Nucleotide sequence of the genes encoding the L30, S12 and S7 equivalent ribosomal proteins from the archaeum Thermococcus celer.
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Archaea↗
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Nucleotide sequence of the genes encoding the three largest subunits of the DNA-dependent RNA polymerase from the archaeum Thermococcus celer.
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Archaea↗
The nomenclatural types of the orders Acholeplasmatales, Halanaerobiales, Halobacteriales, Methanobacteriales, Methanococcales, Methanomicrobiales, Planctomycetales, Prochlorales, Sulfolobales, Thermococcales, Thermoproteales and Verrucomicrobiales are the genera Acholeplasma, Halanaerobium, Halobacterium, Methanobacterium, Methanococcus, Methanomicrobium, Planctomyces, Prochloron, Sulfolobus, Thermococcus, Thermoproteus and Verrucomicrobium, respectively. Opinion 79.
The Judicial Commission of the International Committee on Systematics of Prokaryotes has corrected the nomenclatural types of 12 orders: Acholeplasmatales, Halanaerobiales, Halobacteriales, Methanobacteriales, Methanococcales, Methanomicrobiales, Planctomycetales, Prochlorales, Sulfolobales, Thermococcales, Thermoproteales and Verrucomicrobiales.
Archaea↗
Localization of prefoldin interaction sites in the hyperthermophilic group II chaperonin and correlations between binding rate and protein transfer rate.
Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin for correct folding. The manner by which prefoldin interacts with a group II chaperonin is poorly understood. Here, we have examined the prefoldin interaction site in the archaeal group II chaperonin, comparing the interaction of two Thermococcus chaperonins and their mutants with Pyrococcus prefoldin by surface plasmon resonance. We show that the mutations of Lys250 and Lys256 of Thermococcus alpha chaperonin residues to Glu residues increase the affinity to Pyrococcus prefoldin to the level of Thermococcus beta chaperonin and Pyrococcus chaperonin, indicating that their Glu250 and Glu256 residues of the helical protrusion region are responsible for relatively stronger binding to Pyrococcus prefoldin than Thermococcus alpha chaperonin. Since the putative chaperonin binding sites in the distal ends of Pyrococcus prefoldin are rich in basic residues, electrostatic interaction seems to be important for their interaction. The substrate protein transfer rate from prefoldin correlates well with its affinity for chaperonin.
Amino Acid Sequence↗
Distribution and physiological characteristics of hyperthermophiles in the Kubiki oil reservoir in Niigata, Japan.
The distribution of culturable hyperthermophiles was studied in relation to environmental conditions in the Kubiki oil reservoir in Japan, where the temperature was between 50 and 58 degrees C. Dominant hyperthermophilic cocci and rods were isolated and shown to belong to the genera Thermococcus and Thermotoga, respectively, by 16S rDNA analyses. Using the most-probable-number method, we found that hyperthermophilic cocci were widely distributed in several unconnected fault blocks in the Kubiki oil reservoir. In 1996 to 1997, their populations in the production waters from oil wells were 9.2 x 10(3) to 4.6 x 10(4) cells/ml, or 10 to 42% of total cocci. On the other hand, hyperthermophilic rods were found in only one fault block of the reservoir with populations less than 10 cells/ml. Dominant Thermococcus and Thermotoga spp. grew at reservoir temperatures and utilized amino acids and sugars, respectively, as sole carbon sources. While organic carbon was plentiful in the environment, these hyperthermophiles were unable to grow in the formation water due to lack of essential nutrients. Concentrations of some organic and inorganic substances differed among fault blocks, indicating that the movement of formation water between fault blocks was restricted. This finding suggests that the supply of nutrients via fluid current is limited in this subterranean environment and that the organisms are starved in the oil reservoir. Under starved conditions at 50 degrees C, culturable cells of Thermococcus sp. remained around the initial cell density for about 200 days, while those of Thermotoga sp. decreased exponentially to 0. 01% of the initial cell density after incubation for the same period. The difference in survivability between these two hyperthermophiles seems to reflect their populations in the fault blocks. These results indicate that hyperthermophilic cocci and rods adapt to the subterranean environment of the Kubiki oil reservoir by developing an ability to survive under starved conditions.
