Bifunctional protein ArsRM contributes to arsenite methylation and resistance in Brevundimonas sp. M20.

BACKGROUND: Arsenic (As) with various chemical forms, including inorganic arsenic and organic arsenic, is the most prevalent water and environmental toxin. This metalloid occurs worldwide and many of its forms, especially arsenite [As(III)], cause various diseases including cancer. Organification of arsenite is an effective way for organisms to cope with arsenic toxicity. Microbial communities are vital contributors to the global arsenic biocycle and represent a promising way to reduce arsenite toxicity. METHODS: Brevundimonas sp. M20 with arsenite and roxarsone resistance was isolated from aquaculture sewage. The arsHRNBC cluster and the metRFHH operon of M20 were identified by sequencing. The gene encoding ArsR/methyltransferase fusion protein, arsRM, was amplified and expressed in Escherichia coli BL21 (DE3), and this strain showed resistance to arsenic in the present of 0.25-6 mM As(III), aresenate, or pentavalent roxarsone. The methylation activity and regulatory action of ArsRM were analyzed using Discovery Studio 2.0, and its functions were confirmed by methyltransferase activity analysis and electrophoretic mobility shift assays. RESULTS: The minimum inhibitory concentration of the roxarsone resistant strain Brevundimonas sp. M20 to arsenite was 4.5 mM. A 3,011-bp arsenite resistance ars cluster arsHRNBC and a 5649-bp methionine biosynthesis met operon were found on the 3.315-Mb chromosome. Functional prediction analyses suggested that ArsRM is a difunctional protein with transcriptional regulation and methyltransferase activities. Expression of ArsRM in E. coli increased its arsenite resistance to 1.5 mM. The arsenite methylation activity of ArsRM and its ability to bind to its own gene promoter were confirmed. The As(III)-binding site (ABS) and S-adenosylmethionine-binding motif are responsible for the difunctional characteristic of ArsRM. CONCLUSIONS: We conclude that ArsRM promotes arsenite methylation and is able to bind to its own promoter region to regulate transcription. This difunctional characteristic directly connects methionine and arsenic metabolism. Our findings contribute important new knowledge about microbial arsenic resistance and detoxification. Future work should further explore how ArsRM regulates the met operon and the ars cluster.

Roxarsone inhibits hepatic stellate cell activation and ameliorates liver fibrosis by blocking TGF-ß1/Smad signaling pathway.

Hepatic fibrosis is a pathological change caused by chronic liver injury and self-repair, and it is the inevitable stage of the development of chronic liver disease to cirrhosis or even liver cancer. Activation of hepatic stellate cells (HSCs) is a core event in the development of liver fibrosis and blockage of the activation of HSCs has been shown to alleviate liver fibrosis. Roxarsone, an organoarsenic additive, with antibiotic effect, growth promotion and improving feed efficiency, is widely used in livestock and animal production. The purpose of this study was to evaluate the therapeutic effect of Roxarsone on liver fibrosis and explore the possible mechanism. We found that Roxarsone could inhibit transforming growth factor-ß1 (TGF-ß1) induced the activation of HSCs and weaken the migration ability. Moreover, Roxarsone administration significantly ameliorated CCl4-induced liver fibrosis in mice with improvement of liver function and decreases of deposition of extracellular matrix (ECM). Mechanism investigations revealed that Roxarsone specifically inhibited the activation of TGF-ß1/Smad signaling pathway, but had no effect on MAPK and PI3K/AKT pathways. These results suggest that Roxarsone has a protective effect on liver fibrosis which provides a new candidate for the treatment of liver fibrosis.

Highly efficient persulfate catalyst prepared from modified electrolytic manganese residues coupled with biochar for the roxarsone removal.

