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A hyperthermophilic, strictly anaerobic archaeon, designated strain SY113 T , was isolated from a deep-sea hydrothermal vent chimney on the Southwest Indian Ridge at a water depth of 2770 m. Enrichment and isolation of strain SY113 T were performed at 85 °C at 0.1 MPa. Cells of strain SY113 T were irregular motile cocci with peritrichous flagella and generally 0.8-2.4 µm in diameter. Growth was observed at temperatures between 50 and 90 °C (optimum at 85 °C) and under hydrostatic pressures of 0.1-60 MPa (optimum, 27 MPa). Cells of SY113 T grew at pH 4.0-9.0 (optimum, pH 5.5) and a NaCl concentration of 0.5-5.5 % (w/v; optimum concentration, 3.0 % NaCl). Strain SY113 T was an anaerobic chemoorganoheterotroph and grew on complex proteinaceous substrates such as yeast extract and tryptone, as well as on maltose and starch. Elemental sulphur stimulated growth, but not obligatory for its growth. The G+C content of the genomic DNA was 55.0 mol%. Phylogenetic analysis of the 16S rRNA sequence of strain SY113 T showed that the novel isolate belonged to the genus Thermococcus . On the basis of physiological characteristics, average nucleotide identity values and in silico DNA-DNA hybridization results, we propose a novel species, named Thermococcus aciditolerans sp. nov. The type strain is SY113 T (=MCCC 1K04190 T =JCM 39083 T ).

A coccoid-shaped, strictly anaerobic, hyperthermophilic and piezophilic organoheterotrophic archaeon, strain Iri35c T , was isolated from a hydrothermal chimney rock sample collected at a depth of 2300 m at the Mid-Atlantic Ridge (Rainbow vent field). Cells of strain Iri35c T grew at NaCl concentrations ranging from 1-5 % (w/v) (optimum 2.0 %), from pH 5.0 to 9.0 (optimum 7.0-7.5), at temperatures between 50 and 90 °C (optimum 75-80 °C) and at pressures from 0.1 to at least 50 MPa (optimum: 10-30 MPa). The novel isolate grew on complex organic substrates, such as yeast extract, tryptone, peptone or beef extract, preferentially in the presence of elemental sulphur or l-cystine; however, these molecules were not necessary for growth. Its genomic DNA G+C content was 54.63 mol%. The genome has been annotated and the metabolic predictions are in accordance with the metabolic characteristics of the strain and of Thermococcales in general. Phylogenetic analyses based on 16S rRNA gene sequences and concatenated ribosomal protein sequences showed that strain Iri35c T belongs to the genus Thermococcus, and is closer to the species T. celericrescens and T. siculi . Average nucleotide identity scores and in silico DNA-DNA hybridization values between the genome of strain Iri35c T and the genomes of the type species of the genus Thermococcus were below the species delineation threshold. Therefore, and considering the phenotypic data presented, strain Iri35c T is suggested to represent a novel species, for which the name Thermococcus camini sp. nov. is proposed, with the type strain Iri35c T (=UBOCC M-2026 T =DSM 111003 T ).

A novel extreme thermophilic and piezophilic chemoorganoheterotrophic archaeon, strain EXT12c T , was isolated from a hydrothermal chimney sample collected at a depth of 2496 m at the East Pacific Rise 9° N. Cells were strictly anaerobic, motile cocci. The strain grew at NaCl concentrations ranging from 1 to 5 % (w/v; optimum, 2.0%), from pH 6.0 to 7.5 (optimum, pH 6.5-7.0), at temperatures between 60 and 95 °C (optimum, 80-85 °C), and at pressures from 0.1 to at least 50 MPa (optimum, 30 MPa). Strain EXT12c T grew chemoorganoheterotrophically on complex proteinaceous substrates. Its growth was highly stimulated by the presence of elemental sulphur or l-cystine, which were reduced to hydrogen sulfide. The DNA G+C content was 54.58 mol%. Phylogenetic analyses based on 16S rRNA gene sequences and concatenated ribosomal protein sequences showed that strain EXT12c T falls into the genus Thermococcus and is most closely related to Thermococcus nautili strain 30-1 T . Overall genome relatedness index analyses (average nucleotide identity scores and in silico DNA-DNA hybridizations) showed a sufficient genomic distance between the new genome and the ones of the Thermococcus type strains for the delineation of a new species. On the basis of genotypic and phenotypic data, strain EXT12c T is considered to represent a novel species, for which the name Thermococcus henrietii sp. nov. is proposed, with the type strain EXT12c T (=UBOCC M-2417 T =DSM 111004 T ).

Mobile genetic elements (MGEs) often encode integrases which catalyze the site-specific insertion of their genetic information into the host genome and the reverse reaction of excision. Hyperthermophilic archaea harbor integrases belonging to the SSV-family which carry the MGE recombination site within their open reading frame. Upon integration into the host genome, SSV integrases disrupt their own gene into two inactive pseudogenes and are termed suicidal for this reason. The evolutionary maintenance of suicidal integrases, concurring with the high prevalence and multiples recruitments of these recombinases by archaeal MGEs, is highly paradoxical. To elucidate this phenomenon, we analyzed the wide phylogenomic distribution of a prominent class of suicidal integrases which revealed a highly variable integration site specificity. Our results highlighted the remarkable hybrid nature of these enzymes encoded from the assembly of inactive pseudogenes of different origins. The characterization of the biological properties of one of these integrases, IntpT26-2 showed that this enzyme was active over a wide range of temperatures up to 99 °C and displayed a less-stringent site specificity requirement than comparable integrases. These observations concurred in explaining the pervasiveness of these suicidal integrases in the most hyperthermophilic organisms. The biochemical and phylogenomic data presented here revealed a target site switching system operating on highly thermostable integrases and suggested a new model for split gene reconstitution. By generating fast-evolving pseudogenes at high frequency, suicidal integrases constitute a powerful model to approach the molecular mechanisms involved in the generation of active genes variants by the recombination of proto-genes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

