Arsenic Resistance Research Articles

Influence of pore size and membrane surface properties on arsenic removal by nanofiltration membranes

Nanofiltration (NF) has a great potential in removing arsenate from contaminated water. The performance including arsenate rejection, water permeability and resistance to fouling could however differ substantially among NF membranes. This study was conducted to investigate the influence of membrane pore size and surface properties on these aspects of membrane performance. Four fullyaromatic NF membranes with different physicochemical properties were adopted for this study. The results showed that surface charge, hydrophobicity, roughness and pore size could affect water permeability and/or arsenate rejection considerably. A more negative surface charge was desirable to enhance arsenate rejection rates. NF90 and a non-commercialized membrane (M#1) demonstrated the best performance in terms of arsenate rejection and water permeability. The M#1 membrane showed less membrane fouling than NF90 when used for filtration of real arsenic-containing groundwater. This was mainly due to its distinct chemical composition and surface properties. A severe membrane fouling could lead to a substantial reduction of arsenic rejection. The M#1 membrane showed the best performance, which indicated that membrane modification could indeed enhance the overall membrane performance for water treatment.

Polyextremophilic Bacteria from High Altitude Andean Lakes: Arsenic Resistance Profiles and Biofilm Production.

High levels of arsenic present in the High Altitude Andean Lakes (HAALs) ecosystems selected arsenic-resistant microbial communities which are of novel interest to study adaptations mechanisms potentially useful in bioremediation processes. We herein performed a detailed characterization of the arsenic tolerance profiles and the biofilm production of two HAAL polyextremophiles, Acinetobacter sp. Ver3 (Ver3) and Exiguobacterium sp. S17 (S17). Cellular adherence over glass and polypropylene surfaces were evaluated together with the effect of increasing doses and oxidative states of arsenic over the quality and quantity of their biofilm production. The arsenic tolerance outcomes showed that HAAL strains could tolerate higher arsenic concentrations than phylogenetic related strains belonging to the German collection of microorganisms and cell cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ), which suggest adaptations of HAAL strains to their original environment. On the other hand, the crystal violet method (CV) and SEM analysis showed that Ver3 and S17 were able to attach to solid surfaces and to form the biofilm. The quantification of biofilms production in 48 hours' cultures through CV shows that Ver3 yielded higher production in the treatment without arsenic cultured on a glass support, while S17 yield higher biofilm production under intermediate arsenic concentration on glass supports. Polypropylene supports had negative effects on the biofilm production of Ver3 and S17. SEM analysis shows that the highest biofilm yields could be associated with a larger number of attached cells as well as the development of more complex 3D multicellular structures.

Six New Families of Aerobic Arsenate Reducing Bacteria: Leclercia, Raoultella, Kosakonia, Lelliottia, Yokenella, and Kluyvera

Elevated levels of arsenate can occur in the environment due to processes such as mining activities, and microbes must utilize various detoxification mechanisms to adapt to the associated pressure. The aim of this study was to identify as many aerobic arsenate-reducing bacteria (aARB) as possible in order to investigate their phylogenetic diversity and molecular mechanisms of arsenic resistance. We isolated 24 strains of aARB from a long-standing arsenic contaminated environment and detected the ars genotype in them. All 24 strains could reduce approximately 90% of arsenate, and 23 of them exhibited (6–59%) arsenic removal ability. The 16S rRNA gene analyses revealed aARB representing 16 genera were abundant. The included six genera, namely Leclercia, Raoultella, Kosakonia, Lelliottia, Yokenella, and Kluyvera, that were not previously known to reduce or exhibit resistance to arsenic. Twenty-one of 24 aARB were positive for ars amplification and 17 of them harbored a putative arsC gene, which is well-known for its involvement in arsenate reduction. However, the arsenic resistance associated with aARB strains is not always determined by the ars operon system. These results have provided additional insight into aARB and their potential for arsenic transformation and bioremediation.

