[The thermophilic bacteria hydrolyzing agar: characterization of thermostable agarase].

Three strains of anaerobic thermophilic bacteria capable of growing on agarose as a source of energy and carbon were isolated from hot springs near Lake Baikal (Barguzin National Park) and the caldera Uzon (Kamchatka). Cells of all the three strains were spore bacilli with peritrichous flagellation. These isolates grew at a temperature of 55-60 degrees C and pH 6.5-7.0 and fermented a wide range of organic substrates. Analysis of the 16S rRNA sequences allowed us to ascribe the strains B5 and K14 to the genus Thermoanaerobacter and the strain K67 to the genus Caldoanaerobacter. According to the results of DNA-DNA hybridization, B5 was determined as belonging to the species Thermoanaerobacter wiegelii. Agarase was isolated by preparative PAGE and subsequent gel chromatography from the culture liquid of strain B5 grown on the medium containing 0.5% agarose and 0.3% galactose. The molecular weight of this enzyme amounted to 67 kDa and pI, to 4.2. The T. wiegelii B5 agarase was active in the pH range of 3.5 to 7.0 (optimum, 5.2) and temperature range of 50 to 80 degrees C (optimum, 70 degrees C). The preincubation of this enzyme at 90 degrees C for 60 min did not reduce the agarase activity. This activity increased in the presence of metal ions; the maximal effect was observed in the presence of 5 mM Mg2+ and 25 mM Co2+.

[Metabolism of the thermophilic bacterium Oceanithermus profundus].

The metabolism of the novel facultatively anaerobic thermophilic bacterium Oceanithermus profundus was studied during growth on maltose, acetate, pyruvate, and hydrogen. The utilization of carbohydrates was shown to proceed via the glycolytic pathway. Under microaerobic growth conditions, the metabolism of O. profundus grown on maltose depended on the substrate concentration. At an initial maltose concentration of 1.4 mM, O. profundus carried out oxygen respiration, and in the presence of 3.5 mM maltose, facilitated fermentation occurred, with the formation of acetate and ethanol and limited involvement of oxygen. The use of pyruvate and acetate occurs via the TCA cycle. In cells grown on acetate, the activity of glyoxylate pathway enzymes was revealed. Depending on the energy-yielding process providing for growth (oxygen respiration or nitrate reduction), cells contained cytochromes a and c or b, respectively. The results obtained demonstrate the plasticity of the metabolism of O. profundus, which thus appears to be well-adjusted to the rapidly changing conditions in deep-sea hydrothermal vents.

[Investigation of the catabolism of acetate and peptides in the new anaerobic thermophilic bacterium Caldithrix abyssi].

This work is concerned with the metabolism of Caldithrix abyssi-an anaerobic, moderately thermophilic bacterium isolated from deep-sea hydrothermal vents of the Mid-Atlantic Ridge and representing a new, deeply deviated branch within the domain Bacteria. Cells of C. abyssi grown on acetate and nitrate, which was reduced to ammonium, possessed nitrate reductase activity and contained cytochromes of the b and c types. Utilization of acetate occurred as a result of the operation of the TCA and glyoxylate cycles. During growth of C. abyssi on yeast extract, fermentation with the formation of acetate, propionate, hydrogen, and CO2 occurred. In extracts of cells grown on yeast extract, acetate was produced from pyruvate with the involvement of the following enzymes: pyruvate:ferredoxin oxidoreductase (2.6 micromol/(min mg protein)), phosphate acetyltransferase (0.46 micromol/(min mg protein)), and acetate kinase (0.3 micromol/(min mg protein)). The activity of fumarate reductase (0.14 micromol/(min mg protein)), malate dehydrogenase (0.17 micromol/(min mg protein)), and fumarate hydratase (1.2 micromol/(min mg protein)), as well as the presence of cytochrome b, points to the formation of propionate via the methyl-malonyl-CoA pathway. The activity of antioxidant enzymes (catalase and superoxide dismutase) was detected. Thus, enzymatic mechanisms have been elucidated that allow C. abyssi to switch from fermentation to anaerobic respiration and to exist in the gradient of redox conditions characteristic of deep-sea hydrothermal vents.

Vulcanibacillus modesticaldus gen. nov., sp. nov., a strictly anaerobic, nitrate-reducing bacterium from deep-sea hydrothermal vents.

