World J Microbiol Biotechnol
DOI 10.1007/s11274-007-9370-2
ORIGINAL PAPER
Identification of an arsenic resistance mechanism
in rhizobial strains
Paula Sá-Pereira Æ Mónica Rodrigues Æ
Isabel Videira e Castro Æ Fernanda Simões
Received: 29 November 2006 / Accepted: 30 January 2007

Springer Science+Business Media B.V. 2007
Abstract Arsenic (As) is a very toxic metalloid to a great
number of organisms. It is one of the most important global
environmental pollutants. To resist the arsenate invasion,
some microorganisms have developed or acquired genes
that permit the cell to neutralize the toxic effects of arsenic
through the exclusion of arsenic from the cells. In this
work, two arsenic resistance genes, arsA and arsC, were
identified in three strains of Rhizobium isolated from
nodules of legumes that grew in contaminated soils with
effluents from the chemical and fertilizer industry con-
taining heavy-metals, in the industrial area of Estarreja,
Portugal. The arsC gene was identified in strains of Sino-
rhizobium loti [DQ398936], Rhizobium leguminosarum
[DQ398938] and Mesorhizobium loti [DQ398939]. This is
the first time that arsenic resistance genes, namely arsC,
have been identified in Rhizobium leguminosarum strains.
The search for the arsA gene revealed that not all the
strains with the arsenate reductase gene had a positive
result for ArsA, the ATPase for the arsenite-translocating
system. Only in Mesorhizobium loti was the arsA gene
amplified [DQ398940]. The presence of an arsenate
reductase in these strains and the identification of the arsA
gene in Mesorhizobium loti, confirm the presence of an ars
operon and consequently arsenate resistance.
P. Sá-Pereira (&)
M. Rodrigues
F. Simões
Dept Biotecnologia, Grupo de Biologia Molecular, INETI,
Estrada do Paço do Lumiar, 22, Lisbon 1649-038, Portugal
e-mail: paula.pereira@ineti.pt
I. V. e Castro
Dept Ecologia, Recursos Naturais e Ambiente, EFN-INIA,
Quinta do Marquês, Av. República, Nova Oeiras, Oeiras
2784-505, Portugal
Keywords Arsenic
ars operon
arsC
arsA

Mesorhizobium loti
Rhizobium leguminosarum
Introduction
Nitrogen fixation is a very important reaction in nature and
is a bacterial-dependent process. However, only a few
bacteria are able to do it, particularly soil bacteria from the
Rhizobium, Bradyrhizobium, Azorhizobium, Sinorhizobi-
um, Allorhizobium and Mesorhizobium genus, collectively
called nitrogen-fixing bacteria (Taiz and Zeiger 1998;
Raven et al. 1999; Lindstrom et al. 2002). Rhizobia can
induce nodule formation on legume plant roots, like alfalfa
(Medicago sativa), clover (Trifolium), soybean (Glycine
max L. Merr.), pea (Pisum sativum L.) or bean (Phaseolus)
(Raven et al. 1999; Lindstrom et al. 2002) and this asso-
ciation may be of great ecological value, because bacteria
resistant to toxic compounds present in soil may act in the
bioremediation of those soils.
Arsenic (As) is a semi-metal or metalloid, toxic for most
organisms. It exists naturally in organic and inorganic
forms in environment, but is also produced as a waste
product from a number of industries (Jones et al. 2000;
Oremland and Stolz 2003; Jackson et al. 2003). In the
inorganic form, it mainly exists as arsenate, the oxidized
form (AsO3– 2–
4 or HAsO4 ) and as arsenite, in the reduced
– –
form (AsO2 or H2AsO3), both being toxic for cells (Cullen
and Reimer 1989). The arsenate toxicity is related to its
structural analogy with the phosphate ion (PO3– 4 ), so it
interferes with the intracellular transport of phosphate,
endangering the entire cell metabolism. Because of the
resemblance to phosphate, arsenate enters to the cell
through two types of phosphate transport system. To resist
to the arsenate invasion, some microorganisms have
123

