Identification of genes and proteins involved in the pleiotropic response to arsenic stress in Caenibacter arsenoxydans, a metalloresistant beta-proteobacterium with an unsequenced genome

The effect of high concentrations of arsenic has been investigated in Caenibacter arsenoxydans, a beta-proteobacterium isolated from an arsenic contaminated environment and able to oxidize arsenite to arsenate. As the genome of this bacterium has not yet been sequenced, the use of a specific proteomic approach based on nano-high performance liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) studies and de novo sequencing to perform cross-species protein identifications was necessary. In addition, a random mutational analysis was performed. Twenty-two proteins and 16 genes were shown to be differentially accumulated and expressed, respectively, in cells grown in the presence of arsenite. Two genes involved in arsenite oxidation and one in arsenite efflux as well as two proteins responsible for arsenate reduction were identified. Moreover, numerous genes and proteins belonging to various functional classes including information and regulation pathways, intermediary metabolism, cell envelope and cellular processes were also up- or down-regulated, which demonstrates that bacterial response to arsenic is pleiotropic.     

Arsenic toxicity is not due to a direct effect on the oxidation of reduced inorganic sulfur compounds by Thiobacillus caldus

Abstract Thiobacillus caldus is a moderately thermophilic acidophile which has been implicated in the biooxidation of arsenic containing mineral Sulfides. The toxic effects of arsenic on this bacterium are presented here. Addition of arsenite to a growing culture of T. caldus caused a transient increase in the optical density of the culture while causing a simultaneous decrease in cell viability. The increase in optical density was shown to be due to the formation of extracellular sulfur. The oxidation rates of tetrathionate and thiosulfate were decreased by increasing concentrations of arsenite, while in a culture induced to arsenic resistance the rates were not as adversely effected. Sulfur oxidation was also inhibited to the same extent as tetrathionate oxidation, with the oxidation of solid sulfur being slightly more effected than the oxidation of sulfur dissolved in acetone. Thus, bactericidal arsenite causes a transient formation of extracellular sulfur in the culture supernatant of T. caldus yet the toxicity of arsenite is not due to direct inhibitory effects on reduced inorganic sulfur compound oxidation by these bacteria.     

Brevibacterium sp. strain CS2: A potential candidate for arsenic bioremediation from industrial wastewater

A multiple metal-resistant Brevibacterium sp. strain CS2, isolated from an industrial wastewater, resisted arsenate and arsenate upto 280 and 40 mM. The order of resistance against multiple metals was Arsenate > Arsenite > Selenium = Cobalt > Lead = Nickel > Cadmium = Chromium = Mercury. The bacterium was characterized as per morphological and biochemical characteristics at optimum conditions (37 ℃ and 7 pH). The appearance of brownish color precipitation was due to the interaction of silver nitrate confirming its oxidizing ability against arsenic. The strain showed arsenic processing ability at different temperatures, pH, and initial arsenic concentration which was 37% after 72 h and 48% after 96 h of incubation at optimum conditions with arsenite 250 mM/L (initial arsenic concentration). The maximum arsenic removal ability of strain CS2 was determined for 8 days, which was 32 and 46% in wastewater and distilled water, respectively. The heat-inactivated cells of the isolated strain showed a bioremediation efficiency (E) of 96% after 10 h. Genes cluster (9.6 kb) related to arsenite oxidation was found in Brevibacterium sp. strain CS2 after the genome analysis of isolated bacteria through illumine and nanopore sequencing technology. The arsenite oxidizing gene smaller subunit (aioB) on chromosomal DNA locus (Prokka_01508) was identified which plays a role in arsenite oxidation for energy metabolism. The presence of arsenic oxidizing genes and an efficient arsenic oxidizing potential of Brevibacterium sp. strain CS2 make it a potential candidate for green chemistry to eradicate arsenic from arsenic-contaminated wastewater.     