A strictly anaerobic, dissimilatory Fe(III)-reducing hyperthermophilic archaeon, designated as strain IOH1 T , was isolated from a new deep-sea hydrothermal vent (Onnuri Vent Field) area in the Central Indian Ocean ridge. Strain IOH1 T showed > 99% 16S rRNA gene sequence similarity with Thermococcus celericrescens TS2 T (99.4%) and T. siculi DSM 12349 T (99.2%). Additional three species T. barossii SHCK-94 T (99.0%), T. celer Vu13 T (98.8%), and T. piezophilus (98.6%) showed > 98.6% of 16S rRNA gene sequence similarity, however, the maximum OrthoANI value is 89.8% for the genome of T. celericrescens TS2 T . Strain IOH1 T cells are coccoid, 1.2-1.8 μm in diameter, and motile by flagella. Growth was at 70-82°C (optimum 80°C), pH 5.4-8.0 (optimum pH 6.0) with 2-4% (optimum 3%) NaCl. Growth of strain IOH1T was enhanced by starch, pyruvate, D(+)-maltose and maltodextrin as a carbon sources, and elemental sulfur as an electron acceptor; clearly different from those of related species T. celecrescens DSM 17994 T and T. siculi DSM 12349 T . Strain IOH1 T , T. celercrescence DSM 17994 T , and T. siculi DSM 12349 T reduced soluble Fe(III)-citrate present in the medium, whereas the amount of total cellular proteins increased with the concomitant accumulation of Fe(II). We determined a circular chromosome of 2,234 kb with an extra-chromosomal archaeal plasmid, pTI1, of 7.7 kb and predicted 2,425 genes. The DNA G + C content was 54.9 mol%. Based on physiological properties, phylogenetic, and genome analysis, we proposed that strain IOH1 T (= KCTC 15844 T = JCM 39077 T ) is assigned to a new species in the genus Thermococcus and named Thermococcus indicus sp. nov.

Newly erupted black smokers (hydrothermal vent chimneys) are sterile during their formation, but they house hyperthermophiles in substantial amounts in later stages. No hard data exist on the mechanisms by which hyperthermophiles colonize newly erupted black smokers. Here I propose a scenario - based on various experimental data - for how hyperthermophiles colonize black smokers. Hyperthermophiles which are present in cold sea water in minute amounts are transferred by chance to the outside of black smokers and react within seconds to the high temperature by very fast movements. After reaching an optimal temperature region they scan the surface via a zigzag seek-movement and adhere via their flagella at a suitable place, building up biofilms., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A novel strictly anaerobic, hyperthermophilic archaeon, designated strain CDGS T , was isolated from a deep-sea hydrothermal vent in the Cayman Trough at 4964m water depth. The novel isolate is obligate anaerobe and grows chemoorganoheterotrophically with stimulation of growth by sulphur containing compounds. Its growth is optimal at 75°C, pH 6.0 and under a pressure of 50MPa. It possesses the broadest hydrostatic pressure range for growth that has ever been described for a microorganism. Its genomic DNA G+C content is 51.11mol%. The novel isolate belongs to the genus Thermococcus. Phylogenetic analyses indicated that it is most closely related to Thermococcus barossii DSM17882 T based on its 16S rRNA gene sequence, and to 'Thermococcus onnurineus' NA1 based on its whole genome sequence. The average nucleotide identity scores with these strains are 77.66% for T. barossii and 84.84% for 'T. onnurineus', respectively. Based on the draft whole genome sequence and phenotypic characteristics, strain CDGS T is suggested to be separated into a novel species within the genus Thermococcus, with proposed name Thermococcus piezophilus (type strain CDGS T =ATCC TSD-33 T =UBOCC 3296 T )., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

A conditional piezophilic, hyperthermophilic archaeon showing growth over a wide range of temperature, pH and pressure was isolated from an oil-immersed hydrothermal chimney at a depth of 2006.9 m in the Guaymas Basin. Enrichment and isolation of strain A501(T) were performed at 80 °C at 0.1 MPa. Cells of isolate A501(T) were irregular motile cocci with a polar tuft of flagella and generally 0.6-2.6 µm in diameter. Growth was detected over the range 50-100 °C (optimal growth at 85 °C) at atmospheric pressure and was observed at 102 °C at a pressure of 10 MPa. At 85 °C, growth was observed at a pressure of 0.1-70 MPa (optimum pressure 0.1 MPa-30 MPa), while at 95 °C, the pressure allowing growth ranged from 0.1 MPa to 50 MPa (optimum pressure 10 MPa). Cells of strain A501(T) grew at pH 4-9 (optimum pH 7.0) and a NaCl concentration of 1.0-5.0 % (w/v) (optimum concentration 2.5 % NaCl). This isolate was an anaerobic chemo-organoheterotroph and was able to utilize yeast extract, peptone, tryptone and starch as the single carbon source for growth. Elemental sulfur and cysteine stimulated growth; however, these molecules were not necessary. The DNA G+C content of the complete genome was 53.47 mol%. The results of 16S rRNA gene sequence analysis indicated that strain A501(T) belongs to the genus Thermococcus. There was no significant similarity between strain A501(T) and the phylogenetically related species of the genus Thermococcus based on complete genome sequence alignments and calculation of the average nucleotide identity and the tetranucleotide signature frequency correlation coefficient. These results indicate that strain A501(T) represents a novel species, Thermococcus eurythermalis sp. nov. The type strain is A501(T) ( = CGMCC 7834(T) = JCM 30233(T))., (© 2015 IUMS.)

Two heterotrophic hyperthermophilic strains, ES1(T) and CL1(T), were isolated from Paralvinella sp. polychaete worms collected from active hydrothermal vent chimneys in the north-eastern Pacific Ocean. Both were obligately anaerobic and produced H2S in the presence of elemental sulfur and H2. Complete genome sequences are available for both strains. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strains are more than 97% similar to most other species of the genus Thermococcus. Therefore, overall genome relatedness index analyses were performed to establish that these strains are novel species. For each analysis, strain ES1(T) was determined to be most similar to Thermococcus barophilus MP(T), while strain CL1(T) was determined to be most similar to Thermococcus sp. 4557. The average nucleotide identity scores for these strains were 84% for strain ES1(T) and 81% for strain CL1(T), genome-to-genome direct comparison scores were 23% for strain ES1(T) and 47% for strain CL1(T), and the species identification scores were 89% for strain ES1(T) and 88% for strain CL1(T). For each analysis, strains ES1(T) and CL1(T) were below the species delineation cut-off. Therefore, based on their whole genome sequences, strains ES1(T) and CL1(T) are suggested to represent novel species of the genus Thermococcus for which the names Thermococcus paralvinellae sp. nov. and Thermococcus cleftensis sp. nov. are proposed, respectively. The type strains are ES1(T) ( =DSM 27261(T) =KACC 17923(T)) and CL1(T) ( =DSM 27260(T) =KACC 17922(T))., (© 2014 IUMS.)