Genome analysis of a thermophilic exopolysaccharide-producing bacterium - Geobacillus sp. WSUCF1

Geobacillus sp. WSUCF1 is a Gram-positive, spore-forming, aerobic and thermophilic bacterium, isolated from a soil sample obtained from a compost facility. Strain WSUCF1 demonstrated EPS producing capability using different sugars as the carbon source. The whole-genome analysis of WSUCF1 was performed to disclose the essential genes correlated with nucleotide sugar precursor biosynthesis, assembly of monosaccharide units, export of the polysaccharide chain, and regulation of EPS production. Both the biosynthesis pathway and export mechanism of EPS were proposed based on functional annotation. Additionally, the genome description of strain WSUCF1 suggests sophisticated systems for its adaptation under thermophilic conditions. The presence of genes associated with CRISPR-Cas system, quorum quenching lactonase, polyketide synthesis and arsenic resistance makes this strain a potential candidate for various applications in biotechnology and biomedicine. The present study indicates that strain WSUCF1 has promise as a thermophilic EPS producer for a broad range of industrial applications. To the best of our knowledge, this is the first report on genome analysis of a thermophilic Geobacillus species focusing on its EPS biosynthesis and transportation, which will likely pave the way for both enhanced yield and tailor-made EPS production by thermophilic bacteria.

The Complete Genome and Physiological Analysis of the Eurythermal Firmicute Exiguobacterium chiriqhucha Strain RW2 Isolated From a Freshwater Microbialite, Widely Adaptable to Broad Thermal, pH, and Salinity Ranges.

Members of the genus Exiguobacterium are found in diverse environments from marine, freshwaters, permafrost to hot springs. Exiguobacterium can grow in a wide range of temperature, pH, salinity, and heavy-metal concentrations. We characterized Exiguobacterium chiriqhucha strain RW2 isolated from a permanently cold freshwater microbialite in Pavilion Lake, British Columbia using metabolic assays, genomics, comparative genomics, phylogenetics, and fatty acid composition. Strain RW2 has the most extensive growth range for temperature (4–50°C) and pH (5–11) of known Exiguobacterium isolates. Strain RW2 genome predicts pathways for wide differential thermal, cold and osmotic stress using cold and heat shock cascades (e.g., csp and dnaK), choline and betaine uptake/biosynthesis (e.g., opu and proU), antiporters (e.g., arcD and nhaC Na+/K+), membrane fatty acid unsaturation and saturation. Here, we provide the first complete genome from Exiguobacterium chiriqhucha strain RW2, which was isolated from a freshwater microbialite. Its genome consists of a single 3,019,018 bp circular chromosome encoding over 3,000 predicted proteins, with a GC% content of 52.1%, and no plasmids. In addition to growing at a wide range of temperatures and salinities, our findings indicate that RW2 is resistant to sulfisoxazole and has the genomic potential for detoxification of heavy metals (via mercuric reductases, arsenic resistance pumps, chromate transporters, and cadmium-cobalt-zinc resistance genes), which may contribute to the metabolic potential of Pavilion Lake microbialites. Strain RW2 could also contribute to microbialite formation, as it is a robust biofilm former and encodes genes involved in the deamination of amino acids to ammonia (i.e., L-asparaginase/urease), which could potentially boost carbonate precipitation by lowering the local pH and increasing alkalinity. We also used comparative genomic analysis to predict the pathway for orange pigmentation that is conserved across the entire Exiguobacterium genus, specifically, a C30 carotenoid biosynthesis pathway is predicted to yield diaponeurosporene-4-oic acid as its final product. Carotenoids have been found to protect against ultraviolet radiation by quenching reactive oxygen, releasing excessive light energy, radical scavenging, and sunscreening. Together these results provide further insight into the potential of Exiguobacterium to exploit a wide range of environmental conditions, its potential roles in ecosystems (e.g., microbialites/microbial mats), and a blueprint model for diverse metabolic processes.

Arsenic Disulfide Combined with L-Buthionine-(S, R)-Sulfoximine Induces Synergistic Antitumor Effects in Two-Dimensional and Three-Dimensional Models of MCF-7 Breast Carcinoma Cells.

