RESUMEN
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 Vmax 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.
Asunto(s)
Agrobacterium tumefaciens/enzimología , Agrobacterium tumefaciens/metabolismo , Antimonio/metabolismo , Arsenitos/metabolismo , Oxidorreductasas/metabolismo , Oxidación-Reducción , Rhizobium/enzimología , Rhizobium/metabolismoRESUMEN
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 degrees 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.
Asunto(s)
Arsénico/metabolismo , Eucariontes/metabolismo , Arsenitos/metabolismo , Biotransformación , Ambiente , Células Eucariotas , Calor , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Datos de Secuencia MolecularRESUMEN
Geothermal waters contain numerous potential electron donors capable of supporting chemolithotrophy-based 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.
Asunto(s)
Bacterias/metabolismo , Manantiales de Aguas Termales/química , Manantiales de Aguas Termales/microbiología , Sulfuro de Hidrógeno/metabolismo , Hidrógeno/metabolismo , Microbiología del Agua , Bacterias/genética , Crecimiento Quimioautotrófico , ADN Bacteriano , Ecosistema , Genes Bacterianos , Genes de ARNr , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Consumo de Oxígeno , Filogenia , ARN Ribosómico 16S/genética , Alineación de Secuencia , Temperatura , WyomingRESUMEN
Previous studies in an acid-sulfate-chloride spring in Yellowstone National Park found that microbial arsenite [As(III)] oxidation is absent in regions of the spring outflow channel where H(2)S exceeds approximately 5 microM and served as a backdrop for continued efforts in the present study. Ex situ assays with microbial mat samples demonstrated immediate As(III) oxidation activity when H(2)S was absent or at low concentrations, suggesting the presence of As(III) oxidase enzymes that could be reactivated if H(2)S is removed. Cultivation experiments initiated with mat samples taken from along the H(2)S gradient in the outflow channel resulted in the isolation of an As(III)-oxidizing chemolithotroph from the low-H(2)S region of the gradient. The isolate was phylogenetically related to Acidicaldus and was characterized in vitro for spring-relevant properties, which were then compared to its distribution pattern in the spring as determined by denaturing gradient gel electrophoresis and quantitative PCR. While neither temperature nor oxygen requirements appeared to be related to the occurrence of this organism within the outflow channel, H(2)S concentration appeared to be an important constraint. This was verified by in vitro pure-culture modeling and kinetic experiments, which suggested that H(2)S inhibition of As(III) oxidation is uncompetitive in nature. In summary, the studies reported herein illustrate that H(2)S is a potent inhibitor of As(III) oxidation and will influence the niche opportunities and population distribution of As(III) chemolithotrophs.
Asunto(s)
Alphaproteobacteria/metabolismo , Arsenitos/metabolismo , Manantiales de Aguas Termales/microbiología , Sulfuros/farmacología , Alphaproteobacteria/clasificación , Alphaproteobacteria/genética , Alphaproteobacteria/aislamiento & purificación , Arsenitos/química , Oxidación-Reducción , Sulfuros/metabolismo , Temperatura , WyomingRESUMEN
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.
Asunto(s)
Agrobacterium tumefaciens/metabolismo , Antimonio/metabolismo , Arsénico/metabolismo , Rhodophyta/metabolismo , Oxidación-ReducciónRESUMEN
Sudden infant death syndrome (SIDS) has been associated with the volatilization of arsenic, antimony or phosphorus compounds from infants' bedding material by micro-organisms, the so-called 'toxic gas hypothesis'. The volatilization of arsenic by aerobic micro-organisms isolated from new sheepskin bedding material, as well as on material used by a healthy infant and by an infant who perished of SIDS, was examined. Three fungi were isolated from a piece of sheepskin bedding material on which an infant perished of SIDS, which methylated arsenic to form trimethylarsenic(V) species, precursors to volatile trimethylarsine. These three fungi were identified as Scopulariopsis koningii, Fomitopsis pinicola and Penicillium gladioli by their 26S-ribosomal RNA polymerase chain reaction products. These fungi were not previously known to methylate arsenic. The volatilization of arsenic by these three fungi was then examined. Only P. gladioli volatilized arsenic and only under conditions such that the production of sufficient trimethylarsine to be acutely toxic to an infant is unlikely. S. brevicaulis grew on the sheepskin bedding material and evolved a trace amount of trimethylarsine. Known human pathogens such as Mycobacterium neoaurum and Acinetobacter junii were isolated from used bedding.