Metallosphaera Research Articles

Formation of pharmacosiderite (KFe4(AsO4)3(OH)4·6-7H2O) in the acid-sulfate-chloride (ASC) geothermal spring, Norris Geyser Basin, Yellowstone National Park, USA: Implication of Fe and As redox reaction associated with microbe and clay minerals

Microbially-mediated redox reactions have long been known to participate in mineral formation in geothermal systems. In the current study sediment samples collected from an acid-sulfate-chloride thermal spring (pH = 3.5, 78 °C) in Yellowstone National Park, USA were incubated anaerobically in the presence and absence of nutrient enrichment at in-situ temperature and pH for up to 12 months to investigate biomineralization. Neoformation of pharmacosiderite (KFe4(AsO4)3(OH)4·6-7H2O) through the oxidation of ferrous iron and arsenite (or desorbed phase of arsenate) in the hot spring, occurred in nutrient-amended but not in unamended sediment slurries after 6 months of incubation.These reactions are not favored under abiotic, anoxic, or acidic conditions. The reactions are more favorable, however, when mediated biotically by microorganisms such as arsenic and Fe oxidizers. Indeed, 16S rRNA sequence analysis of the sediment slurries revealed the presence of a diverse community of archaea which included a sulfate/thiosulfate reducing Vulcanisaeta sp., the Fe-oxidizer Metallosphaera yellowstonensis MK-1, and members of the heterotrophic iron-reducing genera Sulfolobus and Sulfurihydrogenibium; the latter known to contain members which possess the aioA gene involved in arsenite oxidation. The results suggest that precipitation of pharmacosiderite under ASC hot spring conditions requires the stimulation of heterotrophic members of the microbial community capable of iron and arsenic transformations. Furthermore, in the clay-rich environment such as the present study, kaolinite also have an important role in oxidizing As(III) through a surface complexation providing the favorable conditions for the biotic pharmacosiderite formation compared to the clay-free condition.

Multi-mode scanning electrochemical microscopic study of microbiologically influenced corrosion mechanism of 304 stainless steel by thermoacidophilic archaea

In this work, different modes of scanning electrochemical microscopy (SECM) were employed to study the biofilm morphology, oxygen concentration distribution and Fe2+ oxidation metabolism during the microbiologically influenced corrosion (MIC) of 304 stainless steel caused by the thermoacidophilic archaeon Metallosphaera cuprina. The results of in situ SECM study suggested that the decrease in the concentration of organic nutrients forced the M. cuprina biofilms to use Fe2+ as an electron donor and consume more Fe2+ from the pitting corrosion, which accelerated anodic dissolution and led to aggravated pitting corrosion of stainless steel.

Investigation of microbiologically influenced corrosion of 304 stainless steel by aerobic thermoacidophilic archaeon Metallosphaera cuprina

In this study, the influence of thermoacidophilic archaeon Metallosphaera cuprina on the corrosion of 304 stainless steel was investigated. 304 stainless steel in M. cuprina-inoculated culture medium exhibited more marked pitting corrosion behavior than that seen in sterile culture medium. After 14 days, the average pit depth under M. cuprina biofilms was nearly twice as great as that in sterile culture medium. Electrochemical measurements also showed that 304 stainless steel had lower charge transfer resistance and smaller pitting potential after 14 days of exposure in inoculated culture medium. The ferrous ion oxidation ability of M. cuprina biofilms can cause a change in the composition of passive films and accelerate the anodic dissolution of the steel substrate, to promote the pitting corrosion process at 304 stainless steel.

Comparative Genomic Analysis Reveals the Metabolism and Evolution of the Thermophilic Archaeal Genus Metallosphaera.

