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Bacteria and Genes Involved in Arsenic Speciation in
Bacteria and Genes Involved in Arsenic Speciation in
Sediment Impacted by Long-Term Gold Mining
Patrı́cia S. Costa1, Larissa L. S. Scholte2, Mariana P. Reis1, Anderson V. Chaves1, Pollyanna L. Oliveira1,
Luiza B. Itabayana1, Maria Luiza S. Suhadolnik1, Francisco A. R. Barbosa1, Edmar Chartone-Souza1,
Andréa M. A. Nascimento1*
1 Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais; Belo Horizonte, Brazil, 2 Grupo de Genômica e Biologia
Computacional, Centro de Pesquisas René Rachou (CPqRR), Fundação Oswaldo Cruz (FIOCRUZ), Belo Horizonte, Brazil
Abstract
The bacterial community and genes involved in geobiocycling of arsenic (As) from sediment impacted by long-term gold
mining were characterized through culture-based analysis of As-transforming bacteria and metagenomic studies of the
arsC, arrA, and aioA genes. Sediment was collected from the historically gold mining impacted Mina stream, located in one
of the world’s largest mining regions known as the ‘‘Iron Quadrangle’’. A total of 123 As-resistant bacteria were recovered
from the enrichment cultures, which were phenotypically and genotypically characterized for As-transformation. A diverse
As-resistant bacteria community was found through phylogenetic analyses of the 16S rRNA gene. Bacterial isolates were
affiliated with Proteobacteria, Firmicutes, and Actinobacteria and were represented by 20 genera. Most were AsV-reducing
(72%), whereas AsIII-oxidizing accounted for 20%. Bacteria harboring the arsC gene predominated (85%), followed by aioA
(20%) and arrA (7%). Additionally, we identified two novel As-transforming genera, Thermomonas and Pannonibacter.
Metagenomic analysis of arsC, aioA, and arrA sequences confirmed the presence of these genes, with arrA sequences being
more closely related to uncultured organisms. Evolutionary analyses revealed high genetic similarity between some arsC
and aioA sequences obtained from isolates and clone libraries, suggesting that those isolates may represent
environmentally important bacteria acting in As speciation. In addition, our findings show that the diversity of arrA
genes is wider than earlier described, once none arrA-OTUs were affiliated with known reference strains. Therefore, the
molecular diversity of arrA genes is far from being fully explored deserving further attention.
Citation: Costa PS, Scholte LLS, Reis MP, Chaves AV, Oliveira PL, et al. (2014) Bacteria and Genes Involved in Arsenic Speciation in Sediment Impacted by LongTerm Gold Mining. PLoS ONE 9(4): e95655. doi:10.1371/journal.pone.0095655
Editor: Celine Brochier-Armanet, Université Claude Bernard - Lyon 1, France
Received June 3, 2013; Accepted March 31, 2014; Published April 22, 2014
Copyright: ß 2014 Costa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Sources of funding: FAPEMIG APQ 00801/12, CNPq nu472411/2012-8, CNPq/INCT no 15206-7. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
more comprehensive knowledge on the structure of the bacterial
community involved in As-transformation in gold-mining sites
remains warranted.
The arsenate (AsV) reducing pathways known are the detoxification (arsC gene) and the dissimilatory respiration (arrA/B
genes). The organization of ars operons varies greatly between
taxa, and the core genes include arsR, arsB and arsC, whereas arsD
and arsA genes can eventually be found [1]. The arsC gene encodes
the enzyme AsV reductase, which is located in the cytoplasm and
is responsible for the biotransformation of AsV to AsIII. This
enzyme together with a transmembrane efflux pump, encoded by
arsA and arsB genes, is the most common As transformation
mechanism in the environment [2,14–16]. Moreover, arrA/B
genes encode a periplasmic AsV reductase that works during
anaerobic respiration using AsV as the final electron acceptor for
energy generation [17]. The AsV dissimilatory respiration
reduction has already been described for many bacterial phyla,
including obligatory and facultative anaerobic bacteria and some
archaea [1].
The microbial oxidation of AsIII was first reported in 1918 and
can be mediated by two distinct enzymes: AioBA, hardly studied,
and ArxAB, recently described by Zargar et al. [18]. Both enzymes
Introduction
Arsenic occurs naturally in the earth’s crust and is widely
distributed in the environment [1,2]. Natural mineralization and
microorganisms enhance arsenic mobilization in the environment,
but human interventions, such as gold mining, have aggravated
the environmental arsenic contamination arousing health concerns. Water pollution by arsenic is one of the major challenges for
public health, primarily due to its carcinogenic potential at low
doses [3,4,5,6]. According to Nordstrom [7] over 50 million
people in the world are at risk from drinking arsenic-contaminated
water. Moreover, given that arsenic has a variety of valence states
(+V, +III, 0, 2III) with different physicochemical properties, the
removal of arsenic from contaminated water bodies is yet a
challenge.