The contamination of organoarsenic is becoming increasingly prominent while SR-AOPs were confirmed to be valid for their remediation. This study has found that the novel metal/carbon catalyst (Fe/C-Mn) prepared by solid waste with hierarchical pores could simultaneously degrade roxarsone (ROX) and remove As(V). A total of 95.6% of ROX (20 mg/L) could be removed at the concentration of 1.0 g/L of catalyst and 0.4 g/L of oxidant in the Fe/C-Mn/PMS system within 90 min. The scavenging experiment and electrochemical test revealed that both single-electron and two-electron pathways contributed to the ROX decomposition. Spectroscopic analysis suggested the ROX has been successfully mineralized while As(V) was fixed with the surface Fe and Mn. Density functional theory (DFT) calculation and chromatographic analysis indicated that the As7, N8, O9 and O10 sites of ROX molecule were vulnerable to being attacked by nucleophilic, electrophilic and radical, resulting in the formation of several intermediates such as phenolic compounds. Additionally, the low metal leaching concentration during recycling and high anti-interference ability in various water matrices manifested the practicability of Fe/C-Mn/PMS system.

The VEGFR2/mTOR/S6K1 pathway involved in the angiogenic effects of roxarsone in vitro and in vivo.

Roxarsone, an organoarsenic compound used in poultry industry to increase weight gain, is widely used as a feed additive in some developing countries. Roxarsone has a low absorption rate and is mostly excreted with feces, which could pose a risk to human health through environmental and animal food routes. Roxarsone has been demonstrated to have tumor-promoting and proangiogenic effects. Herein, we report the role of VEGFR2/mTOR/S6K1 signaling in roxarsone-promoted vessel endothelial cell growth and angiogenesis in the Matrigel plug model and the mouse B16 cell tumor transplantation model. In angiogenesis-related experiments in vitro, 1.0 µM roxarsone significantly increased the activity, proliferation, migration, and tube formation of rat vascular endothelial cells. In addition, 1.0 µM roxarsone upregulated the protein levels of mTOR, phosphorylated mTOR, S6K1, and phosphorylated S6K1 and significantly increase the expression of Mtor and S6k1 mRNA. Rapamycin and SU5416 significantly inhibited the effects of 1.0 µM roxarsone on cell growth. Furthermore, the weight, volume, and CD31 expression of B16-F10 xenografts and Matrigel plugs in mice were upregulated by 25 mg/kg roxarsone. The protein and mRNA levels of mTOR, S6K1 and its phosphorylated protein were significantly increased in the roxarsone treatment group in xenografts. SU5416 and a short hairpin RNA targeting Vegfr2 significantly reduced roxarsone-promoted xenograft and Matrigel plug growth. In summary, this study indicated that the VEGFR2/mTOR/S6K1 signaling plays a regulatory role in roxarsone-mediated promotion angiogenesis and enhanced tumor growth.

Characterization of Two Highly Arsenic-Resistant Caulobacteraceae Strains of Brevundimonas nasdae: Discovery of a New Arsenic Resistance Determinant.

Arsenic (As), distributed widely in the natural environment, is a toxic substance which can severely impair the normal functions in living cells. Research on the genetic determinants conferring functions in arsenic resistance and metabolism is of great importance for remediating arsenic-contaminated environments. Many organisms, including bacteria, have developed various strategies to tolerate arsenic, by either detoxifying this harmful element or utilizing it for energy generation. More and more new arsenic resistance (ars) determinants have been identified to be conferring resistance to diverse arsenic compounds and encoded in ars operons. There is a hazard in mobilizing arsenic during gold-mining activities due to gold- and arsenic-bearing minerals coexisting. In this study, we isolated 8 gold enrichment strains from the Zijin gold and copper mine (Longyan, Fujian Province, China) wastewater treatment site soil, at an altitude of 192 m. We identified two Brevundimonas nasdae strains, Au-Bre29 and Au-Bre30, among these eight strains, having a high minimum inhibitory concentration (MIC) for As(III). These two strains contained the same ars operons but displayed differences regarding secretion of extra-polymeric substances (EPS) upon arsenite (As(III)) stress. B. nasdae Au-Bre29 contained one extra plasmid but without harboring any additional ars genes compared to B. nasdae Au-Bre30. We optimized the growth conditions for strains Au-Bre29 and Au-Bre30. Au-Bre30 was able to tolerate both a lower pH and slightly higher concentrations of NaCl. We also identified folE, a folate synthesis gene, in the ars operon of these two strains. In most organisms, folate synthesis begins with a FolE (GTP-Cyclohydrolase I)-type enzyme, and the corresponding gene is typically designated folE (in bacteria) or gch1 (in mammals). Heterologous expression of folE, cloned from B. nasdae Au-Bre30, in the arsenic-hypersensitive strain Escherichia coli AW3110, conferred resistance to As(III), arsenate (As(V)), trivalent roxarsone (Rox(III)), pentavalent roxarsone (Rox(V)), trivalent antimonite (Sb(III)), and pentavalent antimonate (Sb(V)), indicating that folate biosynthesis is a target of arsenite toxicity and increased production of folate confers increased resistance to oxyanions. Genes encoding Acr3 and ArsH were shown to confer resistance to As(III), Rox(III), Sb(III), and Sb(V), and ArsH also conferred resistance to As(V). Acr3 did not confer resistance to As(V) and Rox(V), while ArsH did not confer resistance to Rox(V).

