Arsenate reductase of Rufibacter tibetensis is a metallophosphoesterase evolved to catalyze redox reactions.

An arsenate reductase (Car1) from the Bacteroidetes species Rufibacter tibetensis 1351T was isolated from the Tibetan Plateau. The strain exhibits resistance to arsenite [As(III)] and arsenate [As(V)] and reduces As(V) to As(III). Here we shed light on the mechanism of enzymatic reduction by Car1. AlphaFold2 structure prediction, active site energy minimization, and steady-state kinetics of wild-type and mutant enzymes give insight into the catalytic mechanism. Car1 is structurally related to calcineurin-like metallophosphoesterases (MPPs). It functions as a binuclear metal hydrolase with limited phosphatase activity, particularly relying on the divalent metal Ni2+. As an As(V) reductase, it displays metal promiscuity and is coupled to the thioredoxin redox cycle, requiring the participation of two cysteine residues, Cys74 and Cys76. These findings suggest that Car1 evolved from a common ancestor of extant phosphatases by incorporating a redox function into an existing MPP catalytic site. Its proposed mechanism of arsenate reduction involves Cys74 initiating a nucleophilic attack on arsenate, leading to the formation of a covalent intermediate. Next, a nucleophilic attack of Cys76 leads to the release of As(III) and the formation of a surface-exposed Cys74-Cys76 disulfide, ready for reduction by thioredoxin.

Arsenic release from microbial reduction of scorodite in the presence of electron shuttle in flooded soil.

Scorodite (FeAsO4·H2O) is a common arsenic-bearing (As-bearing) iron mineral in near-surface environments that could immobilize or store As in a bound state. In flooded soils, microbe induced Fe(III) or As(V) reduction can increase the mobility and bioavailability of As. Additionally, humic substances can act as electron shuttles to promote this process. The dynamics of As release and diversity of putative As(V)-reducing bacteria during scorodite reduction have yet to be investigated in detail in flooded soils. Here, the microbial reductive dissolution of scorodite was conducted in an flooded soil in the presence of anthraquinone-2,6-disulfonate (AQDS). Anaeromyxobacter, Dechloromonas, Geothrix, Geobacter, Ideonella, and Zoogloea were found to be the dominant indigenous bacteria during Fe(III) and As(V) reduction. AQDS increased the relative abundance of dominant species, but did not change the diversity and microbial community of the systems with scorodite. Among these bacteria, Geobacter exhibited the greatest increase and was the dominant Fe(III)- and As(V)-reducing bacteria during the incubation with AQDS and scorodite. AQDS promoted both Fe(III) and As(V) reduction, and over 80% of released As(V) was microbially transformed to As(III). The increases in the abundance of arrA gene and putative arrA sequences of Geobacter were higher with AQDS than without AQDS. As a result, the addition of AQDS promoted microbial Fe(III) and As(V) release and reduction from As-bearing iron minerals into the environment. These results contribute to exploration of the transformation of As from As-bearing iron minerals under anaerobic conditions, thus providing insights into the bioremediation of As-contaminated soil.

Responses of antioxidant enzymes and key resistant substances in perennial ryegrass (Lolium perenne L.) to cadmium and arsenic stresses.

Cadmium (Cd) and arsenic (As) exist simultaneously in soil environment, which poses a serious threat to the safety of agricultural products and forage production. Four Perennial Ryegrass (Lolium perenne L.) cultivars with different accumulation characteristics ('Nicaragua', 'Venus', 'Excellent' and 'Monro') were selected as the material for pot experiment. The coupled responses of key components and related enzyme activities under combined stresses of Cd and As were investigated. key components contents include Non protein sulfhydryl (NPT), glutathione (GSH) and phytochelatins (PCs). The related enzyme includes (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), γ-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GSS), phytochelatin synthetases (PCSase) and arsenate reductase (AR). The results showed that Cd contents of perennial ryegrass were higher than those of As contents with TFCd/As < 1. Cd and As contents in roots were in the higher proportion than those in shoots. Compared to control, POD activities increased by 2.72 folds under 120 mg kg-1 As treatment. The contents of PCs increased by 5.68 folds under 120 mg kg-1 As treatment. Under combined Cd and As stress, the MDA contents and antioxidant enzyme activities of 'Venus' were higher than those of 'Nicaragua'. 'Nicaragua', a high accumulation cultivar. Under the combined stresses of Cd and As, the enzyme activities and the key components were significantly correlated (P < 0.05) with the contents of Cd and As. The tolerance to Cd and As was improved with increase in GSH and PCs contents and γ-ECS, GSS, PCSase and AR activities. In conclusion, the antioxidant enzyme system and key resistant substances of perennial ryegrass have important and antagonistic effects on Cd and As stresses.

