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Arsenic Removal from Aqueous Solutions by Different Bacillus and

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Arsenic Removal from Aqueous Solutions by

Different Bacillus and Lysinibacillus Species

Eman A. H. Mohamed & Azza G. Farag

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Aqueous Solutions by Different Bacillus and Lysinibacillus Species, Bioremediation Journal,
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 Bioremediation Journal, 19(4):269–276, 2015
Copyright Ó 2015 Taylor and Francis Group, LLC
ISSN: 1088-9868 print / 1547-6529 online
DOI: 10.1080/10889868.2014.995375
Arsenic Removal from Aqueous Solutions by
Different Bacillus and Lysinibacillus Species
Eman A. H. Mohamed 1,2 and
Azza G. Farag 2,3
1
Department of Botany,
Damanhour University,
Damanhour, Egypt
2 Saudi Arabia, containing arsenic, vanadium, and boron. The isolates were
Biotechnology Department,
Downloaded by [Taif University] at 01:09 26 October 2015
Faculty of Science, Taif University,
Taif, Kingdom of Saudi Arabia
3
Virus and Phytoplasma
Department, Plant Pathology
Institute, Agriculture Research
Centre, Giza, Egypt
Address correspondence to Eman A. H.
Mohamed, PhD, Biotechnology
Department, Faculty of Science, Taif
University, P.O. Box 888, Taif, Kingdom
of Saudi Arabia. E-mail:
eymanmohamed@yahoo.com
ABSTRACT Removal of toxic and carcinogenic arsenic from underground
water is very essential for the safety of water that may be used for drinking or
irrigation. In this study, six different bacterial strains were recently isolated from
a groundwater sample, routinely used for irrigation at Taif City, Kingdom of
molecularly identified and the 16S rDNA sequencing data revealed their
belonging to two different genera, Bacillus and Lysinibacillus. B. cereus strains
EA4, EA5, and EA6 were able to resist arsenic up to 15 mg/L. B. cereus strain
EA5 and a mixed culture of L. sphaericus EA1, B. fusiformis EA2, and
Lysinibacillus sp. EA3 were found to be efficient in bioremediation of arsenic
oxychloride up to 94.9% and 99.7%, respectively. Due to these near-standard
records, these strains are strongly recommended for bioremediation of the
highly toxic arsenic from the environment. B. cereus EA5 was also effective to
remediate different concentrations of arsenic. High concentrations of arsenic
showed dramatic decrease in the bioremediation activity of this strain.
Reduction in cell size was distinct in scanning electron micrographs when cells
were exposed to arsenic. Besides, protein electrophoresis showed that around
15 different stress proteins were produced when cells of B. cereus EA5 were
exposed to arsenic oxychloride.
KEYWORDS arsenic bioremediation, Bacillus cereus, electron microscopy, Lysinibacillus,
stress proteins
INTRODUCTION
Metals are introduced into aquatic systems as a result of weathering of soils,
volcanic eruptions, and human activities such as extraction of fossil fuels and
extensive use of ground water (Laws 1993). Thus, arsenic can be easily released
by these activities to the environment, causing serious health damages
(Miyatake and Hayashi 2009). This metal is an extremely toxic environmental
pollutant that is widely distributed the earth’s crust (Cullen and Reimer 1989;
Tamaki and Frankenberger 1992; Smedley and Kinniburgh 2002). Therefore,
removal of arsenic from underground water is very essential for the safety of
drinking water (Nickson et al. 2000). Most of the arsenic-bearing minerals, such
as arsenides and sulfarsenides, are considered nontoxic because they are highly
269
 insoluble. Problems arise when these primary minerals
break down and enter into solution or form more solu-
ble species such as oxides (Vaughan 2006). One of such
cases, a common phenomenon in metallurgy industry,
is arsenic release from ores and deposits into the envi-
ronment through mining and smelting operations
(Lukasz et al. 2014).
