Microbial populations responsible for the oxidation and reduction of As were examined in unsaturated (aerobic) soil columns treated with 75 microM arsenite [As(III)] or 250 microM arsenate [As(V)]. Arsenite [As(III)] was rapidly oxidized to As(V) via microbial activity, whereas no apparent reduction of As(V) was observed in the column experiments. Eight aerobic heterotrophic bacteria with varying As redox phenotypes were isolated from the same columns. Three isolates, identified as Agrobacterium tumefaciens-, Pseudomonas fluorescens-, and Variovorax paradoxus-like organisms (based on 16S sequence), were As(III) oxidizers, and all were detected in community DNA fingerprints generated by PCR coupled with denaturing gradient gel electrophoresis. The five other isolates were identified (16S gene sequence) as A. tumefaciens, Flavobacterium sp., Microbacterium sp., and two Arthrobacter sp. -like organisms and were shown to rapidly reduce As(V) under aerobic conditions. Although the two A. tumefaciens-like isolates exhibited opposite As redox activity,their 16S rDNA sequences (approximately 1400 bp) were 100% identical, and both were shown to contain putative arsC genes. Our results support the hypothesis that bacteria capable of either oxidizing As(III) or reducing As(V) coexist and are ubiquitous in soil environments, suggesting that the relative abundance and metabolic activity of specific microbial populations plays an important role in the speciation of inorganic As in soil pore waters.

Seminal regulatory controls of microbial arsenite [As(III)] oxidation are described in this study. Transposon mutagenesis of Agrobacterium tumefaciens identified genes essential for As(III) oxidation, including those coding for a two-component signal transduction pair. The transposon interrupted a response regulator gene (referred to as aoxR), which encodes an ntrC-like protein and is immediately downstream of a gene (aoxS) encoding a protein with primary structural features found in sensor histidine kinases. The structural genes for As(III) oxidase (aoxAB), a c-type cytochrome (cytc 2 ), and molybdopterin biosynthesis (chlE) were downstream of aoxR. The mutant could not be complemented by aoxSR in trans but was complemented by a clone containing aoxS-aoxR-aoxA-aoxB-cytc 2 and consistent with reverse transcriptase (RT) PCR experiments, which demonstrated these genes are cotranscribed as an operon. Expression of aoxAB was monitored by RT-PCR and found to be up-regulated by the addition of As(III) to cell cultures. Expression of aoxAB was also controlled in a fashion consistent with quorum sensing in that (i) expression of aoxAB was absent in As(III)-unexposed early-log-phase cells but was observed in As(III)-unexposed, late-log-phase cells and (ii) treating As(III)-unexposed, early-log-phase cells with ethyl acetate extracts of As(III)-unexposed, late-log-phase culture supernatants also resulted in aoxAB induction. Under inducing conditions, aoxS expression was readily observed in the wild-type strain but significantly reduced in the mutant, indicating that AoxR is autoregulatory and at least partially controls the expression of the aox operon. In summary, regulation of A. tumefaciens As(III) oxidation is complex, apparently being controlled by As(III) exposure, a two-component signal transduction system, and quorum sensing.Arsenic (As) is a known carcinogen, occurring in the environment primarily as the oxyanion arsenate (H 2 AsO 4 Ϫ , HAsO 4 2Ϫ ) [As(V)] and arsenite (H 3 AsO 3 0 ) [As(III)]. As(III) is more toxic and generally the more mobile species (reviewed in reference 6), and hence, there is significant interest in understanding the factors that control As speciation in the environment. Both abiotic and biotic factors are involved; however, it is now generally viewed that microbial As redox transformations are important, if not principal, drivers controlling As speciation in soils, sediments, and natural waters (6, 20).Microbial As(III) oxidation and As(V) reduction activities are cellular strategies for either detoxification or for generating energy. A fairly detailed model explaining the genetics, regulation, and function of detoxification-based As(V) reduction is in place (32), but beyond that little is known. Genes encoding respiratory As(V) reductases have been cloned and enzyme characterizations are under way (recently reviewed in reference 32). Likewise, genes encoding As(III) oxidase have also recently been cloned and characterized (17,29), and extensive enzyme characterization has been achieved (...

