Expression of Genes and Proteins Involved in Arsenic Respiration and Resistance in Dissimilatory Arsenate-Reducing Geobacter sp. Strain OR-1.

The reduction of arsenate [As(V)] to arsenite [As(III)] by dissimilatory As(V)-reducing bacteria, such as Geobacter spp., may play a significant role in arsenic release from anaerobic sediments into groundwater. The biochemical and molecular mechanisms by which these bacteria cope with this toxic element remain unclear. In this study, the expression of several genes involved in arsenic respiration (arr) and resistance (ars) was determined using Geobacter sp. strain OR-1, the only cultured Geobacter strain capable of As(V) respiration. In addition, proteins expressed differentially under As(V)-respiring conditions were identified by semiquantitative proteomic analysis. Dissimilatory As(V) reductase (Arr) of strain OR-1 was localized predominantly in the periplasmic space, and the transcription of its gene (arrA) was upregulated under As(V)-respiring conditions. The transcription of the detoxifying As(V) reductase gene (arsC) was also upregulated, but its induction required 500 times higher concentration of As(III) (500 µM) than did the arrA gene. Comparative proteomic analysis revealed that in addition to the Arr and Ars proteins, proteins involved in the following processes were upregulated under As(V)-respiring conditions: (i) protein folding and assembly for rescue of proteins with oxidative damage, (ii) DNA replication and repair for restoration of DNA breaks, (iii) anaplerosis and gluconeogenesis for sustainable energy production and biomass formation, and (iv) protein and nucleotide synthesis for the replacement of damaged proteins and nucleotides. These results suggest that strain OR-1 copes with arsenic stress by orchestrating pleiotropic processes that enable this bacterium to resist and actively metabolize arsenic.IMPORTANCE Dissimilatory As(V)-reducing bacteria, such as Geobacter spp., play significant roles in arsenic release and contamination in groundwater and threaten the health of people worldwide. However, the biochemical and molecular mechanisms by which these bacteria cope with arsenic toxicity remain unclear. In this study, it was found that both respiratory and detoxifying As(V) reductases of a dissimilatory As(V)-reducing bacterium, Geobacter sp. strain OR-1, were upregulated under As(V)-respiring conditions. In addition, various proteins expressed specifically or more abundantly in strain OR-1 under arsenic stress were identified. Strain OR-1 actively metabolizes arsenic while orchestrating various metabolic processes that repair oxidative damage caused by arsenic. Such information is useful in assessing and identifying possible countermeasures for the prevention of microbial arsenic release in nature.

Comparative community gene expression analysis of Aquificales-dominated geothermal springs.

Members of Sulfurihydrogenibium are often observed as visible filamentous biomass in circumneutral hot springs and play roles in sulfur-cycling, hydrogen oxidation and iron mineralization. To gain insight into the ecophysiology of Sulfurihydrogenibium populations, we conducted preliminary metatranscriptomic analysis of three distinct thermal springs; Calcite Springs (YNP-CS) and Mammoth Springs (YNP-MHS) in Yellowstone National Park, USA, and Furnas Springs (AZ) in Azores, Portugal. Genes to which transcripts were assigned revealed commonly expressed functions among the sites, while several differences were also observed. All three sites, Sulfurihydrogenibium spp. dominate and are obtaining energy via metabolism of sulfur compounds under microaerophilic conditions. Cell motility was one of the expressed functions in two sites (YNP-CS and AZ) with slower stream flow rates and thicker well-formed biofilms. The transcripts from YNP-CS and -MHS exhibited varying levels of sequence divergence from the reference genomes and corresponding metagenomes, suggesting the presence of microdiversity among Sulfurihydrogenibium populations in situ. Conversely, the majority of the AZ transcripts were identical to the S. azorense genome. Our initial results show that the metatranscriptomes in these similar Aquificales-dominated communities can reveal community-level gene function in geochemically distinct thermal environments.

Benz[a]anthracene biotransformation and production of ring fission products by Sphingobium sp. strain KK22.