Colony Count, Microbial↗
Compatible solutes of the hyperthermophile Palaeococcus ferrophilus: osmoadaptation and thermoadaptation in the order thermococcales.
The effect of salinity and growth temperature on the accumulation of intracellular organic solutes was examined in the hyperthermophilic archaeon Palaeococcus ferrophilus. The genus Palaeococcus represents a deep-branching lineage of the order Thermococcales, which diverged before Thermococcus and Pyrococcus. Palaeococcus ferrophilus accumulated mannosylglycerate, glutamate, and aspartate as major compatible solutes. Unlike members of the genera Pyrococcus and Thermococcus, Palaeococcus ferrophilus did not accumulate di-myo-inositol phosphate, a canonical solute of hyperthermophiles. The level of mannosylglycerate increased in response to both heat and salt stress; glutamate increased at supraoptimal growth temperatures, whereas aspartate increased at supraoptimal salt concentration. Proline, alanine, and trehalose were also found in lesser amounts, but their levels did not respond significantly to any of the stresses imposed. Additionally, the genes involved in the synthesis of mannosylglycerate in Palaeococcus ferrophilus and Thermococcus litoralis were identified. In both organisms the synthesis proceeds via the two-step pathway comprising mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). The mpgS and mpgP genes of Palaeococcus ferrophilus were expressed in Escherichia coli and the proteins were characterized. MPGS had maximal activity at 90 degrees C and pH near 7.0, and was strictly dependent on Mg2+. MPGP had optimal activity at 90 degrees C and pH 6.0 and was barely dependent on Mg2+. The half-life values for inactivation of MPGS and MPGP at 83 degrees C were 18 and 25 min, respectively. A comparative discussion of the osmo- and thermoadaptation strategies in these three genera of the Thermococcales is presented.
Acclimatization↗
Nucleotide sequence of a DNA region comprising the gene for elongation factor 1 alpha (EF-1 alpha) from the ultrathermophilic archaeote Pyrococcus woesei: phylogenetic implications.
The gene encoding elongation factor 1 alpha (EF-1 alpha, 1290 bp) of the ultrathermophilic, sulfur-reducing archaeote Pyrococcus woesei was localized within a Bg/II fragment of chromosomal DNA. Sequence analysis showed that the EF-1 alpha gene is the upstream unit of a three-gene cluster comprising the genes for ribosomal protein S10 (306 bp) and transfer RNAser (GGA). The three genes follow each other immediately in the order EF-1 alpha.S10.tRNA(ser) after a putative promoter located 55 bp upstream of the EF-1 alpha gene. Alignment of the derived EF-1 alpha sequence with the corresponding sequences from Eukarya, Bacteria/organelles, and with available archaeal sequences (Sulfolobus, Thermococcus, Methanococcus, Halobacterium) showed that Pyrococcus EF-1 alpha is highly homologous (89% identity) to Thermococcus celer EF-1 alpha, both being strikingly more similar to eukaryotic EF-1 alpha than to bacterial EF-Tu. Unrooted dendrograms computed from aligned sequences by distance matrix and DNA parsimony methods, including evolutionary parsimony, showed the Archaea to be a monophyletic-holophyletic cluster closer to Eukarya than to Bacteria. Both distance matrix and DNA parsimony--although not evolutionary parsimony--support the partition of the known archaeal lineages between the kingdoms Crenarchaeota and Euryarchaeota, and the affiliation of the Pyrococcus-Thermococcus lineage to the Euryarchaeota, of which it is the most primitive offspring. A closer relation of Pyrococcus to Euryarchaeota than to Crenarchaeota was also inferred from sequence analysis of S10 ribosomal proteins.
