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.     

The ars operon of Escherichia coli confers arsenical and antimonial resistance.

The chromosomally encoded arsenical resistance (ars) operon subcloned into a multicopy plasmid was found to confer a moderate level of resistance to arsenite and antimonite in Escherichia coli. When the operon was deleted from the chromosome, the cells exhibited hypersensitivity to arsenite, antimonite, and arsenate. Expression of the ars genes was inducible by arsenite. By Southern hybridization, the operon was found in all strains of E. coli examined but not in Salmonella typhimurium, Pseudomonas aeruginosa, or Bacillus subtilis.     

Alleviation of type I restriction in Escherichia coli K12 in the presence of the arsR gene from pKW301 of Acidiphilium multivorum AIU 301

A study was made of the antirestriction activity of Acidiphilium multivorum AIU 301 ArsR, a repressor of the ars operon which confers resistance to arsenite and arsenate and is on pKW301. In Escherichia coli, arsR cloned under the control of Plac in a multi-copy vector alleviated restriction of nonmodified lambda DNA by a factor of 120, six times more efficiently than its analogs of conjugal plasmids R64 (incI1) and R773 (incFI). Amino acid sequence analysis showed that the three ArsR proteins have a homologous region of 38 residues, including the antirestriction motif, in their N domains, whereas the motif is in the C domain in the Ard proteins. The other regions are nonhomologous, and pKW301 ArsR is 33 residues shorter than R64 and R773 ArsRs. The total charge is -4 in pKW301 ArsR and +2 in R64 and R733 ArsRs. A total negative charge was assumed to contribute to the antirestriction activity.     

Arsenic resistance in the archaeon "Ferroplasma acidarmanus": new insights into the structure and evolution of the ars genes

Arsenic resistance in the acidophilic iron-oxidizing archaeon " Ferroplasma acidarmanus" was investigated. F. acidarmanus is native to arsenic-rich environments, and culturing experiments confirm a high level of resistance to both arsenite and arsenate. Analyses of the complete genome revealed protein-encoding regions related to known arsenic-resistance genes. Genes encoding for ArsR (arsenite-sensitive regulator) and ArsB (arsenite-efflux pump) homologues were found located on a single operon. A gene encoding for an ArsA relative (anion-translocating ATPase) located apart from the arsRB operon was also identified. Arsenate-resistance genes encoding for proteins homologous to the arsenate reductase ArsC and the phosphate-specific transporter Pst were not found, indicating that additional unknown arsenic-resistance genes exist for arsenate tolerance. Phylogenetic analyses of ArsA-related proteins suggest separate evolutionary lines for these proteins and offer new insights into the formation of the arsA gene. The ArsB-homologous protein of F. acidarmanus had a high degree of similarity to known ArsB proteins. An evolutionary analysis of ArsB homologues across a number of species indicated a clear relationship in close agreement with 16S rRNA evolutionary lines. These results support a hypothesis of arsenic resistance developing early in the evolution of life.     

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.     

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.     

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.     

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.