Respiratory arsenate reductase as a bidirectional enzyme

The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe-S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.     

Genome sequence and characterisation of a freshwater photoarsenotroph,Cereibacter azotoformans strain ORIO,isolated from sediments capable of cyclic light–dark arsenic oxidation and reduction

A freshwater photosynthetic arsenite-oxidizing bacterium, Cereibacter azotoformans strain ORIO, was isolated from Owens River, CA, USA. The waters from Owens River are elevated in arsenic and serve as the headwaters to the Los Angeles Aqueduct. The complete genome sequence of strain ORIO is 4.8 Mb genome (68% G + C content) and comprises two chromosomes and six plasmids. Taxonomic analysis placed ORIO within the Cereibacter genus (formerly Rhodobacter). The ORIO genome contains arxB₂AB₁CD (encoding an arsenite oxidase), arxXSR (regulators) and several ars arsenic resistance genes all co-localised on a 136 kb plasmid, named pORIO3. Phylogenetic analysis of ArxA, the molybdenum-containing arsenite oxidase catalytic subunit, demonstrated photoarsenotrophy is likely to occur within members of the Alphaproteobacteria. ORIO is a mixotroph, oxidises arsenite to arsenate (As(V)) photoheterotrophically, and expresses arxA in cultures grown with arsenite. Further ecophysiology studies with Owens River sediment demonstrated the interconversion of arsenite and As(V) was dependent on light–dark cycling. arxA and arrA (As(V) respiratory reductase) genes were detected in the light–dark cycled sediment metagenomes suggesting syntrophic interactions among arsenotrophs. This work establishes C. azotoformans str. ORIO as a new model organism for studying photoarsenotrophy and light–dark arsenic biogeochemical cycling.     

Selenate-dependent anaerobic arsenite oxidation by a bacterium from Mono Lake, California

Arsenate was produced when anoxic Mono Lake water samples were amended with arsenite and either selenate or nitrate. Arsenite oxidation did not occur in killed control samples or live samples with no added terminal electron acceptor. Potential rates of anaerobic arsenite oxidation with selenate were comparable to those with nitrate ( approximately 12 to 15 mumol.liter(-1) h(-1)). A pure culture capable of selenate-dependent anaerobic arsenite oxidation (strain ML-SRAO) was isolated from Mono Lake water into a defined salts medium with selenate, arsenite, and yeast extract. This strain does not grow chemoautotrophically, but it catalyzes the oxidation of arsenite during growth on an organic carbon source with selenate. No arsenate was produced in pure cultures amended with arsenite and nitrate or oxygen, indicating that the process is selenate dependent. Experiments with washed cells in mineral medium demonstrated that the oxidation of arsenite is tightly coupled to the reduction of selenate. Strain ML-SRAO grows optimally on lactate with selenate or arsenate as the electron acceptor. The amino acid sequences deduced from the respiratory arsenate reductase gene (arrA) from strain ML-SRAO are highly similar (89 to 94%) to those from two previously isolated Mono Lake arsenate reducers. The 16S rRNA gene sequence of strain ML-SRAO places it within the Bacillus RNA group 6 of gram-positive bacteria having low G+C content.     

Arsenate respiratory reductase gene (arrA) for Desulfosporosinus sp. strain Y5

Desulfosporosinus sp. strain Y5 is a spore-forming bacterium capable of dissimilatory arsenate reduction coupled to the oxidation of aromatic compounds. In arsenate respiration, the arsenate respiratory reductase (ARR) catalyzes the reduction of arsenate to arsenite. Our objective is to characterize the arrA gene, encoding the ARR, for Desulfosporosinus sp. strain Y5. Oligonucleotide primers were designed based on the few arrA gene sequences available at the time and validated against positive and negative controls. The resulting arrA-amplicon of approximately 2.0kb was cloned and sequenced. The arrA from Desulfosporosinus sp. Y5 is closely related to Desulfitobacterium hafniense (similarity of 77% and 81% at the nucleotide and amino acid levels, respectively). Phylogenetic topology based on the arrA gene was partially congruent with that of 16S rRNA-based analysis. This arrA sequence will support the development of specific tracking probes for Desulfosporosinus sp. Y5 and the molecular characterization and monitoring of dissimilatory arsenate reducing bacteria.     

