Thermococcus aciditolerans sp. nov., a piezotolerant, hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent chimney in the Southwest Indian Ridge.

A hyperthermophilic, strictly anaerobic archaeon, designated strain SY113T, was isolated from a deep-sea hydrothermal vent chimney on the Southwest Indian Ridge at a water depth of 2770 m. Enrichment and isolation of strain SY113T were performed at 85 °C at 0.1 MPa. Cells of strain SY113T were irregular motile cocci with peritrichous flagella and generally 0.8-2.4 µm in diameter. Growth was observed at temperatures between 50 and 90 °C (optimum at 85 °C) and under hydrostatic pressures of 0.1-60 MPa (optimum, 27 MPa). Cells of SY113T grew at pH 4.0-9.0 (optimum, pH 5.5) and a NaCl concentration of 0.5-5.5 % (w/v; optimum concentration, 3.0 % NaCl). Strain SY113T was an anaerobic chemoorganoheterotroph and grew on complex proteinaceous substrates such as yeast extract and tryptone, as well as on maltose and starch. Elemental sulphur stimulated growth, but not obligatory for its growth. The G+C content of the genomic DNA was 55.0 mol%. Phylogenetic analysis of the 16S rRNA sequence of strain SY113T showed that the novel isolate belonged to the genus Thermococcus. On the basis of physiological characteristics, average nucleotide identity values and in silico DNA-DNA hybridization results, we propose a novel species, named Thermococcus aciditolerans sp. nov. The type strain is SY113T (=MCCC 1K04190T=JCM 39083T).

Thermococcus henrietii sp. nov., a novel extreme thermophilic and piezophilic sulfur-reducing archaeon isolated from a deep-sea hydrothermal chimney.

A novel extreme thermophilic and piezophilic chemoorganoheterotrophic archaeon, strain EXT12cT, was isolated from a hydrothermal chimney sample collected at a depth of 2496 m at the East Pacific Rise 9° N. Cells were strictly anaerobic, motile cocci. The strain grew at NaCl concentrations ranging from 1 to 5 % (w/v; optimum, 2.0%), from pH 6.0 to 7.5 (optimum, pH 6.5-7.0), at temperatures between 60 and 95 °C (optimum, 80-85 °C), and at pressures from 0.1 to at least 50 MPa (optimum, 30 MPa). Strain EXT12cT grew chemoorganoheterotrophically on complex proteinaceous substrates. Its growth was highly stimulated by the presence of elemental sulphur or l-cystine, which were reduced to hydrogen sulfide. The DNA G+C content was 54.58 mol%. Phylogenetic analyses based on 16S rRNA gene sequences and concatenated ribosomal protein sequences showed that strain EXT12cT falls into the genus Thermococcus and is most closely related to Thermococcus nautili strain 30-1T. Overall genome relatedness index analyses (average nucleotide identity scores and in silico DNA-DNA hybridizations) showed a sufficient genomic distance between the new genome and the ones of the Thermococcus type strains for the delineation of a new species. On the basis of genotypic and phenotypic data, strain EXT12cT is considered to represent a novel species, for which the name Thermococcus henrietii sp. nov. is proposed, with the type strain EXT12cT (=UBOCC M-2417T=DSM 111004T).

Thermococcus camini sp. nov., a hyperthermophilic and piezophilic archaeon isolated from a deep-sea hydrothermal vent at the Mid-Atlantic Ridge.

A coccoid-shaped, strictly anaerobic, hyperthermophilic and piezophilic organoheterotrophic archaeon, strain Iri35cT, was isolated from a hydrothermal chimney rock sample collected at a depth of 2300 m at the Mid-Atlantic Ridge (Rainbow vent field). Cells of strain Iri35cT grew at NaCl concentrations ranging from 1-5 % (w/v) (optimum 2.0 %), from pH 5.0 to 9.0 (optimum 7.0-7.5), at temperatures between 50 and 90 °C (optimum 75-80 °C) and at pressures from 0.1 to at least 50 MPa (optimum: 10-30 MPa). The novel isolate grew on complex organic substrates, such as yeast extract, tryptone, peptone or beef extract, preferentially in the presence of elemental sulphur or l-cystine; however, these molecules were not necessary for growth. Its genomic DNA G+C content was 54.63 mol%. The genome has been annotated and the metabolic predictions are in accordance with the metabolic characteristics of the strain and of Thermococcales in general. Phylogenetic analyses based on 16S rRNA gene sequences and concatenated ribosomal protein sequences showed that strain Iri35cT belongs to the genus Thermococcus, and is closer to the species T. celericrescens and T. siculi. Average nucleotide identity scores and in silico DNA-DNA hybridization values between the genome of strain Iri35cT and the genomes of the type species of the genus Thermococcus were below the species delineation threshold. Therefore, and considering the phenotypic data presented, strain Iri35cT is suggested to represent a novel species, for which the name Thermococcus camini sp. nov. is proposed, with the type strain Iri35cT (=UBOCC M-2026T=DSM 111003T).