In nature, microorganisms have developed different response
mechanisms to metabolize As, mainly via reduction and oxidation
reactions, leading to its speciation [8]. Previous studies have
regarded As speciation as a result of microbial activity in the
environment, including some derived from gold-mining activities
[1,2]. However, few bacterial genera involved in As transformation have been found at any of the sites studied [9–13]. Thus, a
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Bacteria and Genes Involved in Arsenic Speciation
have been found in several heterotrophic and chemolithoautotrophic bacterial species [19–22]. Aerobic AsIII oxidation is
catalyzed by arsenite oxidase, which uses O2 as terminal electron
acceptor, and is encoded by aioB/A genes, formerly referred to as
aoxA/B, aroB/A and asoB/A genes [20,23]. ArxAB detected in
AsIII oxidizing bacteria in anoxic conditions, in which nitrate or
chlorate reduction is coupled to AsIII oxidation in the chemolithotrophs [24,25]. Interestingly, members of the genus Ectothiorhodospira are able to use AsIII as electron donor for anoxygenic
phototrophic growth [26]. According to Zargar et al. [18] the arxA
gene is more closely related to arrA than to aioA genes.
In this research, we bioprospected As-resistant bacteria from Asenrichment culture of sediments collected from a stream located at
the Brazilian gold mining area known as the Iron Quadrangle (IQ,
Minas Gerais state), one of the world’s largest mining regions.
Much concern exists about As-contamination of gold-mining sites
in this area because it is estimated that at least 390,000 tons of As
have been released into this area since the beginning of goldmining activity in the 17th century [27]. We also investigated the
diversity of As-transforming genes using metagenomic strategies.
This included the genes for arsenite oxidase (aioA) and arsenate
reductases (arsC and arrA).
Arsenic Enrichment and Isolation
Sediment (10 g) samples were added to Erlenmeyer flasks
containing 100 mL of CDM medium (0.012 mM Fe2SO4, 7 mM
Na2SO4, 0.0574 mM K2HPO4, 9.5 mM NaHCO3, 18.7 mM
NH4Cl, 8.12 mM MgSO4, 0.457 mM CaCl2 and 44.6 mM
sodium lactate as organic carbon source, pH 7.2) with either
2 mM sodium arsenite or 10 mM sodium arsenate and incubated
at 28uC for seven days. Then, serial 10-fold dilutions of the
enrichment cultures were plated onto CDM agar media (1.5%
agar) amended with 2 mM sodium arsenite or 10 mM sodium
arsenate to selectively enrich and isolate AsIII- and AsV-resistant
bacteria. Plates were incubated at 28uC for five days. The resulting
colonies were repeatedly streaked on the same medium to
accomplish their purification. The bacterial isolates from AsIIIand AsV-resistant bacteria (named MS-AsIII and MS-AsV,
respectively) were stored at 220uC in 25% glycerol.
DNA Extraction from the Cultures and Sediment
Genomic DNA was extracted and purified from each MS-AsIII
and MS-AsV isolate using a protocol previously described [34].
Additionally, metagenomic DNA was extracted from 10 g (wet
weight) of sediment using the PowerSoil DNA Extraction Kit (MO
BIO Laboratories, USA) according to the manufacturer’s instructions. Total DNA from the MS-AsIII and MS-AsV isolates and
sediment were quantified by absorbance at 260 nm using a
NanoDrop Spectrophotometer (NanoDrop Technologies). DNA
purity was assessed using the A260/A280 and A260/A230 ratios.
DNA was stored at 220uC until further processing.
Materials and Methods
Ethics Statement
For sampling in Mina stream, no specific permit was required
for the described field study. The study location is not privatelyowned or protected in any way and we confirm that the field study
did not involve endangered or protected species.
PCR Amplification and Construction of Clone Libraries
Briefly, touchdown PCR was carried out by amplifying bacterial
MS-AsIII and MS-AsV isolates 16S rRNA gene fragments using
the conditions previously described by Freitas et al. [35]. The
reactions were performed using the bacterial-targeted primer set
8F (59-AGAGTTTGATYMTGGCTCAG-39) and 907R (59CCGTCAATTCMTTTRAGT-39) [36]. Taq DNA polymerase
and dNTPs were purchased from Fermentas (Canada) and used in
all the PCR reactions.
Metagenomic and genomic DNA were used as template for
PCR employing the arsC, arrA and aioA genes for construction of
clone libraries and genotypic characterization of the bacterial MSAsIII and MS-AsV isolates. PCR reactions targeting the arsC, arrA
and aioA genes were carried out using primers and conditions as
previously described by Sun et al. [37], Malasarn et al. [17] and
Hamamura et al. [20], respectively. The arsC gene examined was of
the glutaredoxin-dependent arsenate reductase enzyme, ArsC,
from Escherichia coli R773 plasmid. The primer chosen has been
successfully applied in several investigations of a variety of
environmental samples [37,38].
The amplicons of arsC, arrA, and aioA genes were gel-purified
using the Silica Bead DNA Gel Extraction Kit (Fermentas,
Canada). PCR products were cloned into the vector pJET1.2/
blunt (Fermentas, Canada), and propagated with Escherichia coli
XL1-Blue electrocompetent cells according to the manufacturer’s
instructions.
