Isolation and characterization of Staphylococcus sp. strain NBRIEAG-8 from arsenic contaminated site of West Bengal

Arsenic contaminated rhizospheric soils of West Bengal, India were sampled for arsenic resistant bacteria that could transform different arsenic forms. Staphylococcus sp. NBRIEAG-8 was identified by16S rDNA ribotyping, which was capable of growing at 30,000?mg?l(-1) arsenate [As(V)] and 1,500?mg?l(-1) arsenite [As(III)]. This bacterial strain was also characterized for arsenical resistance (ars) genes which may be associated with the high-level resistance in the ecosystems of As-contaminated areas. A comparative proteome analysis was conducted with this strain treated with 1,000?mg?l(-1) As(V) to identify changes in their protein expression profiles. A 2D gel analysis showed a significant difference in the proteome of arsenic treated and untreated bacterial culture. The change in pH of cultivating growth medium, bacterial growth pattern (kinetics), and uptake of arsenic were also evaluated. After 72?h of incubation, the strain was capable of removing arsenic from the culture medium amended with arsenate and arsenite [12% from As(V) and 9% from As(III)]. The rate of biovolatilization of As(V) was 23% while As(III) was 26%, which was determined indirectly by estimating the sum of arsenic content in bacterial biomass and medium. This study demonstrates that the isolated strain, Staphylococcus sp., is capable for uptake and volatilization of arsenic by expressing ars genes and 8 new upregulated proteins which may have played an important role in reducing arsenic toxicity in bacterial cells and can be used in arsenic bioremediation.     

Biochemistry of arsenic detoxification

All living organisms have systems for arsenic detoxification. The common themes are (a) uptake of As(V) in the form of arsenate by phosphate transporters, (b) uptake of As(III) in the form of arsenite by aquaglyceroporins, (c) reduction of As(V) to As(III) by arsenate reductases, and (d) extrusion or sequestration of As(III). While the overall schemes for arsenic resistance are similar in prokaryotes and eukaryotes, some of the specific proteins are the products of separate evolutionary pathways.     

Synergistic interaction of glyceraldehydes‐3‐phosphate dehydrogenase and ArsJ,a novel organoarsenical efflux permease,confers arsenate resistance

Microbial biotransformations are major contributors to the arsenic biogeocycle. In parallel with transformations of inorganic arsenic, organoarsenicals pathways have recently been recognized as important components of global cycling of arsenic. The well‐characterized pathway of resistance to arsenate is reduction coupled to arsenite efflux. Here, we describe a new pathway of arsenate resistance involving biosynthesis and extrusion of an unusual pentavalent organoarsenical. A number of arsenic resistance (ars) operons have two genes of unknown function that are linked in these operons. One, gapdh, encodes the glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase. The other, arsJ, encodes a major facilitator superfamily (MFS) protein. The two genes were cloned from the chromosome of Pseudomonas aeruginosa. When expressed together, but not alone, in Escherichia coli, gapdh and arsJ specifically conferred resistance to arsenate and decreased accumulation of As(V). Everted membrane vesicles from cells expressing arsJ accumulated As(V) in the presence of purified GAPDH, D‐glceraldehylde 3‐phosphate (G3P) and NAD⁺. GAPDH forms the unstable organoarsenical 1‐arseno‐3‐phosphoglycerate (1As3PGA). We propose that ArsJ is an efflux permease that extrudes 1As3PGA from cells, where it rapidly dissociates into As(V) and 3‐phosphoglycerate (3PGA), creating a novel pathway of arsenate resistance.     

Arsenic-resistant bacteria isolated from agricultural soils of Bangladesh and characterization of arsenate-reducing strains

Aims:  To analyse the arsenic-resistant bacterial communities of two agricultural soils of Bangladesh, to isolate arsenic-resistant bacteria, to study their potential role in arsenic transformation and to investigate the genetic determinants for arsenic resistance among the isolates.
Methods and Results:  Enrichment cultures were performed in a minimal medium in the presence of As(III) and As(V) to isolate resistant bacteria. Twenty-one arsenic-resistant bacteria belonging to different genera of Gram-positive and Gram-negative bacteria were isolated. The isolates, with the exception of Oceanimonas doudoroffii Dhal Rw, reduced 2 mmol l⁻¹ As(V) completely to As(III) in aerobic conditions. Putative gene fragments for arsenite efflux pumps were amplified in isolates from Dhal soil and a putative arsenate reductase gene fragment was amplified from a Bacillus sp. from Rice soil.
Conclusions:  Phylogenetically diverse arsenic-resistant bacteria present in agricultural soils of Bangladesh are capable of reducing arsenate to arsenite under aerobic conditions apparently for detoxification purpose.
Significance and Impact of the Study:  This study provides results on identification, levels of arsenic resistance and reduction of arsenate by the bacterial isolates which could play an important role in arsenic cycling in the two arsenic-contaminated soils in Bangladesh.     