developed or acquired genes that permit to the cell to
neutralize the toxic effects of arsenic through the exclusion
of arsenic from the cell (Gihring et al. 2003). Chromo-
somal and plasmid-based arsenic-resistance genes are
clustered in an operon, commonly known as the ars operon
(Silver and Keach 1982; Cervantes et al. 1994; Xu et al.
1998; Nies 1999). These genes have been identified in
several bacteria like Escherichia coli (Silver et al. 1981),
Staphylococcus aureus (Silver et al. 1981; Ji and Silver
1992a, b), Bacillus subtilis (Tsutomu and Kobayashi 1998),
Shigella sonnei, Citrobacter freundii, Klebsiella pneumo-
niae (Diorio et al. 1995), Thiobacillus ferrooxidans (Mer-
geay 1991; Butcher et al. 2000). Normally, it enters the cell
in periods of phosphate abundance through the Pit system
which is a nutrient transport system, nonspecific, and which
is constitutively expressed (Bruins et al. 2000). Arsenate is
transported into the cell by the Pit system and is subse-
quently reduced to arsenite by a cytoplasmic arsenate
reductase enzyme (EC 1.20.99.1), encoded by the arsC
gene. Arsenite is then effluxed from cell through the
chemiosmotic gradient by the arsenite-specific transmem-
brane protein ArsB, encoded by the arsB gene. The ArsA
protein, encoded by the arsA gene, is an arsenite-stimu-
lated ATPase that couples ATP hydrolysis to the arsenite
efflux through the ArsB protein. So, ArsA and ArsB,
together, form an ATP-driven arsenite pump (Dey et al.
1994; Silver 1996; Suzuki et al. 1998). ArsB can function
with or without the catalytic ArsA ATPase, although
resistance is reportedly enhanced by the ArsA unit (Cer-
vantes et al. 1994). Therefore, arsenic resistance is con-
ferred by the arsRBC genetic system, but if arsA gene is
also expressed, arsenite efflux is more efficient. ars gene
transcription is regulated by two different regulatory pro-
teins, ArsD and ArsR, encoded respectively by the arsD
and arsR genes. Some microorganisms lack the arsD gene,
so it is thought that the ArsD protein is not strictly
necessary in the detoxification process, acting only as a
secondary regulator (Rouch et al. 1995).
The main goal in this study is to search for the ars genes
associated with genetic resistance from three rhizobial
species harvested from root nodules of Lotus corniculatus
L. and Trifolium sp. that had grown in soils contaminated
by effluents from the chemical and fertilizer industry,
located on the Estarreja Chemical Complex.
Materials and methods
Culture media, growth conditions and maintenance
strains of Rhizobium
This study involved 37 rhizobial isolates obtained from
nodules of the leguminous plants Lotus corniculatus
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World J Microbiol Biotechnol
L. and Trifolium sp., harvested from soils contaminated
with effluents from the chemical and fertilizer industry.
We select soils from an area of known pollution prob-
lems, where heavy metals and other pollutants have been
emitted by industry for nearly 40 years. This area is
particularly affected by the release of liquid effluents
from fertilizer industry (mainly sulphuric acid, nitric acid,
mononitrobenzene and heavy metals such as Pb, Zn and
Cd and the metalloid As) and chemical industries (mainly
Hg and sulphuric acid from the manufacture of NaOH
and chlorate, Leitão et al. 1992). Composite soil samples
were collected from an arable field in the centre of
Portugal (Estarreja region). They were taken from 0 to
20 cm depth. One was collected near an industrial
effluent channel (not impermeable and rarely overflow-
ing) and the other was collected 10 m away. When the
soil samples were collected, no clover was present at
either site. However, the soil had been used for annual
forages including a mixture of grass and clover. Both
soils were of identical texture (sandy loam) and similar
organic matter (33 and 32 g kg–1), electrical conductivity
(0.21 and 0.11 dS m–l) and pH (6.00 and 6.17) for pol-
luted and unpolluted soils, respectively. The soil samples
were stored at 50% WHC in loosely-tied plastic bags at
4 C in the dark. The total heavy metal concentrations
(Zn, Cd, Cu, Ni, Pb, As and Cr) in both soils were
measured after extraction with aqua regia. Hg was
determined by an adaptation of the method described by
Stewart and Bettany (1982).
For isolation of rhizobial strains from nodules, the
procedure of Vincent (1970) was followed.
These strains were characterized phenotypically by
the usual microbiological methods, and were identified
as being from the species Mesorhizobium loti, Sino-
rhizobium meliloti and Rhizobium leguminosarum
bv. trifolli (Table 1). They were selected due to their
ability to nodulate their hosts in heavy-metal contami-
nated soils.
The strains were grown in mannitol medium yeast
extract (YEM) at 28 C for one week and preserved in the
same medium containing agar (1.8%, w v–1, YEMA) at
4 C, (Castro et al. 2003).
DNA extraction
For extraction of genomic DNA, methodology of Vogel-
stein and Gillespie (1979) was followed. The bacterial
biomass grown for 5 days in a 5 cm Petri dish was col-
lected with a pipette tip and introduced in a 1 mL micro-
tube. Bacterial DNA was extracted using the kit
Nucleospin Tissue (Macherey-Nagel) according to the
supplier’s instructions.
World J Microbiol Biotechnol

