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.     

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).     

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.     

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.     

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.     

Demethylation of methylarsonic acid by a microbial community

Arsenic is one of the most widespread environmental carcinogens and has created devastating human health problems worldwide, yet little is known about mechanisms of biotransformation in contaminated regions. Methylarsonic acid [MAs(V)], extensively utilized as an herbicide, is largely demethylated to more toxic inorganic arsenite, which causes environmental problems. To understand the process of demethylation of methylarsenicals, soil samples commonly used on Florida golf courses were studied. Several soil extracts were found to demethylate MAs(V) to inorganic arsenite [As(III)]. From these extracts, a bacterial isolate was capable of reducing MAs(V) to MAs(III) but not of demethylating to As(III). A second bacterial isolate was capable of demethylating MAs(III) to As(III) but not of reducing MAs(V). A mixed culture could carry out the complete process of reduction and demethylation, demonstrating that demethylation of MAs(V) to As(III) is a two-step process. Analysis of the 16S ribosomal DNA sequences of the two organisms identified the MAs(V)-reducing and the MAs(III)-demethylating isolates as belong to Burkholderia and Streptomyces species respectively. This is the first report of a novel pathway of degradation of a methylarsenical herbicide by sequential reduction and demethylation in a microbial soil community, which we propose plays a significant role in the arsenic biogeocycle.     

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.     

Arsenic methylation by a novel ArsM As(III) S‐adenosylmethionine methyltransferase that requires only two conserved cysteine residues

Arsenic (As) biomethylation is an important component of the As biogeochemical cycle that can influence As toxicity and mobility in the environment. Biomethylation of As is catalyzed by the enzyme arsenite (As[III]) S‐adenosylmethionine methyltransferase (ArsM). To date, all identified ArsM orthologs with As(III) methylation activities have four conserved cysteine residues, which are thought to be essential for As(III) methylation. Here, we isolated an As(III)‐methylating bacterium, Bacillus sp. CX‐1, and identified a gene encoding a S‐adenosylmethionine methyltranserase termed BlArsM with low sequence similarities (≤ 39%) to other ArsMs. BlArsM has six cysteine residues (Cys10, Cys11, Cys145, Cys193, Cys195 and Cys268), three of which (Cys10, Cys145 and Cys195) align with conserved cysteine residues found in most ArsMs. BlarsM is constitutively expressed in Bacillus sp. CX‐1. Heterologous expression of BlarsM conferred As(III) resistance. Purified BlArsM methylated both As(III) and methylarsenite (MAs[III]), with a final product of dimethylarsenate (DMAs[V]). When all six cysteines were individually altered to serine residues, only C145S and C195S derivatives lost the ability to methylate As(III) and MAs(III). The derivative C10S/C11S/C193S/C268S was still active. These results suggest that BlArsM is a novel As(III) S‐adenosylmethionine methyltransferase requiring only two conserved cysteine residues. A model of As(III) methylation by BlArsM is proposed.     

4种蚯蚓肠道微生物对砷毒性的响应差异研究

蚯蚓肠道是微生物多样性的一个潜在存储库。砷对蚯蚓肠道微生物群落的影响已被证实,但砷在不同蚯蚓肠道菌群中生物转化的差异仍不清楚。为了进一步阐述土壤中广泛存在的低浓度砷(浓度为5,15,25 mg/kg)对不同种类蚯蚓肠道微生物影响的差异,将4种典型蚯蚓暴露于砷污染土壤后,测定其肠道微生物组成变化,并分析砷对不同蚯蚓肠道内砷富集、形态和砷生物转化基因的影响。结果显示,所有蚯蚓组织内均存在明显的砷富集,其富集系数由高到低依次为:安德爱胜蚓(1.93)>加州腔蚓(0.80)>通俗腔蚓(0.78)>湖北远盲蚓(0.52),蚯蚓组织和肠道内砷形态主要以无机砷为主,其中As(III)含量比例> 80%,部分蚯蚓组织内还发现少量有机砷。4种蚯蚓肠道微生物群落在门水平上主要以变形菌、厚壁菌和放线菌为主,并与周围土壤细菌群落组成存在显著差异。同时,在土壤和肠道内共检测到17个砷转化基因,其中蚯蚓肠道内As(V)还原和砷转运相关基因相对丰度较高,而砷(去)甲基化基因丰度较低。此外,低浓度砷污染对蚯蚓生长无显著影响,却能引起蚯蚓肠道微生物群落的紊乱。蚯蚓种类和砷污染是引起蚯蚓肠道微生物...     

Genes involved in arsenic transformation and resistance associated with different levels of arsenic-contaminated soils

Background  

Arsenic is known as a toxic metalloid, which primarily exists in inorganic form [As(III) and As(V)] and can be transformed by microbial redox processes in the natural environment. As(III) is much more toxic and mobile than As(V), hence microbial arsenic redox transformation has a major impact on arsenic toxiCity and mobility which can greatly influence the human health. Our main purpose was to investigate the distribution and diversity of microbial arsenite-resistant species in three different arsenic-contaminated soils, and further study the As(III) resistance levels and related functional genes of these species.     

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.     

