Arsenic is the most common toxic substance in the environment, ranking first on the Superfund list of hazardous substances. It is introduced primarily from geochemical sources and is acted on biologically, creating an arsenic biogeocycle. Geothermal environments are known for their elevated arsenic content and thus provide an excellent setting in which to study microbial redox transformations of arsenic. To date, most studies of microbial communities in geothermal environments have focused on Bacteria and Archaea, with little attention to eukaryotic microorganisms. Here, we show the potential of an extremophilic eukaryotic alga of the order Cyanidiales to influence arsenic cycling at elevated temperatures. Cyanidioschyzon sp. isolate 5508 oxidized arsenite [As(III)] to arsenate [As(V)], reduced As(V) to As(III), and methylated As(III) to form trimethylarsine oxide (TMAO) and dimethylarsenate [DMAs(V)]. Two arsenic methyltransferase genes, CmarsM7 and CmarsM8, were cloned from this organism and demonstrated to confer resistance to As(III) in an arsenite hypersensitive strain of Escherichia coli. The 2 recombinant CmArsMs were purified and shown to transform As(III) into monomethylarsenite, DMAs(V), TMAO, and trimethylarsine gas, with a T opt of 60 -70°C. These studies illustrate the importance of eukaryotic microorganisms to the biogeochemical cycling of arsenic in geothermal systems, offer a molecular explanation for how these algae tolerate arsenic in their environment, and provide the characterization of algal methyltransferases.arsenic detoxification ͉ arsenic methylation ͉ As(III) S-adenosylmethyltransferase ͉ thermophile

Members of the rhodophytan order Cyanidiales are unique among phototrophs in their ability to live in extreme environments that combine low pH levels (ϳ0.2 to 4.0) and moderately high temperatures of 40 to 56°C. These unicellular algae occur in far-flung volcanic areas throughout the earth. Three genera (Cyanidium, Galdieria, and Cyanidioschyzon) are recognized. The phylogenetic diversity of culture isolates of the Cyanidiales from habitats throughout Yellowstone National Park (YNP), three areas in Japan, and seven regions in New Zealand was examined by using the chloroplast RuBisCO large subunit gene (rbcL) and the 18S rRNA gene. Based on the nucleotide sequences of both genes, the YNP isolates fall into two groups, one with high identity to Galdieria sulphuraria (type II) and another that is by far the most common and extensively distributed Yellowstone type (type IA). The latter is a spherical, walled cell that reproduces by internal divisions, with a subsequent release of smaller daughter cells. This type, nevertheless, shows a 99 to 100% identity to Cyanidioschyzon merolae (type IB), which lacks a wall, divides by "fission"-like cytokinesis into two daughter cells, and has less than 5% of the cell volume of type IA. The evolutionary and taxonomic ramifications of this disparity are discussed. Although the 18S rRNA and rbcL genes did not reveal diversity among the numerous isolates of type IA, chloroplast short sequence repeats did show some variation by location within YNP. In contrast, Japanese and New Zealand strains showed considerable diversity when we examined only the sequences of 18S and rbcL genes. Most exhibited identities closer to Galdieria maxima than to other strains, but these identities were commonly as low as 91 to 93%. Some of these Japanese and New Zealand strains probably represent undescribed species that diverged after long-term geographic isolation.The Cyanidiales are an order of asexual, unicellular red algae that are able to grow in low-pH environments and at moderately high temperatures throughout the globe (4, 31, 32). These algae are not red, but blue-green, due to their predominant pigments, c-phycocyanin and chlorophyll a. Many members of this order are known to grow at temperatures as high as 56°C and at pH levels from 0.2 to 4.0. No other photosynthetic microorganisms are known to inhabit this combination of conditions, and these algae often form well-developed mats in acidic geothermal locations. Surprisingly though, little is known about the ecology, biodiversity, and geographical distribution of these organisms. In these acidic habitats, no prokaryotic phototrophs are known to exist below a pH level of ϳ4 and the number of species reaching levels below pH 5 is small (37).The order Cyanidiales consists of three recognized genera, Cyanidium, Galdieria, and Cyanidioschyzon (6, 13, 16), referred to colloquially as "cyanidia" in this work. The genera Cyanidium and Cyanidioschyzon are thought to include a single species each, Cyanidium caldarium and Cyanidioschyzon merolae, respe...