A New Strategy for As(V) Biosensing Based on the Inhibition of the Phosphatase Activity of the Arsenate Reductase from Thermus thermophilus.

Arsenic (As) pollution is a widespread problem worldwide. In recent years, biosensors based on enzymatic inhibition have been developed for arsenic detection, making the study of the effect of inhibitors on the selected enzymatic activity crucial for their setup. The arsenate reductase of Thermus thermophilus HB27, TtArsC, reduces As(V) into As(III), but is also endowed with phosphatase activity. This work investigates the inhibitory effects of As(V) and As(III) on phosphatase activity by taking advantage of a simple colorimetric assay; the results show that both of them are non-competitive inhibitors affecting the Vmax but not the KM of the reaction. However, their Ki values are different from each other (15.2 ± 1.6 µM for As(V) and 394.4 ± 40.3 µm with As(III)), indicating a higher inhibitory effect by As(V). Moreover, the inhibition-based biosystem results to be selective for As(V) since several other metal ions and salts do not affect TtArsC phosphatase activity; it exhibits a sensitivity of 0.53 ± 0.03 mU/mg/µM and a limit of detection (LOD) of 0.28 ± 0.02 µM. The good sensitivity and specificity for As(V) point to consider inhibition of TtArsC phosphatase activity for the setup of a novel biosensor for the detection of As(V).

Targeted expression of the arsenate reductase HAC1 identifies cell type specificity of arsenic metabolism and transport in plant roots.

High Arsenic Concentration 1 (HAC1), an Arabidopsis thaliana arsenate reductase, plays a key role in arsenate [As(V)] tolerance. Through conversion of As(V) to arsenite [As(III)], HAC1 enables As(III) export from roots, and restricts translocation of As(V) to shoots. To probe the ability of different root tissues to detoxify As(III) produced by HAC1, we generated A. thaliana lines expressing HAC1 in different cell types. We investigated the As(V) tolerance phenotypes: root growth, As(III) efflux, As translocation, and As chemical speciation. We showed that HAC1 can function in the outer tissues of the root (epidermis, cortex, and endodermis) to confer As(V) tolerance, As(III) efflux, and limit As accumulation in shoots. HAC1 is less effective in the stele at conferring As(V) tolerance phenotypes. The exception is HAC1 activity in the protoxylem, which we found to be sufficient to restrict As translocation, but not to confer As(V) tolerance. In conclusion, we describe cell type-specific functions of HAC1 that spatially separate the control of As(V) tolerance and As translocation. Further, we identify a key function of protoxylem cells in As(V) translocation, consistent with the model where endodermal passage cells, above protoxylem pericycle cells, form a 'funnel' loading nutrients and potentially toxic elements into the vasculature.

Biochemical, molecular and in silico characterization of arsenate reductase from Bacillus thuringiensis KPWP1 tolerant to salt, arsenic and a wide range of pH.

The present study characterized aresenate reductase of Bacillus thuringiensis KPWP1, tolerant to salt, arsenate and a wide range of pH during growth. Interestingly, it was found that arsC, arsB and arsR genes involved in arsenate tolerance are distributed in the genome of strain KPWP1. The inducible arsC gene was cloned, expressed and the purified ArsC protein showed profound enzyme activity with the KM and Kcat values as 25 µM and 0.00119 s-1, respectively. In silico studies revealed that in spite of 19-26% differences in gene sequences, the ArsC proteins of Bacillus thuringiensis, Bacillus subtilis and Bacillus cereus are structurally conserved and ArsC structure of strain KPWP1 is close to nature. Docking and analysis of the binding site showed that arsenate ion interacts with three cysteine residues of ArsC and predicts that the ArsC from B. thuringiensis KPWP1 reduces arsenate by using the triple Cys redox relay mechanism.

