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.

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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.

The type III ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) present in the archaeon Thermococcus kodakaraensis was found to participate in adenosine 5'-monophosphate (AMP) metabolism, a role that is distinct from that of classical RuBisCOs of the Calvin-Benson-Bassham cycle. Genes annotated as thymidine phosphorylase (deoA) and eucaryal translation initiation factor 2B (e2b2) were found to encode AMP phosphorylase and ribose-1,5-bisphosphate isomerase, respectively. These enzymes supplied the RuBisCO substrate, ribulose-1,5-bisphosphate, from AMP and phosphate. Archaea with type III RuBisCOs all harbor both DeoA and the corresponding E2b2 homologs. In this pathway, adenine was released from AMP and the phosphoribose moiety entered central-carbon metabolism.

We have recently developed a gene disruption system for the hyperthermophilic archaeon Thermococcus kodakaraensis by utilizing a pyrF-deficient mutant, KU25, as a host strain and the pyrF gene as a selectable marker. To achieve multiple genetic manipulations for more advanced functional analyses of genes in vivo, it is necessary to establish multiple host-marker systems or to develop a system in which repeated utilization of one marker gene is possible. In this study, we first constructed a new host strain, KU216 (⌬pyrF), by specific and almost complete deletion of endogenous pyrF through homologous recombination. In this refined host, there is no need to consider unknown mutations caused by random mutagenesis, and unlike in the previous host, KU25, there is little, if any, possibility that unintended recombination between the marker gene and the chromosomal allele occurs. Furthermore, a new host-marker combination of a trpE deletant, KW128 (⌬pyrF ⌬trpE::pyrF), and the trpE gene was developed. This system made it possible to isolate transformants through a more simple selection procedure as well as to deduce the transformation efficiency, overcoming practical disadvantages of the first system. The effects of the transformation conditions were also investigated using this system. Finally, we have also established a system in which repeated utilization of the counterselectable pyrF marker is possible through its excision by pop-out recombination. Both endogenous and exogenous sequences could be applied as tandem repeats flanking the marker pyrF for pop-out recombination. A double deletion mutant, KUW1 (⌬pyrF ⌬trpE), constructed with the pop-out strategy, was demonstrated to be a useful host for the dual markers pyrF and trpE. Likewise, a triple deletion mutant, KUWH1 (⌬pyrF ⌬trpE ⌬hisD), could also be constructed. The transformation systems developed here now provide the means for extensive genetic studies in this hyperthermophilic archaeon.Recent phylogenetic analysis of living organisms based on rRNA sequences has indicated that hyperthermophiles occupy the deepest and shortest branches in the phylogenetic tree, postulating that the origin and evolution of biological systems may have derived from hyperthermophiles (25). Studies on the unique properties of hyperthermophiles are expected to provide valuable perspectives on the mechanisms that enable them to survive and grow in extreme environments (26). They are also important as potential resources for highly thermostable enzymes (2, 28). Many members of hyperthermophiles belong to the third domain of life, Archaea, along with halophiles and methanogens. Archaea exhibit a mosaic of features from the other two domains, Bacteria and Eucarya; intriguingly, their components for information processing are more closely related to those in eucaryotes than those in bacteria. From these interests, genome projects of various types of hyperthermophilic archaea have been performed, and complete genome sequences of more than 10 species have been determined. The genomes of hyp...

Each protein folds into a unique and native structure spontaneously. However, during the unfolding or refolding process, a protein often tends to form aggregates. To establish a method to prevent undesirable protein aggregation and to increase the stability of native protein structures under deterioration conditions, two types of aggregation conditions, thermal unfolding-induced aggregation and dilution-induced aggregation from denatured state, were studied in the presence of additional amino acids and ions using lysozyme as a model protein. Among 15 amino acids tested, arginine exhibited the best results in preventing the formation of aggregates in both cases. Further biophysical studies revealed that arginine did not change the thermal denaturation temperature (T(m)) of the lysozyme. The preventive effect of arginine on aggregation was not dependent on the size or isoelectric point of eight kinds of proteins tested.

A biosurfactant termed arthrofactin produced by Arthrobacter species strain MIS38 was purified and chemically characterized as 3-hydroxydecanoyl-D-leucyl-D-asparagyl-D-threonyl-D-leucyl-D-leucyl-D-seryl-Lleucyl-D-seryl-L-isoleucyl-L-isoleucyl-L-asparagyl lactone. Surface activity of arthrofactin was examined, with surfactin as a control. Critical micelle concentration values of arthrofactin and surfactin were around 1.0 X 10 -5 M and 7.0 x 10-5 M at 25°C, respectively. Arthrofactin was found to be five to seven times more effective than surfactin. The minimum surface tension value of arthrofactin was 24 mN/m at a concentration higher than the critical micelle concentration. According to the oil displacement assay, arthrofactin was a better oil remover than synthetic surfactants, such as Triton X-100 and sodium dodecyl sulfate. Arthrofactin is one of the most effective lipopeptide biosurfactants.Biosurfactants are surface active substances derived from living organisms, mainly from microorganisms (2, 5). The biological function of biosurfactants is thought to be participation in the solubilization of hydrophobic substances (hydrocarbons, lipids, and sterols, etc.), promoting enhanced cell assimilation. Emulsification which increases the surface area between two immiscible phases, results in small oil drops in water. It is generally concluded that microorganisms growing on water-insoluble hydrocarbons benefit from the presence of a surfactant (6,8,13). Biosurfactants are usually complex lipids, with more chemically complicated structures than synthetic surfactants. Lipopeptide biosurfactants are structurally more heterogeneous than glycolipid types.The minimum surface tension and critical micelle concentration are parameters used to measure the efficiency of surfactant systems. The best-known lipopeptide biosurfactant is surfactin, which lowers the surface tension of 0.1 M NaHCO3 from 71.6 to 27.0 mN/m (1). Recently, some strains of Bacillus licheniformis were shown to produce quite similar biosurfactant to surfactin (9,15). Arthrobacter species have been reported to produce extracellular glycolipids (12,20), none of which lowers the surface tension of water to 30 mN/m. We describe the chemical structure of a new lipopeptide-type biosurfactant. MATERIALS AND METHODSScreening of biosurfactant-producing bacteria. Biosurfactant-producing strains were selected as described previously (16), except that the cultivation temperature was 30°C. A slimy colony (strain MIS38) surrounded by a large halo on an oil-agar plate was obtained and used for further experiments.Production and purification of biosurfactant. Strain MIS38 was cultivated in 3 liters of L broth (1% Bacto tryptone, 0.5% yeast extract, 0.5% NaCl [pH 7.2]) at 30°C for 48 h. The culture was centrifuged (10,000 x g for 10 min), and the supernatant fluid was concentrated by ultrafiltration (exclusion molecular size, 10 kDa).The concentrated biosurfactant was extracted three times * Corresponding author.with an equal volume of hexane. After evaporation, the biosur...