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The involvement of prokaryotes in the redox reactions of arsenic occurring between its +5 [arsenate; As(V)] and +3 [arsenite; As(III)] oxidation states has been well established. Most research to date has focused upon circum-neutral pH environments (e.g., freshwater or estuarine sediments) or arsenic-rich “extreme” environments like hot springs and soda lakes. In contrast, relatively little work has been conducted in acidic environments. With this in mind we conducted experiments with sediments taken from the Herman Pit, an acid mine drainage impoundment of a former mercury (cinnabar) mine. Due to the large adsorptive capacity of the abundant Fe(III)-rich minerals, we were unable to initially detect in solution either As(V) or As(III) added to the aqueous phase of live sediment slurries or autoclaved controls, although the former consumed added electron donors (i.e., lactate, acetate, hydrogen), while the latter did not. This prompted us to conduct further experiments with diluted slurries using t...

Brevibacterium linens AE038-8, isolated from As-contaminated groundwater in Tucum an (Argentina), is highly resistant to arsenic oxyanions, being able to tolerate up to 1 M As(V) and 75 mM As(III) in a complex medium. Strain AE038-8 was also able to reduce As(V) to As(III) when grown in complex medium but paradoxically it could not do this in a defined minimal medium with sodium acetate and ammonium sulfate as carbon and nitrogen sources, respectively. No oxidation of As(III) to As(V) was observed under any conditions. Three copies of the ars operon comprising arsenic resistance genes were found on B. linens AE038-8 genome. In addition to the well known arsC, ACR3 and arsR, two copies of the arsO gene of unknown function were detected. Fil: Maizel, Daniela. Universidad Nacional de Tucumán; Argentina. Consejo Nacional de Investigaciónes Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Astronomía y Física del Espacio. - Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Astronomía y Física del Espacio; Argentina Fil: Blum, Jodi Switzer. United States Geological Survey; Estados Unidos Fil: Ferrero, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina Fil: Utturkar, Sagar M.. The University Of Tennessee System; Estados Unidos Fil: Brown, Steven D.. University of Tennessee; Estados Unidos Fil: Rosen, Barry. Florida International University; Estados Unidos Fil: Oremland, Ronald S.. United States Geological Survey; Estados Unidos

Primary production in Mono Lake, a hypersaline soda lake rich in dissolved inorganic arsenic, is dominated by Picocystis strain ML. We set out to determine if this photoautotrophic picoplankter could metabolize inorganic arsenic and in doing so form unusual arsenolipids (e.g., arsenic bound to 2-O-methyl ribosides) as reported in other saline ecosystems and by halophilic algae. We cultivated Picocystis strain ML on a seawater-based medium with either low (37 &micro, M) or high (1000 &micro, M) phosphate in the presence of arsenite (400 &micro, M), arsenate (800 &micro, M), or without arsenic additions (ca 0.025 &micro, M). Cultivars formed a variety of organoarsenic compounds, including a phytyl 2-O-methyl arsenosugar, depending upon the cultivation conditions and arsenic exposure. When the cells were grown at low P, the organoarsenicals they produced when exposed to both arsenite and arsenate were primarily arsenolipids (~88%) with only a modest content of water-soluble organoarsenic compounds (e.g., arsenosugars). When grown at high P, sequestration shifted to primarily water-soluble, simple methylated arsenicals such as dimethylarsinate, arsenolipids still constituted ~32% of organoarsenic incorporated into cells exposed to arsenate but &lt, 1% when exposed to arsenite. Curiously, Picocystis strain ML grown at low P and exposed to arsenate sequestered huge amounts of arsenic into the cells accounting for 13.3% of the dry biomass, cells grown at low P and arsenite exposure sequestered much lower amounts, equivalent to 0.35% of dry biomass. Extraction of a resistant phase with trifluoroacetate recovered most of the sequestered arsenic in the form of arsenate. Uptake of arsenate into low P-cultivated cells was confirmed by X-ray fluorescence, while XANES/EXAFS spectra indicated the sequestered arsenic was retained as an inorganic iron precipitate, similar to scorodite, rather than as an As-containing macromolecule. Samples from Mono Lake demonstrated the presence of a wide variety of organoarsenic compounds, including arsenosugar phospholipids, most prevalent in zooplankton (Artemia) and phytoplankton samples, with much lower amounts detected in the bottom sediments. These observations suggest a trophic transfer of organoarsenicals from the phytoplankton (Picocystis) to the zooplankton (Artemia) community, with efficient bacterial mineralization of any lysis-released organoarsenicals back to inorganic oxyanions before they sink to the sediments.

