Validation of Arsenic Resistance in Bacillus cereus Strain AG27by Comparative Protein Modeling of arsC Gene Product

Sourabh Jain • Bhoomika Saluja • Abhishek Gupta •

Soma S. Marla • Reeta Goel

Published online: 22 January 2011

� Springer Science+Business Media, LLC 2011

Abstract The ars gene system provides arsenic resis-

tance to a variety of microorganisms and can be chromo-

somal or plasmid-borne. The arsC gene, which codes for

an arsenate reductase is essential for arsenate resistance

and transforms arsenate into arsenite, which is extruded

from the cell. Therefore, arsC gene from Bacillus cereus

strain AG27 isolated from soil was amplified, cloned and

sequenced. The strain exhibited a minimum inhibitory

concentration of 40 and 35 mM to sodium arsenate and

sodium arsenite, respectively. Homology of the sequence,

when compared with available database using BLASTn

search showed that 300 bp amplicons obtained possess

partial arsC gene sequence which codes for arsenate

reductase, an enzyme involved in the reduction of arsenate

to arsenite which is then effluxed out of the cell, thereby

indicating the presence of efflux mechanism of resistance

in strain. The efflux mechanism was further confirmed by

atomic absorption spectroscopy and scanning electron

microscopy studies. Moreover, three dimensional structure

of modeled arsC from Bacillus cereus strain shares sig-

nificant structural similarity with arsenate reductase protein

of B.subtilis, consisting of, highly similar overall fold with

single a/b domain containing a central four stranded, par-

allel, open-twisted b-sheet flanked by a-helices on both

sides. The structure harbors the arsenic binding motif AB

loop or P-loop that is highly conserved in arsenate reduc-

tase family.

Keywords arsC � Bacillus cereus � Homology modeling �3D structure

Abbreviations

arsC Arsenate reductase C

3D structure Three dimensional structure

As Arsenic

arsB Arsenate reductase B

MIC Minimum inhibitory concentration

16S rDNA 16S Ribosomal DNA

NCBI National centre of biotechnological

information

BLASTp Basic local alignment search tool protein

PDB Protein database

ExPasy Expert protein analysis system

SIB Swiss institute of bionformatics

SEM Scanning electron microscopy

PMDB Protein model database

Cys Cysteine

Thr Threonine

Ser Serine

His Histidine

Asp Aspartate

Arg Arginine

1 Introduction

Arsenic is a toxic element that is found in both natural

environments such as geothermal springs, and in sites

contaminated by a number of industries. When present in

Sourabh Jain and Bhoomika Saluja have contributed equally.

S. Jain � B. Saluja � A. Gupta � R. Goel (&)

Department of Microbiology, G.B. Pant University of

Agriculture and Technology, Pantnagar 263145, India

e-mail: rg55@rediffmail.com

S. S. Marla

DNA Fingerprinting Division, National Bureau of Plant Genetic

Resources, IARI Campus, New Delhi, India

123

Protein J (2011) 30:91–101

DOI 10.1007/s10930-011-9305-5

insoluble form, which is non-toxic, arsenic is frequently

found as a mineral like arsenopyrite etc. The oxidation of

these insoluble forms, chemically and/or microbiologi-

cally, results in the formation of two valence states of

inorganic arsenic - arsenite As (III) which when oxidized

forms arsenate As (V). Both forms are toxic to microor-

ganisms, with arsenite disrupting enzyme function, and

arsenate behaving as a phosphate analogue and interfering

with phosphate uptake and utilization [54].

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 [16], arsenite

oxidation through the activity of arsenite oxidase [16, 39],

or peroxidation reactions with membrane lipids [1, 2].

Other microorganisms utilize arsenic in metabolism, either

as a terminal electron acceptor in dissimilatory arsenate

respiration [5, 23, 40, 53] or as an electron donor in che-

moautotrophic arsenite oxidation [25, 46]. However, the

most well-characterized microbial arsenic detoxification

pathway involves the ars operon [16, 27] which codes for

(1) a regulatory protein (arsR) arsenite (As (III))-respon-

sive repressor of transcription (2) an arsenate permease

(arsB) which codes for an arsenite specific transmembrane

pump and (3) a gene coding for an arsenate reductase

(arsC) that converts arsenate to arsenite [26, 44, 45].