Study Area and Sampling
Mina stream (19u58946.800S–43u49917.070W) is a natural body
of water located at the Velhas River Basin (IQ, Minas Gerais state,
Brazil) and characterized as backwater (Figure S1). This stream
was chosen because is located near a historically impacted goldmining area. Moreover, previous investigations [28] reported As
concentrations superior to those permitted by Brazilian law
(Conselho Nacional do Meio Ambiente – CONAMA) and by
Canadian Environmental Quality Guidelines (Canadian Council
of Ministers of the Environment– CCME).
Bulk water and superficial sediment samples (up to 1.0 cm
depth) were collected on 13 July 2011, during the dry season. The
typical sediment core can be divided into three zones: oxic,
suboxic and anoxic [29]. According to literature the thick oxic
zone can extend from several mm up to 10 cm [30,31]. In this
work the sampling site was shallow (20 cm) and therefore highly
influenced by the nutrients and oxygen concentrations of the water
body. The analyzed sediment was taken from the upper part,
representing the oxic zone. Samples were collected aseptically at
three points at 1m distance from each other, subsequently pooled
in a single sample, and stored at 4uC for bacterial analysis or at 2
20uC for chemical and molecular analyses.
To assess the bulk water conditions physicochemical characteristics such as temperature, pH, and dissolved oxygen (DO)
concentration were measured in situ with a multiprobe (Horiba,
model U-22) [30]. Concentrations of total nitrogen (TN), total
phosphorus (TP), ammonium (NH4+-N), nitrite (NO2-N), nitrate
(NO3-N), and soluble reactive phosphorus (PO4-P) were measured
as previously described [32,33]. Metal and metalloid concentrations of water and sediment samples were determined by using an
inductively coupled plasma-optical emission spectrometer (ICPOES, Optima 7300 DV, PerkinElmer).
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Sequencing and Phylogenetic Analysis
Partial 16S rRNA, arsC, arrA, and aioA gene sequences were
obtained using BigDye Terminator Cycle Sequencing kit (Life
Technologies, USA) according to the manufacturer’s instructions.
The nucleotide sequences were quality checked and submitted to
GenBank with accession numbers from KC577613 to KC577798.
The 16S rRNA gene sequences were analyzed through blastn
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Bacteria and Genes Involved in Arsenic Speciation
(http://www.ncbi.nlm.nih.gov) and Classifier search tool (http://
rdp.cme.msu.edu) to determine their phylogenetic affiliation. The
arsC, arrA, and aioA gene sequences were compared with those
available at the GenBank databases using blastn and blastx tools
(http://www.ncbi.nlm.nih.gov) to retrieve potential homologs.
Operational taxonomic unities (OTUs) from As gene clone
libraries were defined with DOTUR software [39] using a cutoff threshold of $97% identity. Coverage of the clone libraries was
calculated using the equation C = 12(n/N)6100, where n is the
number of unique OTUs and N is the number of sequences
analyzed in the library [40].
In total, five fasta files were obtained containing arsC, arrA, aioA,
MS-AsIII 16S rRNA, and Ms-AsV 16S rRNA gene sequences.
Due to the short length of arsC and arrAamino acid sequences
obtained in this study, added to the high similarity of some OTUs
and isolates, we decided to reconstruct the phylogenetic relationships of As metabolism genes using nucleotide sequences to
increase the phylogenetic signal and avoid overparameterization.
Sets of nucleotide sequences were independently aligned using
MAFFT 7 with iterative refinement by the G-INS-i strategy [41].
Multiple sequence alignments were manually refined using Jalview
[42]. To optimize the datasets for evolutionary analyses we
removed redundancy and sequences too distantly related using the
Decrease Redundancy tool available as a resource at ExPaSy
(www.expasy.org). The Decrease Redundancy parameters were set
as 99 for ‘‘% max similarity’’ and 30 for ‘‘% min similarity’’.
Identical sequences were clustered as single OTUs and filtered
alignments were further used in phylogenetic analyses. Identifiers
of filtered sequences were later included into the phylogenetic tree.
To reconstruct phylogenetic trees we used the maximum
likelihood method (ML) as implemented in PhyML [43]. For the
phylogenetic reconstruction we tested seven different evolutionary
models (HKY85, JC69, K80, F81, F84, TN93, and GTR) using
the jModelTest 2 software [44]. The evolutionary model best
fitting the data was determined by comparing the likelihood of
tested models according to the Akaike Information Criterion
(AIC). Statistical support value for each node was computed by
approximate likelihood ratio test (aLTR). Trees were visualized
and edited using the FigTree software (tree.bio.ed.ac.uk/software/
figtree).
Results
Environmental Parameters
The physicochemical characteristics of the water and sediment
samples from the Mina stream are presented in Tables 1 and 2.
Data displayed on Table 1 revealed that metal concentrations in
the Mina stream exceeded the maximum allowable concentrations
established by Brazilian and Canadian environmental regulations
[46,47] for sediment and water. Al, Mn, Fe, Cu, As and Zn were
the metals present in the highest concentrations in the sediment
sample analyzed.