A novel anaerobic, moderately thermophilic, spore-forming bacterium, designated strain BRT, was isolated from deep-sea hydrothermal core samples collected at the Rainbow vent field on the Mid-Atlantic Ridge (36 degrees 14' N 33 degrees 54' W). The cells were found to be rod-shaped, non-motile, Gram-positive and spore-forming. The organism grew in the temperature range 37-60 degrees C, with an optimum at 55 degrees C, and at pH values in the range 6-8.5, with an optimum around pH 7. NaCl concentrations for growth were in the range 10-40 g l(-1), with an optimum at 20-30 g l(-1). Strain BRT grew chemo-organoheterotrophically with carbohydrates, proteinaceous substrates and organic acids with nitrate as electron acceptor. The novel isolate was not able to ferment. The G+C content of the genomic DNA was 34.5 mol%. Phylogenetic analysis of the 16S rRNA gene sequence placed strain BRT in the Bacillaceae within the class 'Bacilli'. On the basis of the phenotypic and phylogenetic data, this isolate should be described as a member of a novel genus, for which the name Vulcanibacillus gen. nov. is proposed. The type species is Vulcanibacillus modesticaldus sp. nov., with the type strain BRT (=DSM 14931T=JCM 12998T).

[Thermophilic microbial communities of deep-sea hydrothermal environments]. / Termofil'nye mikrobnye soobshchestva glubokovodnykh gidroterm.

The latest publications on the phylogenetic and functional diversity of thermophilic prokaryotes inhabiting thermal deep-sea environments are reviewed. Along with general physicochemical characterization of the biotope studied, certain adaptation mechanisms are discussed that are peculiar to the microorganisms inhabiting it. A separate chapter addresses phylogenetic analysis of deep-sea hydrothermal microbial communities and uncultivated microorganisms recently discovered therein using molecular biological techniques. Physiological groups of thermophilic microorganisms found in deep-sea hydrotherms are considered: methanogens, sulfate-, iron-, and sulfur-reducers, aerobic hydrogen-oxidizing prokaryotes, aerobic and anaerobic organotrophs. In most cases, the isolates represent novel taxa.

Caminibacter profundus sp. nov., a novel thermophile of Nautiliales ord. nov. within the class 'Epsilonproteobacteria', isolated from a deep-sea hydrothermal vent.

A novel moderately thermophilic, microaerobic to anaerobic, chemolithoautotrophic bacterium, designated strain CRT, was isolated from a deep-sea hydrothermal vent site at 36 degrees N on the Mid-Atlantic Ridge. Cells were Gram-negative, non-motile rods. The organism grew at 45-65 degrees C and pH 6.5-7.4, with optimum growth at 55 degrees C and pH 6.9-7.1. The NaCl range for growth was 5-50 g l(-1) (optimum 30 g l(-1)). Strain CRT was an obligate chemolithoautotroph, growing with H2 as energy source, sulfur, nitrate or oxygen as electron acceptors and CO2 as carbon source. Hydrogen sulfide and ammonium were the respective products of sulfur and nitrate reduction. The G+C content of the genomic DNA was 32.1 mol%. Based on 16S rRNA gene sequence analysis, this organism was most closely related to Caminibacter hydrogeniphilus (94.9% similarity). On the basis of phenotypic and phylogenetic data, it is proposed that the isolate represents a novel species, Caminibacter profundus sp. nov. The type strain is CRT (=DSM 15016T=JCM 11957T). The phylogenetic data also correlate well with the significant phenotypic differences between the lineage encompassing the genera Nautilia and Caminibacter and other members of the class 'Epsilonproteobacteria'. The lineage encompassing the genera Nautilia and Caminibacter is therefore proposed as a new order, Nautiliales ord. nov., represented by a single family, Nautiliaceae fam. nov.

Deferribacter abyssi sp. nov., an anaerobic thermophile from deep-sea hydrothermal vents of the Mid-Atlantic Ridge.