Variations of arsenic forms and the role of arsenate reductase in three hydrophytes exposed to different arsenic species.

In order to understand the mechanisms of arsenic (As) accumulation and detoxification in aquatic plants exposed to different As species, a hydroponic experiment was conducted and the three aquatic plants (Hydrilla verticillata, Pistia stratiotes and Eichhornia crassipes) were exposed to different concentrations of As(III), As(V) and dimethylarsinate (DMA) for 10 days. The biomass, the surface As adsorption and total As adsorption of three plants were determined. Furthermore, As speciation in the culture solution and plant body, as well as the arsenate reductase (AR) activities of roots and shoots, were also analyzed. The results showed that the surface As adsorption of plants was far less than total As absorption. Compared to As(V), the plants showed a lower DMA accumulation. P. stratiotes showed the highest accumulation of inorganic arsenic but E. crassipes showed the lowest at the same As treatment. E. crassipes showed a strong ability to accumulate DMA. Results from As speciation analysis in culture solution showed that As(III) was transformed to As(V) in all As(III) treatments, and the oxidation rates followed as the sequence of H. verticillata>P. stratiotes>E. crassipes>no plant. As(III) was the predominant species in both roots (39.4-88.3%) and shoots (39-86%) of As(III)-exposed plants. As(V) and As(III) were the predominant species in roots (37-94%) and shoots (31.1-85.6%) in As(V)-exposed plants, respectively. DMA was the predominant species in both roots (23.46-100%) and shoots (72.6-100%) in DMA-exposed plants. The As(III) contents and AR activities in the roots of P. stratiotes and in the shoots of H. verticillata were significantly increased when exposed to 1 mg·L-1 or 3 mg·L-1 As(V). Therefore, As accumulation mainly occurred via biological uptake rather than physicochemical adsorption, and AR played an important role in As detoxification in aquatic plants. In the case of As(V)-exposed plants, their As tolerance was attributed to the increase of AR activities.

Arsenic accumulation and speciation in two cultivars of Pteris cretica L. and characterization of arsenate reductase PcACR2 and arsenite transporter PcACR3 genes in the hyperaccumulating cv. Albo-lineata.

Pollution and poisoning with carcinogenic arsenic (As) is of major concern globally. Interestingly, there are ferns that can naturally tolerate remarkably high As concentrations in soils while hyperaccumulating this metalloid in their fronds. Besides Pteris vittata in which As-related traits and molecular determinants have been studied in detail, the As hyperaccumulation status has been attributed also to Pteris cretica. We thus inspected two P. cretica cultivars, Parkerii and Albo-lineata, for As hyperaccumulation traits. The cultivars were grown in soils supplemented with 20, 100, and 250 mg kg-1 of inorganic arsenate (iAsV). Unlike Parkerii, Albo-lineata was confirmed to be As tolerant and hyperaccumulating, with up to 1.3 and 6.4 g As kg-1 dry weight in roots and fronds, respectively, from soils amended with 250 mg iAsV kg-1. As speciation analyses rejected that organoarsenical species and binding with phytochelatins and other proteinaceous ligands would play any significant role in the biology of As in either cultivar. While in Parkerii, the dominating As species, particularly in roots, occurred as iAsV, in Albo-lineata the majority of the root and frond As was apparently converted to iAsIII. Parkerii markedly accumulated iAsIII in its fronds when grown on As spiked soils. Considering the roles iAsV reductase ACR2 and iAsIII transporter ACR3 may have in the handling of iAs, we isolated Albo-lineata PcACR2 and PcACR3 genes closely related to P. vittata PvACR2 and PvACR3. The gene expression analysis in Albo-lineata fronds revealed that the transcription of PcACR2 and PcACR3 was clearly As responsive (up to 6.5- and 45-times increase in transcript levels compared to control soil conditions, respectively). The tolerance and uptake assays in yeasts showed that PcACRs can complement corresponding As-sensitive mutations, indicating that PcACR2 and PcACR3 encode functional proteins that can perform, respectively, iAsV reduction and membrane iAsIII transport tasks in As-hyperaccumulating Albo-lineata.