Therefore, the arsC gene product is the key enzyme in the

arsenate reduction process. Among the several families of

arsC, identified and characterized [19, 21, 37, 38, 45],

Staphylococcus aureus arsC has been extensively studied

[28, 30, 33–36].

Comparative modelling of proteins is done to build a

three-dimensional model on one or more related proteins of

known structure. Further, for deducing the biological

functions involved in the mechanism via structure–function

relationship, structure knowledge is essential for all areas

of protein research such as enzyme kinetics, ligand–protein

binding studies, gene characterization and construction,

structure based molecule design, and rational designing of

proteins. In addition, these models can speed up the process

of experimental structure determination by molecular

replacement phasing in X-ray crystallography.

This study describes arsenic resistance mechanism in

B.cereus strain AG-27 isolated from soil and its validation

by comparative protein modeling of arsC gene. This

includes molecular characterization of the strain to identify

the location of arsC gene coding for arsenate reductase.

Furthermore, an effort has been made to find out whether

the resistance to arsenic was due to efflux mechanism or

due to accumulation of metal in the bacterial cell. Further,

this study is planned to analyse the structure function

relationship of arsC that was cloned and characterized.

A three-dimensional structure of arsC from AG-27 was

built on the basis of comparative homology using the

software SWISS-MODEL. Prediction of conserved resi-

dues and functional/active sites in modelled three-dimen-

sional structure of protein was done by using softwares

ConSurf and Q-SiteFinder, respectively.

2 Materials and Methods

2.1 Bacterial Strains and Growth Conditions

Arsenic resistant strain B.cereus AG27 previously isolated

from the soil of Panki thermal power plant, Kanpur, India

was procured from the departmental culture collection. The

strain was grown in nutrient broth medium (Himedia lab-

oratories private limited, Mumbai, India) and was kept at

30 �C overnight. Furthermore, the grown culture was also

streaked on nutrient agar plates (Himedia laboratories pri-

vate limited, Mumbai, India) and the plates were kept at

4 �C for further use. The minimum inhibitory concentra-

tion was determined by growing the strain at varying

concentrations of sodium arsenate and sodium arsenite,

respectively. The 16SrDNA sequence was previously

submitted in genbank database (Accession Number:

AY970345). The strain was further checked for resistance

to other heavy metals namely cadmium, antimony, chro-

mium, copper and cobalt and MIC of these metals are

determined.

2.2 Plasmid and Genomic DNA Isolation

The plasmid DNA was isolated using previously described

protocol [8] and genomic DNA by Qiagen Bacterial

genomic DNA isolation kit.The isolated DNA was made to

run on 0.8% agarose gel and visualized subsequently.

2.3 Polymerase Chain Reaction

The arsC gene was amplified from both plasmid and

genomic DNA of strain AG27 using degenerate primers

arsC1F (TTT AYT TAT GYA CAG GHA ACT) and

arsC1R (ATC RTC AAA TCC CCA RTG WWN). Poly-

merase chain reaction (50 ll) mixture contained 0.5 lM of

each primer, 200 lM dNTPS, 1.5UTaq DNA Polmerase,

PCR buffer supplied with the enzyme and 1 ll (75 ng) of

template DNA. The total volume of the reaction mixture

was maintained with sterilized triple distilled water. PCR

was performed in ‘BioRad iQTM5, multicolor real time

PCR detection system’ and was carried out as follows: a

single denaturation step at 95 �C for 5 min followed by a

34 cycle programme which include denaturation at 94 �C

for 2 min, annealing at 54 �C for 1 min and extension

72 �C for 1 min and a final extension at 72 �C for 10 min.

92 S. Jain et al.

123

2.4 Cloning and Sequencing of PCR Products

The purified product of arsC gene was ligated with pGEM-T

vector for 1 h using TA cloning kit (Promega,USA) and was

transformed in E.coli DH5a. DNA sequencing of single

strand of arsC gene was done using primer T7. The sequence

thus obtained was analysed using BLASTn search (http://

www.ncbi.nlm.nih.gov/Blast). The arsC gene sequence

of strain AG-27 was deposited in gene bank database

(Accession number: DQ517938).

