Thermococcus acidaminovorans sp. nov., a new hyperthermophilic alkalophilic archaeon growing on amino acids

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° and 93°C (optimum 85°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 strain is isolate AEDII10 (DSM 11906). Received: September 24, 1997 / Accepted: January 1, 1998     

Thermococcus alcaliphilus sp. nov., a new hyperthermophilic archaeum growing on polysulfide at alkaline pH

A novel coccoid-shaped, hyperthermophilic, heterotrophic member of the archaea was isolated from a shallow marine hydrothermal system at Vulcano Island, Italy. The isolate grew between 56 and 90° C with an optimum around 85° C. The pH range for growth was 6.5 to 10.5, with an optimum around 9.0. Polysulfide and elemental sulfur were reduced to H₂S. Sulfur stimulated the growth rate. The isolate fermented yeast extract, peptone, meat extract, tryptone, and casein. Isovalerate, isobutyrate, propionate, acetate, CO₂, NH₃, and H₂S (in the presence of S°) were detected as end products. Growth was not inhibited by H₂. Based on DNA-DNA hybridization and 16S rRNA partial sequences, the new isolate represents a new species of Thermococcus, which we named Thermococcus alcaliphilus. The type strain is isolate AEDII12 (DSM 10322) Received: 7 July 1995 / Accepted: 25 August 1995     

Thermococcus waiotapuensis sp. nov., an extremely thermophilic archaeon isolated from a freshwater hot spring

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 °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 °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. Received: 5 January 1999 / Accepted: 19 May 1999     

Properties of extremely thermostable proteases from anaerobic hyperthermophilic bacteria

Summary Hyperthermostable proteases were characterized from five archaeobacterial species (Thermococcus celer, T. stetteri, Thermococcus strain AN 1, T. litoralis, Staphylothermus marinus) and the hyperthermophilic eubacterium Thermobacteroides proteolyticus. These proteases, which were found to be of the serine type, exhibited a preference for phenylalanine in the carboxylic side of the peptide. The enzymes from Thermococcus stetteri and T. litoralis hydrolysed most substrates (peptides) tested. All proteases were extremely thermostable and demonstrated optimal activities between 80 and 95°C. The pH optimum was either neutral (T. celer, Thermococcus strain AN 1) or alkaline. The protease of Thermobacteroides proteolyticus was optimally active at pH 9.5. Zymogram staining showed the presence of multiple protease bands for all strains investigated.Offprint requests to: G. Antranikian     

Thermococcus peptonophilus sp. nov., a fast-growing,extremely thermophilic archaebacterium isolated from deep-sea hydrothermal vents

Two extremely thermophilic archaebacteria, strains OG-1 and SM-2, were isolated from newly discovered deep-sea hydrothermal vent areas in the western Pacific ocean. These strains were cocci, obligately anaerobic Archaea about 0.7–2 μm in diameter. Optimum growth conditions for OG-1 and SM-2 were at 85–90°C (range 60–100°C), pH 6 (range pH 4–8), a NaCl concentration of 3% (range 1–5%), and a nutrient concentration (tryptone plus yeast extract) of 0.2% (range 0.005–5%). Elemental sulfur stimulated the growth rate fourfold. Ammonium slightly stimulated growth. Both tryptone and yeast extract allowed growth as sole carbon sources; these isolates were not able to utilize or grow exclusively on sucrose, glucose, maltose, succinate, pyruvate, propionate, acetate, or free amino acids. OG-1 showed the fastest growth rate within the genus Thermococcus. Growth was inhibited by rifampicin. The DNA G+C content was 52 mol%. Sequencing of their 16S rDNA gene fragment indicated that these isolates belonged to the genus Thermococcus. OG-1 and SM-2 were different than the described Thermococcus species. We propose that OG-1 belongs to a new species: Thermococcus peptonophilus. Received: 8 March 1995 / Accepted: 24 May 1995     

Thermococcus chitonophagus sp. nov., a novel,chitin-degrading,hyperthermophilic archaeum from a deep-sea hydrothermal vent environment