Yeast strain Debaryomyces hansenii for amelioration of arsenic stress in rice.

Arsenic (As) is a serious threat for environment and human health. Rice, the main staple crop is more prone to As uptake. Bioremediation strategies with heavy metal tolerant rhizobacteria are well known. The main objective of the study was to characterize arsenic-resistant yeast strains, capable of mitigating arsenic stress in rice. Three yeast strains identified as Debaryomyces hansenii (NBRI-Sh2.11), Candida tropicalis (NBRI-B3.4) and Candida dubliniensis (NBRI-3.5) were found to have As reductase activity. D. hansenii with higher As tolerance has As expulsion ability as compared to other two strains. Inoculation of D. hansenii showed improved detoxification through scavenging of reactive oxygen species (ROS) by the modulation of SOD and APX activity under As stress condition in rice. Modulation of defense responsive gene (NADPH, GST, GR) along with arsR and metal cation transporter are the probable mechanism of As detoxification as evident with improved membrane (electrolyte leakage) stability. Reduced grain As (~40% reduction) due to interaction with D. hansenii (NBRI-Sh2.11) further validated it's As mitigation property in rice. To the best of our knowledge D. hansenii has been reported for the first time for arsenic stress mitigation in rice with improved growth and nutrient status of the plant.

Evaluation of arsenic induced toxicity based on arsenic accumulation, translocation and its implications on physio-chemical changes and genomic instability in indica rice (Oryza sativa L.) cultivars.

Arsenic (As) accumulation in rice is a principal route of As exposure for rice based population. We have tested physiochemical and molecular parameters together to identify low As accumulating rice cultivars with normal growth and vigor. The present study examined potential toxicity caused by arsenate (AsV) among four rice cultivars tested that varied with respect to accumulation of total arsenic, arsenite (AsIII) and their differential translocation rate which had deleterious impact on growth and metabolism. Intracellular homeostasis of rice cultivars viz., TN-1, IR-64, IR-20 and Tulaipanji was hampered by 21 days long As(V) treatment due to generation of reactive oxygen species (ROS) and inadequate activity of catalase (CAT; EC 1.11.1.6). Upregulation of oxidative stress markers viz., H2O2, proline and MDA along with alteration in enzymatic antioxidants profile were conspicuously pronounced in cv. Tulaipanji while cv. TN-1 was least affected under As(V) challenged environment. In addition to that genomic template stability and band sharing indices were qualitatively measured by DNA profiling of all tested cultivars treated with 25 µM, 50 µM, and 75 µM As(V). In rice cv. Tulaipanji genetic polymorphism was significantly detected with the application of random amplified polymorphic DNA (RAPD) tool and characterized as susceptible cultivar of As compared to cvs. TN-1, IR-64 and IR-20 that is in correlation with data obtained from cluster analysis. Hence, identified As tolerant cultivars viz., TN-1, IR64 and IR-20 especially TN-1 could be used in As contaminated agricultural field after appropriate field trial. This study could help to gather information regarding cultivar-specific tolerance strategy to avoid pollutant induced toxicity.

An endophytic Kocuria palustris strain harboring multiple arsenate reductase genes.