The physicochemical analysis revealed that the Mina stream
can be characterized as a mesothermal oxidized environment with
highly oxygenated and circum-neutral waters (Table 2). Nitrogen
and phosphorus ratio was greater than nine (Table 2). According
to Salas & Martino [48], this ratio indicates that the phosphorus
was the most limiting nutrient and that the stream can be classified
as eutrophic.
Phylogenetic Affiliation
In total, 123 bacterial isolates were recovered from the
enrichment cultures (68 and 55 from the MS-AsIII and MSAsV, respectively). Partial 16S rRNA gene sequences used for
phylogenetic analysis were approximately 600 bp long and
spanned the V2 to V4 variable regions. MS-AsIII and MS-AsV
isolates were categorized into three phyla: Proteobacteria (56% and
59%, respectively, includes alpha, beta, and gamma-Proteobacteria),
Firmicutes (36% in both enrichment cultures), and Actinobacteria (8%
and 5%). Twenty genera represented these phyla in the Mina
stream sample analyzed. Differences in the bacterial composition
between the MS-AsIII and MS-AsV enrichment cultures were
detected (Table 3 and Figure 1). The resulting Venn diagram
shows that a higher bacterial diversity was observed in the MSAsIII than in the MS-AsV enrichment cultures. Eight genera were
specifically found in MS-AsIII and seven were shared between the
culture systems (Figure 1).
Dominant genera in MS-AsV were Bacillus (26%), Pseudoxanthomonas (18%), and Brevundimonas (16%). The predominant population in MS-AsIII was Bacillus (30%), followed by Pseudoxanthomonas
(25%). The other bacteria related to MS-AsIII and MS-AsV are
listed in Table 3. Although the Proteobacteria phylum was the most
diverse and dominant, the Bacillus (29%) genus was the most
abundant and diverse among the genera because it harbored eight
identified species.
Susceptibility and Arsenic Transformation Tests
Minimum inhibitory concentrations (MIC) were established, in
triplicate, by the agar dilution method in CDM with 16105 CFU
ml21 as standard inoculums. CDM plates were supplemented with
increasing concentrations (from 2 mM to 1024 mM) of AsIII or
AsV and incubated at 28uC for seven days. MIC was defined as
the lowest AsIII or AsV concentration that completely inhibited
bacterial growth.
The ability to oxidize AsIII and reduce AsV was investigated
using a qualitative screening according to [45]. To achieve that,
bacterial MS-AsIII and MS-AsV isolates were grown in CDM
broth with 100 mg l21 2 mM sodium arsenite or 100 mg l21
sodium arsenate until an optical density of 0.4 at 595 nm was
reached. After that, 20 ml of 0.01 mol l21 of potassium permanganate solution were added in 1 ml of bacterial culture. The data
were interpreted according to the change in medium color, i.e., a
pink color indicated a positive oxidation of AsIII and a yellow
color indicated a positive reduction of AsV.
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Characterization of As-reducing and Oxidizing Isolates
and Identification of their Genes Involved in As
Metabolism
The MICs for the MS-AsIII and MS-AsV isolates were
determined. The highest MIC was found for AsV in which 94%
of the isolates exhibited values $256 mM, whereas 90% of the
isolates displayed MICs ranging from 32 mM to 64 mM for the
most toxic AsIII.
The As-transformation ability of the isolates was determined
with a qualitative test that revealed that 72% of the isolates were
AsV-reducing, whereas 20% were AsIII-oxidizing. Of those, 8%
presented AsV-reducing as well as AsIII-oxidizing activities.
Among the 20 genera identified in both MS-AsIII and MS-AsV
enrichment cultures, Acidovorax and Achromobacter presented only
AsIII-oxidizing activity. No As-transformation activity was found
in 8% of the total of MS-AsIII and MS-AsV isolates (123) (Table 3).
The molecular analysis of the MS-AsIII and MS-AsV isolates
unveiled that the arsC gene was the most frequent (85%), followed
by aioA (20%) and arrA (7%) (Table 3). Of those, Bacillus was the
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Bacteria and Genes Involved in Arsenic Speciation
Table 1. Metal concentration from sediment and water of Mina Stream and limits permitted by law.
Metals
Sediment (mg kg21)
CONAMA* (mg kg21)
Water (mg l21)
CONAMA** (mg l21)
Fe
492.8
NE
0.52
15
Ni
9.0
18
,0.1
2
Mn
1284.5
NE
1.45
1
Cu
387.7
35.7
0.19
1
Pb
8.7
35
NE
0.5
Cd
,2.5
0.6
,0.1
0.2
Zn
180.9
123
0.2
5
Al
2343.2
NE
,0.5
NE
As
297.1
5.9
,0.1
0.5
Cr
17.3
37.3
,0.1
1
Hg
,2.5
0.17
,0.1
0.01
NE – Not established.
*CONAMA resolution 344/04.
