For more than a decade, it has been recognized that arsenate [H2AsO4 1-; As(V)] can be used by microorganisms as a terminal electron acceptor in anaerobic respiration. Given the toxicity of arsenic, the mechanistic basis of this process is intriguing, as is its evolutionary origin. Here we show that a two-gene cluster (arrAB; arsenate respiratory reduction) in the bacterium Shewanella sp. strain ANA-3 specifically confers respiratory As(V) reductase activity. Mutants with in-frame deletions of either arrA or arrB are incapable of growing on As(V), yet both are able to grow on a wide variety of other electron acceptors as efficiently as the wild-type. Complementation by the wild-type sequence rescues the mutants' ability to respire As(V). arrA is predicted to encode a 95.2-kDa protein with sequence motifs similar to the molybdenum containing enzymes of the dimethyl sulfoxide reductase family. arrB is predicted to encode a 25.7-kDa iron-sulfur protein. arrA and arrB comprise an operon that contains a twin arginine translocation (Tat) motif in ArrA (but not in ArrB) as well as a putative anaerobic transcription factor binding site upstream of arrA, suggesting that the respiratory As(V) reductase is exported to the periplasm via the Tat pathway and under anaerobic transcriptional control. These genes appear to define a new class of reductases that are specific for respiratory As(V) reduction.T he consumption of arsenic (As)-tainted surface waters and ground waters has created a public health crisis in many countries (1, 2). Although much of the As contamination derives from natural weathering and dissolution of As-bearing minerals, recognition that microorganisms can alter the mobility of As in natural waters through redox transformations (3) drove the discovery of the first arsenate [As(V)]-respiring bacterium nearly a decade ago (4). Since then, many more microorganisms that can reduce As(V) to arsenite [H 3 AsO 3 , As(III)] have been discovered (5-8), but a mechanistic understanding of this metabolism has lagged. To date, only three studies have described the biochemistry of arsenate respiration (5, 9, 10), and detailed biochemical analyses have not been performed. In part, the limitations of these studies can be attributed to the fact that the As(V)-respiring organisms being studied were not genetically tractable. To quantify the geochemical impact of As(V)-respiring microorganisms in a given locale, we must be able to predict when these organisms will be active, and how rapidly they will transform As(V). Identification of the gene(s) that control this process, elucidation of their regulation, and determination of the kinetics of their protein products, are necessary steps toward understanding the specific contribution of As(V)-respiring bacteria to As-cycling in the environment.In response to this need, a new As(V)-respiring species, Shewanella strain ANA-3 that is amenable to genetic analysis, was recently isolated (8). This organism contains two systems for reducing As(V). One is similar to the well conserved...

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Graphene oxide (GO) can be reduced to graphene in a normal aerobic setup under ambient conditions as mediated by microbial respiration of Shewanella cells. The microbially-reduced graphene (MRG) exhibited excellent electrochemical properties. Extracellular electron transfer pathways at the cell/GO interface were systematically investigated, suggesting both direct electron transfer and electron mediators are involved in the GO reduction.

Arsenate [As(V)]-respiring bacteria affect the speciation and mobilization of arsenic in the environment. This can lead to arsenic contamination of drinking water supplies and deleterious consequences for human health. Using molecular genetics, we show that the functional gene for As(V) respiration, arrA, is highly conserved; that it is required for As(V) reduction to arsenite when arsenic is sorbed onto iron minerals; and that it can be used to identify the presence and activity of As(V)-respiring bacteria in arsenic-contaminated iron-rich sediments. The expression of arrA thus can be used to monitor sites in which As(V)-respiring bacteria may be controlling arsenic geochemistry.