Amino Acid Sequence↗
Comparative analysis of Embden-Meyerhof and Entner-Doudoroff glycolytic pathways in hyperthermophilic archaea and the bacterium Thermotoga.
The Embden-Meyerhof (EM) or Entner-Doudoroff (ED) pathways of sugar degradation were analyzed in representative species of the hyperthermophilic archaeal genera Thermococcus, Desulfurococcus, Thermoproteus, and Sulfolobus, and in the hyperthermophilic (eu)bacterial genus Thermotoga. The analyses included (1) determination of 13C-labeling patterns by 1H- and 13C-NMR spectroscopy of fermentation products derived from pyruvate after fermentation of specifically 13C-labeled glucose by cell suspensions, (2) identification of intermediates of sugar degradation after conversion of 14C-labeled glucose by cell extracts, and (3) measurements of enzyme activities in cell extracts. Thermococcus celer and Thermococcus litoralis fermented 13C-glucose to acetate and alanine via a modified EM pathway (100%). This modification involves ADP-dependent hexokinase, 6-phosphofructokinase, and glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (GAP:FdOR). Desulfurococcus amylolyticus fermented 13C-glucose to acetate via a modified EM pathway in which GAP:FdOR replaces GAP-DH/phosphoglycerate kinase. Thermoproteus tenax fermented 13C-glucose to low amounts of acetate and alanine via simultaneous operation of the EM pathway (85%) and the ED pathway (15%). Aerobic Sulfolobus acidocaldarius fermented 13C-labeled glucose to low amounts of acetate and alanine exclusively via the ED pathway. The anaerobic (eu)bacterium Thermotoga maritima fermented 13C-glucose to acetate and lactate via the EM pathway (85%) and the ED pathway (15%). Cell extracts contained glucose-6-phosphate dehydrogenase and 2-keto-3-deoxy-6-phosphogluconate aldolase, key enzymes of the conventional phosphorylated ED pathway, and, as reported previously, all enzymes of the conventional EM pathway. In conclusion, glucose was degraded by hyperthermophilic archaea to pyruvate either via modified EM pathways with different types of hexose kinases and GAP-oxidizing enzymes, by the nonphosphorylated ED pathway, or by a combination of both pathways. In contrast, glucose catabolism in the hyperthermophilic (eu)bacterium Thermotoga involves the conventional forms of the EM and ED pathways. The data are in accordance with various previous reports.
Archaea↗
Vitamin contents of archaebacteria.
The levels of six water-soluble vitamins of seven archaebacterial species were determined and compared with the levels found in a eubacterium, Escherichia coli. Biotin, riboflavin, pantothenic acid, nicotinic acid, pyridoxine, and lipoic acid contents of Halobacterium volcanii, Methanobacterium thermoautotrophicum delta H, "Archaeoglobus fulgidus" VC-16, Thermococcus celer, Pyrodictium occultum, Thermoproteus tenax, and Sulfolobus solfataricus were measured by using bioassays. The archaebacteria examined were found to contain these vitamins at levels similar to or significantly below the levels found in in E. coli. Riboflavin was found at levels comparable to those in E. coli. Pyridoxine was as abundant among the archaebacteria of the methanogenhalophile branch as in E. coli. It was only one-half as abundant in the sulfur-metabolizing branch. "A. fulgidus," however, contained only 4% as much pyridoxine as E. coli. Nicotinic and pantothenic acids were approximately 10-fold less abundant (except for a 200-fold-lower nicotinic acid level in "A. fulgidus"). Nicotinic acid may be replaced by an 8-hydroxy-5-deazaflavin coenzyme (factor F420) in some archaebacteria (such as "A. fulgidus"). Compared with the level in E. coli, biotin was equally as abundant in Thermococcus celer and Methanobacterium thermoautotrophicum, about one-fourth less abundant in P. occultum and "A. fulgidus," and 25 to over 100 times less abundant in the others. The level of lipoic acid was up to 20 times lower in H. volcanii, Methanobacterium thermoautotrophicum, and Thermococcus celer. It was over two orders of magnitude lower among the remaining organisms. With the exception of "A. fulgidus," lipoic acid, pantothenic acid, and pyridoxine were more abundant in the members of the methanogen-halophile branch of the archaebacteria than in the sulfur-metabolizing branch.