To remove arsenic from water, physicochemical
methods are usually used (Wang and Mulligan 2006).
These methods are expensive and produce secondary
metabolites; hence, biological approaches have been
considered as an alternative remediation for arsenic
removal (Wang and Mulligan 2006; Nanda, Sharma,
and Kumar 2011). Amongst the various bioremedia-
tion processes, phytoremediation and bioremediation
by microbes are quite effective (Akhtar, Chali, and
Azam 2013).
Arsenic is not usually adsorbed by microorganisms,
Downloaded by [Taif University] at 01:09 26 October 2015
but it can be altered by them throughout redox trans-
formations (Saltikov et al. 2003; Miyatake and Hayashi
2009). Microorganisms are known to play an important
role in the biochemical cycling of arsenic through its
conversion to species with different solubility, mobil-
ity, bioavailability, and toxicity (Silver and Phung
2005). However, many Bacillus species such as
B. megaterium were reported to remove arsenic through
adsorption (Miyatake and Hayashi 2009). The complex
structure of microorganisms implies that there are
many ways for the metal to be taken up by the micro-
bial cell. According to the dependence on the cell’s
metabolism, biosorption mechanisms, for instance,
can be divided into metabolism dependent and non-
metabolism dependent. According to the location
where the metal removed from solution is found, bio-
sorption can be classified as extracellular accumula-
tion/precipitation, cell surface sorption/precipitation,
and intracellular accumulation (Nanda, Sharma, and
Kumar 2011). Microbial cell walls have abundant metal
binding groups such as carboxyl, sulfate, phosphate,
and amino groups. This non-metabolism-dependent
biosorption is rapid and can be reversible (Kuyucak
and Volesky 1988).
The aim of the present study is to discover, identify,
and characterize some bacterial strains recently isolated
from underground water of Taif City, Kingdom of
Saudi Arabia, that are capable of removing the highest
possible concentration of arsenic from aqueous solu-
tions and thus can be used for bioremediation applica-
tions. Besides, stress proteins and cell morphology of
E. A. H. Mohamed and A. G. Farag
arsenic-treated cells were also investigated using protein
electrophoresis and scanning electron microscopy,
respectively.
MATERIALS AND METHODS
Source of Samples and Bacterial
Isolation
Underground water used in this study was collected
in February 2014 from a deep well that is routinely
used in plant irrigation, Taif City, Kingdom of Saudi
Arabia. Bacteria were isolated from the water samples
by serial dilutions and pour plate method using nutri-
ent agar medium. The isolates were then purified and
maintained in agar slants.
Water Chemical Analyses
Chemical analyses of underground water were per-
formed at Soil, Water and Environment Research Insti-
tution (SWERI), Giza, Egypt. The analyses included
some heavy metals and total pesticides, as will be indi-
cated in Results.
Cell Morphology and Antibiotic
Sensitivity Test
Routine Gram stain reaction was performed, and
cells were examined using bright field microscopy at
100£ magnification. Sensitivity of the isolates toward
some commercially available antibiotic discs was tested
in Muller Hinton agar plates. The plates were incu-
bated at 30 C for 24 h. The antibiotics were as follows
(mg): ampicillin (20), penicillin G (10), cephalothin
(30), amoxicillin (10), and sulfamethoxazole (25).
Bacterial Resistance to Arsenic
The pure bacterial isolates were cultured in nutrient
agar plates supplemented with different concentrations
of arsenic oxychloride, 0.2–15 mg/L. The plates were
incubated at 30 C for 24 h.
Arsenic Adsorption
Bacterial cells obtained from nutrient broth cultures
grown at 30 C for 24 h with agitation at 100 rpm were
harvested by centrifugation. Harvested cells were then
270
 weighed and 0.1 g wet cells were used for inoculation
