Sulfuracidifex tepidarius gen. nov., sp. nov. and transfer of Sulfolobus metallicus Huber and Stetter 1992 to the genus Sulfuracidifex as Sulfuracidifex metallicus comb. nov.

Two novel, strictly aerobic, sulfur-dependent, thermoacidophilic strains, IC-006T and IC-007, were isolated from a solfataric field at Hakone Ohwaku-dani, Kanagawa, Japan. Cells of the two strains were irregular cocci with a diameter of 1.0-1.8 µm. They were strict aerobes and grew in a temperature range between 45 and 69 °C (optimally at 65 °C) and a pH range between 0.4 and 5.5 (optimally at pH 3.5). They required sulfur or a reduced sulfur compound, and sulfur was oxidized to sulfate. They grew autotrophically or mixotrophically utilizing several sugars and complex organic substances as carbon sources. The DNA G+C content was 42.4 mol%. A comparison of the 16S rRNA gene sequences among members of the order Sulfolobales indicated that they were closely related to Sulfolobus metallicus, forming an independent lineage within this order. The two isolates and Sulfolobus metallicus were also diffentiated based on their phenotypic properties from the other members of the order Sulfolobales. Detailed comparisons of the phenotypic properties and DNA-DNA hybridization study illustrated that the two isolates belong to a species different from Sulfolobus metallicus. On the basis of the phylogenetic and phenotypic comparisons, we propose a new genus and species, Sulfuracidifex tepidarius gen. nov., sp. nov. to accommodate strains IC-006T and IC-007. The type strain of Sulfuracidifex tepidarius is IC-006T (=JCM 16833T=DSM 104736T). In addition, Sulfolobus metallicus should be transferred to the new genus as Sulfuracidifex metallicus comb. nov.: the type strain is Kra23T (=DSM 6482T=JCM 9184T=NBRC 15436T).

Complete genome sequence of the Sulfodiicoccus acidiphilus strain HS-1T, the first crenarchaeon that lacks polB3, isolated from an acidic hot spring in Ohwaku-dani, Hakone, Japan.

OBJECTIVE: Sulfodiicoccus acidiphilus HS-1T is the type species of the genus Sulfodiicoccus, a thermoacidophilic archaeon belonging to the order Sulfolobales (class Thermoprotei; phylum Crenarchaeota). While S. acidiphilus HS-1T shares many common physiological and phenotypic features with other Sulfolobales species, the similarities in their 16S rRNA gene sequences are less than 89%. In order to know the genomic features of S. acidiphilus HS-1T in the order Sulfolobales, we determined and characterized the genome of this strain. RESULTS: The circular genome of S. acidiphilus HS-1T is comprised of 2353,189 bp with a G+C content of 51.15 mol%. A total of 2459 genes were predicted, including 2411 protein coding and 48 RNA genes. The notable genomic features of S. acidiphilus HS-1T in Sulfolobales species are the absence of genes for polB3 and the autotrophic carbon fixation pathway, and the distribution pattern of essential genes and sequences related to genomic replication initiation. These insights contribute to an understanding of archaeal genomic diversity and evolution.

Sulfurisphaera javensis sp. nov., a hyperthermophilic and acidophilic archaeon isolated from Indonesian hot spring, and reclassification of Sulfolobus tokodaii Suzuki et al. 2002 as Sulfurisphaera tokodaii comb. nov.