Microbial arsenic: from geocycles to genes and enzymes

Arsenic compounds have been abundant at near toxic levels in the environment since the origin of life. In response, microbes have evolved mechanisms for arsenic resistance and enzymes that oxidize As(III) to As(V) or reduce As(V) to As(III). Formation and degradation of organoarsenicals, for example methylarsenic compounds, occur. There is a global arsenic geocycle, where microbial metabolism and mobilization (or immobilization) are important processes. Recent progress in studies of the ars operon (conferring resistance to As(III) and As(V)) in many bacterial types (and related systems in Archaea and yeast) and new understanding of arsenite oxidation and arsenate reduction by respiratory-chain-linked enzyme complexes has been substantial. The DNA sequencing and protein crystal structures have established the convergent evolution of three classes of arsenate reductases (that is classes of arsenate reductases are not of common evolutionary origin). Proposed reaction mechanisms in each case involve three cysteine thiols and S-As bond intermediates, so convergent evolution to similar mechanisms has taken place.     

An ArsRC fusion protein enhances arsenate sensing and detoxification

Arsenical resistance (ars) operons encode genes for arsenic resistance and biotransformation. The majority are composed of individual genes, but fusion of ars genes is not uncommon, although it is not clear if the fused gene products are functional. Here we report identification of a four-gene ars operon from Paracoccus sp. SY that has two arsR-arsC gene fusions. ArsRC1 and ArsRC2 are related proteins that consist of an N-terminal ArsR arsenite (As(III))-responsive repressor with a C-terminal ArsC arsenate reductase. The other two genes in the operon are gapdh and arsJ. GAPDH, glyceraldehyde 3-phosphate dehydrogenase, forms 1-arseno-3-phosphoglycerate (1As3PGA) from 3-phosphoglyceraldehyde and arsenate (As(V)), ArsJ is an efflux permease for 1As3PGA that dissociates into extracellular As(V) and 3-phosphoglycerate. The net effect is As(V) extrusion and resistance. ArsRs are usually selective for As(III) and do not respond to As(V). However, the substrates and products of this operon are pentavalent, which would not be inducers of the operon. We propose that ArsRC fusions overcome this limitation by channelling the ArsC product into the ArsR binding site without diffusion through the cytosol, a de facto mechanism for As(V) induction. This novel mechanism for arsenate sensing can confer an evolutionary advantage for detoxification of inorganic arsenate.     

A subgroup of plant aquaporins facilitate the bi-directional diffusion of As(OH)<Subscript>3</Subscript> and Sb(OH)<Subscript>3</Subscript>across membranes

Background  

Arsenic is a toxic and highly abundant metalloid that endangers human health through drinking water and the food chain. The most common forms of arsenic in the environment are arsenate (As(V)) and arsenite (As(III)). As(V) is a non-functional phosphate analog that enters the food chain via plant phosphate transporters. Inside cells, As(V) becomes reduced to As(III) for subsequent extrusion or compartmentation. Although much is known about As(III) transport and handling in microbes and mammals, the transport systems for As(III) have not yet been characterized in plants.     

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.     

Arsenic resistance and removal by marine and non-marine bacteria

Arsenic resistance and removal was evaluated in nine bacterial strains of marine and non-marine origins. Of the strains tested, Marinomonas communis exhibited the second-highest arsenic resistance with median effective concentration (EC(50)) value of 510 mg As l(-1), and was capable of removing arsenic from culture medium amended with arsenate. Arsenic accumulation in cells amounted to 2290 microg As g(-1) (dry weight) when incubated on medium containing 5 mg As l(-1) of arsenate. More than half of the arsenic removed was related to metabolic activity: 45% of the arsenic was incorporated into the cytosol fraction and 10% was found in the lipid-bound fraction of the membrane, with the remaining arsenic considered to be adsorbed onto the cell surface. Potential arsenic resistance and removal were also examined in six marine and non-marine environmental water samples. Of the total bacterial colony counts, 28-100% of bacteria showed arsenic resistance. Some of the bacterial consortia, especially those from seawater enriched with arsenate, exhibited higher accumulated levels of arsenic than M. communis under the same condition. These results showed that arsenic resistant and/or accumulating bacteria are widespread in the aquatic environment, and that arsenic-accumulating bacteria such as M. communis are potential candidates for bioremediation of arsenic contaminated water.     