Citrobacter sp. strain TSA-1 is an enteric bacterium isolated from the hindgut of the termite. Strain TSA-1 displays anaerobic growth with selenite, fumarate, tetrathionate, nitrate, or arsenate serving as electron acceptors, and it also grows aerobically. In regards to arsenate, genome sequencing revealed that strain TSA-1 lacks a homolog for respiratory arsenate reductase, arrAB, and we were unable to obtain amplicons of arrA. This raises the question as to how strain TSA-1 achieves As(V)-dependent growth. We show that growth of strain TSA-1 on glycerol, which it cannot ferment, is linked to the electron acceptor arsenate. A series of transcriptomic experiments were conducted to discern which genes were upregulated during growth on arsenate, as opposed to those on fumarate or oxygen. For As(V), upregulation was noted for 1 of the 2 annotated arsC genes, while there was no clear upregulation for tetrathionate reductase (ttr), suggesting that this enzyme is not an alternative to arrAB as occurs in certain hyperthermophilic archaea. A gene-deletion mutant strain of TSA-1 deficient in arsC could not achieve anaerobic respiratory growth on As(V). Our results suggest that Citrobacter sp. strain TSA-1 has an unusual and as yet undefined means of achieving arsenate respiration, perhaps involving its ArsC as a respiratory reductase as well as a detoxifying agent.

Life is mostly composed of the elements carbon, hydrogen, nitrogen, oxygen, sulfur, and phosphorus. Although these six elements make up nucleic acids, proteins, and lipids and thus the bulk of living matter, it is theoretically possible that some other elements in the periodic table could serve the same functions. Here, we describe a bacterium, strain GFAJ-1 of the Halomonadaceae, isolated from Mono Lake, California, that is able to substitute arsenic for phosphorus to sustain its growth. Our data show evidence for arsenate in macromolecules that normally contain phosphate, most notably nucleic acids and proteins. Exchange of one of the major bio-elements may have profound evolutionary and geochemical importance.

Searles Lake occupies a closed basin harboring salt-saturated, alkaline brines that have exceptionally high concentrations of arsenic oxyanions. Strain SLAS-1 T was previously isolated from Searles Lake (R. S. Oremland, T. R. Kulp, J. Switzer Blum, S. E. Hoeft, S. Baesman, L. G. Miller, and J. F. Stolz, Science 308:1305-1308, 2005). We now describe this extremophile with regard to its substrate affinities, its unusual mode of motility, sequenced arrABD gene cluster, cell envelope lipids, and its phylogenetic alignment within the order Halanaerobacteriales , assigning it the name “ Halarsenatibacter silvermanii ” strain SLAS-1 T . We also report on the substrate dynamics of an anaerobic enrichment culture obtained from Searles Lake that grows under conditions of salt saturation and whose members include a novel sulfate reducer of the order Desulfovibriales , the archaeon Halorhabdus utahensis , as well as a close homolog of strain SLAS-1 T .

