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Michael W W Adams - One of the best experts on this subject based on the ideXlab platform.
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Defining Components of the Chromosomal Origin of Replication of the Hyperthermophilic Archaeon Pyrococcus furiosus Needed for Construction of a Stable Replicating Shuttle Vector
Applied and Environmental Microbiology, 2011Co-Authors: Joel Farkas, Megan Debarry, Daehwan Chung, Michael W W Adams, Janet WestphelingAbstract:We report the construction of a series of replicating shuttle vectors that consist of a low-copy-number cloning vector for Escherichia coli and functional components of the origin of replication ( oriC ) of the chromosome of the hyperthermophilic archaeon Pyrococcus furiosus . In the process of identifying the minimum replication origin sequence required for autonomous plasmid replication in P. furiosus , we discovered that several features of the origin predicted by bioinformatic analysis and in vitro binding studies were not essential for stable autonomous plasmid replication. A minimum region required to promote plasmid DNA replication was identified, and plasmids based on this sequence readily transformed P. furiosus . The plasmids replicated autonomously and existed in a single copy. In contrast to shuttle vectors based on a plasmid from the closely related hyperthermophile Pyrococcus abyssi for use in P. furiosus , plasmids based on the P. furiosus chromosomal origin were structurally unchanged after transformation and were stable without selection for more than 100 generations.
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Identification of Protein-Bound Dinitrosyl Iron Complexes by Nuclear Resonance Vibrational Spectroscopy
Journal of the American Chemical Society, 2010Co-Authors: Zachary J. Tonzetich, Francis E. Jenney, Michael W W Adams, Hongxin Wang, Devrani Mitra, Christine E. Tinberg, Loi H., Stephen P. Cramer, Stephen J. LippardAbstract:We have applied 57Fe nuclear resonance vibrational spectroscopy (NRVS) to identify protein-bound dinitrosyl iron complexes. Intense NRVS peaks due to vibrations of the N−Fe−N unit can be observed between 500 and 700 cm−1 and are diagnostic indicators of the type of iron dinitrosyl species present. NRVS spectra for four iron dinitrosyl model compounds are presented and used as benchmarks for the identification of species formed in the reaction of Pyrococcus furiosus ferredoxin D14C with nitric oxide.
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normal mode analysis of Pyrococcus furiosus rubredoxin via nuclear resonance vibrational spectroscopy nrvs and resonance raman spectroscopy
Journal of the American Chemical Society, 2005Co-Authors: Yuming Xiao, Francis E. Jenney, Michael W W Adams, Hongxin Wang, Simon J George, Matt C Smith, W Sturhahn, Jiyong Zhao, Yoshitaka Yoda, Edward I SolomonAbstract:We have used 57Fe nuclear resonance vibrational spectroscopy (NRVS) to study the Fe(Scys)4 site in reduced and oxidized rubredoxin (Rd) from Pyrococcus furiosus (Pf). The oxidized form has also bee...
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Structure of the prolidase from Pyrococcus furiosus.
Biochemistry, 2004Co-Authors: Megan J. Maher, Michael W W Adams, Angeli Lal Menon, Amy M. Grunden, Mousumi Ghosh, Hans C. Freeman, J. Mitchell GussAbstract:The structure of prolidase from the hyperthermophilic archaeon Pyrococcus furiosus (Pfprol) has been solved and refined at 2.0 A resolution. This is the first structure of a prolidase, i.e., a peptidase specific for dipeptides having proline as the second residue. The asymmetric unit of the crystals contains a homodimer of the enzyme. Each of the two protein subunits has two domains. The C-terminal domain includes the catalytic site, which is centered on a dinuclear metal cluster. In the as-isolated form of Pfprol, the active-site metal atoms are Co(II) [Ghosh, M., et al. (1998) J. Bacteriol. 180, 4781−9]. An unexpected finding is that in the crystalline enzyme the active-site metal atoms are Zn(II), presumably as a result of metal exchange during crystallization. Both of the Zn(II) atoms are five-coordinate. The ligands include a bridging water molecule or hydroxide ion, which is likely to act as a nucleophile in the catalytic reaction. The two-domain polypeptide fold of Pfprol is similar to the folds of...