Three-dimensionally (3D) cultured tumor cells (spheroids) exhibit more resistance to therapeutic agents than the cells cultured in traditional two-dimensional (2D) system (monolayers). We previously demonstrated that arsenic disulfide (As2 exerted significant anticancer efficacies in both 2D- and 3D-cultured MCF-7 cells, whereas 3D spheroids were shown to be resistant to the As2S2 treatment. L-buthionine-(S, R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, has been regarded to be a potent candidate for combinatorial treatment due to its GSH modulation function. In the present study, we introduced BSO in combination with As2S2 at a low concentration to investigate the possible enhancing anticancer efficacy by the combinatorial treatment on 2D- and 3D-cultured MCF-7 cells. Our results presented for the first time that the combination of As2S2 and BSO exerted potent anticancer synergism in both MCF-7 monolayers and spheroids. The values of As2S2 in combinatorial treatment were significantly lower than those in treatment of As2S2 alone in both 2D- and 3D-cultured MCF-7 cells (, respectively). In addition, augmented induction of apoptosis and enhanced cell cycle arrest along with the regulation of apoptosis- and cell cycle-related proteins, as well as synergistic inhibitions of PI3K/Akt signals, were also observed following co-treatment of As2S2 and BSO. Notably, the combinatorial treatment significantly decreased the cellular GSH levels in both 2D- and 3D-cultured MCF-7 cells in comparison with each agent alone ( in each). Our results suggest that the combinatorial treatment with As2S2 and BSO could be a promising novel strategy to reverse arsenic resistance in human breast cancer.

Arsenic Resistance and Biosorption by Isolated Rhizobacteria from the Roots of Ludwigia octovalvis

Certain rhizobacteria can be applied to remove arsenic in the environment through bioremediation or phytoremediation. This study determines the minimum inhibitory concentration (MIC) of arsenic on identified rhizobacteria that were isolated from the roots of Ludwigia octovalvis (Jacq.) Raven. The arsenic biosorption capability of the was also analyzed. Among the 10 isolated rhizobacteria, five were Gram-positive (Arthrobacter globiformis, Bacillus megaterium, Bacillus cereus, Bacillus pumilus, and Staphylococcus lentus), and five were Gram-negative (Enterobacter asburiae, Sphingomonas paucimobilis, Pantoea spp., Rhizobium rhizogenes, and Rhizobium radiobacter). R. radiobacter showed the highest MIC of >1,500 mg/L of arsenic. All the rhizobacteria were capable of absorbing arsenic, and S. paucimobilis showed the highest arsenic biosorption capability (146.4 ± 23.4 mg/g dry cell weight). Kinetic rate analysis showed that B. cereus followed the pore diffusion model (R2 = 0.86), E. asburiae followed the pseudo-first-order kinetic model (R2 = 0.99), and R. rhizogenes followed the pseudo-second-order kinetic model (R2 = 0.93). The identified rhizobacteria differ in their mechanism of arsenic biosorption, arsenic biosorption capability, and kinetic models in arsenic biosorption.

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)].

Distribution of Arsenic Resistance Genes in Prokaryotes.

Arsenic is a metalloid that occurs naturally in aquatic and terrestrial environments. The high toxicity of arsenic derivatives converts this element in a serious problem of public health worldwide. There is a global arsenic geocycle in which microbes play a relevant role. Ancient exposure to arsenic derivatives, both inorganic and organic, has represented a selective pressure for microbes to evolve or acquire diverse arsenic resistance genetic systems. In addition, arsenic compounds appear to have been used as a toxin in chemical warfare for a long time selecting for an extended range of arsenic resistance determinants. Arsenic resistance strategies rely mainly on membrane transport pathways that extrude the toxic compounds from the cell cytoplasm. The ars operons, first discovered in bacterial R-factors almost 50 years ago, are the most common microbial arsenic resistance systems. Numerous ars operons, with a variety of genes and different combinations of them, populate the prokaryotic genomes, including their accessory plasmids, transposons, and genomic islands. Besides these canonical, widespread ars gene clusters, which confer resistance to the inorganic forms of arsenic, additional genes have been discovered recently, which broadens the spectrum of arsenic tolerance by detoxifying organic arsenic derivatives often used as toxins. This review summarizes the presence, distribution, organization, and redundance of arsenic resistance genes in prokaryotes.