of arsenic oxychloride solution (15 mg/L). Each strain
was inoculated separately or in a mixed culture. The
total volume of arsenic solution was 20 ml in a 100-ml
conical flask, and pH was adjusted to 7. Cells were sus-
pended in arsenic solution for 12 h at 30 C with agita-
tion rate of 100 rpm. Finally, cells were centrifuged
and clear supernatant was isolated in clean tubes for
quantitative analysis of arsenic using atomic absorption
spectrometer (Conter AA700 Graphite; France Analy-
tik, Jena, Germany). Measurements were performed
twice, and obtained values were in the range of §3%
from average values (results are given as average values).
Effect of Arsenic Concentration on Its
Bioremoval Percentage
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The changes in adsorption efficiency of different
arsenic oxychloride concentrations, 0.2–25 mg/L, by
the bacterial cells were also examined using the same
fixed conditions as described before.
Scanning Electron Microscopy (SEM)
To compare between cell morphology features of
arsenic-treated as well as untreated cells, scanning elec-
tron microscopy (Electron Microscope Unit, Taif Uni-
versity, Kingdom of Saudi Arabia) was used. Cell
suspensions with and without arsenic at the fixed con-
ditions were centrifuged and harvested cells were exam-
ined using SEM.
Stress Proteins Electrophoresis
For estimation of stress proteins in the presence of
arsenic, cells were precultured in nutrient broth over
night at 30 C. This preculture was used to inoculate
fresh nutrient broth medium containing 15 mg/L arse-
nic oxychloride for 12 h at 30 C. Cells were then sepa-
rated from the supernatant by centrifugation and
TABLE 1 Forward and Reverse Primers Used in PCRs and Sequencing
Primer name Primer direction
27F Forward
1492R Reverse
518F Forward
800R Reverse
271
sonicated. Intra- and extracellular stress proteins were
then electrophoresed. The same protocol was used for
cells without arsenic addition. Protein electrophoresis
was done according to Laemmli (1970).
16S rDNA Partial Sequencing
DNA was extracted using Insta Gene Matrix (Bio-
Rad, Hercules, CA, USA). Amplification of the 16S
rDNA was done using 20 ng DNA in 30-ml reaction
mixture using EF-Taq (SolGent, Daejeon, South Korea)
as follows: activation of Taq polymerase at 95 C for
2 min, 35 cycles of 95 C for 1 min, 55 C and 72 C for
1 min, and 10-min step at 72 C. Amplicons were puri-
fied using multiscreen filter plate (Millipore, Bedford,
MA, USA). Sequencing was performed at Macrogene
(Seoul, Korea) using PRISM Big Dye Terminator V3.1
cycle sequencing kit. Amplification products were ana-
lyzed by ABI PRISM 3730XL DNA analyzer (Applied
Biosystems, Foster City, CA, USA). The sequences
were compared with those in the GenBank database
using BLAST search (Altschul et al. 1997). The sequen-
ces were finally deposited in the GenBank and acces-
sion numbers were obtained, as will be indicated in
Results. Forward and reverse primers (Macrogene) used
in the polymerase chain reactions (PCRs) and sequenc-
ing are illustrated in Table 1.
RESULTS
Some chemical analyses were performed to the
groundwater sample that was collected in February
2014, Taif City, Kingdom of Saudi Arabia (Table 2).
The results revealed the presence of boron, arsenic, and
vanadium rather than other heavy metals listed in
Table 2. Besides, the total amount of pesticide in the
sample is 0.4 mg/L. However, arsenic was chosen for
further tests concerning its removal throughout
bioremediation.
Six different bacterial colonies, M1–M6, were picked
up from the environmental sample and subjected to
Primer sequence Purpose
5’-AGAGTTTGATCMTGGCTCAG-3’ For PCR
5’-TAGGGYTACCTTGTTACGACTT-3’
5’-CCAGCAGCCGCGGTAATACG-3’ For sequencing
5’-TACCAGGGTATCTAATCC-3’
As Removal by Bacillus and Lysinibacillus
 TABLE 2 Heavy Metal and Total Pesticide Analyses of the TABLE 3 Resistance of the New Isolates to Different Concen-
Underground Water Sample trations of Arsenic