Thermococcus nautili, strain 30-1T (formerly reported as Thermococcus nautilus), was isolated from a hydrothermal chimney sample collected from the East Pacific Rise at a depth of 2633 m on the 'La chainette PP57' area. Cells were motile, irregular cocci with a polar tuft of flagella (0.8-1.5 µm) and divided by constriction. The micro-organism grew optimally at 87.5 °C (range 55-95 °C), at pH 7 (range pH 4-9) and with 2% NaCl (range 1-4%). Doubling time was 64 min in Zillig's broth medium under optimal conditions. Growth was strictly anaerobic. It grew preferentially in the presence of elemental sulfur or cystine, which are reduced to H2S, on complex organic substrates such as yeast extract, tryptone, peptone, Casamino acids and casein. Slow growth was observed on starch and pyruvate. Strain 30-1T was resistant to chloramphenicol and tetracyclin (at 100 µg ml(-1)) but sensitive to kanamycin and rifampicin. The G+C content of the genomic DNA was 54 mol%. Strain 30-1T harboured three plasmids named pTN1, pTN2 and pTN3 and produced membrane vesicles that incorporate pTN1 and pTN3. As determined by 16S rRNA gene sequence analysis, strain 30-1T is related most closely to Thermococcus sp. AM4 (99.3% similarity) and Thermococcus gammatolerans DSM 15229T (99.2%). DNA-DNA hybridization values (in silico) with these two closest relatives were below the threshold value of 70% (33% with Thermococcus sp. AM4 and 32% with T. gammatolerans DSM 15229T) and confirmed that strain 30-1 represents a novel species. On the basis of the data presented, strain 30-1T is considered to represent a novel species of the genus Thermococcus, for which the name Thermococcus nautili sp. nov. is proposed. The type strain is 30-1T (=CNCM 4275=JCM 19601).

Thermococcales are hyperthermophilic archaea found in deep-sea hydrothermal vents. They have been recently reported to produce membrane vesicles (MVs) into their culture medium. Here, we have characterized the mode of production and determined the biochemical composition of MVs from two species of Thermococcales, Thermococcus gammatolerans and Thermococcus kodakaraensis. We observed that MVs are produced by a budding process from the cell membrane reminiscent of ectosome (microparticle) formation in eukaryotes. MVs and cell membranes from the same species have a similar protein and lipid composition, confirming that MVs are produced from cell membranes. The major protein present in cell membranes and MVs of both species is the oligopeptide binding protein OppA. This protein is also abundant in MVs from cells grown in minimal medium, suggesting that OppA could be involved in processes other than peptides scavenging. We have previously shown that MVs from Thermococcales harbour DNA and protect DNA against thermodegradation. Here, we show that T. kodakaraensis cells transformed with the shuttle plasmid pLC70 release MVs harbouring this plasmid. Notably, these MVs can be used to transfer pLC70 into plasmid-free cells, suggesting that MVs could be involved in DNA transfer between cells at high temperature., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Thermococcus sp. strain CL1 is a hyperthermophilic, anaerobic, and heterotrophic archaeon isolated from a Paralvinella sp. polychaete worm living on an active deep-sea hydrothermal sulfide chimney on the Cleft Segment of the Juan de Fuca Ridge. To further understand the distinct characteristics of this archaeon at the genome level, its genome was completely sequenced and analyzed. Here, we announce the complete genome sequence (1,950,313 bp) of Thermococcus sp. strain CL1, with a focus on H(2)- and energy-producing capabilities and its amino acid biosynthesis and acquisition in an extreme habitat.

Thermococcus zilligii, a thermophilic anaerobe in freshwater, is useful for physiological research and biotechnological applications. Here we report the high-quality draft genome sequence of T. zilligii AN1(T). The genome contains a number of genes for an immune system and adaptation to a microbial biomass-rich environment as well as hydrogenase genes.

Analysis of the complete genome of Thermococcus sp. strain AM4, which was the first lithotrophic Thermococcales isolate described and the first archaeal isolate to exhibit a capacity for hydrogenogenic carboxydotrophy, reveals a proximity with Thermococcus gammatolerans, corresponding to close but distinct species that differ significantly in their lithotrophic capacities.

Thermococcus barophilus is a hyperthermophilic, anaerobic, mixed heterotrophic, and carboxydotrophic euryarchaeon isolated from the deep sea hydrothermal vent Snakepit site on the mid-Atlantic ridge at a depth of 3,550 m. T. barophilus is the first true piezophilic, hyperthermophilic archaeon isolated, having an optimal growth at 40 MPa. Here we report the complete genome sequence of strain MP, the type strain of T. barophilus. The genome data reveal a close proximity with Thermococcus sibiricus, another Thermococcus isolated from the deep biosphere and a possible connection to life in the depths.

The gene encoding a new extracellular amylopullulanase (type II pullulanase) was cloned from an extremely thermophilic anaerobic archaeon Thermococcus siculi strain HJ21 isolated previously from a deep-sea hydrothermal vent. The functional hydrolytic domain of the amylopullulanase (TsiApuN) and its MalE fusion protein (MTsiApuN) were expressed heterologously. The complete amylopullulanase (TsiApu) was also purified from fermentation broth of the strain. The pullulanase and amylase activities of the three enzymes were characterized. TsiApu had optimum temperature of 95°C for the both activities, while MTsiApuN and TsiApuN had a higher optimum temperature of 100°C. The residual total activities of MTsiApuN and TsiApuN were both 89% after incubation at 100°C for 1 h, while that of TsiApu was 70%. For all the three enzymes the optimum pHs for amylase and pullulanase activities were 5.0 and 6.0, respectively. By analyzing enzymatic properties of the three enzymes, this study suggests that the carboxy terminal region of TsiApu might interfere with the thermoactivity. The acidic thermoactive amylopullulanases MTsiApuN and TsiApuN could be further employed for industrial saccharification of starch.