The whole genome insight on condition-specific redox activity and arsenopyrite interaction promoting As-mobilization by strain Lysinibacillus sp. B2A1.

A gram-positive spore former, Lysinibacillus sp. B2A1 was isolated from a high arsenic containing groundwater of Beimen2A well, Chianan Plain area, Southwestern Taiwan. Noteworthy, in the subsurface-mimicking anoxic incubation with a Na-lactate amendment system, this isolate could interact with arsenic-source mineral arsenopyrite and enhance arsenic mobilization. Further, the isolate showed elevated levels of arsenic resistance, 200 mM and 7.5 mM for arsenate and arsenite, respectively. Lysinibacillus sp. B2A1 demonstrated condition-specific redox activities including salient oxic oxidation of arsenite and anoxic reduction of arsenate. The elevated rate of As(III) oxidation (Vmax = 0.13 μM min-1 per 106 cells, Km = 15.3 μM) under oxic conditions was potent. Correlating with stout persistence in an arsenic-rich niche, remarkably, the lesser toxic effects of arsenic ions on bacterial sporulation frequency and germination highlight this strain's ability to thrive under catastrophic conditions. Moreover, the whole genome analysis elucidated diverse metal redox/resistance genes that included a potential arsenite S-adenosylmethyltransferase capable of mitigating arsenite toxicity. Owing to its arsenic resistance, conditional redox activities and ability to interact with arsenic minerals leading to arsenic mobilization, the presence of such spore-forming strains could be a decisive indication towards arsenic mobilization in subsurface aquifers having a high concentration of soluble arsenic or its source minerals.

Ars2 promotes cell proliferation and tumorigenicity in glioblastoma through regulating miR-6798-3p

Arsenic resistance protein 2 (Ars2) is a component of the nuclear RNA cap-binding complex (CBC) that is important for some microRNA biogenesis and it is critical for cell proliferation and tumorigenicity. However, mechanism of Ars2-regulated cellular proliferation and tumorigenicity in glioblastoma has not been fully understood. Western blotting was used to detect the expressions of Ars2, p53, p21, and cleavage/activation of caspases-3 (C-Caspase 3). Microarray and Quantitative Real-time PCR (qRT-PCR) were performed to identify the Ars2-regulated microRNAs. Apoptosis assessed by flow cytometry analysis was used to evaluate the role of Ars2 in cells proliferation. The lentivirus-mediated gene knockdown approach was conducted to determine the function of Ars2. The orthotopic glioblastoma xenograft was used to demonstrate the role of Ars2 in glioblastoma growth in vivo. The high expression of Ars2 was observed in several glioblastoma cell lines and was significantly associated with poorer overall survival. Importantly, the overexpression of Ars2 promoted cell proliferation and colony formation in glioblastoma cells, whereas the depletion of Ars2 inhibited cell proliferation, colony formation, and tumor growth. Mechanistic study revealed that knockdown of Ars2 reduced the expression levels of miR-6798-3p, which was responsible for the up-regulation of p53 and p21, leading to apoptosis. Furthermore, the knockdown of Ars2 suppressed tumor growth in orthotopic glioblastoma xenograft model and significantly prolonged the survival time of the tumor-bearing mice. These findings identify a critical role for Ars2 in regulation of proliferation and tumorigenicity in glioblastoma and suggest that Ars2 could be a critical therapeutic target for glioblastoma intervention.

Genetic mechanisms of arsenic detoxification and metabolism in bacteria.