Heavy metal Concentration (mg/L) Isolate resistance

Zn 0.00 Arsenic concentration (mg/L) M1 M2 M3 M4 M5 M6

P 0.008
0.2–4 C C C C C C
Fe 0.001
6 ¡ C C C C C
B 0.478
8 ¡ C C C C C
Mn 0.00
10 ¡ C ¡ C C C
Cu 0.00
15 ¡ ¡ ¡ C C C
As 0.191
Cd 0.00 Note. C, resistant; ¡, sensitive.
Co 0.00
Ni 0.00
Pb 0.006 (10), and sulfamethoxazole (25), but they showed dif-
Mo 0.00 ferent diameters of inhibition zones (data not shown).
Ag 0.00
The isolates were identified at the molecular level by
Se 0.00
Ti 0.00
partial sequencing of the 16S rRNA gene. The PCR
V 0.128 amplicons, approximately 1500 bp, of this gene
Al 0.00 (Figure 2) were obtained using 27F and 1492R primers
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Total pesticides 0.40
more experiments due to their resistance to different
concentrations of arsenic, 0.2–15 mg/L (Table 3).
Growth of isolate M1 was inhibited upon using 6 mg/
L of arsenic oxychloride, whereas that of M2 was
stopped at 15 mg/L. On the other hand, M3 was resis-
tant till only 8 mg/L of arsenic oxychloride. M4, M5,
and M6 were resistant even at 15 mg/L.
Gram staining indicated that all of the isolates are
gram-positive rods (Table 4), and cell morphology for
one of them, M5, is illustrated further by scanning elec-
tron microscopy (Figure 1). For more characterization,
the antibiotic sensitivity test was applied (Table 4).
This test was generally differential among the 6 isolates.
However, M2 and M6 looked similar in their sensitivity
toward ampicillin (20), penicillin G (10), amoxicillin
(Table 1). The sequencing and BLAST search results
revealed that M1 and M3 belong to two different spe-
cies of Lysinibacillus, whereas M2 is an isolate of Bacillus
fusiformis. Isolates M4, M5, and M6 belong to Bacillus
cereus (Table 5). The sequences of the 16S rRNA gene
were deposited in the GenBank, and the accession
numbers were given in Table 5.
Removal of arsenic from aqueous solutions using
the new Bacillus strains was tested at fixed temperature
(30 C), pH (7), agitation rate (100 rpm), cell weight
(0.1 g wet weight), and contact time (12 h). The initial
concentration of arsenic was 15 mg/L in a total volume
of 20 ml/100-ml conical flask. For each single strain,
the percentage of arsenic removal was higher than 83%
(Table 6). The maximum arsenic adsorption was
recorded by B. cereus EA5 (94.9%) and B. fusiformis
EA2 (94.7%), followed by L. sphaericus EA1 (93%) and
B. cereus EA4 (92.8%). On the other hand, the lowest
arsenic bioremediation percent was recorded by

TABLE 4 Gram Stain and Resistance to Different Antibiotics

Isolate resistance

Antibiotic discs (mg) M1 M2 M3 M4 M5 M6

Ampicillin (20) s s s s s s
Penicillin G (10) s s s r r s
Cephalothin (30) s r s r r r
Amoxicillin (10) s s r s s s
Sulfamethoxazole(25) r s s s r s
Gram stain Gram-positive rods
Note. r, resistant; s, sensitive.

E. A. H. Mohamed and A. G. Farag 272

 FIGURE 2 The 16S rDNA electrophoresis for the six new iso-
lates using 27F and 1492R primers. M, genetic marker in bp.

surface area and shrinking of B. cereus EA5 cells treated

FIGURE 1 Cell morphology of Bacillus cereus strain EA5 using
scanning electron microscopy.
Lysinibacillus sp. EA3 and B. cereus EA6. Mixed cultures
were also used in two different combinations. One
Downloaded by [Taif University] at 01:09 26 October 2015
with arsenic in comparison with untreated cells.
To investigate the global response of B. cereus EA5
cells to arsenic stress, cell total protein was electrophor-
esed on gel (Figure 4). Five proteins are commonly pro-
duced intracellular in absence and presence of arsenic
(10, 12, 13, 14, and 26 kDa). On the other hand, more
than 15 proteins are unique to be produced intra- and