From a hydrothermal vent site off the Mexican west coast (20°50′N, 109°06′W) at a depth of 2,600 m, a novel, hyperthermophilic, anaerobic archaeum was isolated. Cells were round to slightly irregular cocci, 1.2–2.5 μm in diameter and were motile by means of a tuft of flagella. The new isolate grew between 60 and 93°C (optimum: 85°C), from pH 3.5 to 9 (optimum: pH 6.7), and from 0.8 to 8% NaCl (optimum: 2%). The isolate was an obligate organotroph, using chitin, yeast extract, meat extract, and peptone for growth. Chitin was fermented to H₂, CO₂, NH₃, acetate, and formate. H₂S was formed in the presence of sulfur. The chitinoclastic enzyme system was oxygen-stable, cell-associated, and inducible by chitin. The cell wall was composed of a surface layer of hex- americ protein complexes arranged on a p6 lattice. The core lipids consisted of glycerol diphytanyl diethers and acyclic and cyclic glycerol diphytanyl tetraethers. The G+C content was 46.5 mol%. DNA/DNA hybridization and 16S rRNA sequencing indicated that the new isolate belongs to the genus Thermococcus, representing a new species, Thermococcus chitonophagus. The type strain is isolate GC74, DSM 10152. Received: 8 May 1995 / Accepted: 26 June 1995     

Thermococcus siculi sp. nov., a novel hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent at the Mid-Okinawa Trough

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°32.7′N, 126°58.5′E). Cells of this isolate occur singly or in pairs and are about 0.8 to 2 μm in diameter. Growth was observed at temperatures between 50° and 93°C, with an optimum at 85°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 μg/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). Received: May 11, 1998 / Accepted: July 24, 1998     

Isolation and characterization of a novel facultatively alkaliphilic Nitrobacter species, N. alkalicus sp. nov.

Five strains of lithotrophic, nitrite-oxidizing bacteria (AN1-AN5) were isolated from sediments of three soda lakes (Kunkur Steppe, Siberia; Crater Lake and Lake Nakuru, Kenya) and from a soda soil (Kunkur Steppe, Siberia) after enrichment at pH 10 with nitrite as sole electron source. Morphologically, the isolates resembled representatives of the genus Nitrobacter. However, they differed from recognized species of this genus by the presence of an additional S-layer in their cell wall and by their unique capacity to grow and oxidize nitrite under highly alkaline conditions. The influence of pH on growth of one of the strains (AN1) was investigated in detail by using nitrite-limited continuous cultivation. Under such conditions, strain AN1 was able to grow at a broad pH range from 6.5 to 10.2, with an optimum at 9.5. Cells grown at pH higher than 9 exhibited a clear shift in the optimal operation of the nitrite-oxidizing system towards the alkaline pH region with respect to both reaction rates and the affinity. Cells grown at neutral pH values behaved more like neutrophilic Nitrobacter species. These data demonstrated the remarkable potential of the new nitrite-oxidizing bacteria for adaptation to varying alkaline conditions. The 16S rRNA gene sequences of isolates AN1, AN2, and AN4 showed high similarity (≥ 99.8%) to each other, and to sequences of Nitrobacter strain R6 and of Nitrobacter winogradskyi. However, the DNA-DNA homology in hybridization studies was too low to consider these isolates as new strains. Therefore, the new isolates from the alkaline habitats are described as a new species of the genus Nitrobacter, N. alkalicus, on the basis of their substantial morphological, physiological, and genetic differences from the recognized neutrophilic representatives of this genus. Received: 3 April 1998 / Accepted: 2 July 1998     

Formate hydrogenlyase and formate secretion ameliorate H2 inhibition in the hyperthermophilic archaeon Thermococcus paralvinellae

Some hyperthermophilic heterotrophs in the genus Thermococcus produce H₂ in the absence of S° and have up to seven hydrogenases, but their combined physiological roles are unclear. Here, we show which hydrogenases in Thermococcus paralvinellae are affected by added H₂ during growth without S°. Growth rates and steady‐state cell concentrations decreased while formate production rates increased when T. paralvinallae was grown in a chemostat with 65 µM of added H_2(aq). Differential gene expression analysis using RNA‐Seq showed consistent expression of six hydrogenase operons with and without added H₂. In contrast, expression of the formate hydrogenlyase 1 (fhl1) operon increased with added H₂. Flux balance analysis showed H₂ oxidation and formate production using FHL became an alternate route for electron disposal during H₂ inhibition with a concomitant increase in growth rate relative to cells without FHL. T. paralvinellae also grew on formate with an increase in H₂ production rate relative to growth on maltose or tryptone. Growth on formate increased fhl1 expression but decreased expression of all other hydrogenases. Therefore, Thermococcus that possess fhl1 have a competitive advantage over other Thermococcus species in hot subsurface environments where organic substrates are present, S° is absent and slow H₂ efflux causes growth inhibition.     

