Phosphate and glucose accumulation by Pseudomonas cultures in relation to their arsenic resistance

The effect of arsenite and arsenate on 14C-glucose and 32-P-phosphate transport was studied in the cells of Pseudomonas aeruginosa 561 sensitive to arsenite and in the cells of Pseudomonas putida 18 oxidizing arsenite and resistant to arsenic. Transport and accumulation of phosphate and glucose were inhibited in the presence of arsenite in the cells of P. aeruginosa 561 whereas arsenate inhibited only phosphate accumulation. Arsenite and arsenate had hardly any effect at the initial transport rate and on the overall accumulation of phosphate and glucose in the cells of P. putida 18. The resistance to arsenite is supposed to be caused by selective impermeability of the cellular membranes to arsenite and arsenate.     

The <Emphasis Type="Italic">ars</Emphasis> genotype characterization of arsenic-resistant bacteria from arsenic-contaminated gold–silver mines in the Republic of Korea

The isolates were identified on the basis of ars genotype characteristics as well as arsenic oxidation/reduction analysis based on the molecular detection characterization. Diversity, pH range (4.0 to 7.0), location, and ars features were assessed for four arsenic-contaminated pond sites and six arsenic tailings located in the Duck-um mine and Myoung-bong mine areas. The presence of ars genes in the genomes of each bacterial strain was evaluated using polymerase chain reaction. Batch experiment results showed that Pseudomonas putida strains OS-3 and -18 completely oxidized 1 mM of arsenite(III) to arsenate(V) within 35-40 h. In contrast, two arsenate-reducing bacteria isolated from mines, P. putida RS-4 and RS-5, were capable of growing aerobically in growth medium supplemented with up to 66.7 mM arsenate(V), which are significantly higher concentration than those tolerated by other arsenic-resistant bacteria. These results suggest that newly isolated indigenous arsenic-resistant bacteria may provide a better understanding of the molecular geomicrobiology and may be applied to the bioremediation of arsenic-contaminated mines in Korea. Ecologically, the redox potential plays an important role in arsenic toxicity and mobility in As-contaminated mine areas, as it facilitates the biogeochemical cycling activity of Pseudomonas sp. groups.     

Plasmid-determined resistance to arsenic and antimony inPseudomonas aeruginosa

Resistance to arsenic salts in aPseudomonas aeruginosa clinical isolate was shown to be determined by a 100 kb transferable plasmid. The resistance pattern included arsenate, arsenite, and antimonate ions. Arsenate and arsenite resistances were inducible by previous exposure of cultures to subinhibitory amounts of either of the two ions. Phosphate ions protectedP. aeruginosa cells from the toxic effects of arsenate but did not alter arsenite toxicity.     

Toxicity, transformation and accumulation of inorganic arsenic species in a microalga Scenedesmus sp. isolated from soil

Arsenic speciation and cycling in the natural environment are highly impacted via biological processes. Since arsenic is ubiquitous in the environment, microorganisms have developed resistance mechanisms and detoxification pathways to overcome the arsenic toxicity. This study has evaluated the toxicity, transformation and accumulation of arsenic in a soil microalga Scenedesmus sp. The alga showed high tolerance to arsenite. The 72-h 50 % growth inhibitory concentrations (IC₅₀ values) of the alga exposed to arsenite and arsenate in low-phosphate growth medium were 196.5 and 20.6 mg? L^?1, respectively. When treated with up to 7.5 mg? L^?1 arsenite, Scenedesmus sp. oxidised all arsenite to arsenate in solution. However, only 50 % of the total arsenic remained in the solution while the rest was accumulated in the cells. Thus, this alga has accumulated arsenic as much as 606 and 761 μg? g^?1 dry weight when exposed to 750 μg? L^?1 arsenite and arsenate, respectively, for 8 days. To our knowledge, this is the first report of biotransformation of arsenic by a soil alga. The ability of this alga to oxidise arsenite and accumulate arsenic could be used in bioremediation of arsenic from contaminated water and soil.     

