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PubMed1999

X-ray structure of pyrrolidone carboxyl peptidase from the hyperthermophilic archaeon Thermococcus litoralis.

BACKGROUND: Pyrrolidone carboxyl peptidases (pcps) are a group of exopeptidases responsible for the hydrolysis of N-terminal pyroglutamate residues from peptides and proteins. The bacterial and archaeal pcps are members of a conserved family of cysteine proteases. The pcp from the hyperthermophilic archaeon Thermococcus litoralis is more thermostable than the bacterial enzymes with which it has up to 40% sequence identity. The pcp activity in archaea and eubacteria is proposed to be involved in detoxification processes and in nutrient metabolism; eukaryotic counterparts of the enzyme are involved in the processing of biologically active peptides. RESULTS: The crystal structure of pcp has been determined by multiple isomorphous replacement techniques at 1.73 A resolution and refined to an R factor of 18.7% (Rfree = 21.4%). The enzyme is a homotetramer of single open alpha/beta domain subunits, with a prominent hydrophobic core formed from loops coming together from each monomer. The active-site residues have been identified as a Cys143-His167-Glu80 catalytic triad. Structural homology to enzymes of different specificity and mechanism has been identified. CONCLUSIONS: The Thermococcus pcp has no sequence or structural homology with other members of the cysteine protease family. It does, however, show considerable similarities to other hydrolytic enzymes of widely varying substrate specificity and mechanism, suggesting that they are the products of divergent evolution from a common ancestor. The enhanced thermostability of the T. litoralis pcp may arise from hydrophobic interactions between the subunits and the presence of intersubunit disulphide bridges.

Animals↗

PubMed2001

FK506-binding protein of the hyperthermophilic archaeum, Thermococcus sp. KS-1, a cold-shock-inducible peptidyl-prolyl cis-trans isomerase with activities to trap and refold denatured proteins.

The FK506 (tacrolimus)-binding protein (FKBP) type peptidyl-prolyl cis-trans isomerase (PPIase) in the hyperthermophilic archaeum Thermococcus sp. KS-1 was shown to be induced by temperature downshift to growth temperatures lower than the optimum. This PPIase (TcFKBP18) showed chaperone-like protein refolding activity in addition to PPIase activity in vitro. It refolded unfolded citrate synthase (CS) and increased the yield of the refolded protein. At a molar ratio of 15:1 ([TcFKBP18] to [CS]) in the refolding mixture, the recovered yield of folded CS was maximal at 62%, whereas that of spontaneous refolding was 11%. Increasing FKBP above a 15:1 ratio decreased the final yield, whereas the aggregation of unfolded CS was suppressed. A cross-linking analysis showed the formation of a complex between TcFKBP18 and unfolded CS (1:1 complex) at molar ratios of 3:1 to 15:1. However, molar ratios of 15:1 or 60:1 induced the binding of multiple FKBP molecules to an unfolded CS molecule (multimeric complex). Disrupting hydrophobic interaction by adding ethylene glycol at a molar ratio of 60:1 ([TcFKBP18] to [CS]) suppressed the formation of this multimeric complex, simultaneously enhancing CS refolding. FK506 also suppressed the formation of the multimeric complex while increasing the chaperone-like activity. These results suggest that the hydrophobic region of TcFKBP18, probably the FK506-binding pocket, was important for the interaction with unfolded proteins. No cross-linked product was detected between TcFKBP18 and native dimeric CS. TcFKBP18 probably traps the unfolded protein, then refolds and releases it in a native form. This FKBP might be important at growth temperatures lower than the optimum in Thermococcus sp. KS-1 cells.

Citrate (si)-Synthase↗

PubMed2002

TrmB, a sugar-specific transcriptional regulator of the trehalose/maltose ABC transporter from the hyperthermophilic archaeon Thermococcus litoralis.

We report the characterization of TrmB, a protein of 38,800 apparent molecular weight, that is involved in the maltose-specific regulation of a gene cluster in Thermococcus litoralis, malE malF malG orf trmB malK, encoding a binding protein-dependent ABC transporter for trehalose and maltose. TrmB binds maltose and trehalose half-maximally at 20 microm and 0.5 mm sugar concentration, respectively. Binding of maltose but not of trehalose showed indications of sigmoidality and quenched the intrinsic tryptophan fluorescence by 15%, indicating a conformational change on maltose binding. TrmB causes a shift in electrophoretic mobility of DNA fragments harboring the promoter and upstream regulatory motif identified by footprinting. Band shifting by TrmB can be prevented by maltose. In vitro transcription assays with purified components from Pyrococcus furiosus have been established to show pmalE promoter-dependent transcription at 80 degrees C. TrmB specifically inhibits transcription, and this inhibition is counteracted by maltose and trehalose. These data characterize TrmB as a maltose-specific repressor for the trehalose/maltose transport operon of Thermococcus litoralis.

