Arsenic Efflux Research Articles

Efflux Transporter ArsK Is Responsible for Bacterial Resistance to Arsenite, Antimonite, Trivalent Roxarsone, and Methylarsenite.

Arsenic-resistant bacteria have evolved various efflux systems for arsenic resistance. Five arsenic efflux proteins, ArsB, Acr3, ArsP, ArsJ, and MSF1, have been reported. In this study, comprehensive analyses were performed to study the function of a putative major facilitator superfamily gene, arsK, and the regulation of arsK transcriptional expression in Agrobacterium tumefaciens GW4. We found that (i) arsK is located on an arsenic gene island in strain GW4. ArsK orthologs are widely distributed in arsenic-resistant bacteria and are phylogenetically divergent from the five reported arsenic efflux proteins, indicating that it may be a novel arsenic efflux transporter. (ii) Reporter gene assays showed that the expression of arsK was induced by arsenite [As(III)], antimonite [Sb(III)], trivalent roxarsone [Rox(III)], methylarsenite [MAs(III)], and arsenate [As(V)]. (iii) Heterologous expression of ArsK in an arsenic-hypersensitive Escherichia coli strain showed that ArsK was essential for resistance to As(III), Sb(III), Rox(III), and MAs(III) but not to As(V), dimethylarsenite [dimethyl-As(III)], or Cd(II). (iv) ArsK reduced the cellular accumulation of As(III), Sb(III), Rox(III), and MAs(III) but not to As(V) or dimethyl-As(III). (v) A putative arsenic regulator gene arsR2 was cotranscribed with arsK, and (vi) ArsR2 interacted with the arsR2-arsK promoter region without metalloids and was derepressed by As(III), Sb(III), Rox(III), and MAs(III), indicating the repression activity of ArsR2 for the transcription of arsK These results demonstrate that ArsK is a novel arsenic efflux protein for As(III), Sb(III), Rox(III), and MAs(III) and is regulated by ArsR2. Bacteria use the arsR2-arsK operon for resistance to several trivalent arsenicals or antimonials.IMPORTANCE The metalloid extrusion systems are very important bacterial resistance mechanisms. Each of the previously reported ArsB, Acr3, ArsP, ArsJ, and MSF1 transport proteins conferred only inorganic or organic arsenic/antimony resistance. In contrast, ArsK confers resistance to several inorganic and organic trivalent arsenicals and antimonials. The identification of the novel efflux transporter ArsK enriches our understanding of bacterial resistance to trivalent arsenite [As(III)], antimonite [Sb(III)], trivalent roxarsone [Rox(III)], and methylarsenite [MAs(III)].

Influx and efflux of arsenic in cotton fields irrigated with arsenic-contaminated groundwater

Samples of rhizosphere soil, groundwater used for drip irrigation, and mature cotton plants were collected from farms at Kuitun, Xinjiang and analyzed for their arsenic (As) levels to account for the influx and efflux of arsenic in the field. The pH value and the soil-grain-size distribution were found to correlate with the different levels of arsenic accumulated by cotton plants and arable soil layers. The principal component analysis (PCA) indicates that the arsenic level in soil is influenced by the silt content and the arsenic level in groundwater, while the arsenic level in plant roots is affected mainly by the silt content and the arsenic level in soil. As estimated, about 64.6 ± 2.7 μg of arsenic was extracted from soil by one cotton plant, and more than 44% of it was stored in husks. About 0.8-2.6 mg As/(m2 yr) was extracted by plants from soil according to the average dry weight of the plant and the field planting density, while 2.4–28.0 mg As/(m2 yr) entered the soil based on the irrigated groundwater volume and the arsenic level in groundwater. Taking account of the cotton residues left after harvest, up to 85.8% of arsenic in the plants returned to the soil. After calculation, about 0.7–2.2 mg As/(m2 yr) returned to the soil, and thus the actual net arsenic input to the soil could reach 2.3–27.6 mg As/(m2 yr). Because of an obvious rise of the soil arsenic level in Kuitun, Xinjiang, and certain preventive measures should be taken to prevent the propagation of arsenic in the agricultural field.