Two strains of thermophilic, anaerobic, chemolithoautotrophic bacteria, designated JR(T) and DR, were isolated from hydrothermal samples collected on the Mid-Atlantic Ridge from the Rainbow (36 degrees 16' N, 33 degrees 54' W) and Menez Gwen (37 degrees 50' N, 31 degrees 50' W) vent fields, respectively. Cells of both isolates were short, straight- to vibrio-shaped, motile rods with one polar flagellum, and were Gram-negative and non-sporulating. Strain JR(T) was characterized in detail. It was found to grow optimally at pH 6.5-6.7, at 60 degrees C and in the presence of 30 g NaCl l(-1). Strain JR(T) could use molecular hydrogen, acetate, succinate, pyruvate and proteinaceous compounds as electron donors, and elemental sulfur, nitrate or Fe(III) as electron acceptors. No fermentation of organic substrates occurred. The G+C content of the DNA of strain JR(T) was 30.8 mol%. Strain DR (=DSM 14927) possessed the same morphology and pH, temperature and salinity optima and ranges, and used the same electron acceptors as strain JR(T). On the basis of their 16S rDNA sequences (1517 nucleotides), strains JR(T) and DR were identical and distantly related to Deferribacter thermophilus and Deferribacter desulfuricans (95.3 and 95.2 % sequence similarity, respectively). Based on their phenotypic and phylogenetic characteristics, it is proposed that both strains are members of a new species of the genus Deferribacter, for which the name Deferribacter abyssi (type strain JR(T)=DSM 14873(T)=JCM 11955(T)) is proposed.

Vulcanithermus mediatlanticus gen. nov., sp. nov., a novel member of the family Thermaceae from a deep-sea hot vent.

A novel thermophilic, microaerophilic, facultatively chemolithoheterotrophic bacterium designated strain TR(T) was isolated from a sample of a deep-sea hydrothermal chimney collected at the Rainbow vent field on the Mid-Atlantic Ridge (36 degrees 14'N). Gram-negative, non-spore-forming, non-motile rods occurred singly or in pairs. The organism grew in the temperature range 37-80 degrees C with an optimum at 70 degrees C and at pH 5.5-8.4 with an optimum around 6.7. The NaCl range for growth was 10-50 g l(-1) with an optimum of 30 g l(-1). Strain TR(T) grew chemoorganoheterotrophically with carbohydrates, proteinaceous substrates, organic acids and alcohols using oxygen or nitrate as electron acceptors. The isolate was able to grow at oxygen concentrations from 0.5 to 21%. Oxygen concentrations that promoted fastest growth ranged from 4 to 8% under agitation. The novel isolate was able to grow lithoheterotrophically with molecular hydrogen as the energy source. The G + C content of the genomic DNA was 68.4 mol%. Phylogenetic analysis of the 16S rDNA sequence placed strain TR(T) within the phylum Deinococcus-Thermus of the Bacteria. On the basis of phenotypic and phylogenetic data, it is proposed that this isolate should be described as a member of a novel species of a new genus as Vulcanithermus mediatlanticus gen. nov., sp. nov. The type strain is TR(T) (= DSM 14978T = VKM B-2292T = JCM 11956T).

Oceanithermus profundus gen. nov., sp. nov., a thermophilic, microaerophilic, facultatively chemolithoheterotrophic bacterium from a deep-sea hydrothermal vent.

A novel moderately thermophilic, organotrophic, microaerophilic, facultatively chemolithotrophic bacterium, designated strain 506(T), was isolated from a deep-sea hydrothermal vent site at 13 degrees N in the East Pacific Rise. Cells were Gram-negative, non-motile rods. The organism grew in the temperature range 40-68 degrees C, with an optimum at 60 degrees C, and in the pH range 5.5-8.4, with an optimum around pH 7.5. The NaCl concentration for growth was in the range 10-50 g l(-1), with an optimum at 30 g l(-1). Strain 506(T) grew chemoorganoheterotrophically with carbohydrates, proteinaceous substrates, organic acids and alcohols using oxygen or nitrate as electron acceptor. Alternatively, strain 506(T) was able to grow lithoheterotrophically with molecular hydrogen as the energy source. The G +C content of the genomic DNA was 62.9 mol%. Phylogenetic analysis of the 16S rDNA sequence placed strain 506(T) in the family Thermaceae. On the basis of phenotypic and phylogenetic data, strain 506(T) (= DSM 14977(T) = VKM B-2274(T)) is proposed as the type strain of a novel species in a new genus, Oceanithermus profundus gen. nov., sp. nov.

Petrotoga olearia sp. nov. and Petrotoga sibirica sp. nov., two thermophilic bacteria isolated from a continental petroleum reservoir in Western Siberia.