A novel hyperthermophilic, acidophilic and facultatively anaerobic archaeon, strain KD-1T, was isolated from an acidic hot spring in Indonesia and characterized with the phylogenetically related species Sulfurisphaera ohwakuensis Kurosawa et al. 1998, Sulfolobus tokodaii Suzuki et al., 2002 and Sulfolobus yangmingensis Jan et al. 1999. Cells of KD-1T were irregular cocci with diameters of 0.9-1.3 µm. The strain grew at 60-90 °C (optimum 80-85 °C), pH 2.5-6.0 (optimum pH 3.5-4.0) and 0-1.0 % (w/v) NaCl concentration. KD-1T grew anaerobically in the presence of S0 (headspace: H2/CO2) and FeCl3 (headspace: N2). Under aerobic conditions, chemolithoautotrophic growth occurred on S0, pyrite, K2S4O6, Na2S2O3 and H2. This strain utilized various complex substrates, such as yeast extract, but did not grow on sugars and amino acids as the sole carbon source. The main core lipids were calditoglycerocaldarchaeol and caldarchaeol. The DNA G+C content was 30.6 mol%. Analyses of phylogenetic trees based on 16S rRNA and 23S rRNA genes indicated that KD-1T formed an independent lineage near Sulfurisphaera ohwakuensis TA-1T, Sulfolobus tokodaii 7T and Sulfolobus yangmingensis YM1T. On the basis of the results of morphological, physiological, chemotaxonomic and phylogenetic analyses, KD-1T represents a novel species of the genus Sulfurisphaera Kurosawa et al. 1998, for which the name Sulfurisphaera javensis sp. nov. is proposed. The type strain is KD-1T (=JCM 32117T=InaCC Ar81T). Based on the data, we also propose the reclassification of Sulfolobus tokodaii Suzuki et al., 2002 as Sulfurisphaera tokodaii comb. nov. (type strain 7T=JCM 10545T=DSM 16993T).

Sulfodiicoccus acidiphilus gen. nov., sp. nov., a sulfur-inhibited thermoacidophilic archaeon belonging to the order Sulfolobales isolated from a terrestrial acidic hot spring.

A novel thermoacidophilic archaeon, strain HS-1T, was isolated from the Hakone Ohwaku-dani hot spring in Japan. Cells of strain HS-1T in exponential phase were cocci to irregular cocci with a diameter of 0.8-1.5 µm. The strain grew within a temperature range of 50-70 °C (optimal: 65-70 °C), a pH range of pH 1.4-5.5 (optimal: pH 3.0-3.5) and a NaCl concentration range of 0-2.5 % (w/v). The novel strain grew in aerobic conditions but did not grow anaerobically. Moreover, this strain utilized various complex substrates (beef extract, casamino acids, peptone, tryptone and yeast extract) and sugars (arabinose, xylose, galactose, glucose, maltose, sucrose, raffinose and lactose) as sole carbon sources. No chemolithoautotrophic growth occurred on elemental sulfur, pyrite, K2S4O6, Na2S2O3 or FeSO4 . 7H2O; however, growth by the oxidation of hydrogen occurred weakly. The core lipids were calditoglycerocaldarchaeol (CGTE) and caldarchaeol (DGTE). The DNA G+C content of the strain was 52.0 mol%, which was remarkably higher than those of known species of the order Sulfolobales(31-46.2 %). The growth of the strain was significantly inhibited in the presence of elemental sulfur. Analyses of 16S rRNA and 23S rRNA gene sequences showed that HS-1T belonged to the order Sulfolobales; however, it was distantly related to all known species of the order Sulfolobales (less than 89 % sequence similarity). On the basis of these results, we propose the novel genus, Sulfodiicoccus, in the order Sulfolobales (in the family Sulfolobaceae). The type species of the genus is Sulfodiicoccus acidiphilus sp. nov., and the type strain of the species is HS-1T (=JCM 31740T=InaCC Ar79T).

Transcriptomes of the Extremely Thermoacidophilic Archaeon Metallosphaera sedula Exposed to Metal "Shock" Reveal Generic and Specific Metal Responses.