A novel arsenate reductase from the arsenic hyperaccumulating fern Pteris vittata

Pteris vittata sporophytes hyperaccumulate arsenic to 1% to 2% of their dry weight. Like the sporophyte, the gametophyte was found to reduce arsenate [As(V)] to arsenite [As(III)] and store arsenic as free As(III). Here, we report the isolation of an arsenate reductase gene (PvACR2) from gametophytes that can suppress the arsenate sensitivity and arsenic hyperaccumulation phenotypes of yeast (Saccharomyces cerevisiae) lacking the arsenate reductase gene ScACR2. Recombinant PvACR2 protein has in vitro arsenate reductase activity similar to ScACR2. While PvACR2 and ScACR2 have sequence similarities to the CDC25 protein tyrosine phosphatases, they lack phosphatase activity. In contrast, Arath;CDC25, an Arabidopsis (Arabidopsis thaliana) homolog of PvACR2 was found to have both arsenate reductase and phosphatase activities. To our knowledge, PvACR2 is the first reported plant arsenate reductase that lacks phosphatase activity. CDC25 protein tyrosine phosphatases and arsenate reductases have a conserved HCX5R motif that defines the active site. PvACR2 is unique in that the arginine of this motif, previously shown to be essential for phosphatase and reductase activity, is replaced with a serine. Steady-state levels of PvACR2 expression in gametophytes were found to be similar in the absence and presence of arsenate, while total arsenate reductase activity in P. vittata gametophytes was found to be constitutive and unaffected by arsenate, consistent with other known metal hyperaccumulation mechanisms in plants. The unusual active site of PvACR2 and the arsenate reductase activities of cell-free extracts correlate with the ability of P. vittata to hyperaccumulate arsenite, suggesting that PvACR2 may play an important role in this process.     

Comparative effects of single intraperitoneal or oral doses of sodium arsenate or arsenic trioxide during in utero development.

Numerous studies have suggested that single-day intraperitoneal (IP) injection of inorganic arsenic results in failure of neural tube closure and other malformations in rats, hamsters, and mice. Most of these studies involved treatment of limited numbers of animals with maternally toxic doses of arsenic (generally As(V)), without defining a dose-response relationship. In the present Good Laboratory Practice-compliant study, sodium arsenate (As(V)) was administered IP and arsenic trioxide (As(III)) was administered either IP or orally (by gavage) on gestational day 9 to groups of 25 mated Crl:CD(R)(SD)BR rats. Only at dose levels that caused severe maternal toxicity, including lethality, did IP injection of arsenic trioxide produce neural tube and ocular defects; oral administration of higher doses of arsenic trioxide caused some maternal deaths but no treatment-related fetal malformations. In contrast, IP injection of similar amounts of sodium arsenate (based on the molar amount of arsenic) caused mild maternal toxicity but a large increase in malformations, including neural tube, eye, and jaw defects. In summary, neural tube and craniofacial defects were observed after IP injection of both As(V) and As(III); however, no increase in malformations was seen following oral administration of As(III), even at maternally lethal doses. These results demonstrate that the frequently cited association between prenatal exposure to inorganic arsenic and malformations in laboratory animals is dependent on a route of administration that is not appropriate for human risk assessment.     

Transport and detoxification systems for transition metals,heavy metals and metalloids in eukaryotic and prokaryotic microbes

Transition metals, heavy metals and metalloids are usually toxic in excess, but a number of transition metals are essential trace elements. In all cells there are mechanisms for metal ion homeostasis that frequently involve a balance between uptake and efflux systems. This review will briefly describe ATP-coupled resistance pumps. ZntA and CadA are bacterial P-type ATPases that confers resistance to Zn(II), Cd(II) and Pb(II). Homologous copper pumps include the Menkes and Wilson disease proteins and CopA, an Escherichia coli pump that confers resistance to Cu(I). For resistance to arsenicals and antimonials there are several different families of transporters. In E. coli the ArsAB ATPase is a novel system that confers resistance to As(III) and Sb(III). Eukaryotic arsenic resistance transporters include Acr3p and Ycf1p of Saccharomyces cerevisiae. These systems provide resistance to arsenite [As(III)]. Arsenate [As(V)] detoxification involves reduction of As(V) to As(III), a process catalyzed by arsenate reductase enzymes. There are three families of arsenate reductases, two found in bacterial systems and a third identified in S. cerevisiae.     