Members of the genus Metallosphaera are widely found in sulfur-rich and metal-laden environments, but their physiological and ecological roles remain poorly understood. Here, we sequenced Metallosphaera tengchongensis Ric-A, a strain isolated from the Tengchong hot spring in Yunnan Province, China, and performed a comparative genome analysis with other Metallosphaera genomes. The genome of M. tengchongensis had an average nucleotide identity (ANI) of approximately 70% to that of Metallosphaera cuprina. Genes sqr, tth, sir, tqo, hdr, tst, soe, and sdo associated with sulfur oxidation, and gene clusters fox and cbs involved in iron oxidation existed in all Metallosphaera genomes. However, the adenosine-5′-phosphosulfate (APS) pathway was only detected in Metallosphaera sedula and Metallosphaera yellowstonensis, and several subunits of fox cluster were lost in M. cuprina. The complete 3-hydroxypropionate/4-hydroxybutyrate cycle and dicarboxylate/4-hydroxybutyrate cycle involved in carbon fixation were found in all Metallosphaera genomes. A large number of gene family gain events occurred in M. yellowstonensis and M. sedula, whereas gene family loss events occurred frequently in M. cuprina. Pervasive strong purifying selection was found acting on the gene families of Metallosphaera, of which transcription-related genes underwent the strongest purifying selection. In contrast, genes related to prophages, transposons, and defense mechanisms were under weaker purifying pressure. Taken together, this study expands knowledge of the genomic traits of Metallosphaera species and sheds light on their evolution.

Multiscale analysis of autotroph-heterotroph interactions in a high-temperature microbial community

Interactions among microbial community members can lead to emergent properties, such as enhanced productivity, stability, and robustness. Iron-oxide mats in acidic (pH 2–4), high-temperature (> 65 °C) springs of Yellowstone National Park contain relatively simple microbial communities and are well-characterized geochemically. Consequently, these communities are excellent model systems for studying the metabolic activity of individual populations and key microbial interactions. The primary goals of the current study were to integrate data collected in situ with in silico calculations across process-scales encompassing enzymatic activity, cellular metabolism, community interactions, and ecosystem biogeochemistry, as well as to predict and quantify the functional limits of autotroph-heterotroph interactions. Metagenomic and transcriptomic data were used to reconstruct carbon and energy metabolisms of an important autotroph (Metallosphaera yellowstonensis) and heterotroph (Geoarchaeum sp. OSPB) from the studied Fe(III)-oxide mat communities. Standard and hybrid elementary flux mode and flux balance analyses of metabolic models predicted cellular- and community-level metabolic acclimations to simulated environmental stresses, respectively. In situ geochemical analyses, including oxygen depth-profiles, Fe(III)-oxide deposition rates, stable carbon isotopes and mat biomass concentrations, were combined with cellular models to explore autotroph-heterotroph interactions important to community structure-function. Integration of metabolic modeling with in situ measurements, including the relative population abundance of autotrophs to heterotrophs, demonstrated that Fe(III)-oxide mat communities operate at their maximum total community growth rate (i.e. sum of autotroph and heterotroph growth rates), as opposed to net community growth rate (i.e. total community growth rate subtracting autotroph consumed by heterotroph), as predicted from the maximum power principle. Integration of multiscale data with ecological theory provides a basis for predicting autotroph-heterotroph interactions and community-level cellular organization.

Isolation and Characterization of Metallosphaera Turreted Icosahedral Virus, a Founding Member of a New Family of Archaeal Viruses.

Our understanding of archaeal virus diversity and structure is just beginning to emerge. Here we describe a new archaeal virus, tentatively named Metallosphaera turreted icosahedral virus (MTIV), that was isolated from an acidic hot spring in Yellowstone National Park, USA. Two strains of the virus were identified and were found to replicate in an archaeal host species closely related to Metallosphaera yellowstonensis Each strain encodes a 9.8- to 9.9-kb linear double-stranded DNA (dsDNA) genome with large inverted terminal repeats. Each genome encodes 21 open reading frames (ORFs). The ORFs display high homology between the strains, but they are quite distinct from other known viral genes. The 70-nm-diameter virion is built on a T=28 icosahedral lattice. Both single particle cryo-electron microscopy and cryotomography reconstructions reveal an unusual structure that has 42 turret-like projections: 12 pentameric turrets positioned on the icosahedral 5-fold axes and 30 turrets with apparent hexameric symmetry positioned on the icosahedral 2-fold axes. Both the virion structural properties and the genome content support MTIV as the founding member of a new family of archaeal viruses.IMPORTANCE Many archaeal viruses are quite different from viruses infecting bacteria and eukaryotes. Initial characterization of MTIV reveals a virus distinct from other known bacterial, eukaryotic, and archaeal viruses; this finding suggests that viruses infecting Archaea are still an understudied group. As the first known virus infecting a Metallosphaera sp., MTIV provides a new system for exploring archaeal virology by examining host-virus interactions and the unique features of MTIV structure-function relationships. These studies will likely expand our understanding of virus ecology and evolution.