ATP-Binding Cassette Transporters↗

PubMed1999

Thermococcus barophilus sp. nov., a new barophilic and hyperthermophilic archaeon isolated under high hydrostatic pressure from a deep-sea hydrothermal vent.

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.

Atlantic Ocean↗

PubMed2000

Thermococcus aegaeicus sp. nov. and Staphylothermus hellenicus sp. nov., two novel hyperthermophilic archaea isolated from geothermally heated vents off Palaeochori Bay, Milos, Greece.

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).

Base Composition↗

PubMed2004

Bioinformatics of the glycoside hydrolase family 57 and identification of catalytic residues in amylopullulanase from Thermococcus hydrothermalis.

Fifty-nine amino acid sequences belonging to family 57 (GH-57) of the glycoside hydrolases were collected using the CAZy server, Pfam database and blast tools. Owing to the sequence heterogeneity of the GH-57 members, sequence alignments were performed using mainly manual methods. Likewise, five conserved regions were identified, which are postulated to be GH-57 consensus motifs. In the 659 amino acid-long 4-alpha-glucanotransferase from Thermococcus litoralis, these motifs correspond to 13_HQP (region I), 76_GQLEIV (region II), 120_WLTERV (region III), 212_HDDGEKFGVW (region IV), and 350_AQCNDAYWH (region V). The third and fourth conserved regions contain the catalytic nucleophile and the proton donor, respectively. Based on our sequence alignment, residues Glu291 and Asp394 were proposed as the nucleophile and proton donor, respectively, in a GH-57 amylopullulanase from Thermococcus hydrothermalis. To validate this prediction, site-directed mutagenesis was performed. The results of this work reveal that both residues are critical for the pullulanolytic and amylolytic activities of the amylopullulanase. Therefore, these data support the prediction and strongly suggest that the bifunctionality of the amylopullulanase is determined by a single catalytic centre. Despite this positive validation, our alignment also reveals that certain GH-57 members do not possess the Glu and Asp corresponding to the predicted GH-57 catalytic residues. However, the sequences concerned by this anomaly encode putative proteins for which no biochemical or enzymatic data are yet available. Finally, the evolutionary trees generated for GH-57 reveal that the entire family can be divided into several subfamilies that may reflect the different enzyme specificities.

Amino Acid Sequence↗

PubMed2000

Cloning and expression of an alpha-amylase encoding gene from the hyperthermophilic archaebacterium Thermococcus hydrothermalis and biochemical characterisation of the recombinant enzyme.

An alpha-amylase encoding gene from the extremely thermophilic Archaea Thermococcus hydrothermalis was cloned and expressed in Escherichia coli. The encoded alpha-amylase possesses molecular characteristics specific to the Archaea, especially from Pyrococcus species, with biochemical characteristics of the alpha-amylases from Thermococcus. The gene is 1374 bp long and encodes a protein of 457 amino acids composed of a 22 amino acid putative signal peptide and a 435 amino acid mature protein (calculated molecular mass 49236 Da). The T. hydrothermalis recombinant alpha-amylase is optimally active at 75-85 degrees C and at pH 5.0-5.5.

Amino Acid Sequence↗

PubMed2006

Cell structure degradation in Escherichia coli and Thermococcus sp. strain Tc-1-95 associated with thermal death resulting from brief heat treatment.

The thermal death mechanism of microorganisms when heated at lethally high temperatures is still not fully understood. In this study, we examined the relationship between thermal death and degradation of the cell structure in the mesophilic bacterium Escherichia coli strain W3110 and the hyperthermophilic archaeon Thermococcus sp. strain Tc-1-95. By heating the microorganisms at lethally high temperatures only briefly (1.5 s duration) in a flow-type apparatus, we studied the microbial cells at very early and critical stages of the thermal death process. For E. coli, it was found that the loss of viability was not associated with thermal damage to the cell envelope. Deformation of the nucleoid was observed. These results suggest that the thermal death of E. coli is attributed to thermal denaturation or degradation of cytoplasmic molecules. On the other hand, the thermal death of Thermococcus sp. strain Tc-1-95 was strongly associated with rupture of the cell envelope. Furthermore, massive deformation of the S-layer with lethal thermal stress was observed. These results demonstrate that the thermal deaths of the two microorganisms investigated proceed via very different mechanisms. The contrast can be attributed to the difference in their cell envelope structures.

