Mechanisms Of Arsenic Resistance Research Articles

Structural studies of the ArsD arsenic metallochaperone using molecular dynamics

Over the past three decades the occurrence of high environmental concentrations of arsenic has been recognized as a major public health concern. Arsenic is a carcinogen and causative agent for number of diseases. The toxic and carcinogenic effects of arsenic are the result of inactivation of specific enzymes of metabolism, induction of oxidative stress, inhibition of DNA repair mechanisms and deregulation of cell proliferation. Despite its toxicity, arsenic has a long history of usage as chemotherapeutic agent. Today, drugs containing arsenic (and the related metalloid antimony) are used for treating acute promyelocytic leukemia and diseases caused by protozoan parasites. In response to its toxicity, many microorganisms have developed arsenic resistance mechanism. In bacteria, arsenic resistance genes are organized in ars operons. The best characterized, the ars operon from Escherichia coli plasmid R773, has five genes, arsRDABC. ArsC is a reductase that reduces inorganic As(V) to As(III). ArsR is a 117-residue homodimeric As(III)-responsive transcriptional repressor with high affinity for the ars promoter. ArsA is an ATPase, which is the catalytic subunit of the ArsAB efflux pump, and ArsD is an As(III) chaperone to the ArsAB pump. ArsD is a homodimer of two 120-residue subunits with three vicinal cysteine pairs, Cys12-Cys13, Cys112-Cys113 and Cys119-Cys120. A fully-active truncated version of ArsD protein with 109 residues (ArsD109) has been crystallized, and the structure has been solved using X-ray crystallography. Biochemical studies suggested that wild-type ArsD binds three As(III) per monomer, while the derivative with a truncation at residue 109 binds only a single As(III). To understand the secondary structure and folding of the C-terminus of ArsD, which includes two cysteine pairs Cys112-Cys113 and Cys119-Cys120, Molecular Dynamics (MD) simulations were employed using AMBER software in presence or absence of arsenite [As(III)] ligand.

Genetic analysis of arsenic metabolism in Micrococcus luteus BPB1, isolated from the Bengal basin

A highly arsenic-metabolizing bacterial strain was isolated from an agricultural field known for arsenic contamination near Munshiganj (Bangladesh). Based on 16S rRNA gene analysis, the strain was identified as Micrococcus luteus and designated as strain BPB1. Arsenate and arsenite minimal inhibitory concentrations of 650 mM and 7.5 mM, respectively, were observed for strain BPB1, slightly higher than the figures observed in its close relative M. luteus DSM 20030T. Such observations were consistent with the presence of arsenic-metabolizing genes in the genome of M. luteus. We describe this strain as having an MSH/Mrx-dependent class of arsenate reductase, and an arsenite transporter family in the ACR3(1) group. Besides an intracellular arsenic resistance mechanism, experiments carried out using field emission scanning electron microscopy-energy dispersive X-ray spectroscopy (FESEM-EDS) and Fourier transform infrared spectroscopy (FTIR) demonstrated the ability of BPB1 to sequester arsenate in extracellular polymeric substances on its cell surface.

Varying responses of PML-RARA with different genetic mutations to arsenic trioxide

AbstractResistance to arsenic and/or all-trans retinoic acid (ATRA) is a challenging problem in the clinical management of acute promyelocytic leukemia (APL). Acquired genetic mutations in the PML moiety of the PML-RARA fusion gene are found in some patients with relapsed/refractory APL. Whether all of the identified point mutations play a role and have a similar function in the mechanisms of arsenic resistance remains unknown. Here we performed in vitro functional analyses and a retrospective analysis of APL patients to investigate the effect of PML-RARA mutations in mediating resistance to arsenic trioxide. Among the 5-point mutations in the PML part of PML-RARA identified in patients with relapsed APL, we found that A216V, S214L, and A216T mutations could attenuate the negative regulation of arsenic on PML-RARA, resulting in the retention of oncoproteins. In contrast, L217F and S220G mutations functioned weakly in this context. Furthermore, we demonstrated that either increasing the concentration of arsenic trioxide or combining it with ATRA could overcome the mutation-triggered arsenic resistance in vitro. In addition to presenting more evidence to reinforce the correlation of genetic mutations in PML-RARA with arsenic efficacy, we provide novel insight into the functional difference of acquired mutations of PML-RARA both in vitro and in the clinical setting. Our findings may help predict the prognosis and select more effective strategies during APL therapy.