抗砷性微生物及其抗砷分子机制研究进展

砷(Arsenic, As)是一种剧毒类金属(Metalloid), 在自然环境中主要以三价亚砷酸盐[Arsenite, AsO2-, As(III)]和五价砷酸盐[Arsenate, AsO43-, As(V)]的无机形式广泛存在。许多微生物在含砷环境的长期适应过程中, 进化了多种不同的砷解毒抗性机制。目前研究发现主要存在4种类型的砷抗性机理, 包括: As(III)氧化, 细胞质As(V)还原, 呼吸性As(V)还原, As(III)甲基化, 这些机制赋予微生物砷抗性并在砷的转化和地球化学循环中起着极     

Alternative splicing variants of human arsenic (+3 oxidation state) methyltransferase

Arsenic (+3 oxidation state) methyltransferase (As3MT) catalyzes the methylation of trivalent arsenic (As(III)) to monomethylarsonate (MMA(V)) and dimethylarsinic acid (DMA(V)), and plays an important role in the detoxification of arsenicals. Here, we report the identification of two splicing variants of the human As3MT gene. One splicing variant was an exon-3 skipping (Δ3) form which produced a premature stop codon, and the other was an exon-4 and -5 skipping (Δ4,5) form which produced a 31.1 kDa As3MT protein. In addition to the full-length mRNA of As3MT, Δ4,5 mRNAs were detected in HepG2, A549, HL60, K562, and HEK293 cells. The methyltransferase activity of the recombinant Δ4,5 As3MT and wild-type As3MT proteins purified from Escherichia coli was determined. Speciation analysis by HPLC–ICP-MS showed a clear peak of MMA(V) after incubation of As(III) with the wild-type As3MT protein, but not with the Δ4,5 As3MT protein. In addition, COS-7 cells transfected with Δ4,5 As3MT cDNA did not convert As(III) to MMA(V) or DMA(V). The lack of methyltransferase activity of Δ4,5 As3MT seems to be related to the deletion of an S-adenosylmethionine-binding site and a critical cysteine residue. These data suggest that the expression pattern of splicing variants of the As3MT gene may affect the capacity for arsenic methylation in cells.     

Novel Hyper Antimony-Oxidizing Bacteria Isolated from Contaminated Mine Soils in China

Antimony (Sb)-oxidizing bacteria play an important role in environmental Sb bioremediation because of their ability to convert the more toxic Sb(III) to the less toxic Sb(V). So far, the information about the Sb(III)-oxidizing bacteria species is still limited. In this study, three highly Sb(III)-resistant bacterial strains were isolated from contaminated mine soils after aerobic enrichment culturing with Sb(III) (1 mM). The morphological, biochemical, and 16S rRNA gene sequencing analysis suggested that the three novel bacterial isolates fell within Cupriavidus, Moraxella, and Bacillus, respectively. Among the strains, Moraxella sp. S2 isolated from soils with the highest Sb content exhibited the highest minimum inhibitory concentration for Sb(III) but the lowest Sb(III) oxidation efficiency, which could not completely oxidize 50 μM Sb(III) in 15 days. Cupriavidus sp. S1 was able to oxidize 50 μM Sb(III) completely in 12 days, but could not oxidize 100 μM Sb(III) even with extended time of incubation, while Bacillus sp. S3 with the lowest resistance to Sb(III) could aerobically oxidize 100 µM Sb(III) within 2 days, showing high Sb(III) oxidation efficiency. Our research demonstrated that indigenous microorganisms associated with Sb mine soils were capable of Sb oxidation, and the novel bacteria isolated could represent good candidates for Sb remediation in heavily polluted sites.     

ArsH is an organoarsenical oxidase that confers resistance to trivalent forms of the herbicide monosodium methylarsenate and the poultry growth promoter roxarsone

Environmental organoarsenicals are produced by microorganisms and are introduced anthropogenically as herbicides and antimicrobial growth promoters for poultry and swine. Nearly every prokaryote has an ars (arsenic resistance) operon, and some have an arsH gene encoding an atypical flavodoxin. The role of ArsH in arsenic resistance has been unclear. Here we demonstrate that ArsH is an organoarsenical oxidase that detoxifies trivalent methylated and aromatic arsenicals by oxidation to pentavalent species. Escherichia coli, which does not have an arsH gene, is very sensitive to the trivalent forms of the herbicide monosodium methylarsenate [MSMA or MAs(V)] and antimicrobial growth promoter roxarsone [Rox(V)], as well as to phenylarsenite [PhAs(III), also called phenylarsine oxide or PAO]. Pseudomonas putida has two chromosomally encoded arsH genes and is highly resistant to the trivalent forms of these organoarsenicals. A derivative of P. putida with both arsH genes deleted is sensitive to MAs(III), PhAs(III) or Rox(III). P. putida arsH expressed in E. coli conferred resistance to each trivalent organoarsenical. Cells expressing PpArsH oxidized the trivalent organoarsenicals. PpArsH was purified, and the enzyme in vitro similarly oxidized the trivalent organoarsenicals. These results suggest that ArsH catalyzes a novel biotransformation that confers resistance to environmental methylated and aromatic arsenicals.