2.5 Atomic Absorption Spectroscopy and Scanning

Electron Microscopy

To observe possible accumulation or precipitation of

arsenic in AG27; atomic absorption spectroscopy was done

by a combined protocol [43, 56]. Furthermore, the samples

of strain AG27 grown in the presence and absence of

arsenic were drawn for scanning electron microscopy.

2.6 Three-Dimensional Computer Modeling

The comparative modeling of arsC gene from AG27,

accession number, DQ517938, includes following stages:

(a) The target protein sequence was obtained from NCBI-

Genpept database and subsequently submitted to BLASTp

[7]. The database chosen for BLASTp was PDB [13],

which resulted in the identification of 1JL3|B| PDB as

suitable template for creating full atom three-dimensional

model of arsC from AG27. (b) The three dimensional

structure of target protein was predicted by using SWISS-

MODEL SIB [9] service, accessible via ExPASY web

server [18].

SWISS-MODEL workspace is a web-based integrated

service dedicated to protein structure homology modeling.

It assists and guides the user in building protein homology

models at different levels of complexity. A personal

working environment is provided for each user where

several modeling projects can be carried out in parallel.

Protein sequence and structure databases necessary for

modeling are accessible from the workspace and are

updated in regular intervals. Tools for template selection,

model building and structure quality evaluation can be

invoked from within the workspace.

2.7 Recognition of Errors in Three-Dimensional

Structures of Proteins

ProSA-web server [50] is widely used to check three

dimensional models of protein structures for potential

errors. Its range of application includes error recognition in

experimentally determined structures [10, 31, 55], theo-

retical models [32, 41, 47], and protein engineering [11].

2.8 Analysis on the Crucial Residues of Theoretical

Model of Arsenate Reductase Protein from B.

cereus Strain AG27

The theoretical models were submitted to ConSurf [20], an

automated web based server for the identification of

functional region in proteins. The conservation grades are

projected onto the molecular surface of these proteins to

reveal the patches of highly conserved residues that are

often important for biological function.

2.9 Prediction of Ligand Binding Sites of Theoretical

Model of Arsenate Reductase Protein from

B. cereus Strain AG27

Q-SiteFinder uses the interaction energy between the pro-

tein and a simple Vander Waal probe to locate energeti-

cally favorable binding sites. Energetically favorable probe

sites are clustered according to their spatial proximity and

clusters are then ranked according to the sum of interaction

energies for the sites within each cluster [6].

3 Results and Discussion

Strain AG27 possess high resistance to both forms of

arsenic viz arsenate (AsV) and arsenite (AsIII) i.e. 40 and

35 mM, respectively. Multiple metal resistance is impor-

tant for the bioremediation of isolates in metal contami-

nated site(s) with multiple metals. It has been reported that

resistance to oxyanions viz arsenate, arsenite and antimo-

nite is generally found together in gram positive and gram

negative bacteria [29]. Further, high level of resistance to

antimonite has been associated with arsenical resistance

gene cluster [22]. Bacteria that can tolerate various metals

including nickel, chromium and uranium have also been

reported [3, 24]. Further, another gram positive bacterium

strain ADG (Accession no.EU326525) showed resistance

to Ni, Cu, Pb at concentrations of 4, 5 and 6 mM,

respectively [4]. Therefore, the isolate AG27 was also

checked for resistance to other heavy metals and minimum

inhibitory concentration was determined which was found

to be 4, 5, 3 and 2 mM for chromium, cadmium, copper

and cobalt, respectively.

3.1 Gene Characterization

Resistance to arsenic is reported to be encoded by both

plasmid and chromosomal resistance operons [14, 17].

Therefore, to determine the location of resistant gene/s,

genomic composition in terms of presence or absence of

plasmid DNA in strain AG-27 was determined (Fig. 1a).

