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At least 19 recordsLinked to original sources
PubMed1995

Dual mode of energy coupling by the oxyanion-translocating ArsB protein.

The arsA and arsB genes of the ars operon of R-factor R773 confer arsenite resistance in Escherichia coli by coding for an anion-translocating ATPase. Arsenite resistance and the in vivo energetics of arsenite transport were compared in cells expressing the arsA and arsB genes and those expressing just the arsB gene. Cells expressing the arsB gene exhibited intermediate arsenite resistance compared with cells expressing both the arsA and arsB genes. Both types of cells exhibited energy-dependent arsenite exclusion. Exclusion of 73AsO2- from cells expressing only the arsB gene was coupled to electrochemical energy, while in cells expressing both genes, transport was coupled to chemical energy, most likely ATP. These results suggest that the Ars anion transport system can be either an obligatory ATP-coupled primary pump or a secondary carrier coupled to the proton motive force, depending on the subunit composition of the transport complex.

Adenosine Triphosphatases↗
PubMed1993

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.

Adenosine Triphosphatases↗
PubMed1997

Alternate energy coupling of ArsB, the membrane subunit of the Ars anion-translocating ATPase.

The arsenical resistance (ars) operon of the conjugative R-factor R773 confers resistance to arsenical and antimonial compounds in Escherichia coli, where resistance results from active extrusion of arsenite catalyzed by the products of the arsA and arsB genes. Previous in vivo studies on the energetics of arsenite extrusion showed that expression of both genes produced an ATP-coupled arsenite extrusion system that was independent of the electrochemical proton gradient. In contrast, in cells expressing only the arsB gene, arsenite extrusion was coupled to electrochemical energy and independent of ATP, suggesting that the Ars transport system exhibits a dual mode of energy coupling depending on the subunit composition. In vitro the ArsA-ArsB complex has been shown to catalyze ATP-coupled uptake of 73AsO2(-1) in everted membrane vesicles. However, transport catalyzed by ArsB alone has not previously been observed in vitro. In this study we demonstrate everted membrane vesicles prepared from cells expressing only arsB exhibit uptake of 73AsO2(-1) coupled to electrochemical energy.

Adenosine Triphosphatases↗
PubMed2001

Functional analysis of a chromosomal arsenic resistance operon in Pseudomonas fluorescens strain MSP3.

We reported earlier about the detection of a chromosomally located arsenic operon (arsRBC) in a gram-negative bacterium Pseudomonas fluorescens strain MSP3, which showed resistance to elevated levels of sodium arsenate and sodium arsenite. The genes for arsenic resistance were cloned into the HindIII site of pBluescript vector producing three clones MSA1, MSA2 and MSI3 conferring resistance to sodium arsenate and arsenite salts. They were further sub-cloned to delineate the insert size and the sub-clones were designated as MSA11, MSA12 and MSI13. The sub-clone pMSA12 (2.6 kb) fragment was further packaged into EcoRI-PstI site of M13mp19 and sequenced. Nucleotide sequencing revealed the presence of three open reading frames homologous to the arsR, arsB and arsC genes of arsenic resistance. Three cistrons of the ars operon encoded polypeptides ArsR, ArsB and ArsC with molecular weights ranging approximately 12, 37and 24 kDa, respectively. These polypeptides were visualized on SDS-PAGE stained with Coomassie blue and measured in a densitometer. The arsenic resistance operon (arsRBC) of strain MSP3 plasmid pMSA12 consists of 3 genes namely, arsR--encoding a repressor regulatory protein, arsB--the determinant of the membrane efflux protein that confers resistance by pumping arsenic from the cells and arsC--a small cytoplasmic polypeptide required for arsenate resistance only, not for arsenite resistance. ArsB protein is believed to use the cell membrane potential to drive the efflux of intracellular arsenite ions. ArsC encodes for the enzyme arsenate reductase which reduces intracellular As(V) (arsenate) to more toxic As(III) (arsenite) and is subsequently extruded from the cell. The arsenate reductase activity was present in the soluble cytoplasmic fraction in E. coli clones. In the context of specified function of the arsenic operon encoded proteins, uptake and efflux mechanisms were studied in the wild strain and the arsenate/arsenite clones. The cell free filtrates of the arsenate clones (MSA11 and MSA12) obtained from P. fluorescens containing the arsC gene showed that arsenate reduction requires glutathione reductase, glutathione (GSH), glutaredoxin and ArsC protein. The protein was purified in an active form and a spectrophotometric assay was developed in which the oxidation of NADPH was coupled to reduction of arsenate. The molecular weights and the location of the polypeptides were obtained from Coomassie stained SDS-PAGE of extracellular and intracellular fractions of the cells.