A soil bacterium, designated strain KK22, was isolated from a phenanthrene enrichment culture of a bacterial consortium that grew on diesel fuel, and it was found to biotransform the persistent environmental pollutant and high-molecular-weight polycyclic aromatic hydrocarbon (PAH) benz[a]anthracene. Nearly complete sequencing of the 16S rRNA gene of strain KK22 and phylogenetic analysis revealed that this organism is a new member of the genus Sphingobium. An 8-day time course study that consisted of whole-culture extractions followed by high-performance liquid chromatography (HPLC) analyses with fluorescence detection showed that 80 to 90% biodegradation of 2.5 mg liter(-1) benz[a]anthracene had occurred. Biodegradation assays where benz[a]anthracene was supplied in crystalline form (100 mg liter(-1)) confirmed biodegradation and showed that strain KK22 cells precultured on glucose were equally capable of benz[a]anthracene biotransformation when precultured on glucose plus phenanthrene. Analyses of organic extracts from benz[a]anthracene biodegradation by liquid chromatography negative electrospray ionization tandem mass spectrometry [LC/ESI(-)-MS/MS] revealed 10 products, including two o-hydroxypolyaromatic acids and two hydroxy-naphthoic acids. 1-Hydroxy-2- and 2-hydroxy-3-naphthoic acids were unambiguously identified, and this indicated that oxidation of the benz[a]anthracene molecule occurred via both the linear kata and angular kata ends of the molecule. Other two- and single-aromatic-ring metabolites were also documented, including 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid and salicylic acid, and the proposed pathways for benz[a]anthracene biotransformation by a bacterium were extended.

Dynamics of rumen microbiome in sika deer (Cervus nippon yakushimae) from unique subtropical ecosystem in Yakushima Island, Japan.

Yaku sika deer (Cervus nippon yakushimae) are endemic to Yakushima Island, whose landscape covered with primary evergreen forest is recognized as a World Heritage Site. In this study, the rumen bacterial microbiota (RBM) of wild Yaku sika was characterized using high throughput sequencing of bacterial 16S rRNA genes combined with targeted cultivation and functional analyses. Comparative analyses of RBM datasets from other ruminant animals revealed distinct community structure among domesticated and wild ruminants. Wild Yaku sika RBM exhibited higher species richness than other sika deer (i.e. wild Ezo sika and domesticated sika deer), likely reflecting their dietary variations associated with unique ecosystem in the island. The Yaku sika RBM of high deer population density samples exhibited higher diversity and contained higher proportion of Firmicutes than those of lower density samples. Moreover, the highest abundance of tannase gene were observed in individuals from the highest population density area, consistent with the previous observation that Yaku sika in the high density areas expanded their feed to include tannin-rich unpalatable plants. This study indicated that RBM of unique wild Yaku sika contribute to the flexibility of dietary shift and thus maintaining nutritional status of Yaku sika under high density conditions.

Draft Genome Sequence of Cupriavidus sp. Strain IK-TO18, Isolated from Antimony-Contaminated Sediment.

Cupriavidus sp. strain IK-TO18 was isolated from antimony-contaminated sediment. The draft genome sequence of the isolate contains 6,605 predicted protein-coding sequences, including genes associated with heavy metal resistance and the aerobic degradation of aromatic hydrocarbons. This sequence will provide valuable information regarding the functional versatility of the genus Cupriavidus.

Draft Genome Sequence of the Anaerobic Arsenite-Oxidizing Halomonas sp. Strain ANAO-440, Isolated from an Alkaline Saline Lake in Khovsgol, Mongolia.

The draft genome sequence of Halomonas sp. strain ANAO-440 contains 3,866 predicted protein-coding sequences. This strain is capable of anaerobic arsenite oxidation and encodes an arxA-type arsenite oxidase within the arxB2AB1CD gene island. This genome sequence provides valuable information regarding the physiological diversity of Arx-dependent arsenite-oxidizing microorganisms.

Draft Genome Sequence of the Antimony-Oxidizing Pseudomonas sp. Strain SbOxS1, Isolated from Stibnite Mine Tailing Soil.

Pseudomonas sp. strain SbOxS1 was isolated from stibnite mine tailing soil for its ability to oxidize antimonite. We present a draft genome sequence of strain SbOxS1, which contains 6,484 predicted protein-coding sequences. This genome information extends our understanding of the physiological versatility of antimony-transforming microorganisms.