Oxidation of Arsenite by a Soil Isolate of Alcaligenes

A strain of Alcaligenes , isolated from soil and grown in nutrient broth in the presence of arsenite, possessed the ability to oxidize arsenite to arsenate. Washed cell suspensions consumed one-half mol of oxygen/mol of arsenite and produced arsenate. The optimum pH for arsenite oxidation was 7.0. The K_m for arsenite was 1.5 × 10^-4 M and V _max was 6.7 μl of oxygen/min. The arsenite-oxidizing enzyme system was induced by growth in arsenite. Response of the arsenite-oxidizing enzyme system to respiratory inhibitors suggested that electrons resulting from arsenite oxidation by an oxido-reductase with a bound flavin are transferred via cytochrome c and cytochrome oxidase to oxygen. The presence of the cytochromes in crude extract was confirmed by spectral measurements.     

Characterization of arsenic resistant and arsenopyrite oxidizing Acidithiobacillus ferrooxidans from Hutti gold leachate and effluents

Four arsenic resistant ferrous oxidizers were isolated from Hutti Gold Mine Ltd. (HGML) samples. Characterization of these isolates was done using conventional microbiological, biochemical and molecular methods. The ferrous oxidation rates with these isolates were 16, 48, 34 and 34 mg L(-1)h(-1) and 15, 47, 34 and 32 mg L(-1)h(-1) in absence and presence of 20 mM of arsenite (As3+) respectively. Except isolate HGM 8, other three isolates showed 2.9-6.3% inhibition due to the presence of 20 mM arsenite. Isolate HGM 8 was able to grow in presence of 14.7 g L(-1) of arsenite, with 25.77 mg L(-1)h(-1) ferrous oxidation rate. All the four isolates were able to oxidize iron and arsenopyrite from 20 g L(-1) and 40 g L(-1) refractory gold ore and 20 g L(-1) refractory gold concentrate. Once the growth was established pH adjustment was not needed inspite of ferrous oxidation, which could be due to concurrent oxidation of pyrite. Isolate HGM 8 showed the final cell count of as high as 1.12 x 10(8) cells mL(-1) in 40 g L(-1) refractory gold ore. The isolates were grouped into one haplotypes by amplified ribosomal DNA restriction analysis (ARDRA). The phylogenetic position of HGM 8 was determined by 16S rDNA sequencing. It was identified as Acidithiobacillus ferrooxidans and strain name was given as SRHGM 1.     

Glucose consumption and dehydrogenase activity of the cells of the arsenite-oxidizing bacterium Pseudomonas putida

The rates of glucose assimilation and dehydrogenase activity were studied in Pseudomonas putida oxidizing arsenite. The rate of glucose utilization by the cells decreased in the presence of arsenites in the medium at the beginning because of the microbial adaptation to arsenite. The activity of dehydrogenase fell down when the cells were cultivated in the medium with arsenite. An inverse correlation existed between the rate of glucose assimilation and arsenite oxidation. Apparently, arsenites were oxidized under the action of metabolites produced by P. putida in the process of its heterotrophous growth.     

On-line reversed-phase liquid chromatography hydride generation emission spectrometry: speciation of arsenic in urine of patients intravenously treated with As2O3

Hydride generation inductively coupled plasma–atomic emission spectrometry (HG ICP–AES) was used as a continuous detection system for the determination of arsenic in the eluate from a high-performance liquid chromatographic (HPLC) system. Four arsenic species [arsenite As(III), arsenate As(V), monomethylarsonate (MMA), and dimethylarsinate (DMA)] present in the urine samples of patients treated intravenously with arsenite, were analyzed separately by HPLC–HG-ICP–AES using a non-polar C₁₈ column. This analytical method allowed the sensitive determination of the arsenic species in the submicrogram per liter range. Urine samples collected on different days after arsenite administration were found to contain arsenite predominantly – monomethylarsonate and dimethylarsinate were also detected.     