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structural basis for thermostability in aporubredoxins from Pyrococcus furiosus and clostridium pasteurianum
Biochemistry, 2001Co-Authors: Edward R Zartler, Francis E. Jenney, Michael W W Adams, Mark Terrell, Marly K Eidsness, James H PrestegardAbstract:The structures of apo- and holorubredoxins from Pyrococcus furiosus (PfRd) and Clostridium pasteurianum (CpRd) have been investigated and compared using residual dipolar couplings to probe the orig...
Philippe Oger - One of the best experts on this subject based on the ideXlab platform.
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Novel intact polar and core lipid compositions in the Pyrococcus model species, P. furiosus and P. yayanosii, reveal the largest lipid diversity amongst Thermococcales
Biomolecules, 2020Co-Authors: Maxime Tourte, Vanessa Kuentz, Philippe Schaeffer, Vincent Grossi, Anaïs Cario, Philippe OgerAbstract:Elucidating the lipidome of Archaea is essential to understand their tolerance to extreme environmental conditions. Previous characterizations of the lipid composition of Pyrococcus species, a model genus of hyperthermophilic archaea belonging to the Thermococcales order, led to conflicting results, which hindered the comprehension of their membrane structure and the putative adaptive role of their lipids. In an effort to clarify the lipid composition data of the Pyrococcus genus, we thoroughly investigated the distribution of both the core lipids (CL) and intact polar lipids (IPL) of the model Pyrococcus furiosus and, for the first time, of Pyrococcus yayanosii, the sole obligate piezophilic hyperthermophilic archaeon known to date. We showed a low diversity of IPL in the lipid extract of P. furiosus, which nonetheless allowed the first report of phosphatidyl inositol-based glycerol mono-and trialkyl glycerol tetraethers. With up to 13 different CL structures identified, the acid methanolysis of Pyrococcus furiosus revealed an unprecedented CL diversity and showed strong discrepancies with the IPL compositions reported here and in previous studies. By contrast, P. yayanosii displayed fewer CL structures but a much wider variety of polar heads. Our results showed severe inconsistencies between IPL and CL relative abundances. Such differences highlight the diversity and complexity of the Pyrococcus plasma membrane composition and demonstrate that a large part of its lipids remains uncharacterized. Reassessing the lipid composition of model archaea should lead to a better understanding of the structural diversity of their lipidome and of their physiological and adaptive functions.
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Pyrococcus kukulkanii sp nov a hyperthermophilic piezophilic archaeon isolated from a deep sea hydrothermal vent
International Journal of Systematic and Evolutionary Microbiology, 2016Co-Authors: Nolwenn Callac, Philippe Oger, Francoise Lesongeur, Jayne E Rattray, Pauline Vannier, Gregoire Michoud, Mickael BeauvergerAbstract:A novel hyperthermophilic, piezophilic, anaerobic archaeon, designated NCB100T, was isolated from a hydrothermal vent flange fragment collected in the Guaymas basin at the hydrothermal vent site named ‘Rebecca’s Roost’ at a depth of 1997 m. Enrichment and isolation were performed at 100 °C under atmospheric pressure. Cells of strain NCB100T were highly motile, irregular cocci with a diameter of ~1 µm. Growth was recorded at temperatures between 70 and 112 °C (optimum 105 °C) and hydrostatic pressures of 0.1–80 MPa (optimum 40–50 MPa). Growth was observed at pH 3.5–8.5 (optimum pH 7) and with 1.5–7 % NaCl (optimum at 2.5–3 %). Strain NCB100T was a strictly anaerobic chemo-organoheterotroph and grew on complex proteinaceous substrates such as yeast extract, peptone and tryptone, as well as on glycogen and starch. Elemental sulfur was required for growth and was reduced to hydrogen sulfide. The fermentation products from complex proteinaceous substrates were CO2 and H2. The G+C content of the genomic DNA was 41.3 %. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain NCB100T belongs to the genus Pyrococcus , showing 99 % similarity with the other described species of the genus Pyrococcus . On the basis of physiological characteristics, DNA G+C content, similarity level between ribosomal proteins and an average nucleotide identity value of 79 %, strain NCB100T represents a novel species for which the name Pyrococcus kukulkanii sp. nov. is proposed. The type strain is NCB100T (=DSM 101590T=Souchotheque de Bretagne BG1337T).
