Mechanisms Of Arsenic Resistance Research Articles

Arsenate detoxification in a Pseudomonad hypertolerant to arsenic

Pseudomonas sp. strain As-1, obtained from an electroplating industrial effluent, was capable of growing aerobically in growth medium supplemented with up to 65 mM arsenate (As (V)), significantly higher concentrations than those tolerated by other reference arsenic resistant bacteria. The majority of the arsenic was detected in culture supernatants as arsenite (As (III)) and X-ray absorbance spectroscopy suggested that 30% of this cell-bound arsenic was As (V), 65% As (III) and 5% of arsenic was associated with sulphur. PCR analysis using primers designed against arsenic resistance genes of other Gram-negative bacteria confirmed the presence of an arsenic resistance operon comprising of three genes, arsR, arsB and arsC in order of predicted transcription, and consistent with a role in intracellular reduction of As (V) and efflux of As (III). In addition to this classical arsenic resistance mechanism, other biochemical responses to arsenic were implicated. Novel arsenic-binding proteins were purified from cellular fractions, while proteomic analysis of arsenic-induced cultures identified the upregulation of additional proteins not normally associated with the metabolism of arsenic. Cross-talk with a network of proteins involved in phosphate metabolism was suggested by these studies, consistent with the similarity between the phosphate and arsenate anions.

Arsenic resistance in Halobacterium sp. strain NRC-1 examined by using an improved gene knockout system.

The genome sequence of Halobacterium sp. strain NRC-1 encodes genes homologous to those responsible for conferring resistance to arsenic. These genes occur on both the large extrachromosomal replicon pNRC100 (arsADRC and arsR2M) and on the chromosome (arsB). We studied the role of these ars genes in arsenic resistance genetically by construction of gene knockouts. Deletion of the arsADRC gene cluster in a Halobacterium NRC-1 Deltaura3 strain resulted in increased sensitivity to arsenite and antimonite but not arsenate. In contrast, knockout of the chromosomal arsB gene did not show significantly increased sensitivity to arsenite or arsenate. We also found that knockout of the arsM gene produced sensitivity to arsenite, suggesting a second novel mechanism of arsenic resistance involving a putative arsenite(III)-methyltransferase. These results indicate that Halobacterium sp. strain NRC-1 contains an arsenite and antimonite extrusion system with significant differences from bacterial counterparts. Deletion analysis was facilitated by an improved method for gene knockouts/replacements in Halobacterium that relies on both selection and counterselection of ura3 using a uracil dropout medium and 5-fluoroorotic acid. The arsenite and antimonite resistance elements were shown to be regulated, with resistance to arsenic in the wild type inducible by exposure to a sublethal concentration of the metal. Northern hybridization and reverse transcription-PCR analyses showed that arsA, arsD, arsR, arsM, arsC, and arsB, but not arsR2, are inducible by arsenite and antimonite. We discuss novel aspects of arsenic resistance in this halophilic archaeon and technical improvements in our capability for gene knockouts in the genome.

A transposon-located arsenic resistance mechanism from a strain of Acidithiobacillus caldus isolated from commercial, arsenopyrite biooxidation tanks