Table 1 Rhizobial isolates used in the study

Isolates Strains Harvest place

1C, 13C, 15C, 16C, 28C, 30C Mesorhizobium loti Estarreja eucaliptal
2, 3,5, 6, 7, 8, 10, 9,11,12,13,14, 15, 17, 18, 20, 21, 22, 27, 28, 29 Mesorhizobium loti Estarreja Vala de Breja
32 TsIA(C+), 123 Ts2a (C++), 31, 32, 33, 34 Rhizobium leguminosarum bv trifolli Estarreja Póvoa de Baixo
35, 36, 37, 38 Sinorhizobium meliloti

Primer design varying the temperature according to each primer,

The primers designed to amplify arsA, arsB, arsC, arsD and
arsR genes were obtained using the COnsensus-DEgenerate
Hybrid Oligonucleotide Primers strategy (CODEHOP)
(Rose et al. 1998). The CODEHOP program designs a pool
of primers that includes a short 3¢ degenerate core region
and a longer 5¢ consensus clamp region. All the primers
were drawn based on protein sequences alignment using
MultAlign software tool from each gene that was obtained
from NCBI database. After alignment of the protein se-
quences with the BLOCK MAKER program (http://
blocks.fhcrc.org/) (Heinikoff et al. 1995), consensus-
degenerate PCR primers were designed using the software
in http://blocks.fhcrc.org/blocks/codehop.html. The primers
obtained using this strategy are described in the Table 2.
(Table 2), extension (45 s at 72 C) and a final extension
step at 72 C for 10 min. Amplified DNA fragments were
separated using gel electrophoresis: 1–2% agarose (w v–1,
Bioline) in TAE (1X, Qbiogene), with EtBr at 2% (v v–1)
(stock solution, 10 mg ml–1, Macherey-Nagel). After run-
ning the gel, at 100 V, constant current, for 1 h, the frag-
ment migration was analysed and registered.
Cloning procedures
The PCR products were cloned into pTZ57R/T vector
using InsT/AcloneTM PCR Product Cloning Kit according
to supplier’s instructions (Fermentas).
DNA Sequencing