The plasmid DNA profile revealed the presence of plasmid

Validation of Arsenic Resistance in Bacillus cereus Strain AG27 93

123

DNA of approximately 5.14 kb when the strain was grown

in the presence of 5 mM sodium arsenate. Furthermore, the

genomic DNA when visualized on gel showed a band of

21 kb.

3.2 Cloning and Sequencing of arsC Gene

It has been reported that ars system consist of a trans-

membrane pump system and an arsC gene coding for

arsenate reductase which reduces arsenate to arsenite

which is finally pumped out of the cell by membrane

protein encoded by arsB gene [51]. The plasmid DNA

isolated from strain AG27, when subjected to Polymerase

chain reaction for the amplification arsC gene produced an

amplicon (300 bp) while no amplified product was

obtained when the gene was amplified from genomic DNA.

Furthermore, no amplicon was obtained in case of DH5awhich served as a negative control. The presence of

amplified product in case of plasmid DNA indicated the

presence of arsC gene encoding arsenate reductase, thereby

giving a clear indication of efflux mechanism (Fig. 1b).

Homology of the sequence when compared with the

available database using BLASTn search showed that the

amplicons contained a partial arsC gene sequence. The

nucleotide sequence of the cloned gene of strain AG27

showed 98% similarity with arsC gene of B. cereus ATCC

10987 (plasmid PBC 10987) and 99% similarity with B.

cereus CM2K4. Further, the translation of gene product of

DQ517938 showed an 86 amino acid residue. The align-

ment of the translated product showed similarity with

arsenate reductase ATCC 10987 and other Bacillus species

strains. Furthermore, the partial sequencing of 16srDNA of

strain AG27 revealed the isolate to be under genus Bacil-

lus. Thus, from the above stated results, it is clear that

resistance to arsenic in strain AG27 is due to arsC gene

present on plasmid DNA and involves efflux mechanism of

resistance.

3.3 Atomic Absorption Spectroscopy and Scanning

Electron Microscopy

Since, the presence of arsC gene indicated the prevalence

of efflux mechanism of resistance in strain AG27, further

validation of the mechanism was done by atomic absorp-

tion spectroscopy and scanning electron microscopic

analysis of strain. The atomic absorption spectroscopy

results of strain AG27 showed that the concentration of

arsenic increased in the bacterial cells with the start of log

phase (4 h) and reached to a maximum of 10 lg/mL of dry

weight of the cell and then decreased after 12 h i.e. with

the end of the log phase. However, in the supernatant the

concentration of arsenic followed a reverse trend i.e. the

concentration of arsenic decreased with the start of log

phase followed by an increase in the late log phase (Fig. 2).

These results ruled out the possibility of accumulation of

metal in the bacterial cells. The comparative arsenic con-

centration in pellet and supernatant fraction clearly support

the increase in arsenic concentration in pellet and decrease

in supernatant at the beginning of the log phase (till 8 h).

This is followed by a constant zone (8–12 h) in the graph

where arsenic concentration does not change either in

pellet or in supernatent. After 12 h i.e. in the late log phase,

the concentration of arsenic decreased in the pellet and

increased in supernatant. This suggested that once arsenic

(sodium arsenate) was taken inside the cell, an enzyme

arsenate reductase encoded by arsC gene converts arsenate

into arsenite which is then effluxed out of the cell, thereby,

leading to increase in concentration of arsenic in superna-

tant and thus, indicating efflux mechanism of resistance.

Furthermore, the data of atomic absorption spectroscopy

was substantiated by scanning electron microscopy (SEM).