A facultative chemoautotrophic bacterium, strain MLHE-1T, was isolated from Mono Lake, an alkaline hypersaline soda lake in California, USA. Cells of strain MLHE-1T were Gram-negative, short motile rods that grew with inorganic electron donors (arsenite, hydrogen, sulfide or thiosulfate) coupled with the reduction of nitrate to nitrite. No aerobic growth was attained with arsenite or sulfide, but hydrogen sustained both aerobic and anaerobic growth. No growth occurred when nitrite or nitrous oxide was substituted for nitrate. Heterotrophic growth was observed under aerobic and anaerobic (nitrate) conditions. Cells of strain MLHE-1T could oxidize but not grow on CO, while CH4 neither supported growth nor was it oxidized. When grown chemoautotrophically, strain MLHE-1T assimilated inorganic carbon via the Calvin–Benson–Bassham reductive pentose phosphate pathway, with the activity of ribulose 1,5-bisphosphate carboxylase (RuBisCO) functioning optimally at 0.1 M NaCl and at pH 7.3. Strain MLHE-1T grew over broad ranges of pH (7.3–10.0; optimum, 9.3), salinity (15–190 g l−1; optimum 30 g l−1) and temperature (13–40 °C; optimum, 30 °C). Phylogenetic analysis of 16S rRNA gene sequences placed strain MLHE-1T in the class Gammaproteobacteria (family Ectothiorhodospiraceae) and most closely related to Alkalispirillum mobile (98.5 %) and Alkalilimnicola halodurans (98.6 %), although none of these three haloalkaliphilic micro-organisms were capable of photoautotrophic growth and only strain MLHE-1T was able to oxidize As(III). On the basis of physiological characteristics and DNA–DNA hybridization data, it is suggested that strain MLHE-1T represents a novel species within the genus Alkalilimnicola for which the name Alkalilimnicola ehrlichii is proposed. The type strain is MLHE-1T (=DSM 17681T=ATCC BAA-1101T). Aspects of the annotated full genome of Alkalilimnicola ehrlichii are discussed in the light of its physiology.

Searles Lake is a salt-saturated, alkaline brine unusually rich in the toxic element arsenic. Arsenic speciation changed from arsenate [As(V)] to arsenite [As(III)] with sediment depth. Incubated anoxic sediment slurries displayed dissimilatory As(V)-reductase activity that was markedly stimulated by H 2 or sulfide, whereas aerobic slurries had rapid As(III)-oxidase activity. An anaerobic, extremely haloalkaliphilic bacterium was isolated from the sediment that grew via As(V) respiration, using either lactate or sulfide as its electron donor. Hence, a full biogeochemical cycle of arsenic occurs in Searles Lake, driven in part by inorganic electron donors.

A selenite-respiring bacterium, Bacillus selenitireducens, produced significant levels of Se(-II) (as aqueous HSe−) when supplied with Se(0). B. selenitireducens was also able to reduce selenite [Se(IV)] through Se(0) to Se(-II). Reduction of Se(0) by B. selenitireducens was more rapid in cells grown on colloidal sulfur [S(0)] or Se(IV) as their electron acceptor than for cell lines grown on fumarate. In contrast, three cultures of selenate-respiring bacteria, Sulfurospirillum barnesii, B. arsenicoselenatis, and Selenihalanaerobacter shriftii either were unable to reduce Se(0) to Se(-II) or had only a very limited capacity to achieve this reduction. Biological reduction of Se(0) to Se(-II) was observed during incubation of estuarine sediment slurries, while no such activity was noted in formalin-killed controls. The majority of the Se(-II) produced was found in the sediments as a solid precipitate of FeSe, rather than in solution as HSe−. These results demonstrate that certain anaerobic bacteria have the ...

Environmental Significance of Group 15 and 16 Toxic Metalloids Selenium, tellurium, and arsenic are present naturally in aquatic and terrestrial environments and share many similar biogeochemical characteristics. These elements are released into the environment through the weathering and decomposition of minerals contained within a variety of lithologies, with slow release rates resulting in low environmental concentrations. Selenium, tellurium, and arsenic occur in several oxidation states as oxyanions (e.g., selenate [SeO4 2], selenite [SeO3 2], tellurate [TeO4 2], tellurite [TeO3 2], arsenate [HAsO4 2], and arsenite [HAsO3 2]) in their native elemental states [e.g., Se(0), Te(0)] or in their most reduced states as selenide (-II) and telluride (-II) or arsenide/arsines (-III). These elements can be methylated through microbial activity to form compounds such as dimethylselenide (Ehrlich, 2002; Masscheleyn, et al., 1990), dimethyltelluride (Basnayake, et al., 2001; Fleming and Alexander, 1972), and methylarsonous acid (Dopp, et al., 2004) as well as a variety of toxic methylated arsine gases (Yuan, et al., 2008). These elements are also found as analogues of sulfurous proteins such as selenocysteine and selenomethionine (Bock, et al., 1991; Jones, et al., 1979; Stolz, et al., 2006; Zannoni, et al., 2008), tellurocysteine, telluromethionine (Zannoni, et al., 2008), and the arsenic-containingmore » amino acid, arsenomethionine (Dembitsky and Levitsky, 2004).« less

