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.

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.

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).

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.

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.

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.

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.

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.

Metabolomics reveals the potential mechanism of La(III) promoting enrichment of Sodium hydrogen arsenate and Roxarsone in Solanum nigrum L.

The rare earth element lanthanum (La(III)) has been found to effectively enhance crop yields and improve plant growth and development. Arsenic (As), as a class of toxic metals widely found in the environment, poses a serious threat to both ecological and human health. Research on the application of La(III) in phytoremediation to enhance remediation efficiency is currently lacking. This study examined the impact of La(III) on physiological and biochemical indicators of Solanum nigrum L. (S. nigrum) exposed to Sodium hydrogen arsenate (SA) and Roxarsone (ROX) treatments under hydroponic conditions. Results indicated that La(III) treatment increased S. nigrum's aboveground As transport capacity by 58.68 %-213 % compared to no La(III) application. Additionally, foliar spraying of La(III) significantly inhibited the expression of toxic metabolites in the root system of S. nigrum, reducing Benzamide by 99.79 % under SA treatment and ZON by 87.72 % under ROX treatment. La(III) is likely to promote the transport of toxins and nutrients within and out of cells by activating ABC transporters, thereby enhancing S. nigrum's arsenic tolerance and metabolic activity. These findings provide molecular-scale insights into La(III) enhancement of the resilience of hyper-enriched plants and the remediation potential of contaminated sites.

Influence of dissolved organic matter on the anaerobic biotransformation of roxarsone accompanying microbial community response.

Roxarsone (ROX), commonly employed as a livestock feed additive, largely remains unmetabolized and is subsequently excreted via feces. ROX could cause serious environmental risks due to its rapid transformation and high mobility in the anaerobic subsurface environment. Dissolved organic matter (DOM) is an important constituent of fecal organics in livestock waste and could affect the ROX biotransformation. Nonetheless, the underlying mechanisms governing the interaction between DOM and ROX biotransformation have not yet been elucidated in the anaerobic environment. In this study, the changes of ROX, metabolites, and microbial biomass in the solutions with varying DOM concentrations (0, 50, 100, 200, and 400 mg/L) under anaerobic environments were investigated during the ROX (200 mg/L) degradation. EEM-PARAFAC and metagenomic sequencing were combined to identify the dynamic shifts of DOM components and the functional microbial populations responsible for ROX degradation. Results indicated that DOM facilitated the anaerobic biotransformation of ROX and 200 mg/L ROX could be degraded completely in 28 h. The tryptophan-like within DOM functioned as a carbon source to promote the growth of microorganisms, thus accelerating the degradation of ROX. The mixed microflora involved in ROX anaerobic degrading contained genes associated with arsenic metabolism (arsR, arsC, acr3, arsA, nfnB, and arsB), and arsR, arsC, acr3 exhibited high microbial diversity. Variations in DOM concentrations significantly impacted the population dynamics of microorganisms involved in arsenic metabolism (Proteiniclasticum, Exiguobacterium, Clostridium, Proteiniphilum, Alkaliphilus, and Corynebacterium spp.), which in turn affected the transformation of ROX and its derivatives. This study reveals the mechanism of ROX degradation influenced by the varying concentrations of DOM under anaerobic environments, which is important for the prevention of arsenic contamination with elevated levels of organic matter.

Rainwater-borne H2O2 accelerates roxarsone degradation and reduces bioavailability of arsenic in paddy rice soils.

Contamination of rice by arsenic represents a significant human health risk. Roxarsone -bearing poultry manure is a major pollution source of arsenic to paddy soils. A mesocosm experiment plus a laboratory experiment was conducted to reveal the role of rainwater-borne H2O2 in the degradation of roxarsone in paddy rice soils. While roxarsone could be degraded via chemical oxidation by Fenton reaction-derived hydroxyl radical, microbially mediated decomposition was the major mechanism. The input of H2O2 into the paddy soils created a higher redox potential, which favored certain roxarsone-degrading and As(III)-oxidizing bacterial strains and disfavored certain As(V)-reducing bacterial strains. This was likely to be responsible for the enhanced roxarsone degradation and transformation of As(III) to As(V). Fenton-like reaction also tended to enhance the formation of Fe plaque on the root surface, which acted as a filter to retain As. The dominance of As(V) in porewater, combined with the filtering effect of Fe plaque significantly reduced the uptake of inorganic As by the rice plants and consequently its accumulation in the rice grains. The findings have implications for developing management strategies to minimize the negative impacts from the application of roxarsone-containing manure for fertilization of paddy rice soils.

A Sb(III)-specific efflux transporter from Ensifer adhaerens E-60.