The effect of metal on cell morphology was demonstrated

by [42] where transmission electron microscopy analysis of

strain Pseudomonas putida strain 62BN showed an

increase in size of the cells grown in the presence of

Fig. 1 a Plasmid an genomic

DNA profile of arsenic resistant

strain AG-27 lane M- ladder,

lambda DNA/EcoRI/HindIII

double digest, Lane 1- pUC 19

vector (2.85 kb), Lane 2- self

ligated pST blue vector

(3.85 kb) Lane 3- Plasmid DNA

profile, Lane4 -Genomic DNA

profile. b Amplified arsC gene

from plasmid and genomic

DNA of AG-27, lane M-100 bp

ladder, lane1- control (DH5a),

lane2- Amplification profile

from plasmid DNA, lane3-

Amplification profile from

genomic DNA

94 S. Jain et al.

123

cadmium and also showed intracellular and periplasmic

accumulation of metal in the cells. Further, the SEM

analysis of gram positive strain ADG (EU236525) [4]

showed an increase in surface area of cells treated with

copper as compared to untreated cells. SEM analysis of

Pseudomonas aeruginosa strain MCCB 102 showed cad-

mium and lead accumulation in the cell wall and along the

external cell surfaces [59]. However, the scanning electron

micrographs of strain AG27 did not show any change in

cell size and morphology of the cells when the cells were

grown in the presence of 5 mM sodium arsenate thereby

indicating efflux mechanism of resistance (Fig. 3).

3.4 Three Dimensional Computer Modeling

Further, to explore the catalytic mechanism of arsenic in

the strains, 3D structure was modeled to spot the active site

and residues which are taking part in the efflux. The

sequence of arsC from AG27 was obtained from NCBI

database (accession number: DQ517938) which is used as

target for this technique. Closely related sequences or

templates of this target were selected by using protein

sequence similarity search against three-dimensional

structural data banks. BLASTp was used as protein

sequence similarity search engine which accepts input as

protein sequence. Conserved domain sequence was sub-

mitted to this search engine and picked out its homologs

with the help of PDB. Results of BLASTp revealed that

arsC, the major arsenate reductase from B.subtilis, has

79.9% sequence similarity score with the given protein

sequence DQ 517938 and its PDB id 1JL3|B| which was

taken as template.

Three-dimensional structure of modeled arsC from

AG-27 reveals that like arsC from B. subtilis, it consists of

single a/b domain containing a central four stranded, paral-

lel, open-twisted b-sheet flanked by a-helices on both sides

(Fig. 4). Secondary structure assessment shows that arsC

from AG27 encompasses b1 residues 1–4 (YFIC); b2 resi-

dues 29–33 (YSAGI); b3 residues 70–74 (DLVVT); b4

residues 91–95 (KRVHW), whereas arsC from B. subtilis

encompasses b1 residues 2–8 (NKIIYFIC); b2 residues

30–36 (EWKVYSAGIE); b3 residues 75–78 (ADLVVTLC);

b4 residues 95–98 (KREHW). Aromatic amino acid residues

F2, H94 and W95, which are essential for single strand

nucleic acid binding and lysine residue K91, located on the

surface are common in both arsC genes.

Also, both the three dimensional structures consists of 6

hydrogen bonded turns, all of them being b turns. In

1JL3|B|, arsC consists of residues; 9–11 (TGN), 27–29

(GDEW), 37–39 (TEA), 57–60 (ISNQ), 73–74 (AD),

91–94 (PPHV), and in AG27 arsC turns consists of fol-

lowing residues; 5–7 (TGN), 23–26 (GDKW), 33–35

(IEA), 53–56 (ITDQ), 69–70 (AD), 87–90 (PPHV). In both

the structures, same residues are participating in the bstrands and b turns, and no deviation whatsoever was

found, apart from some similar residues (Fig. 5).

Earlier studies revealed that three redox active cysteine

residues (Cys10, Cys82, and Cys89) are critical for arse-

nate reduction. A disulfide bridge (Cys82–Cys89) is

Fig. 2 Arsenic content in pellet and supernatant fraction of Bacilluscereus strain AG27 (grown at 30 �C and at pH 7) at different time

intervals of growth

Fig. 3 Scanning electron

micrographs of Bacillus cereusstrain AG27 (a) in absence and

(b) in presence of 5 mM sodium

arsenate at a magnification of

6000X

Validation of Arsenic Resistance in Bacillus cereus Strain AG27 95

123

formed after a single catalytic reaction cycle, converting

the enzyme into the inactive form. arsC is subsequently

regenerated by thioredoxin, which converts the enzyme

into the reduced form [36].

