Plasmid-encoded resistance to arsenic and antimony.

Resistance determinants to the toxic oxyanionic salts of arsenic and antimony are found on plasmids of both gram-negative and gram-positive organisms. In most cases these provide resistance to both the oxyanions of +III oxidation state, antimonite and arsenite, and the +V oxidation state, arsenate. In both gram-positive and -negative bacteria, resistance is correlated with efflux of the anions from cells. The determinant from the plasmid R773, isolated from a gram-negative organism, has been studied in detail. It encodes an oxyanion-translocating ATPase with three subunits, a catalytic subunit, the ArsA protein, a membrane subunit, the ArsB subunit, and a specificity factor, the ArsC protein. The first two form a membrane-bound complex with arsenite-stimulated ATPase activity. The determinants from gram-positive bacteria have only the arsB and arsC genes and encode an efflux system without the participation of an ArsA homologue.     

A plasmid-encoded anion-translocating ATPase

An anion-translocating ATPase has been identified as the product of the arsenical resistance operon of resistance plasmid R773. When expressed in Escherichia coli this ATP-driven oxyanion pump catalyzes extrusion of the oxyanions arsenite, antimonite and arsenate. Maintenance of a low intracellular concentration of oxyanion produces resistance to the toxic agents. The pump is composed of two polypeptides, the products of the arsA and arsB genes. This two-subunit enzyme produces resistance to arsenite and antimonite. A third gene, arsC, expands the substrate specificity to allow for arsenate pumping and resistance.     

Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and gamma-glutamylcysteine synthetase expression

We have developed a genetics-based phytoremediation strategy for arsenic in which the oxyanion arsenate is transported aboveground, reduced to arsenite, and sequestered in thiol-peptide complexes. The Escherichia coli arsC gene encodes arsenate reductase (ArsC), which catalyzes the glutathione (GSH)-coupled electrochemical reduction of arsenate to the more toxic arsenite. Arabidopsis thaliana plants transformed with the arsC gene expressed from a light-induced soybean rubisco promoter (SRS1p) strongly express ArsC protein in leaves, but not roots, and were consequently hypersensitive to arsenate. Arabidopsis plants expressing the E. coli gene encoding gamma-glutamylcysteine synthetase (gamma-ECS) from a strong constitutive actin promoter (ACT2p) were moderately tolerant to arsenic compared with wild type. However, plants expressing SRS1p/ArsC and ACT2p/gamma-ECS together showed substantially greater arsenic tolerance than gamma-ECS or wild-type plants. When grown on arsenic, these plants accumulated 4- to 17-fold greater fresh shoot weight and accumulated 2- to 3-fold more arsenic per gram of tissue than wild type or plants expressing gamma-ECS or ArsC alone. This arsenic remediation strategy should be applicable to a wide variety of plant species.     

Insights into the structure, solvation, and mechanism of ArsC arsenate reductase, a novel arsenic detoxification enzyme.

BACKGROUND: In Escherichia coli bearing the plasmid R773, resistance to arsenite, arsenate, antimonite, and tellurite is conferred by the arsRDABC plasmid operon that codes for an ATP-dependent anion pump. The product of the arsC gene, arsenate reductase (ArsC), is required to efficiently catalyze the reduction of arsenate to arsenite prior to extrusion. RESULTS: Here, we report the first X-ray crystal structures of ArsC at 1.65 A and of ArsC complexed with arsenate and arsenite at 1.26 A resolution. The overall fold is unique. The native structure shows sulfate and sulfite ions binding in the active site as analogs of arsenate and arsenite. The covalent adduct of arsenate with Cys-12 in the active site of ArsC, which was analyzed in a difference map, shows tetrahedral geometry with a sulfur-arsenic distance of 2.18 A. However, the corresponding adduct with arsenite binds as a hitherto unseen thiarsahydroxy adduct. Finally, the number of bound waters (385) in this highly ordered crystal structure approaches twice the number expected at this resolution for a structure of 138 ordered residues. CONCLUSIONS: Structural information from the adduct of ArsC with its substrate (arsenate) and with its product (arsenite) together with functional information from mutational and biochemical studies on ArsC suggest a plausible mechanism for the reaction. The exceptionally well-defined water structure indicates that this crystal system has precise long-range order within the crystal and that the upper limit for the number of bound waters in crystal structures is underestimated by the structures in the Protein Data Bank.     

Resistance to arsenic compounds in microorganisms

Abstract: 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.     

Construction and purification of His-tagged staphylococcal ArsB protein,an integral membrane protein that is involved in arsenical salt resistance

Bacterial resistance to arsenical salts encoded on plasmid pI258 occurs by active extrusion of toxic oxyanions from cells of Staphylococcus aureus. The operon encodes for three gene products: ArsR, ArsB and ArsC. The gene product of arsB is an integral membrane protein and it is sufficient to provide resistance to arsenite and antimonite. A poly His-ArsB fusion protein was generated to purify the staphylococcal ArsB protein. Cells containing the His-tagged arsB gene were resistant to arsenite and antimonite. The levels of resistance to these toxic oxyanions by the His-tagged construct were greater than the levels obtained with the wild type gene. These data would indicate that the His-tagged protein is functionally active. A new 36 kDa protein band was visualized on 10% SDS-polyacrylamide gel electrophoresis (PAGE), which was confirmed as the His-ArsB protein by immunodetection with polyclonal Hisantibodies. The His-ArsB fusion protein was purified by the use of metal-chelate affinity chromatography with a Ni⁺²-nitrilotriacetic acid column and size-exclusion chromatography suggests that the protein was a homodimer.     