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Pyrococcus yayanosii sp nov an obligate piezophilic hyperthermophilic archaeon isolated from a deep sea hydrothermal vent
International Journal of Systematic and Evolutionary Microbiology, 2011Co-Authors: Joël Querellou, Philippe Oger, Xiang Zeng, Jeanlouis Birrien, Mohamed Jebbar, Marieanne CambonbonavitaAbstract:An obligate piezophilic anaerobic hyperthermophilic archaeon, designated strain CH1(T), was isolated from a hydrothermal vent site named 'Ashadze', which is located on the Mid-Atlantic Ridge at a depth of 4100 m. Enrichment and isolation of the strain were carried out at 95 °C under a hydrostatic pressure of 42 MPa. Cells of strain CH1(T) were highly motile irregular cocci with a diameter of ~1-1.5 µm. Growth was recorded at 80-108 °C (optimum 98 °C) and at pressures of 20-120 MPa (optimum 52 MPa). No growth was observed under atmospheric pressures at 60-110 °C. Growth was observed at pH 6.0-9.5 (optimum 7.5-8.0) and in 2.5-5.5% (w/v) NaCl (optimum 3.5%). Strain CH1(T) was strictly anaerobic and grew on complex proteinaceous substrates, such as yeast extract, Peptone, and casein, as well as on sucrose, starch, chitin, pyruvate, acetate and glycerol without electron acceptors. The G+C content of the genomic DNA was 49.0±0.5 mol%. Analysis of 16S rRNA gene sequences revealed that strain CH1(T) belongs to the genus Pyrococcus. Based on its physiological properties and similarity levels between ribosomal proteins, strain CH1(T) represents a novel species, for which the name Pyrococcus yayanosii sp. nov. is proposed. The type strain is CH1(T) (=JCM 16557). This strain is also available by request from the Souchotheque de Bretagne (catalogue LMBE) culture collection (collection no. 3310).
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Pyrococcus ch1 an obligate piezophilic hyperthermophile extending the upper pressure temperature limits for life
The ISME Journal, 2009Co-Authors: Joël Querellou, Philippe Oger, Xiang Zeng, Jeanlouis Birrien, Yves Fouquet, Georgy Cherkashov, Mohamed Jebbar, Marieanne Cambonbonavita, Xiang XiaoAbstract:A novel hydrothermal site was discovered in March 2007, on the mid-Atlantic ridge during the cruise 'Serpentine'. At a depth of 4100 m, the site 'Ashadze' is the deepest vent field known so far. Smoker samples were collected with the ROV 'Victor 6000' and processed in the laboratory for the enrichment of anaerobic heterotrophic microorganisms under high-temperature and high-hydrostatic pressure conditions. Strain CH1 was successfully isolated and assigned to the genus Pyrococcus, within the Euryarchaeota lineage within the Archaea domain. This organism grows within a temperature range of 80 to 108 degrees C and a pressure range of 20 to 120 MPa, with optima for 98 degrees C and 52 MPa respectively. Pyrococcus CH1 represents the first obligate piezophilic hyperthermophilic microorganism known so far. Comparisons of growth yields obtained under high-temperature/high-pressure conditions for relative organisms isolated from various depths, showed clear relationships between depth at origin and responses to hydrostatic pressure.
Daniel Prieur - One of the best experts on this subject based on the ideXlab platform.