**CONAMA resolution 430/11.
doi:10.1371/journal.pone.0095655.t001
corresponding to arsC were associated with arsC harboring
different bacterial taxa from a variety of environments. The
aioA-OTUs were closely related to uncultured and cultured clones
from As contaminated environments. Furthermore, all arrA-OTUs
were closely related to uncultured clones from rock biofilms of an
ancient gold mine and Cache Valley Land Fill sediments, both
arsenic-contaminated environments.
only genus harboring all three genes, and Shewanella was the only
genus which did not harbor the most common gene (arsC) in the
isolate analyzed (MS-AsIII-61). Achromobacter and Acidovorax both
harbored the aioA gene, confirming the phenotypic data.
Thermomonas and Pannonibacter also harbored As resistance genes.
General Features of Clone Libraries
To unveil the molecular diversity of genes involved in As
metabolism in the Mina stream sediment, three clone libraries for
arsC, arrA, and aioA genes were constructed. One hundred sixtyfour sequences were analyzed after quality control and the
removal of chimeric sequences. The coverage values of the three
libraries (80%, 70% and 63%, respectively for arsC, arrA, and aioA)
indicated that most of the diversity of these genes was detected.
Blastx analysis of arsC, aioA, and arrA-OTUs revealed high
similarity with sequences from glutaredoxin-glutathione arsenate
reductase (from 76 to 100%), molybdopterin-binding arsenite
oxidase (from 71 to 96%), and respiratory arsenate reductase (from
64 to 98%) (Tables S1, S2, and S3 in Tables S1). The sequences
Phylogenetic Analyses of 16S rRNA, arsC, aioA, and arrA
Genes Sequences
In this study we have amplified, sequenced and reconstructed
the evolutionary relationships of 16S rRNA and As metabolism
genes encoded by As-resistant bacteria retrieved from a stream
located at the Brazilian gold mining area and cultivated on Asenrichment sediment’s culture, as well as As metabolism genes of
clone libraries. The phylogeny of the AsIII-resistant bacteria (MSAsIII) 16S rRNA gene sequences was reconstructed from an
alignment containing 57 operational taxonomic units and 719
sites, which represent 99 sequences (Fig. 2). Therefore, 42
sequences were considered redundant by the Decrease Redundancy tool (www.expasy.org). The phylogenetic tree reconstructed
by using the maximum likelihood method as implemented in
PhyML [43], shows sequence’s clear separation into three strongly
supported clades, which have representatives of the Firmicutes,
Actinobacteria, and Proteobacteria phyla (Fig. 2). Similar results were
obtained for the AsV-resistant bacteria (MS-AsV) 16S rRNA
phylogenetic analysis (Fig. 3). The evolutionary history was based
on an alignment containing 40 OTUs and 721 sites, representing
79 sequences (Fig. 3). The Decrease Redundancy tool filtered
about 50% of the initially selected sequences. The resulting
phylogeny also exhibits the presence of three well-supported clades
containing Firmicutes, Actinobacteria, and Proteobacteria phyla representatives (Fig. 3).
Concerning evolutionary histories of As metabolism genes, the
phylogenetic tree of arsC sequences was reconstructed with 48
nucleotide sequences and 352 sites, which represent 142 sequences
(Fig. 4). TrN+I+G+F was selected as best fit model. The resulting
phylogeny supports the hypothesis that horizontal gene transfer
(HGT) seems to have played a role in the widespread distribution
of arsC coding gene in Actinobacteria and Proteobacteria. Similar
findings were retrieved on the phylogeny reconstructed for arrA
Table 2. Physicochemical parameters from water of Mina
Stream.
Parameters
Water
pH
6.2
Conductivity (ms cm21)
2151
Temperature (uC)
18.0
Dissolved Oxygen (mg l21)
9.1
Redox (mV)
215
NO32 -N (mg l21)
3103.8
NO22 -N (mg l21)
161.3
NH4+ -N (mg l21)
829.5
PO432 -P (mg l21)
2.3
Total P (mg l21)
77.6
21
Total N (mg l
)
2916.8
doi:10.1371/journal.pone.0095655.t002
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Bacteria and Genes Involved in Arsenic Speciation
Table 3. Phylogenetic distribution of the bacterial isolates and their As-metabolism phenotype and genotype.
