In this study, new strains were isolated from an environment with elevated arsenic levels, Sainte-Marieaux-Mines (France), and the diversity of aoxB genes encoding the arsenite oxidase large subunit was investigated. The distribution of bacterial aoxB genes is wider than what was previously thought. AoxB subfamilies characterized by specific signatures were identified. An exhaustive analysis of AoxB sequences from this study and from public databases shows that horizontal gene transfer has likely played a role in the spreading of aoxB in prokaryotic communities.Arsenic, which is one of the most toxic metalloids, is distributed ubiquitously but not uniformly around the world. Levels of arsenic differ considerably from one geographical region to another, depending on the geochemical characteristics of the soil (natural contamination) and the industrial activities carried out in the vicinity (anthropogenic contamination) (22). In aquatic environments, arsenic occurs mainly in the form of the inorganic species arsenate [As(V)] and arsenite [As(III)]; the latter species, which is more bioavailable, is usually thought to have more-toxic effects on prokaryotes than As(V) (34). As(III) oxidation leads to the formation of the less available form As(V), which can either precipitate with iron [Fe(III)] or be adsorbed by ferrihydrite. The oxidation process may be mediated by microbial activities, which contribute to the natural remediation processes observed in contaminated environments (21,26,27,34). Consequently, bioprocesses for the treatment of arsenic-contaminated waters have been developed based on the precipitation or adsorption of the As(V) produced by bacteria (4, 9, 21). Some well-known prokaryotes oxidize As(III) into As(V) under aerobic (e.g., Herminiimonas arsenicoxydans, Thiomonas spp., or Rhizobium sp. strain NT26) or anaerobic (e.g., Alkalilimnicola ehrlichii) conditions as part of a detoxification process (12,17,31,32,39). Some chemolithotrophs also use arsenite as an electron donor (e.g., Rhizobium sp. strain NT26 or Thiomonas arsenivorans) (5, 32). The aerobic arsenite oxidases involved in such processes are heterodimers consisting of a large subunit with a molybdenum center and a [3Fe-4S] cluster (AroA, AsoA, and AoxB) and a small subunit containing a Rieske-type [2Fe-2S] cluster (AroB, AsoB, and AoxA) (1, 13). The large subunit in these enzymes is similar to that found in other members of the dimethyl sulfoxide (DMSO) reductase family of molybdenum enzymes but is clearly phylogenetically divergent from the respiratory arsenate reductases (ArrA) or other proteins of the DMSO reductase family of molybdenum oxidoreductases, such as the new arsenite reductase described recently for Alkalilimnicola ehrlichii (25,31,40).aox genes have been identified in 25 bacterial and archaeal genera isolated from various arsenic-rich environments, most of which belong to the Alpha-, Beta-, or Gammaproteobacteria phylum (7,10,12,14,23,25,29,32,37). Recent studies based on environmental DNA extracted from soils, sediments, a...