Strains of hyperthermophilic anaerobic hydrothermal vent archaea maintained in the culture collection assembled by Holger Jannasch at the Woods Hole Oceanographic Institution between 1984 and 1998 were identified and partially characterized by Denaturing Gradient Gel Electrophoresis, 16S rRNA gene sequencing, and by growth tests at different temperatures and on different organic carbon and nitrogen sources. All strains were members of the genera Thermococcus and Pyrococcus. The greatest phylogenetic diversity was found in strains from a single Guaymas Basin core isolated by serial dilution from four different depth horizons of heated sediment incubated at the corresponding in situ temperatures. In contrast, geographically distinct vent locations and sample materials yielded a lower diversity of isolates when enriched under uniform temperature regimes and without prior dilution of the source material.

Thermococcus species are widely distributed in terrestrial and marine hydrothermal areas, as well as in deep subsurface oil reservoirs. Thermococcus sibiricus is a hyperthermophilic anaerobic archaeon isolated from a well of the never flooded oil-bearing Jurassic horizon of a high-temperature oil reservoir. To obtain insight into the genome of an archaeon inhabiting the oil reservoir, we have determined and annotated the complete 1,845,800-base genome of T. sibiricus. A total of 2,061 protein-coding genes have been identified, 387 of which are absent in other members of the order Thermococcales. Physiological features and genomic data reveal numerous hydrolytic enzymes (e.g., cellulolytic enzymes, agarase, laminarinase, and lipases) and metabolic pathways, support the proposal of the indigenous origin of T. sibiricus in the oil reservoir, and explain its survival over geologic time and its proliferation in this habitat. Indeed, in addition to proteinaceous compounds known previously to be present in oil reservoirs at limiting concentrations, its growth was stimulated by cellulose, agarose, and triacylglycerides, as well as by alkanes. Two polysaccharide degradation loci were probably acquired by T. sibiricus from thermophilic bacteria following lateral gene transfer events. The first, a "saccharolytic gene island" absent in the genomes of other members of the order Thermococcales, contains the complete set of genes responsible for the hydrolysis of cellulose and beta-linked polysaccharides. The second harbors genes for maltose and trehalose degradation. Considering that agarose and laminarin are components of algae, the encoded enzymes and the substrate spectrum of T. sibiricus indicate the ability to metabolize the buried organic matter from the original oceanic sediment.

The extremely thermophilic anaerobic archaeon strain, HJ21, was isolated from a deep-sea hydrothermal vent, could produce hyperthermophilic alpha-amylase, and later was identified as Thermococcus from morphological, biochemical, and physiological characteristics and the 16S ribosomal RNA gene sequence. The extracellular thermostable alpha-amylase produced by strain HJ21 exhibited maximal activity at pH 5.0. The enzyme was stable in a broad pH range from pH 5.0 to 9.0. The optimal temperature of alpha-amylase was observed at 95 degrees C. The half-life of the enzyme was 5 h at 90 degrees C. Over 40% and 30% of the enzyme activity remained after incubation at 100 degrees C for 2 and 3 h, respectively. The enzyme did not require Ca(2+) for thermostability. This alpha-amylase gene was cloned, and its nucleotide sequence displayed an open reading frame of 1,374 bp, which encodes a protein of 457 amino acids. Analysis of the deduced amino acid sequence revealed that four homologous regions common in amylases were conserved in the HJ21 alpha-amylase. The molecular weight of the mature enzyme was calculated to be 51.4 kDa, which correlated well with the size of the purified enzyme as shown by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A hyperthermophilic, sulfur-reducing, organo-heterotrophic archaeon, strain OGL-20P(T), was isolated from 'black smoker' chimney material from the Rainbow hydrothermal vent site on the Mid-Atlantic Ridge (36.2 degrees N, 33.9 degrees W). The cells of strain OGL-20P(T) have an irregular coccoid shape and are motile with a single flagellum. Growth was observed within a pH range of 5.0-8.5 (optimum pH 7.0), an NaCl concentration range of 1-5 % (w/v) (optimum 3 %) and a temperature range of 55-94 degrees C (optimum 83-85 degrees C). The novel isolate is strictly anaerobic and obligately dependent upon elemental sulfur as an electron acceptor, but it does not reduce sulfate, sulfite, thiosulfate, Fe(III) or nitrate. Proteolysis products (peptone, bacto-tryptone, Casamino acids and yeast extract) are utilized as substrates during sulfur reduction. Strain OGL-20P(T) is resistant to ampicillin, chloramphenicol, kanamycin and gentamicin, but sensitive to tetracycline and rifampicin. The G+C content of the DNA is 52.9 mol%. The 16S rRNA gene sequence analysis revealed that strain OGL-20P(T) is closely related to Thermococcus coalescens and related species, but no significant homology by DNA-DNA hybridization was observed between those species and the new isolate. On the basis of physiological and molecular properties of the new isolate, we conclude that strain OGL-20P(T) represents a new separate species within the genus Thermococcus, for which we propose the name Thermococcus thioreducens sp. nov. The type strain is OGL-20P(T) (=JCM 12859(T)=DSM 14981(T)=ATCC BAA-394(T)).

Surveys of Champagne Pool, one of New Zealand's largest terrestrial hot springs and rich in arsenic ions and compounds, have been restricted to geological and geochemical descriptions, and a few microbiological studies applying culture-independent methods. In the current investigation, a combination of culture and culture-independent approaches were chosen to determine microbial density and diversity in Champagne Pool. Recovered total DNA and adenosine 5'-triphosphate (ATP) content of spring water revealed relatively low values compared to other geothermal springs within New Zealand and are in good agreement with low cell numbers of 5.6 +/- 0.5 x 10(6) cells/ml obtained for Champagne Pool water samples by 4',6-diamidino-2-phenylindole (DAPI) staining. Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA (small-subunit ribosomal nucleic acid) gene clone library analyses of environmental DNA indicated the abundance of Sulfurihydrogenibium, Sulfolobus, and Thermofilum-like populations in Champagne Pool. From these results, media were selected to target the enrichment of hydrogen-oxidizing and sulfur-dependent microorganisms. Three isolates were successfully obtained having 16S rRNA gene sequences with similarities of approximately 98% to Thermoanaerobacter tengcongensis, 94% to Sulfurihydrogenibium azorense, and 99% to Thermococcus waiotapuensis, respectively.