Characterization of a promiscuous cadmium and arsenic resistance mechanism in Thermus thermophilus HB27 and potential application of a novel bioreporter system

BackgroundThe characterization of the molecular determinants of metal resistance has potential biotechnological application in biosensing and bioremediation. In this context, the bacterium Thermus thermophilus HB27 is a metal tolerant thermophile containing a set of genes involved in arsenic resistance which, differently from other microbes, are not organized into a single operon. They encode the proteins: arsenate reductase, TtArsC, arsenic efflux membrane transporter, TtArsX, and transcriptional repressor, TtSmtB.ResultsIn this work we show that the arsenic efflux protein TtArsX and the arsenic responsive transcriptional repressor TtSmtB are required to provide resistance to cadmium. We analyzed the sensitivity to Cd(II) of mutants lacking TtArsX, finding that they are more sensitive to this metal than the wild type strain. In addition, using promoter probe reporter plasmids, we show that the transcription of TtarsX is also stimulated by the presence of Cd(II) in a TtSmtB-dependent way. Actually, a regulatory circuit composed of TtSmtB and a reporter gene expressed from the TtarsX promoter responds to variation in Cd(II), As(III) and As(V) concentrations.ConclusionsOur results demonstrate that the system composed by TtSmtB and TtArsX is responsible for both the arsenic and cadmium resistance in T. thermophilus. The data also support the use of T. thermophilus as a suitable chassis for the design and development of As-Cd biosensors.

Post-depositional behaviour of mercury and arsenic in submarine mine tailings deposited in Buyat Bay, North Sulawesi, Indonesia

The post-depositional geochemical behaviour of mercury and arsenic in submarine mine tailings from the Mesel Gold Mine in Buyat Bay, North Sulawesi, Indonesia was assessed by in situ sampling of tailings porewaters using dialysis arrays and seawater and fish monitoring. Under steady-state conditions one year after cessation of tailings discharge, the calculated arsenic efflux incrementally added 0.8 μg/L of arsenic to the overlying seawater. The mercury efflux across the tailings-seawater interface was negligible. The arsenic and mercury concentration in seawater bottom samples monitored biannually during a 9-year post-closure program were 1.54 μg/L and <0.05 μg/L, respectively. Analysis of 650 fish tissue samples, from the post-closure monitoring had mean mercury and arsenic concentrations consistently below the FAO/WHO CODEX, and Australian and New Zealand National Food Standards, respectively. The results of the porewater, seawater and fish tissue demonstrate that the arsenic and mercury-bearing bearing compounds in the tailings are geochemically stable.

Comparative Genomics Analysis of a New Exiguobacterium Strain from Salar de Huasco Reveals a Repertoire of Stress-Related Genes and Arsenic Resistance

The Atacama Desert hosts diverse ecosystems including salt flats and shallow Andean lakes. Several heavy metals are found in the Atacama Desert, and microorganisms growing in this environment show varying levels of resistance/tolerance to copper, tellurium, and arsenic, among others. Herein, we report the genome sequence and comparative genomic analysis of a new Exiguobacterium strain, sp. SH31, isolated from an altiplanic shallow athalassohaline lake. Exiguobacterium sp. SH31 belongs to the phylogenetic Group II and its closest relative is Exiguobacterium sp. S17, isolated from the Argentinian Altiplano (95% average nucleotide identity). Strain SH31 encodes a wide repertoire of proteins required for cadmium, copper, mercury, tellurium, chromium, and arsenic resistance. Of the 34 Exiguobacterium genomes that were inspected, only isolates SH31 and S17 encode the arsenic efflux pump Acr3. Strain SH31 was able to grow in up to 10 mM arsenite and 100 mM arsenate, indicating that it is arsenic resistant. Further, expression of the ars operon and acr3 was strongly induced in response to both toxics, suggesting that the arsenic efflux pump Acr3 mediates arsenic resistance in Exiguobacterium sp. SH31.