We isolated an obligately anaerobic halophilic bacterium from the Dead Sea that grew by respiration of selenate. The isolate, designated strain DSSe-1, was a gram-negative, non-motile rod. It oxidized glycerol or glucose to acetate + CO2 with concomitant reduction of selenate to selenite plus elemental selenium. Other electron acceptors that supported anaerobic growth on glycerol were nitrate and trimethylamine-N-oxide; nitrite, arsenate, fumarate, dimethylsulfoxide, thiosulfate, elemental sulfur, sulfite or sulfate could not serve as electron acceptors. Growth on glycerol in the presence of nitrate occurred over a salinity range from 100 to 240 g/l, with an optimum at 210 g/l. Analysis of the 16S rRNA gene sequence suggests that strain DSSe-1 belongs to the order Halanaerobiales, an order of halophilic anaerobes with a fermentative or homoacetogenic metabolism, in which anaerobic respiratory metabolism has never been documented. The highest 16S rRNA sequence similarity (90%) was found with Acetohalobium arabaticum (X89077). On the basis of physiological properties as well as the relatively low homology of 16S rRNA from strain DSSe-1 with known genera, classification in a new genus within the order Halanaerobiales, family Halobacteroidaceae is warranted. We propose the name Selenihalanaerobacter shriftii. Type strain is strain DSSe-1 (ATCC accession number BAA-73).

The stratified (meromictic) water column of alkaline and hypersaline Mono Lake, California, contains high concentrations of dissolved inorganic arsenic (∼200 μmol/L). Arsenic speciation changes from arsenate [As (V)] to arsenite [As (III)] with the transition from oxic surface waters (mixolimnion) to anoxic bottom waters (monimolimnion). A radioassay was devised to measure the reduction of 73 As (V) to 73 As (III) and tested using cell suspensions of the As (V)-respiring Bacillus selenitireducens , which completely reduced the 73 As (V). In field experiments, no significant activity was noted in the aerobic mixolimnion waters, but reduction of 73 As (V) to 73 As (III) was observed in all the monimolimnion samples. Rate constants ranged from 0.02 to 0.3/day, with the highest values in the samples from the deepest depths (24 and 28 m). The highest activities occurred between 18 and 21 m, where As (V) was abundant (rate, ∼5.9 μmol/L per day). In contrast, sulfate reduction occurred at depths below 21 m, with the highest rates attained at 28 m (rate, ∼2.3 μmol/L per day). These results indicate that As (V) ranks second in importance, after sulfate, as an electron acceptor for anaerobic bacterial respiration in the water column. Annual arsenate respiration may mineralize as much as 14.2% of the pelagic photosynthetic carbon fixed during meromixis. When combined with sulfate-reduction data, anaerobic respiration in the water column can mineralize 32–55% of this primary production. As lakes of this type approach salt saturation, As (V) can become the most important electron acceptor for the biogeochemical cycling of carbon.

Two strains of dissimilatory arsenate-reducing vibrio-shaped bacteria are assigned to the genus Sulfurospirillum. These two new species, Sulfurospirillum barnesii strain SES-3T and Sulfurospirillum arsenophilum strain MIT-13T, in addition to Sulfurospirillum sp. SM-5, two strains of Sulfurospirillum deleyianum, and Sulfurospirillum arcachonense, form a distinct clade within the epsilon subclass of the Proteobacteria based on 16S rRNA analysis.