Antimony is pervasive environmental toxic substance, and numerous genes encoding mechanisms to resist, transform and extrude the toxic metalloid antimony have been discovered in various microorganisms. Here we identified a major facilitator superfamily (MFS) transporter, AntB, on the chromosome of the arsenite-oxidizing bacterium Ensifer adhaerens E-60 that confers resistance to Sb(III) and Sb(V). The antB gene is adjacent to gene encoding a LysR family transcriptional regulator termed LysRars, which is an As(III)/Sb(III)-responsive transcriptional repressor that is predicted to control expression of antB. Similar antB and lysRars genes are found in related arsenic-resistant bacteria, especially strains of Ensifer adhaerens, and the lysRars gene adjacent to antB encodes a member of a divergent subgroup of putative LysR-type regulators. Closely related AntB and LysRars orthologs contain three conserved cysteine residues, which are Cys17, Cys99, and Cys350 in AntB and Cys81, Cys289 and Cys294 in LysRars, respectively. Expression of antB is induced by As(III), Sb(III), Sb(V) and Rox(III) (4-hydroxy-3-nitrophenyl arsenite). Heterologous expression of antB in E. coli AW3110 (Δars) conferred resistance to Sb(III) and Sb(V) and reduced the intracellular concentration of Sb(III). The discovery of the Sb(III) efflux transporter AntB enriches our knowledge of the role of the efflux transporter in the antimony biogeochemical cycle.

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.

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.

Uptake and transport of roxarsone and its metabolites in water spinach as affected by phosphate supply.

Roxarsone (ROX) is widely used as a feed additive in intensive animal production. While an animal is fed with ROX, the As compounds in the manure primarily occur as ROX and its metabolites, including arsenate (As[V]), arsenite (As[III]), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA). Animal manure is commonly land applied with phosphorous fertilizers in China. A pot experiment was conducted to investigate the phytoavailability of ROX, As(V), As(III), MMA, and DMA in water spinach (Ipomoea aquatica), with the soil amended with 0, 0.25, 0.50, 1.0, and 2.0 g PO(4)/kg, respectively, plus 2% (w/w manure/soil) chicken manure (CM) bearing ROX and its metabolites. The results indicate that this species of water spinach cannot accumulate ROX and MMA at detectable levels, but As(V), As(III), and DMA were present in all plant samples. Increased phosphorous decreased the shoot As(V) and As(III) in water spinach but did not affect the root As(V). The shoot DMA and root As(III) and DMA were decreased/increased and then increased/decreased by elevated phosphorous. The total phosphorous content (P) in plant tissue did not correlate with the total As or the three As species in tissues. Arsenate, As(III), and DMA were more easily accumulated in the roots, and phosphate considerably inhibited their upward transport. Dimethylarsinic acid had higher transport efficiency than As(V) and As(III), but As(III) was dominant in tissues. Conclusively, phosphate had multiple effects on the accumulation and transport of ROX metabolites, which depended on their levels. However, proper utilization of phosphate fertilizer can decrease the accumulation of ROX metabolites in water spinach when treated with CM containing ROX and its metabolites.

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.

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.

Transformation of inorganic and organic arsenic by Alkaliphilus oremlandii sp. nov. strain OhILAs.

Alkaliphilus oremlandii sp. nov. strain OhILAs is a mesophilic, spore-forming, motile, low mole%GC gram positive. It was enriched from Ohio River sediments on a basal medium with 20 mM lactate and 5 mM arsenate and isolated through passage on medium with increased arsenic concentration (10 and 20 mM), tindalization, and serial dilution. The pH optimal for growth was 8.4 and 16S rRNA gene sequence analysis indicated it is most closely related to species in the genus Alkaliphilus (A. crotonoxidans 95%, A. auruminator 95%, A. metalliredigens, 94%). A strict anaerobe, it can ferment lactate via the acrylate pathway as well as fructose and glycerol. A. oremlandii also has respiratory capability, as it is able to use arsenate and thiosulfate as terminal electron acceptors with acetate, pyruvate, formate, lactate, fumarate, glycerol, or fructose as the electron donor. A respiratory arsenate reductase, which is constitutively expressed, has been identified through biochemical and Western blot analyses and confirmed by cloning and sequencing of the gene encoding the structural subunit arrA. The entire arr operon as well as the ars operon have also been identified in the fully annotated genome. A. oremlandii also transforms the organoarsenical 3-nitro-4-hydroxy benzene arsonic acid (roxarsone). Growth experiments and genomic analysis suggest that it couples the reduction of the nitro group of the organoarsenical to the oxidation of either lactate or fructose in a dissimilatory manner, generating ATP via a sodium dependent ATP synthase.

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.

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.