Identification of an anaerobic bacterial consortium that degrades roxarsone.

The degradation of roxarsone, an extensively used organoarsenic feed additive, occurs quickly under anaerobic conditions with microorganisms playing an important role in its degradation. Here, an anaerobic bacterial consortium that effectively degraded roxarsone was isolated, and its degradation efficiency and community changes along a roxarsone concentration gradient under anaerobic conditions were assessed. We used batch experiments to determine the roxarsone degradation rates, as well as the bacterial community structure and diversity, at initial roxarsone concentrations of 50, 100, 200, and 400 mg/kg. The results showed that roxarsone was degraded completely within 28, 28, 36, and 44 hr at concentrations of 50, 100, 200, and 400 mg/kg, respectively. The anaerobic bacterial consortium displayed considerable potential to degrade roxarsone, as the degradation rate increased with increasing roxarsone concentrations. Roxarsone promoted microbial growth, and in turn, the microorganisms degraded the organoarsenic compound, with the functional bacterial community varying between different roxarsone concentrations. Lysinibacillus, Alkaliphilus, and Proteiniclasticum were the main genera composing the roxarsone-degrading bacterial community.

Soil attribute regulates assimilation of roxarsone metabolites by rice (Oryza sativa L.).

Roxarsone (ROX), an organoarsenic feed additive, and its metabolites, can be present in animal manure used to fertilize rice. Rice is prone to absorb arsenic, and is subject to straighthead disorder, which reduces rice yield and is linked with organic arsenic compounds. This study aims to elucidate how soil property affect arsenic accumulation in rice plants fertilized with chicken manure containing ROX metabolites. Manures of chickens fed without or with ROX, designated as control manure and ROX treated manure (ROXCM), respectively, were applied in eight paddy soils of different origins, to investigate the assimilation of arsenic species in rice plants. The results show that inorganic arsenic (arsenate and arsenite), monomethylarsonic acid and dimethylarsinic acid (DMA) were detected in all brown rice and husk, trace tetramethylarsonium and trimethylarsine oxide were occasionally found in these both parts, whereas all these arsenic species were determined in straw, irrespective of manure type. ROXCM application specifically and significantly increased brown rice DMA (P = 0.002), which remarkably enhanced the risk of straighthead disease in rice. Although soil total As impacted grain biomass, soil free-iron oxides and pH dominated arsenic accumulation by rice plants. The significantly increased grain DMA suggests manure bearing ROX metabolites is not suitable to be used in soils with abundant free-iron oxides and/or high pH, if straighthead disorder is to be avoided in rice.

Biotransformation of arsenic-containing roxarsone by an aerobic soil bacterium Enterobacter sp. CZ-1.

