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     

Identification of a novel acetylated form of branched-chain polyamine from a hyperthermophilic archaeon Thermococcus kodakarensis

Long/branched-chain polyamines are unique polycations found in thermophiles. The hyperthermophilic archaeon Thermococcus kodakarensis contains spermidine and a branched-chain polyamine, N⁴-bis(aminopropyl)spermidine, as major polyamines. The metabolic pathways associated with branched-chain polyamines remain unknown. Here, we used gas chromatography and liquid chromatography-tandem mass spectrometry analyses to identify a new acetylated polyamine, N⁴-bis(aminopropyl)-N¹-acetylspermidine, from T. kodakarensis; this polyamine was not found in other micro-organisms. The amounts of branched-chain polyamine and its acetylated form increased with temperature, indicating that branched-chain polyamines are important for growth at higher temperatures. The amount of quaternary acetylated polyamine produced was associated with the amount of N⁴-bis(aminopropyl)spermidine in the cell. The ratio of acetylated to non-acetylated forms was higher in the stationary phase than in the logarithmic growth phase under high-temperature stress condition.     

深海热液区超嗜热古菌Thermococcus sp. TVG2的培养分离与鉴定

【目的】培养分离大西洋脊热液区的超嗜热古菌,为进一步认识该生态系统中的物种及其特点奠定基础。【方法】将大西洋脊热液区海水样品用YTSV培养基富集培养,选取其中富集效果最佳的TVG2培养物用减绝稀释法分离纯化。对所分离菌株进行形态、生理生化特征等分析,并通过分子生物学手段对其进行初步鉴定。【结果】菌株TVG2属于超嗜热厌氧球菌,直径约1.0 μm;生长温度范围50?88 °C,最适生长温度82 °C;生长pH范围为5.0?9.0,最适生长pH 6.5;生长NaCl浓度为1.0%?4.0% (质量体积比),最适生长浓度为2.5%;元素硫可显著提高菌株TVG2的生物量,但非生长必需;丙酮酸钠能显著促进该菌株生长,但葡萄糖对其生长则有抑制作用。根据其形态特征、生理生化特性及16S rRNA基因序列分析,确定菌株TVG2属于热球菌属。【结论】用YTSV培养基从大西洋脊热液区样品中分离获得超嗜热厌氧菌株TVG2,并确定其为Thermococcus属成员,命名为Thermococcus sp. TVG2。     

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     

Characterization of DNA polymerase from the hyperthermophilic archaeon Thermococcus marinus and its application to PCR

The family B DNA polymerase gene from the archaeon Thermococcus marinus (Tma) contains a long open reading frame of 3,939 bp that encodes 1,312 amino acid residues. The gene is split by one intervening sequence that forms a continuous open reading frame with the two polymerase exteins. In this study, the Tma DNA polymerase gene both with (precursor form) and without (mature form) its intein was expressed in Escherichia coli, purified by heat treatment and HiTrap™ Heparin HP column chromatography and characterized. Primary sequence analysis of the mature Tma polymerase showed high sequence identity with DNA polymerases in the genus Thermococcus. The expressed precursor form was easily spliced during purification steps. The molecular mass of the purified Tma DNA polymerases is about 90 kDa, as estimated by SDS-PAGE. Both Tma DNA polymerases showed the same properties. PCR performed with this enzyme was found to be optimal in the presence of 50 mM Tris–HCl (pH 8.4), 40 mM KCl, 12.5 mM (NH₄)₂SO_4, 2 mM MgCl_2, 0.05% Triton X-100 and 0.0075% BSA. Furthermore, long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tma DNA polymerases (Tma plus DNA polymerase).     

Recovery of ionizing-radiation damage after high doses of gamma ray in the hyperthermophilic archaeon Thermococcus gammatolerans

The recently discovered hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans is of great interest to compare and contrast the impact of its physiology on radioresistance and its ability to repair damaged chromosomes after exposure to gamma irradiation with radioresistant bacteria. We showed that, in contrast to other organisms, cell survival was not modified by the cellular growth phase under optimal growth conditions but nutrient-limited conditions did affect the T. gammatolerans radioresistance. We determined the first kinetics of damaged DNA recovery in an archaeon after exposure to massive doses of gamma irradiation and compared the efficiency of chromosomal DNA repair according to the cellular growth phase, nutrient availability and culture conditions. Chromosomal DNA repair kinetics showed that stationary phase cells reconstitute disrupted chromosomes more rapidly than exponential phase cells. Our data also revealed that this radioresistant archaeon was proficient to reconstitute shattered chromosomes either slowly or rapidly without any loss of viability. These results suggest that rapid DNA repair is not required for the extreme radioresistance of T. gammatolerans. Angels Tapias and Christophe Leplat contributed equally to this work.     