Arsenic, distributed pervasively in the natural environment, is an extremely toxic substance which can severely impair the normal functions of living cells. Research on the genetic mechanisms of arsenic metabolism is of great importance for remediating arsenic-contaminated environments. Many organisms, including bacteria, have developed various strategies to tolerate arsenic, by either detoxifying this harmful element or utilizing it for energy generation. This review summarizes arsenic detoxification as well as arsenic respiratory metabolic pathways in bacteria and discusses novel arsenic resistance pathways in various bacterial strains. This knowledge provides insights into the mechanisms of arsenic biotransformation in bacteria. Multiple detoxification strategies among bacteria imply possible functional relationships among different arsenic detoxification/metabolism pathways. In addition, this review sheds light on the bioremediation of arsenic-contaminated environments and prevention of antibiotic resistance.

Knockdown of arsenic resistance protein 2 inhibits human glioblastoma cell proliferation through the MAPK/ERK pathway.

It is generally known that glioblastoma is the most common primary malignant brain tumor and that it is highly aggressive and deadly. Although surgical and pharmacological therapies have made long-term progress, glioblastoma remains extremely lethal and has an uncommonly low survival rate. Therefore, further elucidation of the molecular mechanisms of glioblastoma initiation and its pathological processes are urgent. Arsenic resistance protein 2 (Ars2) is a highly conserved gene, and it has been found to play an important role in microRNA biosynthesis and cell proliferation in recent years. Furthermore, absence of Ars2 results in developmental death in Drosophila, zebrafish and mice. However, there are few studies on the role of Ars2 in regulating tumor development, and the mechanism of its action is mostly unknown. In the present study, we revealed that Ars2 is involved in glioblastoma proliferation and we identified a potential mechanistic role for it in cell cycle control. Our data demonstrated that Ars2 knockdown significantly repressed the proliferation and tumorigenesis abilities of glioblastoma cells in vitro and in vivo. Further investigation clarified that Ars2 deficiency inhibited the activation of the MAPK/ERK pathway, leading to cell cycle arrest in the G1 phase, resulting in suppression of cell proliferation. These findings support the conclusion that Ars2 is a key regulator of glioblastoma progression.

Analysis of the ars gene cluster from highly arsenic-resistant Burkholderia xenovorans LB400.

The Burkholderia xenovorans LB400 multireplicon genome displays a relatively high proportion of redundant genes, including several genes predicted to be related to arsenic resistance. These comprise an ars gene cluster, composed of the arsR3, acr3, arsC1 and arsH genes, as well as two arsB, arsC2, and seven individual arsR genes. The objective of this work was to elucidate the involvement of the ars gene cluster in arsenic resistance by the LB400 strain. Susceptibility tests showed that B. xenovorans LB400 is highly resistant to arsenate and arsenite. Arsenic resistance was induced by prior exposure of LB400 to arsenate or arsenite. reverse transcription-polymerase chain reaction assays using total RNA from LB400 showed arsenite-induced transcription of the arsR3 gene, suggesting that the ars gene cluster constitutes an arsenite-responsive operon. Transfer of cloned LB400 ars genes to heterologous Escherichia coli or Pseudomonas aeruginosa strains demonstrated that the ArsR3 transcriptional repressor, ArsC1 arsenate reductase, and the Acr3 arsenite efflux pump encoded in the LB400 ars gene cluster, are all associated to the arsenic resistance phenotype of this strain. The ars gene cluster from Burkholderia xenovorans LB400 is responsible for the inducible arsenic-resistance phenotype of the bacterium.

The role of submerged macrophytes in phytoremediation of arsenic from contaminated water: A case study on Vallisneria natans (Lour.) Hara