Roxarsone (3-nitro-4-hydroxyphenylarsonic acid, ROX) is an arsenic-containing compound widely used as a feed additive in poultry industries. ROX excreted in chicken manure can be transformed by microbes to different arsenic species in the environment. To date, most of the studies on microbial transformation of ROX have focused on anaerobic microorganisms. Here, we isolated a pure cultured aerobic ROX-transforming bacterial strain, CZ-1, from an arsenic-contaminated paddy soil. On the basis of 16S rRNA gene sequence, strain CZ-1 was classified as a member of the genus Enterobacter. During ROX biotransformation by strain CZ-1, five metabolites including arsenate (As[V]), arsenite (As[III]), N-acetyl-4-hydroxy-m-arsanilic acid (N-AHPAA), 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA) and a novel sulfur-containing arsenic species (AsC9H13N2O6S) were detected and identified based on high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS), HPLC-ICP-MS/electrospray ionization mass spectrometry (ESI-MS) and HPLC-electrospray ionization hybrid quadrupole time-of-flight mass spectrometry (ESI-qTOF-MS) analyses. N-AHPAA and 3-AHPAA were the main products, and 3-AHPAA could also be transformed to N-AHPAA. Based on the results, we propose a novel ROX biotransformation pathway by Enterobacter. sp CZ-1, in which the nitro group of ROX is first reduced to amino group (3-AHPAA) and then acetylated to N-AHPAA.

Phytotoxicity and uptake of roxarsone by wheat (Triticum aestivum L.) seedlings.

Roxarsone (ROX), the primary aromatic arsenical additive (AAA) used in animal feeding operations, is of increasing concern to environmental and human health due to land application of ROX-laden animal manure. Few studies have investigated the phytotoxicity, uptake mechanisms, and speciation of AAA in crop plants. In this study, wheat seedlings were employed to address these issues under hydroponic conditions. Compared to inorganic arsenic, ROX was less toxic to wheat root elongation. Wheat roots were more sensitive to ROX stress than shoots. For the first time, metabolized inorganic arsenic was detected in plants, although ROX was the predominant detected arsenic species in wheat seedlings. ROX uptake and toxicity to roots were inhibited by humic acid at concentrations higher than 50 mg/L due to interaction with ROX. Phosphate enhanced ROX uptake, but no trends were observed for ROX uptake in the presence of glycerol at concentrations lower than 250 mM. In addition, ROX uptake was significantly decreased by silicate (Si(IV), 0.5-10 mM) and the metabolic inhibitor, 2,4-dinitrophenol (0.5-2 mM), indicating that ROX transport into wheat roots was actively mediated by Si(IV)-sensitive transporters. These findings provide important insights into the fate and speciation of AAA in soil-water-plant systems relevant to human health.

Accumulation and Transport of Roxarsone, Arsenobetaine, and Inorganic Arsenic Using the Human Immortalized Caco-2 Cell Line.

Roxarsone (Rox), an organoarsenic compound, served as a feed additive in the poultry industry for more than 60 years. Residual amounts of Rox present in chicken meat could give rise to potential human exposure to Rox. However, studies on the bioavailability of Rox in humans are scarce. We report here the accumulation and transepithelial transport of Rox using the human colon-derived adenocarcinoma cell line (Caco-2) model. The cellular accumulation and transepithelial passage of Rox in Caco-2 cells were evaluated and compared to those of arsenobetaine (AsB), arsenite (AsIII), and arsenate (AsV). When Caco-2 cells were exposed to 3 µM Rox, AsB, and AsIII separately for 24 h, the maximum accumulation was reached at 12 h. After 24-h exposure, the accumulated Rox was 6-20 times less than AsB and AsIII. The permeability of Rox from the apical to basolateral side of Caco-2 monolayers was similar to AsV but less than AsIII and AsB. The results of lower bioavailability of Rox are consistent with previous observations of relatively lower amounts of Rox retained in the breast meat of Rox-fed chickens. These data provide useful information for assessing human exposure to and intestinal bioavailability of Roxarsone.

Delivery of roxarsone via chicken diet→chicken→chicken manure→soil→rice plant.