The Influence of Pyrite on Galvanic Assisted Bioleaching of Low Grade Chalcopyrite Ores

The experiment focused on the effect of pyrite on the dissolution of the ore from Saindak copper mine in Pakistan. The MLA results showed that the ore mainly consisted of chalcopyrite, which was associated closely with pyrite. The bioleaching process and galvanic promotion experiments of pyrite on the copper extraction were investigated. The data revealed that copper recovery rate could be up to 97.8% after the bioleaching process by extreme thermophilic acidophilic organisms for 30 days at 65 OC. The temperature and redox potential were the controlled factors to recover copper from the Saindak mineral. The phylogenetic analyses of 16S rRNA gene fragments revealed that organisms related to Metallosphaera cuprina strain Ar-4 and Sulfolobus sp. HB59 were most dominant. Pyrite could accelerate the dissolution of chalcopyrite as one galvanic assistant substance, which would be strengthened by elevating the temperature or in the presence of microorganisms.

Hydrogen Peroxide Cycling in High-Temperature Acidic Geothermal Springs and Potential Implications for Oxidative Stress Response

Hydrogen peroxide (H2O2), superoxide (O2•-), and hydroxyl radicals (OH•) are produced in natural waters via ultraviolet (UV) light-induced reactions between dissolved oxygen (O2) and organic carbon, and further reaction of H2O2 and Fe(II) (i.e., Fenton chemistry). The temporal and spatial dynamics of H2O2 and other dissolved compounds (Fe(II), Fe(III), H2S, O2) were measured during a diel cycle (dark/light) in surface waters of three acidic geothermal springs (Beowulf Spring, One Hundred Springs Plain, and Echinus Geyser Spring; pH = 3-3.5, T = 68-80 °C) in Norris Geyser Basin, Yellowstone National Park. In situ analyses showed that H2O2 concentrations were lowest (ca. 1 μM) in geothermal source waters containing high dissolved sulfide (and where oxygen was below detection) and increased by two-fold (ca. 2-3 μM) in oxygenated waters corresponding to Fe(III)-oxide mat formation down the water channel. Small increases in dissolved oxygen and H2O2 were observed during peak photon flux, but not consistently across all springs sampled. Iron-oxide microbial mats were sampled for molecular analysis of ROS gene expression in two primary autotrophs of acidic Fe(III)-oxide mat ecosystems: Metallosphaera yellowstonensis (Archaea) and Hydrogenobaculum sp. (Bacteria). Expression (RT-qPCR) assays of specific stress-response genes (e.g., superoxide dismutase, peroxidases) of the primary autotrophs were used to evaluate possible changes in transcription across temporal, spatial and/or seasonal samples. Data presented here documented the presence of H2O2 and general correlation with dissolved oxygen. Moreover, two dominant microbial populations expressed ROS response genes throughout the day, but showed less expression of key genes during peak sunlight. Oxidative stress response genes (especially external peroxidases) were highly-expressed in microorganisms within Fe(III)-oxide mat communities, suggesting a significant role for these proteins during survival and growth in situ.

Formaldehyde as a carbon and electron shuttle between autotroph and heterotroph populations in acidic hydrothermal vents of Norris Geyser Basin, Yellowstone National Park.