Archaea↗
Glycine at the 65th position plays an essential role in ATP-dependent protein folding by Archael group II chaperonin.
In the previous study, we have found that G65C and I125T double mutant of alpha chaperonin homo-oligomer from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1, lacks ATP-dependent protein refolding activity despite showing ATPase activity and the ability to bind the denatured proteins. In this study, we have characterized several mutant Thermococcus chaperonin homo-oligomers with the amino acid substitutions of Gly-65 or Ile-125. The results showed that amino acid residue at 65th position should be a small amino acid such as glycine or alanine for the ATP-dependent refolding activity. The alpha chaperonin homo-oligomers with amino acid substitution of Gly-65 by amino acids whose side chains are larger than the methyl group did not have ATP-dependent protein refolding activity, but exhibited an increase of the binding affinity for unfolded proteins in the presence of ATP or AMP-PNP. (c)2001 Elsevier Science.
Adenosine Triphosphatases↗
Archaeal group II chaperonin mediates protein folding in the cis-cavity without a detachable GroES-like co-chaperonin.
Group II chaperonins of archaea and eukaryotes are distinct from group I chaperonins of bacteria. Whereas group I chaperonins require the co-chaperonin Cpn-10 or GroES for protein folding, no co-chaperonin has been known for group II. The protein folding mechanism of group II chaperonins is not yet clear. To understand this mechanism, we examined protein refolding by the recombinant alpha or beta-subunit chaperonin homo-oligomer (alpha16mer and beta16mer) from a hyperthermoplilic archaeum, Thermococcus strain KS-1, using a model substrate, green fluorescent protein (GFP). The alpha16mer and beta16mer captured the non-native GFP and promoted its refolding without any co-chaperonin in an ATP dependent manner. A non-hydrolyzable ATP analog, AMP-PNP, induced the GFP refolding mediated by beta16mer but not by the alpha16mer. A mutant alpha-subunit chaperonin homo-oligomer (trap-alpha) could capture the non-native protein but lacked the ability to refold it. Although trap-alpha suppressed ATP-dependent refolding of GFP mediated by alpha16mer or beta16mer, it did not affect the AMP-PNP-dependent refolding. This indicated that the GFP refolding mediated by beta16mer with AMP-PNP was not accessible to the trap-alpha. Gel filtration chromatography and a protease protection experiment revealed that this refolded GFP, in the presence of AMP-PNP, was associated with beta16mer. After the completion of GFP refolding mediated by beta16mer with AMP-PNP, addition of ATP induced an additional refolding of GFP. Furthermore, the beta16mer preincubated with AMP-PNP showed the ability to capture the non-native GFP. These suggest that AMP-PNP induced one of two chaperonin rings (cis-ring) to close and induced protein refolding in this ring, and that the other ring (trans-ring) could capture the unfolded GFP which was refolded by adding ATP. The present data indicate that, in the group II chaperonin of Thermococcus strain KS-1, the protein folding proceeds in its cis-ring in an ATP-dependent fashion without any co-chaperonin.
Adenosine Triphosphate↗
Continuous enrichment culture and molecular monitoring to investigate the microbial diversity of thermophiles inhabiting deep-sea hydrothermal ecosystems.