We investigated the effect of temperature on the binding specificity of the recombinant d-trehalose/d-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis (TMBP). Importantly, we found that TMBP can bind d-glucose (Glc). The Glc binding was characterized by means of fluorescence spectroscopy in the temperature range of 25 degrees C-85 degrees C. Our results show that at 25 degrees C the binding of Glc to TMBP is well represented by a bimodal model with apparent K(d) of 20 muM and approximately 3-8 mM for the first and the second binding step, respectively. At 60 degrees C the binding of Glc to TMBP is represented by a simple hyperbolic model with an apparent K(d) value of about 40 muM. Finally, at 85 degrees C Glc did not bind to TMBP. Molecular dynamics (MD) simulations were used to shed light on the molecular mechanism of the Glc binding. Our results suggest that after proper fluorescent labeling TMBP can be used as a highly thermostable and non-consuming analyte biosensor for monitoring the level of glucose in fluids (e.g. human blood) where other sugars are not present.

A cell-fusing hyperthermophilic archaeon was isolated from hydrothermal fluid obtained from Suiyo Seamount of the Izu-Bonin Arc. The isolate, TS1(T), is an irregular coccus, usually 0.5-2 microm in diameter and motile with a polar tuft of flagella. Cells in the exponential phase of growth fused at room temperature in the presence of DNA-intercalating dye to become as large as 5 microm in diameter. Fused cells showed dark spots that moved along in the cytoplasm. Large cells with a similar appearance were also observed upon culture at 87 degrees C, suggesting the occurrence of similar cell fusions during growth. Transmission electron microscopy revealed that cells in the exponential phase possessed a thin and electron-lucent cell envelope that could be lost subsequently during culture. The fragile cell envelope must be related to cell fusion. The cells grew at 57-90 degrees C, pH 5.2-8.7 and at NaCl concentrations of 1.5-4.5 %, with the optima being 87 degrees C, pH 6.5 and 2.5 % NaCl. The isolate was an anaerobic chemo-organotroph that grew on either yeast extract or tryptone as the sole growth substrate. The genomic DNA G+C content was 53.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the isolate was closely related to Thermococcus species. However, no significant DNA-DNA hybridization was observed between genomic DNA of strain TS1(T) and phylogenetically related Thermococcus species. We propose that isolate TS1(T) represents a novel species, Thermococcus coalescens sp. nov., with the name reflecting the cell fusion activity observed in the strain. The type strain is TS1(T) (=JCM 12540T=DSM 16538T).

The microflora developing during a continuous enrichment culture from a hydrothermal chimney sample was investigated by molecular methods. The culture was performed in a gas-lift bioreactor under anaerobic conditions, at 90 degrees C and pH 6.5, on a complex medium containing sulfur as the terminal electron acceptor. Archaeal and bacterial diversity was studied. Microorganisms affiliated with the genera Pyrococcus, Marinitoga, and Bacillus were detected through DGGE analysis of 16S rDNA. Additional sequences phylogenetically related to Thermococcus and epsilon-Proteobacteria were detected by cloning and sequencing of 16S rDNA from two samples of the enrichment culture. In comparison, the sequences retrieved from cloning analysis from an enrichment culture performed in a flask (batch condition) using the same culture medium showed that only members of the genus Thermococcus were cultivated. Therefore, continuous enrichment culture using the gas-lift bioreactor can be considered as an efficient and improved method for investigating microbial communities originating from deep-sea hydrothermal vents.

A hyperthermophilic archaeal strain, KOD1, isolated from a solfatara on Kodakara Island, Japan, has previously been reported as Pyrococcus sp. KOD1. However, a detailed phylogenetic tree, made possible by the recent accumulation of 16S rRNA sequences of various species in the order Thermococcales, indicated that strain KOD1 is a member of the genus Thermococcus. We performed DNA-DNA hybridization tests against species that displayed high similarity in terms of 16S ribosomal DNA sequences, including Thermococcus peptonophilus and Thermococcus stetteri. Hybridization results and differences in growth characteristics and substrate utilization differentiated strain KOD1 from T. peptonophilus and T. stetteri at the species level. Our results indicate that strain KOD1 represents a new species of Thermococcus, which we designate as Thermococcus kodakaraensis KOD1 sp. nov.

From 24 samples of hydrothermal venting structures collected at the East Pacific Rise (13 degrees N), 13 enrichments of coccoid cells were obtained which grew on CO, producing H2 and CO2 at 80 degrees C. A hyperthermophilic archaeon capable of lithotrophic growth on CO coupled with equimolar production of H2 was isolated. Based on its 16S rRNA sequence analysis, this organism was affiliated with the genus Thermococcus. Other strains of Thermococcales species ( Pyrococcus furiosus, Thermococcus peptonophilus, T. profundus, T. chitonophagus, T. stetteri, T. gorgonarius, T. litoralis, and T. pacificus) were shown to be unable to grow on CO. Searches in sequence databases failed to reveal deposited sequences of genes related to CO metabolism in Thermococcales. Our work provides the first evidence of anaerobic CO oxidation coupled with H2 production performed by an archaeon as well as the first documented case of lithotrophic growth of a Thermococcales representative.