Draft Genome Sequence of Stenotrophomonas sp. Strain SbOxS2, an Antimony-Oxidizing Bacterium Isolated from Stibnite Mine Tailing Soil.

The antimony-oxidizing Stenotrophomonas sp. strain SbOxS2 was isolated from stibnite mine tailing soil. The draft genome sequence of strain SbOxS2 comprises 4.76 Mbp with 4,211 predicted protein-coding sequences. This genome will provide useful information for characterizing the molecular mechanisms associated with heavy metal resistance within the genus Stenotrophomonas.

Influence of the Chemical Form of Antimony on Soil Microbial Community Structure and Arsenite Oxidation Activity.

In the present study, the influence of the co-contamination with various chemical forms of antimony (Sb) with arsenite (As[III]) on soil microbial communities was investigated. The oxidation of As(III) to As(V) was monitored in soil columns amended with As(III) and three different chemical forms of Sb: antimony potassium tartrate (Sb[III]-tar), antimony(III) oxide (Sb2O3), and potassium antimonate (Sb[V]). Soil microbial communities were examined qualitatively and quantitatively using 16S rDNA- and arsenite oxidase gene (aioA)-targeted analyses. Microbial As(III) oxidation was detected in all soil columns and 90-100% of added As(III) (200 µmol L-1) was oxidized to As(V) in 9 d, except in the Sb(III)-tar co-amendments that only oxidized 30%. 16S rDNA- and aioA-targeted analyses showed that the presence of different Sb chemical forms significantly affected the selection of distinct As(III)-oxidizing bacterial populations. Most of the 16S rRNA genes detected in soil columns belonged to Betaproteobacteria and Gammaproteobacteria, and some sequences were closely related to those of known As(III) oxidizers. Co-amendments with Sb(III)-tar and high concentrations of Sb2O3 significantly increased the ratios of aioA-possessing bacterial populations, indicating the enrichment of As(III) oxidizers resistant to As and Sb toxicity. Under Sb co-amendment conditions, there was no correlation between aioA gene abundance and the rates of As(III) oxidation. Collectively, these results demonstrated that the presence of different Sb chemical forms imposed a strong selective pressure on the soil bacterial community and, thus, the co-existing metalloid is an important factor affecting the redox transformation of arsenic in natural environments.

Molecular and physiological approaches to understanding the ecology of pollutant degradation.

Pollutant biodegradation in the environment occurs in the context of various interactions among microorganisms. To understand this ecological process, identification of functionally important populations is considered to be the primary step, which can be followed by isolation and laboratory pure-culture studies of the important organisms. Laboratory studies can then proceed to the analysis of in situ activity and interactions with other organisms. Such studies will shape a deeper understanding of the ecology of pollutant degradation and facilitate the development of new bioremediation strategies.

Cellular Response of Sinorhizobium sp. Strain A2 during Arsenite Oxidation.

Arsenic (As) is a widely distributed toxic element in the environment and microorganisms have developed resistance mechanisms in order to tolerate it. The cellular response of the chemoorganotrophic arsenite (As[III])-oxidizing α-Proteobacteria, Sinorhizobium sp. strain A2, to arsenic was examined in the present study. Several proteins associated with arsenite oxidase and As resistance were shown to be accumulated in the presence of As(III). A shift in central carbon metabolism from the tricarboxylic acid pathway to glyoxylate pathway was also observed in response to oxidative stress. Our results revealed the strategy of the As(III)-oxidizing Sinorhizobium strain to mitigate arsenic toxicity and oxidative damage by multiple metabolic adaptations.

Application of DNA adductomics to soil bacterium Sphingobium sp. strain KK22.