Arsenates↗
PubMed1992

Reduction of arsenate to arsenite by the ArsC protein of the arsenic resistance operon of Staphylococcus aureus plasmid pI258.

The arsenic resistance operon of Staphylococcus aureus plasmid pI258 consists of three genes, arsR (encoding the repressor regulatory protein), arsB (the determinant of the membrane efflux protein that confers resistance by pumping arsenic from the cells), and arsC (the small gene whose protein product is required for arsenate resistance only, not for arsenite resistance). ArsC has now been shown to be an arsenate reductase, converting intracellular arsenate [As(V)] to arsenite [As(III)], which is then exported from the cells by an energy-dependent efflux process. The arsenate reductase activity was found in the soluble cytoplasmic fraction in Escherichia coli (and not associated with the periplasmic fraction or the sedimentable cell envelope). Purified ArsC protein coupled in vitro with thioredoxin plus dithiothreitol (but not 2-mercaptoethanol or reduced glutathione) to reduce arsenate to arsenite.

Adenosine Triphosphatases↗
PubMed1995

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.

Adenosine Triphosphatases↗
PubMed1996

Soft metal thiol chemistry is not involved in the transport of arsenite by the Ars pump.

The single cysteine in the ArsB protein subunit of the arsenite resistance pump was changed to serine and alanine residues. Resistance in cells expressing the two mutant arsB genes was the same as in the wild type, and the serine substitution had no effect on the arsenite transport properties. These results eliminate possible thiol chemistry in translocation. Thus, the pump uses soft metal chemistry for metalloactivation and nonmetal chemistry for oxyanion transport.

Adenosine Triphosphatases↗
PubMed1993

Orphan enzyme or patriarch of a new tribe: the arsenic resistance ATPase of bacterial plasmids.

The plasmid-determined arsenite and antimonite efflux ATPase of bacteria differs from other membrane transport ATPases, which are classified into several families (such as the F0F1-type H(+)-translocating ATP synthases, the related vacuolar H(+)-translocating ATPases, the P-type cation-translocating ATPases, and the superfamily which includes the periplasmic binding-protein-dependent systems in Gram-negative bacteria, the human multidrug resistance P-glycoprotein, and the cystic fibrosis transport regulator). The amino acid sequences of the components of the arsenic resistance system are not similar to known ATPase proteins. New findings with the arsenic resistance operons of bacterial plasmids suggest that instead of being an orphan the Ars system will now be the first recognized member of a new class of ATPases. Furthermore, fundamental questions of energy-coupling (ATP-driven or chemiosmotic) have recently been raised and the finding that the arsC gene product is a soluble enzyme that reduces arsenate to arsenite changes the previous picture of the functioning of this widespread bacterial system.

Adenosine Triphosphatases↗
PubMedDate not supplied

[Bacterial resistance to arsenic compounds].

Arsenic compounds, often present as environmental pollutants, are highly toxic for most microorganisms. Some microbial strains possess genetic determinants conferring resistance to arsenic derivatives. In bacteria, these determinants are usually located on plasmids, which has facilitated their analysis with molecular detail. Bacterial plasmids conferring arsenic resistance encode specific pumps that extrude arsenite (AsIII). In Gram-negative bacteria, the efflux pump consists of a complex formed by an ATPase (ArsA) associated with a membrane anion channel (ArsB). Arsenate (AsV) is converted to arsenite by a soluble reductase (ArsC). Proteins ArsB and ArsC, but not the ATPase, are also found in Gram-positive bacteria. Besides the widely spread plasmid arsenic resistance determinants, some bacteria possess the ability to enzimatically oxidize arsenite to less toxic arsenate.

Adenosine Triphosphatases↗
PubMed1994

A functional chimeric membrane subunit of an ion-translocating ATPase.

A chimeric transport protein was made by expression of a fusion of the arsB genes from Escherichia coli plasmid R773 and Staphylococcus aureus plasmid pI258. The two genes were fused to encode a functional protein with first eight membrane spanning alpha-helices of the S. aureus and the last four helices of the E. coli protein. The hybrid protein provided arsenite resistance and transport. When an arsA gene was expressed in trans with the ArsB proteins encoded by the R773, pI258 and fusion genes, arsenite efflux was dependent on chemical but not electrochemical energy. The Ars system is hypothesized to be a novel transport system that functions as a primary ATP-driven pump or a secondary carrier, depending on the subunit composition of the complex.