Thermococcus indicus sp. nov., a Fe(III)-reducing hyperthermophilic archaeon isolated from the Onnuri Vent Field of the Central Indian Ocean ridge.

A strictly anaerobic, dissimilatory Fe(III)-reducing hyperthermophilic archaeon, designated as strain IOH1T, was isolated from a new deep-sea hydrothermal vent (Onnuri Vent Field) area in the Central Indian Ocean ridge. Strain IOH1T showed > 99% 16S rRNA gene sequence similarity with Thermococcus celericrescens TS2T (99.4%) and T. siculi DSM 12349T (99.2%). Additional three species T. barossii SHCK-94T (99.0%), T. celer Vu13T (98.8%), and T. piezophilus (98.6%) showed > 98.6% of 16S rRNA gene sequence similarity, however, the maximum OrthoANI value is 89.8% for the genome of T. celericrescens TS2T. Strain IOH1T cells are coccoid, 1.2-1.8 µm in diameter, and motile by flagella. Growth was at 70-82°C (optimum 80°C), pH 5.4-8.0 (optimum pH 6.0) with 2-4% (optimum 3%) NaCl. Growth of strain IOH1T was enhanced by starch, pyruvate, D(+)-maltose and maltodextrin as a carbon sources, and elemental sulfur as an electron acceptor; clearly different from those of related species T. celecrescens DSM 17994T and T. siculi DSM 12349T. Strain IOH1T, T. celercrescence DSM 17994T, and T. siculi DSM 12349T reduced soluble Fe(III)-citrate present in the medium, whereas the amount of total cellular proteins increased with the concomitant accumulation of Fe(II). We determined a circular chromosome of 2,234 kb with an extra-chromosomal archaeal plasmid, pTI1, of 7.7 kb and predicted 2,425 genes. The DNA G + C content was 54.9 mol%. Based on physiological properties, phylogenetic, and genome analysis, we proposed that strain IOH1T (= KCTC 15844T = JCM 39077T) is assigned to a new species in the genus Thermococcus and named Thermococcus indicus sp. nov.

Microbe Profile: Thermococcus kodakarensis: the model hyperthermophilic archaeon.

Thermococcus kodakarensis is a hyperthermophilic Euryarchaeon that grows well under laboratory conditions and, being naturally competent for genetic transformation, it has become a widely studied experimental model species. With the genome sequence available since 2004, combining genetic, enzymological and structural biochemical approaches has revealed previously unknown and unanticipated features of archaeal molecular biology and metabolism. T. kodakarensis DNA polymerase is already commercialized and with the details of metabolism and hydrogenase available, generating H2 from biopolymers solubilized at high temperatures, most notably chitin, now seems a very attractive possibility as a renewable energy bioprocess.

Identification of Branched-Chain Polyamines in Hyperthermophiles.

Thermophiles are organisms that grow optimally at temperatures higher than 55 °C. They contain two types of unusual longer/branched-chain polyamines in addition to common polyamines such as spermidine and putrescine. These unusual polyamines contribute to the survival of hyperthermophiles at high temperatures. Recently, the novel aminopropyltransferase BpsA was found to be responsible for the biosynthesis of branched-chain polyamines in the hyperthermophilic archaeon Thermococcus kodakarensis, which contains N 4-bis(aminopropyl)spermidine as the major polyamine. This compound is synthesized by the sequential addition of decarboxylated S-adenosylmethionine (dcSAM) aminopropyl groups to spermidine via the bifunctional catalytic action of BpsA. In this chapter, methods for the extraction and identification of branched-chain polyamines are presented, along with methods for the production and characterization of recombinant T. kodakarensis BpsA as a model aminopropyltransferase.

Analysis of the complete genome sequence of the archaeon Pyrococcus chitonophagus DSM 10152 (formerly Thermococcus chitonophagus).