Roxarsone (ROX), a widely used feed additive, occurs as itself and its metabolites in animal manure. Rice is prone to accumulate As than other staple food. Four diets with 0, 40, 80 and 120mgROXkg(-1) were fed in chickens, and four chicken manures (CMs) were collected to fertilize rice plants in a soil culture experiment. Linear regression analysis shows that the slopes of As species including 4-hydroxy-phenylarsonic acid, As(V), As(III), monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) in CM versus dietary ROX were 0.033, 0.314, 0.033, 0.054 and 0.138, respectively. Both As(III) and DMA were determined in all rice grains, and As(III), As(V), MMA and DMA in rice hull, but detectable As forms in rice straws and soils increased with increasing ROX dose. Grain As(III) was unrelated to ROX dose but exceeded the Chinese rice As limit (0.15mgAs(III)kg(-1)). Dietary ROX enhanced straw As(III) mostly, with the slope of 0.020, followed by hull DMA (0.006) and grain DMA (0.002). The slopes of soil As(V) and As(III) were 0.003 and 0.001. This is the first report illustrating the quantitative delivery of ROX via food chain, which helps to evaluate health and environmental risks caused by ROX use in animal production.

Shewanella oneidensis MR-1-Induced Fe(III) Reduction Facilitates Roxarsone Transformation.

Although microbial activity and associated iron (oxy)hydroxides are known in general to affect the environmental dynamics of 4-hydroxy-3-nitrobenzenearsonic acid (roxarsone), the mechanistic understanding of the underlying biophysico-chemical processes remains unclear due to limited experimental information. We studied how Shewanella oneidensis MR-1 -a widely distributed metal-reducing bacterium, in the presence of dissolved Fe(III), affects roxarsone transformations and biogeochemical cycling in a model aqueous system. The results showed that the MR-1 strain was able to anaerobically use roxarsone as a terminal electron acceptor and to convert it to a single product, 3-amino-4-hydroxybenzene arsonic acid (AHBAA). The presence of Fe(III) stimulated roxarsone transformation via MR-1-induced Fe(III) reduction, whereby the resulting Fe(II) acted as an efficient reductant for roxarsone transformation. In addition, the subsequent secondary Fe(III)/Fe(II) mineralization created conditions for adsorption of organoarsenic compounds to the yielded precipitates and thereby led to arsenic immobilization. The study provided direct evidence of Shewanella oneidensis MR-1-induced direct and Fe(II)-associated roxarsone transformation. Quantitative estimations revealed a candidate mechanism for the early-stage environmental dynamics of roxarsone in nature, which is essential for understanding the environmental dynamics of roxarsone and successful risk assessment.

Biodegradation of roxarsone by a bacterial community of underground water and its toxic impact.

Roxarsone is included in chicken food as anticoccidial and mainly excreted unchanged in faeces. Microorganisms biotransform roxarsone into toxic compounds that leach and contaminate underground waters used for human consumption. This study evaluated roxarsone biotransformation by underground water microorganisms and the toxicity of the resulting compounds. Underground water from an agricultural field was used to prepare microcosms, containing 0.05 mM roxarsone, and cultured under aerobic or anaerobic conditions. Bacterial communities of microcosms were characterized by PCR-DGGE. Roxarsone degradation was measured by HPLC/HG/AAS. Toxicity was evaluated using HUVEC cells and the Toxi-ChromoTest kit. Roxarsone degradation analysis, after 15 days, showed that microcosms of underground water with nutrients degraded 90 and 83.3% of roxarsone under anaerobic and aerobic conditions, respectively. Microcosms without nutrients degraded 50 and 33.1% under anaerobic and aerobic conditions, respectively. Microcosms including nutrients showed more roxarsone conversion into toxic inorganic arsenic species. DGGE analyses showed the presence of Proteobacteria, Firmicutes, Actinobacteria, Planctomycetes and Spirochaetes. Toxicity assays showed that roxarsone biotransformation by underground water microorganisms in all microcosms generated degradation products toxic for eukaryotic and prokaryotic cells. Furthermore, toxicity increased when roxarsone leached though a soil column and was further transformed by the bacterial community present in underground water. Therefore, using underground water from areas where roxarsone containing manure is used as fertilizer might be a health risk.

Earthworms as agents for ecotoxicity in roxarsone-contaminated soil ecosystem: a modeling study of ultrastructure and proteomics.