Adenosine Triphosphatases↗
PubMed2000

Trimeric ring-like structure of ArsA ATPase.

ArsA protein is the soluble subunit of the Ars anion pump in the Escherichia coli membrane which extrudes arsenite or antimonite from the cytoplasm. The molecular weight of the subunit is 63 kDa. In the cell it hydrolyzes ATP, and the energy released is used by the membrane-bound subunit ArsB to transport the substrates across the membrane. We have obtained two-dimensional crystals of ArsA in the presence of arsenite on negatively-charged lipid monolayer composed of DMPS and DOPC. These crystals have been studied using electron microscopy of negatively-stained specimens followed by image processing. The projection map obtained at 2.4 nm resolution reveals a ring-like structure with threefold symmetry. Many molecular assemblies with the same ring-shape and dimensions were also seen dispersed on electron microscopy grids, prepared directly from purified ArsA protein solution. Size-exclusion chromatography of the protein sample with arsenite present revealed that the majority of the protein particles in solution have a molecular weight of about 180 kDa. Based on these experiments, we conclude that in solution the ArsA ATPase with substrate bound is mainly in a trimeric form.

Adenosine Triphosphatases↗
PubMed1992

Construction of a chimeric ArsA-ArsB protein for overexpression of the oxyanion-translocating ATPase.

Resistance to toxic oxyanions of arsenic and antimony in Escherichia coli is conferred by the conjugative R-factor R773, which encodes an ATP-driven anion extrusion pump. The ars operon is composed of three structural genes, arsA, arsB, and arsC. Although transcribed as a single unit, the three genes are differentially expressed as a result of translational differences, such that the ArsA and ArsC proteins are produced in high amounts relative to the amount of ArsB protein made. Consequently, biochemical characterization of the ArsB protein, which is an integral membrane protein containing the anion-conducting pathway, has been limited, precluding studies of the mechanism of this oxyanion pump. To overexpress the arsB gene, a series of changes were made. First, the second codon, an infrequently used leucine codon, was changed to a more frequently utilized codon. Second, a GC-rich stem-loop (delta G = -17 kcal/mol) between the third and twelfth codons was destabilized by changing several of the bases of the base-paired region. Third, the re-engineered arsB gene was fused 3' in frame to the first 1458 base pairs of the arsA gene to encode a 914-residue chimeric protein (486 residues of the ArsA protein plus 428 residues of the mutated ArsB protein) containing the entire re-engineered ArsB sequence except for the initiating methionine. The ArsA-ArsB chimera has been overexpressed at approximately 15-20% of the total membrane proteins. Cells producing the chimeric ArsA-ArsB protein with an arsA gene in trans excluded 73AsO2- from cells, demonstrating that the chimera can function as a component of the oxyanion-translocating ATPase.

Adenosine Triphosphatases↗
PubMed2005

Analysis of genes involved in arsenic resistance in Corynebacterium glutamicum ATCC 13032.

Corynebacterium glutamicum is able to grow in media containing up to 12 mM arsenite and 500 mM arsenate and is one of the most arsenic-resistant microorganisms described to date. Two operons (ars1 and ars2) involved in arsenate and arsenite resistance have been identified in the complete genome sequence of Corynebacterium glutamicum. The operons ars1 and ars2 are located some distance from each other in the bacterial chromosome, but they are both composed of genes encoding a regulatory protein (arsR), an arsenite permease (arsB), and an arsenate reductase (arsC); operon ars1 contains an additional arsenate reductase gene (arsC1') located immediately downstream from arsC1. Additional arsenite permease and arsenate reductase genes (arsB3 and arsC4) scattered on the chromosome were also identified. The involvement of ars operons in arsenic resistance in C. glutamicum was confirmed by gene disruption experiments of the three arsenite permease genes present in its genome. Wild-type and arsB3 insertional mutant C. glutamicum strains were able to grow with up to 12 mM arsenite, whereas arsB1 and arsB2 C. glutamicum insertional mutants were resistant to 4 mM and 9 mM arsenite, respectively. The double arsB1-arsB2 insertional mutant was resistant to only 0.4 mM arsenite and 10 mM arsenate. Gene amplification assays of operons ars1 and ars2 in C. glutamicum revealed that the recombinant strains containing the ars1 operon were resistant to up to 60 mM arsenite, this being one of the highest levels of bacterial resistance to arsenite so far described, whereas recombinant strains containing operon ars2 were resistant to only 20 mM arsenite. Northern blot and reverse transcription-PCR analysis confirmed the presence of transcripts for all the ars genes, the expression of arsB3 and arsC4 being constitutive, and the expression of arsR1, arsB1, arsC1, arsC1', arsR2, arsB2, and arsC2 being inducible by arsenite.