Two gram-positive anaerobic bacteria (strains E1H and MLS10) were isolated from the anoxic muds of Mono Lake, California, an alkaline, hypersaline, arsenic-rich water body. Both grew by dissimilatory reduction of As(V) to As(III) with the concomitant oxidation of lactate to acetate plus CO2. Bacillus arsenicoselenatis (strain E1H) is a spore-forming rod that also grew by dissimilatory reduction of Se(VI) to Se(IV). Bacillus selenitireducens (strain MLS10) is a short, non-spore-forming rod that grew by dissimilatory reduction of Se(IV) to Se(0). When the two isolates were cocultured, a complete reduction of Se(VI) to Se(0) was achieved. Both isolates are alkaliphiles and had optimal specific growth rates in the pH range of 8.5-10. Strain E1H had a salinity optimum at 60 g l-1 NaCl, while strain MLS10 had optimal growth at lower salinities (24-60 g l-1 NaCl). Both strains have limited abilities to grow with electron donors and acceptors other than those given above. Strain MLS10 demonstrated weak growth as a microaerophile and was also capable of fermentative growth on glucose, while strain E1H is a strict anaerobe. Comparative 16S rRNA gene sequence analysis placed the two isolates with other Bacillus spp. in the low G+C gram-positive group of bacteria.

The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b554 (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b556 (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth.

A gram-negative, strictly anaerobic, motile vibrio was isolated from a selenate-respiring enrichment culture. The isolate, designated strain SES-3, grew by coupling the oxidation of lactate to acetate plus CO 2 with the concomitant reduction of selenate to selenite or of nitrate to ammonium. No growth was observed on sulfate or selenite, but cell suspensions readily reduced selenite to elemental selenium (Se 0 ). Hence, SES-3 can carry out a complete reduction of selenate to Se 0 . Washed cell suspensions of selenate-grown cells did not reduce nitrate, and nitrate-grown cells did not reduce selenate, indicating that these reductions are achieved by separate inducible enzyme systems. However, both nitrate-grown and selenate-grown cells have a constitutive ability to reduce selenite or nitrite. The oxidation of [ 14 C]lactate to 14 CO 2 coupled to the reduction of selenate or nitrate by cell suspensions was inhibited by CCCP (carbonyl cyanide m -chlorophenylhydrazone), cyanide, and azide. High concentrations of selenite (5 mM) were readily reduced to Se 0 by selenate-grown cells, but selenite appeared to block the synthesis of pyruvate dehydrogenase. Tracer experiments with [ 75 Se]selenite indicated that cell suspensions could achieve a rapid and quantitative reduction of selenite to Se 0 . This reduction was totally inhibited by sulfite, partially inhibited by selenate or nitrite, but unaffected by sulfate or nitrate. Cell suspensions could reduce thiosulfate, but not sulfite, to sulfide. These results suggest that reduction of selenite to Se 0 may proceed, in part, by some of the components of a dissimilatory system for sulfur oxyanions.

Bovine rumen fluid and slurried hamster feces completely reduced millimolar levels of arsenate to arsenite upon incubation under anoxic conditions. This activity was strongly inhibited by autoclaving or aerobic conditions, and partially inhibited by tungstate or chloramphenicol. The rate of arsenate reduction was faster in feces from a population of arsenate-watered (100 ppm) hamsters compared to a control group watered without arsenate. Using radioisotope methods, arsenate reductase activity in hamster feces was also detected at very low concentrations of added arsenate ( approximately 10 muM). Bacterial cultures were isolated from these materials, as well as from the termite hindgut, that grew using H(2) as their electron donor, acetate as their carbon source, and arsenate as their respiratory electron acceptor. The three cultures aligned phylogenetically either with well-established enteric bacteria, or with an organism associated with feedlot fecal wastes. Because arsenite is transported across the gut epithelium more readily than arsenate, microbial dissimilatory reduction of arsenate in the gut may promote the body's absorption of arsenic and hence potentiate its toxicity.