Strictly anaerobic, thermophilic bacteria (strains SL24T, SL25T, SL27, SL29 and SL32) were isolated from a deep, continental oil reservoir in Western Siberia (Russia). These motile, rod-shaped organisms were surrounded by a sheath-like structure, a feature characteristic of the Thermotogales. On the basis of partial 16S rDNA sequences (500 nucleotides), strains SL25T, SL27, SL29 and SL32 were identical. Therefore, only strains SL24T and SL25T were studied in detail. The optimum temperature for growth of both strains was 55 degrees C. Their optimum pH for growth was 7.5 and their optimum NaCl concentration was between 20 and 30 g l(-1). The novel isolates reduced elemental sulfur and cystine, but not thiosulfate or sulfate, to hydrogen sulfide. The G+C contents of the genomic DNA of strains SL24T and SL25T were respectively 35 and 33 mol%. Phylogenetically, both strains are most closely related to Petrotoga miotherma, there being 98.9-99.4% similarity between their 16S rDNA sequences. Phenotypic properties and DNA-DNA hybridization experiments indicate that the strains belong to two novel species, for which the names Petrotoga olearia (type strain SL24T = DSM 13574T = JCM 11234T) and Petrotoga sibirica (type strain SL25T= DSM 13575T = JCM 11235T) are proposed.

Nautilia lithotrophica gen. nov., sp. nov., a thermophilic sulfur-reducing epsilon-proteobacterium isolated from a deep-sea hydrothermal vent.

A novel, strictly anaerobic, thermophilic sulfur-reducing bacterium, strain 525T, was isolated from tubes of the deep-sea hydrothermal vent polychaete Alvinella pompejana, collected on the East Pacific Rise (13 degrees N). This organism grew in the temperature range 37-68 degrees C, the optimum being 53 degrees C, and in the pH range 6.4-7.4, the optimum being 6.8-7.0. The NaCl range for growth was 0.8-5.0%, the optimum being 3.0%. Strain 525T grew lithoautotrophically with H2 as energy source, S0 as electron acceptor and CO2 as carbon source. Alternatively, strain 525T was able to use formate as an energy source. The G+C content of the genomic DNA was 34.7 mol%. Phylogenetic analysis of the 16S rDNA gene sequence placed strain 525T in the epsilon-subclass of the Proteobacteria, where it forms a deep cluster with recently isolated relatives. On the basis of phenotypic and phylogenetic differences between strain 525T and its closest phylogenetic relatives, it is proposed that the new isolate should be described as a member of a new genus, Nautilia, for which the name Nautilia lithotrophica gen. nov., sp. nov. is proposed. The type strain is strain 525T (= DSM 13520T).

Thermosipho geolei sp. nov., a thermophilic bacterium isolated from a continental petroleum reservoir in Western Siberia.

Three strictly anaerobic, thermophilic bacteria (SL31T, SL30 and MLM39636) were isolated from a deep continental oil reservoir in Western Siberia (Russia). Following the mid-exponential phase of growth, the non-motile rod-shaped organisms were surrounded by a sheath-like structure. As DNA-DNA hybridizations showed that these strains were highly related genomically, only strain SL31T was studied in detail. The temperature range for growth of strain SL31T was between 45 and 75 degrees C, with optimum growth at 70 degrees C. Its optimum pH and NaCl concentration for growth were pH 7.5 and 20-30 g l(-1), respectively. The novel isolate reduced elemental sulfur and cystine, but not thiosulfate or sulfate, to hydrogen sulfide. The G+C content of the genomic DNA was 30.0 mol %. As determined by 16S rDNA sequence analysis, this organism belonged to the genus Thermosipho. DNA-DNA hybridization levels between strain SL31T and type strains of the previously described species of Thermosipho were less than 10%. On the basis of physiological and molecular properties, it is proposed that this organism should be placed in a new species, Thermosipho geolei sp. nov. The novel organism represents the first species of the genus Thermosipho that has been isolated from a petroleum reservoir. The type strain is SL31T ( = DSM 13256T = JCM 10986T).

Isolation and characterization of Thermococcus sibiricus sp. nov. from a Western Siberia high-temperature oil reservoir.

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.

