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PubMed2006

Enhanced arsenate reduction by a CDC25-like tyrosine phosphatase explains increased phytochelatin accumulation in arsenate-tolerant Holcus lanatus.

Decreased arsenate [As(V)] uptake is the major mechanism of naturally selected As(V) hypertolerance in plants. However, As(V)-hypertolerant ecotypes also show enhanced rates of phytochelatin (PC) accumulation, suggesting that improved sequestration might additionally contribute to the hypertolerance phenotype. Here, we show that enhanced PC-based sequestration in As(V)-hypertolerant Holcus lanatus is not due to an enhanced capacity for PC synthesis as such, but to increased As(V) reductase activity. Vacuolar transport of arsenite-thiol complexes was equal in both ecotypes. Based on homology with the yeast As(V) reductase, Acr2p, we identified a Cdc25-like plant candidate, HlAsr, and confirmed the As(V) reductase activity of both HlAsr and the homologous protein from Arabidopsis thaliana. The gene appeared to be As(V)-inducible and its expression was enhanced in the As(V)-hypertolerant H. lanatus ecotype, compared with the non-tolerant ecotype. Homologous ectopic overexpression of the AtASR cDNA in A. thaliana produced a dual phenotype. It improved tolerance to mildly toxic levels of As(V) exposure, but caused hypersensitivity to more toxic levels. Arabidopsis asr T-DNA mutants showed increased As(V) sensitivity at low exposure levels and enhanced arsenic retention in the root. It is argued that, next to decreased uptake, enhanced expression of HlASR might act as an additional determinant of As(V) hypertolerance and As transport in H. lanatus.

Amino Acid Sequence↗
PubMed1993

luxAB gene fusions with the arsenic and cadmium resistance operons of Staphylococcus aureus plasmid pI258.

pC101, a novel shuttle vector between Escherichia coli and Staphylococcus aureus carrying the lux genes encoding luciferase from Vibrio harveyi, selectable ampicillin and chloramphenicol markers and origins of replication for Gram-negative and Gram-positive bacteria has been constructed. The inducibility of the arsenic and cadmium operon from S. aureus plasmid pI258 to different ions has been tested in E. coli and in S. aureus with two fusions in pC101: an arsB-luxAB and a cadA-luxAB transcriptional gene fusion. Patterns of induction are influenced by the host strain and are slightly different from previous reports using the blaZ gene as reporter gene.

Adenosine Triphosphatases↗
PubMed1996

The arsenical resistance operon of IncN plasmid R46.

The arsenical resistance operon of the IncN plasmid R46 consists of 4696 bp and starts with predicted transcriptional control and initiation signals, followed by five genes, arsD, arsA, and arsC. The corresponding Escherichia coli chromosomal ars operon and two staphylococcal ars operons lack arsA and arsD genes. The R46 system contains only the second known versions of arsA and arsD, after those of plasmid R773. Western blot analysis identified the R46 proteins using antibodies against R773 ArsA, ArsD and ArsR.

Adenosine Triphosphatases↗
PubMed1998

Saccharomyces cerevisiae ACR2 gene encodes an arsenate reductase.

The ACR2 gene of Saccharomyces cerevisiae was disrupted by insertion of a HIS3 gene. Cells with the disruption were sensitive to arsenate. This phenotype could be complemented by ACR2 on a plasmid. The ACR2 gene was cloned and expressed in Escherichia coli as a malE gene fusion with a C-terminal histidine tag. The combination of chimeric MBP-Acr2-6H protein and yeast cytosol from an ACR2-disrupted strain exhibited arsenate reductase activity.

Adenosine Triphosphatases↗
PubMed1999

Bacterial respiration of arsenic and selenium.