The P-loop, which is conserved CX5R anion-binding

motif containing Cys10, is proposed to be the arsenate-

binding site [52]. Four cysteine residues (Cys10, Cys15,

Cys82, and Cys89) are conserved between the two arsC

proteins. In case of arsC AG27, the P-loop consists of

CTGNSCR residues which are probably acting as arsenate

binding site which are accordant with the 1JL3|B| P-loop

residues. These motifs facilitate recognition/binding of

arsenic, thereby enhancing reduction of arsenate to

arsenite.

3.5 Recognition of Errors in 3D Structure

The stereochemistry of the theoretical model of arsC was

done by PROCHECK, which is used for verification and

evaluation of three-dimensional structure of proteins. The

main output of PROCHECK is the Ramachandran plot. In

the Ramachandran plot analysis, the residues were classi-

fied according to their regions in the quadrangle. The

Ramachandran map for AG27 arsC is represented in

Fig. 6. By the plot analysis, it can be noticed that more than

98.0% of the residues are in allowed regions, leading to a

good validation for the model. The residues that are in bad

quadrangles are a reflex from the protein used as template.

A good quality model would be expected to have over 90%

in the most favored regions. Finally, the model was

deposited into PMDB and ID number given to submission

is PM0076501.

A major problem in structural biology is the recognition

of errors in experimental and theoretical models of protein

structures. ProSA program (Protein Structure Analysis) is

an established tool which has a large user base and is

frequently employed in the refinement and validation of

experimental protein structures and in structure prediction

and modeling [57]. ProSA uses knowledge-based potentials

of mean force to evaluate model accuracy [50]. After

parsing the coordinates, the energy of the structure is

evaluated using a distance-based pair potential [48, 50] and

a potential that captures the solvent exposure of protein

residues [50]. From these energies, two characteristics of

the inputs structure are derived and displayed on the

webpage: its Z-score and a plot of its residues energies.

Fig. 4 Alignment of sequences

of arsC from Bacillus cereusstrain AG27 and arsC from

Bacillus subtilis by SWISS-

MODEL showing consensus

regions and secondary structure

Fig. 5 Three dimensional

structural representation

(YASARA view) ribbon

display. a arsC from Bacillussubtilis and, b arsC from

Bacillus cereus strain AG27

(DQ51798)

96 S. Jain et al.

123

The Z-score indicates overall model quality and measure

the deviation of the total energy of the structure with

respect to an energy distribution derived from random

conformation [49, 50].

Z-score outside range characteristics for native proteins

indicate erroneous structures. Groups of structures from

different sources (X-ray, NMR) are distinguished by dif-

ferent colors. This plot can be used to check whether the

Z-score of the protein in question is within the range of the

proteins of similar size belonging to one of these groups.

The value, -5.89 (Fig. 7) is in the range of native con-

formation. Overall, the residues energies are largely neg-

ative with the exception of some peaks in N-terminal part.

3.6 Analysis of Conserved Residues

The ConSurf server was used to extract information about

important residues, which are of functional value. This

server provides evolutionary related conservation scores

for residues, which could be correlated with biological

function. The continuous conservation scores of each of

the amino acid position are available (Table 1) that are

divided into a discrete scale of 9 grades for visualization

purpose. Grade 1 contains the most variable positions and

is colored light grey; grade 5 contains intermediately

conserved position and colored white; and grade 9 con-

tains the most conserved positions and is colored dark

grey.

The color conservation grades are projected onto the

three dimensional structure of the query protein and arsC

from B.subtilis. ConSurf server shows that 44 amino acid

residues in arsC from AG27 and 52 amino acid residues in

B.subtilis are with high conservation score. In Table 1,

amino acid residues (shown as bold), of arsC from

B.subtilis and modeled arsC from B.cereus strain AG27

shows the conservation score of 8 and 9.

Fig. 6 Quality check of

modeled arsC from AG27.