Acidilobus aceticus gen. nov., sp. nov., a novel anaerobic thermoacidophilic archaeon from continental hot vents in Kamchatka.

New thermoacidophilic organisms that were able to grow anaerobically on starch were isolated from the acidic hot springs of Kamchatka. Strain 1904T, isolated from a hot spring of the Moutnovski volcano, was characterized in detail. Its cells were regular or irregular cocci that were 1-2 microm in diameter, non-motile, and had a cell envelope consisting of one layer of subunits. The new organism was a hyperthermophile, growing in the temperature range 60-92 degrees C (with an optimum at 85 degrees C), an acidophile, having the pH range for growth of 2.0-6.0 (with an optimum at 3.8), and an obligate anaerobe. It fermented starch, forming acetate as the main growth product. Other growth substrates were yeast extract, beef extract and soya extract. Growth on yeast extract, beef extract and soya extract was stimulated by elemental sulfur, which was reduced to H2S. Acetate, arabinose, cellulose, formate, fructose, galactose, glucose, glycine, guar gum, lichenan, malate, maltose, methanol, pectin, pyruvate, propionate, xylan, xylose or a mixture of amino acids failed to support growth both in the presence and the absence of sulfur. When starch was used as the growth substrate, yeast extract (100 mg l(-1)) was required as a growth factor. The G+C content of the DNA was found to be 53.8 mol%. Comparison of the complete 16S rDNA sequence with databases revealed that the new isolate belonged to the kingdom Crenarchaeota. It was not closely related to any described genera (showing sequence similarity below 90.8%) and formed a separate branch of the Crenarchaeota. On the basis of physiological differences and rRNA sequence data, a new genus--Acidilobus--is proposed, the type species being Acidilobus aceticus strain 1904T (= DSM 11585T).

Investigation of structure and antigenic capacities of Thermococcales cell envelopes and reclassification of "Caldococcus litoralis" Z-1301 as Thermococcus litoralis Z-1301.

Fourteen strains of hyperthermophilic organotrophic anaerobic marine Archaea were isolated from shallow water and deep-sea hot vents, and four of them were characterized. These isolates, eight previously published strains, and six type strains of species of the order Thermococcales were selected for the study of cell wall components by means of thin sectioning or freeze-etching electron microscopy. The cell envelopes of most isolates were shown to consist of regularly arrayed surface protein layers, either single or double, with hexagonal lattice (p6) symmetry, as the exclusive constituents outside the cytoplasmic membrane. The S-layers studied differed in center-to-center spacing and molecular mass of the constituent protein subunits. Polyclonal antisera raised against the cells of 10 species were found to be species-specific and allowed 12 new isolates from shallow water hot vents to be identified as representatives of the species Thermococcus litoralis, Thermococcus stetteri, Thermococcus chitonophagus, and Thermococcus pacificus. Of the 7 deep-sea isolates, only 1 was identified as a T. litoralis strain. Thus, hyperthermophilic marine organotrophic isolates obtained from deep-sea hot vents showed greater diversity with regard to their S-layer proteins than shallow water isolates.

Hippea maritima gen. nov., sp. nov., a new genus of thermophilic, sulfur-reducing bacterium from submarine hot vents.

Three strains of moderately thermophilic, sulfur-reducing bacteria were isolated from shallow-water hot vents of the Bay of Plenty (New Zealand) and Matupi Harbour (Papua New Guinea). Cells of all isolates were short, Gram-negative, motile rods with one polar flagellum. All strains were obligate anaerobes and grew optimally at pH 5.8-6.2, 52-54 degrees C and 2.5-3% (w/v) NaCl. Growth substrates were molecular hydrogen, acetate and saturated fatty acids; one of the strains, isolated from Matupi Harbour, was able to utilize ethanol. Elemental sulfur was required for growth. H2S and CO2 were the only growth products. No growth occurred in the absence of 100 mg yeast extract I-1. The G+C content of the DNA determined for the type strain MH2T was 40.4 mol%. Results of 16S rDNA sequencing indicated that these strains represent a distinct lineage most closely related to the genus Desulfurella. On the basis of the results of morphological, physiological and phylogenetic studies, a new genus, Hippea gen. nov., is proposed with the type species Hippea maritima gen. nov., sp. nov., of which the type strain is MH2T (= DSM 10411T).