UNLABELLED: The extremely thermoacidophilic archaeon Metallosphaera sedula mobilizes metals by novel membrane-associated oxidase clusters and, consequently, requires metal resistance strategies. This issue was examined by "shocking" M. sedula with representative metals (Co(2+), Cu(2+), Ni(2+), UO2 (2+), Zn(2+)) at inhibitory and subinhibitory levels. Collectively, one-quarter of the genome (554 open reading frames [ORFs]) responded to inhibitory levels, and two-thirds (354) of the ORFs were responsive to a single metal. Cu(2+) (259 ORFs, 106 Cu(2+)-specific ORFs) and Zn(2+) (262 ORFs, 131 Zn(2+)-specific ORFs) triggered the largest responses, followed by UO2 (2+) (187 ORFs, 91 UO2 (2+)-specific ORFs), Ni(2+) (93 ORFs, 25 Ni(2+)-specific ORFs), and Co(2+) (61 ORFs, 1 Co(2+)-specific ORF). While one-third of the metal-responsive ORFs are annotated as encoding hypothetical proteins, metal challenge also impacted ORFs responsible for identifiable processes related to the cell cycle, DNA repair, and oxidative stress. Surprisingly, there were only 30 ORFs that responded to at least four metals, and 10 of these responded to all five metals. This core transcriptome indicated induction of Fe-S cluster assembly (Msed_1656-Msed_1657), tungsten/molybdenum transport (Msed_1780-Msed_1781), and decreased central metabolism. Not surprisingly, a metal-translocating P-type ATPase (Msed_0490) associated with a copper resistance system (Cop) was upregulated in response to Cu(2+) (6-fold) but also in response to UO2 (2+) (4-fold) and Zn(2+) (9-fold). Cu(2+) challenge uniquely induced assimilatory sulfur metabolism for cysteine biosynthesis, suggesting a role for this amino acid in Cu(2+) resistance or issues in sulfur metabolism. The results indicate that M. sedula employs a range of physiological and biochemical responses to metal challenge, many of which are specific to a single metal and involve proteins with yet unassigned or definitive functions. IMPORTANCE: The mechanisms by which extremely thermoacidophilic archaea resist and are negatively impacted by metals encountered in their natural environments are important to understand so that technologies such as bioleaching, which leverage microbially based conversion of insoluble metal sulfides to soluble species, can be improved. Transcriptomic analysis of the cellular response to metal challenge provided both global and specific insights into how these novel microorganisms negotiate metal toxicity in natural and technological settings. As genetics tools are further developed and implemented for extreme thermoacidophiles, information about metal toxicity and resistance can be leveraged to create metabolically engineered strains with improved bioleaching characteristics.

Metallosphaera tengchongensis sp. nov., an acidothermophilic archaeon isolated from a hot spring.

Two novel acidothermophilic archaea, strains Ric-A(T) and Ric-F, were isolated from muddy water samples of a sulfuric hot spring located in Tengchong County, Yunnan Province, PR China. The strains were aerobic and facultatively chemolithoautotrophic. Both strains could oxidize S(0) and K2S4O6 for autotrophic growth, and could use organic materials for heterotrophic growth. Growth was observed at 55-75 °C and pH 1.5-6.5. The strains could oxidize metal sulfide ores, showing their potential in bioleaching. The DNA G+C contents of strains Ric-A(T) and Ric-F were 41.8 and 41.6 mol%, respectively. Analysis of 16S rRNA gene sequences showed that the two strains shared 99.8 % sequence similarity to each other, but <97 % to other known species of the genus Metallosphaera. DNA-DNA hybridization indicated that the isolates were different strains of a novel species of the genus Metallosphaera. Strains Ric-A(T) and Ric-F also shared a number of physiological and biochemical characteristics that distinguished them from recognized species of the genus Metallosphaera. On the basis of phenotypic, chemotaxonomic and phylogenetic comparisons with their closest relatives, it was concluded that strains Ric-A(T) and Ric-F represent a novel species of the genus Metallosphaera, for which the name Metallosphaera tengchongensis sp. nov. is proposed. The type strain is Ric-A(T) ( = NBRC 109472(T) = CGMCC 1.12287(T)).

Carbon dioxide fixation by Metallosphaera yellowstonensis and acidothermophilic iron-oxidizing microbial communities from Yellowstone National Park.