The microflora developing during a continuous enrichment culture from a hydrothermal chimney sample was investigated by molecular methods. The culture was performed in a gas-lift bioreactor under anaerobic conditions, at 90 degrees C and pH 6.5, on a complex medium containing sulfur as the terminal electron acceptor. Archaeal and bacterial diversity was studied. Microorganisms affiliated with the genera Pyrococcus, Marinitoga, and Bacillus were detected through DGGE analysis of 16S rDNA. Additional sequences phylogenetically related to Thermococcus and epsilon-Proteobacteria were detected by cloning and sequencing of 16S rDNA from two samples of the enrichment culture. In comparison, the sequences retrieved from cloning analysis from an enrichment culture performed in a flask (batch condition) using the same culture medium showed that only members of the genus Thermococcus were cultivated. Therefore, continuous enrichment culture using the gas-lift bioreactor can be considered as an efficient and improved method for investigating microbial communities originating from deep-sea hydrothermal vents.
Atlantic Ocean↗
The first evidence of anaerobic CO oxidation coupled with H2 production by a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent.
From 24 samples of hydrothermal venting structures collected at the East Pacific Rise (13 degrees N), 13 enrichments of coccoid cells were obtained which grew on CO, producing H2 and CO2 at 80 degrees C. A hyperthermophilic archaeon capable of lithotrophic growth on CO coupled with equimolar production of H2 was isolated. Based on its 16S rRNA sequence analysis, this organism was affiliated with the genus Thermococcus. Other strains of Thermococcales species ( Pyrococcus furiosus, Thermococcus peptonophilus, T. profundus, T. chitonophagus, T. stetteri, T. gorgonarius, T. litoralis, and T. pacificus) were shown to be unable to grow on CO. Searches in sequence databases failed to reveal deposited sequences of genes related to CO metabolism in Thermococcales. Our work provides the first evidence of anaerobic CO oxidation coupled with H2 production performed by an archaeon as well as the first documented case of lithotrophic growth of a Thermococcales representative.
Anaerobiosis↗
The ribosomal gene spacer region in archaebacteria.
Sequences for the spacer regions that separate the 16S and 23S ribosomal RNA genes have been determined for four more (strategically placed) archaebacteria. These confirm the general rule that methanogens and extreme halophiles have spacers that contain a single tRNAala gene, while tRNA genes are not found in the spacer region of the true extreme thermophiles. The present study also shows that the spacer regions from the sulfate reducing Archaeglobus and the extreme thermophile Thermococcus (both of which cluster phylogenetically with the methanogens and extreme halophiles) contain each a tRNAala gene. Thus, not only all methanogens and extreme halophiles show this characteristic, but all organisms on the "methanogen branch" of the archaebacterial tree appear to do so. The finding of a tRNA gene in the spacer region of the extreme thermophile Thermococcus celer is the first known phenotypic property that links this organism with its phylogenetic counterparts, the methanogens, rather than with its phenotypic counterparts, the sulfur-dependent extreme thermophiles.
Alanine↗
Isolation of new plasmids from hyperthermophilic Archaea of the order Thermococcales.
A collection of 57 strains of hyperthermophilic Archaea from the order Thermococcales was screened for the presence of plasmids; 9 plasmids present in six of these strains were isolated and characterized in terms of size and cross-hybridization. The Notl macrorestriction patterns of genomic DNA of strains harbouring these plasmids were obtained. Pyrococcus abyssi strains GE27 and GE23 as well as Thermococcus sp. GE31 contained a single plasmid of 3.5 kb (pGN27), 16.8 kb (pGN23) and 5.3 kb (pGN31), respectively, whilst the three strains I559, I560 and I690 all contained two plasmids of 3.5 kb (pSN559, pSN560, pSN690) and 24 kb (pLN559, pLN560, pLN690), respectively. Plasmid pGN27 strongly cross-hybridized with the previously described plasmid pGT5 from P. abyssi strain GE5, whilst plasmids pGN23 and pGN31 did not cross-hybridize with each other, nor with any other plasmid. The three small plasmids of strains I559, I560 and I690 cross-hybridized, as well as their three large plasmids. Macrorestriction pattern analysis and the results of plasmid cross-hybridization experiments indicated that these three strains were different but closely related, and likely belonged to the genus Thermococcus. This study shows that plasmids are widespread in hyperthermophilic archaea, and significantly increases the number and diversity of plasmids available for laboratory work.
