A new primer set was designed to specifically amplify ca. 1,100 bp of aoxB genes encoding the As(III) oxidase catalytic subunit from taxonomically diverse aerobic As(III)-oxidizing bacteria. Comparative analysis of AoxB protein sequences showed variable conservation levels and highlighted the conservation of essential amino acids and structural motifs. AoxB phylogeny of pure strains showed well-discriminated taxonomic groups and was similar to 16S rRNA phylogeny. Alphaproteobacteria-, Betaproteobacteria-, and Gammaproteobacteria-related sequences were retrieved from environmental surveys, demonstrating their prevalence in mesophilic Ascontaminated soils. Our study underlines the usefulness of the aoxB gene as a functional marker of aerobic As(III) oxidizers.Arsenic (As) exists mainly in two toxic soluble forms, arsenite, As(III), and arsenate, As(V), with the latter tending to associate with some oxyhydroxides and clay minerals. The bacterial oxidation of As(III) can thus contribute to a natural attenuation of As contamination by decreasing As bioavailability. These properties have recently been used to develop a bioprocess for removing As from a mining effluent by using the activity of As-metabolizing bacteria indigenous to the contaminated site (4). The feasibility of such a process depends on a good knowledge of the ability of the indigenous microflora to oxidize As(III) and requires reliable methods for detecting, identifying, and monitoring As(III) oxidizers in the environment.More than 50 phylogenetically diverse As(III)-oxidizing strains distributed among 25 genera have been isolated from various environments so far. Bacterial aerobic As(III) oxidation is performed by a dedicated enzyme, the As(III) oxidase (1,36,40), which belongs to the dimethyl sulfoxide (DMSO) reductase of the molybdenum family (9). In Alcaligenes faecalis, it is an ␣ 1  1 heterodimer comprising a large subunit incorporating a molybdenum center and a [3Fe-4S] cluster and a small subunit incorporating a Rieske-type [2Fe-2S] cluster (9). Genes encoding these subunits are cotranscribed as an operon and have been successively characterized in Herminiimonas arsenicoxydans (26), Rhizobium sp. strain , and Agrobacterium tumefaciens (21). They have also been found in the genome of Chloroflexus aurantiacus, on a plasmid in Thermus thermophilus, in two aerobic thermophilic As(III) oxidizers, and in the genome of strains for which the ability to oxidize As(III) has not been experimentally proven (27).Due to the polyphyly of As(III)-oxidizing bacteria, the aoxB gene encoding the catalytic subunit of the enzyme seems to be a valuable molecular marker for investigating its ecology and the potential of As(III) oxidation in the environment. To this end, a recent study described primers targeting the first quarter of the aoxB gene to detect its presence and expression in the environment and suggested that the gene is widely distributed among the Bacteria and also is widespread in soil-water systems containing As (16).In our present study, we designed...
Aims: To select an autotrophic arsenic(III)-oxidizing population, named CASO1, and to evaluate the performance of the selected bacteria in reactors. Methods and Results: An As(III)-containing medium without organic substrate was used to select CASO1 from a mining environment. As(III) oxidation was studied under batch and continuous conditions. The main organisms present in CASO1 were identified with molecular biology tools. CASO1 exhibited significant As(III)-oxidizing activity between pH 3 and 8. The optimum temperature was 25°C. As(III) oxidation was still observed in the presence of 1000 mg l )1 As(III). In continuous culture mode, the As(III) oxidation rate reached 160 mg l )1 h )1 . The CASO1 consortium contains at least two organisms -strain b3, which is phylogenetically close to Ralstonia picketii, and strain b6, which is related to the genus Thiomonas. The divergence in 16S rDNA sequences between b6 and the closest related organism was 5AE9%, suggesting that b6 may be a new species. Conclusions: High As(III)-oxidizing activity can be obtained without organic nutrient supply, using a bacterial population from a mining environment. Significance and Impact of the Study: The biological oxidation of arsenite by the CASO1 population is of particular interest for decontamination of arsenic-contaminated waste or groundwater.