The arsenic resistance gene cluster of Microbacterium sp. A33 contains a novel pair of genes (arsTX) encoding a thioredoxin system that are cotranscribed with an unusual arsRC2 fusion gene, ACR3, and arsC1 in an operon divergent from arsC3. The whole ars gene cluster is required to complement an Escherichia coli ars mutant. ArsRC2 negatively regulates the expression of the pentacistronic operon. ArsC1 and ArsC3 are related to thioredoxin-dependent arsenate reductases; however, ArsC3 lacks the two distal catalytic cysteine residues of this class of enzymes.Arsenic is widely dispersed in the environment and occurs primarily in two oxidation states, arsenate [As(V)] and arsenite [As(III)], and both are toxic to the majority of living organisms. The frequent abundance of arsenic in all environmental compartments has guided the evolution of detoxification systems in almost all microorganisms. Of these, the arsenic resistance system (ars) appears to be widely distributed among prokaryotes. It involves an arsenate reductase (ArsC), an arsenite efflux pump (ArsB or ACR3), and a transcriptional repressor (ArsR) (32), encoded by a set of genes that display large variations in their number and genomic organization. The early identified ars system of Escherichia coli plasmid R773 (41) has two additional components, ArsA, which acts as the catalytic subunit of the ArsAB arsenite extrusion pump (33), and ArsD, a metallochaperone protein that transfers As(III) to ArsA (18). In addition to these well-studied ars components, a variety of ars clusters contain additional genes whose functions in arsenic resistance have not been clearly established in many cases (31).Members of the Microbacterium lineage of actinobacteria that can tolerate various metals, including nickel, chromium, and uranium (1,16,25), have been isolated from metal-rich environments. New examples of arsenic-resistant isolates of Microbacterium are continuously being reported (1,2,8,10,12,21). In each case, however, the tolerance mechanism was not investigated, probably due to the lack of efficient genetic systems in this genus. Among actinobacteria, only Streptomyces sp. FR-008 (40) and Corynebacterium glutamicum ATCC 13032 (28) have been subjected to molecular characterization of determinants of defense against arsenic. In the former, the linear plasmid pHZ227 carries an arsenic resistance gene cluster with two novel genes, the arsO and arsT genes, which encode a putative flavin-binding monooxygenase and a putative thioredoxin reductase, respectively (40). The latter strain was recently shown to possess two members of a new class of arsenate reductases (Cg_ArsC1 and Cg_ArsC2) (30) and a transcriptional repressor (Cg_ArsR1) with a metalloid binding site unrelated to other previously characterized members of the ArsR/SmtB metalloregulatory proteins (29).The present study focuses on Microbacterium sp. strain A33, a soil isolate previously shown to tolerate high concentrations of arsenite and arsenate (2). Here, we report on the isolation and functional characteriz...

Arsenic-resistant prokaryote diversity is far from being exhaustively explored. In this study, the arsenic-adapted prokaryotic community present in a moderately arsenic-contaminated site near Sainte-Marie-aux-Mines (France) was characterized, using metaproteomic and 16S rRNA-encoding gene amplification. High prokaryotic diversity was observed, with a majority of Proteobacteria, Acidobacteria and Bacteroidetes, and a large archaeal community comprising Euryarchaeaota and Thaumarchaeota. Metaproteomic analysis revealed that Proteobacteria, Planctomycetes and Cyanobacteria are among the active bacteria in this ecosystem. Taken together, these results highlight the unsuspected high diversity of the arsenic-adapted prokaryotic community, with some phyla never having been described in highly arsenic-exposed sites.

A collection of 219 bacterial arsenic-resistant isolates was constituted from neutral arsenic mine drainage sediments. Isolates were grown aerobically or anaerobically during 21 days on solid DR2A medium using agar or gelan gum as gelling agent, with 7 mM As(III) or 20 mM As(V) as selective pressure. Interestingly, the sum of the different incubation conditions used (arsenic form, gelling agent, oxygen pressure) results in an overall increase of the isolate diversity. Isolated strains mainly belonged to Proteobacteria (63%), Actinobacteria (25%), and Bacteroidetes (10%). The most representative genera were Pseudomonas (20%), Acinetobacter (8%), and Serratia (15%) among the Proteobacteria; Rhodococcus (13%) and Microbacterium (5%) among Actinobacteria; and Flavobacterium (13%) among the Bacteroidetes. Isolates were screened for the presence of arsenic-related genes (arsB, ACR3(1), ACR3(2), aioA, arsM, and arrA). In this way, 106 ACR3(1)-, 74 arsB-, 22 aioA-, 14 ACR3(2)-, and one arsM-positive PCR products were obtained and sequenced. Analysis of isolate sensitivity toward metalloids (arsenite, arsenate, and antimonite) revealed correlations between taxonomy, sensitivity, and genotype. Antimonite sensitivity correlated with the presence of ACR3(1) mainly present in Bacteroidetes and Actinobacteria, and arsenite or antimonite resistance correlated with arsB gene presence. The presence of either aioA gene or several different arsenite carrier genes did not ensure a high level of arsenic resistance in the tested conditions.