The Norris Geyser Basin in Yellowstone National Park contains a large number of hydrothermal systems, which host microbial populations supported by primary productivity associated with a suite of chemolithotrophic metabolisms. We demonstrate that Metallosphaera yellowstonensis MK1, a facultative autotrophic archaeon isolated from a hyperthermal acidic hydrous ferric oxide (HFO) spring in Norris Geyser Basin, excretes formaldehyde during autotrophic growth. To determine the fate of formaldehyde in this low organic carbon environment, we incubated native microbial mat (containing M. yellowstonensis) from a HFO spring with (13)C-formaldehyde. Isotopic analysis of incubation-derived CO2 and biomass showed that formaldehyde was both oxidized and assimilated by members of the community. Autotrophy, formaldehyde oxidation, and formaldehyde assimilation displayed different sensitivities to chemical inhibitors, suggesting that distinct sub-populations in the mat selectively perform these functions. Our results demonstrate that electrons originally resulting from iron oxidation can energetically fuel autotrophic carbon fixation and associated formaldehyde excretion, and that formaldehyde is both oxidized and assimilated by different organisms within the native microbial community. Thus, formaldehyde can effectively act as a carbon and electron shuttle connecting the autotrophic, iron oxidizing members with associated heterotrophic members in the HFO community.

Assembly and Succession of Iron Oxide Microbial Mat Communities in Acidic Geothermal Springs.

Biomineralized ferric oxide microbial mats are ubiquitous features on Earth, are common in hot springs of Yellowstone National Park (YNP, WY, USA), and form due to direct interaction between microbial and physicochemical processes. The overall goal of this study was to determine the contribution of different community members to the assembly and succession of acidic high-temperature Fe(III)-oxide mat ecosystems. Spatial and temporal changes in Fe(III)-oxide accretion and the abundance of relevant community members were monitored over 70 days using sterile glass microscope slides incubated in the outflow channels of two acidic geothermal springs (pH = 3–3.5; temperature = 68–75°C) in YNP. Hydrogenobaculum spp. were the most abundant taxon identified during early successional stages (4–40 days), and have been shown to oxidize arsenite, sulfide, and hydrogen coupled to oxygen reduction. Iron-oxidizing populations of Metallosphaera yellowstonensis were detected within 4 days, and reached steady-state levels within 14–30 days, corresponding to visible Fe(III)-oxide accretion. Heterotrophic archaea colonized near 30 days, and emerged as the dominant functional guild after 70 days and in mature Fe(III)-oxide mats (1–2 cm thick). First-order rate constants of Fe(III)-oxide accretion ranged from 0.046 to 0.05 day−1, and in situ microelectrode measurements showed that the oxidation of Fe(II) is limited by the diffusion of O2 into the Fe(III)-oxide mat. The formation of microterracettes also implicated O2 as a major variable controlling microbial growth and subsequent mat morphology. The assembly and succession of Fe(III)-oxide mat communities follows a repeatable pattern of colonization by lithoautotrophic organisms, and the subsequent growth of diverse organoheterotrophs. The unique geochemical signatures and micromorphology of extant biomineralized Fe(III)-oxide mats are also useful for understanding other Fe(II)-oxidizing systems.

Novel Transcriptional Regulons for Autotrophic Cycle Genes in Crenarchaeota.