A novel bacterium, strain b6(T) (T=type strain), was isolated from a disused mine site by growth using arsenite [As(III)] as energy source in a simple mineral medium. Cells of strain b6(T) were rod-shaped, Gram-negative, non-sporulating and motile. Optimum growth occurred at temperatures between 20 and 30 degrees C, and at pH between 4.0 and 7.5. Strain b6(T) grew chemoautotrophically on As(III), sulphur and thiosulphate, and also heterotrophically on yeast extract and a variety of defined organic compounds. Several other Thiomonas strains, including the type species Thiomonas (Tm.) intermedia, were able to oxidize As(III), though only strain b6(T) and strain NO115 could grow using As(III) as sole energy source in the absence of any organic compound. The G+C content of the DNA of strain b6(T) was 65.1 mol %. Comparative small subunit (SSU) ribosomal RNA (rRNA) analysis indicated that strain b6(T) belongs to the genus Thiomonas in the beta-subdivision of the Proteobacteria. It was closely related to an unnamed Thiomonas strain (NO115) isolated from a Norwegian mining site, though sequence identities between strain b6(T) and characterized Thiomonas species were less than 95%. DNA-DNA hybridization between strain b6(T) and the type species of the genus Tm. intermedia showed less than 50% homology. On the basis of phylogenetic and phenotypic characteristics, strain b6(T) (DSM 16361(T), LMG 22795(T)) is proposed as the type strain of the new species Thiomonas arsenivorans, sp. nov.
Denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR) were successfully developed to monitor functional aoxB genes as markers of aerobic arsenite oxidizers. DGGE profiles showed a shift in the structure of the aoxB-carrying bacterial population, composed of members of the Alpha-, Beta-and Gammaproteobacteria, depending on arsenic (As) and E h levels in Upper Isle River Basin waters. The highest aoxB gene densities were found in the most As-polluted oxic surface waters but without any significant correlation with environmental factors. Arsenite oxidizers seem to play a key role in As mobility in As-impacted waters.Arsenic (As) occurs naturally as a local geological constituent of the soils surrounding the Upper Isle River Basin (Massif Central, France) due to natural geochemical anomalies but is also released from Au/As deposits of disused gold mines (4,11,12,33). Important variations in dissolved As concentrations are found in the Isle River and depend on the hydrogeological season, with maximum values in spring and summer generally detected during low-flow conditions (12,22), and probably on temperature-controlled microbial As(V) reduction and/or microbial dissolution of solid As carrier phases (22). Two toxic inorganic forms of As are usually detected in aquatic system: arsenite, As(III), which is found mainly under anaerobic conditions and is more mobile than arsenate, As(V), which typically occurs under aerobic conditions and tends to associate with oxyhydroxides and clay minerals (11,34). Although bacteria are known to play a key role in speciation, mobility, and bioavailability of As in the environment, they have never been considered in previous studies of As mobility in the Isle River system. Indeed, former investigations of As cycling were focused on geochemical studies (4,11,12,22,33).As(III)-oxidizing bacteria can contribute to a natural attenuation of As pollution by decreasing its bioavailability and can help remove As from mine wastewaters through bioprocessing (1, 2). Many As(III) oxidizers have been isolated from various environments, especially mesophilic ecosystems (3,5,8,16,25,27,32,38). They belong to more than 25 genera, mainly of the Proteobacteria phylum (3, 32, 38), and are related to organisms unable to oxidize As(III) based on 16S rRNA phylogeny. Diverse primer sets have been successfully developed to specifically target the functional aoxB gene (9,14,17,25,26), encoding the large molybdenum-bearing catalytic subunit of As(III)-oxidase (EC 1.20.98.1), an enzyme of the dimethyl sulfoxide (DMSO) reductase family. Using cloning-sequencing approaches, the aoxB gene has proven to be a reliable molecular marker for diversity studies of the polyphyletic aerobic As(III) oxidizers in As-impacted soil and water systems (17, 25). The genetic fingerprinting denaturing gradient gel electrophoresis (DGGE) technique is one useful tool for spatial, temporal, and geographical monitoring of complex bacterial population structure (23, 24). Quantitative real-time PCR (qPCR) p...
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