Enriched culture
Phylum
Genus
N6 of isolates*
Phenotype**
Genotype***
MS-AsV
Proteobacteria
Acinetobacter
1
reducer
arsC aioA
Brevundimonas
9
reducer (3)
arsC[9] aioA [1]
Diaphorobacter
1
reducer
arsC
Firmicutes
Actinobacteria
MS-AsIII
Proteobacteria
Firmicutes
Actinobacteria
Pannonibacter
2
reducer (2)
arsC[2]
Pseudomonas
6
reducer (2)
arsC[6] aioA [2]
Pseudoxanthomonas
10
reducer (6)
arsC[7] aioA [1]
Stenotrophomonas
5
reducer (5)
arsC[5] aioA [2]
arsC
Thermomonas
1
reducer
Bacillus
14
reducer (9), oxidizer (8)
arsC[12] aioA [6] arrA [1]
Exiguobacterium
1
reducer
arsC aioA
Microbacterium
4
reducer (4) oxidizer (1)
arsC[3] aioA [1]
Rhodococcus
1
reducer
arsC
Achromobacter
1
oxidizer
arsC aioA
Acidovorax
2
oxidizer(2)
arsC[2] aioA [1]
Acinetobacter
4
reducer (3)
arsC[2] aioA [1]
Comamonas
5
reducer (5)
arsC[5] arrA [2]
Ochrobactrum
1
reducer
arsC
Pseudomonas
3
reducer (3)
arsC[1] aioA[1]
Pseudoxanthomonas
17
reducer (9) oxidizer (5)
arsC[14] aioA[2] arrA [1]
Shewanella
1
reducer
arrA
Stenotrophomonas
2
reducer (2) oxidizer (1)
arsC[1] aioA [1]
Thermomonas
2
reducer (1)
arsC[2] arrA [1]
Bacillus
21
reducer (19) oxidizer (5)
arsC[21] aioA[2] arrA [3]
Paenibacillus
1
reducer oxidizer (1)
arsC
Staphylococcus
4
reducer (4) oxidizer (1)
arsC[3]
Micrococcus
3
reducer (2) oxidizer (1)
arsC[1] aioA[1]
Microbacterium
1
reducer
-
*The number represents the total of bacterial isolates identified.
**Values in parentheses indicate the number of As-redox isolates.
***Values in bracket indicate the number of isolates harboring As-metabolism genes.
doi:10.1371/journal.pone.0095655.t003
sequences based on 47 nucleotide sequences and 242 sites where
GTR+I+G+F was selected as best fit model (Fig. 5). On the other
hand, the phylogenetic analysis of aioA sequences based on 72
nucleotide sequences and 543 sites shows two clades strongly
supported: alpha- and beta-proteobacteria (Fig. 6) without clear
evidence of HGT. For this analysis GTR+I+G+F was selected as
best fit model. Interestingly, all putative arrA sequences obtained in
this study (arrA- OTU) were more closely related to themselves or
to sequences from uncultured bacteria, showing that more studies
involving arrA sequences will be relevant to better understand the
molecular diversity of those genes (Fig. 5).
Discussion
The environmental impact of gold mining is presently a major
concern because its processes release toxic metals such as As in
both soil and groundwater. Considering the relevance of bacteria
in the speciation of As in aquatic environments, we bioprospected
As-resistant bacteria and As-transforming genes originated from
sediments impacted by long-term gold mining. Although some
studies have focused in the identification of As-resistant bacterial
communities in a long-term As-contaminated environment [9–
Figure 1. Venn diagram showing the exclusive and shared
bacterial genera retrieved from MS-AsIII and MS-AsV enrichment cultures.
doi:10.1371/journal.pone.0095655.g001
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Bacteria and Genes Involved in Arsenic Speciation
Figure 2. Evolutionary relationships of AsIII-resistant bacteria (MS-AsIII) 16S rRNA sequences. A total of 57 nucleotide sequences and
719 sites were analyzed. The phylogeny was reconstructed by maximum likelihoodand TIM3+I+G+F was selected as best fit model. Support values for
each node were estimated using the Akaike Likelihood Ratio Test (aLRT). Only support values higher than 70% are shown. Reference sequences
retrieved from the non-redundant database of the NCBI are shown in black, bacterial isolates (MS-AsIII and MS-AsV) in green. Different background
colors highlight three well-supported clades: Firmicutes, Actinobacteria, and Proteobacteria. Thermodesulfobacteria was used as outgroup.
doi:10.1371/journal.pone.0095655.g002
possible use of these natural isolates in future bioremediation
projects.
A recent study of our group [56], using culture-independent
approach to assess the prokaryotic diversity in Mina stream
sediment, revealed the presence of the Thermomonas, Acidovorax,
Acinetobacter and Ochobactrum genera also detected in the present
study. Moreover, Bandyopadhyay et al. [57] have proposed a novel
species of the Pannonibacter genus, Pannonibacterindica, which is able
to grow in high concentrations of AsV. However, it should be
noted that Thermomonas and Panonnibacter were not previously
reported in the literature as As-transforming genera.
The phenotypic and genotypic characterization of the MS-AsIII
and MS-AsV bacterial isolates revealed their ability to reduce and
oxidize As. Most bacteria (85%) were AsV-resistant bacteria (ARB)
harboring the arsC gene, responsible for the reduction of AsV to
13,49–52], the employment of combination of culture-based
physiological and genomic approaches with metagenomic analysis
in sediments collected from these areas are scarce [53,54]. In this
study, we reveal a large number of phylogenetically distinct Asresistant bacterial genera retrieved from sediment collected from a
stream in a long-term gold-mining area.