Here we analyze the first complete genome sequence of Pyrococcus chitonophagus. The archaeon was previously suggested to belong to the Thermococcus rather than the Pyrococcus genus. Whole genome phylogeny as well as whole proteome comparisons using all available complete genomes in Thermococcales clearly showed that the species belongs to the Pyrococcus genus. P. chitonophagus was originally isolated from a hydrothermal vent site and it has been described to effectively degrade chitin debris, and therefore is considered to play a major role in the sea water ecology and metabolic activity of microbial consortia within hot sea water ecosystems. Indeed, an obvious feature of the P. chitonophagus genome is that it carries proteins showing complementary activities for chitin degradation, i.e. endo- and exo-chitinase, diacetylchitobiose deacetylase and exo-ß-D glucosaminidase activities. This finding supports the hypothesis that compared to other Thermococcales species P. chitonophagus is adapted to chitin degradation.

Hydrogenogenic and Sulfidogenic Growth of Thermococcus Archaea on Carbon Monoxide and Formate.

Enrichment and pure cultures of hyperthermophilic archaea capable of anaerobic growth on one- carbon compounds (CO and/or formate) were obtained from deep-sea sites of hydrothermal activity at the Mid-Atlantic Ridge, Lau Basin, and Guaymas Basin. All isolates belonged to the T barophilus-T paralvi- .nellae group within the genus Thermococcus. In all cases available for analysis, the genomes of Thermococcus strains capable of growth by hydrogenogenic utilization of CO and/or formate contained clusters of genes en- coding energy-converting hydrogenase and either CO dehydrogenase or formate dehydrogenase and formate transporter. Apart from the previously known processes of hydrogenogenic oxidation of CO and formate, the oxidation of these substrates coupled to sulfur reduction was observed, processes previously unknown among archaea. The capacities for hydrogenogenic or sulfidogenic oxidation of CO and formate occurred in the studied strains in all possible combinations, which could only in part be explained by peculiarities of organi- zation of genetic determinants revealed in the genomes. Investigation of CO and formate consumption kinet- ics revealed that T barophilus strain Ch5 was able to grow at concentrations close to the environmental ones. Thus, it was shown that hyperthermophilic archaea from deep-sea hydrothermal vents are able to utilize one- carbon substrates of abiotic origin both in the presence of an electron acceptor (sulfur) and in its absence. These processes were probably of importance under the conditions of the early Earth biosphere.

Structural basis for the substrate selectivity of a HAD phosphatase from Thermococcus onnurineus NA1.

Proteins in the haloalkaloic acid dehalogenase (HAD) superfamily, which is one of the largest enzyme families, is generally composed of a catalytic core domain and a cap domain. Although proteins in this family show broad substrate specificities, the mechanisms of their substrate recognition are not well understood. In this study, we identified a new substrate binding motif of HAD proteins from structural and functional analyses, and propose that this motif might be crucial for interacting with hydrophobic rings of substrates. The crystal structure of TON_0338, one of the 17 putative HAD proteins identified in a hyperthermophilic archaeon, Thermococcus onnurineus NA1, was determined as an apo-form at 2.0 Å resolution. In addition, we determined the crystal structure TON_0338 in complex with Mg(2+) or N-cyclohexyl-2-aminoethanesulfonic acid (CHES) at 1.7 Å resolution. Examination of the apo-form and CHES-bound structures revealed that CHES is sandwiched between Trp58 and Trp61, suggesting that this Trp sandwich might function as a substrate recognition motif. In the phosphatase assay, TON_0338 was shown to have high activity for flavin mononucleotide (FMN), and the docking analysis suggested that the flavin of FMN may interact with Trp58 and Trp61 in a way similar to that observed in the crystal structure. Moreover, the replacement of these tryptophan residues significantly reduced the phosphatase activity for FMN. Our results suggest that WxxW may function as a substrate binding motif in HAD proteins, and expand the diversity of their substrate recognition mode.

Identification and characterization of bifunctional proline racemase/hydroxyproline epimerase from archaea: discrimination of substrates and molecular evolution.