Enrichments for anaerobic, organotrophic hyperthermophiles were performed with hydrothermal chimney samples collected from the Mid-Atlantic Ridge at a depth of 3,550 m (23 degrees 22'N, 44 degrees 57'W) and the Guaymas Basin (27 degrees 01'N, 111 degrees 24'W) at a depth of 2,616 m. Positive enrichments were submitted to gamma-irradiation at doses of 20 and 30 kGy. Two hyperthermophilic, anaerobic, sulfur-metabolizing archaea were isolated. Strain EJ1T was isolated from chimney samples collected from the Mid-Atlantic Ridge after gamma-irradiation at 20 kGy, and strain EJ2T was isolated from the Guaymas Basin after gamma-irradiation at 30 kGy. Only strain EJ2T was motile, and both formed regular cocci. These new strains grew between 55 and 95 degrees C with the optimal temperature being 88 degrees C. The optimal pH for growth was 6.0, and the optimal NaCl concentration for growth was around 20 g l(-1). These strains were obligate anaerobic heterotrophs that utilized yeast extract, tryptone, and peptone as a carbon source for growth. Ten amino acids were essential for the growth of strain EJ1), such as arginine, aspartic acid, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tyrosine, and valine, while strain EJ2T was unable to grow on a mixture of amino acids. Elemental sulfur or cystine was required for EJ2T growth and was reduced to hydrogen sulfide. Rifampicin inhibited growth for both strains EJ1T and EJ2T. The G + C contents of the genomic DNA were 52.3 and 54.5 mol% for EJ1T and EJ2T, respectively. As determined by 16S rRNA gene sequence analysis, these strains were more closely related to Thermococcus gorgonarius, T. celer, T. guaymasensis, T. profundus, and T. hydrothermalis. However, no significant homology was observed between them with DNA-DNA hybridization. These novel organisms also possess phenotypic traits that differ from those of its closest phylogenetic relatives. Therefore, it is proposed that these isolates, which are amongst the most radioresistant hyperthermophilic archaea known to date with T. gammatolerans (Jolivet et al. 2003a), should be described as novel species T. marinus sp. nov. and T. radiotolerans sp. nov. The type strain of T. marinus is strain EJ1T (= DSM 15227T = JCM 11825T) and the type strain of T. radiotolerans is strain EJ2T (= DSM 15228T = JCM 11826T)., (Copyright 2004 Springer-Verlag)

Members of the Thermococcales are anaerobic Archaea belonging to the kingdom Euryarchaea that are studied in many laboratories as model organisms for hyperthermophiles. We describe here a molecular analysis of 86 new Thermococcales isolates collected from six different chimneys of a single hydrothermal field located in the 13 degrees N 104 degrees W segment of the East Pacific ridge at a depth of 2,330 m. These isolates were sorted by randomly amplified polymorphic DNA (RAPD) fingerprinting into nine groups, and nine unique RAPD profiles were obtained. One RAPD group corresponds to new isolates of Thermococcus hydrothermalis, whereas all other groups and isolates with unique profiles are different from the 22 reference strains included in this study. Analysis of 16S rRNA gene sequences of representatives of each RAPD group and unique profiles showed that one group corresponds to Pyrococcus strains, whereas all the other isolates are Thermococcus strains. We estimated that our collection may contain at least 11 new species. These putative species, isolated from a single area of hydrothermal deep-sea vents, are dispersed in the 16S rRNA tree among the reference strains previously isolated from diverse hot environments (terrestrial, shallow water, hydrothermal vents) located around the world, suggesting that there is a high degree of dispersal of Thermococcales: About one-half of our isolates contain extrachromosomal elements that could be used to search for novel replication proteins and to develop genetic tools for hyperthermophiles.

Enrichments for anaerobic organotrophic hyperthermophiles were performed with hydrothermal chimney samples collected at the Guaymas Basin (27 degrees 01' N, 111 degrees 24' W). Positive enrichments were submitted to gamma-irradiation at a dose of 30 kGy. One of the resistant strains, designated strain EJ3(T), formed regular motile cocci. The new strain grew between 55 and 95 degrees C, with an optimum growth temperature of 88 degrees C. The optimal pH for growth was 6.0, and the optimum NaCl concentration for growth was around 20 g l(-1). Strain EJ3(T) was an obligately anaerobic heterotroph that utilized yeast extract, tryptone and peptone. Elemental sulfur or cystine was required for growth and reduced to hydrogen sulfide. The G + C content of the genomic DNA was 51.3 mol%. As determined by 16S rRNA gene sequence analysis, the organism was most closely related to Thermococcus celer, Thermococcus guaymasensis, Thermococcus hydrothermalis, Thermococcus profundus and Thermococcus gorgonarius. However, no significant homology was observed between them by DNA-DNA hybridization. The novel organism also possessed phenotypic traits that differ from those of its closest phylogenetic relatives. Therefore, it is proposed that this isolate, which constitutes the most radioresistant hyperthermophilic archaeon known to date, should be described as the type strain of a novel species, Thermococcus gammatolerans sp. nov. The type strain is EJ3(T) (= DSM 15229(T) = JCM 11827(T)).

An extremely thermophilic archaeon, strain MA898, was isolated from a deep-sea hydrothermal vent on the Mid-Atlantic Ridge. This strain is a strictly anaerobic coccus of approximately 0.7-1.2 microm in diameter. Optimal temperature, pH, and NaCl concentration for growth are around 85 degrees C, pH 7, and 3%, respectively. Strain MA898 grows preferentially in the presence of elemental sulfur, polysulfur, cystine, or L-cysteine. The microorganism requires rich proteinaceous substrates. BHI-S medium supports rapid growth, with a final concentration of more than 1.2 x 10(9) cells ml(-1), but strain MA898 exhibits poor growth on 2216S medium (yeast/peptone) and poor growth on starch. Growth is inhibited by rifampicin and chloramphenicol at a concentration of 100 microg/ml. The DNA G+C content is 50 mol%. Sequencing of the 16S rRNA gene indicates that strain MA898 belongs to the Thermococcusgenus, and from DNA/DNA hybridization data it is proposed as a new species: Thermococcus atlanticus. The deposition numbers are CIP-107420T and DSM15226.

The starvation survivability of seven Thermococcus strains isolated from four Japanese oil reservoirs was compared with that of Thermococcus strains from marine hydrothermal fields. 16S rDNA analyses showed the isolates to be closely related to Thermococcus litoralis. Growth of the isolates was dependent on amino acids, which were present at low concentrations in the oil reservoirs. At 80 degrees C in the formation water, strain CKU-1 from the oil reservoir showed a higher starvation survivability than strain KS-1 from the marine hydrothermal field. Crude oil did not affect the starvation survivability of strain CKU-1, but it reduced that of strain KS-1. These results indicate that strain CKU-1 could survive longer than stain KS-1 under the conditions of an oil reservoir. At 90 degrees C in artificial seawater without organic nutrients, the half-lives of the isolates were between 7.7 and 25.1 days. However, those of the strains from marine hydrothermal fields, except Thermococcus litoralis and Thermococcus chitonophagus, were less than 1.0 day. The higher starvation survivability is probably important for the hyperthermophiles to continue to exist in a hot subterranean oil reservoir where the supply of nutrients seems to be limited.

