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Arsenate respiration by bacteria was discovered over two decades ago and is catalyzed by diverse organisms using the well-conserved Arr enzyme complex. Until now, the mechanisms underpinning this metabolism have been relatively opaque. Here, we report the structure of an Arr complex (solved by X-ray crystallography to 1.6-Å resolution), which was enabled by an improved Arr expression method in the genetically tractable arsenate respirer Shewanella sp. ANA-3. We also obtained structures bound with the substrate arsenate (1.8 Å), the product arsenite (1.8 Å), and the natural inhibitor phosphate (1.7 Å). The structures reveal a conserved active-site motif that distinguishes Arr [(R/K)GRY] from the closely related arsenite respiratory oxidase (Arx) complex (XGRGWG). Arr activity assays using methyl viologen as the electron donor and arsenate as the electron acceptor display two-site ping-pong kinetics. A Mo(V) species was detected with EPR spectroscopy, which is typical for proteins with a pyranopterin guanine dinucleotide cofactor. Arr is an extraordinarily fast enzyme that approaches the diffusion limit (K m = 44.6 ± 1.6 μM, k cat = 9,810 ± 220 seconds−1), and phosphate is a competitive inhibitor of arsenate reduction (K i = 325 ± 12 μM). These observations, combinedwith knowledge of typical sedimentary arsenate and phosphate concentrations and known rates of arsenate desorption from minerals in the presence of phosphate, suggest that (i) arsenate desorption limits microbiologically induced arsenate reductive mobilization and (ii) phosphate enhances arsenic mobility by stimulating arsenate desorption rather than by inhibiting it at the enzymatic level.

Arsenic (As) contamination in paddy soil can cause phytotoxicity and elevated As accumulation in rice grain. Rice varieties vary in As uptake and tolerance, but the underlying mechanisms remain unclear. In this study, the aus variety Kasalath was found to be more tolerant to arsenate [As(V)] than the japonica variety Nipponbare, but the two varieties showed similar arsenite [As(III)] tolerance. Nipponbare took up more phosphate (Pi) and As(V) than Kasalath. The expression of genes for Pi transporters or Pi homeostasis regulation was quantified. Nipponbare showed 2- to 3-fold higher expression of the Pi transporter genes OsPT2 and OsPT8 than Kasalath. Two ospt8 mutants were isolated from the Kasalath background and compared with an ospt8 mutant in the Nipponbare background. Mutation in OsPT8 in both backgrounds decreased As(V) uptake by 33–57%, increased As(V) tolerance assayed by root elongation by >100-fold, and abolished the varietal differences in As(V) uptake and tolerance. The results show that OsPT8 plays a key role in As(V) uptake and that As(V) uptake mediated by OsPT8 exerts a profound toxic effect on root elongation. The results also suggest that differential OsPT8 expression explains the varietal differences in As(V) uptake and tolerance between Kasalath and Nipponbare.

Soil contamination with arsenic (As) can cause phytotoxicity and elevated As accumulation in rice grain. Here, we used a forward genetics approach to investigate the mechanism of arsenate (As(V)) tolerance and accumulation in rice. A rice mutant hypersensitive to As(V), but not to As(III), was isolated. Genomic resequencing and complementation tests were used to identify the causal gene. The function of the gene, its expression pattern and subcellular localization were characterized. OsHAC4 is the causal gene for the As(V)-hypersensitive phenotype. The gene encodes a rhodanase-like protein that shows As(V) reductase activity when expressed in Escherichia coli. OsHAC4 was highly expressed in roots and was induced by As(V). In OsHAC4pro-GUS transgenic plants, the gene was expressed exclusively in the root epidermis and exodermis. OsHAC4-eGFP was localized in the cytoplasm and the nucleus. Mutation in OsHAC4 resulted in decreased As(V) reduction in roots, decreased As(III) efflux to the external medium and markedly increased As accumulation in rice shoots. Overexpression of OsHAC4 increased As (V) tolerance and decreased As accumulation in rice plants. OsHAC4 is an As(V) reductase that is critical for As(V) detoxification and for the control of As accumulation in rice. As(V) reduction, followed by As(III) efflux, is an important mechanism of As(V) detoxification.