PCR amplification Sequencing reactions were carried out according to

Amplifications were performed in a reaction mixture with
1 mM MgCl2, 0.2 mM dNTP’s, 1· PCR buffer (NH4)2SO4,
1 U Taq DNA polymerase (Fermentas), 0.4 pmol lL–1 of
each primer (forward and reverse) and 50 ng of DNA
template in a final volume of 25 lL. Negative controls
were performed by addition of 1 lL of water to the reac-
tion mixture. DNA amplification was performed in a iCy-
cler BioRad thermocycler with the following program:
initial denaturation for 3 min at 95 C, 35 cycles consisting
of: denaturation (1 min at 95 C), annealing (1 min,
standard protocols (ABI Sequencing Reaction Kit V3.1,
Applied Biosystems), using either the original PCR primers
(see Table 2), or the InsT/AcloneTM cloning kit sequencing
primers. Nucleotide sequencing was performed with an
ABI Prism 310 automated sequencer (Applied Biosys-
tems).
Data analysis
Sequences were assembled with the BioEdit sequence
alignment editor (Hall 1999) with local BLAST (Altschul

Table 2 Degenerated arsA, arsB, arsC, arsD and arsR primers using the CODEHOP strategy
Primers Sequence Ta (C) Degeneracy degree

arsA (F) 5¢-GGCAAGGGCGGCGTNGGNAARAC-3¢ 50 32

arsA R) 5¢-GGGTATGGCCGGTCGGNGCNGTRT-3¢ 32
arsB (F) 5¢-TTCACCATCGTCCTGGTCATHTGGCARCC-3¢ 50 6
arsB (R) 5¢-CGCAGGGCCAGGGCNGCNARNGT-3¢ 128
arsC (F) 5¢-ATGACCGTCACCATHTAYCAYAAYC-3¢ 48 24
arsC R) 5¢-ACGACCGCCTCGCCRTCYTCYTT-3¢ 8
arsD (F) 5¢-TGGTGTTCGACCCCGCNATGTCYTG-3¢ 50 8
arsD R) 5¢-GCAGCAGGAGGTGTTGCCNCCRCARCA-3¢ 16
arsR (F) 5¢-CGCCACGGCCGAGTCNCARCCNAARA-3¢ 50 64
arsR (R) 5¢-CGTGTCGATGATCTCGGCNGCCCANGC-3¢ 16

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 World J Microbiol Biotechnol

et al. 1990) and CLUSTAL W algorithms (Thompson et al.

1994). The BLAST server at http://www.ch.embnet.org/
software/bBLAST.html was also used. All sequences were
obtained from the European Molecular Biology Laboratory
nucleotide sequence database (EMBL-Bank) at http://
srs6.ebi.ac.uk/. Accession numbers are indicated in brackets.

Results

PCR reactions with arsA primers were performed and it

were obtained fragments whose size varied between 250
and 400 bp according to the strains analysed (Fig. 1). Two
strains from each species with positive amplification were
selected to study the arsA gene. All fragments were cloned
and sequenced. Only the Mesorhizobium loti strains
showed homology with ArsA, the catalytic ATPase subunit
of the arsenite-translocating system [DQ398940].
For amplification of the arsB gene, PCR reactions were
performed with all strains but there was no fragment
amplification.
To look for the arsC gene, PCR reactions were
performed and a single fragment was amplified with about
400 bp (Fig. 2). The strains with positive amplification
using the ArsA primers were selected to continue the study
of the arsC gene.
These fragments were cloned and sequenced. The
sequences amplified from Sinorhizobium loti [DQ398936],
Rhizobium leguminosarum [DQ398938] and Mesorhizobi-
um loti [DQ398939] were shown to have homology with
arsenate reductases.
To amplify the regulatory genes, arsD and arsR, PCR
reactions were performed but using the arsD primer no
fragment was amplified, with these assay conditions. To
amplify the arsR gene, PCR reactions were performed
using the strains positive for arsA and arsC primers (28c,
33, 34, 36 and 37). PCR showed the presence of several
bands for each strain, corresponding to fragments which
size ranging from 250 bp to 1500 bp. The fragments
obtained were re-amplified with the purpose to investigate
if any of them matched the arsR gene (results not shown).
The fragments sequenced did not show homology with
regulatory proteins.
Fig. 2 Electrophoresis for the PCR amplification with arsC primers,
Ta = 48 C, visualized by 1% agarose gel in TAE 1· buffer; M: DNA
ladder 1 kb; C: negative control
Discussion and conclusion
Metal resistance is a common process in many microor-
ganisms that deal with these toxic compounds in their
habitats. In the last few years, investigation about the cel-
lular mechanisms involved in metal resistance has in-
creased our knowledge of many of those mechanisms
which are encoded by genes clustered in operons. The ars
operon of arsenic resistance is one of those.
The arsC gene that encodes a cytoplasmic arsenate
reductase, is a highly conserved gene among microorgan-
isms (Mukhopadhyay et al. 2002), which could be related
to its essential function in the arsenic resistance mecha-
nism. Arsenate reduction to arsenite is the first step of the
detoxification process of this metalloid in cells. The high
homology of the arsC gene among different bacterial
species has made possible primer design for this study.
The arsC gene was identified in strains of Sinorhizobium
loti [DQ398936], Rhizobium leguminosarum [DQ398938]
and Mesorhizobium loti [DQ398939]. The presence of an
arsenate reductase in these strains confirms the presence of

