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

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

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

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

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

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

Thermococcus 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     

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.     

<Emphasis Type="Italic">Pyrolycus manusanus</Emphasis>, a new genus and species of deep-sea eelpout from a hydrothermal vent field in the Manus Basin,Papua New Guinea (Zoarcidae,Lycodinae)

A new genus and species of deep-sea zoarcid fish, Pyrolycus manusanus, is described on the basis of eight specimens collected from a hydrothermal vent field in the Manus Basin, Papua New Guinea, at depths of 1623–1971 m. This genus is distinguished from other scaleless eelpout genera in the subfamily Lycodinae by the following combination of characters: six suborbital bones; six to seven suborbital pores; flesh gelatinous; gill slit large, extending ventrally beyond pectoral fin base; three postorbital pores; single occipital pore; two to three pelvic fin rays; palatopterygoid series weak; vomer and palatine teeth present; pseudobranch, scales, lateral line absent; pyloric caeca present; 16–17 pectoral fin rays; 22–23 + 56–59 = 78–81 vertebrae. Received: August 5, 1999 / Revised: July 5, 2001 / Accepted: August 26, 2001     

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.     

Expansion of the geographic distribution of a novel lineage of epsilon-Proteobacteria to a hydrothermal vent site on the Southern East Pacific Rise

The diversity associated with a microbial mat sample collected from a deep-sea hydrothermal vent on the Southern East Pacific Rise was determined using a molecular phylogenetic approach based on the comparison of sequences from the small subunit ribosomal RNA gene (16S rDNA). The DNA was extracted from the sample and the 16S rDNA was amplified by PCR. Sixteen different phylotypes were identified by restriction fragment length polymorphism analysis; four phylotypes were later identified as putative chimeras. Analysis of the 16S rDNA sequences placed all the phylotypes within the Proteobacteria. The majority of the sequences (98%) were most closely related to a new clade of epsilon-Proteobacteria that were initially identified from an in situ growth chamber deployed on a deep-sea hydrothermal vent on the Mid-Atlantic Ridge in 1995. The similarity between phylotypes identified from Atlantic and Pacific deep-sea hydrothermal vent sites indicates that this new clade of Proteobacteria may be endemic to and widely distributed among deep-sea hydrothermal vents.     

History of discovery of the first hyperthermophiles

Hyperthermophiles, growing optimally at 80°C and above had been discovered in 1981. They represent the upper temperature border of life and are found within high temperature environments. In their basically anaerobic surroundings, they gain energy mainly by inorganic redox reactions. Within the phylogenetic tree, hyperthermophiles occupy all the short deep branches closest to the root. The earliest archaeal phylogenetic lineage is represented by the extremely tiny members of the novel kingdom of Nanoarchaeota.     

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.     

<Emphasis Type="Italic">Thermococcus marinus</Emphasis> sp. nov. and<Emphasis Type="Italic"> Thermococcus radiotolerans</Emphasis> sp. nov., two hyperthermophilic archaea from deep-sea hydrothermal vents that resist ionizing radiation