mixed culture includes L. sphaericus EA1, B. fusiformis extracellularly under arsenic stress. However, these arse-
EA2, and Lysinibacillus sp. EA3, and the other includes nic stress proteins are not generally common intra- and
B. cereus EA4, B. cereus EA5, and B. cereus EA6. The for- extracellular. For example, a protein of approximately
mer showed the highest arsenic removal activity, 99.7% 35 kDa was produced only inside the cells when treated
(Table 6). with arsenic and was not found in the cell supernatant.
The effect of arsenic concentration on its biore- On the other side, a protein of about 150 kDa is
moval percentage by B. cereus EA5 was also investigated excreted in the supernatant of arsenic-treated cells and
(Table 7). When B. cereus EA5 cells were added to arse- was not found intracellular.
nic oxychloride solution (15 mg/L) at the experiment
conditions described before, 94.9% of arsenic was
removed. However, the maximum adsorption effi-
ciency was found when 0.5–2 mg/L arsenic was used.
DISCUSSION
At high arsenic concentrations (20 and 25 mg/L), bac- Arsenic was chosen in this study due to its high envi-
terial cells showed dramatic decrease in its arsenic bio- ronmental toxicity, especially to the groundwater
remediation efficiency. (Nickson et al. 2000). Using different arsenic concen-
The effect of arsenic on B. cereus EA5 cells morphol- trations revealed that the highest recorded resistance
ogy was demonstrated throughout scanning electron among the strains was for Bacillus cereus. In another
microscopy (SEM) (Figure 3). Distinct changes in cell study of Raja and Omine (2011), B. safensis MS11 was
size and morphology were observed when cells sub- found to resist arsenic and boron as well. Moreover,
jected to arsenic. SEM image showed decrease in arsC gene from B. cereus strain AG27 provides arsenic
resistance (Jain et al. 2011). This gene system provides
arsenic resistance to a variety of microorganisms and
TABLE 5 Accession Numbers of the Newly Isolated Strains
can be chromosomal or plasmid borne (Jain et al.
Accession 2011).
Code Strain number In an experiment of arsenic adsorption by dried cells
M1 Lysinibacillus sphaericus strain EA1 KJ781355 of B. megaterium strain UM-123 (Miyatake and Hayashi
M2 Bacillus fusiformis strain EA2 KJ781356 2009), As(III) was well adsorbed. The maximum
M3 Lysinibacillus sp. strain EA3 KJ781357 adsorption capacity was estimated to 0.127 mg As/g
M4 Bacillus cereus strain EA4 KJ781358 (dry weight). Surprisingly, using mixed cultures of Lysi-
M5 Bacillus cereus strain EA5 KJ781359
nibacillus and Bacillus fusiformis reduced arsenic concen-
M6 Bacillus cereus strain EA6 KJ781360
tration from 15 to 0.05 mg/L. This final concentration

273 As Removal by Bacillus and Lysinibacillus

 TABLE 6 Arsenic Removal in Single and Mixed Cultures
Remaining
arsenic
concentration % Arsenic % Arsenic
Bacteria Code (mg/L) remaining removed

Lysinibacillus sphaericus strain EA1 M1 1.06 7.0 93.0

Bacillus fusiformis strain EA2 M2 0.79 5.3 94.7
Lysinibacillus sp. strain EA3 M3 2.45 16.4 83.6
Bacillus cereus strain EA4 M4 1.08 7.2 92.8
Bacillus cereus strain EA5 M5 0.77 5.1 94.9
Bacillus cereus strain EA6 M6 2.43 16.2 83.8
Mixed culture 1 M1, M2, and M3 0.05 0.3 99. 7
Mixed culture 2 M4, M5, and M6 1.81 12.1 87.9
Control (no bacteria) 14.98 100 00.0

is the nearest to the environmental standard for arsenic, EA5 at the same fixed conditions. Based on these near
0.01 mg/L (Miyatake and Hayashi 2009). Using mixed standard records, B. cereus EA5 and mixed cultures of
bacterial cultures was also recorded by Teclu, Laing, Lysinibacillus sphaericus EA1, Bacillus fusiformis EA2,
Downloaded by [Taif University] at 01:09 26 October 2015