Arsenic efflux governed by the arsenic resistance determinant of Staphylococcus aureus plasmid pI258.

The arsenic resistance operon of Staphylococcus aureus plasmid pI258 determined lowered net cellular uptake of 73As by an active efflux mechanism. Arsenite was exported from the cells; intracellular arsenate was first reduced to arsenite and then transported out of the cells. Resistant cells showed lower accumulation of 73As originating from both arsenate and arsenite. Active efflux from cells loaded with arsenite required the presence of the plasmid-determined arsB gene. Efflux of arsenic originating as arsenate required the presence of the arsC gene and occurred more rapidly with the addition of arsB. Inhibitor studies with S. aureus loaded with arsenite showed that arsenite efflux was energy dependent and appeared to be driven by the membrane potential. With cells loaded with 73AsO4(3-), a requirement for ATP for energy was observed, leading to the conclusion that ATP was required for arsenate reduction. When the staphylococcal arsenic resistance determinant was cloned into Escherichia coli, lowered accumulation of arsenate and arsenite and 73As efflux from cells loaded with arsenate were also found. Cloning of the E. coli plasmid R773 arsA gene (the determinant of the arsenite-dependent ATPase) in trans to the S. aureus gene arsB resulted in increased resistance to arsenite.     

Isolation and Characterization of Arsenate-Reducing Bacteria from Arsenic-Contaminated Sites in New Zealand

Two environmental sites in New Zealand were sampled (e.g., water and sediment) for bacterial isolates that could use either arsenite as an electron donor or arsenate as an electron acceptor under aerobic and anaerobic growth conditions, respectively. These two sites were subjected to widespread arsenic contamination from mine tailings generated from historic gold mining activities or from geothermal effluent. No bacteria were isolated from these sites that could utilize arsenite or arsenate under the respective growth conditions tested, but a number of chemoheterotrophic bacteria were isolated that could grow in the presence of high concentrations of arsenic species. In total, 17 morphologically distinct arsenic-resistant heterotrophic bacteria isolates were enriched from the sediment samples, and analysis of the 16S rRNA gene sequence of these bacteria revealed them to be members of the genera Exiguobacterium, Aeromonas, Bacillus, Pseudomonas, Escherichia, and Acinetobacter. Two isolates, Exiguobacterium sp. WK6 and Aeromonas sp. CA1, were of particular interest because they appeared to gain metabolic energy from arsenate under aerobic growth conditions, as demonstrated by an increase in cellular growth yield and growth rate in the presence of arsenate. Both bacteria were capable of reducing arsenate to arsenite via a non-respiratory mechanism. Strain WK6 was positive for arsB, but the pathway of arsenate reduction for isolate CA1 was via a hitherto unknown mechanism. These isolates were not gaining an energetic advantage from arsenate or arsenite utilization, but were instead detoxifying arsenate to arsenite. As a subsidiary process to arsenate reduction, the external pH of the growth medium increased (i.e., became more alkaline), allowing these bacteria to grow for extended periods of time.     

Investigation of the structure and function of a Shewanella oneidensis arsenical-resistance family transporter

The toxic metalloid arsenic is an abundant element and most organisms possess transport systems involved in its detoxification. One such family of arsenite transporters, the ACR3 family, is widespread in fungi and bacteria. To gain a better understanding of the molecular mechanism of arsenic transport, we report here the expression and characterization of a family member, So_ACR3, from the bacterium Shewanella oneidensis MR-1. Surprisingly, expression of this transporter in the arsenic-hypersensitive Escherichia coli strain AW3110 conferred resistance to arsenate, but not to arsenite. Purification of a C-terminally His-tagged form of the protein allowed the binding of putative permeants to be directly tested: arsenate but not arsenite quenched its intrinsic fluorescence in a concentration-dependent fashion. Fourier transform infrared spectroscopy showed that the purified protein was predominantly α-helical. A mutant bearing a single cysteine residue at position 3 retained the ability to confer arsenate resistance, and was accessible to membrane impermeant thiol reagents in intact cells. In conjunction with successful C-terminal tagging with oligohistidine, this finding is consistent with the experimentally-determined topology of the homologous human apical sodium-dependent bile acid transporter, namely 7 transmembrane helices and a periplasmic N-terminus, although the presence of additional transmembrane segments cannot be excluded. Mutation to alanine of the conserved residue proline 190, in the fourth putative transmembrane region, abrogated the ability of the transporter to confer arsenic resistance, but did not prevent arsenate binding. An apparently increased thermal stability is consistent with the mutant being unable to undergo the conformational transitions required for permeant translocation.     