Oxyanions of arsenic and selenium can be used in microbial anaerobic respiration as terminal electron acceptors. The detection of arsenate and selenate respiring bacteria in numerous pristine and contaminated environments and their rapid appearance in enrichment culture suggest that they are widespread and metabolically active in nature. Although the bacterial species that have been isolated and characterized are still few in number, they are scattered throughout the bacterial domain and include Gram-positive bacteria, beta, gamma and epsilon Proteobacteria and the sole member of a deeply branching lineage of the bacteria, Chrysiogenes arsenatus. The oxidation of a number of organic substrates (i.e. acetate, lactate, pyruvate, glycerol, ethanol) or hydrogen can be coupled to the reduction of arsenate and selenate, but the actual donor used varies from species to species. Both periplasmic and membrane-associated arsenate and selenate reductases have been characterized. Although the number of subunits and molecular masses differs, they all contain molybdenum. The extent of the environmental impact on the transformation and mobilization of arsenic and selenium by microbial dissimilatory processes is only now being fully appreciated.

Adenosine Triphosphatases↗
PubMed2002

Microbial arsenic: from geocycles to genes and enzymes.

Arsenic compounds have been abundant at near toxic levels in the environment since the origin of life. In response, microbes have evolved mechanisms for arsenic resistance and enzymes that oxidize As(III) to As(V) or reduce As(V) to As(III). Formation and degradation of organoarsenicals, for example methylarsenic compounds, occur. There is a global arsenic geocycle, where microbial metabolism and mobilization (or immobilization) are important processes. Recent progress in studies of the ars operon (conferring resistance to As(III) and As(V)) in many bacterial types (and related systems in Archaea and yeast) and new understanding of arsenite oxidation and arsenate reduction by respiratory-chain-linked enzyme complexes has been substantial. The DNA sequencing and protein crystal structures have established the convergent evolution of three classes of arsenate reductases (that is classes of arsenate reductases are not of common evolutionary origin). Proposed reaction mechanisms in each case involve three cysteine thiols and S-As bond intermediates, so convergent evolution to similar mechanisms has taken place.

Arsenate Reductases↗
PubMed1998

Expression and regulation of the arsenic resistance operon of Acidiphilium multivorum AIU 301 plasmid pKW301 in Escherichia coli.

The arsenic resistance (ars) operon from plasmid pKW301 of Acidiphilium multivorum AIU 301 was cloned and sequenced. This DNA sequence contains five genes in the following order: arsR, arsD, arsA, arsB, arsC. The predicted amino acid sequences of all of the gene products are homologous to the amino acid sequences of the ars gene products of Escherichia coli plasmid R773 and IncN plasmid R46. The ars operon cloned from A. multivorum conferred resistance to arsenate and arsenite on E. coli. Expression of the ars genes with the bacteriophage T7 RNA polymerase-promoter system allowed E. coli to overexpress ArsD, ArsA, and ArsC but not ArsR or ArsB. The apparent molecular weights of ArsD, ArsA, and ArsC were 13,000, 64,000, and 16,000, respectively. A primer extension analysis showed that the ars mRNA started at a position 19 nucleotides upstream from the arsR ATG in E. coli. Although the arsR gene of A. multivorum AIU 301 encodes a polypeptide of 84 amino acids that is smaller and less homologous than any of the other ArsR proteins, inactivation of the arsR gene resulted in constitutive expression of the ars genes, suggesting that ArsR of pKW301 controls the expression of this operon.

Adenosine Triphosphatases↗
PubMed2000

The chromosomal arsenic resistance genes of Thiobacillus ferrooxidans have an unusual arrangement and confer increased arsenic and antimony resistance to Escherichia coli.