Insights into arsenic multi-operons expression and resistance mechanisms in Rhodopseudomonas palustris CGA009.

Arsenic (As) is widespread in the environment and causes numerous health problems. Rhodopseudomonas palustris has been regarded as a good model organism for studying arsenic detoxification since it was first demonstrated to methylate environmental arsenic by conversion to soluble or gaseous methylated species. However, the detailed arsenic resistance mechanisms remain unknown though there are at least three arsenic-resistance operons (ars1, ars2, and ars3) in R. palustris. In this study, we investigated how arsenic multi-operons contributed to arsenic detoxification in R. palustris. The expression of ars2 or ars3 operons increased with increasing environmental arsenite (As(III)) concentrations (up to 1.0 mM) while transcript of ars1 operon was not detected in the middle log-phase (55 h). ars2 operon was actively expressed even at the low concentration of As(III) (0.01 μM), whereas the ars3 operon was expressed at 1.0 μM of As(III), indicating that there was a differential regulation mechanism for the three arsenic operons. Furthermore, ars2 and ars3 operons were maximally transcribed in the early log-phase where ars2 operon was 5.4-fold higher than that of ars3 operon. A low level of ars1 transcript was only detected at 43 h (early log-phase). Arsenic speciation analysis demonstrated that R. palustris could reduce As(V) to As(III). Collectively, strain CGA009 detoxified arsenic by using arsenic reduction and methylating arsenic mechanism, while the latter might occur with the presence of higher concentrations of arsenic.

Characterization, real-time quantification and in silico modeling of arsenate reductase (arsC) genes in arsenic-resistant Herbaspirillum sp. GW103

This study investigated the mechanism of arsenic resistance in the diazotrophic bacterium Herbaspirillum sp. GW103 isolated from rhizosphere soil of Phragmites austrails. The isolate Herbaspirillum sp. GW103 exhibited maximum tolerance to arsenic (550 mg/L). Four different arsenate reductase (arsC) genes (arsC1, arsC2, arsC3 and arsC4) were located in the genome of the isolate Herbaspirillum sp. GW103. The expression pattern of the arsC1 differed from other genes. All four types of arsC genes had different protein secondary structures and stereochemical properties. Molecular modeling and structural analysis of arsC genes revealed close structural homology with arsC family proteins from Escherichia coli (PDB ID: 1I9D) and Pseudomonas aeruginosa (PDB ID: 1RW1).

Exploring the diversity of arsenic resistance genes from acid mine drainage microorganisms.

The microbial communities from the Tinto River, a natural acid mine drainage environment, were explored to search for novel genes involved in arsenic resistance using a functional metagenomic approach. Seven pentavalent arsenate resistance clones were selected and analysed to find the genes responsible for this phenotype. Insights about their possible mechanisms of resistance were obtained from sequence similarities and cellular arsenic concentration. A total of 19 individual open reading frames were analysed, and each one was individually cloned and assayed for its ability to confer arsenic resistance in Escherichia coli cells. A total of 13 functionally active genes involved in arsenic resistance were identified, and they could be classified into different global processes: transport, stress response, DNA damage repair, phospholipids biosynthesis, amino acid biosynthesis and RNA-modifying enzymes. Most genes (11) encode proteins not previously related to heavy metal resistance or hypothetical or unknown proteins. On the other hand, two genes were previously related to heavy metal resistance in microorganisms. In addition, the ClpB chaperone and the RNA-modifying enzymes retrieved in this work were shown to increase the cell survival under different stress conditions (heat shock, acid pH and UV radiation). Thus, these results reveal novel insights about unidentified mechanisms of arsenic resistance.