Molecular cloning and characterization of the srdBCA operon, encoding the respiratory selenate reductase complex, from the selenate-reducing bacterium Bacillus selenatarsenatis SF-1

Previously, we isolated a selenate- and arsenate-reducing bacterium, designated strain SF-1, from selenium-contaminated sediment and identified it as a novel species, Bacillus selenatarsenatis. B. selenatarsenatis strain SF-1 independently reduces selenate to selenite, arsenate to arsenite, and nitrate to nitrite by anaerobic respiration. To identify the genes involved in selenate reduction, 17 selenate reduction-defective mutant strains were isolated from a mutant library generated by random insertion of transposon Tn916. Tn916 was inserted into the same genome position in eight mutants, and the representative strain SF-1AM4 did not reduce selenate but did reduce nitrate and arsenate to the same extent as the wild-type strain. The disrupted gene was located in an operon composed of three genes designated srdBCA, which were predicted to encode a putative oxidoreductase complex by the BLASTX program. The plasmid vector pGEMsrdBCA, containing the srdBCA operon with its own promoter, conferred the phenotype of selenate reduction in Escherichia coli DH5α, although E. coli strains containing plasmids lacking any one or two of the open reading frames from srdBCA did not exhibit the selenate-reducing phenotype. Domain structure analysis of the deduced amino acid sequence revealed that SrdBCA had typical features of membrane-bound and molybdopterin-containing oxidoreductases. It was therefore proposed that the srdBCA operon encoded a respiratory selenate reductase complex. This is the first report of genes encoding selenate reductase in gram-positive bacteria.     

Redox cycling of arsenic by the hydrothermal marine bacterium Marinobacter santoriniensis

Marinobacter santoriniensis NKSG1^T is a mesophilic, dissimilatory arsenate-reducing and arsenite-oxidizing bacterium isolated from an arsenate-reducing enrichment culture. The inoculum was obtained from arsenic-rich shallow marine hydrothermal sediment from Santorini, Greece, with evidence of arsenic redox cycling. Growth studies demonstrated M. santoriniensis NKSG1^T is capable of conserving energy from the reduction of arsenate [As(V)] with acetate or lactate as the electron donor, and of oxidizing arsenite [As(III)] heterotrophically with oxygen as the electron acceptor. The oxidation of As(III) coincided with the expression of the aoxB gene encoding for the catalytic molybdopterin subunit of the heterodimeric arsenite oxidase operon, indicating the reaction is enzymatically controlled, and M. santoriniensis NKSG1^T is a heterotrophic As(III)-oxidizing bacterium. Although it is clear that this organism also performs dissimilatory As(V) reduction, no amplification of the arrA arsenate reductase gene was attained using a range of primers and PCR conditions. Marinobacter santoriniensis NKSG1^T belongs to a genus of bacteria widely occurring in marine environments, including hydrothermal sediments, and is among the first marine bacteria shown to be capable of either anaerobic As(V) respiration or aerobic As(III) oxidation.     

Isolation and characterization of an arsenate-reducing bacterium and its application for arsenic extraction from contaminated soil

A Gram-negative anaerobic bacterium, Citrobacter sp. NC-1, was isolated from soil contaminated with arsenic at levels as high as 5,000 mg As kg⁻¹. Strain NC-1 completely reduced 20 mM arsenate within 24 h and exhibited arsenate-reducing activity at concentrations as high as 60 mM. These results indicate that strain NC-1 is superior to other dissimilatory arsenate-reducing bacteria with respect to arsenate reduction, particularly at high concentrations. Strain NC-1 was also able to effectively extract arsenic from contaminated soils via the reduction of solid-phase arsenate to arsenite, which is much less adsorptive than arsenate. To characterize the reductase systems in strain NC-1, arsenate and nitrate reduction activities were investigated using washed-cell suspensions and crude cell extracts from cells grown on arsenate or nitrate. These reductase activities were induced individually by the two electron acceptors. This may be advantageous during bioremediation processes in which both contaminants are present.     

