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At least 19 recordsLinked to original sources

PubMed1998

Cloning and expression in Escherichia coli of the recombinant his-tagged DNA polymerases from Pyrococcus furiosus and Pyrococcus woesei.

Complete PCR-derived DNA fragments containing the structural genes for DNA polymerases of the archaeons Pyrococcus furiosus and Pyrococcus woesei were cloned into an expression vector. The clones expressing thermostable His-tagged DNA polymerases were selected. The cloned fragments were sequenced. The DNA sequences were verified to be authentic by sequencing several clones. The nucleotide (nt) sequence revealed that DNA polymerase of P. woesei (Pwo DNA polymerase) consists of 775 amino acids and has a molecular weight of 90,566. It shows 100% nucleotide identity to the nucleotide sequence of DNA polymerase from P. furiosus (Pfu DNA polymerase). The results confirm that nucleotide sequences of both archaeons (P. furiosus and P. woesei) are highly similar. The recombinant DNA polymerases (His-tagged Pfu and His-tagged Pwo) contained a polyhistidine tag at the N-terminus (43 additional amino acids) that allowed single-step isolation by Ni-affinity chromatography. We found that recombinant plasmids are toxic or unstable in the expressing strain BL21(DE3), even in the absence of the inducing agent, IPTG. However, the plasmids were stable in BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and His-tagged proteins were expressed upon IPTG addition. The proteins were purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Ni2+-Sepharose columns. The enzymes were characterized and displayed high DNA polymerase activity and thermostability. This bacterial expression system appears to be the method of choice for production of Pfu or Pwo DNA polymerases.

Amino Acid Sequence↗

PubMed2000

Comparison between Pyrococcus horikoshii and Pyrococcus abyssi genome sequences reveals linkage of restriction-modification genes with large genome polymorphisms.

Recent work suggests that restriction-modification gene complexes are mobile genetic elements that insert themselves into the genome and cause various genome rearrangements. In the present work, the complete genome sequences of Pyrococcus horikoshii and Pyrococcus abyssi, two species in a genus of hyperthermophilic archaeon (archaebacterium), were compared to detect large genome polymorphisms linked with restriction-modification gene homologs. Sequence alignments, GC content analysis, and codon usage analysis demonstrated the diversity of these homologs and revealed a possible case of relatively recent acquisition (horizontal transfer). In two cases out of the six large polymorphisms identified, there was insertion of a DNA segment with a modification gene homolog, accompanied by target deletion (simple substitution). In two other cases, homologous DNA segments carrying a modification gene homolog were present at different locations in the two genomes (transposition). In both cases, substitution (insertion/deletion) in one of the two loci was accompanied by inversion of adjacent chromosomal segment. In the fifth case, substitution by a DNA segment carrying type I restriction, modification, and specificity gene homologs was likewise accompanied by adjacent inversion. In the last case, two homologous DNA segments, were found at different loci in the two genomes (transposition), but only one of them had insertion of a modification homolog and an unknown ORF. The possible relationship of these polymorphisms to attack by restriction enzymes on the chromosome will be discussed.

Chromosome Inversion↗

PubMed1993

Purification and properties of extremely thermostable glutamate dehydrogenases from two hyperthermophilic archaebacteria, Pyrococcus woesei and Pyrococcus furiosus.

Glutamate dehydrogenase (L-glutamate: NADP oxidoreductase, deaminating, EC 1.4.1.4) from the hyperthermophilic archaebacteria Pyrococcus woesei and P. furiosus were purified to homogeneity from crude extracts. The enzymes had similar enzymological properties: molecular mass, subunit structure, optimum pHs for the oxidative deamination and reductive amination, substrate specificity and coenzyme specificity as well as thermostability; the activity was not lost after incubation at 105 degrees C for 30 min. However, some differences were detected in resistance to urea denaturation and effects of salts on their activity and stability. The N-terminal 20 amino acid sequences of the two enzymes were identical.