Desulfurella kamchatkensis sp. nov. and desulfurella propionica sp. nov., new sulfur-respiring thermophilic bacteria from Kamchatka thermal environments.

Two strains of moderately thermophilic bacteria, which reduce elemental sulfur to hydrogen sulfide, were isolated from volcanic sources in Kamchatka. Strain K-119T was obtained from a thermophilic microbial community associated with Thermothrix thiopara, and strain U-8T was isolated from a cyanobacterial mat inhabiting a sulfide-rich hot spring. Cells of both strains are short Gram-negative rods, motile with one polar flagellum (strain K-119T) or non-motile (strain U-8T). Both strains are obligate anaerobes, have temperature otima of 54-55 degrees C and pH optima of 6.9-7.2. Molecular hydrogen, acetate, fumarate, malate, pyruvate, lactate and long-chain saturated fatty acids served as growth substrates for both species; strain U-8T was also able to grow on propionate. All substrates were oxidized completely, H2S and CO2 being the only metabolic products. Elemental sulfur was obligately required for growth of strain K-119T, whereas strain U-8T was able to grow also with thiosulfate as electron acceptor and on pyruvate without an external electron acceptor. The DNA G + C contents of strains K-119T and U-8T were 31.6 and 32.2 mol%, respectively. Phenotypic features and the results of 16S rRNA sequencing indicate the affiliation of the new isolates to the genus Desulfurella. The DNA-DNA hybridization with Desulfurella acetivorans was 40% for strain K-119T and 55% for strain U-8T; the DNA-DNA hybridization between the new isolates was 32%. Based on the results of morphological, physiological and phylogenetic studies the following two new species are proposed: Desulfurella kamchatkensis sp. nov. with the type strain K-119T (= DSM 10409T) and Desulfurella propionica sp. nov. with the type strain U-8T (= DSM 10410T).

Thermococcus gorgonarius sp. nov. and Thermococcus pacificus sp. nov.: heterotrophic extremely thermophilic archaea from New Zealand submarine hot vents.

Two extremely thermophilic archaea, designated W-12 and P-4, were isolated from a geothermal vent in the tidal zone of Whale Island, New Zealand, and from geothermally heated bottom deposits of the Bay of Plenty, New Zealand, respectively. Cells of isolate W-12 are irregular cocci, 0.3-1.2 microns in diameter, motile with polar flagella. The cell envelope consists of one layer of subunits with a major protein of M(r) 75,000. Cells produce protrusions of different kinds: prostheca-like, chains of bubbles, or network of fimbriae. Cells of isolate P-4 are regular cocci, 0.7-1.0 micron in diameter, motile with polar flagella. The cell envelope consists of two layers of subunits; its major protein has an M(r) of 56,000. Both organisms are obligate anaerobes, fermenting peptides in the case of strain W-12, or peptides and starch in the case of P-4. Elemental sulfur is required for growth and is reduced to hydrogen sulfide. The optimal growth temperature of the new isolates is in the range 80-88 degrees C. The optimal growth pH is 6.5-7.2. The G + C content of the DNA of strain W-12 is 50.6 mol%, and of strain P-4 is 53.3 mol%. Based on physiological characteristics, 165 rDNA sequence comparison and DNA base composition, the new isolates were considered to be members of the genus Thermococcus. The low level of DNA-DNA hybridization with the type strains of other Thermococcus species confirms the novel species status of the new isolates. The new isolates are described as Thermococcus gorgonarius sp. nov., with type strain W-12 (= DSM 10395T), and Thermococcus pacificus sp. nov., with type strain P-4 (= DSM 10394T).

Isolation of thermostable phosphatase from the hyperthermophilic archaeon Thermococcus pacificus by immobilized metal affinity chromatography.

Phosphatase was isolated from cells of the hyperthermophilic marine archaeon Thermococcus pacificus by a procedure including chromatography on Butyl-Fractogel TSK-650 and Ni(2+)-iminodiacetic-agarose. Enzyme activity is maximal at 90 degrees C, and the enzyme half-life time at this temperature is 1 h. The pH optimum of phosphatase activity is 6.0. Electrophoresis under denaturating conditions yielded a subunit molecular weight of 45 kDa. On gel-filtration on Sephacryl S-300 HR three peak corresponding to 295, 85 and 45 kDa were observed, suggesting that the enzyme is a homohexamer.