Pteris vittata exhibits enhanced arsenic uptake, but the corresponding mechanisms are not well known. The prevalent form of arsenic in most soils is arsenate, which is a phosphate analog and a substrate for Phosphate transporter 1 (Pht1) transporters. Herein we identify and characterize three P. vittata Pht1 transporters. Pteris vittata Pht1 cDNAs were isolated and characterized via heterologous expression in Saccharomyces cerevisiae (yeast) and Nicotiana benthamiana leaves. Expression of the PvPht1 loci in P. vittata gametophytes was also examined in response to phosphate deficiency and arsenate exposure. Expression of each of the PvPht1 cDNAs complemented the phosphate uptake defect of a yeast mutant. Compared with yeast cells expressing Arabidopsis thaliana Pht1;5, cells expressing PvPht1;3 were more sensitive to arsenate, and accumulated more arsenic. Uptake assays with yeast cells and radiolabeled ³²P revealed that PvPht1;3 and AtPht1;5 have similar affinities for phosphate, but the affinity of PvPht1;3 for arsenate is much greater. In P. vittata gametophytes, PvPht1;3 transcript levels increased in response to phosphate (Pi) deficiency and arsenate exposure. PvPht1;3 is induced by Pi deficiency and arsenate, and encodes a phosphate transporter that has a high affinity for arsenate. PvPht1;3 probably contributes to the enhanced arsenate uptake capacity and affinity exhibited by P. vittata.

Background: Assessment of soil arsenic (As) bioavailability may profoundly affect the extent of remediation required at contaminated sites by improving human exposure estimates. Because small adjustments in soil As bioavailability estimates can significantly alter risk assessments and remediation goals, convenient, rapid, reliable, and inexpensive tools are needed to determine soil As bioavailability. Objectives: We evaluated inexpensive methods for assessing As bioavailability in soil as a means to improve human exposure estimates and potentially reduce remediation costs. Methods: Nine soils from residential sites affected by mining or smelting activity and two National Institute of Standards and Technology standard reference materials were evaluated for As bioavailability, bioaccessibility, and speciation. Arsenic bioavailability was determined using an in vivo mouse model, and As bioaccessibility was determined using the Solubility/Bioavailability Research Consortium in vitro assay. Arsenic speciation in soil and selected soil physicochemical properties were also evaluated to determine whether these parameters could be used as predictors of As bioavailability and bioaccessibility. Results: In the mouse assay, we compared bioavailabilities of As in soils with that for sodium arsenate. Relative bioavailabilities (RBAs) of soil As ranged from 11% to 53% (mean, 33%). In vitro soil As bioaccessibility values were strongly correlated with soil As RBAs (R² = 0.92). Among physicochemical properties, combined concentrations of iron and aluminum accounted for 80% and 62% of the variability in estimates of RBA and bioaccessibility, respectively. Conclusion: The multifaceted approach described here yielded congruent estimates of As bioavailability and evidence of interrelations among physicochemical properties and bioavailability estimates.

Arsenate [As(V)] reduction is a major cause of arsenic (As) release from soils, which threatens more than 200 million people worldwide. While heterotrophic As(V) reduction has been investigated extensively, the mechanism of chemolithotrophic As(V) reduction is less studied. Since As is frequently found as a sulfidic mineral in the environment, microbial mediated sulfur oxidation coupled to As(V) reduction (SOAsR), a chemolithotrophic process, may be more favorable in sites impacted by oligotrophic mining (e.g. As-contaminated mine tailings). While SOAsR is thermodynamically favorable, knowledge regarding this biogeochemical process is still limited. The current study suggested that SOAsR was a more prevalent process than heterotrophic As(V) reduction in oligotrophic sites, such as mine tailings. The water-soluble reduced sulfur concentration was predicted to be one of the major geochemical parameters that had a substantial impact on SOAsR potentials. A combination of DNA stable isotope probing and metagenome binning revealed members of the genera Sulfuricella, Ramlibacter, and Sulfuritalea as sulfur oxidizing As(V)-reducing bacteria (SOAsRB) in mine tailings. Genome mining further expanded the list of potential SOAsRB to diverse phylogenetic lineages such as members associated with Burkholderiaceae and Rhodocyclaceae. Metagenome analysis using multiple tailing samples across southern China confirmed that the putative SOAsRB were the dominant As(V) reducers in these sites. Together, the current findings expand our knowledge regarding the chemolithotrophic As(V) reduction process, which may be harnessed to facilitate future remediation practices in mine tailings.

