Arginine 60 in the ArsC arsenate reductase of E. coli plasmid R773 determines the chemical nature of the bound As(III) product

Arsenic is a ubiquitous environmental toxic metal. Consequently, organisms detoxify arsenate by reduction to arsenite, which is then excreted or sequestered. The ArsC arsenate reductase from Escherichia coli plasmid R773, the best characterized arsenic-modifying enzyme, has a catalytic cysteine, Cys 12, in the active site, surrounded by an arginine triad composed of Arg 60, Arg 94, and Arg 107. During the reaction cycle, the native enzyme forms a unique monohydroxyl Cys 12-thiol-arsenite adduct that contains a positive charge on the arsenic. We hypothesized previously that this unstable intermediate allows for rapid dissociation of the product arsenite. In this study, the role of Arg 60 in product formation was evaluated by mutagenesis. A total of eight new structures of ArsC were determined at resolutions between 1.3 A and 1.8 A, with R(free) values between 0.18 and 0.25. The crystal structures of R60K and R60A ArsC equilibrated with the product arsenite revealed a covalently bound Cys 12-thiol-dihydroxyarsenite without a charge on the arsenic atom. We propose that this intermediate is more stable than the monohydroxyarsenite intermediate of the native enzyme, resulting in slow release of product and, consequently, loss of activity.     

Investigation of the structure and function of a Shewanella oneidensis arsenical-resistance family transporter

The toxic metalloid arsenic is an abundant element and most organisms possess transport systems involved in its detoxification. One such family of arsenite transporters, the ACR3 family, is widespread in fungi and bacteria. To gain a better understanding of the molecular mechanism of arsenic transport, we report here the expression and characterization of a family member, So_ACR3, from the bacterium Shewanella oneidensis MR-1. Surprisingly, expression of this transporter in the arsenic-hypersensitive Escherichia coli strain AW3110 conferred resistance to arsenate, but not to arsenite. Purification of a C-terminally His-tagged form of the protein allowed the binding of putative permeants to be directly tested: arsenate but not arsenite quenched its intrinsic fluorescence in a concentration-dependent fashion. Fourier transform infrared spectroscopy showed that the purified protein was predominantly α-helical. A mutant bearing a single cysteine residue at position 3 retained the ability to confer arsenate resistance, and was accessible to membrane impermeant thiol reagents in intact cells. In conjunction with successful C-terminal tagging with oligohistidine, this finding is consistent with the experimentally-determined topology of the homologous human apical sodium-dependent bile acid transporter, namely 7 transmembrane helices and a periplasmic N-terminus, although the presence of additional transmembrane segments cannot be excluded. Mutation to alanine of the conserved residue proline 190, in the fourth putative transmembrane region, abrogated the ability of the transporter to confer arsenic resistance, but did not prevent arsenate binding. An apparently increased thermal stability is consistent with the mutant being unable to undergo the conformational transitions required for permeant translocation.     

Effects of protein deficient diets on the developmental toxicity of inorganic arsenic in mice

BACKGROUND: Inorganic arsenic, when given by injection to pregnant laboratory animals (mice, rats, hamsters), has been shown to induce malformations. Arsenic methylation may be a detoxification step, and diets deficient in protein are a poor source of methyl donors and may possibly result in impaired arsenic methylation. Human health effects from chronic arsenic exposure have been reported mainly in populations with low socioeconomic status. Individuals in such populations are likely to suffer from malnutrition, which can compromise embryonic/fetal development and diminish arsenic methylating capacity. We sought to determine if dietary protein deficiency affects the developmental toxicity of inorganic arsenic. METHODS: Mated females were randomly assigned to one of 12 treatment groups. Experimental groups received either AsIII or AsV i.p. on Gestation Day 8 (GD 8, plug=GD 0) and were maintained on a 5%, 10%, or 20% protein custom mixed diet from GD 1 until sacrifice. Controls received the custom diets alone, were given AsIII or AsV i.p. on GD 8 with Teklad LM-485 rodent diet, or were fed the LM-485 diet alone. Test females were sacrificed on GD 17, and their litters were examined for mortality and developmental defects. RESULTS: Arsenic plus dietary protein deficiency decreased maternal weight gain and increased the incidences of exencephaly, ablepharia, and skeletal defects, such as malformed vertebral centra, fused ribs, and abnormal sternebrae (bipartite, rudimentary, or unossified). CONCLUSIONS: These results demonstrate that dietary protein deficiency enhances the developmental toxicity of inorganic arsenic, possibly by impairment of arsenic methylation.     