Fig. 1 Electrophoresis for the

PCR amplification with arsA
primers, Ta = 50 C, visualized
by 1% agarose gel in TAE 1·
buffer; M: DNA ladder 1 kb;
C: negative control

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World J Microbiol Biotechnol
an ars operon and consequently arsenate resistance. This
was the first time that arsenic resistance genes, namely
arsC, were identified in Rhizobium leguminosarum strains.
However, not all the strains with the arsenate reductase
gene had a positive result for ArsA, the ATPase for the
arsenite-translocating system. Only in Mesorhizobium loti
was this gene amplified [DQ398940]. The sequence has
homology with the ArsA-catalytic ATPase subunit of the
arsenite-translocating system in many organisms known as
metal resistant, including Magnetospirillum magnetotacti-
cum [ZP_00208147], Shewanella sp. [AAO31598, Saltikov
et al. 2003], Burkholderia vietnamiensis [ZP_00422772],
Rhodospirillum rubrum [ZP_00269302], Bacillus cereus
[NP_982163, Rasko et al. 2004] and Rhodopirellula bal-
tica [CAD76332, Glockner et al. 2003]. The fact that only
in the Mesorhizobium loti strains was this gene amplified,
suggests that the other strains may have a different struc-
ture for the ars operon, that may be similar to the Staph-
ylococcus aureus pI258 (Ji and Silver 1992b), in which the
ars operon is composed only by arsBCR genes. This
operon structure without asrA can be due to ArsB proteins
that can also function as primary ATP-driven arsenite
pumps by interacting with an arsenite-stimulated ATPase
(Rosen et al. 1999). However, Galibert et al. (2001),
detected arsA gene in composite genome of the legume
symbiont Sinorhizobium meliloti, but the information
obtained from in the NCBI database concerning Sinorhiz-
obium meliloti genome, does not show any arsA gene or
homologous sequence [NC_003047], confirming the results
obtained in this study for this strain.
In spite of two genes of the operon being identified,
arsB, arsD and arsR genes were not found in any of the
rhizobial species. This could be related to some
unspecificity of the designed primers, since these genes
show a high variability in the sequences among different
species, making difficult the good alignment for the
primer design.
For the three rhizobia species in this study, arsenic
resistance was confirmed at molecular level, and for the
first time, arsenic resistance genes were identified in Rhi-
zobium leguminosarum, particularly the arsC gene. These
species are capable of reducing arsenate to arsenite via
transcription of the arsC arsenate reductase gene, a com-
ponent of a fairly widespread arsenic detoxification system.
The presence of the arsA gene was also demonstrated in
Mesorhizobium loti. These results confirm the presence of
an ars operon and consequently arsenate resistance, in these
strains.
As expected, the strains isolated from soils contami-
nated by effluents from chemical and fertilizer industry,
with high arsenic content, have the genetic information that
allows them to survive in these harsh habitats.
Acknowledgements We are grateful to Eng. Diogo Mendonça from
Molecular Biology Group-INETI and Fátima Sobral from Direcção
Geral de Veterinária, for DNA sequencing procedures.
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