Mechanistic Modeling of Genetic Circuits for ArsR Arsenic Regulation.

Bioreporters are living cells that generate an easily measurable signal in the presence of a chemical compound. They acquire their functionality from synthetic gene circuits, the configuration of which defines the response signal and signal-to-noise ratio. Bioreporters based on the Escherichia coli ArsR system have raised significant interest for quantifying arsenic pollution, but they need to be carefully optimized to accurately work in the required low concentration range (1-10 μg arsenite L-1). To better understand the general functioning of ArsR-based genetic circuits, we developed a comprehensive mechanistic model that was empirically tested and validated in E. coli carrying different circuit configurations. The model accounts for the different elements in the circuits (proteins, DNA, chemical species), and their detailed affinities and interactions, and predicts the (fluorescent) output from the bioreporter cell as a function of arsenite concentration. The model was parametrized using existing ArsR biochemical data, and then complemented by parameter estimations from the accompanying experimental data using a scatter search algorithm. Model predictions and experimental data were largely coherent for feedback and uncoupled circuit configurations, different ArsR alleles, promoter strengths, and presence or absence of arsenic efflux in the bioreporters. Interestingly, the model predicted a particular useful circuit variant having steeper response at low arsenite concentrations, which was experimentally confirmed and may be useful as arsenic bioreporter in the field. From the extensive validation we expect the mechanistic model to further be a useful framework for detailed modeling of other synthetic circuits.

Arsenic Detoxification by Geobacter Species.

This study examines arsenic transformation mechanisms utilized by Geobacter, a genus of iron-reducing bacteria that are predominant in many anoxic iron-rich subsurface environments. Geobacter species play a major role in microbially mediated arsenic release from metal hydroxides in the subsurface. This release raises arsenic concentrations in drinking water to levels that are high enough to cause major health problems. Therefore, information obtained from studies of Geobacter should shed light on arsenic cycling in iron-rich subsurface sedimentary environments, which may help reduce arsenic-associated illnesses. These studies should also help in the development of biosensors that can be used to detect arsenic contaminants in anoxic subsurface environments. We examined 14 different Geobacter genomes and found that all of these species possess genes coding for an arsenic detoxification system (ars operon), and some also have genes required for arsenic respiration (arr operon) and arsenic methylation (arsM).

NRF2 Is a Potential Modulator of Hyperresistance to Arsenic Toxicity in Stem-Like Keratinocytes.

Arsenic is a well-known human carcinogen. Stem cells are indicated to be involved in arsenic carcinogenesis and have a survival selection advantage during arsenic exposure with underlying mechanisms undefined. In the present study, we demonstrated that CD34high-enriched cells derived from HaCaT human keratinocytes showed stem-like phenotypes. These cells were more resistant to arsenic toxicity and had higher arsenic efflux ability than their mature compartments. The master transcription factor in antioxidant defense, nuclear factor erythroid 2-related factor 2 (NRF2) with its downstream genes, was highly expressed in CD34high-enriched cells. Stable knockdown of NRF2 abolished the hyperresistance to arsenic toxicity and holoclone-forming ability of CD34high-enriched cells. Our results suggest that skin epithelial stem/progenitor cells are more resistant to arsenic toxicity than mature cells, which is associated with the high innate expression of NRF2 in skin epithelial stem/progenitor cells.