The fate and transport of arsenic is regulated, in part, by its strong affinity for iron (hydr)oxides. A transition from aerobic to anaerobic conditions resulting in concomitant reduction of both As(V) and iron (hydr)oxides can thus have a pronounced influence on As partitioning. However, it is presently unclear whether As desorption under anaerobic conditions results predominantly from a transformation from As(V) to As(III) or from mineralogical changes as a consequence of iron and manganese reduction. Here, we examine desorption of both As(III) and As(V) from ferrihydrite-, goethite-, and hematite-coated sand under hydrodynamic conditions. Furthermore, to resolve the relative role of Fe(III) and/or As(V) reduction in regulating dissolved As concentrations, we also examined As desorption from ferrihydrite- and goethite-coated sands presorbed with As(V) using wild type or mutants of Shewanella sp. ANA-3, capable of Fe(III)- and/or As(V)-reduction. We reveal substantial differences in As(III) and As(V) desorption from ferrihydrite, goethite, and hematite. Despite being adsorbed to a greater extent than As(V), As(III) is desorbed more rapidly and extensively from all oxides, suggesting weaker binding of As(III) than As(V). When As(V) and Fe(III) reduction are decoupled, As(V) reduction appears to be the dominant process controlling As release. Our results also suggest the importance of appreciating physical properties of specific Fe (hydr)oxides when predicting the potential for As desorption.

Because arsenate [As(V)] reduction by bacteria can significantly enhance arsenic mobility in the environment, it is important to be able to predict when this activity will occur. Currently, two bacterial systems are known that specifically reduce As(V), namely, a respiratory system (encoded by the arr genes) and a detoxification system (encoded by the ars genes). Here we analyze the conditions under which these two systems are expressed in Shewanella sp. strain ANA-3. The ars system is expressed under both aerobic and anaerobic conditions, whereas the arr system is only expressed anaerobically and is repressed by oxygen and nitrate. When cells are grown on As(V), the arr system is maximally induced during exponential growth, with peak expression of the ars system occurring at the beginning of stationary phase. Both the arr and ars systems are specifically induced by arsenite [As(III)], but the arr system is activated by a concentration of As(III) that is 1,000 times lower than that required for the arsC system (<100 nM versus <100 M, respectively). A double mutant was constructed that does not reduce As(V) under any growth conditions. In this strain background, As(V) is capable of inducing the arr system at low micromolar concentrations, but it does not induce the ars system. Collectively, these results demonstrate that the two As(V) reductase systems in ANA-3 respond to different amounts and types of inorganic arsenic.In sediments and groundwaters throughout the world, microorganisms affect the geochemistry of arsenic (As), which can lead to As contamination of drinking water supplies and poisoning of epidemic proportions (18,20,28). The mechanism for As release into drinking water typically involves the reductive dissolution of ferric (hydr)oxide minerals and/or the reduction of arsenate [HAsO 4 2Ϫ , or As(V)] to arsenite [H 3 AsO 3 , or As(III)] (19). Although both As(V) and As(III) strongly adsorb to ferric (hydr)oxides at the pH of most natural waters, microbial reduction of ferric (hydr)oxide and As(V) can liberate As(III) into sedimentary pore waters, which under the appropriate hydrological conditions can be drawn down into sandy aquifers, where As(III) is mobile in the aqueous phase (6, 7). In light of this, it is important to be able to predict when As(V)-reducing microorganisms will be active.Two different arsenate reduction pathways exist in bacteria, namely, the ars and the arr systems. The ars genes are present in many bacteria and archaea and are diverse in their sequence and genomic organization (17, 21). The Escherichia coli and Staphylococcus sp. ars operons have been well characterized at the molecular level with respect to both their biochemistry and their regulation (17, 22). Because As(V) is structurally similar to phosphate (HPO 4 2Ϫ ), it enters the cytosol via a low-affinity phosphate transporter (such as the Pit system in E. coli) (23,29). When this happens, the cell employs a 12-to 15-kDa soluble reductase, ArsC, that couples the oxidation of thiols from either glutathione/glutaredoxin...

