42 .
23. Gale GR , Smith AB , Jones MM , Singh PK . Meso 2-3 dimercaptosuccinic acid monoalkyl esters: effects on mercury levels in mice . Toxicology . 1993 ; 81 : 49 - 56 .
24. Xu C , Holscher MA , Jones MM , Singh PK . Effect of monoisoamyl meso-2 ,3 -dimercaptosuccinate on the pathology of acute cadmium intoxication . J Toxicol Environ Health . 1995 ; 45 : 261 - 77 .
25. Sharma S , Sundaram CS , Luthra PM , Singh Y , Sirdeshmukh R , Gade WN . Role of proteins in resistance mechanism of Pseudomonas fluorescens against heavy metal induced stress with proteomics approach . J Biotechnol . 2006 ; 126 : 374 - 82 .