Construction of an Escherichia coli strain for sensitive detection of arsenite ion in water

In order to construct an Escherichia coli strain with high sensitivity and specificity to detect arsenic ion using fluorescence as reporter, a sensitive strain to arsenic ion was obtained by knocking out the gene arsB that acts as an arsenic efflux pump. The pET28b vector containing arsenite detecting cassette Pars-arsR-egfp was constructed and then transformed into arsB deleted mutant. Measuring conditions of this constructed whole-cell biosensor were optimized and its linear concentration range, limit of detection and specificity were determined. This modified biosensor was much more sensitive than that using wild-type strain as host. The optimal detection range of As³⁺ concentration was 0.013 to 42.71 μmol/L, and the limit concentration of detection was as low as 5.13 nmol/L. Thus we successfully improved the sensitivity of arsenite detecting biosensor by modification of E. coli genome, which may provide useful strategies for development and optimization of microbial sensors to detect heavy metals.

Arbuscular mycorrhiza detoxifying response against arsenic and pathogenic fungus in soybean

Uptake of Arsenic (As) in plant tissues can affect metabolism, causing physiological disorders, even death. As toxicity, but also pathogen infections trigger a generalised stress response called oxidative stress; however knowledge on the response of soybean (Glycine max L.) under multiple stressors is limited so far. Arbuscular mycorrhizal fungi (AMF) enhance the tolerance of host plants to abiotic and biotic stress. Thus, we investigated the effects of the AMF Rhizophagus intraradices on soybean grown in As-contaminated soils as well as in the presence of the pathogen Macrophomina phaseolina (charcoal rot of the stem). Plant parameters and degree of mycorrhizal colonization under the different assessed treatments were analyzed. Content of As in roots and leaves was quantified. Increasing As level in the soil stopped plant growth, but promoted plant As uptake. Inoculation of soybean plants with M. phaseolina accentuated As effect at all physiological levels. In the presence of mycorrhizal symbiosis biomass dramatically increased, and significantly reduced the As concentration in plant tissues. Mycorrhization decreased oxidative damage in the presence of both As and the pathogen. Furthermore, transcription analysis revealed that the high-affinity phosphate transporter from R. intraradices RiPT and the gene encoding a putative arsenic efflux pump RiArsA were up-regulated under higher As doses. These results suggest that R. intraradices is most likely to get involved in the defense response against M. phaseolina, but also in the reduction of arsenate to arsenite as a possible detoxification mechanism in AMF associations in soybean. Capsule abstractR. intraradices actively participates in the soybean antioxidant defense response against arsenic stress and M. phaseolina infection.

Nrf2 activation ameliorates cytotoxic effects of arsenic trioxide in acute promyelocytic leukemia cells through increased glutathione levels and arsenic efflux from cells

Carnosic acid (CA), a phenolic diterpene isolated from Rosmarinus officinalis, has been shown to activate nuclear transcription factor E2-related factor 2 (Nrf2), which plays a central role in cytoprotective responses to oxidative and electrophilic stress.Recently, the Nrf2-Kelch ECH associating protein 1 (Keap1) pathway has been associated with cancer drug resistance attributable to modulation of the expression and activation of antioxidant and detoxification enzymes. However, the exact mechanisms by which Nrf2 activation results in chemoresistance are insufficiently understood to date.This study investigated the mechanisms by which the cytotoxic effects of arsenic trioxide (ATO), an anticancer drug, were decreased in acute promyelocytic leukemia cells treated with CA, a typical activator of Nrf2 used to stimulate the Nrf2/Keap1 system.Our findings suggest that arsenic is non-enzymatically incorporated into NB4 cells and forms complexes that are dependent on intracellular glutathione (GSH) concentrations. In addition, the arsenic complexes are recognized as substrates by multidrug resistance proteins and subsequently excreted from the cells. Therefore, Nrf2-associated activation of the GSH biosynthetic pathway, followed by increased levels of intracellular GSH, are key mechanisms underlying accelerated arsenic efflux and attenuation of the cytotoxic effects of ATO.

Roles of Extracellular Polysaccharides and Biofilm Formation in Heavy Metal Resistance of Rhizobia.