Autotrophic microorganisms are able to utilize carbon dioxide as their only carbon source, or, alternatively, many of them can grow heterotrophically on organics. Different variants of autotrophic pathways have been identified in various lineages of the phylum Crenarchaeota. Aerobic members of the order Sulfolobales utilize the hydroxypropionate-hydroxybutyrate cycle (HHC) to fix inorganic carbon, whereas anaerobic Thermoproteales use the dicarboxylate-hydroxybutyrate cycle (DHC). Knowledge of transcriptional regulation of autotrophic pathways in Archaea is limited. We applied a comparative genomics approach to predict novel autotrophic regulons in the Crenarchaeota. We report identification of two novel DNA motifs associated with the autotrophic pathway genes in the Sulfolobales (HHC box) and Thermoproteales (DHC box). Based on genome context evidence, the HHC box regulon was attributed to a novel transcription factor from the TrmB family named HhcR. Orthologs of HhcR are present in all Sulfolobales genomes but were not found in other lineages. A predicted HHC box regulatory motif was confirmed by in vitro binding assays with the recombinant HhcR protein from Metallosphaera yellowstonensis. For the DHC box regulon, we assigned a different potential regulator, named DhcR, which is restricted to the order Thermoproteales. DhcR in Thermoproteus neutrophilus (Tneu_0751) was previously identified as a DNA-binding protein with high affinity for the promoter regions of two autotrophic operons. The global HhcR and DhcR regulons reconstructed by comparative genomics were reconciled with available omics data in Metallosphaera and Thermoproteus spp. The identified regulons constitute two novel mechanisms for transcriptional control of autotrophic pathways in the Crenarchaeota. Little is known about transcriptional regulation of carbon dioxide fixation pathways in Archaea. We previously applied the comparative genomics approach for reconstruction of DtxR family regulons in diverse lineages of Archaea. Here, we utilize similar computational approaches to identify novel regulatory motifs for genes that are autotrophically induced in microorganisms from two lineages of Crenarchaeota and to reconstruct the respective regulons. The predicted novel regulons in archaeal genomes control the majority of autotrophic pathway genes and also other carbon and energy metabolism genes. The HhcR regulon was experimentally validated by DNA-binding assays in Metallosphaera spp. Novel regulons described for the first time in this work provide a basis for understanding the mechanisms of transcriptional regulation of autotrophic pathways in Archaea.

Thiosulfate Transfer Mediated by DsrE/TusA Homologs from Acidothermophilic Sulfur-oxidizing Archaeon Metallosphaera cuprina

Conserved clusters of genes encoding DsrE and TusA homologs occur in many archaeal and bacterial sulfur oxidizers. TusA has a well documented function as a sulfurtransferase in tRNA modification and molybdenum cofactor biosynthesis in Escherichia coli, and DsrE is an active site subunit of the DsrEFH complex that is essential for sulfur trafficking in the phototrophic sulfur-oxidizing Allochromatium vinosum. In the acidothermophilic sulfur (S(0))- and tetrathionate (S4O6(2-))-oxidizing Metallosphaera cuprina Ar-4, a dsrE3A-dsrE2B-tusA arrangement is situated immediately between genes encoding dihydrolipoamide dehydrogenase and a heterodisulfide reductase-like complex. In this study, the biochemical features and sulfur transferring abilities of the DsrE2B, DsrE3A, and TusA proteins were investigated. DsrE3A and TusA proved to react with tetrathionate but not with NaSH, glutathione persulfide, polysulfide, thiosulfate, or sulfite. The products were identified as protein-Cys-S-thiosulfonates. DsrE3A was also able to cleave the thiosulfate group from TusA-Cys(18)-S-thiosulfonate. DsrE2B did not react with any of the sulfur compounds tested. DsrE3A and TusA interacted physically with each other and formed a heterocomplex. The cysteine residue (Cys(18)) of TusA is crucial for this interaction. The single cysteine mutants DsrE3A-C(93)S and DsrE3A-C(101)S retained the ability to transfer the thiosulfonate group to TusA. TusA-C(18)S neither reacted with tetrathionate nor was it loaded with thiosulfate with DsrE3A-Cys-S-thiosulfonate as the donor. The transfer of thiosulfate, mediated by a DsrE-like protein and TusA, is unprecedented not only in M. cuprina but also in other sulfur-oxidizing prokaryotes. The results of this study provide new knowledge on oxidative microbial sulfur metabolism.

Quartet Analysis of Putative Horizontal Gene Transfer in Crenarchaeota

Horizontal gene transfers (HGT) between four Crenarchaeota species (Metallosphaera cuprina Ar-4T, Acidianus hospitalis W1T, Vulcanisaeta moutnovskia 768-28T, and Pyrobaculum islandicum DSM 4184T) were investigated with quartet analysis. Strong support was found for individual genes that disagree with the phylogeny of the majority, implying genomic mosaicism. One such gene, a ferredoxin-related gene, was investigated further and incorporated into a larger phylogeny, which provided evidence for HGT of this gene from the Vulcanisaeta lineage to the Acidianus lineage. This is the first application of quartet analysis of HGT for the phylum Crenarchaeota. The results have shown that quartet analysis is a powerful technique to screen homologous sequences for putative HGTs and is useful in visually describing genomic mosaicism and HGT within four taxa.