A novel arsenate reductase from the bacterium Thermus thermophilus HB27: Its role in arsenic detoxification

Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20mM and 15mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram+bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a kcat/KM value of 1.2×104M−1s−1. It also exhibited weak phosphatase activity with a kcat/KM value of 2.7×10−4M−1s−1. The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural–functional characterization of a thermophilic arsenate reductase.

Draft Genome Sequence of Ochrobactrum pseudogrignonense Strain CDB2, a Highly Efficient Arsenate-Resistant Soil Bacterium from Arsenic-Contaminated Cattle Dip Sites

We report the 4.97-Mb draft genome sequence of a highly efficient arsenate-resistant bacterium, Ochrobactrum sp. strain CDB2. It contains a novel arsenic resistance (ars) operon (arsR-arsC1-ACR3-arsC2-arsH-mfs) and two non-operon-associated ars genes, arsC3 and arsB. The genome information will aid in the understanding of the arsenic resistance mechanism of this and other bacterial species.

The Contribution of ArsB to Arsenic Resistance in Campylobacter jejuni

Arsenic, a toxic metalloid, exists in the natural environment and its organic form is approved for use as a feed additive for animal production. As a major foodborne pathogen of animal origin, Campylobacter is exposed to arsenic selection pressure in the food animal production environments. Previous studies showed that Campylobacter isolates from poultry were highly resistant to arsenic compounds and a 4-gene operon (containing arsP, arsR, arsC, and acr3) was associated with arsenic resistance in Campylobacter. However, this 4-gene operon is only present in some Campylobacter isolates and other arsenic resistance mechanisms in C. jejuni have not been characterized. In this study, we determined the role of several putative arsenic resistance genes including arsB, arsC2, and arsR3 in arsenic resistance in C. jejuni and found that arsB, but not the other two genes, contributes to the resistance to arsenite and arsenate. Inactivation of arsB in C. jejuni resulted in 8- and 4-fold reduction in the MICs of arsenite and arsenate, respectively, and complementation of the arsB mutant restored the MIC of arsenite. Additionally, overexpression of arsB in C. jejuni 11168 resulted in a 16-fold increase in the MIC of arsenite. PCR analysis of C. jejuni isolates from different animals hosts indicated that arsB and acr3 (the 4-gene operon) are widely distributed in various C. jejuni strains, suggesting that Campylobacter requires at least one of the two genes for adaptation to arsenic-containing environments. These results identify ArsB as an alternative mechanism for arsenic resistance in C. jejuni and provide new insights into the adaptive mechanisms of Campylobacter in animal food production environments.

Arsenic-transforming microbes and their role in biomining processes

It is well known that microorganisms can dissolve different minerals and use them as sources of nutrients and energy. The majority of rock minerals are rich in vital elements (e.g., P, Fe, S, Mg and Mo), but some may also contain toxic metals or metalloids, like arsenic. The toxicity of arsenic is disclosed after the dissolution of the mineral, which raises two important questions: (1) why do microorganisms dissolve arsenic-bearing minerals and release this metal into the environment in a toxic (also for themselves) form, and (2) How do these microorganisms cope with this toxic element? In this review, we summarize current knowledge about arsenic-transforming microbes and their role in biomining processes. Special consideration is given to studies that have increased our understanding of how microbial activities are linked to the biogeochemistry of arsenic, by examining (1) where and in which forms arsenic occurs in the mining environment, (2) microbial activity in the context of arsenic mineral dissolution and the mechanisms of arsenic resistance, (3) the minerals used and technologies applied in the biomining of arsenic, and (4) how microbes can be used to clean up post-mining environments.

Arsenic accumulation and resistance mechanism in Panax notoginseng, a traditional rare medicinal herb