Proline racemase (ProR) is a member of the pyridoxal 5'-phosphate-independent racemase family, and is involved in the Stickland reaction (fermentation) in certain clostridia as well as the mechanisms underlying the escape of parasites from host immunity in eukaryotic Trypanosoma. Hydroxyproline epimerase (HypE), which is in the same protein family as ProR, catalyzes the first step of the trans-4-hydroxy-L-proline metabolism of bacteria. Their substrate specificities were previously considered to be very strict, in spite of similarities in their structures and catalytic mechanisms, and no racemase/epimerase from the ProR superfamily has been found in archaea. We here characterized the ProR-like protein (OCC_00372) from the hyperthermophilic archaeon, Thermococcus litoralis (TlProR). This protein could reversibly catalyze not only the racemization of proline, but also the epimerization of 4-hydroxyproline and 3-hydroxyproline with similar kinetic constants. Among the four (putative) ligand binding sites, one amino acid substitution was detected between TlProR (tryptophan at the position of 241) and natural ProR (phenylalanine). The W241F mutant showed a significant preference for proline over hydroxyproline, suggesting that this (hydrophobic and bulky) tryptophan residue played an importance role in the recognition of hydroxyproline (more hydrophilic and bulky than proline), and substrate specificity for hydroxyproline was evolutionarily acquired separately between natural HypE and ProR.　A phylogenetic analysis indicated that such unique broad substrate specificity was derived from an ancestral enzyme of this superfamily.

Thermococcus eurythermalis sp. nov., a conditional piezophilic, hyperthermophilic archaeon with a wide temperature range for growth, isolated from an oil-immersed chimney in the Guaymas Basin.

A conditional piezophilic, hyperthermophilic archaeon showing growth over a wide range of temperature, pH and pressure was isolated from an oil-immersed hydrothermal chimney at a depth of 2006.9 m in the Guaymas Basin. Enrichment and isolation of strain A501(T) were performed at 80 °C at 0.1 MPa. Cells of isolate A501(T) were irregular motile cocci with a polar tuft of flagella and generally 0.6-2.6 µm in diameter. Growth was detected over the range 50-100 °C (optimal growth at 85 °C) at atmospheric pressure and was observed at 102 °C at a pressure of 10 MPa. At 85 °C, growth was observed at a pressure of 0.1-70 MPa (optimum pressure 0.1 MPa-30 MPa), while at 95 °C, the pressure allowing growth ranged from 0.1 MPa to 50 MPa (optimum pressure 10 MPa). Cells of strain A501(T) grew at pH 4-9 (optimum pH 7.0) and a NaCl concentration of 1.0-5.0 % (w/v) (optimum concentration 2.5 % NaCl). This isolate was an anaerobic chemo-organoheterotroph and was able to utilize yeast extract, peptone, tryptone and starch as the single carbon source for growth. Elemental sulfur and cysteine stimulated growth; however, these molecules were not necessary. The DNA G+C content of the complete genome was 53.47 mol%. The results of 16S rRNA gene sequence analysis indicated that strain A501(T) belongs to the genus Thermococcus. There was no significant similarity between strain A501(T) and the phylogenetically related species of the genus Thermococcus based on complete genome sequence alignments and calculation of the average nucleotide identity and the tetranucleotide signature frequency correlation coefficient. These results indicate that strain A501(T) represents a novel species, Thermococcus eurythermalis sp. nov. The type strain is A501(T) ( = CGMCC 7834(T) = JCM 30233(T)).

The X-ray crystal structure of the euryarchaeal RNA polymerase in an open-clamp configuration.

The archaeal transcription apparatus is closely related to the eukaryotic RNA polymerase II (Pol II) system. Archaeal RNA polymerase (RNAP) and Pol II evolved from a common ancestral structure and the euryarchaeal RNAP is the simplest member of the extant archaeal-eukaryotic RNAP family. Here we report the first crystal structure of euryarchaeal RNAP from Thermococcus kodakarensis (Tko). This structure reveals that the clamp domain is able to swing away from the main body of RNAP in the presence of the Rpo4/Rpo7 stalk by coordinated movements of these domains. More detailed structure-function analysis of yeast Pol II and Tko RNAP identifies structural additions to Pol II that correspond to the binding sites of Pol II-specific general transcription factors including TFIIF, TFIIH and Mediator. Such comparisons provide a framework for dissecting interactions between RNAP and these factors during formation of the pre-initiation complex.

Thermococcus paralvinellae sp. nov. and Thermococcus cleftensis sp. nov. of hyperthermophilic heterotrophs from deep-sea hydrothermal vents.