On archaeal homologs of the human RNase P proteins Pop5 and Rpp30 in the hyperthermophilic archaeon Thermococcus kodakarensis

The ribonuclease P (RNase P) proteins TkoPop5 and TkoRpp30, homologs of human Pop5 and Rpp30, respectively, in the hyperthermophilic archaeon Thermococcus kodakarensis were prepared and characterized with respect to pre-tRNA cleavage activity using the reconstitution system of the well-studied Pyrococcus horikoshii RNase P. The reconstituted particle containing TkoPop5 in place of the P. horikoshii counterpart PhoPop5 retained pre-tRNA cleavage activity comparable to that of the reconstituted P. horikoshii RNase P, while that containing TkoRpp30 instead of its corresponding protein PhoRpp30 had slightly lower activity than the P. horikoshii RNase P. Moreover, we determined crystal structures of TkoRpp30 alone and in complex with TkoPop5. Like their P. horikoshii counterparts, whose structures were solved previously, TkoRpp30 and TkoPop5 fold into TIM barrel and RRM-like fold, respectively. This finding demonstrates that RNase P proteins in T. kodakarensis and P. horikoshii are interchangeable and that their three-dimensional structures are highly conserved.     

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     

Enzymatic and structural characterization of type II isopentenyl diphosphate isomerase from hyperthermophilic archaeon Thermococcus kodakaraensis

Enzymatic and thermodynamic characteristics of type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase (Tk-IDI) from Thermococcus kodakaraensis, which catalyzes the interconversion of IPP and DMAPP, were examined. FMN was tightly bound to Tk-IDI, and the enzyme required NADPH and Mg2+ for the isomerization in both directions. The melting temperature (Tm), the change of enthalpy (deltaH(m)), and the heat capacity change (deltaC(p)) of Tk-IDI were 88.0 degrees C, 444 kJ mol(-1), and 13.2 kJ mol(-1) K(-1), respectively, indicating that Tk-IDI is fairly thermostable. Kinetic parameters dramatically changed when the temperature crossed 80 degrees C even though its native overall structure was stably maintained up to 90 degrees C, suggesting that local conformational change would occur around 80 degrees C. This speculation was supported by the result of the circular dichroism analysis that showed the shift of the alpha-helical content occurred at 80 degrees C.     

Culturability and survival of an extreme thermophile isolated from deep-sea hydrothermal vents

The culturability of a strictly anaerobic, extremely thermophilic archaeon, Thermococcus peptonophilus (optimal growth temperature: 85° C), was studied during survival stages at various temperatures (98, 85, 70, and 4° C). Total cell number (determined by DAPI staining), active cells (rhodamine-stained cells), and culturable cells (using most-probable-number) were counted over time. The number of culturable cells decreased under each condition tested. The total number of cells significantly decreased only at temperatures close to the maximum for growth (98° C); at this temperature, the cells spontaneously lysed. Our results suggested that survival at 4° C in oxygenated waters might be a mechanism for the dispersion of extreme thermophiles in the ocean. In addition, we proved the existence of T. peptonophilus cells in several physiological states: culturable cells, active non-culturable cells, inactive non-culturable cells, and dead cells. Cell death was caused by cellular lysis. Received: 5 February 1996 / Accepted: 16 April 1996     

Characterization of a thermophilic DNA ligase from the archaeon Thermococcus fumicolans

A PCR protocol was used to identify and sequence a gene encoding a DNA ligase from Thermococcus fumicolans (Tfu). The recombinant enzyme, expressed in Escherichia coli BL21(DE3) pLysS, was purified to homogeneity and characterized. The optimum temperature and pH of Tfu DNA ligase were 65 degrees C and 7.0, respectively. The optimum concentration of MgCl2, which is indispensable for the enzyme activity, was 2 mM. We showed that Tfu DNA ligase displayed nick joining and blunt-end ligation activity using either ATP or NAD+, as a cofactor. In addition, our results would suggest that Tfu DNA ligase is likely to use the same catalytic residues with the two cofactors. The ability for DNA ligases, to use either ATP or NAD+, as a cofactor, appears to be specific of DNA ligases from Thermococcales, an order of hyperthermophilic microorganisms that belongs to the euryarchaeotal branch of the archaea domain.     

Cloning, expression, and characterization of pyrrolidone carboxyl peptidase from the archaeon Thermococcus litoralis

The gene encoding pyrrolidone carboxyl peptidase (Pcp) has been cloned from the hyperthermophilic archaeon Thermococcus litoralis. The recombinant enzyme has been expressed in Escherichia coli, purified, and char-acterized. The T. litoralis Pcp demonstrates strong sequence homology to previously characterized bacterial Pcps. Some investigations have been carried out on enzyme substrate specificity and stability. Received: July 4, 2000 / Accepted: July 21, 2000     

1H NMR investigation of the secondary structure, tertiary contacts and cluster environment of the four-iron ferredoxin from the hyperthermophilic archaeon Thermococcus litoralis