The chromosomal arsenic resistance genes of the acidophilic, chemolithoautotrophic, biomining bacterium Thiobacillus ferrooxidans were cloned and sequenced. Homologues of four arsenic resistance genes, arsB, arsC, arsH, and a putative arsR gene, were identified. The T. ferrooxidans arsB (arsenite export) and arsC (arsenate reductase) gene products were functional when they were cloned in an Escherichia coli ars deletion mutant and conferred increased resistance to arsenite, arsenate, and antimony. Therefore, despite the fact that the ars genes originated from an obligately acidophilic bacterium, they were functional in E. coli. Although T. ferrooxidans is gram negative, its ArsC was more closely related to the ArsC molecules of gram-positive bacteria. Furthermore, a functional trxA (thioredoxin) gene was required for ArsC-mediated arsenate resistance in E. coli; this finding confirmed the gram-positive ArsC-like status of this resistance and indicated that the division of ArsC molecules based on Gram staining results is artificial. Although arsH was expressed in an E. coli-derived in vitro transcription-translation system, ArsH was not required for and did not enhance arsenic resistance in E. coli. The T. ferrooxidans ars genes were arranged in an unusual manner, and the putative arsR and arsC genes and the arsBH genes were translated in opposite directions. This divergent orientation was conserved in the four T. ferrooxidans strains investigated.

Acidithiobacillus thiooxidans↗
PubMed2002

Homology of Escherichia coli R773 arsA, arsB, and arsC genes in arsenic-resistant bacteria isolated from raw sewage and arsenic-enriched creek waters.

The occurrence and diversity of the Escherichia coli R773 ars operon were investigated among arsenic-resistant enteric and nonenteric bacteria isolated from raw sewage and arsenic-enriched creek waters. Selected isolates from each creek location were screened for ars genes by colony hybridization and PCR. The occurrence of arsA, arsB, and arsC determined by low-stringency colony hybridization (31 to 53% estimated mismatch) was 81, 87, and 86%, respectively, for 84 bacteria isolated on arsenate- and arsenite-amended media from three locations. At moderate stringency (21 to 36% estimated mismatch), the occurrence decreased to 42, 56, and 63% for arsA, arsB, and arsC, respectively. PCR results showed that the ars operon is conserved in some enteric bacteria isolated from creek waters and raw sewage. The occurrence of the arsBC genotype was about 50% in raw sewage enteric bacteria, while arsA was detected in only 9.4% of the isolates (n = 32). The arsABC and arsBC genotypes occurred more frequently in enteric bacteria isolated from creek samples: 71.4 and 85.7% (n = 7), respectively. Average sequence divergence within arsB for six creek enteric bacteria was 20% compared to that of the E. coli R773 ars operon. Only 1 of 11 pseudomonads screened by PCR was positive for arsB. The results from this study suggest that significant divergence has occurred in the ars operon among As-resistant E. coli strains and in Pseudomonas spp.

Adenosine Triphosphatases↗
PubMed2003

The ars detoxification system is advantageous but not required for As(V) respiration by the genetically tractable Shewanella species strain ANA-3.

Arsenate [As(V); HAsO(4)(2-)] respiration by bacteria is poorly understood at the molecular level largely due to a paucity of genetically tractable organisms with this metabolic capability. We report here the isolation of a new As(V)-respiring strain (ANA-3) that is phylogenetically related to members of the genus Shewanella and that also provides a useful model system with which to explore the molecular basis of As(V) respiration. This gram-negative strain stoichiometrically couples the oxidation of lactate to acetate with the reduction of As(V) to arsenite [As(III); HAsO(2)]. The generation time and lactate molar growth yield (Y(lactate)) are 2.8 h and 10.0 g of cells mol of lactate(-1), respectively, when it is grown anaerobically on lactate and As(V). ANA-3 uses a wide variety of terminal electron acceptors, including oxygen, soluble ferric iron, oxides of iron and manganese, nitrate, fumarate, the humic acid functional analog 2,6-anthraquinone disulfonate, and thiosulfate. ANA-3 also reduces As(V) to As(III) in the presence of oxygen and resists high concentrations of As(III) (up to 10 mM) when grown under either aerobic or anaerobic conditions. ANA-3 possesses an ars operon (arsDABC) that allows it to resist high levels of As(III); this operon also confers resistance to the As-sensitive strains Shewanella oneidensis MR-1 and Escherichia coli AW3110. When the gene encoding the As(III) efflux pump, arsB, is inactivated in ANA-3 by a polar mutation that also eliminates the expression of arsC, which encodes an As(V) reductase, the resulting As(III)-sensitive strain still respires As(V); however, the generation time and the Y(lactate) value are two- and threefold lower, respectively, than those of the wild type. These results suggest that ArsB and ArsC may be useful for As(V)-respiring bacteria in environments where As concentrations are high, but that neither is required for respiration.