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Experimental silicification of the extremophilic Archaea Pyrococcus abyssi and Methanocaldococcus jannaschii: applications in the search for evidence of life in early Earth and extraterrestrial rocks
Geobiology, 2009Co-Authors: François Orange, Daniel Prieur, M. Le Romancer, Francès Westall, Jean-robert Disnar, N. Bienvenu, Christian DéfargeAbstract:Hydrothermal activity was common on the early Earth and associated micro-organisms would most likely have included thermophilic to hyperthermophilic species. 3.5–3.3 billion-year-old, hydrothermally influenced rocks contain silicified microbial mats and colonies that must have been bathed in warm to hot hydrothermal emanations. Could they represent thermophilic or hyperthermophilic micro-organisms and if so, how were they preserved? We present the results of an experiment to silicify anaerobic, hyperthermophilic micro-organisms from the Archaea Domain Pyrococcus abyssi and Methanocaldococcus jannaschii, that could have lived on the early Earth. The micro-organisms were placed in a silica-saturated medium for periods up to 1 year. Pyrococcus abyssi cells were fossilized but the M. jannaschii cells lysed naturally after the exponential growth phase, apart from a few cells and cell remains, and were not silicified although their extracellular polymeric substances were. In this first simulated fossilization of archaeal strains, our results suggest that differences between species have a strong influence on the potential for different micro-organisms to be preserved by fossilization and that those found in the fossil record represent probably only a part of the original diversity. Our results have important consequences for biosignatures in hydrothermal or hydrothermally influenced deposits on Earth, as well as on early Mars, as environmental conditions were similar on the young terrestrial planets and traces of early Martian life may have been similarly preserved as silicified microfossils.
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Physiological Responses of the Hyperthermophilic Archaeon “Pyrococcus abyssi” to DNA Damage Caused by Ionizing Radiation
Journal of Bacteriology, 2003Co-Authors: Edmond Jolivet, Yoshizumi Ishino, Patrick Forterre, Daniel Prieur, Fujihiko Matsunaga, Hannu MyllykallioAbstract:The mechanisms by which hyperthermophilic Archaea, such as “Pyrococcus abyssi” and Pyrococcus furiosus, survive high doses of ionizing gamma irradiation are not thoroughly elucidated. Following gamma-ray irradiation at 2,500 Gy, the restoration of “P. abyssi” chromosomes took place within chromosome fragmentation. DNA synthesis in irradiated “P. abyssi” cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism. We also found evidence for transient export of damaged DNA out of irradiated “P. abyssi” cells prior to a restart of chromosomal DNA synthesis. Our cell fractionation assays further suggest that “P. abyssi” contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation.
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An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi
Molecular Microbiology, 2003Co-Authors: Georges N. Cohen, Daniel Prieur, Didier Flament, Valérie Barbe, Michael Y. Galperin, Roland Heilig, Odile Lecompte, Olivier Poch, Joël Querellou, Raymond RippAbstract:Summary 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 phylo- geny, 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.
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Construction of a shuttle vector for, and spheroplast transformation of, the hyperthermophilic archaeon Pyrococcus abyssi.
Applied and Environmental Microbiology, 2002Co-Authors: S. Lucas, Yvan Zivanovic, Patrick Forterre, Daniel Prieur, Laurent Toffin, Daniel Charlier, Hélène Moussard, Gaël ErausoAbstract: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 (102 to 103 transformants per μg 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.
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DNA protection mechanisms are not involved in the radioresistance of the hyperthermophilic archaea Pyrococcus abyssi and P. furiosus.
Molecular Genetics and Genomics, 2001Co-Authors: E. Gérard, Edmond Jolivet, Daniel Prieur, Patrick ForterreAbstract: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.
Patrick Forterre - One of the best experts on this subject based on the ideXlab platform.
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pGT5 replication initiator protein Rep75 from Pyrococcus abyssi.