and Wallis (2009). In their study, a consortium of bac- and Lysinibacillus sp. EA3 are strongly recommended
teria was successfully used to investigate the treatment for effective removal of arsenic, which is highly toxic
of synthetic groundwater containing either As(III) or and carcinogenic to human and animals (Bahar,
As(V) at initial concentrations of 20, 10, 5, and 1 mg/ Megharai, and Naidu 2013).
L. B. cereus strain W2, which was isolated from the soil The effect of arsenic concentration on its bioremedi-
by Miyatake and Hayashi (2009), was also examined ation capacity was also tested. Lower arsenic concentra-
for its potential to remove arsenic. Dried cells of this tions, almost up to 15 mg/L, were effectively removed
strain adsorbed As(III) in a range from 97.3% to 99.1% from aqueous solutions by B. cereus EA5 cells. Higher
in solutions containing up to 1 mg/L As at pH 7. Even concentrations led to arsenic accumulation and dra-
in cultures containing E. coli genetically modified to matic decrease in the efficiency of its adsorption by B.
express high levels of ArsR, the protein regulating ars cereus EA5. Similar results were recorded by Miyatake
operon or Arabidopsis thaliana phytochelatin, the and Hayashi (2009). They have reported that 18.2 and
amount of arsenic removed ranges from 0.11 to 10.3 mg/L of As(III) and As(V), respectively, remained
0.173 mg/g dry weight. In our study, only 0.05 mg/L when 20 mg/L As was used. In their study, they esti-
As remained after 12 h of adsorption by mixed cultures mated that the optimum pH, temperature, and contact
of two different Lysinibacillus species and Bacillus time for arsenic removal were 7, 30 C, and 12 h,
fusiformis and 94.9% of As was adsorbed by B. cereus respectively. Moreover, Teclu, Laing, and Wallis (2009)
estimated that percentage removal of As(III) improved
TABLE 7 The Effect of Different Arsenic Concentrations on Its from 10% to 47% when the concentration was reduced
Bioremoval Percentage Using Cells of Bacillus cereus EA5 from 20 to 1 mg/L, whereas the corresponding
Initial arsenic Remaining arsenic
improvement for As(V) was from 39% to 92%. Arsenic
concentration concentration stress causes negative cell responses such as decrease in
(mg/L) (mg/L) % of arsenic removal surface area and shrinking. This alteration in surface
features may be explained as a negative reaction of B.
0.5 0.01 98
1 0.015 98.5
cereus EA5 against further adsorption of arsenic by
2 0.04 98 decreasing the area of contact with the metal (Adarsh
5 0.24 95.2 et al. 2007). Similar observations were also found by
10 0.53 94.7 He et al. (2010). Other mode of arsenic response by
15 0.76 94.9 bacteria was recently noticed by Rahman et al. (2014)
20 14.7 26.5
using SEM. They observed a long chainlike structure
25 22.6 9.6
formed by Lysinibacillus strain B1-CDA when exposed

E. A. H. Mohamed and A. G. Farag 274

 FIGURE 3 Scanning electron micrographs for arsenic-treated (left) and untreated cells (right) of Bacillus cereus strain EA5. The ampli-
fication power is 5000£ at 5 mm and 15 kV.
to arsenic. In addition, Focardi et al. (2010) also
noticed arsenic precipitation by Desulfosporosinus strain
063 using SEM. The walls of bacteria are efficient metal
chelators, although a wide spectrum of uptake capaci-
ties may be exhibited. Metal binding may be at least a
two-stage process; the first involving interaction
between metal ions and reactive groups, followed by
inorganic deposition of increased amounts of metal.
Downloaded by [Taif University] at 01:09 26 October 2015
The carboxyl groups of glutamic acid of peptidoglycan
are the major site of metal deposition (Zouboulis et al.
2010).
Some genes are cotranscriped and translated in the
presence of arsenic (Zhang et al. 2007). arsP and arsB,
and similarly arsB and arsC, are cotranscriped. This
indicates that the three genes belong to the same tran-
scriptional unit. In acidophilic Ferroplasma acidarmanus
stain Fer1, synthesis of heat-shock proteins HSP60 and
FIGURE 4 Electrophoresis of proteins in Bacillus cereus strain
EA5 cultures. M, protein marker in kDa; C, cells with no arsenic;
Sup, cells supernatant with no arsenic; AC, cells under arsenic
stress; AS, supernatant of cells under arsenic stress.
275
HSP70, which are involved in protein refolding, was
enhanced when cells were exposed to As(III) (Baker-
Austin et al. 2007). In Pseudomonas aeruginosa, exposure
to arsenite resulted in an oxidative-stress-like response
(Parvatiyar et al. 2005). In our study, around 15 pro-
teins were expressed when B. cereus EA5 cells were sub-
jected to arsenic. These proteins may be those that
were indicated by Zhang et al. (2007) in their study.
They identified 18 unique proteins to be responsive to
arsenate. These proteins are phosphate transporters,
heat shock proteins involved in protein refolding,
and enzymes participating in carbon and energy
metabolism.
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