ArsP: a methylarsenite efflux permease

Trivalent organoarsenic compounds are far more toxic than either pentavalent organoarsenicals or inorganic arsenite. Many microbes methylate inorganic arsenite (As(III)) to more toxic and carcinogenic methylarsenite (MAs(III)). Additionally, monosodium methylarsenate (MSMA or MAs(V)) has been used widely as an herbicide and is reduced by microbial communities to MAs(III). Roxarsone (3‐nitro‐4‐hydroxybenzenearsonic acid) is a pentavalent aromatic arsenical that is used as antimicrobial growth promoter for poultry and swine, and its active form is the trivalent species Rox(III). A bacterial permease, ArsP, from Campylobacter jejuni, was recently shown to confer resistance to roxarsone. In this study, C. jejuni arsP was expressed in Escherichia coli and shown to confer resistance to MAs(III) and Rox(III) but not to inorganic As(III) or pentavalent organoarsenicals. Cells of E. coli expressing arsP did not accumulate trivalent organoarsenicals. Everted membrane vesicles from those cells accumulated MAs(III) > Rox(III) with energy supplied by NADH oxidation, reflecting efflux from cells. The vesicles did not transport As(III), MAs(V) or pentavalent roxarsone. Mutation or modification of the two conserved cysteine residues resulted in loss of transport activity, suggesting that they play a role in ArsP function. Thus, ArsP is the first identified efflux system specific for trivalent organoarsenicals.     

Organoarsenical tolerance in Sphingobacterium wenxiniae,a bacterium isolated from activated sludge

Organoarsenicals enter the environment from biogenic and anthropogenic sources. Trivalent inorganic arsenite (As(III)) is microbially methylated to more toxic methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)) that oxidize in air to MAs(V) and DMAs(V). Sources include the herbicide monosodium methylarsenate (MSMA or MAs(V)), which is microbially reduced to MAs(III), and the aromatic arsenical roxarsone (3-nitro-4-hydroxybenzenearsonic acid or Rox), an antimicrobial growth promoter for poultry and swine. Here we show that Sphingobacterium wenxiniae LQY-18^T, isolated from activated sludge, is resistant to trivalent MAs(III) and Rox(III). Sphingobacterium wenxiniae detoxifies MAs(III) and Rox(III) by oxidation to MAs(V) and Rox(V). Sphingobacterium wenxiniae has a novel chromosomal gene, termed arsU1. Expressed in Escherichia coli arsU1 confers resistance to MAs(III) and Rox(III) but not As(III) or pentavalent organoarsenicals. Purified ArsU1 catalyses oxidation of trivalent methylarsenite and roxarsone. ArsU1 has six conserved cysteine residues. The DNA sequence for the three C-terminal cysteines was deleted, and the other three were mutated to serines. Only C45S and C122S lost activity, suggesting that Cys45 and Cys122 play a role in ArsU1 function. ArsU1 requires neither FMN nor FAD for activity. These results demonstrate that ArsU1 is a novel MAs(III) oxidase that contributes to S. wenxiniae tolerance to organoarsenicals.     

ArsV and ArsW provide synergistic resistance to the antibiotic methylarsenite

Toxic organoarsenicals enter the environment from biogenic and anthropogenic activities such as microbial methylation of inorganic arsenic and pentavalent herbicides such as monosodium methylarsenate (MSMA or MAs(V)). Trivalent MAs(III) is considerably more toxic than arsenite or arsenate. Microbes have evolved mechanisms to detoxify organoarsenicals. We previously identified ArsV, a flavin-linked monooxygenase and demonstrated that it confers resistance to methylarsenite by oxidation to methylarsenate. The arsV gene is usually in an arsenic resistance (ars) operon controlled by an ArsR repressor and adjacent to a methylarsenite efflux gene, either arsK or a gene for a putative transporter. Here we show that Paracoccus sp. SY oxidizes methylarsenite. It has an ars operon with three genes, arsR, arsV and a transport gene termed arsW. Heterologous expression of arsV in Escherichia coli conferred resistance to MAs(III), while arsW did not. Co-expression of arsV and arsW increased resistance compared with either alone. The cells oxidized methylarsenite and accumulated less methylarsenate. Everted membrane vesicles from E. coli cells expressing arsW-accumulated methylarsenate. We propose that ArsV is a monooxygenase that oxidizes methylarsenite to methylarsenate, which is extruded by ArsW, one of only a few known pentavalent organoarsenical efflux permeases, a novel pathway of organoarsenical resistance.     

