Metabolic Versatility and Indigenous Origin of the Archaeon Thermococcus sibiricus,Isolated from a Siberian Oil Reservoir,as Revealed by Genome Analysis

Thermococcus species are widely distributed in terrestrial and marine hydrothermal areas, as well as in deep subsurface oil reservoirs. Thermococcus sibiricus is a hyperthermophilic anaerobic archaeon isolated from a well of the never flooded oil-bearing Jurassic horizon of a high-temperature oil reservoir. To obtain insight into the genome of an archaeon inhabiting the oil reservoir, we have determined and annotated the complete 1,845,800-base genome of T. sibiricus. A total of 2,061 protein-coding genes have been identified, 387 of which are absent in other members of the order Thermococcales. Physiological features and genomic data reveal numerous hydrolytic enzymes (e.g., cellulolytic enzymes, agarase, laminarinase, and lipases) and metabolic pathways, support the proposal of the indigenous origin of T. sibiricus in the oil reservoir, and explain its survival over geologic time and its proliferation in this habitat. Indeed, in addition to proteinaceous compounds known previously to be present in oil reservoirs at limiting concentrations, its growth was stimulated by cellulose, agarose, and triacylglycerides, as well as by alkanes. Two polysaccharide degradation loci were probably acquired by T. sibiricus from thermophilic bacteria following lateral gene transfer events. The first, a “saccharolytic gene island” absent in the genomes of other members of the order Thermococcales, contains the complete set of genes responsible for the hydrolysis of cellulose and β-linked polysaccharides. The second harbors genes for maltose and trehalose degradation. Considering that agarose and laminarin are components of algae, the encoded enzymes and the substrate spectrum of T. sibiricus indicate the ability to metabolize the buried organic matter from the original oceanic sediment.Thermococcus sibiricus is a hyperthermophilic anaerobic archaeon isolated from a well of the never flooded oil-bearing Jurassic horizon of the high-temperature Samotlor oil reservoir (Western Siberia) (32). The sampling site had a temperature of 84°C and was located at a depth of 2,350 m. Thermococcus species are widely distributed in terrestrial and marine hydrothermal areas (4), as well as in deep subsurface oil reservoirs (38, 53). Close relatives of T. sibiricus with a 16S rRNA gene sequence similarity of >99% were identified in high-temperature oil wells of Japan (54) and China (36). Together with the genera Pyrococcus and Palaeococcus, Thermococcus spp. form the euryarchaeal order Thermococcales (4). Most of these hyperthermophilic archaea are organoheterotrophs that utilize proteins, starch, and maltose with elemental sulfur (S°) or protons as electron acceptors (4, 47). Two exceptions are Thermococcus strain AM4 (51) and Thermococcus onnurineus (23), which are also capable of lithotrophic CO-dependent hydrogenogenic growth (52).Genomic sequences of T. onnurineus (23), Thermococcus kodakaraensis (11), Pyrococcus horikoshii (18), Pyrococcus furiosus (44), and Pyrococcus abyssi (7) provided information on the genetic and metabolic machinery of these closely related organisms. However, none of them originated from deep subsurface oil reservoirs. These habitats contain low levels of dissolved organic carbon and trace amounts of free amino acids (53) but harbor high numbers of anaerobic organisms, reaching 1.4 × 10⁶ cells ml⁻¹ (38). T. sibiricus was originally reported to grow exclusively on peptides (32). The organism was obtained from a sample of an oil-bearing Jurassic horizon that had never been flooded. The temperature, pH, and salinity characteristics for growth correlated with the natural conditions at the sampling site. Therefore, an indigenous origin was suggested for T. sibiricus, which might have survived over geologic time by metabolizing buried organic matter from the original oceanic sediment (32).Here we present the genome of T. sibiricus and show that it encodes numerous hydrolytic enzymes and metabolic pathways which may allow the utilization of diverse organic polymers from the original oceanic sediment. Our experiments provide evidence for these new physiological features and support the suggestion of the indigenous origin of T. sibiricus.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Phylogeography of mitochondrial DNA in South Asian Dolly Varden char Salvelinus curilus Pallas, 1814 (Salmoniformes, Salmonidae): mediated gene introgression?

In 41 individuals of South Asian Dolly Varden char Salvelinus curilus, nucleotide sequences of tRNA-Pro gene fragment (27 bp) and mtDNA control region (483-484 bp) were analyzed. The fish were collected in 20 localities covering virtually the whole range of the species: Kuril Islands, Sakhalin Island. and Primorye. In addition, six individuals of three other char species (S. albus, S. malma, and S. leucomaenis), which are closely related to S. curilus and inhabit the Russian Far East, were examined. In all, we detected 12 different variants of mtDNA haplotypes that formed three distinct groups differing in 14--20 nucleotide positions. The first group consisted of six haplotypes found in S. curilus in Kuril Islands, Sakhalin, and Primorye (mtDNA phylogroup OKHOTSKIA). The second group comprised four haplotypes representing the mtDNA phylogroup BERING, which had been described earlier (Brunner et al, 2001); they were found in S. curilus in Kuril Islands and Sakhalin, as well as in S. albus and S. malma in Kamchatka and northern Kurils. The third group included two haplotypes detected in S. leucomaenis. The existence of two mtDNA lineages (OKHOTSKIA and BERING) in S. curilus from Kurils and Sakhalin was explained by hybridization and DNA transfer from S. malma to S. curilus. The absence of the BERING haplotypes in S. curilus from Primorye water reservoirs is related to the physical isolation of the Sea of Okhotsk and Sea of Japan basins in past epochs. On the basis of comparing phylogenetic trees, constructed from the data on allozyme and mtDNA variation, we suggest that in this case, an indirect transfer of mtDNA in Alpinoid chars--> S. malma-->S. curilus chain could occur.     