An arsenate-activated glutaredoxin from the arsenic hyperaccumulator fern Pteris vittata L. regulates intracellular arsenite

To elucidate the mechanisms of arsenic resistance in the arsenic hyperaccumulator fern Pteris vittata L., a cDNA for a glutaredoxin (Grx) Pv5-6 was isolated from a frond expression cDNA library based on the ability of the cDNA to increase arsenic resistance in Escherichia coli. The deduced amino acid sequence of Pv5-6 showed high homology with an Arabidopsis chloroplastic Grx and contained two CXXS putative catalytic motifs. Purified recombinant Pv5-6 exhibited glutaredoxin activity that was increased 1.6-fold by 10 mm arsenate. Site-specific mutation of Cys(67) to Ala(67) resulted in the loss of both GRX activity and arsenic resistance. PvGrx5 was expressed in E. coli mutants in which the arsenic resistance genes of the ars operon were deleted (strain AW3110), a deletion of the gene for the ArsC arsenate reductase (strain WC3110), and a strain in which the ars operon was deleted and the gene for the GlpF aquaglyceroporin was disrupted (strain OSBR1). Expression of PvGrx5 increased arsenic tolerance in strains AW3110 and WC3110, but not in OSBR1, suggesting that PvGrx5 had a role in cellular arsenic resistance independent of the ars operon genes but dependent on GlpF. AW3110 cells expressing PvGrx5 had significantly lower levels of arsenite when compared with vector controls when cultured in medium containing 2.5 mm arsenate. Our results are consistent with PvGrx5 having a role in regulating intracellular arsenite levels, by either directly or indirectly modulating the aquaglyceroporin. To our knowledge, PvGrx5 is the first plant Grx implicated in arsenic metabolism.     

Molecular analysis of an anion pump: purification of the ArsC protein.

The ars operon of resistance plasmid R773 encodes an anion-translocating ATPase which catalyzes extrusion of the oxyanions arsenite, antimonite, and arsenate, thus providing resistance to the toxic compounds. Although both arsenite and arsenate contain arsenic, they have different chemical properties. In the absence of the arsC gene the pump transports arsenite and antimonite, oxyanions with the +III oxidation state of arsenic or antimony. The complex neither transports nor provides resistance to arsenate, the oxyanion of the +V oxidation state of arsenic. The arsC gene encodes a 16-kDa polypeptide, the ArsC protein, which alters the substrate specificity of the pump to allow for recognition and transport of the alternate substrate arsenate. The arsC gene was cloned behind a strong promoter and expressed at high levels. The ArsC protein was purified and crystallized.     

Heterologous expression of the yeast arsenite efflux system ACR3 improves Arabidopsis thaliana tolerance to arsenic stress