ArsH protects Pseudomonas putida from oxidative damage caused by exposure to arsenic

The two As resistance arsRBC operons of Pseudomonas putida KT2440 are followed by a downstream gene called arsH that encodes an NADPH-dependent flavin mononucleotide reductase. In this work, we show that the arsH1 and (to a lesser extent) arsH2 genes of P. putida KT2440 strengthened its tolerance to both inorganic As(V) and As(III) and relieved the oxidative stress undergone by cells exposed to either oxyanion. Furthermore, overexpression of arsH1 and arsH2 endowed P. putida with a high tolerance to the oxidative stress caused by diamide (a drainer of metabolic NADPH) in the absence of any arsenic. To examine whether the activity of ArsH was linked to a direct action on the arsenic compounds tested, arsH1 and arsH2 genes were expressed in Escherichia coli, which has an endogenous arsRBC operon but lacks an arsH ortholog. The resulting clones both deployed a lower production of reactive oxygen species (ROS) when exposed to As salts and had a superior endurance to physiological redox insults. These results suggest that besides the claimed direct action on organoarsenicals, ArsH contributes to relieve toxicity of As species by mediating reduction of ROS produced in vivo upon exposure to the oxyanion, e.g. by generating FMNH₂ to fuel ROS-quenching activities.     

The antibiotic action of methylarsenite is an emergent property of microbial communities

Arsenic is the most ubiquitous environmental toxin. Here, we demonstrate that bacteria have evolved the ability to use arsenic to gain a competitive advantage over other bacteria at least twice. Microbes generate toxic methylarsenite (MAs(III)) by methylation of arsenite (As(III)) or reduction of methylarsenate (MAs(V)). MAs(III) is oxidized aerobically to MAs(V), making methylation a detoxification process. MAs(V) is continually re‐reduced to MAs(III) by other community members, giving them a competitive advantage over sensitive bacteria. Because generation of a sustained pool of MAs(III) requires microbial communities, these complex interactions are an emergent property. We show that reduction of MAs(V) by Burkholderia sp. MR1 produces toxic MAs(III) that inhibits growth of Escherichia coli in mixed culture. There are three microbial mechanisms for resistance to MAs(III). ArsH oxidizes MAs(III) to MAs(V). ArsI degrades MAs(III) to As(III). ArsP confers resistance by efflux. Cells of E. coli expressing arsI, arsH or arsP grow in mixed culture with Burkholderia sp. MR1 in the presence of MAs(V). Thus MAs(III) has antibiotic properties: a toxic organic compound produced by one microbe to kill off competitors. Our results demonstrate that life has adapted to use environmental arsenic as a weapon in the continuing battle for dominance.     

Conserved cysteine residues determine substrate specificity in a novel As(III) S‐adenosylmethionine methyltransferase from Aspergillus fumigatus

Methylation of inorganic arsenic is a central process in the organoarsenical biogeochemical cycle. Members of every kingdom have ArsM As(III) S‐adenosylmethionine (SAM) methyltransferases that methylates inorganic As(III) into mono‐ (MAs(III)), di‐ (DMAs(III)) and tri‐ (TMAs(III)) methylarsenicals. Every characterized ArsM to date has four conserved cysteine residues. All four cysteines are required for methylation of As(III) to MAs(III), but methylation of MAs(III) to DMAs(III) requires only the two cysteines closest to the C‐terminus. Fungi produce volatile and toxic arsines, but the physiological roles of arsenic methylation and the biochemical basis is unknown. Here they demonstrate that most fungal species have ArsM orthologs with only three conserved cysteine residues. The genome of Aspergillus fumigatus has four arsM genes encoding ArsMs with only the second, third and fourth conserved cysteine residues. AfArsM1 methylates MAs(III) but not As(III). Heterologous expression of AfarsM1 in an Escherichia coli conferred resistance to MAs(III) but not As(III). The existence of ArsMs with only three conserved cysteine residues suggest that the ability to methylate MAs(III) may be an evolutionary step toward enzymes capable of methylating As(III), the result of a loss of function mutation in organisms with infrequent exposure to inorganic As(III) or as a resistance mechanism for MAs(III).     