Enrichments for anaerobic, organotrophic hyperthermophiles were performed with hydrothermal chimney samples collected from the Mid-Atlantic Ridge at a depth of 3,550 m (23°22N, 44°57W) and the Guaymas Basin (27°01N, 111°24W) at a depth of 2,616 m. Positive enrichments were submitted to -irradiation at doses of 20 and 30 kGy. Two hyperthermophilic, anaerobic, sulfur-metabolizing archaea were isolated. Strain EJ1^T was isolated from chimney samples collected from the Mid-Atlantic Ridge after -irradiation at 20 kGy, and strain EJ2^T was isolated from the Guaymas Basin after -irradiation at 30 kGy. Only strain EJ2^T was motile, and both formed regular cocci. These new strains grew between 55 and 95 °C with the optimal temperature being 88 °C. The optimal pH for growth was 6.0, and the optimal NaCl concentration for growth was around 20 g l^–1. These strains were obligate anaerobic heterotrophs that utilized yeast extract, tryptone, and peptone as a carbon source for growth. Ten amino acids were essential for the growth of strain EJ1^T, such as arginine, aspartic acid, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tyrosine, and valine, while strain EJ2^T was unable to grow on a mixture of amino acids. Elemental sulfur or cystine was required for EJ2^T growth and was reduced to hydrogen sulfide. Rifampicin inhibited growth for both strains EJ1^T and EJ2^T. The G+C contents of the genomic DNA were 52.3 and 54.5 mol% for EJ1^T and EJ2^T, respectively. As determined by 16S rRNA gene sequence analysis, these strains were more closely related to Thermococcus gorgonarius, T. celer, T. guaymasensis, T. profundus, and T. hydrothermalis. However, no significant homology was observed between them with DNA–DNA hybridization. These novel organisms also possess phenotypic traits that differ from those of its closest phylogenetic relatives. Therefore, it is proposed that these isolates, which are amongst the most radioresistant hyperthermophilic archaea known to date with T. gammatolerans (Jolivet et al. 2003a), should be described as novel species T. marinus sp. nov. and T. radiotolerans sp. nov. The type strain of T. marinus is strain EJ1^T (=DSM 15227^T=JCM 11825^T) and the type strain of T. radiotolerans is strain EJ2^T (=DSM 15228^T=JCM 11826^T).Communicated by J. WiegelThe GenBank accession numbers for the 16S rRNA sequence of Thermococcus marinus strain EJ1^T and Thermococcus radiotolerans EJ2^T are AF479012 and AF479013, respectively.     

A novel NADPH-dependent oxidoreductase with a unique domain structure in the hyperthermophilic Archaeon, Thermococcus litoralis

Thermococcus litoralis , a hyperthermophilic Archaeon, is able to reduce elemental sulfur during fermentative growth. An unusual gene cluster ( nsoABCD ) was identified in this organism. In silico analysis suggested that three of the genes ( nsoABC ) probably originated from Eubacteria and one gene ( nsoD ) from Archaea. The putative NsoA and NsoB are similar to NuoE- and NuoF-type electron transfer proteins, respectively. NsoC has a unique domain structure and contains a GltD domain, characteristic of glutamate synthase small subunits, which seems to be integrated into a NuoG-type sequence. Flavin and NAD(P)H binding sites and conserved cysteines forming iron–sulfur clusters binding motifs were identified in the protein sequences deduced. The purified recombinant NsoC contains one FAD cofactor per protein molecule and catalyzes the reduction of polysulfide with NADPH as an electron donor and it also reduces oxygen. It was concluded that the Nso complex is a new type of NADPH-oxidizing enzyme using sulfur and/or oxygen as an electron acceptor.     

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.     

Lebetimonas natsushimae sp. nov., a novel strictly anaerobic,moderately thermophilic chemoautotroph isolated from a deep-sea hydrothermal vent polychaete nest in the Mid-Okinawa Trough

A moderately thermophilic, strictly anaerobic, chemoautotrophic bacterium, designated strain HS1857^T, was isolated from a deep-sea hydrothermal vent at the Noho site in the Mid-Okinawa Trough. Strain HS1857^T grew between 35 and 63 °C (optimum 55 °C), in the presence of 10–55 g l^?1 NaCl (optimum 25 g l^?1), and pH 5.5–7.1 (optimum 6.4). Growth occurred with molecular hydrogen as the electron donor and elemental sulfur, nitrate, or selenate as the electron acceptors. Formate could serve as an alternative electron donor with nitrate as an electron acceptor. During growth with nitrate as the electron acceptor, strain HS1857^T produced ammonium and formed a biofilm. CO₂ was utilized as the sole carbon source. The G + C content of the genomic DNA was 33.2 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain HS1857^T is a member of the order Nautiliales, showing a sequence similarity of 95.0% with Lebetimonas acidiphila Pd55^T. The fatty acid composition was similar to that of L. acidiphila, which was dominated by C_18:0 (47.0%) and C_18:1 (23.7%). Based on the genomic, chemotaxonomic, phenotypic characteristics, the name Lebetimonas natsushimae sp. nov., is proposed. The type strain is HS1857^T (= NBRC 112478^T = DSM 104102^T).