Toward the development of ecotoxicology methods to investigate microbial markers of impacts of hydrocarbon processing activities, DNA adductomic analyses were conducted on a sphingomonad soil bacterium. From growing cells that were exposed or unexposed to acrolein, a commonly used biocide in hydraulic fracturing processes, DNA was extracted, digested to 2'-deoxynucleosides and analyzed by liquid chromatography-positive ionization electrospray-tandem mass spectrometry in selected reaction monitoring mode transmitting the [M + H](+) > [M + H - 116](+) transition over 100 transitions. Overall data shown as DNA adductome maps revealed numerous putative DNA adducts under both conditions with some occurring specifically for each condition. Adductomic analyses of triplicate samples indicated that elevated levels of some targeted putative adducts occurred in exposed cells. Two exposure-specific adducts were identified in exposed cells as 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-6-hydroxy-(and 8-hydroxy-)pyrimido[1,2-a]- purine-(3H)-one (6- and 8-hydroxy-PdG) following synthesis of authentic standards of these compounds and subsequent analyses. A time course experiment showed that 6- and 8-hydroxy-PdG were detected in bacterial DNA within 30 min of acrolein exposure but were not detected in unexposed cells. This work demonstrated the first application of DNA adductomics to examine DNA damage in a bacterium and sets a foundation for future work.

Identification of anaerobic arsenite-oxidizing and arsenate-reducing bacteria associated with an alkaline saline lake in Khovsgol, Mongolia.

Microbial arsenic transformation pathways associated with a saline lake located in northern Mongolia were examined using molecular biological and culturing approaches. Bacterial 16S rRNA gene sequences recovered from saline lake sediments and soils were affiliated with haloalkaliphiles, including Bacillus and Halomonas spp. Diverse sequences of arsenate respiratory reductase (arrA) and a new group of arsenite oxidase (arxA) genes were also identified. Pure cultures of arsenate-reducing Nitrincola strain and anaerobic arsenite-oxidizing Halomonas strain were isolated. The chemoorganotrophic Halomonas strain contains arxA gene similar to that of a chemoautotrophic arsenite-oxidizing Alkalilimnicola ehrlichii strain MLHE-1. These results revealed the diversity of arsenic transformation pathways associated with a geographically distinct saline system and the potential contribution of arx-dependent arsenite oxidation by heterotrophic bacteria.

9,10-Phenanthrenedione biodegradation by a soil bacterium and identification of transformation products by LC/ESI-MS/MS.

Transformation of 9,10-phenanthrenedione, a cytotoxic derivative of phenanthrene, was shown to occur by a soil bacterium belonging to the genus Sphingobium. Phenanthrene-grown cells of this strain were exposed to 50mgL(-1) 9,10-phenanthrenedione in liquid cultures, extracted, and extracts were analyzed by liquid chromatography electrospray ionization mass spectrometry in negative ionization mode. Full scan analyses of exposed cells over the range from m/z 50 to m/z 500 were compared to abiotic and biotic controls. Product and precursor ion scan mode analyses indicated that at least three aromatic ring-cleavage transformation products of 9,10-phenanthrenedione were present and structures for these products, corresponding to [M-H](-)=271, [M-H](-)=241, and [M-H](-)=339 were proposed to be 4-(1-hydroxy-3,4-dioxo-2-naphthyl)-2-oxo-but-3-enoic acid, 2,2'-diphenic acid and 2-[(6-carboxy-2,3-dihydroxy-phenyl)-hydroxy-methyl]-5-oxo-hex-3-enedioic acid. The identity of 2,2'-diphenic acid was confirmed by comparison to an authentic standard and when the strain was exposed to 50mgL(-1) 2,2'-diphenic acid in separate assays, a transformation product with a similar mass spectrum as 9,10-phenanthrenedione-derived [M-H](-)=339 was revealed. Based upon these results, pathways for the transformation of 9,10-phenanthrenedione by strain KK22 were proposed. Strain KK22 appeared unable to use 9,10-phenanthrenedione as a growth substrate under these conditions. This is the first report of potential biotransformation pathways of 9,10-phenanthrenedione by a bacterium.

Identification of antimony- and arsenic-oxidizing bacteria associated with antimony mine tailing.

Antimony (Sb) is a naturally occurring toxic element commonly associated with arsenic (As) in the environment and both elements have similar chemistry and toxicity. Increasing numbers of studies have focused on microbial As transformations, while microbial Sb interactions are still not well understood. To gain insight into microbial roles in the geochemical cycling of Sb and As, soils from Sb mine tailing were examined for the presence of Sb- and As-oxidizing bacteria. After aerobic enrichment culturing with As(III) (10 mM) or Sb(III) (100 µM), pure cultures of Pseudomonas- and Stenotrophomonas-related isolates with Sb(III) oxidation activities and a Sinorhizobium-related isolate capable of As(III) oxidation were obtained. The As(III)-oxidizing Sinorhizobium isolate possessed the aerobic arsenite oxidase gene (aioA), the expression of which was induced in the presence of As(III) or Sb(III). However, no Sb(III) oxidation activity was detected from the Sinorhizobium-related isolate, suggesting the involvement of different mechanisms for Sb and As oxidation. These results demonstrate that indigenous microorganisms associated with Sb mine soils are capable of Sb and As oxidation, and potentially contribute to the speciation and mobility of Sb and As in situ.