Arsenates↗
PubMed1988

Molecular characterization of an anion pump. The arsA gene product is an arsenite(antimonate)-stimulated ATPase.

The products of the arsenical resistance operon of resistance plasmid R733 form an efflux system for arsenicals. Detoxification results from active efflux of the oxyanions, preventing their concentration from reaching toxic levels. The largest polypeptide encoded by the ars operon was purified. From N-terminal sequencing the purified protein, termed the ArsA protein, was shown to correspond to the product of the arsA gene. The purified protein was demonstrated to bind ATP by two methods. First, a photoadduct of the protein with [alpha-32P]ATP was formed by irradiation at 254 nm. Second, the purified protein bound a fluorescent ATP analogue, 2',3'-o-(2,4,6)trinitrophenyl ATP, with a half-maximal affinity of 2 microM. By both assays competition was observed with ATP or ADP, but not with AMP, GTP, CTP, or UTP. In both nucleotide binding assays, Mg2+ was required, but neither arsenite nor antimonate had any affect. In contrast, the ArsA protein exhibited an ATPase activity which was dependent on the presence of arsenite or antimonate. The results suggest that the ArsA protein is the catalytic subunit of an oxyanion-translocating ATPase.

Adenosine Diphosphate↗
PubMed1991

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.

Adenosine Triphosphatases↗
PubMed2003

The Saccharomyces cerevisiae Arr4p is involved in metal and heat tolerance.

Homologues of the bacterial ArsA ATPase are found in nearly every organism. While the enzyme is involved in arsenic detoxification in bacteria, the roles of eukaryotic homologues have not been identified. This article reports the function of the Saccharomyces cerevisiae homologue encoded by ARR4 gene (YDL100c ORF). Disruption of ARR4 was not lethal, but the disrupted strain displayed increased sensitivity to As3+, As5+, Co2+, Cr3+, Cu2+ or VO4(3-) salts and temperature. A plasmid-encoded copy of a wild-type ARR4 gene could complement the heat- or metal-related stress responses. Mutation of a codon within the consensus sequence for the nucleotide-binding site resulted in loss of complementation of the disrupted strain and produced a dominant negative phenotype in a wild type strain. Wild type and mutant Arr4p were purified from Escherichia coli. The wild type protein exhibited a low level of ATPase activity, and the mutant was inactive. The purified ATPase eluted as a dimer of 80-kDa species. A fusion of ARR4 and the GFP (green fluorescent protein) gene was constructed. The gene fusion was able to complement stress-related phenotype of the ARR4 disruption. Under non-stress conditions, GFP fluorescence was found diffusely in the cytosol. Under stress conditions GFP was localized in a few punctate bodies resembling late endosomes. It is proposed that under heat or metal stress, the soluble ATPase becomes membrane-associated, perhaps through interaction with a partner protein, and that this complex is involved in stress tolerance.

Adaptation, Physiological↗
PubMed2000

The linker peptide of the ArsA ATPase.

Plasmid R773 encodes an As(III)/Sb(III)-translocating ATPase that confers resistance to those metalloids in Escherichia coli. The catalytic subunit of the pump, the ArsA ATPase, consists of homologous N- and C-terminal nucleotide-binding domains connected by a 25-residue linker. The role of this linker sequence was examined by deletion of five, 10, 15 or 23 residues or insertion of five glycine residues. Cells expressing arsA with the 5-residue insertion had wild-type arsenite resistance. Resistance of cells expressing modified arsA genes with deletions was dependent on the linker length. Cells with five or 10 deleted residues exhibited slightly reduced resistance. Deletion of 15 or 23 residues resulted in further decreases in resistance. Each altered ArsA was purified. The enzyme with the 5-residue insertion had the same affinity for ATP and Sb(III) as the wild-type enzyme. Enzymes with 5-, 10-, 15- or 23-residue deletions exhibited decreased affinity for both Sb(III) and ATP. The enzyme with a 23-residue deletion exhibited only basal ATPase activity and was unable to be allosterically activated by Sb(III). These results suggest that the linker has evolved to a length optimal for bringing the two halves of the protein into proper contact with each other, facilitating catalysis.

Adenosine Triphosphatases↗