Aiming at revealing the arsenic (As) resistance of the endophytic Kocuria strains isolated from roots and stems of Sphaeralcea angustifolia grown at mine tailing, four strains belonging to different clades of Kocuria based upon the phylogeny of 16S rRNA genes were screened for minimum inhibitory concentration (MIC). Only the strain NE1RL3 was defined as an As-resistant bacterium with MICs of 14.4/0.0125 mM and 300/20.0 mM for As3+ and As5+, respectively, in LB/mineral media. This strain was identified as K. palustris based upon analyses of cellular chemical compositions (cellular fatty acids, isoprenoides, quinones, and sugars), patterns of carbon source, average nucleotide identity of genome and digital DNA-DNA relatedness. Six genes coding to enzymes or proteins for arsenate reduction and arsenite-bumping were detected in the genome, demonstrating that this strain is resistant to As possibly by reducing As5+ to As3+, and then bumping As3+ out of the cell. However, this estimation was not confirmed since no arsenate reduction was detected in a subsequent assay. This study reported for the first time the presence of phylogenetically distinct arsenate reductase genes in a Kocuria strain and evidenced the possible horizontal transfer of these genes among the endophytic bacteria.

Characterization of siderophore producing arsenic-resistant Staphylococcus sp. strain TA6 isolated from contaminated groundwater of Jorhat, Assam and its possible role in arsenic geocycle.

BACKGROUND: Microorganisms specifically bacteria play a crucial role in arsenic mobilization and its distribution in aquatic systems. Although bacteria are well known for their active participation in the different biogeochemical cycles, the role of these bacteria in regulating the concentration of arsenic in Brahmaputra valley has not been investigated in detail. RESULTS: In this paper, we report the isolation of an arsenic resistant bacterium TA6 which can efficiently reduce arsenate. The isolate identified as Staphylococcus sp. TA6 based on the molecular and chemotaxonomic identification (FAME) showed resistance to the high concentration of both arsenate and arsenite (As(III) = 30 mM; As(V) = 250 mM), along with cross-tolerance to other heavy metals viz., Hg2+, Cd2+, Co2+, Ni2+, Cr2+. The bacterium also had a high siderophore activity (78.7 ± 0.004 µmol) that positively correlated with its ability to resist arsenic. The isolate, Staphylococcus sp. TA6 displayed high bio-transformation ability and reduced 2 mM As(V) initially added into As(III) in a period of 72 h with 88.2% efficiency. The characterization of arsenate reductase enzyme with NADPH coupled assay showed the highest activity at pH 5.5 and temperature of 50 °C. CONCLUSIONS: This study demonstrates the role of an isolate, Staphylococcus sp. TA6, in the biotransformation of arsenate to arsenite. The presence of ars operon along with the high activity of the arsenate reductase and siderophore production in this isolate may have played an important role in mobilizing arsenate to arsenite and thus increasing the toxicity of arsenic in the aquatic systems of the Brahmaputra valley.

Dissecting the components controlling root-to-shoot arsenic translocation in Arabidopsis thaliana.

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.

Modulation of growth, ascorbate-glutathione cycle and thiol metabolism in rice (Oryza sativa L. cv. MTU-1010) seedlings by arsenic and silicon.

Arsenic is a carcinogenic metalloid, exists in two important oxidation states-arsenate (As-V) and arsenite (As-III). The influence of arsenate with or without silicate on the growth and thiol metabolism in rice (Oryza sativa L. cv. MTU-1010) seedlings were investigated. Arsenate was more toxic for root growth than shoot growth where the root lengths were short, characteristically fragile and root tips turned brown. The multiple comparison analysis using Tukey's HSD (honest significant difference) tests indicated that the rate of arsenate accumulation and its conversion to arsenite by arsenate reductase were significantly increased in all arsenate treated seedlings while in seedlings treated jointly with arsenate and silicate, arsenate accumulation and its conversion to arsenite decreased. Silicate content was detected in the seedlings treated with silicate alone and under co-application of arsenate with silicate. In the test seedlings arsenic toxicity increased ascorbate and glutathione contents along with the activities of their regulatory enzymes, viz., ascorbate peroxidase, glutathione reductase, glutathione peroxidase and glutathione-s-transferase to reduce the toxicity level induced by arsenic whereas ascorbate oxidase activity was decreased to maintain sufficient ascorbate pool under arsenate treatment. Phytochelatins production were increased in both root and shoot of the test seedlings under arsenate exposure to alter the detrimental effects of arsenic by chelation with arsenite and their subsequent sequestration into vacuole. Thus, joint application of silicate along with arsenate showed significant alterations on all the parameters tested compared to arsenate treatment alone due to less availability of arsenic in the tissue leading to better growth and metabolism in rice seedlings. Thus use of silicon in arsenic contaminated medium may help to grow rice with improved vigour.