Two heterotrophic hyperthermophilic strains, ES1(T) and CL1(T), were isolated from Paralvinella sp. polychaete worms collected from active hydrothermal vent chimneys in the north-eastern Pacific Ocean. Both were obligately anaerobic and produced H2S in the presence of elemental sulfur and H2. Complete genome sequences are available for both strains. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strains are more than 97% similar to most other species of the genus Thermococcus. Therefore, overall genome relatedness index analyses were performed to establish that these strains are novel species. For each analysis, strain ES1(T) was determined to be most similar to Thermococcus barophilus MP(T), while strain CL1(T) was determined to be most similar to Thermococcus sp. 4557. The average nucleotide identity scores for these strains were 84% for strain ES1(T) and 81% for strain CL1(T), genome-to-genome direct comparison scores were 23% for strain ES1(T) and 47% for strain CL1(T), and the species identification scores were 89% for strain ES1(T) and 88% for strain CL1(T). For each analysis, strains ES1(T) and CL1(T) were below the species delineation cut-off. Therefore, based on their whole genome sequences, strains ES1(T) and CL1(T) are suggested to represent novel species of the genus Thermococcus for which the names Thermococcus paralvinellae sp. nov. and Thermococcus cleftensis sp. nov. are proposed, respectively. The type strains are ES1(T) ( =DSM 27261(T) =KACC 17923(T)) and CL1(T) ( =DSM 27260(T) =KACC 17922(T)).

The temperature gradient-forming device, an accessory unit for normal light microscopes to study the biology of hyperthermophilic microorganisms.

To date, the behavior of hyperthermophilic microorganisms in their biotope has been studied only to a limited degree; this is especially true for motility. One reason for this lack of knowledge is the requirement for high-temperature microscopy-combined, in most cases, with the need for observations under strictly anaerobic conditions-for such studies. We have developed a custom-made, low-budget device that, for the first time, allows analyses in temperature gradients up to 40°C over a distance of just 2 cm (a biotope-relevant distance) with heating rates up to ∼5°C/s. Our temperature gradient-forming device can convert any upright light microscope into one that works at temperatures as high as 110°C. Data obtained by use of this apparatus show how very well hyperthermophiles are adapted to their biotope: they can react within seconds to elevated temperatures by starting motility-even after 9 months of storage in the cold. Using the temperature gradient-forming device, we determined the temperature ranges for swimming, and the swimming speeds, of 15 selected species of the genus Thermococcus within a few months, related these findings to the presence of cell surface appendages, and obtained the first evidence for thermotaxis in Archaea.

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

Thermococcus nautili, strain 30-1T (formerly reported as Thermococcus nautilus), was isolated from a hydrothermal chimney sample collected from the East Pacific Rise at a depth of 2633 m on the 'La chainette PP57' area. Cells were motile, irregular cocci with a polar tuft of flagella (0.8-1.5 µm) and divided by constriction. The micro-organism grew optimally at 87.5 °C (range 55-95 °C), at pH 7 (range pH 4-9) and with 2% NaCl (range 1-4%). Doubling time was 64 min in Zillig's broth medium under optimal conditions. Growth was strictly anaerobic. It grew preferentially in the presence of elemental sulfur or cystine, which are reduced to H2S, on complex organic substrates such as yeast extract, tryptone, peptone, Casamino acids and casein. Slow growth was observed on starch and pyruvate. Strain 30-1T was resistant to chloramphenicol and tetracyclin (at 100 µg ml(-1)) but sensitive to kanamycin and rifampicin. The G+C content of the genomic DNA was 54 mol%. Strain 30-1T harboured three plasmids named pTN1, pTN2 and pTN3 and produced membrane vesicles that incorporate pTN1 and pTN3. As determined by 16S rRNA gene sequence analysis, strain 30-1T is related most closely to Thermococcus sp. AM4 (99.3% similarity) and Thermococcus gammatolerans DSM 15229T (99.2%). DNA-DNA hybridization values (in silico) with these two closest relatives were below the threshold value of 70% (33% with Thermococcus sp. AM4 and 32% with T. gammatolerans DSM 15229T) and confirmed that strain 30-1 represents a novel species. On the basis of the data presented, strain 30-1T is considered to represent a novel species of the genus Thermococcus, for which the name Thermococcus nautili sp. nov. is proposed. The type strain is 30-1T (=CNCM 4275=JCM 19601).

Complete genome sequence of hyperthermophilic archaeon Thermococcus sp. ES1.

Thermococcus sp. strain ES1 is an anaerobic, hyperthermophilic archaeon from a hydrothermal vent that catabolizes sugars and peptides and produces H2S from S°, H2, acetate and CO2 as its primary metabolites. We present the complete genome sequence of this strain (1,957,742bp) with a focus on its substrate utilization and metabolite production capabilities. The sequence will contribute to the development of heterotrophic archaea for bioenergy production and biogeochemical modeling in hydrothermal environments.