Cell suspensions of H2/CO2-grown Sporomusa termitida catalyzed an H2-supported synthesis of acetate from CO2 at rates of about 1 μmol acetate x h-1 x mg protein-1. Cells pre-grown on methanol, mannitol, lactate, or glycine also displayed H2-supported acetogenesis from CO2, although at rates 5–85% that of H2/CO2-grown cells. With methanol-grown cell suspensions: the presence of methanol greatly stimulated the rate of H2-supported conversion of 14CO2 to 14C-acetate (which became labeled mainly in the COOH-group); and like-wise the presence of H2 stimulated the conversion of 14CH3OH+CO2 to 14C-acetate (which became labeled mainlyan the CH3-group). Analogous stimulatory effects were observed for cell suspensions pre-grown on methanol + CO2+H2. Furthermore, when H2 (+CO2) was included as a growth substrate with either methanol or lactate: both substrates were used simultaneously; there was no diauxie in the growth of cells or in acetate production; and the molar growth yield of S. termitida was close to that predicted from summation of the yields observed when grown with each substrate alone. These data indicated that S. termitida can grow by mixotrophy, i.e. by the simultaneous use of H2/CO2 and organic compounds for energy. Results are discussed in light of the ability of H2/CO2 acetogens to outprocess methanogens in H2 consumption in the hindgut fermentation of wood-feeding termites.

Certain anaerobes grow by respiring selenium oxyanions, which are in turn reduced to elemental selenium [Se(0)]. From the study of 3 different species of Se-respiring bacteria we report that, while they can form large (/spl sim/500 nm) intracellular particles of Se, most of the Se granules accumulated is extracellular. These external /spl sim/300 nm granules consist of stable, uniform nanospheres whose structure, as determined by electron diffraction and EDS, corresponds to monoclinic Se(0). The optical properties of the purified particles revealed large differences between Se(0) produced by the different bacteria, which in turn was distinct from Se(0) formed via abiotic means. Our study points to the bacterial synthesis of unique nanospheres that internally contain different compacted forms and/ or chain arrangements of Se.

Certain anaerobic bacteria respire toxic selenium oxyanions and in doing so produce extracellular accumulations of elemental selenium [Se(0)]. We examined three physiologically and phylogenetically diverse species of selenate- and selenite-respiring bacteria,Sulfurospirillum barnesii,Bacillus selenitireducens, andSelenihalanaerobacter shriftii, for the occurrence of this phenomenon. When grown with selenium oxyanions as the electron acceptor, all of these organisms formed extracellular granules consisting of stable, uniform nanospheres (diameter, ∼300 nm) of Se(0) having monoclinic crystalline structures. Intracellular packets of Se(0) were also noted. The number of intracellular Se(0) packets could be reduced by first growing cells with nitrate as the electron acceptor and then adding selenite ions to washed suspensions of the nitrate-grown cells. This resulted in the formation of primarily extracellular Se nanospheres. After harvesting and cleansing of cellular debris, we observed large differences in the optical properties (UV-visible absorption and Raman spectra) of purified extracellular nanospheres produced in this manner by the three different bacterial species. The spectral properties in turn differed substantially from those of amorphous Se(0) formed by chemical oxidation of H2Se and of black, vitreous Se(0) formed chemically by reduction of selenite with ascorbate. The microbial synthesis of Se(0) nanospheres results in unique, complex, compacted nanostructural arrangements of Se atoms. These arrangements probably reflect a diversity of enzymes involved in the dissimilatory reduction that are subtly different in different microbes. Remarkably, these conditions cannot be achieved by current methods of chemical synthesis.