Amino Acid Sequence↗

PubMed1996

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↗

PubMed1999

Divergence of the hyperthermophilic archaea Pyrococcus furiosus and P. horikoshii inferred from complete genomic sequences.

Divergence of the hyperthermophilic Archaea, Pyrococcus furiosus and Pyrococcus horikoshii, was assessed by analysis of complete genomic sequences of both species. The average nucleotide identity between the genomic sequences is 70-75% within ORFs. The P. furiosus genome (1.908 mbp) is 170 kbp larger than the P. horikoshii genome (1.738 mbp) and the latter displays significant deletions in coding regions, including the trp, his, aro, leu-ile-val, arg, pro, cys, thr, and mal operons. P. horikoshii is auxotrophic for tryptophan and histidine and is unable to utilize maltose, unlike P. furiosus. In addition, the genomes differ considerably in gene order, displaying displacements and inversions. Six allelic intein sites are common to both Pyrococcus genomes, and two intein insertions occur in each species and not the other. The bacteria-like methylated chemotaxis proteins form a functional group in P. horikoshii, but are absent in P. furiosus. Two paralogous families of ferredoxin oxidoreductases provide evidence of gene duplication preceding the divergence of the Pyrococcus species.

Archaeal Proteins↗

PubMed2001

DNA protection mechanisms are not involved in the radioresistance of the hyperthermophilic archaea Pyrococcus abyssi and P. furiosus.

Hyperthermophilic archaea of the genus Pyrococcus are resistant to gamma radiation, suggesting that efficient mechanisms for DNA repair exist in these organisms. To determine whether protective mechanisms might also be implicated in this radioresistance, we have estimated the linear density of DNA double-stranded breaks caused by gamma irradiation in the genomic DNA of two Pyrococcus species, using Escherichia coli and the radioresistant bacterium Deinococcus radiodurans as controls. The linear density of double-stranded breaks was essentially the same in all four microorganisms when irradiation was carried under similar anaerobic conditions, indicating that no specific DNA protection mechanisms exist in Pyrococcus species. Using one- and two-dimensional gel electrophoresis we compared the protein patterns from Pyrococcus abyssi and P. furiosus cells that had or had not been exposed to gamma rays. We did not detect any significant protein induction following DNA damage in either species.

DNA Damage↗

PubMed2003

An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi.

The hyperthermophilic euryarchaeon Pyrococcus abyssi and the related species Pyrococcus furiosus and Pyrococcus horikoshii, whose genomes have been completely sequenced, are presently used as model organisms in different laboratories to study archaeal DNA replication and gene expression and to develop genetic tools for hyperthermophiles. We have performed an extensive re-annotation of the genome of P. abyssi to obtain an integrated view of its phylogeny, molecular biology and physiology. Many new functions are predicted for both informational and operational proteins. Moreover, several candidate genes have been identified that might encode missing links in key metabolic pathways, some of which have unique biochemical features. The great majority of Pyrococcus proteins are typical archaeal proteins and their phylogenetic pattern agrees with its position near the root of the archaeal tree. However, proteins probably from bacterial origin, including some from mesophilic bacteria, are also present in the P. abyssi genome.

Adaptation, Physiological↗

PubMed1991

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↗

PubMed1996

UV and ethyl methanesulfonate effects in hyperthermophilic archaea and isolation of auxotrophic mutants of Pyrococcus strains.

The lethal and mutagenic effects of ethyl methanesulfonate (EMS) and UV on nine archaeal strains belonging to each of the two described genera of Thermococcales, Pyrococcus and Thermococcus, were investigated. To test the efficiency of the EMS and UV mutagenesis under a variety of experimental conditions, we chose Pyrococcus abyssi strain GE5 as a model strain. We observed a strong induced mutagenicity in both cases, since the spontaneous mutation frequency (expressed as the frequency of resistance to 5-fluoroorotic acid) increased up to 150-fold with EMS and 400-fold with UV, after mutagen exposure. Although a heterogeneous response to the induced effects caused after EMS and UV exposures was detected for all the other sulfothermophilic archaea tested, an efficient mutagenicity of Pyrococcus-like isolates GE27, GE23, and GE9 was observed. Optimal procedures described for UV mutagenesis yielded a number of useful uracil auxotrophic mutant strains of Pyrococcus abyssi.