Mytilus trossulus hsp70 as a biomarker for arsenic exposure in the marine environment: Laboratory and real-world results

Acute renal papillary necrosis (RPN) in animals is characterized by increased renal lipid accumulation. The excretion of renal lipids into urine has been determined to evaluate their possible use as sensitive early biomarkers for the diagnosis of RPN. This study investigates injury induced by two model nephrotoxins, mefenamic acid (MFA), a non-steroidal anti-inflammatory drug (NSAID), and its analogue N-phenylanthranilic acid (NPAA). Oral NPAA was given repeatedly at doses of 100, 250 and 500 mg kg^-1 daily for 5 days, followed by a 2 day respite over the weekend, and then four further daily doses. The same dosing procedure was used with MFA, but at doses of 75, 150 and 300 mgkg^-1. The control groups were given vehicle orally using the same volume given to the test groups. Urinary phospholipids (PLs), notably sphingomyelin (SPM), phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were measured and compared with other urinary parameters. Histopathological investigations were also performed to confirm the presence or absence of RPN. Following MFA treatment, PC, PI and PE were raised significantly (p < 0.001) on days 1 and 3 and for the remaining part of the experiment. After NPAA treatment, PI showed a transient elevation, and PC and PE levels were significantly increased from day 2 onwards. Both drugs caused a dose-related increase in PLs. There was no significant increase in the level of other urinary parameters. However, histopathological examination of the kidney on day 11 revealed lesions in the medulla and papilla following treatment with the two papillotoxins. These findings demonstrate the potential of urinary PLs as diagnostic non-invasive biomarkers for early renal injury associated with RPN, which may provide an important improvement in the approach to the therapeutic management of analgesic nephropathy.     

Arsenic in the rhizosphere soil solution of ferns

The aim of this study was to explore the evidence of arsenic hyperaccumulation in plant rhizosphere solutions. Six common fern plants were selected and grown in three types of substrate: arsenic (As) -tailings, As-spiked soil, and soil-As-tailing composites. A rhizobox was designed with an in-situ collection of soil solutions to analyze changes in the As concentration and valence as well as the pH, dissolved organic carbon (DOC) and total nitrogen (TN). Arsenite composed less than 20% of the total As, and As depletion was consistent with N depletion in the rhizosphere solutions of the various treatments. The As concentrations in the rhizosphere and non-rhizosphere solutions in the presence of plants were lower than in the respective controls without plants, except for in the As-spiked soils. The DOC concentrations were invariably higher in the rhizosphere versus non-rhizosphere solutions from the various plants; however, no significant increase in the DOC content was observed in Pteris vittata, in which only a slight decrease in pH appeared in the rhizosphere compared to non-rhizosphere solutions. The results showed that As reduction by plant roots was limited, acidification-induced solubilization was not the mechanism for As hyperaccumulation.     

Rapid reduction of arsenate in the medium mediated by plant roots

Microbes detoxify arsenate by reduction and efflux of arsenite. Plants have a high capacity to reduce arsenate, but arsenic efflux has not been reported. Tomato (Lycopersicon esculentum) and rice (Oryza sativa) were grown hydroponically and supplied with 10 microm arsenate or arsenite, with or without phosphate, for 1-3 d. The chemical species of As in nutrient solutions, roots and xylem sap were monitored, roles of microbes and root exudates in As transformation were investigated and efflux of As species from tomato roots was determined. Arsenite remained stable in the nutrient solution, whereas arsenate was rapidly reduced to arsenite. Microbes and root exudates contributed little to the reduction of external arsenate. Arsenite was the predominant species in roots and xylem sap. Phosphate inhibited arsenate uptake and the appearance of arsenite in the nutrient solution, but the reduction was near complete in 24 h in both -P- and +P-treated tomato. Phosphate had a greater effect in rice than tomato. Efflux of both arsenite and arsenate was observed; the former was inhibited and the latter enhanced by the metabolic inhibitor carbonylcyanide m-chlorophenylhydrazone. Tomato and rice roots rapidly reduce arsenate to arsenite, some of which is actively effluxed to the medium. The study reveals a new aspect of As metabolism in plants.     