Anaerobic organotrophic hyperthermophilic Archaea were isolated from five of eight samples from oil wells of the Samotlor oil reservoir (depth, 1,799-2,287 m; temperature, 60 degrees-84 degrees C). Three strains were isolated in pure cultures and characterized phylogenetically on the basis of comparison of the 16S rRNA gene sequences. All strains belonged to a new species of the genus Thermococcus, with Thermococcus litoralis, Thermococcus aggregans, Thermococcus fumicolans, and Thermococcus alcaliphilus being the nearest relatives (range of sequence similarity, 97.2%-98.8%). Strain MM 739 was studied in detail. The new isolate grew on peptides but not on carbohydrates. Elemental sulfur had a stimulatory effect on growth. The temperature range for growth was between 40 degrees and 88 degrees C, with the optimum at 78 degrees C; the pH range was 5.8 to 9.0, with the optimum around 7.3; and the salinity range was 0.5% to 7.0%, with the optimum at 1.8%-2.0%. The doubling time at optimal growth conditions was about 43 min. The G+C content of the DNA was 38.4 mol%. The DNA-DNA relatedness between strain MM 739 and T. litoralis was 27%; between strain MM 739 and T. aggregans, it was 22%. Based on the phenotypic and genomic differences with known Thermococcus species, the new species Thermococcus sibiricus is proposed. The isolation of a hyperthermophilic archaeum from a deep subsurface environment, significantly remote from shallow or abyssal marine hot vents, indicates the existence of a subterranean biosphere inhabited by indigenous hyperthermophilic biota.

Two novel, hyperthermophilic, anaerobic, heterotrophic archaea were isolated from shallow hydrothermal vents off Palaeochori Bay, Milos, Greece. Strain P5T (BK17S6-3-b2T) is an irregular coccus, with a single polar flagellum, growing optimally at 90 degrees C, pH 6 and 2% NaCl. The DNA G+C content was 45 mol%. Due to its morphology, phylogenetic analyses based on 16S rRNA gene sequencing, DNA-DNA hybridization experiments, physiological properties and nutritional features, this strain represents a new species within the genus Thermococcus for which the name Thermococcus aegaeicus is proposed. The type strain is P5T (= DSM 12767T = JCM 10828T). Strain p8T (BK20S6-10-b1T) is a coccus that forms aggregates. It grew optimally at 85 degrees C, pH 6 and 3% NaCl. The DNA G+C content was 38 mol%. Physiological properties and sequence analysis of the 165 rRNA gene, as well as DNA-DNA hybridization experiments, indicate that this strain is a new species belonging to the genus Staphylothermus for which the name Staphylothermus hellenicus is proposed. The type strain is P8T (= DSM 12710T = JCM 10830T).

The distribution of culturable hyperthermophiles was studied in relation to environmental conditions in the Kubiki oil reservoir in Japan, where the temperature was between 50 and 58 degrees C. Dominant hyperthermophilic cocci and rods were isolated and shown to belong to the genera Thermococcus and Thermotoga, respectively, by 16S rDNA analyses. Using the most-probable-number method, we found that hyperthermophilic cocci were widely distributed in several unconnected fault blocks in the Kubiki oil reservoir. In 1996 to 1997, their populations in the production waters from oil wells were 9.2 x 10(3) to 4.6 x 10(4) cells/ml, or 10 to 42% of total cocci. On the other hand, hyperthermophilic rods were found in only one fault block of the reservoir with populations less than 10 cells/ml. Dominant Thermococcus and Thermotoga spp. grew at reservoir temperatures and utilized amino acids and sugars, respectively, as sole carbon sources. While organic carbon was plentiful in the environment, these hyperthermophiles were unable to grow in the formation water due to lack of essential nutrients. Concentrations of some organic and inorganic substances differed among fault blocks, indicating that the movement of formation water between fault blocks was restricted. This finding suggests that the supply of nutrients via fluid current is limited in this subterranean environment and that the organisms are starved in the oil reservoir. Under starved conditions at 50 degrees C, culturable cells of Thermococcus sp. remained around the initial cell density for about 200 days, while those of Thermotoga sp. decreased exponentially to 0. 01% of the initial cell density after incubation for the same period. The difference in survivability between these two hyperthermophiles seems to reflect their populations in the fault blocks. These results indicate that hyperthermophilic cocci and rods adapt to the subterranean environment of the Kubiki oil reservoir by developing an ability to survive under starved conditions.

An extremely thermophilic, sulfur-dependent archaeon, strain WT1, was isolated from a freshwater hot spring in the Lake Taupo area of North Island, New Zealand. The cells are flagellated, strictly anaerobic cocci that grow optimally at 85 degrees C and 5.4 g NaCl l(-1). The strain grows heterotrophically on complex proteinaceous substrates or on appropriate salts plus amino acid mixtures and is also able to utilize maltose, starch, and pyruvate. Elemental sulfur could be replaced by cystine or thioglycollate. The range of temperatures allowing growth is from 60 to 90 degrees C; the pH supporting growth ranges from 5 to 8 (optimum, pH 7). Strain WT1 grew in a defined medium containing amino acids as the sole carbon and energy sources. The required amino acids were: Arg, His, Ile, Leu, Phe, Ser, Thr, Trp, Tyr, and Val. Strain WT1 showed sensitivity to rifampicin. DNA G+C content was 50.4 mol%. Phylogenetic analysis of the sequence encoding the 16S rRNA gene indicated that this isolate is a member of the Thermococcales. DNA/DNA hybridization studies revealed no similarity to several species of Thermococcus and Pyrococcus, with the exception of Thermococcus zilligii. Based on the reported results, we propose strain WT1 as a new species to be named Thermococcus waiotapuensis sp. nov.