Thermophilic microbial communities of deep-sea hydrothermal environments

The latest publications on the phylogenetic and functional diversity of thermophilic prokaryotes inhabiting thermal deep-sea environments are reviewed. Along with general physicochemical characterization of the biotope studied, certain adaptation mechanisms are discussed that are peculiar to the microorganisms inhabiting it. A separate chapter addresses phylogenetic analysis of deep-sea hydrothermal microbial communities and uncultivated microorganisms recently discovered therein using molecular biological techniques. Physiological groups of thermophilic microorganisms found in deep-sea hydrotherms are considered: methanogens, sulfate-, iron-, and sulfur-reducers, aerobic hydrogen-oxidizing prokaryotes, aerobic and anaerobic organotrophs. In most cases, the isolates represent novel taxa.     

The unusual estrogen-binding protein (UEBP) of male rat liver: structural determinants of ligands

The unusual estrogen-binding protein (UEBP) found in a male rat liver is a sex dependent protein which differs from other known receptor and transport proteins by the high lability of its complexes with estradiol (E2) and also the unique specificity of affinity for hormones. In this work values of relative binding affinity (RBA) of the UEBP for 57 steroids and their analogs were determined. The affinity of steroids was characterised by the amount of the unlabeled compound needed for 50% inhibition of [3H]-E2 binding with the UEBP. A number of derivatives of estrane and androstane possess an ability to interact with this protein, in contrast to the derivatives of pregnane, stilbene and triphenylethane. Characterized by RBA values, natural steroids are found to have the following order: estriol larger than or equal to E2 greater than 16 alpha-hydroxyestrone = 2 alpha-hydroxytestosterone greater than 16-epiestriol greater than or equal to estetrol greater than or equal to 17-epiestriol greater than or equal to 2-methoxyestradiol greater than or equal to 5 alpha-androstane-3 alpha,17 beta-diol greater than or equal to estrone greater than testosterone greater than or equal to 2 beta-hydroxytestosterone greater than 5 alpha-dihydrotestosterone. Affinity of estrogens and androgens for the UEBP diminishes abruptly after removal of 3- and 17-hydroxy groups, masking of these by ether bonds or changing of 17 beta-hydroxyl to 17 alpha. All the investigated 17 oxo-C19-steroids, 5 beta-derivatives of testosterone, its 6 beta- and 16 alpha-hydroxy metabolites as well as 5 alpha-androstane-3 beta,17 beta-diol and 19-nortestosterone exhibit no essential affinity for the protein. On the basis of the results obtained it is suggested that the binding sites for estrogens and androgens in the UEBP molecule overlap but do not completely coincide.     

On the systematics and phylogeography of eight-barbel loaches the genus Lefua (Cobitoidea: Nemacheilidae): mtDNA typing of L. pleskei

Comparative analysis of personal sequence data for the mtDNA control region (926 to 928 bp) from eight-barbel loaches inhabiting eight localities in the Amur River basin (4) and the Sea of Japan (4) and the GeneBank/NCBI data for the Lefua individuals from the other regions of the world showed that eight-barbel loaches from Primorskii krai water basins were marked by a specific group of mtDNA haplotypes. This finding is considered as supporting the species status of L. pleskei. Genetic distances within L. pleskei are small (on average 0.355) and close to those within L. nikkonis (on average 0.48%). The distances between this species pair are the least (on average 2.15%) among all other pairs compared. In MP, ML, and Bayesian trees, L. pleskei and L. nikkonis haplotypes formed a common clade with high statistically significant support. In all tree variants, L. costata mtDNA haplotypes were located out of the group of interest. A clade consisting of highly diverged lineages of Lefua sp. and L. echigonia haplotypes occupied even more independent position. The mtDNA haplotypes of L. pleskei and L. costata from the Amur River basin were evolutionary young and derived from the haplotypes found in these species from the Sea of Japan (L. pleskei) or the Yellow Sea (L. costata) basins. It is thereby suggested that both species rather recently migrated into the Amur River system. According to the molecular clock data, basal diversification of the eight-barbel loach lineages took place at the end of middle Miocene (about 11 to 12 Myr ago), while divergence of L. pleskei and L. costata ancestral forms probably occurred approximately, 5 Myr ago. Since all main lineages of eight-barbel loaches were found in the Sea of Japan basin (continental coastline and the islands), the divergence order and dispersal patterns of the Lafua species might have been largely determined by the geological development pattern of this water body and the adjacent territories.