Contamination of roxarsone has been recognized as a potential environmental hazard. In this study, Eisenia fetida samples were collected after roxarsone exposures to analyze their intestinal epithelium ultrastructure, expression levels of stress-related genes, and proteomics. Our results showed that mitochondria and endoplasmic reticulum in roxarsone-treated earthworms demonstrated variety of damages. Furthermore, 149 proteins were displayed in 2-DE, and 36 of them were identified by MALDI-TOF/TOF-MS. Those identified proteins are involved in several important processes including cell immunity, cell stress responses, and cell genetic behaviors. Our study demonstrates the toxicity responses of earthworms toward arsenic-based animal drug roxarsone with practical usefulness and demonstrates a proteomic profile change that may be critical for the roxarsone stress survival mechanisms of E. fetida. Graphical Abstract Inspiration of this referred to the form of Fig. 4 in the article "Proteomic analysis of a high aluminum tolerant yeast Rhodotorula taiwanensis RS1 in response to aluminum stress" of Chao, W et al.

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.

Phosphate enhances uptake of As species in garland chrysanthemum (C. coronarium) applied with chicken manure bearing roxarsone and its metabolites.

Roxarsone (ROX), a world widely used feed organoarsenic additive in animal production, can be excreted as itself and its metabolites in animal manure. Animal manure is commonly land applied with phosphorous (P) fertilizer to enhance the P phytoavailability in agriculture. We investigated the accumulation of As species in garland chrysanthemum (C. coronarium) plants fertilized with 1% (w/w, manure/soil) chicken manure bearing ROX and its metabolites, plus 0, 0.05, 0.1, 0.2, 0.4, and 0.8 g P2O5/kg, respectively. The results show that As(III) was the sole As compound in garland chrysanthemum shoots, and As(III) and As(V) were detectable in roots. Elevated phosphate level supplied more As(V) for garland chrysanthemum roots through competitive desorption in rhizosphere, leading to significantly enhanced accumulation of As species in plants. As(III) was the predominant As form in plants (85.0∼90.6%). Phosphate could not change the allocation of As species in plants. Hence, the traditional practice that animal manure is applied with P fertilizer may inadvertently increase the potential risk of As contamination in crop via the way ROX → animal → animal manure → soil → crop.

Mapping the protein profile involved in the biotransformation of organoarsenicals using an arsenic metabolizing bacterium.

Alkaliphilus oremlandii strain OhILAs, a gram-positive bacterium, has been shown to ferment lactate as well as use arsenate and roxarsone as a terminal electron acceptor. This study examines the proteome expressed under four growth conditions to further elucidate the bacterial metabolism of inorganic and organic arsenic. The four growth conditions include, sodium lactate (as fermentative control), sodium lactate with 3-nitro-4-hydroxybenzenearsonic acid (roxarsone), sodium lactate with 3-amino-4-hydroxybenzenearsonic acid (3A4HBAA), and sodium lactate with sodium arsenate. Shotgun proteomics using LC-MS/MS was performed on the soluble cytoplasm as well as solubilized membrane proteins using perfluorooctanoic acid, a surfactant with properties similar to sodium dodecyl sulfate. The MS/MS data were analyzed using the Spectrum Mills Proteomic Workbench. Positive protein matches were confirmed with protein scores of 20 or greater and the presence of two or more peptides among the three technical replicates. A total of 1357 proteins (out of 2836 predicted) were identified with 791 in sodium lactate, 816 in sodium lactate and roxarsone, 715 in sodium lactate and 3A4HBAA, and 733 in sodium lactate and arsenate. The relative abundance of each protein was determined using a method called normalized spectral abundance factor (NSAF). Proteins that were identified in both the control and the experimental conditions were compared using the Power Law Global Error Model (PLGEM) to determine proteins that were significantly up or down regulated. All putative proteins were assigned functions and pathways using the COG databases. However, a large number of proteins were classified as hypothetical or had unknown function. Using the statistical information and known functionalities of the identified proteins, a pathway for the degradation of roxarsone and 3A4HBAA by A. oremlandii strain OhILAs is proposed.

Degradation of roxarsone in a silt loam soil and its toxicity assessment.