Archaea↗

PubMed1996

Protein-encoding genes in the sulfothermophilic archaea Sulfolobus and Pyrococcus.

A number of unrelated protein-encoding genes from sulfothermophilic archaea, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Pyrococcus furiosus and Pyrococcus woesei, has been analyzed. In the Sulfolobus genus, the content of A + T is significantly higher than that of C + G and the base usage follows the order, A > T > G > C. In Pyrococcus, the A + T content is also higher than that of C + G, but with lower values; in the order of base usage, G precedes T. The codon usage of these sulfothermophiles has been determined; alternative start codons are frequently used in both genera; codon preferences reflect the rich A + T composition of the corresponding genomes; for both genera the codon bias is particularly evident within the different arginine triplets, where AGA and AGG are predominant. From the similarities in the codon usage, close taxonomic relationships become evident within the Sulfolobus or the Pyrococcus genus; a lower, but significant similarity is also clear between these genera. The synonymous codon usage of these sulfothermophiles shows similarities with that of Saccharomyces cerevisiae and bovine mitochondria, whereas clear divergences are observed with the halophilic archaeal genus, Halobacterium, or the eubacterium, Escherichia coli. The unrelated proteins of the considered sulfothermophiles have been analyzed for the content of hydrophobic residues; the comparison with mesophiles reveals a significant increase in the average hydrophobicity of amino acid residues. This finding could indicate a mechanism of adaptation of proteins in organisms living under extreme environments. It is noteworthy that an opposite trend, i.e. a decreased average hydrophobicity, occurs in unrelated halophilic proteins.

Animals↗

PubMed2000

Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes.

Ribose methylation is a prevalent type of nucleotide modification in rRNA. Eukaryotic rRNAs display a complex pattern of ribose methylations, amounting to 55 in yeast Saccharomyces cerevisiae and about 100 in vertebrates. Ribose methylations of eukaryotic rRNAs are each guided by a cognate small RNA, belonging to the family of box C/D antisense snoRNAs, through transient formation of a specific base-pairing at the rRNA modification site. In prokaryotes, the pattern of rRNA ribose methylations has been fully characterized in a single species so far, Escherichia coli, which contains only four ribose methylated rRNA nucleotides. However, the hyperthermophile archaeon Sulfolobus solfataricus contains, like eukaryotes, a large number of (yet unmapped) rRNA ribose methylations and homologs of eukaryotic box C/D small nucleolar ribonuclear proteins have been identified in archaeal genomes. We have therefore searched archaeal genomes for potential homologs of eukaryotic methylation guide small nucleolar RNAs, by combining searches for structured motifs with homology searches. We have identified a family of 46 small RNAs, conserved in the genomes of three hyperthermophile Pyrococcus species, which we have experimentally characterized in Pyrococcus abyssi. The Pyrococcus small RNAs, the first reported homologs of methylation guide small nucleolar RNAs in organisms devoid of a nucleus, appear as a paradigm of minimalist box C/D antisense RNAs. They differ from their eukaryotic homologs by their outstanding structural homogeneity, extended consensus box motifs and the quasi-systematic presence of two (instead of one) rRNA antisense elements. Remarkably, for each small RNA the two antisense elements always match rRNA sequences close to each other in rRNA structure, suggesting an important role in rRNA folding. Only a few of the predicted P. abyssi rRNA ribose methylations have been detected so far. Further analysis of these archaeal small RNAs could provide new insights into the origin and functions of methylation guide small nucleolar RNAs and illuminate the still elusive role of rRNA ribose methylations.

Base Sequence↗

PubMed1999

Rapid extraction of plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi.