Effects of petroleum mixture types on soil bacterial population dynamics associated with the biodegradation of hydrocarbons in soil environments.

Soil bacterial population dynamics were examined to assess patterns in microbial response to contamination by different petroleum mixtures with variation in n-alkane profiles or toxic constituents such as pentachlorophenol (PCP). Three soil types from distinct areas of the United States (Montana, Oregon, and Arizona) were used in controlled perturbation experiments containing crude oil, kerosene, diesel, or diesel plus PCP spiked with (14)C-hexadecane or (14)C-tridecane. After a 50-day incubation, 30-70% of added (14)C-alkanes were mineralized to (14)CO2 in Montana and Oregon soils. In contrast, significantly lower mineralization was observed with diesel or kerosene (< 5%) compared to crude-oil treatment (~45%) in the Arizona soil. Different hydrocarbon mixtures selected both unique and common microbial populations across all three soils. Conversely, the contamination of different soils with the same mixture selected for distinct microbial populations. The most consistent genotype observed, a Rhodococcus-like population, was present in the Montana soil with all mixture types. The addition of PCP selected for PCP-tolerant alkane-degrading specialist populations. The results indicated that petroleum mixture type influenced hydrocarbon degradation rates and microbial population selection and that soil characteristics, especially organic content, could also be an important determinant of community responses to hydrocarbon perturbation.

Metagenome sequence analysis of filamentous microbial communities obtained from geochemically distinct geothermal channels reveals specialization of three aquificales lineages.

The Aquificales are thermophilic microorganisms that inhabit hydrothermal systems worldwide and are considered one of the earliest lineages of the domain Bacteria. We analyzed metagenome sequence obtained from six thermal "filamentous streamer" communities (∼40 Mbp per site), which targeted three different groups of Aquificales found in Yellowstone National Park (YNP). Unassembled metagenome sequence and PCR-amplified 16S rRNA gene libraries revealed that acidic, sulfidic sites were dominated by Hydrogenobaculum (Aquificaceae) populations, whereas the circum-neutral pH (6.5-7.8) sites containing dissolved sulfide were dominated by Sulfurihydrogenibium spp. (Hydrogenothermaceae). Thermocrinis (Aquificaceae) populations were found primarily in the circum-neutral sites with undetectable sulfide, and to a lesser extent in one sulfidic system at pH 8. Phylogenetic analysis of assembled sequence containing 16S rRNA genes as well as conserved protein-encoding genes revealed that the composition and function of these communities varied across geochemical conditions. Each Aquificales lineage contained genes for CO2 fixation by the reverse-TCA cycle, but only the Sulfurihydrogenibium populations perform citrate cleavage using ATP citrate lyase (Acl). The Aquificaceae populations use an alternative pathway catalyzed by two separate enzymes, citryl-CoA synthetase (Ccs), and citryl-CoA lyase (Ccl). All three Aquificales lineages contained evidence of aerobic respiration, albeit due to completely different types of heme Cu oxidases (subunit I) involved in oxygen reduction. The distribution of Aquificales populations and differences among functional genes involved in energy generation and electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, H2, O2) have resulted in niche specialization among members of the Aquificales.

The membrane-associated monooxygenase in the butane-oxidizing Gram-positive bacterium Nocardioides sp. strain CF8 is a novel member of the AMO/PMO family.