Arsenates↗
PubMed1995

An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and is conserved in gram-negative bacteria.

Arsenic is a known toxic metalloid, whose trivalent and pentavalent ions can inhibit many biochemical processes. Operons which encode arsenic resistance have been found in multicopy plasmids from both gram-positive and gram-negative bacteria. The resistance mechanism is encoded from a single operon which typically consists of an arsenite ion-inducible repressor that regulates expression of an arsenate reductase and inner membrane-associated arsenite export system. Using a lacZ transcriptional gene fusion library, we have identified an Escherichia coli operon whose expression is induced by cellular exposure to sodium arsenite at concentrations as low as 5 micrograms/liter. This chromosomal operon was cloned, sequenced, and found to consist of three cistrons which we named arsR, arsB, and arsC because of their strong homology to plasmid-borne ars operons. Mutants in the chromosomal ars operon were found to be approximately 10- to 100-fold more sensitive to sodium arsenate and arsenite exposure than wild-type E. coli, while wild-type E. coli that contained the operon cloned on a ColE1-based plasmid was found to be at least 2- to 10-fold more resistant to sodium arsenate and arsenite. Moreover, Southern blotting and high-stringency hybridization of this operon with chromosomal DNAs from a number of bacterial species showed homologous sequences among members of the family Enterobacteriaceae, and hybridization was detectable even in Pseudomonas aeruginosa. These results suggest that the chromosomal ars operon may be the evolutionary precursor of the plasmid-borne operon, as a multicopy plasmid location would allow the operon to be amplified and its products to confer increased resistance to this toxic metalloid.

Adenosine Triphosphatases↗
PubMed2003

Arsenic sensing and resistance system in the cyanobacterium Synechocystis sp. strain PCC 6803.

Arsenic is one of the most important global environmental pollutants. Here we show that the cyanobacterium Synechocystis sp. strain PCC 6803 contains an arsenic and antimony resistance operon consisting of three genes: arsB, encoding a putative arsenite and antimonite carrier, arsH, encoding a protein of unknown function, and arsC, encoding a putative arsenate reductase. While arsB mutant strains were sensitive to arsenite, arsenate, and antimonite, arsC mutants were sensitive only to arsenate. The arsH mutant strain showed no obvious phenotype under the conditions tested. In vivo the arsBHC operon was derepressed by oxyanions of arsenic and antimony (oxidation state, +3) and, to a lesser extent, by bismuth (oxidation state, +3) and arsenate (oxidation state, +5). In the absence of these effectors, the operon was repressed by a transcription repressor of the ArsR/SmtB family, encoded by an unlinked gene termed arsR. Thus, arsR null mutants showed constitutive derepression of the arsBHC operon. Expression of the arsR gene was not altered by the presence of arsenic or antimony compounds. Purified recombinant ArsR protein binds to the arsBHC promoter-operator region in the absence of metals and dissociates from the DNA in the presence of Sb(III) or As(III) but not in the presence of As(V), suggesting that trivalent metalloids are the true inducers of the system. DNase I footprinting experiments indicate that ArsR binds to two 17-bp direct repeats, with each one consisting of two inverted repeats, in the region from nucleotides -34 to + 17 of the arsBHC promoter-operator.

Antimony↗
PubMed2003

An arsenate reductase from Synechocystis sp. strain PCC 6803 exhibits a novel combination of catalytic characteristics.