Washed-cell suspensions ofSulfurospirillum barnesiireduced selenate [Se(VI)] when cells were cultured with nitrate, thiosulfate, arsenate, or fumarate as the electron acceptor. When the concentration of the electron donor was limiting, Se(VI) reduction in whole cells was approximately fourfold greater in Se(VI)-grown cells than was observed in nitrate-grown cells; correspondingly, nitrate reduction was ∼11-fold higher in nitrate-grown cells than in Se(VI)-grown cells. However, a simultaneous reduction of nitrate and Se(VI) was observed in both cases. At nonlimiting electron donor concentrations, nitrate-grown cells suspended with equimolar nitrate and selenate achieved a complete reductive removal of nitrogen and selenium oxyanions, with the bulk of nitrate reduction preceding that of selenate reduction. Chloramphenicol did not inhibit these reductions. The Se(VI)-respiring haloalkaliphileBacillus arsenicoselenatisgave similar results, but its Se(VI) reductase was not constitutive in nitrate-grown cells. No reduction of Se(VI) was noted forBacillus selenitireducens, which respires selenite. The results of kinetic experiments with cell membrane preparations ofS. barnesiisuggest the presence of constitutive selenate and nitrate reduction, as well as an inducible, high-affinity nitrate reductase in nitrate-grown cells which also has a low affinity for selenate. The simultaneous reduction of micromolar Se(VI) in the presence of millimolar nitrate indicates that these organisms may have a functional use in bioremediating nitrate-rich, seleniferous agricultural wastewaters. Results with75Se-selenate tracer show that these organisms can lower ambient Se(VI) concentrations to levels in compliance with new regulations proposed for release of selenium oxyanions into the environment.

The selenate-respiring bacterial strain SES-3 was able to use a variety of inorganic electron acceptors to sustain growth. SES-3 grew with the reduction of arsenate to arsenite, Fe(III) to Fe(II), or thiosulfate to sulfide. It also grew in medium in which elemental sulfur, Mn(IV), nitrite, trimethylamine N-oxide, or fumarate was provided as an electron acceptor. Growth on oxygen was microaerophilic. There was no growth with arsenite or chromate. Washed suspensions of cells grown on selenate or nitrate had a constitutive ability to reduce arsenate but were unable to reduce arsenite. These results suggest that strain SES-3 may occupy a niche as an environmental opportunist by being able to take advantage of a diversity of electron acceptors.

Concerns have been raised about our recent study suggesting that arsenic (As) substitutes for phosphorus in major biomolecules of a bacterium that tolerates extreme As concentrations. We welcome the opportunity to better explain our methods and results and to consider alternative interpretations. We maintain that our interpretation of As substitution, based on multiple congruent lines of evidence, is viable.

An anaerobic, freshwater enrichment grew with either nitrate or selenate as an electron acceptor. With both ions present, nitrate reduction preceded selenate reduction. An isolate from the enrichment grew on either ion, but the presence of nitrate precluded the reduction of selenate. Stock cultures of denitrifiers grew anaerobically on nitrate but not on selenate.

A haloalkaliphilic sulfate-respiring bacterium, strain SLSR-1, was isolated from a lactate-fed stable enrichment culture originally obtained from the extreme environment of Searles Lake, California. The isolate proved capable of growth via sulfate-reduction over a broad range of salinities (125-330 g/L), although growth was slowest at salt-saturation. Strain SLSR-1 was also capable of growth via dissimilatory arsenate-reduction and displayed an even broader range of salinity tolerance (50-330 g/L) when grown under these conditions. Strain SLSR-1 could also grow via dissimilatory nitrate reduction to ammonia. Growth experiments in the presence of high borate concentrations indicated a greater sensitivity of sulfate-reduction than arsenate-respiration to this naturally abundant anion in Searles Lake. Strain SLSR-1 contained genes involved in both sulfate-reduction (dsrAB) and arsenate respiration (arrA). Amplicons of 16S rRNA gene sequences obtained from DNA extracted from Searles Lake sediment revealed the presence of close relatives of strain SLSR-1 as part of the flora of this ecosystem despite the fact that sulfate-reduction activity could not be detected in situ. We conclude that strain SLSR-1 can only achieve growth via arsenate-reduction under the current chemical conditions prevalent at Searles Lake. Strain SLSR-1 is a deltaproteobacterium in the family Desulfohalobiacea of anaerobic, haloalkaliphilic bacteria, for which we propose the name Desulfohalophilus alkaliarsenatis gen. nov., sp. nov.