Oxidation of organoarsenicals and antimonite by a novel flavin monooxygenase widely present in soil bacteria

Arsenic can be biomethylated to form a variety of organic arsenicals differing in toxicity and environmental mobility. Trivalent methylarsenite (MAs(III)) produced in the methylation process is more toxic than inorganic arsenite (As(III)). MAs(III) also serves as a primitive antibiotic and, consequently, some environmental microorganisms have evolved mechanisms to detoxify MAs(III). However, the mechanisms of MAs(III) detoxification are not well understood. In this study, we identified an arsenic resistance (ars) operon consisting of three genes, arsRVK, that contribute to MAs(III) resistance in Ensifer adhaerens ST2. ArsV is annotated as an NADPH-dependent flavin monooxygenase with unknown function. Expression of arsV in the arsenic hypersensitive Escherichia coli strain AW3110Δars conferred resistance to MAs(III) and the ability to oxidize MAs(III) to MAs(V). In the presence of NADPH and either FAD or FMN, purified ArsV protein was able to oxidize both MAs(III) to MAs(V) and Sb(III) to Sb(V). Genes with arsV-like sequences are widely present in soils and environmental bacteria. Metagenomic analysis of five paddy soils showed the abundance of arsV-like sequences of 0.12–0.25 ppm. These results demonstrate that ArsV is a novel enzyme for the detoxification of MAs(III) and Sb(III) and the genes encoding ArsV are widely present in soil bacteria.     

The ArsQ permease and transport of the antibiotic arsinothricin

The pentavalent organoarsenical arsinothricin (AST) is a natural product synthesized by the rhizosphere bacterium Burkholderia gladioli GSRB05. AST is a broad-spectrum antibiotic effective against human pathogens such as carbapenem-resistant Enterobacter cloacae. It is a non-proteogenic amino acid and glutamate mimetic that inhibits bacterial glutamine synthetase. The AST biosynthetic pathway is composed of a three-gene cluster, arsQML. ArsL catalyzes synthesis of reduced trivalent hydroxyarsinothricin (R-AST-OH), which is methylated by ArsM to the reduced trivalent form of AST (R-AST). In the culture medium of B. gladioli, both trivalent species appear as the corresponding pentavalent arsenicals, likely due to oxidation in air. ArsQ is an efflux permease that is proposed to transport AST or related species out of the cells, but the chemical nature of the actual transport substrate is unclear. In this study, B. gladioli arsQ was expressed in Escherichia coli and shown to confer resistance to AST and its derivatives. Cells of E. coli accumulate R-AST, and exponentially growing cells expressing arsQ take up less R-AST. The cells exhibit little transport of their pentavalent forms. Transport was independent of cellular energy and appears to be equilibrative. A homology model of ArsQ suggests that Ser320 is in the substrate binding site. A S320A mutant exhibits reduced R-AST-OH transport, suggesting that it plays a role in ArsQ function. The ArsQ permease is proposed to be an energy-independent uniporter responsible for downhill transport of the trivalent form of AST out of cells, which is oxidized extracellularly to the active form of the antibiotic.     