Panax notoginseng, a traditional rare Chinese medicinal herb, was recently found to bring health risk to consumers, mainly because soil in its major plantation area was contaminated by arsenic (As). We investigated the effect of soil As pollution on the growth and As uptake of pot-cultured P. notoginseng, and the associated mechanisms of As stressed response. Results showed that, comparing with P. notoginseng growing in a low-As soil, the root, stem, and leaf biomasses of those growing in a high-As soil significantly reduced by 0.75, 0.09 and 0.21gseedling−1, respectively. Arsenic concentrations in roots, stems and leaves of the seedlings growing in high-As soil were 22, 15 and 3times higher than those growing in low-As soil, respectively. Regardless of the soil As concentration, As existed in plants mainly as As(III), suggesting that the reduction of As(V) is a key step in As metabolism. Arsenic was distributed primarily in cell walls (51.7% for plants growing in the low-As soil, and 51.5% in the high-As soil), followed by cytoplasm supernatant, with cell organelles containing the least As. Compared with plants growing in the low-As soil, those in the high-As soil had increased superoxide dismutase and peroxidase activities in their roots, stems, and leaves, which would be associate with improving the resistance of P. notoginseng to As stress. The results suggest that there exists some special mechanisms of As-tolerance in P. notoginseng and the study is of significance in developing measures to reduce As in the herb.

Mechanism of arsenic resistance prevalent in Bacillus species isolated from soil and ground water sources of India

Arsenic is a naturally occurring element and its contamination of soil and groundwater is a major threat to the environment and human health. The ubiquity of arsenic in the environment has led to the evolution of the arsenic defence mechanism in certain microbes. The present study highlights identical mechanisms of arsenic resistance in two bacterial strains, namely AG24 and AGM13, which were taken from two different ecosources and states of India. The phylogenetic analysis of the strains using 16S rDNA sequence revealed both strains to be under the genus ‘Bacillus’. Furthermore, arsC gene was amplified from the strains, cloned and sequenced. The homology of the sequences of the two strains, when compared with available database using BLASTn search, showed that the 300bp amplicons obtained in both strains possess a 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. Nevertheless, atomic absorption spectroscopy and scanning electron microscopy analysis of both strains also strengthen the presence of the efflux mechanism. Thus, the presence of a similar mechanism of resistance in both strains suggests the possible role of lateral gene transfer from soil to water system and vice versa which is an alarming situation for global concern.

Purification and enzymatic properties of arsenic resistance protein ArsH from heterogeneous expression in E.coli BL21

Four arsenic-resistance genes ( arsB, arsC, arsH, arsR) have been discovered in Acidithiobacillus ferrooxidans. Their gene sequences have been identified and three different arsenic-resistance mechanisms have been elucidated. However, the function of the arsH gene in At. ferrooxidans remains unclear. In order to evaluate the function of the arsH gene, we cloned it and expressed it in Escherichia coli. The protein was purified and its relative molecular mass was determined by SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The results indicated that the relative molecular mass of the purified ArsH was approximately 29 kDa. The purified protein ArsH from E.coli BL21 was a flavoprotein that oxidized in vitro NADPH with an optimal pH of 6.4.

Disruption of ptLPD1 or ptLPD2, genes that encode isoforms of the plastidial lipoamide dehydrogenase, confers arsenate hypersensitivity in Arabidopsis.

Arsenic is a ubiquitous environmental poison that inhibits root elongation and seed germination to a variable extent depending on the plant species. To understand the molecular mechanisms of arsenic resistance, a genetic screen was developed to isolate arsenate overly sensitive (aos) mutants from an activation-tagged Arabidopsis (Arabidopsis thaliana) population. Three aos mutants were isolated, and the phenotype of each was demonstrated to be due to an identical disruption of plastidial LIPOAMIDE DEHYDROGENASE1 (ptLPD1), a gene that encodes one of the two E3 isoforms found in the plastidial pyruvate dehydrogenase complex. In the presence of arsenate, ptlpd1-1 plants exhibited reduced root and shoot growth and enhanced anthocyanin accumulation compared with wild-type plants. The ptlpd1-1 plants accumulated the same amount of arsenic as wild-type plants, indicating that the aos phenotype was not due to increased arsenate in the tissues but to an increase in the innate sensitivity to the poison. Interestingly, a ptlpd1-4 knockdown allele produced a partial aos phenotype. Two loss-of-function alleles of ptLPD2 in Arabidopsis also caused elevated arsenate sensitivity, but the sensitivity was less pronounced than for the ptlpd1 mutants. Moreover, both the ptlpd1 and ptlpd2 mutants were more sensitive to arsenite than wild-type plants, and the LPD activity in isolated chloroplasts from wild-type plants was sensitive to arsenite but not arsenate. These findings show that the ptLPD isoforms are critical in vivo determinants of arsenite-mediated arsenic sensitivity in Arabidopsis and possible strategic targets for increasing arsenic tolerance.