? Arsenic contamination has a negative impact on crop cultivation and on human health. As yet, no proteins have been identified in plants that mediate the extrusion of arsenic. Here, we heterologously expressed the yeast (Saccharomyces cerevisiae) arsenite efflux transporter ACR3 into Arabidopsis to evaluate how this affects plant tolerance and tissue arsenic contents. ? ACR3 was cloned from yeast and transformed into wild-type and nip7;1 Arabidopsis. Arsenic tolerance was determined at the cellular level using vitality stains in protoplasts, in intact seedlings grown on agar plates and in mature plants grown hydroponically. Arsenic efflux was measured from protoplasts and from intact plants, and arsenic levels were measured in roots and shoots of plants exposed to arsenate. ? At the cellular level, all transgenic lines showed increased tolerance to arsenite and arsenate and a greater capacity for arsenate efflux. With intact plants, three of four stably transformed lines showed improved growth, whereas only transgenic lines in the wild-type background showed increased efflux of arsenite into the external medium. The presence of ACR3 hardly affected tissue arsenic levels, but increased arsenic translocation to the shoot. ? Heterologous expression of yeast ACR3 endows plants with greater arsenic resistance, but does not lower significantly arsenic tissue levels.     

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.     

Relative toxicity of arsenite and arsenate on germination and early seedling growth of rice (Oryza sativa L.)

Elevated soil arsenic levels resulting from long-term use of arsenic contaminated ground for irrigation in Bangladesh may inhibit seed germination and seedling establishment of rice, the country's main food crop. A germination study on rice seeds and a short-term toxicity experiment with different concentrations of arsenite and arsenate on rice seedlings were conducted. Percent germination over control decreased significantly with increasing concentrations of arsenite and arsenate. Arsenite was found to be more toxic than arsenate for rice seed germination. There were varietal differences among the test varieties in response to arsenite and arsenate exposure. The performance of the dry season variety Purbachi was the best among the varieties. Germination of Purbachi was not inhibited at all up to 4 mg l^–1 arsenite and 8 mg l^–1 arsenate treatment. Root tolerance index (RTI) and relative shoot height (RSH) for rice seedlings decreased with increasing concentrations of arsenite and arsenate. Reduction of RTI caused by arsenate was higher than that of arsenite. In general, dry season varieties have more tolerance to arsenite or arsenate than the wet season varieties.     

Resistance determinants of a highly arsenic-resistant strain of Leptospirillum ferriphilum isolated from a commercial biooxidation tank

Two sets of arsenic resistance genes were isolated from the highly arsenic-resistant Leptospirillum ferriphilum Fairview strain. One set is located on a transposon, TnLfArs, and is related to the previously identified TnAtcArs from Acidithiobacillus caldus isolated from the same arsenopyrite biooxidation tank as L. ferriphilum. TnLfArs conferred resistance to arsenite and arsenate and was transpositionally active in Escherichia coli. TnLfArs and TnAtcArs were sufficiently different for them not to have been transferred from one type of bacterium to the other in the biooxidation tank. The second set of arsenic resistance genes conferred very low levels of resistance in E. coli and appeared to be poorly expressed in both L. ferriphilum and E. coli.     

Effect of arsenite on the physiological and cytological properties of Pseudomonas putida strains capable of degrading polycyclic aromatic hydrocarbons]

Some physiological and cytological properties of Pseudomonas putida strains resistant to arsenite and capable of degrading polycyclic aromatic hydrocarbons were studied. The resistance of P. putida BS202 (NPL-1) to arsenite proved to be determined by chromosomal genes, while the arsenite resistance of P. putida BS238 (pBS2; pBS3031) was by plasmid-borne genes. Arsenite affected the pattern and rate of growth of strain BS202 (NPL-1) in media with naphthalene or salicylate as carbon sources; particularly, it lengthened the lag phase. Electron-microscope analysis of the strains studied did not reveal any arsenite-induced destructive changes in the cell envelope. At the same time, arsenite in the growth medium induced some alterations in the structure of the outer membrane of strain BS202 (NPL-1) and the cytoplasmic membrane of strain BS238 (pBS2; pBS3031) and, in both strains, led to an increase in the density of intramembrane particles on the EF face of the freeze-fractured cytoplasmic membrane. Arsenite resistance probably evidently protects cells of both strains from greater damage. Physiological and cytological data suggest that the mechanisms of arsenite resistance in the strains studied are different.