We found Bacillus as the dominant genus in both MS-AsIII and
MS-AsV enrichment cultures. Members of Bacillus are often found
in As-contaminated environments [9,11,16,54] being related to
As-reduction and -oxidation, indicating that they are an essential
component of As speciation in nature [54,55]. The observed
abundance of Bacillus isolates harboring the arsC and aioA genes
confirmed its ubiquity and high As-resistance in As-rich environments, as it is the case of Mina stream sediment. This suggests an
important role for Bacillus in As speciation. It also points to a
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Bacteria and Genes Involved in Arsenic Speciation
Figure 3. Evolutionary relationships of AsV-resistant bacteria (MS-AsV) 16S rRNA sequences. A total of 40 nucleotide sequences and 721
sites were analyzed. The phylogeny was reconstructed by maximum likelihood and TrN+G+F was selected as best fit model. Support values for each
node were estimated using the Akaike Likelihood Ratio Test (aLRT). Only support values higher than 70% are shown. Reference sequences retrieved
from the non-redundant database of the NCBI are shown in black, bacterial isolates (MS-AsIII and MS-AsV) in green. Different background colors
highlight three well-supported clades: Firmicutes, Actinobacteria, and Proteobacteria. Thermodesulfobacteria was used as outgroup.
doi:10.1371/journal.pone.0095655.g003
performers in As-contaminated environments because they
promote transformation from AsIII into AsV.
In a few isolates (8%), oxidizing and reducing As-transformation
activities were not observed in their phenotype and genotype.
There are several possible explanations for this. First, the Astransformation gene expression observed in isolates grown in the
laboratory is likely to be different from that encountered in these
isolates in nature, because of the different conditions of these
environments. Second, these differences may reflect mutations in
the As-resistance genes studied. Third, alternative resistance genes
may be expressed by these isolates [60].
The high diversity and adaptability of the bacterial community
disclosed herein could be explained by the presence of multiple
copies of As-resistance genes either on bacterial chromosomes or
on plasmids as a consequence of pressure created by the long-term
contamination that occurs in the Mina stream area. Nevertheless,
further studies will be needed to establish this.
AsIII, which is the most frequent detoxification reaction among
bacteria in the environment [8]. Although the aerobic enrichment
culture condition employed in this study could inhibit the growth
of dissimilatory arsenate-reducing bacteria (DARB), it is likely that
these bacteria were present because the arrA gene was detected.
Several reports have evidenced DARB bioremediation potential of
As-contaminated samples [2,54,58,59].
AsIII-oxidizing bacterial isolates were minority (20%). This
result is in agreement with those reported by Silver & Phung [14],
who suggest that most isolates from natural environments lack
AsIII-oxidizing ability. In this study, all AsIII-oxidizing isolates
were classified as heterotrophic AsIII oxidizers (HAO) spanning 11
genera. However, only isolates belonging to Bacillus, Pseudoxanthomonas, Stenotrophomonas, Micrococcus, Achromobacter, and Acidovorax
genera co-presented the oxidizing phenotype and genotype. From
an ecological perspective, oxidizing bacteria are important
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Bacteria and Genes Involved in Arsenic Speciation
Figure 4. Evolutionary relationships of arsC sequences. A total of 48 nucleotide sequences and 352 sites were analyzed. The phylogeny was
reconstructed by maximum likelihoodand TrN+I+G+F was selected as best fit model. Support values for each node were estimated using the Akaike
Likelihood Ratio Test (aLRT). Only support values higher than 70% are shown. Reference sequences retrieved from the non-redundant database of the
NCBI are shown in black, bacterial isolates (MS-AsIII and MS-AsV)in green, and operational taxonomic unities (OTUs) from As gene clone librariesin
blue. Different background colors highlight Actinobacteria and three Proteobacteria classes – Gamma-, Beta, and alpha-proteobacteria.
doi:10.1371/journal.pone.0095655.g004
were similar to those previously reported [37,62,63]. The primers
used in this study amplified aioA-like sequences [20,64]. The aioA
sequences were similar to several aioA genes of the Proteobacteria
phylum. This finding is in agreement with Quéméneur et al. [65],
who reported prevalence of AsIII-oxidizing Proteobacteria in
mesophilic As-contaminated soils. However, it should be noted
that aioA genes have been also detected in non-proteobacterial
lineages [53,69].
Phylogenetic analyses’ findings performed for MS-AsIII 16S
rRNA, Ms-AsV 16S rRNA and As metabolism genes were
consistent with findings obtained by similarity searches (blastx and
blastn, respectively). Overall, the phylogenetic trees reconstructed
Previous studies on As-resistance genes are associated with Asresistant cultivable isolates [10,16,59,61]. Considering that the vast
majority of bacteria are uncultivable, this traditional approach has
limited our understanding of the extreme functional diversity in
natural bacterial communities. Therefore, a metagenomic approach to investigate the functional genes associated with Astransformation in nature is essential to further our current
knowledge on this matter. The analysis of arrA sequences revealed
that all of them exhibited similarity with those from uncultured
organisms. This predominance of uncultured organisms indicates
that arrA gene present in Mina stream sediment is expressed by
unidentified DARB. The arsC sequences detected in the sediment
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Bacteria and Genes Involved in Arsenic Speciation
Figure 5. Evolutionary relationships of arrA sequences. A total of 47 nucleotide sequences and 242 sites were analyzed. The phylogeny was
reconstructed by maximum likelihoodand GTR+I+G+F was selected as best fit model. Support values for each node were estimated using the Akaike
Likelihood Ratio Test (aLRT). Only support values higher than 70% are shown. Reference sequences retrieved from the non-redundant database of the
NCBI are shown in black, bacterial isolates (MS-AsIII and MS-AsV) in green, and operational taxonomic unities (OTUs) from As gene clone libraries in
blue. Different background colors highlight three bacterial phyla - Proteobacteria, Firmicutes, and Chrysiogenetes.