The fixation of inorganic carbon has been documented in all three domains of life and results in the biosynthesis of diverse organic compounds that support heterotrophic organisms. The primary aim of this study was to assess carbon dioxide fixation in high-temperature Fe(III)-oxide mat communities and in pure cultures of a dominant Fe(II)-oxidizing organism (Metallosphaera yellowstonensis strain MK1) originally isolated from these environments. Protein-encoding genes of the complete 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) carbon dioxide fixation pathway were identified in M. yellowstonensis strain MK1. Highly similar M. yellowstonensis genes for this pathway were identified in metagenomes of replicate Fe(III)-oxide mats, as were genes for the reductive tricarboxylic acid cycle from Hydrogenobaculum spp. (Aquificales). Stable-isotope ((13)CO2) labeling demonstrated CO2 fixation by M. yellowstonensis strain MK1 and in ex situ assays containing live Fe(III)-oxide microbial mats. The results showed that strain MK1 fixes CO2 with a fractionation factor of ∼2.5‰. Analysis of the (13)C composition of dissolved inorganic C (DIC), dissolved organic C (DOC), landscape C, and microbial mat C showed that mat C is from both DIC and non-DIC sources. An isotopic mixing model showed that biomass C contains a minimum of 42% C of DIC origin, depending on the fraction of landscape C that is present. The significance of DIC as a major carbon source for Fe(III)-oxide mat communities provides a foundation for examining microbial interactions that are dependent on the activity of autotrophic organisms (i.e., Hydrogenobaculum and Metallosphaera spp.) in simplified natural communities.

Complete genome sequence of Metallosphaera cuprina, a metal sulfide-oxidizing archaeon from a hot spring.

The genome of the metal sulfide-oxidizing, thermoacidophilic strain Metallosphaera cuprina Ar-4 has been completely sequenced and annotated. Originally isolated from a sulfuric hot spring, strain Ar-4 grows optimally at 65°C and a pH of 3.5. The M. cuprina genome has a 1,840,348-bp circular chromosome (2,029 open reading frames [ORFs]) and is 16% smaller than the previously sequenced Metallosphaera sedula genome. Compared to the M. sedula genome, there are no counterpart genes in the M. cuprina genome for about 480 ORFs in the M. sedula genome, of which 243 ORFs are annotated as hypothetical protein genes. Still, there are 233 ORFs uniquely occurring in M. cuprina. Genome annotation supports that M. cuprina lives a facultative life on CO(2) and organics and obtains energy from oxidation of sulfidic ores and reduced inorganic sulfuric compounds.

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)).

An improved method for single cell isolation of prokaryotes from meso-, thermo- and hyperthermophilic environments using micromanipulation.

This study presents an improved system that enables isolation of single viable prokaryotic cells from a mixture of cells. The system is based on an inverted microscope, a microinjector and a micromanipulator. The isolated cell is captured in a microcapillary from a volume of 400 mul and transferred to an appropriate growth medium. Validation of the system was performed using two fluorescent strains: Pseudomonas putida expressing red fluorescent protein (DsRed), and Escherichia coli expressing green fluorescent protein (GFP). A mixture (100:1) of the constructed fluorescent strains was subjected to isolation experiments and nine out of ten individually isolated cells yielded axenic cultures of E. coli. Upon construction and validation, the system was used to isolate and subsequently cultivate axenic cultures of the thermophilic Archaeon Metallosphaera sedula and the hyperthermophilic Archaeon Sulfolobus solfataricus from enriched hot spring samples. The high efficiency of single-cell isolation and cultivation demonstrated over a range of temperatures-90% (30 degrees C), 85% (70 degrees C) and 95% (80 degrees C)-from different environments is probably due to the elimination of osmotic stress and limitation of temperature fluctuations during the isolation process, as a result of the large sample volume from which the cells are isolated.

Cloning and heterologous expression of a sulfur oxygenase/reductase gene from the thermoacidophilic archaeon Acidianus sp. S5 in Escherichia coli.

A thermoacidophilic, obligately chemolithotrophic, facultatively aerobic archaebacterium, Acidianus sp. S5, was isolated from acidothermal springs in southwest China. The sulfur oxygenase/reductase (SOR) gene of Acidianus sp. S5 was cloned and expressed in Escherichia coli. Several primers were designed and successfully applied for detection and cloning of the sor gene. A 3.7-kb EcoRI fragment containing the sor gene and three neighboring open reading frames was sequenced. Sequence analysis indicated that the sor gene of Acidianus sp. S5 showed 81% identity to the sor gene of Acidianus ambivalens. E. coli cells carrying the sor gene on pBV220SOR were able to overproduce SOR upon a temperature shift from 30 to 42 degrees C. SOR produced in E. coli catalyzes the oxidation of elemental sulfur and concomitant production of sulfite, thiosulfate and hydrogen sulfide. The recombinant enzyme exhibits the same catalytic properties as the one from Acidianus S5.