Oxidation of Arsenite by a Soil Isolate of Alcaligenes

A strain of Alcaligenes , isolated from soil and grown in nutrient broth in the presence of arsenite, possessed the ability to oxidize arsenite to arsenate. Washed cell suspensions consumed one-half mol of oxygen/mol of arsenite and produced arsenate. The optimum pH for arsenite oxidation was 7.0. The K_m for arsenite was 1.5 × 10^-4 M and V _max was 6.7 μl of oxygen/min. The arsenite-oxidizing enzyme system was induced by growth in arsenite. Response of the arsenite-oxidizing enzyme system to respiratory inhibitors suggested that electrons resulting from arsenite oxidation by an oxido-reductase with a bound flavin are transferred via cytochrome c and cytochrome oxidase to oxygen. The presence of the cytochromes in crude extract was confirmed by spectral measurements.     

Dissimilatory arsenate reduction with sulfide as electron donor: experiments with mono lake water and Isolation of strain MLMS-1, a chemoautotrophic arsenate respirer

Anoxic bottom water from Mono Lake, California, can biologically reduce added arsenate without any addition of electron donors. Of the possible in situ inorganic electron donors present, only sulfide was sufficiently abundant to drive this reaction. We tested the ability of sulfide to serve as an electron donor for arsenate reduction in experiments with lake water. Reduction of arsenate to arsenite occurred simultaneously with the removal of sulfide. No loss of sulfide occurred in controls without arsenate or in sterilized samples containing both arsenate and sulfide. The rate of arsenate reduction in lake water was dependent on the amount of available arsenate. We enriched for a bacterium that could achieve growth with sulfide and arsenate in a defined, mineral medium and purified it by serial dilution. The isolate, strain MLMS-1, is a gram-negative, motile curved rod that grows by oxidizing sulfide to sulfate while reducing arsenate to arsenite. Chemoautotrophy was confirmed by the incorporation of H(14)CO(3)(-) into dark-incubated cells, but preliminary gene probing tests with primers for ribulose-1,5-biphosphate carboxylase/oxygenase did not yield PCR-amplified products. Alignment of 16S rRNA sequences indicated that strain MLMS-1 was in the delta-Proteobacteria, located near sulfate reducers like Desulfobulbus sp. (88 to 90% similarity) but more closely related (97%) to unidentified sequences amplified previously from Mono Lake. However, strain MLMS-1 does not grow with sulfate as its electron acceptor.     

Arsenic-resistant proteobacterium from the phyllosphere of arsenic-hyperaccumulating fern (Pteris vittata L.) reduces arsenate to arsenite

An arsenic-resistant bacterium, AsRB1, was isolated from the fronds of Pteris vittata grown in a site contaminated with copper chromium arsenate. The bacterium exhibited resistance to arsenate, arsenite, and antimony in the culture medium. AsRB1, like Pseudomonas putida, grew on MacConkey and xylose-lactose-desoxycholate agars and utilized citrate but, unlike P. putida, was positive for indole test and negative for oxidase test. A phylogenetic analysis of the 16S rRNA gene showed that AsRB1 is a proteobacterium of the beta subclass, related to Pseudomonas saccharophila and Variovorax paradoxus. Following an exogenous supply of arsenate, most arsenic occurred as arsenite in the medium and the cell extracts, suggesting reduction and extrusion of arsenic as the mechanism for arsenic resistance in AsRB1.     

