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.     

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.     

Growth requirements of hyperthermophilic sulfur-dependent heterotrophic archaea isolated from a shallow submarine geothermal system with reference to their essential amino acids.

Three hyperthermophilic sulfur-dependent heterotrophs were isolated from a shallow submarine hydrothermal system at an inlet of Kodakara-jima island, Kagoshima, Japan. The isolates grew at 60 to 97 degrees C, with the optimum temperatures at 85 to 90 degrees C. Sensitivity to rifampin and the existence of ether lipids indicated that the isolates are hyperthermophilic archaea. Partial sequencing of the genes coding for 16S rRNA showed that the three isolates are closely related to the genus Thermococcus. They grew on proteinaceous mixtures, such as yeast extract, Casamino Acids, and purified proteins (e.g., casein and gelatin), but not on carbohydrates or organic acids as sole carbon and energy sources. Nine amino acids were essential for growth of isolate KS-1 (Thr, Leu, Ile, Val, Met, Phe, His, Tyr, and Arg). Isolate KS-2 required Lys in addition to the nine amino acids, and KS-8 required Lys instead of Tyr. In comparative studies, it was shown that Thermococcus celer DSM 2476 required 10 amino acids (Thr, Leu, Ile, Val, Met, Phe, Tyr, Trp, Lys, and Arg) while Pyrococcus furiosus DSM 3638 required only Ile and Val. The hyperthermophilic fermentative eubacterium Thermotoga neapolitana DSM 4359 did not require any amino acids for growth.     

Diversity among three novel groups of hyperthermophilic deep-sea Thermococcus species from three sites in the northeastern Pacific Ocean

Eight new strains of deep-sea hyperthermophilic sulfur reducers were isolated from hydrothermal vent fields at 9 degrees 50'N East Pacific Rise (EPR) and at the Cleft and CoAxial segments along the Juan de Fuca Ridge (JdFR). 16S rRNA gene sequence analysis showed that each strain belongs to the genus Thermococcus. Restriction fragment length polymorphism patterns of the 16S/23S rRNA intergenic spacer region revealed that these isolates fell into three groups: those from the EPR, those from fluid and rock sources on the JdFR, and those isolated from Paralvinella spp. polychaete vent worms from the JdFR. The optimum-temperature specific growth rates and the temperature ranges for growth were significantly higher and broader for those strains isolated from worms relative to those isolated from low-temperature diffuse hydrothermal fluids. Furthermore, the worm-derived isolates generally produced a larger array of proteases and amylases based on zymogram analyses. The zymogram patterns also changed with growth temperature suggesting that these organisms alter their lytic protein suites in response to changes in temperature. This study suggests that there is significant phenotypic diversity in Thermococcus that is not apparent from their highly conserved 16S rRNA nucleotide sequences.     

Distribution and physiological characteristics of hyperthermophiles in the Kubiki oil reservoir in Niigata, Japan

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.     

Natural chaperonin of the hyperthermophilic archaeum, Thermococcus strain KS-1: a hetero-oligomeric chaperonin with variable subunit composition

To study the difference in expression of the chaperonin alpha- and beta-subunits in Thermococcus strain KS-1 (T. KS-1), we measured their intracellular contents at various growth temperatures using subunit-specific antibodies. The beta-subunit was significantly more abundant with increasing temperature (maximum at 93 degrees C), whereas the alpha-subunit was not. Native PAGE with Western blot analysis indicated that the natural chaperonins in the crude extracts of T. KS-1 cells grown between 65 degrees C and 95 degrees C migrate as single bands with different mobility. The recombinant alpha- and beta-subunit homo-oligomers migrated differently from each other and from natural chaperonins. Immunoprecipitation also showed that the natural chaperonin was the hetero-oligomer. These results indicate that chaperonin in T. KS-1 formed a hetero-oligomer with variable subunit composition, and that the beta-subunit may be adapted to a higher temperature than the alpha-subunit. T. KS-1 probably changes its chaperonin subunit composition to acclimatize to the ambient temperature.     