A novel bacterium was cultivated from an extreme thermal soil in Yellowstone National Park, Wyoming, USA, that at the time of sampling had a pH of 3.9 and a temperature range of 65-92 degrees C. This organism was found to be an obligate aerobic, non-spore-forming rod, and formed pink-colored colonies. Phylogenetic analysis of the 16S rRNA gene sequence placed this organism in a clade composed entirely of environmental clones most closely related to the phyla Chloroflexi and Thermomicrobia. This bacterium stained gram-positive, contained a novel fatty-acid profile, had cell wall muramic acid content similar to that of Bacillus subtilis (significantly greater than Escherichia coli), and failed to display a lipopolysaccharide profile in SDS-polyacrylamide gels that would be indicative of a gram-negative cell wall structure. Ultrastructure examinations with transmission electron microscopy showed a thick cell wall (approximately 34 nm wide) external to a cytoplasmic membrane. The organism was not motile under the culture conditions used, and electron microscopic examination showed no evidence of flagella. Genomic G+C content was 56.4 mol%, and growth was optimal at 67 degrees C and at a pH of 7.0. This organism was able to grow heterotrophically on various carbon compounds, would use only oxygen as an electron acceptor, and its growth was not affected by light. A new species of a novel genus is proposed, with YNP1(T) (T=type strain) being Thermobaculum terrenum gen. nov., sp. nov. (16S rDNA gene GenBank accession AF391972). This bacterium has been deposited in the American Type Culture Collection (ATCC BAA-798) and the University of Oregon Culture Collection of Microorganisms from Extreme Environments (CCMEE 7001).

Transposon Tn5-B22 mutagenesis was used to identify genetic determinants required for arsenite [As(III)] oxidation in an Agrobacterium tumefaciens soil isolate, strain 5A. In one mutant, the transposon interrupted modB, which codes for the permease component of a high-affinity molybdate transporter. In a second mutant, the transposon insertion occurred in mrpB, which is part of a seven-gene operon encoding an Mrp-type Na ؉ :H ؉ antiporter complex. Complementation experiments with mod and mrp operons PCR cloned from the genomesequenced A. tumefaciens strain C58 resulted in complementation back to an As(III)-oxidizing phenotype, confirming that these genes encode activities essential for As(III) oxidation in this strain of A. tumefaciens. As expected, the mrp mutant was extremely sensitive to NaCl and LiCl, indicating that the Mrp complex in A. tumefaciens is involved in Na ؉ circulation across the membrane. Gene expression studies (lacZ reporter and reverse transcriptase PCR experiments) failed to show evidence of transcriptional regulation of the mrp operon in response to As(III) exposure, whereas expression of the mod operon was found to be up-regulated by As(III) exposure. In each mutant, the loss of As(III)-oxidizing capacity resulted in conversion to an arsenate [As(V)]-reducing phenotype. Neither mutant was more sensitive to As(III) than the parental strain.Microbe-arsenic interactions are viewed as a major driver of arsenic (As) chemical speciation in nature, which in turn is a critical factor in determining As fate and transport in the environment (reviewed in references 19 and 38). Many types of As transformations have been documented in various microorganisms (6,14,18,34,41,47), although those currently seen to dominate As speciation in the environment involve either arsenite [H 3 AsO 3 , As(III)] oxidation or arsenate [HAsO 4 2Ϫ , As(V)] reduction. These redox transformation reactions are generally thought to be used by microorganisms either for detoxification or for generating cellular energy to support growth (see reviews in references 19, 38, 49 and 50).Detoxification-based As(V) reduction has been documented to occur in microorganisms throughout the domains Bacteria and Archaea (38, 49), and involves As(V) reduction to As(III) via an As(V) reductase, with the As(III) then extruded by the ArsB efflux pump that is efficient at removing As(III) and antimonite [Sb(III)]. This process, as well as the genes encoding the enzymes and regulatory proteins involved, has been extensively studied and recently reviewed by Silver and Phung (49). Dissimilatory As(V) reduction has also been documented to occur in numerous prokaryotes in both prokaryotic domains (38), although its original discovery was more recent (2), and as a consequence, far less is known about the genetic determinants required for anaerobic As(V) respiration. Dissimilatory As(V) reductases from Chrysiogenes arsenatis (25) and Bacillus selenitireducens (1) have been purified and characterized, and the arr genes have been identified in Shewanella...