Dissimilatory Arsenate Reduction with Sulfide as Electron Donor: Experiments with Mono Lake Water and Isolation of Strain MLMS-1, a Chemoautotrophic Arsenate Respirer

Anoxic bottom water from Mono Lake, California, can biologically reduce added arsenate without any addition of electron donors. Of the possible in situ inorganic electron donors present, only sulfide was sufficiently abundant to drive this reaction. We tested the ability of sulfide to serve as an electron donor for arsenate reduction in experiments with lake water. Reduction of arsenate to arsenite occurred simultaneously with the removal of sulfide. No loss of sulfide occurred in controls without arsenate or in sterilized samples containing both arsenate and sulfide. The rate of arsenate reduction in lake water was dependent on the amount of available arsenate. We enriched for a bacterium that could achieve growth with sulfide and arsenate in a defined, mineral medium and purified it by serial dilution. The isolate, strain MLMS-1, is a gram-negative, motile curved rod that grows by oxidizing sulfide to sulfate while reducing arsenate to arsenite. Chemoautotrophy was confirmed by the incorporation of H¹⁴CO₃⁻ into dark-incubated cells, but preliminary gene probing tests with primers for ribulose-1,5-biphosphate carboxylase/oxygenase did not yield PCR-amplified products. Alignment of 16S rRNA sequences indicated that strain MLMS-1 was in the δ-Proteobacteria, located near sulfate reducers like Desulfobulbus sp. (88 to 90% similarity) but more closely related (97%) to unidentified sequences amplified previously from Mono Lake. However, strain MLMS-1 does not grow with sulfate as its electron acceptor.     

Characterization of As(III) oxidizing <Emphasis Type="Italic">Achromobacter</Emphasis> sp. strain N2: effects on arsenic toxicity and translocation in rice

Achromobacter sp. strain N2 was isolated from a pyrite-cinder-contaminated soil and presented plant growth promoting traits (ACC deaminase activity, production of indole-3-acetic and jasmonic acids, siderophores secretion, and phosphate solubilization) and arsenic transformation abilities. Achromobacter sp. strain N2 was resistant to different metals and metalloids, including arsenate (100 mM) and arsenite (5 mM). The strain was resistant to ionic stressors (i.e., arsenate and NaCl), whereas bacterial growth was impaired by osmotic stress. Strain N2 was able to oxidize 1.0 mmol L^?1 of arsenite to arsenate in 72 h. This evidence was supported by the retrieval of an arsenite oxidase AioA gene highly homologous to arsenite oxidases of Achromobacter and Alcaligenes species. Rice seeds of Oryza sativa (var. Loto) were bio-primed with ACCD-induced and non-induced cells in order to evaluate the effect of inoculation on rice seedlings growth and arsenic uptake. The bacterization with ACCD-induced cells significantly improved seed germination and seedling heights if compared with the seeds inoculated with non-induced cells and non-primed seeds. Enhanced arsenic uptake was evidenced in the presence of ACCD-induced cells, suggesting a role of ACCD activity on the mitigation of the toxicity of arsenic accumulated by the plant. This kind of responses should be taken into account when proposing PGP strains for improving plant growth in arsenic-rich soils.     

Release of Arsenic from Soil by a Novel Dissimilatory Arsenate-Reducing Bacterium,Anaeromyxobacter sp. Strain PSR-1

A novel arsenate-reducing bacterium, designated strain PSR-1, was isolated from arsenic-contaminated soil. Strain PSR-1 was phylogenetically closely related to Anaeromyxobacter dehalogenans 2CP-1^T with 16S rRNA gene similarity of 99.7% and coupled the oxidation of acetate with the reduction of arsenate. Arsenate reduction was inhibited almost completely by respiratory inhibitors such as dicumarol and 2-heptyl-4-hydroxyquinoline N-oxide. Strain PSR-1 also utilized soluble Fe(III), ferrihydrite, nitrate, oxygen, and fumarate as electron acceptors. Strain PSR-1 catalyzed the release of arsenic from arsenate-adsorbed ferrihydrite. In addition, inoculation of washed cells of strain PSR-1 into sterilized soil successfully reproduced arsenic release. Arsenic K-edge X-ray absorption near-edge structure (XANES) analysis revealed that the proportion of arsenite in the soil solid phase actually increased from 20% to 50% during incubation with washed cells of strain PSR-1. These results suggest that strain PSR-1 is capable of reducing not only dissolved arsenate but also arsenate adsorbed on the soil mineral phase. Arsenate reduction by strain PSR-1 expands the metabolic versatility of Anaeromyxobacter dehalogenans. Considering its distribution throughout diverse soils and anoxic sediments, Anaeromyxobacter dehalogenans may play a role in arsenic release from these environments.     