The deduced protein product of open reading frame slr0946 from Synechocystis sp. strain PCC 6803, SynArsC, contains the conserved sequence features of the enzyme superfamily that includes the low-molecular-weight protein-tyrosine phosphatases and the Staphylococcus aureus pI258 ArsC arsenate reductase. The recombinant protein product of slr0946, rSynArsC, exhibited vigorous arsenate reductase activity (V(max) = 3.1 micro mol/min. mg), as well as weak phosphatase activity toward p-nitrophenyl phosphate (V(max) = 0.08 micro mol/min. mg) indicative of its phosphohydrolytic ancestry. pI258 ArsC from S. aureus is the prototype of one of three distinct families of detoxifying arsenate reductases. The prototypes of the others are Acr2p from Saccharomyces cerevisiae and R773 ArsC from Escherichia coli. All three have converged upon catalytic mechanisms involving an arsenocysteine intermediate. While SynArsC is homologous to pI258 ArsC, its catalytic mechanism exhibited a unique combination of features. rSynArsC employed glutathione and glutaredoxin as the source of reducing equivalents, like Acr2p and R773 ArsC, rather than thioredoxin, as does the S. aureus enzyme. As postulated for Acr2p and R773 ArsC, rSynArsC formed a covalent complex with glutathione in an arsenate-dependent manner. rSynArsC contains three essential cysteine residues like pI258 ArsC, whereas the yeast and E. coli enzymes require only one cysteine for catalysis. As in the S. aureus enzyme, these "extra" cysteines apparently shuttle a disulfide bond to the enzyme's surface to render it accessible for reduction. SynArsC and pI258 ArsC thus appear to represent alternative branches in the evolution of their shared phosphohydrolytic ancestor into an agent of arsenic detoxification.

Amino Acid Sequence↗
PubMed2005

Novel pathway for arsenic detoxification in the legume symbiont Sinorhizobium meliloti.

We report a novel pathway for arsenic detoxification in the legume symbiont Sinorhizobium meliloti. Although a majority of ars operons consist of three genes, arsR (transcriptional regulator), arsB [As(OH)3/H+ antiporter], and arsC (arsenate reductase), the S. meliloti ars operon includes an aquaglyceroporin (aqpS) in place of arsB. The presence of AqpS in an arsenic resistance operon is interesting, since aquaglyceroporin channels have previously been shown to adventitiously facilitate uptake of arsenite into cells, rendering them sensitive to arsenite. To understand the role of aqpS in arsenic resistance, S. meliloti aqpS and arsC were disrupted individually. Disruption of aqpS resulted in increased tolerance to arsenite but not arsenate, while cells with an arsC disruption showed selective sensitivity to arsenate. The results of transport experiments in intact cells suggest that AqpS is the only protein of the S. meliloti ars operon that facilitates transport of arsenite. Coexpression of S. meliloti aqpS and arsC in a strain of E. coli lacking the ars operon complemented arsenate but not arsenite sensitivity. These results imply that, when S. meliloti is exposed to environmental arsenate, arsenate enters the cell through phosphate transport systems and is reduced to arsenite by ArsC. Internally generated arsenite flows out of the cell by downhill movement through AqpS. Thus, AqpS confers arsenate resistance together with ArsC-catalyzed reduction. This is the first report of an aquaglyceroporin with a physiological function in arsenic resistance.

Aquaporins↗
PubMed1995

Neither reduced uptake nor increased efflux is encoded by tellurite resistance determinants expressed in Escherichia coli.