Isolation and characterization of the thermostable DNA polymerase of the hyperthermophilic archaeum Thermococcus litoralis Sh1AM

Overall, 30 strains of hyperthermophilic archaea, representing seven species of the genera Thermococcus, Desulfurococcus, Thermoproteus, and Acidilobus, were tested for the presence of thermostable DNA polymerases. Thermostabilities of the polymerases varied distinctly among the strains within one species. Polymerases of five strains retained 60-100% activity upon incubation of the preparations at 95 degrees C for 120 min. A new DNA polymerase was isolated from the strain Thermococcus litoralis Sh1AM, possessing the enzyme with the most promising properties, and characterized. Molecular weight of the enzyme is 90-100 kDa. The purified DNA polymerase preserved 50% of the initial activity upon incubation at 95 degrees C for 120 min. The polymerase isolated displayed an associated 3'-5' exonuclease activity. The error rate when extending DNA strand was at least twofold lower compared with Taq polymerase. The main physicochemical and enzymatic properties of the new polymerase are similar to the known DNA polymerases of family B.     

Molecular diversity of new Thermococcales isolates from a single area of hydrothermal deep-sea vents as revealed by randomly amplified polymorphic DNA fingerprinting and 16S rRNA gene sequence analysis

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.     

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. Received: February 5, 1999 / Accepted: May 11, 1999     

Three novel halotolerant and thermophilic Geobacillus strains from shallow marine vents

During a polyphasic taxonomic analysis performed on isolates from shallow marine hydrothermal vents of Eolian Islands (Italy), three thermophilic, halotolerant bacilli, designated as strain 1bw, strain 5-2 and strain 10-1, could not be affiliated to any described species. Physiological and biochemical characteristics, membrane lipids composition, mol % G+C content, and phylogenetic relationships determined on the basis of the 16S rRNA gene sequence analysis, placed these strains within the genus Geobacillus. The three strains were only moderately related to species of Geobacillus and their relatives, members of Saccharococcus. Determination of the relatedness among each other at a higher taxonomic level by DNA-DNA reassociation experiments demonstrated the three isolates to represent three different novel Geobacillus genomospecies. The taxonomic novelty of these three marine strains was substantiated by their physiological properties and by fatty acid patterns that did not match closely those of any Geobacillus type strain. These three novel strains could be of interest to biotechnology because of their ability to produce exopolysaccharides and to adhere on polystirene, characteristics undescribed so far for other Geobacillus species. They are also able to utilise hydrocarbons such as gas oil, kerosene and mineral lubricating oil. Strain 5-2 is tolerant to zinc.     

Two kinds of archaeal group II chaperonin subunits with different thermostability in Thermococcus strain KS-1

The thermostability of the recombinant alpha- and beta-subunit homo-oligomers (alpha16mer and beta16mer) and of natural chaperonins purified from cultured Thermococcus strain KS-1 cells was measured to understand the mechanism for the thermal acclimatization of T. KS-1. The beta-subunit content of the natural chaperonin from cells grown at 90 degrees C was higher than that at 80 degrees C. The optimum temperature for ATPase activity of the natural chaperonins was 80-90 degrees C, whereas that for alpha16mer and beta16mer was 60 degrees C and over 90 degrees C respectively. Judging from the ATPase activity, beta16mer was more thermostable than alpha16mer. The thermostabilities of the natural chaperonins were intermediate between alpha16mer and beta16mer, whereas the natural chaperonin with a higher beta-subunit content was more stable than that with a lower beta-subunit content. Native polyacrylamide gel electrophoresis (PAGE) revealed that the chaperonin oligomers thermally dissociated to their ATPase-inactive monomers. The thermal denaturation process monitored by circular dichroism showed that the free beta-subunit was more stable than the free alpha-subunit, and that the secondary structure of the chaperonin monomer in the oligomer was more stable than that in the free monomer. These results suggest that the structure of these subunits was stabilized in the oligomer, and that an increase in the beta-subunit content conferred higher thermostability to the natural hetero-oligomeric chaperonin.     

Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase--an extremely heat stable enzyme with proofreading activity

We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3'----5' proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30 x 10(-6). These values were 5-10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15-35 x 10(-4) being 2-4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis in vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.     