Arsenic contamination of water is a global concern due to its heavy threat to human health. In this study, the submerged macrophyte Vallisneria natans (Lour.) Hara was used to remove environmentally relevant concentrations of arsenic in the binary As(III)/As(V) system. The concentrations of total arsenic (tAs) and As(III) in water dropped rapidly within 3 days, while As(V) first increased slightly within 3 days and then gradually decreased. About 1.2% dimethylarsinate (DMA) was detected at the 14th day of treatment. These findings indicated that As(III) could be oxidized to As(V) and methylated to DMA in water with V. natans. In relation to V. natans, both tAs and As(V) were much higher in roots compared to leaves. Arsenate was the predominant species (≥ 95.65 ± 0.10%) in roots, and As(III) was only found at the 14th day (3.45–6.96 mg kg–1). In leaves, As(III) significantly increased (P < 0.05) as the treatment duration increased. The proportions of As(V) (27.99–40.03%) were lower than those of As(III) and arsenobetaine (AsB) was detected (0.52–1.87 mg kg–1) after 7 d. The results of arsenic speciation demonstrated that the transformation of arsenic species in V. natans included As(V) reduction and As(III) methylation to AsB. There were a decrease in chlorophyll content, and an increase in MDA level and antioxidant enzymes (SOD, CAT, and POD) activities. The MDA level was much higher in leaves than roots, whereas the activities of SOD, CAT, and POD were the opposite, suggesting their possible role in arsenic resistance and detoxification. Our results indicate the potential of V. natans in phytoremediation of arsenic-contaminated water.

Exposure to Arsenite in CD-1 Mice during Juvenile and Adult Stages: Effects on Intestinal Microbiota and Gut-Associated Immune Status.

ABSTRACTIntestinal microbiota composition and gut-associated immune response can contribute to the toxicity of arsenic. We investigated the potential toxicity of short-term arsenic exposure on gut microbiome composition, intestinal immune status, microbial arsenic resistance gene, and arsenic metabolic profiles in adult and developmental stages of CD-1 mice. The potential toxicity of arsenite [As(III)] was determined for two life stages: (i) adult animals at 24 or 48 h after single gavage (0.05 mg/kg body weight [b.w.] [low dose], 0.1 mg/kg b.w. [medium dose], and 0.2 mg/kg b.w. [high dose]) and repeated exposure at 1 mg/liter for 8 days and (ii) postnatal day 10 (PND10) and PND21 after single gavage (0.05 mg/kg b.w.). Dose- and time-dependent responses in bacterial recovery/microbial composition were observed in adults after a single gavage. Repeated exposure caused a transient decrease in the recovery of intestinal bacteria, a shift in the bacterial population with abundance of arsenic resistance genes, and evidence for host metabolism of arsenite into less-reactive trivalent methylated species. Arsenic exposure in adult animals induced high levels of CC chemokines and of proinflammatory and anti-inflammatory cytokine secretion in intestine. Arsenic exposure at PND21 resulted in the development of distinct bacterial populations. Results of this study highlight significant changes in the intestinal microbiome and gut-associated immune status during a single or repeated exposure to arsenic in juvenile and adult animals. The data warrant investigation of the long-term effects of oral arsenic exposure on the microbiome and of immune system development and responses.

ABCA1 Is Coordinated with ABCB1 in the Arsenic-Resistance of Human Cells.

Arsenic is one of the most widespread global environmental toxicants associated with endemic poisoning. ATP-binding cassette (ABC) proteins are transmembrane channels that transport and dispose of lipids and metabolic products across the plasma membrane. The majority of ABC family members (including ABCB1 and ABCC1) are reported to play a role in the development of arsenic and drug resistance in mammals. Previously, we established a human arsenic-resistant ECV-304 (AsRE) cell line and identified ABCA1 as a novel arsenic resistance gene. In the current study, we further investigated the potential contribution of ABCA1, ABCB1, and ABCC1 to arsenic resistance through measurement of survival rates and arsenic accumulation in AsRE cells with RNA interference. The arsenic resistance capacity of ABCC1 was the strongest among the three genes, while those of ABCA1 and ABCB1 were similar. Double or triple gene knockdown of ABCA1, ABCB1, and ABCC1 via RNA interference led to a decrease significant in arsenic resistance when ABCA1/ABCB1 or ABCB1/ABCC1 were simultaneously silenced. Interestingly, no differences were evident between cells with ABCA1/ABCC1 and ABCC1 only knockdown. Our findings suggest that ABCA1 and ABCB1 proteins display similar arsenic resistance capabilities and possibly coordinate to promote arsenic resistance in AsRE cells.

Functional annotation of hypothetical proteins from the Exiguobacterium antarcticum strain B7 reveals proteins involved in adaptation to extreme environments, including high arsenic resistance.