The effects of arsenate and arsenite on the growth and morphology of the marine unicellular algae Tetraselmis chui (Chlorophyta) and Hymenomonas carterae (Chrysophyta)

The marine phytoplanktonic algae, Tetraselmis chui Stein and Hymenomonas carterae (Braarud and Fagerland) Braarud, were grown in media containing various concentrations of arsenate or arsenite. The effects of arsenic on the algae varied with the oxidation state of the element, its concentration, and the degree of illumination. Arsenate affected mainly algal growth but also cell morphology, whereas arsenite caused only morphological changes. Studies on the incorporation of ⁷⁴As-arsenate into cells grown in artificial sea water indicated that arsenate was incorporated and later partially released by both T. chui and H. carterae. Both arsenate influx and efflux seemed to be energy-dependent phenomena, because they varied with the degree of illumination. Differences between the rates of uptake and release of arsenic suggested that arsenate undergoes chemical changes after having been transported into the algal cells.     

Influence of sulphur on arsenic accumulation and metabolism in rice seedlings

The influence of sulphur on the accumulation and metabolism of arsenic in rice was investigated. Rice seedlings were grown in nutrient solutions with low sulphate (1.8 μM SO₄²⁻) or high sulphate (0.7 mM SO₄²⁻) for 12 or 14 d, before being exposed to 10 μM arsenite or arsenate for 2 or 1 d, respectively. In the arsenite exposure treatment, low sulphate-pretreated rice accumulated less arsenite than high sulphate pretreated plants, but the arsenite concentrations in shoots of low sulphate pretreated rice were higher than those of high sulphate pretreated. In the arsenate exposure treatment, the low sulphate pre-treatments also resulted in less arsenite accumulation in rice roots. Sulphur deprivation in nutrient solution decreased the concentrations of non-protein thiols in rice roots exposed to either arsenite or arsenate. The low sulphate-pretreated plants had a higher arsenic transfer factor than the high sulphate-pretreated plants. The results suggest that rice sulphate nutrition plays an important role in regulating arsenic translocation from roots to shoots, possibly through the complexation of arsenite-phytochelatins.     

Selenate-dependent anaerobic arsenite oxidation by a bacterium from Mono Lake, California

Arsenate was produced when anoxic Mono Lake water samples were amended with arsenite and either selenate or nitrate. Arsenite oxidation did not occur in killed control samples or live samples with no added terminal electron acceptor. Potential rates of anaerobic arsenite oxidation with selenate were comparable to those with nitrate ( approximately 12 to 15 mumol.liter(-1) h(-1)). A pure culture capable of selenate-dependent anaerobic arsenite oxidation (strain ML-SRAO) was isolated from Mono Lake water into a defined salts medium with selenate, arsenite, and yeast extract. This strain does not grow chemoautotrophically, but it catalyzes the oxidation of arsenite during growth on an organic carbon source with selenate. No arsenate was produced in pure cultures amended with arsenite and nitrate or oxygen, indicating that the process is selenate dependent. Experiments with washed cells in mineral medium demonstrated that the oxidation of arsenite is tightly coupled to the reduction of selenate. Strain ML-SRAO grows optimally on lactate with selenate or arsenate as the electron acceptor. The amino acid sequences deduced from the respiratory arsenate reductase gene (arrA) from strain ML-SRAO are highly similar (89 to 94%) to those from two previously isolated Mono Lake arsenate reducers. The 16S rRNA gene sequence of strain ML-SRAO places it within the Bacillus RNA group 6 of gram-positive bacteria having low G+C content.     

Arsenate, arsenite and dimethyl arsinic acid (DMA) uptake and tolerance in maize (Zea mays L.)

The influx of arsenate, arsenite and dimethyl arsinic acid (DMA) were studied in 7-day-old excised maize roots (Zea mays L.), and then related to arsenate, arsenite and DMA toxicity. Arsenate, arsenite and DMA influx was all found concentration dependent with significant genotypic differences for arsenite and DMA. Arsenate influx in phosphate starved plants best fitted the four-parameter Michaelis–Menten model corresponding to an additive high and low affinity uptake system, while the uptake of phosphate replete plants followed the two parameter model of Michaelis–Menten kinetics. Arsenite influx was well described by the two parameter model of ‘Michaelis–Menten’ kinetics. DMA influx was comprised of linear phase and a hyperbolic phase. DMA influx was much lower than that for arsenite and arsenate. Arsenate and DMA influx decreased when phosphate was given as a pre-treatment as opposed to phosphate starved plants. The +P treatment tended to decrease influx by 50% for arsenate while this figure was 90% for DMA. Arsenite influx increasing slightly at higher arsenite concentrations in P starved plants but at lower arsenite concentrations, there was little or no difference in arsenite uptake. Low toxicity was found for DMA on maize compared with arsenate and arsenite and the relative toxicity of arsenic species was As(V) > As(III) >> DMA.     