Although arsenic is highly toxic to most organisms, certain prokaryotes are known to grow on and respire toxic metalloids of arsenic (i.e., arsenate and arsenite). Two enzymes are known to be required for this arsenic-based metabolism: (i) the arsenate respiratory reductase (ArrA) and (ii) arsenite oxidase (AoxB). Both catalytic enzymes contain molybdopterin cofactors and form distinct phylogenetic clades (ArrA and AoxB) within the dimethyl sulfoxide (DMSO) reductase family of enzymes. Here we report on the genetic identification of a "new" type of arsenite oxidase that fills a phylogenetic gap between the ArrA and AoxB clades of arsenic metabolic enzymes. This "new" arsenite oxidase is referred to as ArxA and was identified in the genome sequence of the Mono Lake isolate Alkalilimnicola ehrlichii MLHE-1, a chemolithoautotroph that can couple arsenite oxidation to nitrate reduction. A genetic system was developed for MLHE-1 and used to show that arxA (gene locus ID mlg_0216) was required for chemoautotrophic arsenite oxidation. Transcription analysis also showed that mlg_0216 was only expressed under anaerobic conditions in the presence of arsenite. The mlg_0216 gene is referred to as arxA because of its greater homology to arrA relative to aoxB and previous reports that implicated Mlg_0216 (ArxA) of MLHE-1 in reversible arsenite oxidation and arsenate reduction in vitro. Our results and past observations support the position that ArxA is a distinct clade within the DMSO reductase family of proteins. These results raise further questions about the evolutionary relationships between arsenite oxidases (AoxB) and arsenate respiratory reductases (ArrA).Arsenic is toxic to most organisms and is known to cause cancer in humans. However, bacteria have adapted several biotransformation pathways that function to either couple the reduction or oxidation of arsenicals to energy conservation and growth (1). The enzymologies of these two pathways have several features in common. The arsenate respiratory reductase (ArrAB) and arsenite oxidase (AoxAB) enzymes are usually composed of at least two subunits, a small iron-sulfur cluster-containing subunit (ArrB and AoxA) and a larger molybdopterin-containing catalytic subunit (ArrA and AoxB). Although they catalyze arsenic redox chemistry, ArrA and AoxB form distinct phylogenetic clades within the dimethyl sulfoxide (DMSO) reductase family of molybdenum-containing enzymes (16,24).Culture-dependent approaches have resulted in the isolation of a variety of diverse bacteria that metabolize arsenic (reviewed in reference 26). Many of these isolates have had their genomes sequenced, which has been insightful for understanding the composition and diversity of arr and aox gene clusters. In the arsenite-oxidizing nitrate reducer Alkalilimnicola ehrlichii strain MLHE-1 (a haloalkaliphile isolated from Mono Lake [CA]) (10, 15), bioinformatic analysis of its genome revealed the absence of genes homologous to the arsenite oxidase genes of the aoxB type. Instead, two genes (mlg_0216 and mlg_24...

Arsenotrophy, growth coupled to autotrophic arsenite oxidation or arsenate respiratory reduction, occurs only in the prokaryotic domain of life. The enzymes responsible for arsenotrophy belong to distinct clades within the DMSO reductase family of molybdenum-containing oxidoreductases: specifically arsenate respiratory reductase, ArrA, and arsenite oxidase, AioA (formerly referred to as AroA and AoxB). A new arsenite oxidase clade, ArxA, represented by the haloalkaliphilic bacterium Alkalilimnicola ehrlichii strain MLHE-1 was also identified in the photosynthetic purple sulfur bacterium Ectothiorhodospira sp. strain PHS-1. A draft genome sequence of PHS-1 was completed and an arx operon similar to MLHE-1 was identified. Gene expression studies showed that arxA was strongly induced with arsenite. Microbial ecology investigation led to the identification of additional arxA-like sequences in Mono Lake and Hot Creek sediments, both arsenic-rich environments in California. Phylogenetic analyses placed these sequences as distinct members of the ArxA clade of arsenite oxidases. ArxA-like sequences were also identified in metagenome sequences of several alkaline microbial mat environments of Yellowstone National Park hot springs. These results suggest that ArxA-type arsenite oxidases appear to be widely distributed in the environment presenting an opportunity for further investigations of the contribution of Arx-dependent arsenotrophy to the arsenic biogeochemical cycle.