Bacterial surface components and extracellular compounds, particularly flagella, lipopolysaccharides (LPSs), and exopolysaccharides (EPSs), in combination with environmental signals and quorum-sensing signals, play crucial roles in bacterial autoaggregation, biofilm development, survival, and host colonization. The nitrogen-fixing species Sinorhizobium meliloti (S. meliloti) produces two symbiosis-promoting EPSs: succinoglycan (or EPS I) and galactoglucan (or EPS II). Studies of the S. meliloti/alfalfa symbiosis model system have revealed numerous biological functions of EPSs, including host specificity, participation in early stages of host plant infection, signaling molecule during plant development, and (most importantly) protection from environmental stresses. We evaluated functions of EPSs in bacterial resistance to heavy metals and metalloids, which are known to affect various biological processes. Heavy metal resistance, biofilm production, and co-culture were tested in the context of previous studies by our group. A range of mercury (Hg II) and arsenic (As III) concentrations were applied to S. meliloti wild type strain and to mutant strains defective in EPS I and EPS II. The EPS production mutants were generally most sensitive to the metals. Our findings suggest that EPSs are necessary for the protection of bacteria from either Hg (II) or As (III) stress. Previous studies have described a pump in S. meliloti that causes efflux of arsenic from cells to surrounding culture medium, thereby protecting them from this type of chemical stress. The presence of heavy metals or metalloids in culture medium had no apparent effect on formation of biofilm, in contrast to previous reports that biofilm formation helps protect various microorganism species from adverse environmental conditions. In co-culture experiments, EPS-producing heavy metal resistant strains exerted a protective effect on AEPS-non-producing, heavy metal-sensitive strains; a phenomenon termed “rescuing” of the non-resistant strain.

Paralogous Regulators ArsR1 and ArsR2 of Pseudomonas putida KT2440 as a Basis for Arsenic Biosensor Development.

The remarkable metal resistance of many microorganisms is related to the presence of multiple metal resistance operons. Pseudomonas putida KT2440 can be considered a model for these microorganisms since its arsenic resistance is due to the action of proteins encoded by the two paralogous arsenic resistance operons ARS1 and ARS2. Both operons contain the genes encoding the transcriptional regulators ArsR1 and ArsR2 that control operon expression. We show here that purified ArsR1 and ArsR2 bind the trivalent salt of arsenic (arsenite) with similar affinities (~30 μM), whereas no binding is observed for the pentavalent salt (arsenate). Furthermore, trivalent salts of bismuth and antimony showed binding to both paralogues. The positions of cysteines, found to bind arsenic in other homologues, indicate that ArsR1 and ArsR2 employ different modes of arsenite recognition. Both paralogues are dimeric and possess significant thermal stability. Both proteins were used to construct whole-cell, lacZ-based biosensors. Whereas responses to bismuth were negligible, significant responses were observed for arsenite, arsenate, and antimony. Biosensors based on the P. putida arsB1 arsB2 arsenic efflux pump double mutant were significantly more sensitive than biosensors based on the wild-type strain. This sensitivity enhancement by pump mutation may be a convenient strategy for the construction of other biosensors. A frequent limitation found for other arsenic biosensors was their elevated background signal and interference by inorganic phosphate. The constructed biosensors show no interference by inorganic phosphate, are characterized by a very low background signal, and were found to be suitable to analyze environmental samples. Arsenic is at the top of the priority list of hazardous compounds issued by the U.S. Agency for Toxic Substances and Disease. The reason for the stunning arsenic resistance of many microorganisms is the existence of paralogous arsenic resistance operons. Pseudomonas putida KT2440 is a model organism for such bacteria, and their duplicated ars operons and in particular their ArsR transcription regulators have been studied in depth by in vivo approaches. Here we present an analysis of both purified ArsR paralogues by different biophysical techniques, and data obtained provide valuable insight into their structure and function. Particularly insightful was the comparison of ArsR effector profiles determined by in vitro and in vivo experimentation. We also report the use of both paralogues to construct robust and highly sensitive arsenic biosensors. Our finding that the deletion of both arsenic efflux pumps significantly increases biosensor sensitivity is of general relevance in the biosensor field.