Biological characterization of Bacillus flexus strain SSAI1 transforming highly toxic arsenite to less toxic arsenate mediated by periplasmic arsenite oxidase enzyme encoded by aioAB genes

Bacillus flexus strain SSAI1 isolated from agro-industry waste, Tuem, Goa, India displayed high arsenite resistance as minimal inhibitory concentration was 25 mM in mineral salts medium. This bacterial strain exposed to 10 mM arsenite demonstrated rapid arsenite oxidation and internalization of 7 mM arsenate within 24 h. The Fourier transformed infrared (FTIR) spectroscopy of cells exposed to arsenite revealed important functional groups on the cell surface interacting with arsenite. Furthermore, scanning electron microscopy combined with electron dispersive X-ray spectroscopy (SEM-EDAX) of cells exposed to arsenite revealed clumping of cells with no surface adsorption of arsenite. Transmission electron microscopy coupled with electron dispersive X-ray spectroscopic (TEM-EDAX) analysis of arsenite exposed cells clearly demonstrated ultra-structural changes and intracellular accumulation of arsenic. Whole-genome sequence analysis of this bacterial strain interestingly revealed the presence of large number of metal(loid) resistance genes, including aioAB genes encoding arsenite oxidase responsible for the oxidation of highly toxic arsenite to less toxic arsenate. Enzyme assay further confirmed that arsenite oxidase is a periplasmic enzyme. The genome of strain SSAI1 also carried glpF, aioS and aioE genes conferring resistance to arsenite. Therefore, multi-metal(loid) resistant arsenite oxidizing Bacillus flexus strain SSAI1 has potential to bioremediate arsenite contaminated environmental sites and is the first report of its kind.

     

Comparative proteomic study of arsenic-induced differentially expressed proteins in rice roots reveals glutathione plays a central role during As stress

While the phytotoxic responses of arsenic (As) on plants have been studied extensively, based on physiological and biochemical aspects, very little is known about As stress-elicited changes in plants at the proteome level. Hydroponically grown 2-wk-old rice seedlings were exposed to different doses of arsenate, and roots were collected after 4 days of treatment, as well as after a recovery period. To gain a comprehensive understanding of the precise mechanisms underlying As toxicity, metabolism, and the defense reactions in plants, a comparative proteomic analysis of rice roots has been conducted in combination with physiological and biochemical analyses. Arsenic treatment resulted in increases of As accumulation, lipid peroxidation, and in vivo H(2)O(2) contents in roots. A total of 23 As-regulated proteins including predicted and novel ones were identified using 2-DE coupled with MS analyses. The expression levels of S-adenosylmethionine synthetase (SAMS), GSTs, cysteine synthase (CS), GST-tau, and tyrosine-specific protein phosphatase proteins (TSPP) were markedly up-regulated in response to arsenate, whereas treatment by H(2)O(2) also regulated the levels of CS suggesting that its expression was certainly regulated by As or As-induced oxidative stress. In addition, an omega domain containing GST was induced only by arsenate. However, it was not altered by treatment of arsenite, copper, or aluminum, suggesting that it may play a particular role in arsenate stress. Analysis of the total glutathione (GSH) content and enzymatic activity of glutathione reductase (GR) in rice roots during As stress revealed that their activities respond in a dose-dependent manner of As. These results suggest that SAMS, CS, GSTs, and GR presumably work synchronously wherein GSH plays a central role in protecting cells against As stress.     