Arsenic in the human food chain,biotransformation and toxicology – Review focusing on seafood arsenic

Fish and seafood are main contributors of arsenic (As) in the diet. The dominating arsenical is the organoarsenical arsenobetaine (AB), found particularly in finfish. Algae, blue mussels and other filter feeders contain less AB, but more arsenosugars and relatively more inorganic arsenic (iAs), whereas fatty fish contain more arsenolipids. Other compounds present in smaller amounts in seafood include trimethylarsine oxide (TMAO), trimethylarsoniopropionate (TMAP), dimethylarsenate (DMA), methylarsenate (MA) and sulfur-containing arsenicals. The toxic and carcinogenic arsenical iAs is biotransformed in humans and excreted in urine as the carcinogens dimethylarsinate (DMA) and methylarsonate (MA), producing reactive intermediates in the process. Less is known about the biotransformation of organoarsenicals, but new insight indicates that bioconversion of arsenosugars and arsenolipids in seafood results in urinary excretion of DMA, possibly also producing reactive trivalent arsenic intermediates. Recent findings also indicate that the pre-systematic metabolism by colon microbiota play an important role for human metabolism of arsenicals. Processing of seafood may also result in transformation of arsenicals.     

Liberation and analysis of protein-bound arsenicals

Protein-bound arsenicals were liberated from binding sites on liver cytosolic proteins by exposure to 0.1M CuCl at pH 1. This method released greater than 90% of the arsenicals associated with biological matrices. Ultrafiltrates of CuCl-treated cytosols were subjected to thin-layer chromatography to speciate and quantify inorganic and methylated arsenicals. For rat liver cytosol in an in vitro methylation assay and for liver and kidney cytosols from arsenite-treated mice, most inorganic arsenic was protein bound. Appreciable fractions of the organoarsenical metabolites present in these cytosols were also protein bound. Therefore, CuCl treatment of cytosols releases protein-bound arsenicals, permitting more accurate estimates of the pattern and extent of arsenic methylation in vitro and in vivo.     

Synergistic interaction of glyceraldehydes‐3‐phosphate dehydrogenase and ArsJ,a novel organoarsenical efflux permease,confers arsenate resistance

Microbial biotransformations are major contributors to the arsenic biogeocycle. In parallel with transformations of inorganic arsenic, organoarsenicals pathways have recently been recognized as important components of global cycling of arsenic. The well‐characterized pathway of resistance to arsenate is reduction coupled to arsenite efflux. Here, we describe a new pathway of arsenate resistance involving biosynthesis and extrusion of an unusual pentavalent organoarsenical. A number of arsenic resistance (ars) operons have two genes of unknown function that are linked in these operons. One, gapdh, encodes the glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase. The other, arsJ, encodes a major facilitator superfamily (MFS) protein. The two genes were cloned from the chromosome of Pseudomonas aeruginosa. When expressed together, but not alone, in Escherichia coli, gapdh and arsJ specifically conferred resistance to arsenate and decreased accumulation of As(V). Everted membrane vesicles from cells expressing arsJ accumulated As(V) in the presence of purified GAPDH, D‐glceraldehylde 3‐phosphate (G3P) and NAD⁺. GAPDH forms the unstable organoarsenical 1‐arseno‐3‐phosphoglycerate (1As3PGA). We propose that ArsJ is an efflux permease that extrudes 1As3PGA from cells, where it rapidly dissociates into As(V) and 3‐phosphoglycerate (3PGA), creating a novel pathway of arsenate resistance.     

Engineering the Soil Bacterium Pseudomonas putida for Arsenic Methylation

Accumulation of arsenic has potential health risks through consumption of food. Here, we inserted the arsenite [As(III)] S-adenosylmethionine methyltransferase (ArsM) gene into the chromosome of Pseudomonas putida KT2440. Recombinant bacteria methylate inorganic arsenic into less toxic organoarsenicals. This has the potential for bioremediation of environmental arsenic and reducing arsenic contamination in food.     