Extremely thermostable glutamate dehydrogenase (GDH) from the freshwater archaeon Thermococcus waiotapuensis: cloning and comparison with two marine hyperthermophilic GDHs

Glutamate dehydrogenases (GDHs) from fresh-water and marine hyperthermophilic Archaea were compared with respect to their responses to different salt concentrations. A gene encoding GDH from the terrestrial hyperthermophilic archaeon Thermococcus waiotapuensis (Twaio) was cloned, sequenced, and expressed at a high level in Escherichia coli. The deduced amino acid sequence, which consists of 418 amino acid residues, revealed a high degree of similarity with GDHs from related marine strains such as Thermococcus litoralis (Tl) and Pyrococcus furiosus (Pfu). Recombinant Twaio GDH was purified 27-fold to homogeneity. The enzyme is hexameric with a molecular weight of 259,000. The effects of several salts (KCl, CaCl, MgSO4), temperature, and pH on enzyme activity were determined and compared in three hyperthermophilic GDHs, including T. waiotapuensis, and GDHs from two marine species, T. litoralis and P. furiosus. Kinetic studies suggested a biosynthetic role for the nicotinamide adenine dinucleotide phosphate- (NADP-) specific Twaio GDH in the cell. Interestingly, Twaio GDH revealed no salt responses, whereas the two marine GDHs showed substantial enhancement of activity as well as thermostability at increasing salt concentrations. Because electrostatic interactions between charged amino acid residues are thought to be a key feature of structural integrity and thermostability in hyperthermophilic GDHs, salt availability and its effects on marine enzymes could partially explain a higher thermal stability in marine species than in phyletically related fresh-water species.     

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     

Genome sequence of the model hyperthermophilic archaeon Thermococcus litoralis NS-C

The hyperthermophilic archaeon Thermococcus litoralis strain NS-C, first isolated in 1985, has been a foundational organism for archaeal research in biocatalysis, DNA replication, metabolism, and the discovery of inteins. Here, we present the genome sequence of T. litoralis with a focus on the replication machinery and inteins.     

Characterization of Amylolytic Enzymes, Having Both alpha-1,4 and alpha-1,6 Hydrolytic Activity, from the Thermophilic Archaea Pyrococcus furiosus and Thermococcus litoralis

Extracellular pullulanases were purified from cell-free culture supernatants of the marine thermophilic archaea Thermococcus litoralis (optimal growth temperature, 90 degrees C) and Pyrococcus furiosus (optimal growth temperature, 98 degrees C). The molecular mass of the T. litoralis enzyme was estimated at 119,000 Da by electrophoresis, while the P. furiosus enzyme exhibited a molecular mass of 110,000 Da under the same conditions. Both enzymes tested positive for bound sugar by the periodic acid-Schiff technique and are therefore glycoproteins. The thermoactivity and thermostability of both enzymes were enhanced in the presence of 5 mM Ca, and under these conditions, enzyme activity could be measured at temperatures of up to 130 to 140 degrees C. The addition of Ca also affected substrate binding, as evidenced by a decrease in K(m) for both enzymes when assayed in the presence of this metal. Each of these enzymes was able to hydrolyze, in addition to the alpha-1,6 linkages in pullulan, alpha-1,4 linkages in amylose and soluble starch. Neither enzyme possessed activity against maltohexaose or other smaller alpha-1,4-linked oligosaccharides. The enzymes from T. litoralis and P. furiosus appear to represent highly thermostable amylopullulanases, versions of which have been isolated from less-thermophilic organisms. The identification of these enzymes further defines the saccharide-metabolizing systems possessed by these two organisms.     

Characterization of 2-ketoisovalerate ferredoxin oxidoreductase, a new and reversible coenzyme A-dependent enzyme involved in peptide fermentation by hyperthermophilic archaea.

Cell extracts of the proteolytic and hyperthermophilic archaea Thermococcus litoralis, Thermococcus sp. strain ES-1, Pyrococcus furiosus, and Pyrococcus sp. strain ES-4 contain an enzyme which catalyzes the coenzyme A-dependent oxidation of branched-chain 2-ketoacids coupled to the reduction of viologen dyes or ferredoxin. This enzyme, termed VOR (for keto-valine-ferredoxin oxidoreductase), has been purified from all four organisms. All four VORs comprise four different subunits and show amino-terminal sequence homology. T. litoralis VOR has an M(r) of ca. 230,000, with subunit M(r) values of 47,000 (alpha), 34,000 (beta), 23,000 (gamma), and 13,000 (delta). It contains about 11 iron and 12 acid-labile sulfide atoms and 13 cysteine residues per heterotetramer (alpha beta gamma delta), but thiamine pyrophosphate, which is required for catalytic activity, was lost during purification. The most efficient substrates (kcat/Km > 1.0 microM-1 s-1; Km < 100 microM) for the enzyme were the 2-ketoacid derivatives of valine, leucine, isoleucine, and methionine, while pyruvate and aryl pyruvates were very poor substrates (kcat/Km < 0.2 microM-1 s-1) and 2-ketoglutarate was not utilized. T. litoralis VOR also functioned as a 2-ketoisovalerate synthase at 85 degrees C, producing 2-ketoisovalerate and coenzyme A from isobutyryl-coenzyme A (apparent Km, 250 microM) and CO2 (apparent Km, 48 mM) with reduced viologen as the electron donor. The rate of 2-ketoisovalerate synthesis was about 5% of the rate of 2-ketoisovalerate oxidation. The optimum pH for both reactions was 7.0. A mechanism for 2-ketoisovalerate oxidation based on data from substrate-induced electron paramagnetic resonance spectra is proposed, and the physiological role of VOR is discussed.     