Summary The solution molecular structure of the four-iron ferredoxin (Fd) from the hyperthermophilic archaeon Thermococcus litoralis (Tl) has been investigated by ¹H NMR spectroscopy. TOCSY and NOESY experiments in H₂O, tailored to detect both weakly and strongly relaxed resonances, together with steady-state NOEs in both H₂O and D₂O, allowed the identification of 58 of the 59 residues, with one residue near the paramagnetic center undetected. It is shown that the contact shifted and strongly relaxed signals for all four cysteines ligated to the paramagnetic cluster can be assigned by standard backbone connectivities that do not require any assumptions about the tertiary structure. Secondary structural elements identified in Tl Fd are a three-stranded antiparallel -strand involving the termini of the protein, a double -strand (also antiparallel), two -helices and four turns. The existence of a disulfide bridge between the nonligated cysteines is also proposed. Dipolar contacts observed in the NOESY maps and by steady-state NOEs between the ligated cysteines and the diamagnetic protein matrix indicate that the overall folding pattern of Tl Fd is very similar to that of the 3Fe ferredoxin from the mesophilic bacterium Desulfovibrio gigas [Kissinger et al. (1991) J. Mol. Biol., 219, 693–723]. The influence of the paramagnetism of the cluster on the relaxation properties of the proton signals of nonligated residues near the cluster, as well as on the ligated cysteines, correlates well with the proximity to the cluster iron(s), as predicted from the crystal structures for homologous protons of other single-cluster ferredoxins. Finally, the potential role of the various identified structural factors in contributing to the hyperthermostability of this protein is discussed.Abbreviations Fd ferredoxin - HiPiP high-potential iron-sulfur proteins - Dg Desulfovibrio gigas - Av Azotobacter vinelandii - Pf Pyrococcus furiosus - Tl Thermococcus litoralis - Pa Peptostreptococcus asaccharolyticus - Bt Bacillus thermoproteolyticus - Cp Clostridium pasteurianum - Ca Clostridium acidi urici - Da Desulfovibrio africanus - Tm Thermatoga maritima - NOE nuclear Overhauser effect - NOESY 2D NOE spectroscopy - MCOSY 2D magnitude correlation spectroscopy - TOCSY total correlation spectroscopy To whom correspondence should be addressed.     

Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans

The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30°C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170 kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity. Received: September 17, 1999 / Accepted: March 21, 2000     

Interaction of pressure, time and temperature of pressurization on viability loss of Listeria innocua

In this study, the combined effectiveness of pressures of 137.9–344.7MPa, temperatures of 25–50°C and exposure times of 5–15min on Listeria innocua viability in peptone solution is examined. The results showed that under the study conditions only the combination of 344.7MPa, 50°C and 9.1min can reduce the viability of this species by 7 logs, with a z value of 173.1MPa.     

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.     

Spectroscopic studies of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Thermococcus litoralis

Thermococcus litoralis (Tl) have been investigated by using the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. The results reveal a [Fe₄S₄]^2+,+ cluster (E _m=−368 mV) that undergoes redox cycling between an oxidized form with an S=0 ground state and a reduced form that exists as a pH- and medium-dependent mixture of S=3/2 (g=5.4; E/D=0.33) and S=1/2 (g=2.03, 1.93, 1.86) ground states, with the former dominating in the presence of 50% (v/v) glycerol. Three distinct types of W(V) EPR signals have been observed during dye-mediated redox titration of as-isolated Tl FOR. The initial resonance observed upon oxidation, termed the “low-potential” W(V) species (g=1.977, 1.898, 1.843), corresponds to approximately 25–30% of the total W and undergoes redox cycling between W(IV)/W(V) and W(V)/W(VI) states at physiologically relevant potentials (E _m=−335 and −280 mV, respectively). At higher potentials a minor “mid-potential” W(V) species, g=1.983, 1.956, 1.932, accounting for less than 5% of the total W, appears with a midpoint potential of −34 mV and persists up to at least +300 mV. At potentials above 0 mV, a major “high-potential” W(V) signal, g=1.981, 1.956, 1.883, accounting for 30–40% of the total W, appears at a midpoint potential of +184 mV. As-isolated samples of Tl FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na₂S under reducing conditions and the sulfide-activated Tl FOR was partially inactivated by cyanide. The spectroscopic and redox properties of the sulfide-activated Tl FOR are quite distinct from those of the as-isolated enzyme, with loss of the low-potential species and changes in both the mid-potential W(V) species (g=1.981, 1.950, 1.931; E _m=−265 mV) and high-potential W(V) species (g=1.981, 1.952, 1.895; E _m=+65 mV). Taken together, the W(V) species in sulfide-activated samples of Tl FOR maximally account for only 15% of the total W. Both types of high-potential W(V) species were lost upon incubation with cyanide and the sulfide-activated high-potential species is converted into the as-isolated high-potential species upon exposure to air. Structural models are proposed for each of the observed W(V) species and both types of mid-potential and high-potential species are proposed to be artifacts of ligand-based oxidation of W(VI) species. A W(VI) species with terminal sulfido or thiol ligands is proposed to be responsible for the catalytic activity in sulfide-activated samples of Tl FOR. Received: 9 September 1999 / Accepted: 17 February 2000