The moderately thermophilic, sulfur-oxidizing bacterium Acidithiobacillus caldus is an important role player in continuous-flow biooxidation processes associated with biomining. Biooxidation of arsenopyrite concentrates by a combination of Leptospirillum and At. caldus results in the release of soluble arsenic into the surrounding environment. Arsenate and arsenite are toxic to most organisms, yet At. caldus is able to thrive in this harsh environment. The purpose of this study was to identify and investigate arsenic resistance genes in At. caldus and compare its ars operon with those previously studied in other bacteria. A gene bank of a South African At. caldus strain #6 was constructed and transformed into an Escherichia coli arsenic-sensitive mutant strain to identify clones containing At. caldus arsenic resistance genes. A clone with an insert of 6.4 kb was selected and after two cycles of chromosome-walking a further 5.6 kb of DNA upstream of the original clone was isolated. This region was sequenced and found to have an unusual ars operon with a functional arsB gene, two arsD-like and two arsA-like genes. The arsenic resistance mechanism appears to be located on a Tn 21-like transposon and includes transposase and resolvase-like genes with the entire assemblage being flanked by Tn 21-like 38 bp inverted repeats (IR). Southern hybridization experiments using the arsB gene as a probe against total DNA from six different At. caldus strains indicated that the three South African strains which had been previously exposed to arsenic gave a positive hybridization signal, while three other At. caldus strains (KU, BC13 and C-SH12) did not. Southern hybridization of pulse field gels indicated that the arsB gene was chromosomally encoded. Genes encoding for NADH oxidase-like and IMP dehydrogenase-like enzymes were situated between the arsA and arsB genes. PCR and Southern hybridization experiments revealed that this unusual arrangement was not an artefact of cloning but that the 2.1 kb NADH oxidase-like and IMP dehydrogenase-like gene containing region was present between the arsA and arsB genes in all three South African strains. When transformed into the E. coli arsenic-sensitive mutant, the transposon-located system conferred increased resistance to arsenite, but not to arsenate. This indicated that the NADH oxidase-like gene did not function as an arsenate reductase in E. coli. It is possible that increased exposure to arsenic placed selection pressure on the three South African strains to acquire a Tn 21-like transposon containing an ars operon to enhance their arsenic resistance.

Arsenic inhibition of telomerase transcription leads to genetic instability.

Arsenic is effective in the treatment of acute promyelocytic leukemia. Paradoxically, it is also carcinogenic. In the process of elucidating a mechanism of arsenic resistance in a leukemia cell line, NB4, we discovered that arsenic exposure causes chromosomal abnormalities, with a preponderance of end-to-end fusions. These chromosomal end fusions suggested that telomerase activity may be inhibited by arsenic. We found that arsenic inhibits transcription of the hTERT gene, which encodes the reverse transcriptase subunit of human telomerase. This effect may in part be explained by decreased c-Myc and Sp1 transcription factor activities. Decreased telomerase activity leads to chromosomal end lesions, which promote either genomic instability and carcinogenesis or cancer cell death. These phenomena may explain the seemingly paradoxical carcinogenic and antitumor effects of arsenic.

The effect of As(III) and As(V) on the batch bioleaching of a pyrite-arsenopyrite concentrate

The bioleaching of arsenical gold-bearing sulphide ores and concentrates solubilises iron, arsenic and sulphur. Previous work has shown that high concentrations of iron and arsenic in solution inhibit bacterial growth, with As(III) reported to inhibit bacteria to a greater degree than As(V). Batch bioleaching experiments were carried out over periods of one month. Varying quantities of either 0.020–0.040 M As(III) or 0.107–0.220 M As(V), were added to a slurry, consisting of a pyrite-arsenopyrite concentrate (20% solids (m.v −1)) in a nutrient solution. The slurry was inoculated with a culture, consisting primarily of Leptospirillum ferrooxidans and Thiobacillus thiooxidans. The culture was obtained from a continuous bioleaching mini-plant treating the same concentrate. The results obtained were compared with those of a culture to which no arsenic was added. The effect of the added arsenic was determined by monitoring three parameters: the oxygen utilisation rate, r O 2 , of the culture, the rate at which the arsenic in the concentrate was solubilised and the speciation of the dissolved arsenic. The results suggest that the nature of the As(III) and As(V) toxicity is different. The addition of the culture to a slurry containing As(III) resulted in a reduced rate of bacterial oxidation. However, the addition of the culture to a slurry containing As(V) resulted in both a lag phase and a reduced rate of bacterial oxidation. At sufficiently high dosages of As(III) and As(V) the maximum oxygen utilisation rate, r O 2 max, of the culture was also affected. The results indicate that As(V) toxicity, and the relative toxicity of As(III) and As(V) to a mixed culture, appear to be affected by the availability of an energy source. Hence the toxicity of As(III) is not necessarily in the region of three times that of As(V). Furthermore, the results suggest that the mechanism of arsenic resistance may be attributed to the Pst + Pit − mutations and an energy dependent efflux pump.