Uptake and toxicity of arsenite and arsenate in cultured brain astrocytes

Inorganic arsenicals are environmental toxins that have been connected with neuropathies and impaired cognitive functions. To investigate whether such substances accumulate in brain astrocytes and affect their viability and glutathione metabolism, we have exposed cultured primary astrocytes to arsenite or arsenate. Both arsenicals compromised the cell viability of astrocytes in a time- and concentration-dependent manner. However, the early onset of cell toxicity in arsenite-treated astrocytes revealed the higher toxic potential of arsenite compared with arsenate. The concentrations of arsenite and arsenate that caused within 24 h half-maximal release of the cytosolic enzyme lactate dehydrogenase were around 0.3 mM and 10 mM, respectively. The cellular arsenic contents of astrocytes increased rapidly upon exposure to arsenite or arsenate and reached after 4 h of incubation almost constant steady state levels. These levels were about 3-times higher in astrocytes that had been exposed to a given concentration of arsenite compared with the respective arsenate condition. Analysis of the intracellular arsenic species revealed that almost exclusively arsenite was present in viable astrocytes that had been exposed to either arsenate or arsenite. The emerging toxicity of arsenite 4 h after exposure was accompanied by a loss in cellular total glutathione and by an increase in the cellular glutathione disulfide content. These data suggest that the high arsenite content of astrocytes that had been exposed to inorganic arsenicals causes an increase in the ratio of glutathione disulfide to glutathione which contributes to the toxic potential of these substances.     

Arsenite oxidation by the heterotroph Hydrogenophaga sp. str. NT-14: the arsenite oxidase and its physiological electron acceptor

Heterotrophic arsenite oxidation by Hydrogenophaga sp. str. NT-14 is coupled to the reduction of oxygen and appears to yield energy for growth. Purification and partial characterization of the arsenite oxidase revealed that it (1). contains two heterologous subunits, AroA (86 kDa) and AroB (16 kDa), (2). has a native molecular mass of 306 kDa suggesting an alpha(3)beta(3) configuration, and (3). contains molybdenum and iron as cofactors. Although the Hydrogenophaga sp. str. NT-14 arsenite oxidase shares similarities to the arsenite oxidases purified from NT-26 and Alcaligenes faecalis, it differs with respect to activity and overall conformation. A c-551-type cytochrome was purified from Hydrogenophaga sp. str. NT-14 and appears to be the physiological electron acceptor for the arsenite oxidase. The cytochrome can also accept electrons from the purified NT-26 arsenite oxidase. A hypothetical electron transport chain for heterotrophic arsenite oxidation is proposed.     

Molecular analysis of microbial community structure in an arsenite-oxidizing acidic thermal spring

Electron microscopy (EM), denaturing gradient gel electrophoresis (DGGE) and 16S rDNA sequencing were used to examine the structure and diversity of microbial mats present in an acid-sulphate–chloride (pH 3.1) thermal (58–62°C) spring in Norris Basin, Yellowstone National Park, WY, USA, exhibiting rapid rates of arsenite oxidation. Initial visual assessments, scanning EM and geochemical measurements revealed the presence of three distinct mat types. Analysis of 16S rDNA fragments with DGGE confirmed the presence of different bacterial and archaeal communities within these zones. Changes in the microbial community appeared to coincide with arsenite oxidation activity. Phylogenetic analysis of 1400 bp 16S rDNA sequences revealed that clone libraries prepared from both arsenic redox active and inactive bacterial communities were dominated by sequences phylogenetically related to Hydrogenobacter acidophilus and Desulphurella sp. The appearance of archaeal 16S rDNA sequences coincided with the start of arsenite oxidation, and sequences were obtained showing affiliation with both Crenarchaeota and Euryarchaeota . The majority of archaeal sequences were most similar to sequences obtained from marine hydrothermal vents and other acidic hot springs, although the level of similarity was typically just 90%. Arsenite oxidation in this system may result from the activities of these unknown archaeal taxa and/or the previously unreported arsenic redox activity of H. acidophilus - or Desulphurella -like organisms. If the latter, arsenite oxidation must be inhibited in the initial high-sulphide zone of the spring, where no change in the distribution of arsenite versus arsenate was observed.     