The mechanisms of detoxification of As(III), dimethylarsinic acid (DMA) and As(V) in the microalga Chlorella vulgaris

The response of Chlorella vulgaris when challenged by As(III), As(V) and dimethylarsinic acid (DMA) was assessed through experiments on adsorption, efflux and speciation of arsenic (reduction, oxidation, methylation and chelation with glutathione/phytochelatin [GSH/PC]). Our study indicates that at high concentrations of phosphate (1.62mM of HPO42−), upon exposure to As(V), cells are able to shift towards methylation of As(V) rather than PC formation. Treatment with As(V) caused a moderate decrease in intracellular pH and a strong increase in the concentration of free thiols (GSH). Passive surface adsorption was found to be negligible for living cells exposed to DMA and As(V). However, adsorption of As(III) was observed to be an active process in C. vulgaris, because it did not show saturation at any of the exposure periods. Chelation of As(III) with GS/PC and to a lesser extent hGS/hPC is a major detoxification mechanism employed by C. vulgaris cells when exposed to As(III). The increase of bound As-GS/PC complexes was found to be strongly related to an increase in concentration of As(III) in media. C. vulgaris cells did not produce any As-GS/PC complex when exposed to As(V). This may indicate that a reduction step is needed for As(V) complexation with GSH/PC. C. vulgaris cells formed DMASV-GS upon exposure to DMA independent of the exposure period. As(III) triggers the formation of arsenic complexes with PC and homophytochelatins (hPC) and their compartmentalisation to vacuoles. A conceptual model was devised to explain the mechanisms involving ABCC1/2 transport. The potential of C. vulgaris to bio-remediate arsenic from water appeared to be highly selective and effective without the potential hazard of reducing As(V) to As(III), which is more toxic to humans.

High-Up: A Remote Reservoir of Microbial Extremophiles in Central Andean Wetlands.

The Central Andes region displays unexplored ecosystems of shallow lakes and salt flats at mean altitudes of 3700 m. Being isolated and hostile, these so-called “High-Altitude Andean Lakes” (HAAL) are pristine and have been exposed to little human influence. HAAL proved to be a rich source of microbes showing interesting adaptations to life in extreme settings (poly-extremophiles) such as alkalinity, high concentrations of arsenic and dissolved salts, intense dryness, large daily ambient thermal amplitude, and extreme solar radiation levels. This work reviews HAAL microbiodiversity, taking into account different microbial niches, such as plankton, benthos, microbial mats and microbialites. The modern stromatolites and other microbialites discovered recently at HAAL are highlighted, as they provide unique modern—though quite imperfect—analogs of environments proxy for an earlier time in Earth's history (volcanic setting and profuse hydrothermal activity, low atmospheric O2 pressure, thin ozone layer and high UV exposure). Likewise, we stress the importance of HAAL microbes as model poly-extremophiles in the study of the molecular mechanisms underlying their resistance ability against UV and toxic or deleterious chemicals using genome mining and functional genomics. In future research directions, it will be necessary to exploit the full potential of HAAL poly-extremophiles in terms of their biotechnological applications. Current projects heading this way have yielded detailed molecular information and functional proof on novel extremoenzymes: i.e., DNA repair enzymes and arsenic efflux pumps for which medical and bioremediation applications, respectively, are envisaged. But still, much effort is required to unravel novel functions for this and other molecules that dwell in a unique biological treasure despite its being hidden high up, in the remote Andes.