Cloning, Expression, and Characterization of a DNA Ligase from a Hyperthermophilic Archaeon Thermococcus Sp.

Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. NA1, revealed an ORF of 1689 bases encoding 562 amino acids that showed a high similarity to DNA ligases from other hyperthermophilic archaea. The ligase, which was designated TNA1_lig (Thermococcus sp. NA1 ligase), was cloned and expressed in Escherichia coli. The recombinant TNA1_lig was purified by metal affinity chromatography. The optimum ligase activity of the recombinant TNA1_lig occurred at 80 °C and pH 7.5. The enzyme was activated by MgCl₂ and ZnCl₂ but was inhibited by MnCl₂ and NiCl₂. Additionally, the enzyme was activated by either ATP or NAD⁺. Revisions requested 27 October 2005; Revisions received 14 December 2005     

Biochemical and phylogenetic characterization of two novel deep-sea Thermococcus isolates with potentially biotechnological applications

The partial 16S rDNA gene sequences of two thermophilic archaeal strains, TY and TYS, previously isolated from the Guaymas Basin hydrothermal vent site were determined. Lipid analyses and a comparative analysis performed with 16S rDNA sequences of similar thermophilic species showed that the strains isolated from deep-sea vents were not identical to the other species belonging to the genus Thermococcus. On the basis of the results of the phylogenetic analyses, lipid analyses, and previously reported physiological data, we believe that strains TY and TYS are significantly different from the previously described Thermococcus species. According to specific physiological and molecular features, we propose the use of these isolates as potential tools for the development of biotechnological applications in the field of starch processing and DNA technology. Received: 19 August 1996 / Accepted: 6 November 1996     

Functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1

Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two α subunits and four β subunits with the structure of a double β-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (α1, α2, β1, and β2) from T. KS-1. All of them (α1-β1, α2-β1, α1-β2, and α2-β2) exist as α₂β₄ heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate chaperonins. Prefoldin complexes containing the β1 subunit interacted with the chaperonins more strongly than those with the β2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as chaperonins.     

Purification and characterization of a histone-like protein from the Archaeal isolate AN1, a member of the Thermococcales

We have purified and characterized the histone-like protein, termed HAN1, and an HAN1-associated DNA-binding protein (hDBP) from nucleoids of the hyper-thermophilic Thermococcus-like AN1. HAN1 is shown to be composed of two subunits, to be thermally stable and to compact DNA in a reversible manner. The N-terminal sequence of HAN1 shares a high degree of homology with HMf, the histone-like protein from Methanothermus fervidus. Consistent with this, the toroidal wrapping of DNA by HAN1 resembles that described for HMf. However, significant differences in both twist and writhe components of these complexes are indicated by the 12.0 bp helical repeat produced during hydroxyl radical footprinting with HAN1. Furthermore, the increased stability of HAN1: DNA complexes allows DNA to be protected from thermal denaturation and cleavage by the restriction enzyme TaqI at 65°C. The hDBP, which co-purified with HAN1, is shown to represent a major portion of the acid-washed nucleoid protein in AN1 and to enhance the mobility of DNA directly, yet decrease the mobility of HAN1:DNA complexes.     

Cloning, Expression, and Characterization of a Methionyl Aminopeptidase from a Hyperthermophilic Archaeon Thermococcus sp. NA1

Genomic analysis of a hyperthermophilic archaeon Thermococcus sp. NA1 revealed the presence of an 885-bp open reading frame encoding a protein of 295 amino acids with a calculated molecular mass of 32,981 Da. Analysis of the deduced amino acid sequence showed that amino acid residues important for catalytic activity and the metal binding ligands conserved in all of methionyl aminopeptidases (MetAP) were also conserved and belonged to type IIa MetAP. The protein, designated TNA1_MetAP (Thermococcus sp. NA1 MetAP), was cloned and expressed in Escherichia coli. The recombinant enzyme was a Mn²⁺-, Ni²⁺-, Fe²⁺-, or Co²⁺-dependent metallopeptidase. Optimal MetAP activity against l-methionine p-nitroanilide (Met-pNA) (K _m = 0.68 mM) occurred at pH 7.0 and 80 to 90°C. The MetAP was very unstable compared to Pyrococcus furiosus MetAP, which was completely inactivated by heating at 80°C for 5 min. It seemed likely that the cysteine residue (Cys53) played a critical role in regulating the thermostability of TNA1_MetAP.     