arsRBOCT arsenic resistance system encoded by linear plasmid pHZ227 in Streptomyces sp. strain FR-008

In the arsenic resistance gene cluster from the large linear plasmid pHZ227, two novel genes, arsO (for a putative flavin-binding monooxygenase) and arsT (for a putative thioredoxin reductase), were coactivated and cotranscribed with arsR1-arsB and arsC, respectively. Deletion of the ars gene cluster on pHZ227 in Streptomyces sp. strain FR-008 resulted in sensitivity to arsenic, and heterologous expression of the ars gene cluster in the arsenic-sensitive Streptomyces strains conferred resistance on the new hosts. The pHZ227 ArsB protein showed homology to the yeast arsenite transporter Acr3p. The pHZ227 ArsC appears to be a bacterial thioredoxin-dependent ArsC-type arsenate reductase with four conserved cysteine thioredoxin-requiring motifs.     

Molecular analysis of an ATP-dependent anion pump

The plasmid-borne arsenical resistance operon encodes an ATP-driven oxyanion pump for the extrusion of the oxyanions arsenite, antimonite and arsenate from bacterial cells. The catalytic component of the pump, the 63 kDa ArsA protein, hydrolyses ATP in the presence of its anionic substrate antimonite (SbO2-). The ATP analogue 5'-p-fluorosulphonylbenzoyladenosine was used to modify the ATP binding site(s) of the ArsA protein. From sequence analysis there are two potential nucleotide binding sites. Mutations were introduced into the N-terminal site. Purified mutant proteins were catalytically inactive and incapable of binding nucleotides. Conformational changes produced upon binding of substrates to the ArsA protein were investigated by measuring the effects of substrates on trypsin inactivation. The hydrophobic 45.5 kDa ArsB protein forms the membrane anchor for the ArsA protein. The presence of the ArsA protein on purified inner membrane can be detected immunologically. In the absence of the arsB gene no ArsA is found on the membrane. Synthesis of the ArsB protein is limiting for formation of the pump. Analysis of mRNA structure suggests a potential translational block to synthesis of the ArsB protein. Northern analysis of the ars message demonstrates rapid degradation of the mRNA in the arsB region.     

Kinetics and active site dynamics of Staphylococcus aureus arsenate reductase

Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular arsenate(V) to the more toxic arsenite(III), which is subsequently extruded from the cell. It couples to thioredoxin, thioredoxin reductase and NADPH to be enzymatically active. ArsC is extremely sensitive to oxidative inactivation, has a very dynamic character hampering resonance assignments in NMR and produces peculiar biphasic Michaelis-Menten curves with two V(max) plateaus. In this study, methods to control ArsC oxidation during purification have been optimized. Next, application of Selwyn's test of enzyme inactivation was applied to progress curves and reveals that the addition of tetrahedral oxyanions (50 mM sulfate, phosphate or perchlorate) allows the control of ArsC stability and essentially eliminates the biphasic character of the Michaelis-Menten curves. Finally, 1H-15N HSQC NMR spectroscopy was used to establish that these oxyanions, including the arsenate substrate, exert their stabilizing effect on ArsC through binding with residues located within a C-X5-R sequence motif, characteristic for phosphotyrosine phosphatases. In view of this need for a tetrahedral oxyanion to structure its substrate binding site in its active conformation, a reappraisal of basic kinetic parameters of ArsC was necessary. Under these new conditions and in contrast to previous observations, ArsC has a high substrate specificity, as only arsenate could be reduced ( Km=68 microM, k(cat)/ Km =5.2 x 10(4 )M-1s-1), while its product, arsenite, was identified as a mixed inhibitor ( K*iu=534 microM, K*ic=377 microM).     

Regulation of arsenate resistance in Desulfovibrio desulfuricans G20 by an arsRBCC operon and an arsC gene

Desulfovibrio desulfuricans G20 grows and reduces 20 mM arsenate to arsenite in lactate-sulfate media. Sequence analysis and experimental data show that D. desulfuricans G20 has one copy of arsC and a complete arsRBCC operon in different locations within the genome. Two mutants of strain G20 with defects in arsenate resistance were generated by nitrosoguanidine mutagenesis. The arsRBCC operons were intact in both mutant strains, but each mutant had one point mutation in the single arsC gene. Mutants transformed with either the arsC1 gene or the arsRBCC operon displayed wild-type arsenate resistance, indicating that the two arsC genes were equivalently functional in the sulfate reducer. The arsC1 gene and arsRBCC operon were also cloned into Escherichia coli DH5alpha independently, with either DNA fragment conferring increased arsenate resistance. The recombinant arsRBCC operon allowed growth at up to 50 mM arsenate in LB broth. Quantitative PCR analysis of mRNA products showed that the single arsC1 was constitutively expressed, whereas the operon was under the control of the arsR repressor protein. We suggest a model for arsenate detoxification in which the product of the single arsC1 is first used to reduce arsenate. The arsenite formed is then available to induce the arsRBCC operon for more rapid arsenate detoxification.