Cell Membrane↗

PubMed2001

Polyamines of the hyperthermophilic archaebacteria belonging to the genera Thermococcus and Methanothermus and two new genera Caldivirga and Palaeococcus.

Cellular polyamines of eight new thermophilic archaebacteria were investigated to determine the chemotaxonomic significance of polyamine distribution profiles. Hyperthermoacidophilic Caldivirga maquilingensis belonging to the family Thermoproteaceae of the Crenarchaeota have a unique polyamine profile comprising spermidine, norspermidine and norspermine as the major polyamines. Within the order Thermococcales of the Euryarchaeota, the major polyamines of an extremely thermophilic terrestrial species of Thermococcus, T. zilligii, were spermidine and agmatine, whereas hyperthermophilic submarine species of Thermococcus and hyperthermophilic submarine Palaeococcus ferrophilus contained a quaternary branched penta-amine, N4-bis(aminopropyl)spermidine, as a major polyamine. A hyperthermophilic methanogen, Methanothermus sociabilis, belonging to Euryarchaeota, contained spermidine and spermine as the major polyamine.

Polyamines↗

PubMedDate not supplied

[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.

Base Sequence↗

PubMed1997

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.

Archaea↗

PubMed1996

Thermococcus fumicolans sp. nov., a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent in the north Fiji Basin.

An extremely thermophilic archaeon, strain ST557T (T = type strain), was isolated from a deep-sea hydrothermal vent in the North Fiji Basin. This strain is a strictly anaerbic coccus whose cells are about 0.8 to 2 microns in diameter. The optimum temperature, pH and sea salt concentration for growth are 85 degrees C, 8.5, and 20 to 40 g/liter, respectively. Strain ST557T grows preferentially in the presence of elemental sulfur on proteinaceous substrates and on a mixture of 20 amino acids. It grows slowly on pyruvate and maltose. Growth is inhibited by rifampin. The DNA G + C content is 54 to 55 mol%. Sequencing of the 16S rRNA gene revealed that strain ST557T belongs to the genus Thermococcus. We propose that this organism should be placed in a new species, Thermococcus fumicolans.

Archaea↗

PubMed1997

Thermococcus hydrothermalis sp. nov., a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent.

An extremely thermophilic archaeon, strain AL662T, was isolated from a deep-sea hydrothermal vent located on the East Pacific Rise at a latitude of 21 degrees N. This strain is a strictly anaerobic coccus, and its cells range from 0.8 to 2 microns in diameter. The optimum temperature, pH, and Sea Salt concentration for growth are 85 degrees C, 6, and 20 to 40 g/liter, respectively. Strain AL662T grows preferentially on proteolysis products, on a mixture of 20 amino acids, and on maltose in the presence of elemental sulfur. The membrane lipids consist of di- and tetraether glycerol lipids. The DNA G+C content is 58 mol%. Sequencing of the 16S rRNA gene showed that strain AL662T belongs to the genus Thermococcus. On the basis of hybridization results, we propose that this strain should be placed in a new species, Thermococcus hydrothermalis.

Acids↗

PubMed2001

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.

Journal Article↗

PubMed1995

A hyperthermophilic sulfur-reducing archaebacterium, Thermococcus sp. DT1331, isolated from a deep-sea hydrothermal vent.

A hyperthermophilic archaebacterium was isolated from a deep-sea black smoker chimney (depth, 760 m) at the Minami-ensei Knoll (28 degrees 23'N, 127 degrees 38'E). The strain, designated DT1331, was a coccoid shaped bacterium about 0.5 to 1.0 microns in diameter. The cells were surrounded by a cell envelope. The temperature for growth was between 55 degrees C and 93 degrees C with an optimum 80 degrees C. The growth occurred from pH 4.5 to 8.5 and the optimum pH was 6.0. DT1331 required 1% to 5% NaCl for growth and cell lysis was observed below 1% NaCl concentration. The strain was an anaerobic chemoorganotroph requiring elemental sulfur obligately. Organic substrates used included tryptone, peptone, soytone, casein, gelatin, and yeast extract. Under the optimal conditions, DT1331 had a generation time of 50 min and could reach densities of about 1.5 x 10(8) cells/ml. DT1331 was resistant to ampicillin, chloramphenicol, erythromycin, kanamycin, streptomycin, and tetracycline, which was one of the common characteristics of archaebacteria. The G+C content of DT1331 was 52.3 mol%. Analysis of the 16S rRNA gene by restriction enzymes coincided with those of Thermococcus celer, indicating that this strain belonged to the genus Thermococcus.