<Emphasis Type="Italic">Acinetobacter brisouii</Emphasis> sp. nov., isolated from a wetland in Korea

A bacterial strain 5YN5-8^T was isolated from peat layer on Yongneup in Korea. Cells of strain 5YN5-8^T were strictly aerobic, Gram-negative, coccobacilli, non-spore forming, and non-motile. The isolate exhibited optimal growth at 28°C, pH 7.0, and 0–1% NaCl. Results of 16S rRNA gene sequence analyses indicated a close relationship of this isolate to Acinetobacter calcoaceticus (97.8% similarity for strain DSM 30006^T). It also exhibited 94.4–97.8% 16S rRNA gene sequence similarities to the validly published Acinetobacter species. The value for DNA-DNA hybridization between strain 5YN5-8^T and other members of the genus Acinetobacter ranged from 16 to 28%. Predominant cellular fatty acids were C_18:1 ω9c, summed feature 4 containing C_15:0 iso 2-OH and/or C_16:1 ω7c, and C_16:0. The DNA G+C content was 43.9 mol%. Phylogenetic, phenotypic, and chemotaxonomic data accumulated in this study revealed that the isolate could be classified in a novel species of the genus Acinetobacter. The name Acinetobacter brisouii sp. nov. is proposed for the novel species, with 5YN5-8^T (=KACC 11602^T = DSM 18516^T) as the type strain.     

Molecular characterization of the recombinant iron-containing alcohol dehydrogenase from the hyperthermophilic Archaeon, Thermococcus strain ES1

The gene encoding a thermostable iron-containing alcohol dehydrogenase from Thermococcus Strain ES1 (ES1 ADH) was cloned, sequenced and expressed in Escherichia coli. The recombinant and native ES1 ADHs were purified using multistep column chromatography under anaerobic conditions. Both enzymes appeared to be homotetramers with a subunit size of 45 ± 1 kDa as revealed by SDS-PAGE, which was close to the calculated value (44.8 kDa). The recombinant ADH contained 1.0 ± 0.1 g-atom iron per subunit. Both enzymes were sensitive to oxygen with a half-life upon exposure to air of about 4 min. The recombinant enzyme exhibited a specific activity of 105 ± 2 U mg⁻¹, which was very similar to that of the native enzyme (110 ± 3 U mg⁻¹). The optimal pH-values for both enzymes for ethanol oxidation and acetaldehyde reduction were 10.4 and 7.0, respectively. Both enzymes also showed similar temperature-dependent activities, and catalyzed the oxidation of primary alcohols, but there was no activity towards methanol and secondary alcohols. Kinetic parameters of the enzymes showed lower K _m-values for acetaldehyde and NADPH and higher K _m-values for ethanol and NADP⁺. It is concluded that the gene encoding ES1 ADH was expressed successfully in E. coli. This is the first report of a fully active recombinant version of an iron-containing ADH from a hyperthermophile.     

Sulfobacillus sibiricus sp. nov., a New Moderately Thermophilic Bacterium

In the course of pilot industrial testing of a biohydrometallurgical technology for processing gold-arsenic concentrate obtained from the Nezhdaninskoe ore deposit (East Siberia, Sakha (Yakutiya)), a new gram-positive rod-shaped spore-forming moderately thermophilic bacterium (designated as strain N1) oxidizing Fe²⁺, S⁰, and sulfide minerals in the presence of yeast extract (0.02%) was isolated from a dense pulp. Physiologically, strain N1 differs from previously described species of the genus Sulfobacillus in having a somewhat higher optimal growth temperature (55°C). Unlike the type strain of S. thermosulfidooxidans, strain N1 could grow on a medium with 1 mM thiosulfate or sodium tetrathionate as a source of energy only within several passages and failed to grow in the absence of an inorganic energy source on media with sucrose, fructose, glucose, reduced glutathione, alanine, cysteine, sorbitol, sodium acetate, or pyruvate. The G+C content of the DNA of strain N1 was 48.2 mol %. The strain showed 42% homology after DNA–DNA hybridization with the type strain of S. thermosulfidooxidans and 10% homology with the type strain of S. acidophilus. The isolate differed from previously studied strains of S. thermosulfidooxidans in the structure of its chromosomal DNA (determined by the method of pulsed-field gel electrophoresis), which remained stable as growth conditions were changed. According to the results of the 16S rRNA gene analysis, the new strain forms a single cluster with the bacteria of the species Sulfobacillus thermosulfidooxidans (sequence similarity of 97.9–98.6%). Based on these genetic and physiological features, strain N1 is described as a new species Sulfobacillus sibiricus sp. nov.     