Impact of arsenite and its methylated metabolites on PARP-1 activity, PARP-1 gene expression and poly(ADP-ribosyl)ation in cultured human cells

The underlying mechanisms of arsenic carcinogenicity are still not fully understood. Mechanisms currently discussed include the induction of oxidative DNA damage and the interference with DNA repair pathways. Still unclear is the role of biomethylation, which has long been considered to be one major detoxification process. Methylated arsenicals have recently been shown to interfere with DNA repair in cellular and subcellular systems, but up to now no DNA repair protein has been identified being particular sensitive towards methylated arsenicals in cultured cells. Here we report that the trivalent methylated metabolites MMA(III) and DMA(III) inhibit poly(ADP-ribosyl)ation in cultured human HeLa S3 cells at concentrations as low as 1nM, thereby showing for the first time an inactivation of an enzymatic reaction related to DNA repair by the trivalent methylated arsenicals at very low environmentally relevant concentrations. In contrast the pentavalent metabolites MMA(V) and DMA(V) showed no such effects up to high micromolar concentrations. All investigated arsenicals did not alter gene expression of PARP-1. However, all trivalent arsenicals were able to inhibit the activity of isolated PARP-1, indicating that the observed decrease in poly(ADP-ribosyl)ation in cultures human cells, predominantly mediated by PARP-1, is likely due to changes in the activity of PARP-1. Since poly(ADP-ribosyl)ation plays a major role in DNA repair, cell cycle control and thus in the maintenance of genomic stability, these findings could in part explain DNA repair inhibition and the genotoxic and carcinogenic effects of arsenic.     

Identification of catalytic residues in the As(III) S-adenosylmethionine methyltransferase

The enzyme As(III) S-adenosylmethionine methyltransferase (EC 2.1.1.137) (ArsM or AS3MT) is found in members of every kingdom, from bacteria to humans. In these enzymes, there are three conserved cysteine residues at positions 72, 174, and 224 in the CmArsM orthologue from the thermophilic eukaryotic alga Cyanidioschyzon sp. 5508. Substitution of any of the three led to loss of As(III) methylation. In contrast, a C72A mutant still methylated trivalent methylarsenite [MAs(III)]. Protein fluorescence of a single-tryptophan mutant reported binding of As(III) or MAs(III). As(GS)(3) and MAs(GS)(2) bound significantly faster than As(III), suggesting that the glutathionylated arsenicals are preferred substrates for the enzyme. Protein fluorescence also reported binding of Sb(III), and the purified enzyme methylated and volatilized Sb(III). The results suggest that all three cysteine residues are necessary for the first step in the reaction, As(III) methylation, but that only Cys174 and Cys224 are required for the second step, methylation of MAs(III) to dimethylarsenite [DMAs(III)]. The rate-limiting step was identified as the conversion of DMAs(III) to trimethylarsine, and DMAs(III) accumulates as the principal product.     

Arsenic biotransformation and volatilization in transgenic rice

? Biotransformation of arsenic includes oxidation, reduction, methylation, and conversion to more complex organic arsenicals. Members of the class of arsenite (As(III)) S-adenosylmethyltransferase enzymes catalyze As(III) methylation to a variety of mono-, di-, and trimethylated species, some of which are less toxic than As(III) itself. However, no methyltransferase gene has been identified in plants. ? Here, an arsM gene from the soil bacterium Rhodopseudomonas palustris was expressed in Japonica rice (Oryza sativa) cv Nipponbare, and the transgenic rice produced methylated arsenic species, which were measured by inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS). ? Both monomethylarsenate (MAs(V)) and dimethylarsenate (DMAs(V)) were detected in the roots and shoots of transgenic rice. After 12 d exposure to As(III), the transgenic rice gave off 10-fold greater volatile arsenicals. ? The present study demonstrates that expression of an arsM gene in rice induces arsenic methylation and volatilization, theoretically providing a potential stratagem for phytoremediation.     

Determination of trivalent methylated arsenicals in rat urine by liquid chromatography-inductively coupled plasma mass spectrometry after solvent extraction

A method for the determination of trivalent arsenicals in urine was examined. Trivalent arsenicals, extracted as complexes with diethylammonium diethyldithiocarbamate (DDDC) into carbon tetrachloride, were determined by liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS). The trivalent methylated arsenicals monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), and trimethylarsine (TMA) were detected in urine of rats that had received dimethylarsinic acid (DMA(V)) or monomethylarsonic acid (MMA(V)) at concentration of 200 microg ml(-1) in drinking water for 24 weeks. This method is the first to permit quantification of trivalent methylated arsenicals in urine without significant changes in concentration during storage or pretreatment.