Statistical optimization of arsenic biosorption by microbial enzyme via Ca-alginate beads.

Bioremediation of arsenic using green technology via microbial enzymes has attracted scientists due to its simplicity and cost effectiveness. Statistical optimization of arsenate bioremediation was conducted by the enzyme arsenate reductase extracted from arsenic tolerant bacterium Pseudomonas alcaligenes. Response surface methodology based on Box-Behnken design matrix was performed to determine the optimal operational conditions of a multivariable system and their interactive effects on the bioremediation process. The highest biosorptive activity of 96.2 µg gm-1 of beads was achieved under optimized conditions (pH = 7.0; As (V) concentration = 1000 ppb; time = 2 h). SEM analysis showed the morphological changes on the surface of enzyme immobilized gluteraldehyde crosslinked Ca-alginate beads. The immobilized enzyme retained its activity for 8 cycles. ANOVA with a high correlation coefficient (R2 > 0.99) and lower "Prob > F"value (<0.0001) corroborated the second-order polynomial model for the biosorption process. This study on the adsorptive removal of As (V) by enzyme-loaded biosorbent revealed a possible way of its application in large scale treatment of As (V)-contaminated water bodies.

New Arsenate Reductase Gene (arrA) PCR Primers for Diversity Assessment and Quantification in Environmental Samples.

The extent of arsenic contamination in drinking water and its potential threat to human health have resulted in considerable research interest in the microbial species responsible for arsenic reduction. The arsenate reductase gene (arrA), an important component of the microbial arsenate reduction system, has been widely used as a biomarker to study arsenate-reducing microorganisms. A new primer pair was designed and evaluated for quantitative PCR (qPCR) and high-throughput sequencing of the arrA gene, because currently available PCR primers are not suitable for these applications. The primers were evaluated in silico and empirically tested for amplification of arrA genes in clones and for amplification and high-throughput sequencing of arrA genes from soil and groundwater samples. In silico, this primer pair matched (≥90% DNA identity) 86% of arrA gene sequences from GenBank. Empirical evaluation showed successful amplification of arrA gene clones of diverse phylogenetic groups, as well as amplification and high-throughput sequencing of independent soil and groundwater samples without preenrichment, suggesting that these primers are highly specific and can amplify a broad diversity of arrA genes. The arrA gene diversity from soil and groundwater samples from the Cache Valley Basin (CVB) in Utah was greater than anticipated. We observed a significant correlation between arrA gene abundance, quantified through qPCR, and reduced arsenic (AsIII) concentrations in the groundwater samples. Furthermore, we demonstrated that these primers can be useful for studying the diversity of arsenate-reducing microbial communities and the ways in which their relative abundance in groundwater may be associated with different groundwater quality parameters. IMPORTANCE: Arsenic is a major drinking water contaminant that threatens the health of millions of people worldwide. The extent of arsenic contamination and its potential threat to human health have resulted in considerable interest in the study of microbial species responsible for the reduction of arsenic, i.e., the conversion of AsV to AsIII In this study, we developed a new primer pair to evaluate the diversity and abundance of arsenate-reducing microorganisms in soil and groundwater samples from the CVB in Utah. We observed significant arrA gene diversity in the CVB soil and groundwater samples, and arrA gene abundance was significantly correlated with the reduced arsenic (AsIII) concentrations in the groundwater samples. We think that these primers are useful for studying the ecology of arsenate-reducing microorganisms in different environments.

OsHAC4 is critical for arsenate tolerance and regulates arsenic accumulation in rice.

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.

Arsenic resistance genes of As-resistant purple nonsulfur bacteria isolated from As-contaminated sites for bioremediation application.