Enhanced arsenate reduction by a CDC25-like tyrosine phosphatase explains increased phytochelatin accumulation in arsenate-tolerant Holcus lanatus

Decreased arsenate [As(V)] uptake is the major mechanism of naturally selected As(V) hypertolerance in plants. However, As(V)-hypertolerant ecotypes also show enhanced rates of phytochelatin (PC) accumulation, suggesting that improved sequestration might additionally contribute to the hypertolerance phenotype. Here, we show that enhanced PC-based sequestration in As(V)-hypertolerant Holcus lanatus is not due to an enhanced capacity for PC synthesis as such, but to increased As(V) reductase activity. Vacuolar transport of arsenite-thiol complexes was equal in both ecotypes. Based on homology with the yeast As(V) reductase, Acr2p, we identified a Cdc25-like plant candidate, HlAsr, and confirmed the As(V) reductase activity of both HlAsr and the homologous protein from Arabidopsis thaliana. The gene appeared to be As(V)-inducible and its expression was enhanced in the As(V)-hypertolerant H. lanatus ecotype, compared with the non-tolerant ecotype. Homologous ectopic overexpression of the AtASR cDNA in A. thaliana produced a dual phenotype. It improved tolerance to mildly toxic levels of As(V) exposure, but caused hypersensitivity to more toxic levels. Arabidopsis asr T-DNA mutants showed increased As(V) sensitivity at low exposure levels and enhanced arsenic retention in the root. It is argued that, next to decreased uptake, enhanced expression of HlASR might act as an additional determinant of As(V) hypertolerance and As transport in H. lanatus.     

Effect of silicate on the growth and arsenate uptake by rice (Oryza sativa L.) seedlings in solution culture

A solution culture experiment was conducted to investigate the effect of silicate on the yield and arsenate uptake by rice. Rice seedlings (Oryza sativaL. cv. Weiyou 77) were cultured in modified Hoagland nutrient solution containing three arsenate levels (0, 0.5 and 1.0 mg L ^–1 As) and four silicate levels (0, 14, 28 and 56 mg L ^–1 Si). Addition of Si significantly increased shoot dry weight (P=0.001) but had little effect on root dry weight (P=0.43). Addition of As had no significant effect on shoot dry weight (P=0.43) but significantly increased root dry weight (P=0.01). Silicon concentrations in shoots and roots increased proportionally to increasing amounts of externally supplied Si (P < 0.001). The presence of As in the nutrient solution had little effect on shoot Si concentration (P=0.16) but significantly decreased root Si concentration (P=0.005). Increasing external Si concentration significantly decreased shoot and root As concentrations and total As uptake by rice seedlings (P <0.001). In addition, Si significantly decreased shoot P concentration and shoot P uptake (P <0.001). The data clearly demonstrate a beneficial effect of Si on the growth of rice seedlings. Addition of Si to the growth medium also inhibited the uptake of arsenate and phosphate by the rice seedlings.     

Effects of arsenate and copper on the algal communities in polluted lakes in the northern parts of Sweden assayed by PICT (Pollution-Induced Community Tolerance)

The tolerance for arsenate and copper in the carbon dioxide fixation activity of phytoplankton communities coming from lakes around the smelter at Rönnskär at the Swedish east-coast were measured during three years (1989–1991). The smelter have for several decades discharged arsenic and heavy metals into the air, and their concentrations in the lakes were clearly correlated with the distance from the smelter. The tolerance of communities from the three most polluted lakes were higher than communities from reference lakes with background concentrations of arsenic and copper. In accordance with the PICT concept it is indicated that those communities have been affected by these substances. These communities also had lower diversity than the others, but no clear correlation could be done with phytoplankton species number, or between phytoplankton biomass and pollution levels.     

A plasmid-encoded arsenite pump produces arsenite resistance in Escherichia coli

The arsenate resistance operon of R-factor R773, a conjugative resistance plasmid, has two functional regions, a promoter-proximal region encoding resistance to arsenite and antimonate, and a promoter-distal one encoding arsenate resistance. Cells bearing arsenite resistance plasmids exhibited reduced accumulation of 74AsO2-. When resistant cells were depleted of endogenous energy reserves and then loaded with 74AsO2-, active extrusion of the ion was observed when an energy source was supplied. Intracellular ATP was required for extrusion, but a proton motive force was neither necessary nor sufficient. An arsenite-sensitive mutant was unable to extrude arsenite, while an arsenate-sensitive mutant had normal arsenite transport. These results suggest that the action of a plasmid-encoded primary arsenite efflux pump is the mechanism of arsenite resistance.     

Hydroponic Screening of Shrub Willow (Salix Spp.) for Arsenic Tolerance and Uptake

Shrub willows have demonstrated potential in many types of phytoremediation applications. Hydroponic culture was used to assess arsenic (As) tolerance and uptake by four shrub willow clones and to determine the effects of phosphate on As accumulation. After 4 weeks of growth in the absence of As, plants received one of four treatments: 0.25X Hoagland's minus P (?P), 0.25X Hoagland's minus P plus 100 μM arsenate (As100(?P)), 0.25X Hoagland's minus P plus 250 μM arsenate (As250(?P)), and 0.25X Hoagland's plus 250 μM arsenate (As250(+P)). Except for treatment As250(+P), phosphate was excluded due to its tendency to interfere with As uptake. After 3 weeks of treatment, plants were separated into root, leaf, and stem tissues. Biomass production and transpiration were used to quantify As tolerance. There was wide variation among clones in As tolerance and uptake. The presence of phosphate in solution alleviated the negative impacts of As on biomass and transpiration and also increased aboveground As accumulation, suggesting that phosphate may play a role in reducing toxicity and enhancing As uptake by willow shrubs. These findings offer insight into As tolerance and uptake in Salix spp. and add to the growing body of evidence supporting the use of shrub willow for phytoremediation.     