Evidence for extensive gene flow and Thermotoga subpopulations in subsurface and marine environments

Oil reservoirs represent a nutrient-rich ecological niche of the deep biosphere. Although most oil reservoirs are occupied by microbial populations, when and how the microbes colonized these environments remains unanswered. To address this question, we compared 11 genomes of Thermotoga maritima-like hyperthermophilic bacteria from two environment types: subsurface oil reservoirs in the North Sea and Japan, and marine sites located in the Kuril Islands, Italy and the Azores. We complemented our genomes with Thermotoga DNA from publicly available subsurface metagenomes from North America and Australia. Our analysis revealed complex non-bifurcating evolutionary history of the isolates'' genomes, suggesting high amounts of gene flow across all sampled locations, a conjecture supported by numerous recombination events. Genomes from the same type of environment tend to be more similar, and have exchanged more genes with each other than with geographically close isolates from different types of environments. Hence, Thermotoga populations of oil reservoirs do not appear isolated, a requirement of the ‘burial and isolation'' hypothesis, under which reservoir bacteria are descendants of the isolated communities buried with sediments that over time became oil reservoirs. Instead, our analysis supports a more complex view, where bacteria from subsurface and marine populations have been continuously migrating into the oil reservoirs and influencing their genetic composition. The Thermotoga spp. in the oil reservoirs in the North Sea and Japan probably entered the reservoirs shortly after they were formed. An Australian oil reservoir, on the other hand, was likely colonized very recently, perhaps during human reservoir development.     

Molecular characterization and heterologous expression of hypCD, the first two [NiFe] hydrogenase accessory genes of Thermococcus litoralis

The hypCD genes, encoding the counterparts of mesophilic proteins involved in the maturation of [NiFe] hydrogenases, were isolated from the hyperthermophilic archaeon Thermococcus litoralis. The deduced gene products showed 30-40% identity to the corresponding mesophilic proteins. HypC and HypD were synthesized by the T7 expression system. Heterologous complementation experiments were done in Escherichia coli and Ralstonia eutropha strains lacking functionally active hypC and hypD genes. Only the cytoplasmic hydrogenase of R. eutropha could be processed by HypD from T. litoralis. This was the first demonstration of mesophilic hydrogenase processing using a hyperthermophilic archaeal accessory protein to produce an active enzyme.     

A barophilic response by two hyperthermophilic, hydrothermal vent Archaea: An upward shift in the optimal temperature and acceleration of growth rate at supra-optimal temperatures by elevated pressure

Abstract The influence of elevated hydrostatic pressure on the growth rates of two hyperthermophilic Archaea isolated from hydrothermal vent environments (strains ES1 and ES4) was investigated over their entire temperature range for growth. Thermococcus celer , a shallow marine hyperthermophile was included in the study for comparative purposes. For one strain (ES4), the pressure at the site of collection (22 MPa) caused an upward shift in the optimal growth temperature of about 6°C compared to growth at 1 MPa. Although the optimal temperature for ES1 was unaffected by 22 MPa, elevated pressure stimulated the growth rate at supra-optimal temperatures. The temperature range for growth for both organisms was extended upward 2°C at 22 MPa pressure. For both strains 22 MPa had little effect on growth rates at sub-optimal temperatures. Growth was observed at pressures as high as 89 MPa for ES1 and 67 MPa for ES4, but with these higher pressures the temperature range for growth was narrowed, and the optimal temperature was shifted downward. Growth of Thermococcus celer was slightly stimulated by 22 MPa at its reported optimal temperature of 88°C, but was inhibited by higher pressure.