The Gram-positive bacterium Nocardioides sp. strain CF8 uses a membrane-associated monooxygenase (pBMO) to grow on butane. The nucleotide sequences of the genes encoding this novel monooxygenase were revealed through analysis of a de novo assembled draft genome sequence determined by high-throughput sequencing of the whole genome. The pBMO genes were in a similar arrangement to the genes for ammonia monooxygenase (AMO) from the ammonia-oxidizing bacteria and for particulate methane monooxygenase (pMMO) from the methane-oxidizing bacteria. The pBMO genes likely constitute an operon in the order bmoC, bmoA and bmoB. The nucleotide sequence was less than 50% similar to the genes for AMO and pMMO. The operon for pBMO was confirmed to be a single copy in the genome by Southern and computational analyses. In an incubation on butane the increase of transcriptional activity of the pBmoA gene was congruent with the increase of pBMO activity and suggested correspondence between gene expression and the utilization of butane. Phylogenetic comparison revealed distant but significant similarity of all three pBMO subunits to homologous members of the AMO/pMMO family and indicated that the pBMO represents a deeply branching third lineage of this group of particulate monooxygenases. No other bmoCAB-like genes were found to cluster with pBMO lineage in phylogenetic analysis by database searches including genomic and metagenomic sequence databases. pBMO is the first example of the AMO/pMMO-like monooxygenase from Gram-positive bacteria showing similarities to proteobacterial pMMO and AMO sequences.

Community ecology of hot spring cyanobacterial mats: predominant populations and their functional potential.

Phototrophic microbial mat communities from 60°C and 65°C regions in the effluent channels of Mushroom and Octopus Springs (Yellowstone National Park, WY, USA) were investigated by shotgun metagenomic sequencing. Analyses of assembled metagenomic sequences resolved six dominant chlorophototrophic populations and permitted the discovery and characterization of undescribed but predominant community members and their physiological potential. Linkage of phylogenetic marker genes and functional genes showed novel chlorophototrophic bacteria belonging to uncharacterized lineages within the order Chlorobiales and within the Kingdom Chloroflexi. The latter is the first chlorophototrophic member of Kingdom Chloroflexi that lies outside the monophyletic group of chlorophototrophs of the Order Chloroflexales. Direct comparison of unassembled metagenomic sequences to genomes of representative isolates showed extensive genetic diversity, genomic rearrangements and novel physiological potential in native populations as compared with genomic references. Synechococcus spp. metagenomic sequences showed a high degree of synteny with the reference genomes of Synechococcus spp. strains A and B', but synteny declined with decreasing sequence relatedness to these references. There was evidence of horizontal gene transfer among native populations, but the frequency of these events was inversely proportional to phylogenetic relatedness.

Metagenomes from high-temperature chemotrophic systems reveal geochemical controls on microbial community structure and function.

The Yellowstone caldera contains the most numerous and diverse geothermal systems on Earth, yielding an extensive array of unique high-temperature environments that host a variety of deeply-rooted and understudied Archaea, Bacteria and Eukarya. The combination of extreme temperature and chemical conditions encountered in geothermal environments often results in considerably less microbial diversity than other terrestrial habitats and offers a tremendous opportunity for studying the structure and function of indigenous microbial communities and for establishing linkages between putative metabolisms and element cycling. Metagenome sequence (14-15,000 Sanger reads per site) was obtained for five high-temperature (>65 degrees C) chemotrophic microbial communities sampled from geothermal springs (or pools) in Yellowstone National Park (YNP) that exhibit a wide range in geochemistry including pH, dissolved sulfide, dissolved oxygen and ferrous iron. Metagenome data revealed significant differences in the predominant phyla associated with each of these geochemical environments. Novel members of the Sulfolobales are dominant in low pH environments, while other Crenarchaeota including distantly-related Thermoproteales and Desulfurococcales populations dominate in suboxic sulfidic sediments. Several novel archaeal groups are well represented in an acidic (pH 3) Fe-oxyhydroxide mat, where a higher O2 influx is accompanied with an increase in archaeal diversity. The presence or absence of genes and pathways important in S oxidation-reduction, H2-oxidation, and aerobic respiration (terminal oxidation) provide insight regarding the metabolic strategies of indigenous organisms present in geothermal systems. Multiple-pathway and protein-specific functional analysis of metagenome sequence data corroborated results from phylogenetic analyses and clearly demonstrate major differences in metabolic potential across sites. The distribution of functional genes involved in electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, Fe, O2) control microbial community structure and function in YNP geothermal springs.