doi:10.1371/journal.pone.0095655.g005
[53,69], our findings show two strongly supported clades clustering
alpha- and beta-proteobacteria homologs. Such results probably reflect
the bias existing on GenBank databases where most aioAsequences
available are from proteobacterial lineages.
Overall, evolutionary analyses revealed high genetic similarity
between some arsC and aioA sequences obtained from isolates and
clone libraries, suggesting that those isolates may represent
environmentally important bacteria acting in As speciation. In
addition, some arsC, aioA, and arrA sequences were found to be
closely related to homologs from uncultured bacteria. Thus, it may
be hypothesized that these divergent sequences could represent
novel variants of the As-resistance genes or other genes with
related function. In addition, our findings show that the diversity
of arrA genes is wider than earlier described, once none arrA-OTUs
were affiliated with known reference strains. Therefore, the
for MS-AsIII and MS-AsV 16S rRNA sequences show very similar
evolutionary histories where the relationships among Firmicutes,
Actinobacteria, Proteobacteria, and Thermodesulfobacteria phyla members
reflect the current knowledge regarding their evolution [66].
As previously described on the literature, the evolutionary
relationships of arsC and arrA homologs (Figs.4 and 5) support the
role of horizontal gene transfer (HGT) on the evolution of arsenate
oxidases e.g. [67,68]. The phylogeny reconstructed for arsC
homologs (Fig. 4) clearly shows two Ochrobactrum sequences
clustered in different well-supported clades suggesting that these
two homologs were acquired by HGT from unrelated donors.
Although it is known that due to HGT events aioA sequences are
not a suitable marker for microbial diversity studies [53], it was not
observed on the aioA phylogeny here presented (Fig. 6). Albeit aioA
sequences have been detected in non-proteobacterial lineages
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Bacteria and Genes Involved in Arsenic Speciation
Figure 6. Evolutionary relationships of aioA sequences. A total of 72 nucleotide sequences and 543 sites were analyzed. The phylogeny was
reconstructed by maximum likelihood and GTR+I+G+F was selected as best fit model. Support values for each node were estimated using the Akaike
Likelihood Ratio Test (aLRT). Only support values higher than 70% are shown. Reference sequences retrieved from the non-redundant database of the
NCBI are shown in black, bacterial isolates (MS-AsIII and MS-AsV) in green, and operational taxonomic unities (OTUs) from As gene clone libraries in
blue. Different background colors highlight two Proteobacteria classes – beta- and alpha-proteobacteria.
doi:10.1371/journal.pone.0095655.g006
molecular diversity of arrA genes is far from being fully explored
deserving further attention.
Altogether, this study is a bioprospection of AsIII-oxidizing and
AsV-reducing bacteria and As-transforming genes in sediments
impacted by long-term gold mining. Our culture efforts successfully identified a large number of phylogenetically distinct arsenicresistant bacterial genera and revealed two novel As-transformation genera, Thermomonas and Pannonibacter. Our heterotrophic
arsenite oxidizers and DARB isolates open new opportunities for
their use in bioremediation of long-term gold-mining impacted
areas. Furthermore, metagenomic analysis of As functional genes
revealed a predominance of previously unidentified DARB.
PLOS ONE | www.plosone.org
Supporting Information
Figure S1 Map showing the sampling site. Crosshatch,
red and yellow areas represent mining, urban, and
sampling areas, respectively.
(TIF)
Tables S1 This file includes Table S1, S2 and S3. Table
S1. Phylogenetic affiliation of aioA OTUs based on blastx protein
database. Table S2. Phylogenetic affiliation of arsC OTUs based
on blastx protein database. Table S3. Phylogenetic affiliation of
arrA OTUs based on blastx protein database.
(DOCX)
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Bacteria and Genes Involved in Arsenic Speciation
Acknowledgments
Author Contributions
We appreciate the technical supportf from Laboratório de Análises
Quı́micas/DEMET/UFMG do Instituto Nacional de Ciência e Tecnologia em Recursos Minerais, Água e Biodiversidade – INCT –ACQUA in
the chemical analyses. The authors acknowledge the use of the computing
resources of the Center for Excellence in Bioinformatics (CEBio//
CPqRR/Fiocruz, Brazil).
Conceived and designed the experiments: PSC ECS AMAN. Performed
the experiments: PSC MPR PLO LBI MLSS FARB. Analyzed the data:
PSC LLSS MPR AVC ECS AMAN. Contributed reagents/materials/
analysis tools: FARB AMAN. Wrote the paper: PSC LLSS MPR ECS
AMAN.
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