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

A novel hyperthermophilic, anaerobic archaeon, strain Bio-pl-0405IT2(T), was isolated from a hydrothermal chimney sample collected from the East Pacific Rise at 2700 m depth in the 'Sarah Spring' area (7° 25' 24" S 107° 47' 66" W). Cells were irregular, motile cocci (0.8-1.5 µm in diameter) and divided by constriction. Growth was observed at temperatures between 60 °C and 95 °C with an optimum at 80 °C. The pH range for growth was between pH 4.0 and pH 8.0 with an optimum around pH 7.0. Strain Bio-pl-0405IT2(T) grew at salt concentrations of 1-5 % (w/v) NaCl with an optimum at 2 %. The novel isolate grew by fermentation or sulphur respiration on a variety of organic compounds. It was a chemoorganoheterotrophic archaeon growing preferentially with yeast extract, peptone and tryptone as carbon and energy sources and sulphur and organic compounds as electron acceptors; it also grew on maltose and starch. Sulphur or l-cystine were required for growth and were reduced to hydrogen sulfide. The strain was resistant to rifampicin, chloramphenicol, vancomycin and kanamycin (all at 100 µg ml(-1)) but was sensitive to tetracycline. The G+C content of its genomic DNA was 53.6 mol%. Phylogenetic analysis of the almost complete 16S rRNA gene sequence (1450 bp) of strain Bio-pl-0405IT2(T) showed that the novel isolate belonged to the genus Thermococcus. DNA-DNA hybridization values with the two closest relatives Thermococcus hydrothermalis AL662(T) and Thermococcus celer JCM 8558(T) were below the threshold value of 70 %. On the basis of the physiological and genotypic distinctness, we propose a novel species, Thermococcus prieurii sp. nov. The type strain is Bio-pl-0405IT2(T) ( = CSUR P577(T)= JCM 16307(T)).

A simple procedure to determine the infectivity and host range of viruses infecting anaerobic and hyperthermophilic microorganisms.

Plaque assay is the method traditionally used to isolate and purify lytic viruses, to determine the viral titer and host range. Whereas most bacterioviruses are either temperate or lytic, the majority of known archeoviruses are not lytic (i.e. they are temperate or chronic). In view of the widespread occurrence of such viruses in extreme environments, we designed an original method, called the inverted spot test, to determine the host range and infectivity of viruses isolated from anaerobic hyperthermophilic and sulfur-reducing microorganisms. Here, we used this approach to prove for the first time the infectivity of Pyrococcus abyssi virus 1 (PAV1) and to confirm the host range of Thermococcus prieurii virus 1 (TPV1), the only two viruses isolated so far from any of the described marine hyperthermophilic archaea (Euryarchaeota phylum, Thermococcales order).

ATP-dependent DNA ligase from Thermococcus sp. 1519 displays a new arrangement of the OB-fold domain.

DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini. Their function is essential for maintaining genome integrity in the replication, recombination and repair of DNA. High flexibility is important for the function of DNA ligase molecules. Two types of overall conformations of archaeal DNA ligase that depend on the relative position of the OB-fold domain have previously been revealed: closed and open extended conformations. The structure of ATP-dependent DNA ligase from Thermococcus sp. 1519 (LigTh1519) in the crystalline state determined at a resolution of 3.02 Šshows a new relative arrangement of the OB-fold domain which is intermediate between the positions of this domain in the closed and the open extended conformations of previously determined archaeal DNA ligases. However, small-angle X-ray scattering (SAXS) measurements indicate that in solution the LigTh1519 molecule adopts either an open extended conformation or both an intermediate and an open extended conformation with the open extended conformation being dominant.

Characterization of thermostable deblocking aminopeptidases of archaeon Thermococcus onnurineus NA1 by proteomic and biochemical approaches.

Thermococcus onnurineus NA1 is a hyperthermophilic archaeon that grows optimally at >80°C. The deblocking aminopeptidase (DAP) (TNA1-DAP1) encoded in Ton_1032 of T. onnurineus NA1 is considered a major DAP. However, four genes encoding putative DAP have been identified from a genomic analysis of T. onnurineus NA1. A proteomic analysis revealed that all four DAPs were differentially induced in YPS culture medium and, particularly, two DAPs (TNA1-DAP1 and TNA1-DAP2) were dominantly expressed in T. onnurineus NA1. The biochemical properties and enzyme activity of DAPs induced in an E. coli expression system suggested that the two major DAPs play complementary roles in T. onnurineus NA1.