The land application of poultry or swine litter, containing large amounts of roxarsone, causes serious arsenic pollution in soil. Understanding biotransformation process of roxarsone and its potential risks favors proper disposal of roxarsone-contaminated animal litter, yet remains not achieved. We report an experimental study of biotransformation process of roxarsone in a silt loam soil under various soil moisture and temperature conditions, and the toxicity of roxarsone and its products from degradation. Results showed that soil moisture and higher temperature promoted roxarsone degradation, associating with emergent pentavalent arsenic. Analysis of fluorescein diacetate (FDA) hydrolysis activity revealed that roxarsone does not exert acute toxic on soil microbes. With the release of inorganic arsenic, FDA hydrolysis activity was inhibited gradually, as evidenced by ecotoxicological assessment using Photobacterium leiognathi. The results shade new lights on the dynamic roxarsone biotransformation processes in soil, which is important for guiding appropriate disposal of poultry or swine litter in the environment.

Electrochemical stimulation of microbial roxarsone degradation under anaerobic conditions.

Roxarsone (4-hydroxy-3-nitrophenylarsonic acid) has been commonly used in animal feed as an organoarsenic additive, most of which is excreted in manure. Roxarsone is easily biodegraded to 4-hydroxy-3-aminophenylarsonic acid (HAPA) under anaerobic conditions, but HAPA persists for long periods in the environment, increasing the risk of arsenic contamination through diffusion. We investigated the electrochemical stimulation of the microbial degradation of roxarsone under anaerobic conditions. After the carbon sources in the substrate were depleted, HAPA was slowly degraded to form arsenite under anaerobic conditions. The degradation rate of HAPA was significantly increased when 0.5 V was applied without adding a carbon source. The two-cell membrane reactor assays reveal that the HAPA was degraded in the anode chambers, confirming that the anode enhanced the electron transfer process by acting as an electron acceptor. The degradation product formed with electrochemical stimulation was arsenate, which facilitates the removal of arsenic from wastewater. Based on the high performance liquid chromatography-ultraviolet-hydride generation-atomic fluorescence spectrometry (HPLC-UV-HG-AFS) and gas chromatography-mass spectrometry (GC-MS) data, the pathway for the biodegradation of roxarsone and the mechanisms for the electrochemically stimulated degradation are proposed. This method provides a potential solution for the removal of arsenic from organoarsenic-contaminated wastewater.

A C⋠As lyase for degradation of environmental organoarsenical herbicides and animal husbandry growth promoters.

Arsenic is the most widespread environmental toxin. Substantial amounts of pentavalent organoarsenicals have been used as herbicides, such as monosodium methylarsonic acid (MSMA), and as growth enhancers for animal husbandry, such as roxarsone (4-hydroxy-3-nitrophenylarsonic acid) [Rox(V)]. These undergo environmental degradation to more toxic inorganic arsenite [As(III)]. We previously demonstrated a two-step pathway of degradation of MSMA to As(III) by microbial communities involving sequential reduction to methylarsonous acid [MAs(III)] by one bacterial species and demethylation from MAs(III) to As(III) by another. In this study, the gene responsible for MAs(III) demethylation was identified from an environmental MAs(III)-demethylating isolate, Bacillus sp. MD1. This gene, termed arsenic inducible gene (arsI), is in an arsenic resistance (ars) operon and encodes a nonheme iron-dependent dioxygenase with C ⋅ As lyase activity. Heterologous expression of ArsI conferred MAs(III)-demethylating activity and MAs(III) resistance to an arsenic-hypersensitive strain of Escherichia coli, demonstrating that MAs(III) demethylation is a detoxification process. Purified ArsI catalyzes Fe(2+)-dependent MAs(III) demethylation. In addition, ArsI cleaves the C ⋅ As bond in trivalent roxarsone and other aromatic arsenicals. ArsI homologs are widely distributed in prokaryotes, and we propose that ArsI-catalyzed organoarsenical degradation has a significant impact on the arsenic biogeocycle. To our knowledge, this is the first report of a molecular mechanism for organoarsenic degradation by a C ⋅ As lyase.