A novel barophilic, hyperthermophilic, anaerobic sulfur-metabolizing archaeon, strain MPT (T = type strain), was isolated from a hydrothermal vent site (Snakepit) on the Mid-Atlantic Ridge (depth, 3550 m). Enrichments and isolation were done under 40 MPa hydrostatic pressure at 95 degrees C. Strain MPT was barophilic at 75, 80, 85, 90, 95 and 98 degrees C, and was an obligate barophile between 95 and 100 degrees C (Tmax). For growth above 95 degrees C, a pressure of 15.0-17.5 MPa was required. The strain grew at 48-95 degrees C under atmospheric pressure. The optimal temperature for growth was 85 degrees C at both high (40 MPa) and low (0.3 MPa) pressures. The growth rate was twofold higher at 85 degrees C under in situ hydrostatic pressure compared to at low pressure. Strain MPT cells were motile, coccoid, 0.8-2.0 microns in diameter and covered by a hexagonal S-layer lattice. The optimum pH and NaCl concentration for growth at low pressure were 7.0 and 20-30 g l-1, respectively. The new isolate was an obligate heterotroph and utilized yeast extract, beef extract and peptone for growth. Growth was optimal in the presence of elemental sulfur. Rifampicin and chloramphenicol inhibited growth. The core lipids consisted of a major archaeol and a complex lipid pattern consisting of a major phospholipid. The DNA G + C content was 37.1 mol%. Sequencing of the 16S rRNA gene revealed that strain MPT belonged to the genus Thermococcus and it is proposed that this isolate should be designated as a new species, Thermococcus barophilus.

A novel coccoid-shaped, hyperthermophilic, anaerobic archaeon, strain RG-20, was isolated from a deep-sea hydrothermal vent fluid sample taken at 1394-m depth at the Mid-Okinawa Trough (27 degrees 32.7'N, 126 degrees 58.5'E). Cells of this isolate occur singly or in pairs and are about 0.8 to 2 microm in diameter. Growth was observed at temperatures between 50 degrees and 93 degrees C, with an optimum at 85 degrees C. The pH range for growth is 5.0-9.0, with an optimum around 7.0. Strain RG-20 requires 1%-4% of NaCl for growth, and cell lysis occurs at concentrations below 1%. The newly isolated strain grows preferentially in the presence of elemental sulfur on proteinaceous substrates such as yeast extract, peptone, or tryptone, and no growth was observed on carbohydrates, carboxylic acids, alcohols, or lipids. This microorganism is resistant to streptomycin, chloramphenicol, ampicillin, and kanamycin at concentrations up to 150 microg/ ml, but is susceptible to rifampicin. Analysis of the hydrolyzed core lipids by thin-layer chromatography (TLC) revealed the presence of archaeol and caldarchaeol. The mol% G+C content of the DNA is 55.8. Partial sequencing of the 16S rDNA indicates that strain RG-20 belongs to the genus Thermococcus. Considering these data and on the basis of the results from DNA-DNA hybridization studies, we propose that this strain should be classified as a new species named Thermococcus siculi (si'cu.li. L. gen. n. siculi, of the deep-sea [siculum, deep-sea in literature of Ovid], referring to the location of the sample site, a deep-sea hydrothermal vent). The type strain is isolate RG-20 (DSM No. 12349).

Thermococcus strains TYST and TYT isolated from the Guaymas Basin hydrothermal vent site and previously described were compared by DNA-DNA hybridization analysis with the closest Thermococcus species in terms of physiology and nutritional aspects. On the basis of the new data and taking into consideration the molecular, physiological and morphological traits published previously, it is proposed that strains TYT and TYST should be classified as new species named Thermococcus aggregans sp. nov. and Thermococcus guaymasensis sp. nov., respectively. The type strain of T. aggregans is strain TYT (= DSM 10597T) and the type strain of T. guaymasensis is strain TYST (= DSM 11113T).

From a shallow marine hydrothermal system at Vulcano (Italy), a new hyperthermophilic member of the Archaea was isolated. The cells are coccoid - shaped and possess up to five flagella. They grow between 56 degrees and 93 degrees C (optimum 85 degrees C) and pH 5.0-9.5 (optimum 9.0). The organism is strictly anaerobic and grows heterotrophically on defined amino acids and complex organic substrates such as casamino acids, yeast extract, peptone, meat extract, tryptone, and casein. Polysulfide and elemental sulfur are reduced to H2S. In the absence of polysulfide or elemental sulfur, the isolate grows at a significantly reduced rate. Growth is not influenced by the presence of H2. DNA- DNA hybridization and 16S rRNA partial sequences indicated that the new isolate belongs to the genus Thermococcus, and represents a new species, Thermococcus acidaminovorans. The type stain is isolate AEDII10 (DSM 11906).

A new hyperthermophilic microorganism, Thermococcus barossii, was isolated from rock fragments of a hydrothermal vent flange formation, located along the East Pacific Rise of the Juan de Fuca Ridge. This organism is obligately anaerobic and grows over a temperature range of at least 60-92 degrees C in artificial seawater-based media, containing elemental sulfur, tryptone and yeast extract. The addition of a maltooligosaccharide mixture and tungsten to this medium improved growth to some extent. At the Topt for growth (82.5 degrees C), cell densities as high as 4 x 10(8) cells/ml could be obtained in 18-liter batch fermentations, with a doubling time of approximately 40 minutes, if culture access to elemental sulfur was sufficient. In continuous culture at the same temperature, comparable cell densities could be obtained but only at slower growth rates. Morphologically, T. barossii is coccoid-shaped, forming irregularly-shaped spheres; under optimal conditions, these coccoids become more regular and smaller, a characteristic of other hyperthermophilic archaea. Negatively-stained preparations showed no pili or flagella associated with the cell surface. 16S rRNA sequencing reveals that T. barossii is most similar to Thermococcus celer (99.7%). Yet, further comparisons with T. celer showed that T. barossii is a new Thermococcus species: different growth temperature optimum (82.5 degrees C vs. 88 degrees C), obligate requirement for sulfur, higher G + C content (60% vs. 56.7%) and 47.7% DNA-DNA hybridization. The nucleotide and translated amino acid sequence for the gene encoding a DNA polymerase from T. barossii was compared to sequences of related genes from other Thermacoccales. The polymerase phylogenies were congruent with those obtained from the 16S rRNA phylogenetic analyses. Based on the high degree of similarity among members of the genus Termococcus for the criteria used thus far, aspects of enzymology may be an important mechanism of differenting one species from another.