The genus Thermococcus, comprised of sulfur-reducing hyperthermophilic archaea, belongs to the order Thermococcales in Euryarchaeota along with the closely related genus Pyrococcus. The members of Thermococcus are ubiquitously present in natural high-temperature environments, and are therefore considered to play a major role in the ecology and metabolic activity of microbial consortia within hot-water ecosystems. To obtain insight into this important genus, we have determined and annotated the complete 2,088,737-base genome of Thermococcus kodakaraensis strain KOD1, followed by a comparison with the three complete genomes of Pyrococcus spp. A total of 2306 coding DNA sequences (CDSs) have been identified, among which half (1165 CDSs) are annotatable, whereas the functions of 41% (936 CDSs) cannot be predicted from the primary structures. The genome contains seven genes for probable transposases and four virus-related regions. Several proteins within these genetic elements show high similarities to those in Pyrococcus spp., implying the natural occurrence of horizontal gene transfer of such mobile elements among the order Thermococcales. Comparative genomics clarified that 1204 proteins, including those for information processing and basic metabolisms, are shared among T. kodakaraensis and the three Pyrococcus spp. On the other hand, among the set of 689 proteins unique to T. kodakaraensis, there are several intriguing proteins that might be responsible for the specific trait of the genus Thermococcus, such as proteins involved in additional pyruvate oxidation, nucleotide metabolisms, unique or additional metal ion transporters, improved stress response system, and a distinct restriction system.

The domain Archaea has historically been divided into two phyla, the Crenarchaeota and Euryarchaeota. Although regarded as members of the Crenarchaeota based on small subunit rRNA phylogeny, environmental genomics and efforts for cultivation have recently revealed two novel phyla/divisions in the Archaea; the ‘Thaumarchaeota’ and ‘Korarchaeota’. Here, we show the genome sequence of Candidatus ‘Caldiarchaeum subterraneum’ that represents an uncultivated crenarchaeotic group. A composite genome was reconstructed from a metagenomic library previously prepared from a microbial mat at a geothermal water stream of a sub-surface gold mine. The genome was found to be clearly distinct from those of the known phyla/divisions, Crenarchaeota (hyperthermophiles), Euryarchaeota, Thaumarchaeota and Korarchaeota. The unique traits suggest that this crenarchaeotic group can be considered as a novel archaeal phylum/division. Moreover, C. subterraneum harbors an ubiquitin-like protein modifier system consisting of Ub, E1, E2 and small Zn RING finger family protein with structural motifs specific to eukaryotic system proteins, a system clearly distinct from the prokaryote-type system recently identified in Haloferax and Mycobacterium. The presence of such a eukaryote-type system is unprecedented in prokaryotes, and indicates that a prototype of the eukaryotic protein modifier system is present in the Archaea.

In contrast to the high accumulation in sequence data for hyperthermophilic archaea, methodology for genetically manipulating these strains is still at an early stage. This study aimed to develop a gene disruption system for the hyperthermophilic euryarchaeon Thermococcus kodakaraensis KOD1. Uracil-auxotrophic mutants with mutations in the orotidine-5-monophosphate decarboxylase gene (pyrF) were isolated by positive selection using 5-fluoroorotic acid (5-FOA) and used as hosts for further transformation experiments. We then attempted targeted disruption of the trpE locus in the host strain by homologous recombination, as disruption of trpE was expected to result in tryptophan auxotrophy, an easily detectable phenotype. A disruption vector harboring the pyrF marker within trpE was constructed for double-crossover recombination. The host cells were transformed with the exogenous DNA using the CaCl 2 method, and several transformants could be selected based on genetic complementation. Genotypic and phenotypic analyses of a transformant revealed the unique occurrence of targeted disruption, as well as a phenotypic change of auxotrophy from uracil to tryptophan caused by integration of the wild-type pyrF into the host chromosome at trpE. As with the circular plasmid, gene disruption with linear DNA was also possible when the homologous regions were relatively long. Shortening these regions led to predominant recombination between the pyrF marker in the exogenous DNA and the mutated allele on the host chromosome. In contrast, we could not obtain trpE disruptants by insertional inactivation using a vector designed for single-crossover recombination. The gene targeting system developed in this study provides a long-needed tool in the research on hyperthermophilic archaea and will open the way to a systematic, genetic approach for the elucidation of unknown gene function in these organisms.Among the living organisms, hyperthermophiles belonging to the third domain of life, Archaea, are attractive subjects of research from various standpoints. Many efforts to understand the strategies for adaptation to extremely high temperature environments have revealed numerous physiologically intriguing and unique properties of hyperthermophilic archaea (30,36,37). They have also attracted attention from an industrial point of view as potential resources for highly thermostable enzymes (3,23,40).Along with the classical biochemistry and molecular cloning of structural genes, complete genome analyses in recent years have provided a new means of understanding the hyperthermophilic archaea. So far, entire genome sequences of 16 archaeal strains, including 10 hyperthermophiles, have been determined. This has revealed that genomes of hyperthermophilic archaea are rather small, and the small number of genes makes them ideal targets for studying the basic principles and evolution of life.In spite of the many attractive aspects of hyperthermophilic archaea, progress of research on these organisms has been constantly hampered by the limitation...

Although a common reaction in anaerobic environments, the conversion of formate and water to bicarbonate and H(2) (with a change in Gibbs free energy of ΔG° = +1.3 kJ mol(-1)) has not been considered energetic enough to support growth of microorganisms. Recently, experimental evidence for growth on formate was reported for syntrophic communities of Moorella sp. strain AMP and a hydrogen-consuming Methanothermobacter species and of Desulfovibrio sp. strain G11 and Methanobrevibacter arboriphilus strain AZ. The basis of the sustainable growth of the formate-users is explained by H(2) consumption by the methanogens, which lowers the H(2) partial pressure, thus making the pathway exergonic. However, it has not been shown that a single strain can grow on formate by catalysing its conversion to bicarbonate and H(2). Here we report that several hyperthermophilic archaea belonging to the Thermococcus genus are capable of formate-oxidizing, H(2)-producing growth. The actual ΔG values for the formate metabolism are calculated to range between -8 and -20 kJ mol(-1) under the physiological conditions where Thermococcus onnurineus strain NA1 are grown. Furthermore, we detected ATP synthesis in the presence of formate as a sole energy source. Gene expression profiling and disruption identified the gene cluster encoding formate hydrogen lyase, cation/proton antiporter and formate transporter, which were responsible for the growth of T. onnurineus NA1 on formate. This work shows formate-driven growth by a single microorganism with protons as the electron acceptor, and reports the biochemical basis of this ability.