The overexpression of multidrug resistance-associated proteins and gankyrin contribute to arsenic trioxide resistance in liver and gastric cancer cells

Arsenic trioxide has been used as a therapeutic agent for acute promyelocytic leukemia and recently for some solid tumors. Although arsenic trioxide has been shown to significantly inhibit the growth of solid tumor cells in vitro, clinical trials indicate that arsenic trioxide alone is pool active against non-hematologic malignant diseases. To understand the mechanisms of arsenic resistance in solid tumor cells, we established two arsenic-resistant solid tumor cell lines, HepG2/AS and SGC7901/AS, isolated from human liver cancer cell line HepG2 and human gastric cancer cell line SGC7901, respectively, by a series of stepwise selections via treatment with increasing concentrations of arsenic trioxide. Three ABC transporter proteins, ABCB1, ABCC1 and ABCC2, were expressed increasingly and differently in two arsenic-resistant cell lines. Further, tumor suppressor p53 was overexpressed in two arsenic-resistant cell lines, but the levels of p53 mediators MDM2 and gankyrin, which regulate the ubiquitination of p53, increased simultaneously. In addition, an increase in the phosphorylation of Rb at Ser795 in the two cell lines might also result from the presence of MDM2 and gankyrin, which suggest that the inactivation of p53 and Rb contribute to drug resistance. These two arsenic-resistant solid tumor cell lines, HepG2/AS and SGC7901/AS, may be useful for studying the mechanism of arsenic resistance in solid tumors and may provide a way to overcome it.

Leptospirilli from different continents have acquired related arsenic-resistance transposons

The microbial consortium in continuous-flow, stirred tank processes used to treat gold-bearing arsenopyrite concentrates in South Africa has become adapted to high concentrations of arsenic over several years. The dominant microorganisms, Acidithiobacillus caldus and Leptospirillum ferriphilum, were found to contain two sets of arsenic-resistance genes. One set was present in all isolates of a species irrespective of whether they were highly arsenic resistant or not, while a second, transposon-located set was present in only those strains that had been adapted to high concentrations of arsenic. The arsenopyrite treatment tanks from Tamboraque (near Lima, Peru) have not been inoculated with an arsenic-adapted consortium from South Africa. Isolation of a Leptospirillum ferrooxidans from the Tamboraque consortium allowed us to determine whether these microorganisms had acquired similar arsenic-resistance mechanisms as contained on the transposons in the highly arsenic resistant South African cultures. Several isolates of both L. ferriphilum and L. ferrooxidans from Europe as well as a “Leptospirillum ferrodiazotrophum” were also screened to detect whether they contained similar arsenic-resistance transposons even though they had not been selected for enhanced arsenic resistance. Transposons containing arsenic-resistance genes that were identical or closely related to those from South Africa were found in both L. ferrooxidans and L. ferriphilum isolates from South America and Europe. The widespread occurrence of arsenic-resistance transposons suggests that it should be possible to select for highly arsenic resistant biomining microorganisms from many different sources and therefore, unnecessary to use pre-adapted arsenic resistant consortia.

An Arsenate-activated Glutaredoxin from the Arsenic Hyperaccumulator Fern Pteris vittata L. Regulates Intracellular Arsenite