Hyperthermophilic archaea, specifically Pyrococcus spp., are the target of current efforts in developing heterologous expression systems. However, the published plasmid purification and plasmid screening protocols are long and tedious. We describe a fast, simple protocol for plasmid purification from Pyrococcus spp. developed while extracting the plasmid pGT5 from Pyrococcus abyssi cells. The protocol is modified from the procedures for commercial plasmid minipreps and is completed in about 20 min. The DNA is easily digested by restriction enzymes and can be used in sequencing reactions without additional purification.

DNA Restriction Enzymes↗

PubMed1998

Pyrococcus horikoshii sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa Trough.

A hyperthermophilic, anaerobic archaeon was isolated from hydrothermal fluid samples obtained at the Okinawa Trough vents in the NE Pacific Ocean, at a depth of 1395m. The strain is obligately heterotrophic, and utilizes complex proteinaceous media (peptone, tryptone, or yeast extract), or a 21-amino-acid mixture supplemented with vitamins, as growth substrates. Sulfur greatly enhances growth. The cells are irregular cocci with a tuft of flagella, growing optimally at 98 degrees C (maximum growth temperature 102 degrees C), but capable of prolonged survival at 105 degrees C. Optimum growth was at pH 7 (range 5-8) and NaCl concentration 2.4% (range 1%-5%). Tryptophan was required for growth, in contrast to the closely related strains Pyrococcus furiosus and P. abyssi. Thin sections of the cell, viewed by transmission electron microscopy, revealed a periplasmic space similar in appearance to the envelope of P. furiosus. The predominant cell membrane component was tetraether lipid, with minor amounts of diether lipids. Treatment of the cells by mild osmotic shock released an extract that contained a Zn(2+)-dependent alkaline phosphatase. Phylogenetic analysis of the sequences encoding 16S rRNA and glutamate dehydrogenase places the isolate with certainty within the genus Pyrococcus although there is relatively low DNA-DNA hybridization (< 63%) with described species of this genus. Based on the reported results, we propose a new species, to be named Pyrococcus horikoshii sp.nov.

Japan↗

PubMed2002

A solution NMR molecular model for the aspartate-ligated, cubane cluster containing ferredoxin from the hyperthermophilic archeaon Pyrococcus furiosus.

A solution molecular model for the conformationally dynamically heterogeneous Pyrococcus furiosus ferredoxin with an intact disulfide bond has been constructed on the basis of reported (1)H NMR spectral parameters using distance geometry and simulated annealing protocols. Conventional long-mixing time NOESY and H-bonding constraints have been augmented by previously reported short-mixing time NOESY, steady-state NOE, and cluster paramagnetism-induced relaxation. The family of 15 structures with inconsequential violations exhibited low rms deviations for backbone atoms for the overwhelming majority of the residues, including the cluster ligating loop with the unprecedented ligated Asp14. Larger rms deviations were observed across the disulfide bond, but closer inspection revealed that the 15 structures can be factored into 10 substructures exhibiting an "S" or right-handed disulfide orientation and 5 exhibiting an "R" or left-handed disulfide orientation. The remainder of the structure is indistinguishable for the two disulfide orientations but confirms stabilizing extensions of secondary structural elements in the lengthening of the long helix and both the lengthening and incorporation of a third strand into the beta-sheet involving the termini, with these extensions interacting strongly in a modular fashion through the rings of Tyr46 and Trp2. These extensions of stabilizing interactions in Pyrococcus furiosus Fd, however, lead to strong destabilization of the disulfide bond and destabilization of the highly conserved first and last beta-turns in the sequence. It is concluded that the structural alternations in Pyrococcus Fd relative to other hyperthermostable Fds are not to increase thermostability but to place "stress" on the disulfide bond and render it more reducible. The possible physiological implications of this unique reducible disulfide bond are discussed.

Amino Acid Sequence↗

PubMed1997

A novel DNA polymerase in the hyperthermophilic archaeon, Pyrococcus furiosus: gene cloning, expression, and characterization.