A strain of Halomonas bacteria, GFAJ-1, has been claimed to be able to use arsenate as a nutrient when phosphate is limiting and to specifically incorporate arsenic into its DNA in place of phosphorus. However, we have found that arsenate does not contribute to growth of GFAJ-1 when phosphate is limiting and that DNA purified from cells grown with limiting phosphate and abundant arsenate does not exhibit the spontaneous hydrolysis expected of arsenate ester bonds. Furthermore, mass spectrometry showed that this DNA contains only trace amounts of free arsenate and no detectable covalently bound arsenate.

Stress constantly challenges plant adaptation to the environment. Of all stress types, arsenic was a major threat during the early evolution of plants. The most prevalent chemical form of arsenic is arsenate, whose similarity to phosphate renders it easily incorporated into cells via the phosphate transporters. Here, we found that arsenate stress provokes a notable transposon burst in plants, in coordination with arsenate/phosphate transporter repression, which immediately restricts arsenate uptake. This repression was accompanied by delocalization of the phosphate transporter from the plasma membrane. When arsenate was removed, the system rapidly restored transcriptional expression and membrane localization of the transporter. We identify WRKY6 as an arsenate-responsive transcription factor that mediates arsenate/phosphate transporter gene expression and restricts arsenate-induced transposon activation. Plants therefore have a dual WRKY-dependent signaling mechanism that modulates arsenate uptake and transposon expression, providing a coordinated strategy for arsenate tolerance and transposon gene silencing.

Arsenic (As) is an important environmental and food-chain toxin. We investigated the key components controlling As accumulation and tolerance in Arabidopsis thaliana. We tested the effects of different combinations of gene knockout, including arsenate reductase (HAC1), γ-glutamyl-cysteine synthetase (γ-ECS), phytochelatin synthase (PCS1) and phosphate effluxer (PHO1), and the heterologous expression of the As-hyperaccumulator Pteris vittata arsenite efflux (PvACR3), on As tolerance, accumulation, translocation and speciation in A. thaliana. Heterologous expression of PvACR3 markedly increased As tolerance and root-to-shoot As translocation in A. thaliana, with PvACR3 being localized to the plasma membrane. Combining PvACR3 expression with HAC1 mutation led to As hyperaccumulation in the shoots, whereas combining HAC1 and PHO1 mutation decreased As accumulation. Mutants of γ-ECS and PCS1 were hypersensitive to As and had higher root-to-shoot As translocation. Combining γ-ECS or PCS1 with HAC1 mutation did not alter As tolerance or accumulation beyond the levels observed in the single mutants. PvACR3 and HAC1 have large effects on root-to-shoot As translocation. Arsenic hyperaccumulation can be engineered in A. thaliana by knocking out the HAC1 gene and expressing PvACR3. PvACR3 and HAC1 also affect As tolerance, but not to the extent of γ-ECS and PCS1.

Enzymatic reduction of arsenate to arsenite is the first step in arsenate metabolism in all organisms studied. The rice genome contains two ACR2-like genes, OsACR2.1 and OsACR2.2, which may be involved in regulating arsenic metabolism in rice. Here, we cloned both OsACR2 genes and expressed them in an Escherichia coli strain in which the arsC gene was deleted and in a yeast (Saccharomyces cerevisiae) strain with a disrupted ACR2 gene. OsACR2.1 complemented the arsenate hypersensitive phenotype of E. coli and yeast. OsACR2.2 showed much less ability to complement. The gene products were purified and demonstrated to reduce arsenate to arsenite in vitro, and both exhibited phosphatase activity. In agreement with the complementation results, OsACR2.1 exhibited higher reductase activity than OsACR2.2. Mutagenesis of cysteine residues in the putative active site HC(X)₅R motif led to nearly complete loss of both phosphatase and arsenate reductase activities. In planta expression of OsACR2.1 increased dramatically after exposure to arsenate. OsACR2.2 was observed only in roots following arsenate exposure, and its expression was less than OsACR2.1.