Oceanobacillus kimchii sp. nov. Isolated from a traditional Korean fermented food

A moderate halophile, strain X50^T, was isolated from mustard kimchi, a traditional Korean fermented food. The organism grew under conditions ranging from 0–15.0% (w/v) NaCl (optimum: 3.0%), pH 7.0–10.0 (optimum: pH 9.0) and 15–45°C (optimum: 37°C). The morphological, physiological, and biochemical features and the 16S rRNA gene sequences of strain X50^T were characterized. Colonies of the isolate were creamcolored and the cells were rod-shaped. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain X50^T belongs to the genus Oceanobacillus and is closely related phylogenetically to the type strain O. iheyensis HTE831^T (98.9%) and O. oncorhynchi subsp. oncorhynchi R-2^T (97.0%). The cellular fatty acid profiles predominately included anteiso-C_15:0 and iso-C_15:0. The G+C content of the genomic DNA of the isolate was 37.9 mol% and the major isoprenoid quinone was MK-7. Analysis of the 16S rRNA gene sequences, DNA-DNA relatedness and physiological and biochemical tests indicated genotypic and phenotypic differences among strain X50^T and reference species in the genus Oceanobacillus. Therefore, strain X50^T was proposed as a novel species and named Oceanobacillus kimchii. The type strain of the new species is X50^T (=JCM 16803^T =KACC 14914^T =DSM 23341^T).     

Streptomyces bullii sp. nov., isolated from a hyper-arid Atacama Desert soil

A Streptomyces strain isolated from a hyper-arid Atacama Desert soil was characterised using a polyphasic taxonomic approach. The strain, designated C2^T, had chemical and morphological properties typical of the genus Streptomyces. The isolate formed a branch in the Streptomyces 16S rRNA gene tree together with the type strain of Streptomyces chromofuscus and was also loosely related to Streptomyces fragilis NRRL 2424^T. DNA:DNA relatedness values between the isolate and its two phylogenetic neighbours showed that it formed a distinct genomic species. The strain was readily distinguished from these organisms using a combination of morphological and phenotypic data. Based on the genotypic and phenotypic results, isolate C2^T represents a novel species in the genus Streptomyces, for which the name Streptomyces bullii sp. nov. is proposed. The type strain is C2^T (=CGMCC 4.7019^T = KACC 15426^T).     

Purification and Characterization of an Extremely Thermostable Cyclomaltodextrin Glucanotransferase from a Newly Isolated Hyperthermophilic Archaeon, a Thermococcus sp.

The extremely thermophilic anaerobic archaeon strain B1001 was isolated from a hot-spring environment in Japan. The cells were irregular cocci, 0.5 to 1.0 μm in diameter. The new isolate grew at temperatures between 60 and 95°C (optimum, 85°C), from pH 5.0 to 9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%). The G+C content of the genomic DNA was 43.0 mol%. The 16S rRNA gene sequencing of strain B1001 indicated that it belongs to the genus Thermococcus. During growth on starch, the strain produced a thermostable cyclomaltodextrin glucanotransferase (CGTase). The enzyme was purified 1,750-fold, and the molecular mass was determined to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Incubation at 120°C with SDS and 2-mercaptoethanol was required for complete unfolding. The optimum temperatures for starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100°C, respectively. The optimum pH for enzyme activity was pH 5.0 to 5.5. At pH 5.0, the half-life of the enzyme was 40 min at 110°C. The enzyme formed mainly α-cyclodextrin with small amounts of β- and γ-cyclodextrins from starch. This is the first report on the presence of the extremely thermostable CGTase from hyperthermophilic archaea.