Regions are nomenclatured as:

most favoured regions [A, B, L],

additional allowed regions [a, b,

l, p], genereously allowed

[-a, -b, -l, -p]

Fig. 7 Investigation of arsC

from AG27 using the ProSA-

web service. a The ProSA web

result indicate that protein

structure has features

characterstics for native

structures The Z-score of

protein structure is -5.89,

highlighted as large dot. b The

energy plot of protein structure

Validation of Arsenic Resistance in Bacillus cereus Strain AG27 97

123

From comparative analysis of residues, it is clear that

most conserved residue with conservation score 8 and 9

and having functional value are conserved in both arsC

from B.subtilis and modeled arsC from B.cereus. The polar

residues known to facilitate the entry of arsenate ions to the

active site (Thr-11, Ser-14 and His-42) in B.subtilis

arsenate reductase are conserved in modeled arsenate

reductase too (Thr-4, Ser-8 and His-46). Further, triple

cysteine redox relay system residues; Cys-10, Cys-82, and

Cys-89 which have been identified as key residues in the

arsenate reductase redox reaction in S. aureus [58],

are located close to the protein surface in modeled arsC

Table 1 The amino acid conservation score of arsC from B. Subtilis (PDB id 1JL3|B|) and rasC from B. Subtilis strain AG27 by Consurf server

Residue no. arsCAG27 arsC 1JL3|B| Residue no. arsC AG27 arsC 1JL3|B| Residue no. arsC AG27 arsC 1JL3|B|