Induction of p53 protein expression by sodium arsenite

Arsenic is carcinogen for humans and has been shown to act as an enhancer in initiated animal models. In a previous work we found impairment of lymphocyte proliferation in arsenic-exposed individuals and in vitro we obtained dose-related inhibition of mitotic response and lymphocyte proliferation. Intrigued by these effects and based on the role of p53 on cell proliferation, we tested different concentrations of sodium arsenite for their ability to induce the expression of tumor suppressor gene p53 in different cell lines (HeLa, C-33A, Jurkat) and a lymphoblast cell line transformed with Epstein–Barr virus (LCL-EBV). We also evaluated changes in their viability after 24 h arsenic treatment; C-33A cells showed the higher sensitivity to arsenic treatment while HeLa, Jurkat and LCL-EBV cells showed similar cytotoxicity curves. Immunoblots showed an increased expression of p53 gene with 1 μM sodium arsenite in Jurkat cells and 10 μM sodium arsenite in HeLa and LCL-EBV cells. In addition, we transfected Jurkat cells and human lymphocytes with wild-type and mutated p53 genes; lymphocytes and Jurkat cells that received the mutated p53 showed increased sensitivity to arsenic cytotoxicity. Data obtained indicate that arsenic induces p53 expression and that cells with a functional p53 contend better with damage induced by this metalloid.     

Comutagenesis of sodium arsenite with ultraviolet radiation in Chinese hamster V79 cells

Summary Solar ultraviolet radiation has been associated with the induction of skin cancer. Recent studies have indicated that near-ultraviolet, especially UVB, is mutagenic. Exposure to trivalent inorganic arsenic compounds has also been associated with increased skin cancer prevalence. Trivalent arsenic compounds are not mutagenicper se, but are comutagenic with a number of cancer agents. Here, we test the hypothesis that arsenite enhances skin cancer via its comutagenic action with solar ultraviolet radiation. Irradiation of Chinese hamster V79 cells with UVA (360 nm), UVB (310 nm) and UVC (254 nm) caused a fluence-dependent increase in mutations at thehprt locus. On an energy basis, UVC was the most mutagenic and UVA the least. However, when expressed as a function of toxicity, UVB was more mutagenic than UVC. Nontoxic concentrations of arsenite increased the toxicity of UVA, UVB and UVC. Arsenite acted as a comutagen at the three wavelengths; however, higher concentrations of arsenite were required to produce a significant (P < 0.05) comutagenic response with UVB. The increased mutagenicity of UVB and UVA by arsenite may play a role in arsenite-related skin cancers.     

Elevation of glutathione levels and glutathione S-transferase activity in arsenic-resistant chinese hamster ovary cells

Summary Arsenic-resistant Chinese hamster ovary (CHO) cells were established by progressively increasing the concentration of sodium arsenite in culture medium. One of the resistant clones, SA7, was also cross-resistant to As(V), Zn, Fe(II), Co, and Hg. The susceptibilities to sodium arsenite in parental CHO cells, revertant SA7N cells, and resistant SA7 cells were correlated with their intracellular glutathione (GSH) levels and glutathione S-transferase (GST) activity. The resistance in SA7 cells was diminished by depletion of GSH in cells after treatment with buthionine sulfoximine. Furthermore, after reexposure of revertant SA7N cells to sodium arsenite, the intracellular GSH levels, GST activity, and resistance to sodium arsenite were raised to the same levels as SA7 cells. These data indicate that the elevation of intracellular GSH levels and GST activity in SA7 cells may be responsible for the resistance to arsenite. A p25 protein, which could be a monomer subunit of GST, accumulated in SA7 cells. In addition, an outward transport inhibitor, verapamil, indiscriminately increased the arsenite toxicity in resistant and parental cells. This work was supported in part by grant NSC77-0201-B001-31 from the National Science Council, Republic of China.