Exiguobacterium antarcticum strain B7 is a psychrophilic Gram-positive bacterium that possesses enzymes that can be used for several biotechnological applications. However, many proteins from its genome are considered hypothetical proteins (HPs). These functionally unknown proteins may indicate important functions regarding the biological role of this bacterium, and the use of bioinformatics tools can assist in the biological understanding of this organism through functional annotation analysis. Thus, our study aimed to assign functions to proteins previously described as HPs, present in the genome of E. antarcticum B7. We used an extensive in silico workflow combining several bioinformatics tools for function annotation, sub-cellular localization and physicochemical characterization, three-dimensional structure determination, and protein-protein interactions. This genome contains 2772 genes, of which 765 CDS were annotated as HPs. The amino acid sequences of all HPs were submitted to our workflow and we successfully attributed function to 132 HPs. We identified 11 proteins that play important roles in the mechanisms of adaptation to adverse environments, such as flagellar biosynthesis, biofilm formation, carotenoids biosynthesis, and others. In addition, three predicted HPs are possibly related to arsenic tolerance. Through an in vitro assay, we verified that E. antarcticum B7 can grow at high concentrations of this metal. The approach used was important to precisely assign function to proteins from diverse classes and to infer relationships with proteins with functions already described in the literature. This approach aims to produce a better understanding of the mechanism by which this bacterium adapts to extreme environments and to the finding of targets with biotechnological interest.

Characterization of arsenite-oxidizing bacteria to decipher their role in arsenic bioremediation

High arsenic groundwater contamination causes serious health risks in many developing countries, particularly in India and Bangladesh. The arsenic fluxes in aquifers are primarily controlled by bacterial populations through biogeochemical cycle. In this present study, two gram-positive rod-shaped bacteria were isolated from shallow aquifers of Bhojpur district in Bihar during the early winter season, able to withstand arsenite (As3+) concentration upto 70 mM and 1000 mM of arsenate (As5+) concentration. They showed high resistance to heavy metals up to 30 mM and utilized some complex sugars along with different carbon sources. Growth at wide range of temperature, pH and salinity were observed. Both these isolates showed high efficiency in converting As3+ into less toxic concentrations of As5+ respectively from arsenic enriched culture media. Along with superior arsenic transformation and arsenic resistance abilities, the isolates showed a wide variety of metabolic capacity in terms of utilizing a variety of carbon sources under aerobic conditions, respectively. This study reports the potential As3+-oxidizing bacteria that can play an important role in subsurface arsenic transformation that will aid in designing future bioremediation strategy for the arsenic affected areas.

Differential protein expression in a marine-derived Staphylococcus sp. NIOSBK35 in response to arsenic(III).

Peptide mass fingerprinting of Gram-positive marine-derived Staphylococcus cohnii #NIOSBK35 gave us an insight into the proteins involved in conferring arsenic resistance as well as the probable metabolic pathways affected under metal stress. Analysis of the protein profiles obtained from LC/MS QToF (Liquid Chromatography-Mass Spectrometry-Quadrupole Time of Flight) resulted in the identification of 689 proteins. Further grouping of these proteins based on the arsenic concentration (0, 250, 500 and 850ppm) and the time points (6, 9, 12, 18, 24 and 36h) in growth phase showed that a total of 13 proteins were up-regulated, while 178 proteins were down-regulated across all the concentrations and time points. Arsenic specific proteins like arsenical pump-driving ATPase, ArsR family transcriptional regulator and arsenic operon resistance repressor were found to be highly up-regulated throughout all the conditions indicating their possible involvement in the tolerance to arsenic. MBL fold metallo-hydrolase, a known stress protein, was the only protein that was up-regulated at all time points across all arsenic concentrations. Metabolic pathways like translation, carbohydrate metabolism, amino acid metabolism, membrane transport, metabolism of cofactors and vitamins, replication and repair, nucleotide metabolism along with stress proteins and hypothetical proteins were found to be significantly expressed. Our results also suggest that arsenic stress at higher levels is negatively affecting the expression of many normal functional proteins required for cell survival.