In situ analysis of oxygen consumption and diffusive transport in high‐temperature acidic iron‐oxide microbial mats

The role of dissolved oxygen as a principal electron acceptor for microbial metabolism was investigated within Fe(III)-oxide microbial mats that form in acidic geothermal springs of Yellowstone National Park (USA). Specific goals of the study were to measure and model dissolved oxygen profiles within high-temperature (65-75°C) acidic (pH = 2.7-3.8) Fe(III)-oxide microbial mats, and correlate the abundance of aerobic, iron-oxidizing Metallosphaera yellowstonensis organisms and mRNA gene expression levels to Fe(II)-oxidizing habitats shown to consume oxygen. In situ oxygen microprofiles were obtained perpendicular to the direction of convective flow across the aqueous phase/Fe(III)-oxide microbial mat interface using oxygen microsensors. Dissolved oxygen concentrations dropped from ∼ 50-60 μM in the bulk-fluid/mat surface to below detection (< 0.3 μM) at a depth of ∼ 700 μm (∼ 10% of the total mat depth). Net areal oxygen fluxes into the microbial mats were estimated to range from 1.4-1.6 × 10(-4) μmol cm(-2) s(-1) . Dimensionless parameters were used to model dissolved oxygen profiles and establish that mass transfer rates limit the oxygen consumption. A zone of higher dissolved oxygen at the mat surface promotes Fe(III)-oxide biomineralization, which was supported using molecular analysis of Metallosphaera yellowstonensis 16S rRNA gene copy numbers and mRNA expression of haem Cu oxidases (FoxA) associated with Fe(II)-oxidation.

Microbial Iron Cycling in Acidic Geothermal Springs of Yellowstone National Park: Integrating Molecular Surveys, Geochemical Processes, and Isolation of Novel Fe-Active Microorganisms

Geochemical, molecular, and physiological analyses of microbial isolates were combined to study the geomicrobiology of acidic iron oxide mats in Yellowstone National Park. Nineteen sampling locations from 11 geothermal springs were studied ranging in temperature from 53 to 88°C and pH 2.4 to 3.6. All iron oxide mats exhibited high diversity of crenarchaeal sequences from the Sulfolobales, Thermoproteales, and Desulfurococcales. The predominant Sulfolobales sequences were highly similar to Metallosphaera yellowstonensis str. MK1, previously isolated from one of these sites. Other groups of archaea were consistently associated with different types of iron oxide mats, including undescribed members of the phyla Thaumarchaeota and Euryarchaeota. Bacterial sequences were dominated by relatives of Hydrogenobaculum spp. above 65–70°C, but increased in diversity below 60°C. Cultivation of relevant iron-oxidizing and iron-reducing microbial isolates included Sulfolobus str. MK3, Sulfobacillus str. MK2, Acidicaldus str. MK6, and a new candidate genus in the Sulfolobales referred to as Sulfolobales str. MK5. Strains MK3 and MK5 are capable of oxidizing ferrous iron autotrophically, while strain MK2 oxidizes iron mixotrophically. Similar rates of iron oxidation were measured for M. yellowstonensis str. MK1 and Sulfolobales str. MK5. Biomineralized phases of ferric iron varied among cultures and field sites, and included ferric oxyhydroxides, K-jarosite, goethite, hematite, and scorodite depending on geochemical conditions. Strains MK5 and MK6 are capable of reducing ferric iron under anaerobic conditions with complex carbon sources. The combination of geochemical and molecular data as well as physiological observations of isolates suggests that the community structure of acidic Fe mats is linked with Fe cycling across temperatures ranging from 53 to 88°C.