不同比例盐生小球藻-枯草芽孢杆菌共生体对砷酸盐耐性及富集差异

采用盐生小球藻与枯草芽孢杆菌为供试材料,构建不同藻菌比(1∶0、1∶1、1∶2、1∶3、1∶4)的共生体,在不同浓度砷酸盐[As(Ⅴ)]处理7 d后,测定藻菌共生体生长及其对砷的富集、吸附、吸收和形态转化。结果表明: 在As(Ⅴ)处理下,随着枯草芽孢杆菌比例的增加,藻菌共生体叶绿素含量、干重、比增长率显著提高,在750 μg·L^-1As(Ⅴ)处理时,1∶4的藻菌共生体分别达到1.81 mg·L^-1、125.0 mg、0.28 mg·L^-1·d^-1。藻菌比例由1∶0变为1∶4时,藻菌共生体对砷的富集和吸收呈下降趋势;砷的富集方式随砷浓度的增加而变化,在75～150 μg·L^-1As(Ⅴ)处理下以吸收为主,在300～750 μg·L^-1 As(Ⅴ)处理下以吸附为主。藻菌共生体内存在As(Ⅴ)和As(Ⅲ)两种形态,且随枯草芽孢杆菌比例的上升,As(Ⅴ)还原率增大(最高达到12.6%)。综上,枯草芽孢杆菌的添加提升了藻菌共生体对As(Ⅴ)的耐性和还原,但减少了对As(Ⅴ)的富集。     

Enhanced arsenate uptake in Saccharomyces cerevisiae overexpressing the Pho84 phosphate transporter

Arsenate is a major toxic constituent in arsenic-contaminated water supplies. Saccharomyces cerevisiae was engineered as a potential biosorbent for enhanced arsenate accumulation. The phosphate transporter, Pho84p, known to import arsenate, was overexpressed using a 2μ-based vector carrying PHO84 under the control of the late-phase ADH2 promoter. Arsenate uptake was then evaluated using a resting cell system. In buffer solutions containing high arsenate concentrations (12,000 and 30,000 ppb), the engineered strains internalized up to 750 μg of arsenate per gram of cells, a 50% improvement over control strains. Increasing the cell mass 2.5-fold yielded a proportional increase in the volumetric arsenate uptake, while maintaining the same level of specific uptake. At high levels of arsenate, loss from the intact cells to the medium was observed with time; knockouts of two known arsenic extrusion genes, ACR3 and FPS1, did not prevent this loss. At trace level concentrations (120 ppb), rapid and total arsenate removal was observed. The presence of 50 μM phosphate reduced uptake by approximately 15% in buffer containing 80 μM (6,000 ppb) arsenate. At trace levels of arsenate (70 ppb), the phosphate reduced the initial rate of uptake, but not the total amount removed. PHO84 mRNA levels were nearly 30 times higher in the engineered strains relative to the control strains. Uptake may no longer be a limiting factor in the engineered system and further increases should be possible by upregulating the downstream reduction and sequestration pathways.     

ACCUMULATION AND REDUCTION OF ARSENATE BY THE FRESHWATER GREEN ALGA CHLORELLA SP. (CHLOROPHYTA)

Arsenate accumulation and reduction kinetics at both high and low phosphate concentrations were investigated in the green alga Chlorella sp, isolated from the arsenic-contaminated Upper Mystic Lake near Boston, MA. Growth rate, accumulated cellular arsenic, and release of As(III) were determined over a range of arsenate concentrations. Arsenate inhibited growth and reduced final cell yield at high phosphate concentration. However, growth rate, final cell yield, and cellular arsenic content were all enhanced by higher arsenate concentrations in cultures grown at a low concentration of phosphate. The traditional view that phosphate-limited cells are necessarily more sensitive to As(V) toxicity may not be correct. The reduction rates of As(V) by Chlorella sp. obtained in our laboratory were similar to net reduction rates measured in epilimnetic water from the Upper Mystic Lake, demonstrating the importance of phytoplankton in arsenic reduction in freshwater.