Blotting, Southern↗
Molecular diversity of new Thermococcales isolates from a single area of hydrothermal deep-sea vents as revealed by randomly amplified polymorphic DNA fingerprinting and 16S rRNA gene sequence analysis.
Members of the Thermococcales are anaerobic Archaea belonging to the kingdom Euryarchaea that are studied in many laboratories as model organisms for hyperthermophiles. We describe here a molecular analysis of 86 new Thermococcales isolates collected from six different chimneys of a single hydrothermal field located in the 13 degrees N 104 degrees W segment of the East Pacific ridge at a depth of 2,330 m. These isolates were sorted by randomly amplified polymorphic DNA (RAPD) fingerprinting into nine groups, and nine unique RAPD profiles were obtained. One RAPD group corresponds to new isolates of Thermococcus hydrothermalis, whereas all other groups and isolates with unique profiles are different from the 22 reference strains included in this study. Analysis of 16S rRNA gene sequences of representatives of each RAPD group and unique profiles showed that one group corresponds to Pyrococcus strains, whereas all the other isolates are Thermococcus strains. We estimated that our collection may contain at least 11 new species. These putative species, isolated from a single area of hydrothermal deep-sea vents, are dispersed in the 16S rRNA tree among the reference strains previously isolated from diverse hot environments (terrestrial, shallow water, hydrothermal vents) located around the world, suggesting that there is a high degree of dispersal of Thermococcales: About one-half of our isolates contain extrachromosomal elements that could be used to search for novel replication proteins and to develop genetic tools for hyperthermophiles.
Base Sequence↗
A cell-free transcription system for the hyperthermophilic archaeon Pyrococcus furiosus.
We describe here the establishment of a cell-free transcription system for the hyperthermophilic Archaeon Pyrococcus furiosus using the cloned glutamate dehydrogenase (gdh) gene as template. The in vitro system that operated up to a temperature of 85 degrees C initiated transcription 23 bp downstream of a TATA box located 45 bp upstream of the translational start codon of gdh mRNA, at the same site as in Pyrococcus cells. Mutational analyses revealed that this TATA box is essential for in vitro initiation of transcription. Pyrococcus transcriptional components were separated into at least two distinct transcription factor activities and RNA polymerase. One of these transcription factors could be functionally replaced by Methanococcus aTFB and Thermococcus TATA bind- ing protein (TBP). Immunochemical analyses demonstrated a structural relationship between Pyrococcus aTFB and Thermococcus TBP. These findings indicate that a TATA box and a TBP are essential components of the Pyrococcus transcriptional machinery.
Archaea↗
Evidence for the presence of thermophilic Fe(III)-reducing microorganisms in deep-sea hydrothermal vents at 13 degrees N (East Pacific Rise).
Microorganisms capable of dissimilatory Fe(III) reduction in the temperature range of 52-90 degrees C were enriched from outer and inner parts of chimney-like structures, tubes of the polychaetous annelid Alvinella sp., and hydrothermal fluids collected at 13 degrees N hydrothermal vent sites on the East Pacific Rise at a depth of 2650 m. Numbers of culturable Fe(III)-reducing thermophiles estimated by the serial dilution technique varied from 10 to 10(7) cells per cm(3) of sample. Phylogenetic analysis of bacterial and archaeal PCR-amplified 16S rDNA genes obtained from Fe(III)-reducing enrichments and separated by denaturing gradient gel electrophoresis revealed sequences related to Deferibacter, Thermotogales (Bacteria) and Thermococcus (Archaea) for which the capacity for Fe(III) reduction had been reported. This was confirmed by isolating a hyperthermophilic iron reducer that belongs to the genus Thermococcus. Other bacterial thermophiles found in the enrichments were related to so far uncultured members of the Clostridiaceae, and epsilon-subdivision of the Proteobacteria.
Journal Article↗