Resistance to arsenic compounds in microorganisms

Arsenic ions, frequently present as environmental pollutants, are very toxic for most microorganisms. Some microbial strains possess genetic determinants that confer resistance. In bacteria, these determinants are often found on plasmids, which has facilitated their study at the molecular level. Bacterial plasmids conferring arsenic resistance encode specific efflux pumps able to extrude arsenic from the cell cytoplasm thus lowering the intracellular concentration of the toxic ions. In Gram-negative bacteria, the efflux pump consists of a two-component ATPase complex. ArsA is the ATPase subunit and is associated with an integral membrane subunit, ArsB. Arsenate is enzymatically reduced to arsenite (the substrate of ArsB and the activator of ArsA) by the small cytoplasmic ArsC polypeptide. In Gram-positive bacteria, comparable arsB and arsC genes (and proteins) are found, but arsA is missing. In addition to the wide spread plasmid arsenic resistance determinant, a few bacteria confer resistance to arsenite with a separate determinant for enzymatic oxidation of more-toxic arsenite to less-toxic arsenate. In contrast to the detailed information on the mechanisms of arsenic resistance in bacteria, little work has been reported on this subject in algae and fungi.

Expression of heme oxygenase in arsenic-resistant human lung adenocarcinoma cells : Lee T-C, Ho I-C. Institute of Biomedical Sciences, Academia Sinica, Taipei 115. Cancer Res 1994;54:1660-4

We have established arsenic-resistant cells (CL3R) and their subclones from a human lung adenocarcinoma cell line (CL3). CL3R cells and their subclones were maintained in the presence of 4 microM sodium arsenite. They were 6-fold more resistant than CL3 cells to arsenite. Heme oxygenase was expressed in CL3R cells and their subclones, as demonstrated by electrophoretic analysis, Northern blotting, and enzyme activity assay. When CL3R15 cells were grown in arsenite-free medium, their arsenite resistance declined in parallel with their decreasing heme oxygenase activity. Tin-protoporphyrin, a heme oxygenase inhibitor, was found to increase the toxicity of arsenic to CL3R cells. Expression of heme oxygenase might therefore be involved in the mechanism of arsenic resistance. CL3R cells were also shown to be cross-resistant to oxygen-radical generating agents, such as menadione and Adriamycin. Furthermore, sodium arsenite treatment dose-dependently increased the dichlorofluorescein fluorescence in CL3 cells but not in CL3R15 cells. These results suggest that heme oxygenase plays an important role in reducing cellular oxidants that are enhanced by sodium arsenite treatment.

Resistance to arsenic compounds in microorganisms

Arsenic ions, frequently present as environmental pollutants, are very toxic for most microorganisms. Some microbial strains possess genetic determinants that confer resistance. In bacteria, these determinants are often found on plasmids, which has facilitated their study at the molecular level. Bacterial plasmids conferring arsenic resistance encode specific efflux pumps able to extrude arsenic from the cell cytoplasm thus lowering the intracellular concentration of the toxic ions. In Gram-negative bacteria, the efflux pump consists of a two-component ATPase complex. ArsA is the ATPase subunit and is associated with an integral membrane subunit, ArsB. Arsenate is enzymatically reduced to arsenite (the substrate of ArsB and the activator of ArsA) by the small cytoplasmic ArsC polypeptide. In Gram-positive bacteria, comparable arsB and arsC genes (and proteins) are found, but arsA is missing. In addition to the wide spread plasmid arsenic resistance determinant, a few bacteria confer resistance to arsenite with a separate determinant for enzymatic oxidation of more-toxic arsenite to less-toxic arsenate. In contrast to the detailed information on the mechanisms of arsenic resistance in bacteria, little work has been reported on this subject in algae and fungi.