Anti-Bacterial Agents↗

PubMed2003

Cellular polyamines of the acidophilic, thermophilic and thermoacidophilic archaebacteria, Acidilobus, Ferroplasma, Pyrobaculum, Pyrococcus, Staphylothermus, Thermococcus, Thermodiscus and Vulcanisaeta.

Cellular polyamines of newly isolated acidophilic, thermophilic and thermoacidophilic archaebacteria were investigated for the chemotaxonomic significance of polyamine distribution profiles. In addition to spermidine, spermine and agmatine, a quaternary branched penta-amine, N(4)-bis(aminopropyl)spermidine, was found in thermophilic Thermococcus waiotapuensis, Thermococcus aegaeus and Pyrococcus glycovorans belonging to the order Thermococcales. An acidophilic euryarchaeon, Ferroplasma acidiphilum located in the order Thermoplasmatales, contained spermidine and agmatine. Norspermidine, spermidine, norspermine and spermine were found in thermoacidophilic Acidilobus aceticus and thermophilic Thermodiscus maritimus located in the order Desulfurococcales, and in thermophilic Pyrobaculum arsenaticum, Pyrobaculum oguniense, Vulcanisaeta distributa and Vulcanisaeta souniana belonging to the order Thermoproteales; however, the four genera differ on their tetra- and penta-amine levels. Thermophilic Staphylothermus hellenicus belonging to Desulfurococcales contained caldopentamine, caldohexamine and N1-acetylcaldopentamine in addition to norspermidine, spermidine and norspermine. This is the first report on the occurrence of acetylated penta-amine in nature.

Chromatography, High Pressure Liquid↗

PubMed1995

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 degrees C with an optimum around 85 degrees 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 degrees ) 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).

Archaea↗

PubMed2000

Effect of carbon and nitrogen sources on growth dynamics and exopolysaccharide production for the hyperthermophilic archaeon Thermococcus litoralis and bacterium Thermotoga maritima.

Batch and continuous cultures were used to compare specific physiological features of the hyperthermophilic archaeon, Thermococcus litoralis (T(opt) of 85 degrees to 88 degrees C), to another fermentative hyperthermophile that reduces S degrees facultatively, that is, the bacterium Thermotoga maritima (T(opt) of 80 degrees to 85 degrees C). Under nutritionally optimal conditions, these two hyperthermophiles had similar growth yields on maltose and similar cell formula weights based on elemental analysis: CH(1.7)O(0. 7)N(0.2)S(0.006) for T. litoralis and CH(1.6)O(0.6)N(0.2)S(0.005) for T. maritima. However, they differed with respect to nitrogen source, fermentation product patterns, and propensity to form exopolysaccharides (EPS). T. litoralis could be cultured in the absence or presence of maltose on an amino acid-containing defined medium in which amino acids served as the sole nitrogen source. T. maritima, on the other hand, did not utilize amino acids as carbon, energy, or nitrogen sources, and could be grown in a similar defined medium only when supplemented with maltose and ammonium chloride. Not only was T. litoralis unable to utilize NH(4)Cl as a nitrogen source, its growth was inhibited at certain levels. At 1 g/L ( approximately 20 mM) NH(4)Cl, the maximum growth yield (Y(x/s(max))) for T. litoralis was reduced to 13 g cells dry weight (CDW)/mol glucose from 40 g CDW/mol glucose in media lacking NH(4)Cl. Alanine production increased with increasing NH(4)Cl concentrations and was most pronounced if growth on NH(4)Cl was carried out in an 80% H(2) atmosphere. In T. maritima cultures, which would not grow in an 80% H(2) atmosphere, alanine and EPS were produced at much lower levels, which did not change with NH(4)Cl concentration. EPS production rose sharply at high dilution rates for both organisms, such that maltose utilization plots were biphasic. Wall growth effects were also noted, because cultures failed to wash out at dilution rates significantly above maximum growth rates determined from batch growth experiments. This study illustrates the importance of effective cultivation methods for addressing physiological issues related to the growth of hyperthermophilic heterotrophs.

Amino Acids↗