To elucidate the mechanisms of arsenic resistance in the arsenic hyperaccumulator fern Pteris vittata L., a cDNA for a glutaredoxin (Grx) Pv5-6 was isolated from a frond expression cDNA library based on the ability of the cDNA to increase arsenic resistance in Escherichia coli. The deduced amino acid sequence of Pv5-6 showed high homology with an Arabidopsis chloroplastic Grx and contained two CXXS putative catalytic motifs. Purified recombinant Pv5-6 exhibited glutaredoxin activity that was increased 1.6-fold by 10 mm arsenate. Site-specific mutation of Cys(67) to Ala(67) resulted in the loss of both GRX activity and arsenic resistance. PvGrx5 was expressed in E. coli mutants in which the arsenic resistance genes of the ars operon were deleted (strain AW3110), a deletion of the gene for the ArsC arsenate reductase (strain WC3110), and a strain in which the ars operon was deleted and the gene for the GlpF aquaglyceroporin was disrupted (strain OSBR1). Expression of PvGrx5 increased arsenic tolerance in strains AW3110 and WC3110, but not in OSBR1, suggesting that PvGrx5 had a role in cellular arsenic resistance independent of the ars operon genes but dependent on GlpF. AW3110 cells expressing PvGrx5 had significantly lower levels of arsenite when compared with vector controls when cultured in medium containing 2.5 mm arsenate. Our results are consistent with PvGrx5 having a role in regulating intracellular arsenite levels, by either directly or indirectly modulating the aquaglyceroporin. To our knowledge, PvGrx5 is the first plant Grx implicated in arsenic metabolism.

Related Arsenic Resistance Transposons Occur in Leptospirilli Isolated from Different Continents

Acidithiobacillus caldus and Leptospirillum ferriphilum dominate the microbial consortium used in continuous-flow, stirred tank processes used to treat gold-bearing arsenopyrite concentrates in South Africa. These microorganisms were adapted to high concentrations of arsenic over several years and both types of microorganisms were found to contain two sets of arsenic resistance genes. One set was present on all isolates of a species irrespective of whether they were highly arsenic resistant or not, while a second, transposon-located set was present in only those strains that had been adapted to high concentrations of arsenic. We isolated a Leptospirillum ferrooxidans from the microbial consortium present in arsenopyrite treatment tanks from Tamboraque (near Lima, Peru) that had not been inoculated with an arsenic-adapted consortium from South Africa. This allowed us to determine whether these microorganisms had acquired similar arsenic resistance mechanisms as contained on the transposons in the highly arsenic resistant South African cultures. Several isolates of both Leptospirillum ferriphilum and L. ferrooxidans from Europe as well as a "Leptospirillum ferrodiazotrophum" were also screened to detect whether they contained similar arsenic resistance transposons even though they had not been selected for enhanced arsenic resistance. Transposons containing arsenic resistance genes that were identical or closely related to those from South Africa were found in both L. ferrooxidans and L. ferriphilum isolates from South America and Europe. The widespread occurrence of arsenic resistance transposons suggests that it should be possible to select for highly arsenic resistant biomining microorganisms from many different sources and therefore, unnecessary to acquire pre-adapted arsenic resistant consortia.

Identification of an arsenic resistance mechanism in rhizobial strains

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 containing heavy-metals, in the industrial area of Estarreja, Portugal. The arsC gene was identified in strains of Sinorhizobium 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.

Arsenate detoxification in a Pseudomonad hypertolerant to arsenic

Pseudomonas sp. strain As-1, obtained from an electroplating industrial effluent, was capable of growing aerobically in growth medium supplemented with up to 65 mM arsenate (As (V)), significantly higher concentrations than those tolerated by other reference arsenic resistant bacteria. The majority of the arsenic was detected in culture supernatants as arsenite (As (III)) and X-ray absorbance spectroscopy suggested that 30% of this cell-bound arsenic was As (V), 65% As (III) and 5% of arsenic was associated with sulphur. PCR analysis using primers designed against arsenic resistance genes of other Gram-negative bacteria confirmed the presence of an arsenic resistance operon comprising of three genes, arsR, arsB and arsC in order of predicted transcription, and consistent with a role in intracellular reduction of As (V) and efflux of As (III). In addition to this classical arsenic resistance mechanism, other biochemical responses to arsenic were implicated. Novel arsenic-binding proteins were purified from cellular fractions, while proteomic analysis of arsenic-induced cultures identified the upregulation of additional proteins not normally associated with the metabolism of arsenic. Cross-talk with a network of proteins involved in phosphate metabolism was suggested by these studies, consistent with the similarity between the phosphate and arsenate anions.