e Arsenic and antimony are toxic metalloids and are considered priority environmental pollutants by the U.S. Environmental Protection Agency. Significant advances have been made in understanding microbe-arsenic interactions and how they influence arsenic redox speciation in the environment. However, even the most basic features of how and why a microorganism detects and reacts to antimony remain poorly understood. Previous work with Agrobacterium tumefaciens strain 5A concluded that oxidation of antimonite [Sb(III)] and arsenite [As(III)] required different biochemical pathways. Here, we show with in vivo experiments that a mutation in aioA [encoding the large subunit of As(III) oxidase] reduces the ability to oxidize Sb(III) by approximately one-third relative to the ability of the wild type. Further, in vitro studies with the purified As(III) oxidase from Rhizobium sp. strain NT-26 (AioA shares 94% amino acid sequence identity with AioA of A. tumefaciens) provide direct evidence of Sb(III) oxidation but also show a significantly decreased V max compared to that of As(III) oxidation. The aioBA genes encoding As(III) oxidase are induced by As(III) but not by Sb(III), whereas arsR gene expression is induced by both As(III) and Sb(III), suggesting that detection and transcriptional responses for As(III) and Sb(III) differ. While Sb(III) and As(III) are similar with respect to cellular extrusion (ArsB or Acr3) and interaction with ArsR, they differ in the regulatory mechanisms that control the expression of genes encoding the different Ars or Aio activities. In summary, this study documents an enzymatic basis for microbial Sb(III) oxidation, although additional Sb(III) oxidation activity also is apparent in this bacterium. T he metalloids arsenic (As) and antimony (Sb) are members of group 15 of the periodic table and are ubiquitous in the environment. Both are poisonous and have oxidation states of Ϫ3, 0, ϩ3, and ϩ5, with the last two being the most prevalent in the environment (1-5). The release of both As and Sb into the environment can occur either naturally or anthropogenically (e.g., mining), and both are considered by the U.S. Environmental Protection Agency to be priority environmental pollutants (6), with maximum drinking water standards of 10 ppb and 6 ppb for As and Sb, respectively (7). As has received more publicity due to As poisoning that has occurred and that continues (4, 8). However, Sb has emerged as a major contaminant in environments that contain mine tailings, such as those in China, Australia, New Zealand, and parts of Europe (for example, see references 5 and 9-11).Microorganisms are fundamental to elemental cycling in all environments, and this includes As (12, 13) and presumably Sb, although information for the latter is quite sparse. As cycling has been well documented and at present is thought primarily to involve arsenite [As(III)]Narsenate [As(V)] redox transformations and As methylation and demethylation reactions. As(V) is reduced for detoxification purposes (via ArsC) or respiratory...