Molybdenum-containing arsenite oxidase of the chemolithoautotrophic arsenite oxidizer NT-26

The chemolithoautotroph NT-26 oxidizes arsenite to arsenate by using a periplasmic arsenite oxidase. Purification and preliminary characterization of the enzyme revealed that it (i) contains two heterologous subunits, AroA (98 kDa) and AroB (14 kDa); (ii) has a native molecular mass of 219 kDa, suggesting an alpha2beta2 configuration; and (iii) contains two molybdenum and 9 or 10 iron atoms per alpha2beta2 unit. The genes that encode the enzyme have been cloned and sequenced. Sequence analyses revealed similarities to the arsenite oxidase of Alcaligenes faecalis, the putative arsenite oxidase of the beta-proteobacterium ULPAs1, and putative proteins of Aeropyrum pernix, Sulfolobus tokodaii, and Chloroflexus aurantiacus. Interestingly, the AroA subunit was found to be similar to the molybdenum-containing subunits of enzymes in the dimethyl sulfoxide reductase family, whereas the AroB subunit was found to be similar to the Rieske iron-sulfur proteins of cytochrome bc1 and b6f complexes. The NT-26 arsenite oxidase is probably exported to the periplasm via the Tat secretory pathway, with the AroB leader sequence used for export. Confirmation that NT-26 obtains energy from the oxidation of arsenite was obtained, as an aroA mutant was unable to grow chemolithoautotrophically with arsenite. This mutant could grow heterotrophically in the presence of arsenite; however, the arsenite was not oxidized to arsenate.     

Arsenate reduction: thiol cascade chemistry with convergent evolution

The frequent abundance of arsenic in the environment has guided the evolution of enzymes for the reduction of arsenate. The arsenate reductases (ArsC) from different sources have unrelated sequences and structural folds, and can be divided into different classes on the basis of their structures, reduction mechanisms and the locations of catalytic cysteine residues. The thioredoxin-coupled arsenate reductase class is represented by Staphylococcus aureus pI258 ArsC and Bacillus subtilis ArsC. The ArsC from Escherichia coli plasmid R773 and the eukaryotic ACR2p reductase from Saccharomyces cerevisiae represent two distinct glutaredoxin-linked ArsC classes. All are small cytoplasmic redox enzymes that reduce arsenate to arsenite by the sequential involvement of three different thiolate nucleophiles that function as a redox cascade. In contrast, the ArrAB complex is a bacterial heterodimeric periplasmic or a surface-anchored arsenate reductase that functions as a terminal electron acceptor and transfers electrons from the membrane respiratory chain to arsenate. Finally, the less well documented arsenate reductase activity of the monomeric arsenic(III) methylase, which is an S-adenosylmethionine (AdoMet)-dependent methyltransferase. After each oxidative methylation cycle and before the next methylation step, As(V) is reduced to As(III). Methylation by this enzyme is also considered an arsenic-resistance mechanism for bacteria, fungi and mammals.     

Enzyme phylogenies as markers for the oxidation state of the environment: The case of respiratory arsenate reductase and related enzymes

Background  

Phylogenies of certain bioenergetic enzymes have proved to be useful tools for deducing evolutionary ancestry of bioenergetic pathways and their relationship to geochemical parameters of the environment. Our previous phylogenetic analysis of arsenite oxidase, the molybdopterin enzyme responsible for the biological oxidation of arsenite to arsenate, indicated its probable emergence prior to the Archaea/Bacteria split more than 3 billion years ago, in line with the geochemical fact that arsenite was present in biological habitats on the early Earth. Respiratory arsenate reductase (Arr), another molybdopterin enzyme involved in microbial arsenic metabolism, serves as terminal oxidase, and is thus situated at the opposite end of bioenergetic electron transfer chains as compared to arsenite oxidase. The evolutionary history of the Arr-enzyme has not been studied in detail so far.