抗砷性微生物及其抗砷分子机制研究进展

砷(Arsenic, As)是一种剧毒类金属(Metalloid), 在自然环境中主要以三价亚砷酸盐[Arsenite, AsO2-, As(III)]和五价砷酸盐[Arsenate, AsO43-, As(V)]的无机形式广泛存在。许多微生物在含砷环境的长期适应过程中, 进化了多种不同的砷解毒抗性机制。目前研究发现主要存在4种类型的砷抗性机理, 包括: As(III)氧化, 细胞质As(V)还原, 呼吸性As(V)还原, As(III)甲基化, 这些机制赋予微生物砷抗性并在砷的转化和地球化学循环中起着极     

Enumeration and characterization of culturable arsenate resistant bacteria in a large estuary

Arsenic is a toxic element that exists in two major inorganic forms, arsenate and arsenite. A number of bacteria have been shown to resist arsenic exposure, and even more bacteria appear to possess the genes for arsenic resistance. In this study, the numbers of culturable arsenate-resistant bacteria present in water at three coastal sites in the Lake Pontchartrain estuary, Louisiana, was determined. Despite insignificant (less than 1.33 μM) levels of arsenic in this system, 20–50% of the viable count of bacteria showed appreciable arsenate resistance, suggesting that arsenic-resistant bacteria are common and widespread. A diverse array of arsenate-resistant isolates was obtained, with 16S rRNA sequence analysis indicating 37 different bacterial strains, representing six major bacterial groups. Many of these isolates were affiliated with groups of bacteria that have been poorly characterized in terms of arsenic resistance, such as the Betaproteobacteria or Flavobacteria. Some isolates were capable of tolerating very high (>100 mM) levels of arsenate, although arsenite resistance was generally much lower. The results suggest that arsenic-resistant bacteria are common, even in environments with insignificant arsenic contamination, and that many different groups of aquatic bacteria show appreciable arsenic resistance.     

Maize response to acute arsenic toxicity as revealed by proteome analysis of plant shoots

Aerial parts (shoots) of maize seedlings fed hydroponically with 300 muM sodium arsenate [As(V)] or 250 muM sodium arsenite [As(III)] for 24 h were analyzed for differentially expressed proteins by 2-DE and digital image analysis. About 15% of total detected proteins (74 out of 500) were up- or, mainly, down-regulated by arsenic, among which 14 were selected as being those most affected by the metalloid. These proteins were analyzed by MALDI-TOF MS and 7 of them were identified: translation initiation factor eIF-5A, ATP synthase, cysteine synthase, malate dehydrogenase, protein kinase C inhibitor, Tn10 transposase-like protein, and guanine nucleotide binding protein. Each of these proteins was completely repressed by As(V) and/or As(III), except protein kinase C inhibitor, which was newly detected after exposure to As(V).     

The distribution of arsenate and arsenite in shoots and roots of Holcus lanatus is influenced by arsenic tolerance and arsenate and phosphate supply

The recent discovery that phytochelatins are important for arsenic (As) detoxification in terrestrial plants results in the necessity to understand As speciation and metabolism in plant material. A hydroponic study was therefore conducted to examine the effects of different levels of phosphate and arsenate [As(V)] on As speciation and distribution in tolerant and non-tolerant clones of Holcus lanatus. Speciation of As in tissue (using high-performance liquid chromatography-inductively coupled plasma mass spectrometry) revealed that the predominant species present were the inorganic As species (As(V) and arsenite [As(III)]), although small levels (<1%) of organic As species (dimethylarsinic acid and monomethylarsonic acid) were detected in shoot material. In roots, the proportion of total As present as As(III) generally increased with increasing levels of As(V) in the nutrient solution, whereas in shoots, the proportion of total As present as As(III) generally decreased with increasing levels of As(V). H. lanatus plants growing in the high-phosphorus (P) (100 micro M) solution contained a higher proportion of As(V) (with regard to total As) in both roots and shoots than plants supplied with low P (10 micro M); in addition, tolerant clones generally contained a higher proportion of As(V) with regard to total As than non-tolerant clones. The study further revealed that As(V) can be reduced to As(III) in both roots and shoots. Although the reduction capacity was limited, the reduction was closely regulated by As influx for all treatments. The results therefore provide a new understanding about As metabolism in H. lanatus.