BACKGROUND: In many respects Archaea are much more like eukaryotes than prokaryotes with respect to the conservation of many of the components involved in transcription, translation and DNA replication. So far, only a few DNA polymerases with structures similar to those of eukaryotic DNA polymerase a have been found in Archaea. The identification and characterization of all the DNA polymerases of one archaeon would add considerably to our knowledge of the basic mechanisms of DNA replication in these organisms. RESULTS: We have identified a novel DNA polymerase composed of two proteins, DP1 and DP2, with molecular weights of 69294 Da and 143161 Da, respectively, in the hyperthermophilic archaeon, Pyrococcus furiosus, and have cloned the corresponding genes which are tandemly arranged on the Pyrococcus genome. No significant sequence homology was found between these two proteins and other known DNA polymerases. The pol genes were transcribed as part of a single operon that additionally contained genes homologous to the cdc18+/CDC6 and Dmc1/Rad51 family of proteins. We purified the Pyrococcus DNA polymerase from Escherichia coli strains expressing the cloned genes and characterized its activity. It possesses strong 3' --> 5' exonucleolytic activity and has a template-primer preference which is characteristic of a replicative DNA polymerase. CONCLUSION: In P. furiosus, we identified a second DNA polymerase encoded by two genes, neither of which display significant homology to any other known DNA polymerase. Both the enzymatic properties of the enzyme and the gene organization raise the possibility that this enzyme might be the replicative DNA polymerase of P. furiosus.

Antigens, CD↗

PubMed2002

Construction of a shuttle vector for, and spheroplast transformation of, the hyperthermophilic archaeon Pyrococcus abyssi.

Our understanding of the genetics of species of the best-studied hyperthermophilic archaea, Pyrococcus spp., is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon Pyrococcus abyssi. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from P. abyssi strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for Pyrococcus spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from P. abyssi strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the pyrE and/or pyrF genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two pyrE mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform P. abyssi GE9 pyrE mutants to prototrophy, though with low frequency (10(2) to 10(3) transformants per micro g of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both Escherichia coli and P. abyssi.

BH3 Interacting Domain Death Agonist Protein↗

PubMed1995

Chromosomal organization and nucleotide sequence of the fus-gene encoding elongation factor 2 (EF-2) of the hyperthermophilic archaeum Pyrococcus woesei.

A Pyrococcus woesei EcoRI DNA fragment (3400 bp) harbouring the gene fus for elongation factor 2 (EF-2) was cloned and almost completely sequenced. Unlike Methanococcus vannielii (which displays the 'str operon'-like fus and tuf gene context, 5'-rps12-rps7-fus-tuf-3'), and similar to Sulfolobus acidocaldarius and Desulfurococcus mobilis, the Pyrococcus fus gene (732 codons) is unlinked to the rps and tuf genes, and is immediately followed (57 bp intergenic spacing) by an ORF of 106 codons. Both ORFs are preceded by potential archaeal promoters located 52 bp (for fus) and 37 bp (for ORF106) upstream of the putative start codons. The Pyrococcus EF-2(G) equivalent factor is somewhat closer to the eukaryal than to the bacterial homolog, and also shares with the former the C-terminal sequence required for ADP ribosylation of EF-2 by Diphtheria toxin.

Amino Acid Sequence↗

PubMed2002

The X-ray crystal structure of pyrrolidone-carboxylate peptidase from hyperthermophilic archaea Pyrococcus horikoshii.

The crystal structure of pyrrolidone-carboxylate peptidase (PCP) from hyperthermophilic archaea Pyrococcus horikoshii (PhoPCP) has been determined at 1.6-A resolution by X-ray crystallography. PCP belongs to the C15 family of cysteine protease, and specifically removes the amino terminal pyroglutamate residue from a wide range of N-terminal-blocking peptides. The crystal structure is very similar to that of other hyperthermophiles, Pyrococcus furiosus and Thermococcus litoralis, and even that from the mesophile, Bacillus amyloliquefaciens. The inter-subunit disulfide bonds, which have been proposed as one of the thermostabilizing factors of the PCP from such hyperthermophiles, was not present in PhoPCP. The result suggests that the thermostability of PhoPCP may be obtained by the accumulation of many weak factors.

Amino Acid Sequence↗