Arsenic metabolism in cyanobacteria

Environmental context Cyanobacteria are ecologically important, photosynthetic organisms that are widely distributed throughout the environment. They play a central role in arsenic transformations in terms of both mineralisation and formation of organoarsenic species as the primary producers in aquatic ecosystems. In this review, arsenic resistance, transport and biotransformation in cyanobacteria are reviewed and compared with those in other organisms. Abstract Arsenic is a toxic element that is widely distributed in the lithosphere, hydrosphere and biosphere. Some species of cyanobacteria can grow in high concentrations of arsenate (pentavalent inorganic arsenic compound) (100mM) and in low-millimolar concentrations of arsenite (trivalent inorganic arsenic compound). Arsenate, which is a molecular analogue of phosphate, is taken up by cells through phosphate transporters, and inhibits oxidative phosphorylation and photophosphorylation. Arsenite, which enters the cell through a concentration gradient, shows higher toxicity than arsenate by binding to sulfhydryl groups and impairing the functions of many proteins. Detoxification mechanisms for arsenic in cyanobacterial cells include efflux of intracellular inorganic arsenic compounds, and biosynthesis of methylarsonic acid and dimethylarsinic acid through methylation of intracellular inorganic arsenic compounds. In some cyanobacteria, ars genes coding for an arsenate reductase (arsC), a membrane-bound protein involved in arsenic efflux (arsB) and an arsenite S-adenosylmethionine methyltransferase (arsM) have been found. Furthermore, cyanobacteria can produce more complex arsenic species such as arsenosugars. In this review, arsenic metabolism in cyanobacteria is reviewed, compared with that in other organisms. Knowledge gaps remain regarding both arsenic transport (e.g. uptake of methylated arsenicals and excretion of arsenate) and biotransformation (especially production of lipid-soluble arsenicals). Further studies in these areas are required, not only for a better understanding of the role of cyanobacteria in the circulation of arsenic in aquatic environments, but also for their application to arsenic bioremediation.

Genomic potential for arsenic efflux and methylation varies among global Prochlorococcus populations.

The globally significant picocyanobacterium Prochlorococcus is the main primary producer in oligotrophic subtropical gyres. When phosphate concentrations are very low in the marine environment, the mol:mol availability of phosphate relative to the chemically similar arsenate molecule is reduced, potentially resulting in increased cellular arsenic exposure. To mediate accidental arsenate uptake, some Prochlorococcus isolates contain genes encoding a full or partial efflux detoxification pathway, consisting of an arsenate reductase (arsC), an arsenite-specific efflux pump (acr3) and an arsenic-related repressive regulator (arsR). This efflux pathway was the only previously known arsenic detox pathway in Prochlorococcus. We have identified an additional putative arsenic mediation strategy in Prochlorococcus driven by the enzyme arsenite S-adenosylmethionine methyltransferase (ArsM) which can convert inorganic arsenic into more innocuous organic forms and appears to be a more widespread mode of detoxification. We used a phylogenetically informed approach to identify Prochlorococcus linked arsenic genes from both pathways in the Global Ocean Sampling survey. The putative arsenic methylation pathway is nearly ubiquitously present in global Prochlorococcus populations. In contrast, the complete efflux pathway is only maintained in populations which experience extremely low PO4:AsO4, such as regions in the tropical and subtropical Atlantic. Thus, environmental exposure to arsenic appears to select for maintenance of the efflux detoxification pathway in Prochlorococcus. The differential distribution of these two pathways has implications for global arsenic cycling, as their associated end products, arsenite or organoarsenicals, have differing biochemical activities and residence times.

Characterization of arsenic hepatobiliary transport using sandwich-cultured human hepatocytes.