This study aimed to identify arsenic resistant mechanisms in As-resistant purple nonsulfur bacteria (PNSB) by screening them for presence of As-resistance genes and related enzymes. Resistance to As(III) and As(V) of four As-resistant PNSB determined in terms of median inhibition concentration (IC50 values) were in the order of strains Rhodopseudomonas palustris C1 > R. palustris AB3 > Rubrivivax benzoatilyticus C31 > R. palustris L28 which corresponded to the presence of As-resistance genes in these bacteria. The strain C1 showed all As-marker genes; arsC, arsM, aioA, and acr3, while aioA was not detected in strain AB3. Strains C31 and L28 had only Arsenite-transporter gene, acr3. Translation of all these detected gene sequences of strain C1 to amino acid sequences showed that these proteins have vicinal cysteine; Cys126, Cys105, and Cys178 of Acr3, ArsC, AioA, respectively. Tertiary structure of proteins Acr3, ArsC, AioA, and ArsM showed strain C1 exhibits the high activities of arsenite oxidase and arsenate reductase enzymes that are encoded by aioA and arsC genes, respectively. Moreover, strain C1 with arsM gene produced volatile-methylated As-compounds; monomethylarsonic acid (MMA), dimethylarsenic acid (DMA), and arsenobetaine (AsB) in the presence of either As(III) or As(V). In conclusion, the strain C1 has great potential for its application in bioremediation of As-contaminated sites.

Identification of Arsenic Resistance Genes from Marine Sediment Metagenome.

A metagenomic library of sea sediment metagenome containing 245,000 recombinant clones representing ~ 2.45 Gb of sea sediment microbial DNA was constructed. Two unique arsenic resistance clones, A7 and A12, were identified by selection on sodium arsenite containing medium. Clone A7 showed a six-fold higher resistance to arsenate [As(V)], a three-fold higher resistance to arsenite [As(III)] and significantly increased resistance to antimony [Sb(III)], while clone A12 showed increased resistance only to sodium arsenite and not to the other two metalloids. The clones harbored inserts of 8.848 Kb and 6.771 Kb, respectively. Both the clones possess A + T rich nucleotide sequence with similarity to sequences from marine psychrophilic bacteria. Sequence and transposon-mutagenesis based analysis revealed the presence of a putative arsenate reductase (ArsC), a putative arsenite efflux pump (ArsB/ACR) and a putative NADPH-dependent FMN reductase (ArsH) in both the clones and also a putative transcriptional regulatory protein (ArsR) in pA7. The increased resistance of clone A7 to As(V), As(III) and Sb(III) indicates functional expression of ArsC and ArsB proteins from pA7. The absence of increased As(V) resistance in clone A12 may be due to the expression of a possible inactive ArsC, as conserved Arg60 residue in this protein was replaced by Glu60, while the absence of Sb(III) resistance may be due to the presence of an ACR3p-type arsenite pump, which is known to lack antimony transport ability.

A Hybrid Mechanism for the Synechocystis Arsenate Reductase Revealed by Structural Snapshots during Arsenate Reduction.

Evolution of enzymes plays a crucial role in obtaining new biological functions for all life forms. Arsenate reductases (ArsC) are several families of arsenic detoxification enzymes that reduce arsenate to arsenite, which can subsequently be extruded from cells by specific transporters. Among these, the Synechocystis ArsC (SynArsC) is structurally homologous to the well characterized thioredoxin (Trx)-coupled ArsC family but requires the glutaredoxin (Grx) system for its reactivation, therefore classified as a unique Trx/Grx-hybrid family. The detailed catalytic mechanism of SynArsC is unclear and how the "hybrid" mechanism evolved remains enigmatic. Herein, we report the molecular mechanism of SynArsC by biochemical and structural studies. Our work demonstrates that arsenate reduction is carried out via an intramolecular thiol-disulfide cascade similar to the Trx-coupled family, whereas the enzyme reactivation step is diverted to the coupling of the glutathione-Grx pathway due to the local structural difference. The current results support the hypothesis that SynArsC is likely a molecular fossil representing an intermediate stage during the evolution of the Trx-coupled ArsC family from the low molecular weight protein phosphotyrosine phosphatase (LMW-PTPase) family.