Rates of uptake of the TeO3(2-) oxyanion were investigated in Escherichia coli cells containing tellurite resistance determinants from both plasmid (RK2Ter, R478, pMER610, MIP233, pHH1508a, pMUR) and chromosomal (tehAB) sources. The uptake was investigated to determine whether or not reduced uptake or increased efflux is involved in the tellurite resistance mechanism. Reduced TeO3(2-) uptake generated by cultures harboring arsABC from the plasmid R773, which has been previously shown to be an oxyanion efflux transporter, was used as the standard. Uptake curves were found to be essentially identical among E. coli cultures harboring the tellurite resistance plasmids RK2Ter, pMER610, pHH1508a, and pMUR and cultures harboring tellurite-sensitive control plasmids. Cultures harboring clones of the tehAB operon from E. coli showed no change in the TeO3(2-) accumulation. Cultures harboring R478 demonstrated reduced uptake. However, a subclone containing only the tellurite resistance determinant displayed no reduced uptake. This suggests that there may be another determinant on R478 other than the primary tellurite resistance determinant that gives rise to TeO3(2-) efflux. These results demonstrate that neither reduced uptake nor increased efflux is responsible for the tellurite resistance in the resistance determinants investigated here.

Adenosine Triphosphatases↗
PubMed1996

Expression of the Escherichia coli chromosomal ars operon.

A chromosomally located operon (ars) of Escherichia coli has been previously shown to be functional in arsenic detoxification. DNA sequencing revealed three open reading frames homologous to the arsR, arsB, and arsC open reading frames of plasmid-based arsenic resistance operons isolated from both E. coli and staphylococcal species. To examine the outline of transcriptional regulation of the chromosomal ars operon, several transcriptional fusions, using the luciferase-encoding luxAB genes of Vibrio harveyi, were constructed. Measurement of the expression of these gene fusions demonstrated that the operon was rapidly induced by sodium arsenite and negatively regulated by the trans-acting arsR gene product. Northern blotting and primer extension analyses revealed that the chromosomal ars operon is most likely transcribed as a single mRNA of approximately 2100 nucleotides in length and processed into two smaller mRNA products in a manner similar to that found in the E. coli R773 plasmid-borne ars operon. However, transcription was found to initiate at a position that is relatively further upstream of the initiation codon of the arsR coding sequence than that determined for the E. coli R773 plasmid's ars operon.

Adenosine Triphosphatases↗
PubMed2003

Phylogenetic analysis of bacterial and archaeal arsC gene sequences suggests an ancient, common origin for arsenate reductase.

BACKGROUND: The ars gene system provides arsenic resistance for a variety of microorganisms and can be chromosomal or plasmid-borne. The arsC gene, which codes for an arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite, which is extruded from the cell. A survey of GenBank shows that arsC appears to be phylogenetically widespread both in organisms with known arsenic resistance and those organisms that have been sequenced as part of whole genome projects. RESULTS: Phylogenetic analysis of aligned arsC sequences shows broad similarities to the established 16S rRNA phylogeny, with separation of bacterial, archaeal, and subsequently eukaryotic arsC genes. However, inconsistencies between arsC and 16S rRNA are apparent for some taxa. Cyanobacteria and some of the gamma-Proteobacteria appear to possess arsC genes that are similar to those of Low GC Gram-positive Bacteria, and other isolated taxa possess arsC genes that would not be expected based on known evolutionary relationships. There is no clear separation of plasmid-borne and chromosomal arsC genes, although a number of the Enterobacteriales (gamma-Proteobacteria) possess similar plasmid-encoded arsC sequences. CONCLUSION: The overall phylogeny of the arsenate reductases suggests a single, early origin of the arsC gene and subsequent sequence divergence to give the distinct arsC classes that exist today. Discrepancies between 16S rRNA and arsC phylogenies support the role of horizontal gene transfer (HGT) in the evolution of arsenate reductases, with a number of instances of HGT early in bacterial arsC evolution. Plasmid-borne arsC genes are not monophyletic suggesting multiple cases of chromosomal-plasmid exchange and subsequent HGT. Overall, arsC phylogeny is complex and is likely the result of a number of evolutionary mechanisms.

Adenosine Triphosphatases↗