Terminal Oxidase Diversity and Function in “ Metallosphaera yellowstonensis ”: Gene Expression and Protein Modeling Suggest Mechanisms of Fe(II) Oxidation in the Sulfolobales

"Metallosphaera yellowstonensis" is a thermoacidophilic archaeon isolated from Yellowstone National Park that is capable of autotrophic growth using Fe(II), elemental S, or pyrite as electron donors. Analysis of the draft genome sequence from M. yellowstonensis strain MK1 revealed seven different copies of heme copper oxidases (subunit I) in a total of five different terminal oxidase complexes, including doxBCEF, foxABCDEFGHIJ, soxABC, and the soxM supercomplex, as well as a novel hypothetical two-protein doxB-like polyferredoxin complex. Other genes found in M. yellowstonensis with possible roles in S and or Fe cycling include a thiosulfate oxidase (tqoAB), a sulfite oxidase (som), a cbsA cytochrome b(558/566), several small blue copper proteins, and a novel gene sequence coding for a putative multicopper oxidase (Mco). Results from gene expression studies, including reverse transcriptase (RT) quantitative PCR (qPCR) of cultures grown autotrophically on either Fe(II), pyrite, or elemental S showed that the fox gene cluster and mco are highly expressed under conditions where Fe(II) is an electron donor. Metagenome sequence and gene expression studies of Fe-oxide mats confirmed the importance of fox genes (e.g., foxA and foxC) and mco under Fe(II)-oxidizing conditions. Protein modeling of FoxC suggests a novel lysine-lysine or lysine-arginine heme B binding domain, indicating that it is likely the cytochrome component of a heterodimer complex with foxG as a ferredoxin subunit. Analysis of mco shows that it encodes a novel multicopper blue protein with two plastocyanin type I copper domains that may play a role in the transfer of electrons within the Fox protein complex. An understanding of metabolic pathways involved in aerobic iron and sulfur oxidation in Sulfolobales has broad implications for understanding the evolution and niche diversification of these thermophiles as well as practical applications in fields such as bioleaching of trace metals from pyritic ores.

Metallosphaera cuprina sp. nov., an acidothermophilic, metal-mobilizing archaeon

A novel acidothermophilic archaeon, strain Ar-4(T), was isolated from a sulfuric hot spring in Tengchong, Yunnan, China. Cells of strain Ar-4(T) were Gram-staining-negative, irregular cocci and motile by means of flagella. Strain Ar-4(T) grew over a temperature range of 55-75 °C (optimum, 65 °C), a pH range of 2.5-5.5 (optimum, pH 3.5) and a NaCl concentration range of 0-1 % (w/v). The novel strain was aerobic and facultatively chemolithoautotrophic. The strain could extract metal ions from sulfidic ore. It was also able to oxidize reduced sulfur compounds. In addition, it was able to use heterogeneous organic materials for organotrophic growth. The main cellular lipids were calditoglycerocaldarchaeol (CGTE) and caldarchaeol (DGTE). The DNA G+C content of the strain was 40.2 mol%. Analysis of 16S rRNA gene sequences showed that strain Ar-4(T) was phylogenetically related to members of the genus Metallosphaera and had sequence similarities of 97.7 %, 97.0 % and 96.8 % with Metallosphaera hakonensis DSM 7519(T), Metallosphaera sedula DSM 5348(T) and Metallosphaera prunae DSM 10039(T), respectively. Strain Ar-4(T) showed DNA-DNA relatedness values of 47.5 %, 30.8 % and 29.1 % with M. hakonensis DSM 7519(T), M. sedula DSM 5348(T) and M. prunae DSM 10039(T), respectively. The differences in cell motility, the temperature and pH ranges for growth, the ability to utilize carbon sources, the DNA G+C content, and the low DNA-DNA relatedness values distinguished strain Ar-4(T) from recognized species of the genus Metallosphaera. On the basis of these results, it was concluded that strain Ar-4(T) represents a novel species of the genus Metallosphaera, for which the name Metallosphaera cuprina is proposed. The type strain is Ar-4(T) ( = JCM 15769(T) = CGMCC 1.7082(T)).