Geothermal waters contain numerous potential electron donors capable of supporting chemolithotrophybased primary production. Thermodynamic predictions of energy yields for specific electron donor and acceptor pairs in such systems are available, although direct assessments of these predictions are rare. This study assessed the relative importance of dissolved H 2 and H 2 S as energy sources for the support of chemolithotrophic metabolism in an acidic geothermal spring in Yellowstone National Park. H 2 S and H 2 concentration gradients were observed in the outflow channel, and vertical H 2 S and O 2 gradients were evident within the microbial mat. H 2 S levels and microbial consumption rates were approximately three orders of magnitude greater than those of H 2 . Hydrogenobaculum-like organisms dominated the bacterial component of the microbial community, and isolates representing three distinct 16S rRNA gene phylotypes (phylotype ‫؍‬ 100% identity) were isolated and characterized. Within a phylotype, O 2 requirements varied, as did energy source utilization: some isolates could grow only with H 2 S, some only with H 2 , while others could utilize either as an energy source. These metabolic phenotypes were consistent with in situ geochemical conditions measured using aqueous chemical analysis and in-field measurements made by using gas chromatography and microelectrodes. Pure-culture experiments with an isolate that could utilize H 2 S and H 2 and that represented the dominant phylotype (70% of the PCR clones) showed that H 2 S and H 2 were used simultaneously, without evidence of induction or catabolite repression, and at relative rate differences comparable to those measured in ex situ field assays. Under in situ-relevant concentrations, growth of this isolate with H 2 S was better than that with H 2 . The major conclusions drawn from this study are that phylogeny may not necessarily be reliable for predicting physiology and that H 2 S can dominate over H 2 as an energy source in terms of availability, apparent in situ consumption rates, and growth-supporting energy.Thermophiles dominate the deepest and shortest branches of the Bacteria and Archaea domains in the tree of life, suggesting that they are likely ancestors of Earth's contemporary microbial populations (8,35). Consequently, these organisms have attracted considerable attention due to interest in the origin of enzymes and metabolic pathways that are thought to have evolved from such organisms. Chemolithotrophic metabolism is foundational to primary productivity in geothermal environments where temperatures exceed the limit of photosynthesis. The bioenergetics of such systems have been examined from the perspective of theoretical energy yield as a way of discussing the relative importance of the various electron donors and acceptors that could support primary productivity (3)(4)(5)22). Other studies have sought to link the inferred physiology of microbial populations with the predicted energy yields obtainable from the inorganic constituents present (4...

Sb(III) oxidation was documented in an Agrobacterium tumefaciens isolate that can also oxidize As(III). Equivalent Sb(III) oxidation rates were observed in the parental wild-type organism and in two well-characterized mutants that cannot oxidize As(III) for fundamentally different reasons. Therefore, despite the literature suggesting that Sb(III) and As(III) may be biochemical analogs, Sb(III) oxidation is catalyzed by a pathway different than that used for As(III). Sb(III) and As(III) oxidation was also observed for an eukaryotic acidothermophilic alga belonging to the order Cyanidiales, implying that the ability to oxidize metalloids may be phylogenetically widespread.Antimony (Sb) occurs globally in fresh and marine waters and in soils, with the most common mineral form being stibnite (Sb 2 S 3 ) (6). Sb is typically found with arsenic (As), another group V element having similar chemistry and toxicity, and is recognized by the U.S. Environmental Protection Agency as a priority pollutant (8,14). Unlike microbe-As interactions, for which significant progress has been made in defining the genetics and physiology of microbial arsenite [As(III)] oxidation and arsenate [As(V)] reduction (reviewed in references 19 and 20), microbe-Sb interactions are poorly understood, and as a consequence, the geomicrobiology of Sb is as yet essentially undefined. Sb(III) biomethylation has been reported (5), but otherwise, reports of microbe-Sb redox interactions are restricted to studies of an organism referred to as Stibiobacter senarmontii and described as being capable of oxidizing the mineral senarmontite to form Sb 2 O 5 (reviewed in reference 6). In the intervening 3 decades, no further characterization or confirmation of this organism has been reported, nor have there been reports of other microorganisms having the capacity to oxidize Sb(III).Given the structural similarities between As and Sb, the presence of one could influence the biological interactions of the other (4). Indeed, both As(III) and Sb(III) will induce the microbial ars-based arsenic defense response (11), both are taken up via the same aquaglyceroporin channel, GlyF (16), and in bacteria they are both extruded by the same porter, ArsB (16). These observations suggest the possibility that the same enzymatic pathways used for As(III) oxidation may also be used for Sb(III). In recent studies regarding the genetics underlying As(III) oxidation in Agrobacterium tumefaciens, we discovered regulatory (aoxR) and Na ϩ :H ϩ antiporter (mrpB) mutants which are defective in As(III) oxidation (12, 13). In subsequent investigations, we found the wild-type A. tumefaciens strain to also be capable of oxidizing Sb(III). This provided an exceptional opportunity to directly determine whether As(III) oxidation and Sb(III) oxidation share similar enzymes.As and Sb speciation and analysis. Borohydride reductionbased speciation was used for both As and Sb analysis. As(III) speciation protocols were as previously described (10). At near-neutral pH, Sb(III) and methyl-Sb species...