ATOM Score ATOM Score ATOM Score ATOM Score ATOM Score ATOM Score

1 TYR 8 TYR 9 41 ASN 6 ASN 6* 81 ASP 1 ASP 1

2 PHE 9 PHE 9 42 ALA 9 ALA 9 82 VAL 1* VAL 1

3 ILE 5 LEU 7 43 ILE 6 VAL 5 83 CYS 8 CYS 9

4 CYS 9 CYS 9 44 LYS 4* LYS 3* 84 PRO 9 PRO 9

5 THR 8 THR 8 45 ALA 8 ALA 8 85 THR 1 MET 1

6 GLY 9 GLY 9 46 MET 8 MET 9 86 THR 5* THR 6*

7 ASN 9 ASN 9 47 LYS 1 LYS 1* 87 PRO 9 PRO 9

8 SER 8 SER 9 48 GLU 9 GLU 9 88 PRO 1 PRO 1

9 CYS 9 CYS 9 49 VAL 3* VAL 6* 89 HIS 3* HIS 5

10 ARG 9 ARG 9 50 ASP 1 GLY 1* 90 VAL 7 VAL 8

11 SER 9 SER 9 51 ILE 8 ILE 8 91 LYS 1* LYS 3*

12 GLN 8 GLN 9 52 ASP 8 ASP 9 92 ARG 7 ARG 8

13 MET 9 MET 9 53 ILE 8 ILE 9 93 VAL 1* GLU 4*

14 ALA 9 ALA 9 54 THR 5* SER 5* 94 HIS 9 HIS 9

15 GLU 9 GLU 9 55 ASP 1* ASN 1 95 TRP 8 TRP 8

16 ALA 6* GLY 7* 56 GLN 8 GLN 8 96 GLY 9 GLY 9

17 TRP 1* TRP 1* 57 THR 8 THR 9 97 PHE 6* PHE 7*

18 GLY 1 ALA 2* 58 SER 9 SER 9 98 ASP 6* ASP 6*

19 LYS 8 LYS 5* 59 ASP 6 ASP 8 99 ASP 9 ASP 9

20 LYS 1 GLN 1 60 ILE 1 ILE 1

21 TYR 2* TYR 1* 61 ILE 8 ILE 9

22 LEU 6* LEU 9 62 ASP 4* ASP 6*

23 GLY 1* GLY 1 63 ARG 1 SER 1

24 ASP 1* ASP 2* 64 ASP 1 ASP 1

25 LYS 1 GLU 1 65 ILE 5* ILE 6*

26 TRP 5* TRP 8 66 LEU 5* LEU 7*

27 ASN 1* LYS 1 67 ASP 1 ASN 1

28 VAL 9 VAL 9 68 LYS 1 ASN 1

29 LEU 1 TYR 1* 69 ALA 8 ALA 8

30 SER 9 SER 9 70 ASP 8 ASP 8

31 ALA 8 ALA 8 71 LEU 3* LEU 4*

32 GLY 9 GLY 9 72 VAL 8 VAL 8

33 ILE 5 ILE 6* 73 VAL 7 VAL 8

34 GLU 7* GLU 9 74 THR 8 THR 9

35 ALA 7 ALA 7* 75 LEU 7 LEU 9

36 HIS 8 FITS 9 76 CYS 8 CYS 9

37 GLY 6* GLY 9 77 GLY 6* GLY 6*

38 VAL 5 LEU 6* 78 HIS 6* ASP 4*

39 ASN 8 ASN 9 79 ALA 9 ALA 9

40 PRO 9 PRO 9 80 ASN 4* ALA 4*

* Less conserved amino acid residues

98 S. Jain et al.

123

(Cys-4, Cys-76, Cys-83), with presence of Cys-10 in AB or

P-loop, and depicts high conservation values. Residues

Asp-105 and Arg-16, which are involved in catalytic

mechanism of arsenic, where latter works as a general acid

to facilitate the leaving of a water molecule, thus stabiliz-

ing the ES complex, and former is a positively charged

residue which has the roles of stabilizing the P-loop and

binding the arsenate ion by lowering the pKa values of all

three cysteine residues to activate them for the reaction

[12], are in conservation score of 9 (Asp-99) and (Arg-10).

Thus, the finding are analogous with the proposed arsenate

reduction mechanism [58], in which the first step (ES

complex formation) is mediated by the activated Cys-10

thiolate which attacks the arsenate to form complex with

the help of Asp-105 residue. Second step (arsenate reduc-

tion) involves three catalytic cysteines so called triple

cysteine redox relay system to produce the Cys-82–Cys-89

disulfide bond and an arsenite ion with the help of Arg-16

residue. Since, the arsenite ion is more toxic than the

arsenate ion, thus, it is most likely coupled to the mem-

brane arsenite-specific transporter and exported immedi-

ately rather than being released into the cell, showing the

existence of efflux mechanism for arsenic detoxification in

B. cereus strain.

3.7 Prediction of Ligand Binding Sites

The function of a protein is defined by the interaction it

makes with other proteins and ligands. Computational

methods for the detection and characterization of functional

sites on protein have increasingly become an area of interest

[15]. There is at least one successful prediction in the top

Table 2 Qsitefinder analysis of

arsC from strain AG27. The

energetically most favored site

with residue involved in

interactions are shown

Validation of Arsenic Resistance in Bacillus cereus Strain AG27 99

123

three predicted sites in 90% of proteins tested when using

Q-SiteFinder. Generally, ligand binding site prediction

method analyzes the protein surface for pockets. The ligand

binding sites are usually in the largest pocket. The pockets

are defined only by energetic criteria. The method calcu-

lates the Vander Waals interaction energies of probe with

protein. Probes with favorable interaction energies are

retained and clusters of these probes are ranked according to

their total interactions energies. The energetically most

favorable cluster is then ranked first. The results of QSite-

Finder studies are summarized in Table 2. Most favorable

binding sites contain amino acids with high conservation

residue score. These residues may be involved with main

binding site.

4 Conclusions

B.cereus strain AG27 isolated from soil, resistant to both

forms of arsenic showing multiple metal resistance was

found to be presenting partial arsC gene sequence on their

plasmid DNA. Moreover, the data obtained after atomic

absorption spectroscopy and scanning electron microscopy

also proved the prevalence of efflux mechanism in AG27.

By explanation of mechanism at 3D level, from the modeled

structure of arsC, it can be concluded that arsenate reduc-

tase protein arsC from B.cereus AG27 strain isolated from

soil is structurally similar to arsC, a major arsenate reduc-

tase protein from B.subtilis and contains CX5R motif, also

found in major arsenic resistant micro-organisms. Further,

residues essential for arsenate reductase mechanism

observed from sequence alignment are found to be well

conserved in structure, thus, validating the built model.

Acknowledgments This study was supported by DBT- India grant

to R.G. Senior Author SJ and BS also acknowledge JRF during course

of this study.

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