Arsenic is a proven human carcinogen and is associated with a myriad of other adverse health effects. This metalloid is methylated in human liver to monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), dimethylarsinic acid (DMA(V)), and dimethylarsinous acid (DMA(III)) and eliminated predominantly in urine. Hepatic basolateral transport of arsenic species is ultimately critical for urinary elimination; however, these pathways are not fully elucidated in humans. A potentially important human hepatic basolateral transporter is the ATP-binding cassette (ABC) transporter multidrug resistance protein 4 (MRP4/ABCC4) that in vitro is a high-affinity transporter of DMA(V) and the diglutathione conjugate of MMA(III) [MMA(GS)(2)]. In rats, the related canalicular transporter Mrp2/Abcc2 is required for biliary excretion of arsenic as As(GS)(3) and MMA(GS)(2). The current study used sandwich cultured human hepatocytes (SCHH) as a physiological model of human arsenic hepatobiliary transport. Arsenic efflux was detected only across the basolateral membrane for 9 out of 14 SCHH preparations, 5 had both basolateral and canalicular efflux. Basolateral transport of arsenic was temperature- and GSH-dependent and inhibited by the MRP inhibitor MK-571. Canalicular efflux was completely lost after GSH depletion suggesting MRP2-dependence. Treatment of SCHH with As(III) (0.1-1 µM) dose-dependently increased MRP2 and MRP4 levels, but not MRP1, MRP6, or aquaglyceroporin 9. Treatment of SCHH with oltipraz (Nrf2 activator) increased MRP4 levels and basolateral efflux of arsenic. In contrast, oltipraz increased MRP2 levels without increasing biliary excretion. These results suggest arsenic basolateral transport prevails over biliary excretion and is mediated at least in part by MRPs, most likely including MRP4.

Effects of Arsenite Resistance on the Growth and Functional Gene Expression ofLeptospirillum ferriphilumandAcidithiobacillus thiooxidansin Pure Culture and Coculture

The response of iron-oxidizing Leptospirillum ferriphilum YSK and sulfur-oxidizing Acidithiobacillus thiooxidans A01 to arsenite under pure culture and coculture was investigated based on biochemical characterization (concentration of iron ion and pH value) and related gene expression. L. ferriphilum YSK and At. thiooxidans A01 in pure culture could adapt up to 400 mM and 800 mM As(III) after domestication, respectively, although arsenite showed a negative effect on both strains. The coculture showed a stronger sulfur and ferrous ion oxidation activity when exposed to arsenite. In coculture, the pH value showed no significant difference when under 500 mM arsenite stress, and the cell number of At. thiooxidans was higher than that in pure culture benefiting from the interaction with L. ferriphilum. The expression profile showed that the arsenic efflux system in the coculture was more active than that in pure culture, indicating that there is a synergetic interaction between At. thiooxidans A01 and L. ferriphilum YSK. In addition, a model was proposed to illustrate the interaction between arsenite and the ars operon in L. ferriphilum YSK and At. thiooxidans A01. This study will facilitate the effective application of coculture in the bioleaching process by taking advantage of strain-strain communication and coordination.

Arsenic efflux from Microcystis aeruginosa under different phosphate regimes.

Phytoplankton plays an important role in arsenic speciation, distribution, and cycling in freshwater environments. Little information, however, is available on arsenic efflux from the cyanobacteria Microcystis aeruginosa under different phosphate regimes. This study investigated M. aeruginosa arsenic efflux and speciation by pre-exposing it to 10 µM arsenate or arsenite for 24 h during limited (12 h) and extended (13 d) depuration periods under phosphate enriched (+P) and phosphate depleted (−P) treatments. Arsenate was the predominant species detected in algal cells throughout the depuration period while arsenite only accounted for no greater than 45% of intracellular arsenic. During the limited depuration period, arsenic efflux occurred rapidly and only arsenate was detected in solutions. During the extended depuration period, however, arsenate and dimethylarsinic acid (DMA) were found to be the two predominant arsenic species detected in solutions under −P treatments, but arsenate was the only species detected under +P treatments. Experimental results also suggest that phosphorus has a significant effect in accelerating arsenic efflux and promoting arsenite bio-oxidation in M. aeruginosa. Furthermore, phosphorus depletion can reduce arsenic efflux from algal cells as well as accelerate arsenic reduction and methylation. These findings can contribute to our understanding of arsenic biogeochemistry in aquatic environments and its potential environmental risks under different phosphorus levels.