Methods in Enzymology, 2004Co-Authors: Stéphanie Marsin, Patrick ForterreAbstract:Publisher Summary Plasmids are extrachromosomal elements that can replicate autonomously in host cells. They are well known and studied in bacteria and have allowed the development of essential genetic tools in many bacterial species. Plasmids have also been very useful in studying fundamental cellular mechanisms, such as DNA replication. The plasmid pGT5 (3.4 kb), isolated from the euryarchaeon Pyrococcus abyssi , was the first plasmid studied in hyperthermophilic archaea.1 It was completely sequenced and shown to replicate via the rolling circle (RC) mechanism. RC replicons encode a replication initiator protein (usually named Rep) that exhibits a site-specific endonuclease/ligase activity. Several Rep protein encoded by bacterial or eukaryal RC replicons have been studied in vivo and in vitro , and they all share common characteristics. The purified Rep protein of pGT5 (Rep75) exhibits a highly thermophilic and specific nicking-closing (NC) activity on single-stranded oligonucleotides containing the pGT5 double-stranded origin (dso) Sequence. Rep75 exhibits an unusual site-specific nucleotidyl-terminal transferase (NTT) activity, never described before for a Rep protein. The protein Rep75 can be overproduced in Escherichia coli and purified to near homogeneity. The method used, as well as the activity tests, is described in detail here and could be relevant to the study of other hyperthermophilic Rep proteins.
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Physiological Responses of the Hyperthermophilic Archaeon “Pyrococcus abyssi” to DNA Damage Caused by Ionizing Radiation
Journal of Bacteriology, 2003Co-Authors: Edmond Jolivet, Yoshizumi Ishino, Patrick Forterre, Daniel Prieur, Fujihiko Matsunaga, Hannu MyllykallioAbstract:The mechanisms by which hyperthermophilic Archaea, such as “Pyrococcus abyssi” and Pyrococcus furiosus, survive high doses of ionizing gamma irradiation are not thoroughly elucidated. Following gamma-ray irradiation at 2,500 Gy, the restoration of “P. abyssi” chromosomes took place within chromosome fragmentation. DNA synthesis in irradiated “P. abyssi” cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism. We also found evidence for transient export of damaged DNA out of irradiated “P. abyssi” cells prior to a restart of chromosomal DNA synthesis. Our cell fractionation assays further suggest that “P. abyssi” contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation.
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Construction of a shuttle vector for, and spheroplast transformation of, the hyperthermophilic archaeon Pyrococcus abyssi.
Applied and Environmental Microbiology, 2002Co-Authors: S. Lucas, Yvan Zivanovic, Patrick Forterre, Daniel Prieur, Laurent Toffin, Daniel Charlier, Hélène Moussard, Gaël ErausoAbstract: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 (102 to 103 transformants per μg 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.
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Pyrococcus genome comparison evidences chromosome shuffling driven evolution
Nucleic Acids Research, 2002Co-Authors: Yvan Zivanovic, Philippe Lopez, Hervé Philippe, Patrick ForterreAbstract:The genomes of three Pyrococcus species, P.abyssi, P.furiosus and P.horikoshii, were compared at the DNA level, taking advantage of our identification of their replication origins. Three types of rearrangements have been identified: (i) inversion and translation across the replication axis (origin/terminus), (ii) inversion and translocation restricted to a replichore (the half chromosome divided by the replication axis) and (iii) apparent mobility of long clusters of repeated sequences. Rearrangements restricted within a replichore were more common between P.furiosus and the two other Pyrococcus species than between P.horikoshii and P.abyssi. A strong correlation was found between 23 homologous insertion sequence elements, present only in P.furiosus, and recombined segment boundaries, suggesting that transposition events have been a major cause of genomic disruption in this species. Moreover, gene orientation bias was much more disrupted than strand composition biases in fragments that switched their orientation within a replichore upon recombination. This allowed us to conclude that one reversion and one translation occurred in P.abyssi after its divergence from P.horikoshii, and that a smaller segment has specifically recombined in P.furiosus. Whereas a majority of genes are transcribed in the same direction as DNA replication in P.horikoshii and P.abyssi, the colinearity of transcription and replication is only maintained for highly transcribed genes in P.furiosus. We discuss the implications of genomic rearrangements on gene orientation and composition biases, and their consequences on sequence evolution.
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DNA protection mechanisms are not involved in the radioresistance of the hyperthermophilic archaea Pyrococcus abyssi and P. furiosus.
Molecular Genetics and Genomics, 2001Co-Authors: E. Gérard, Edmond Jolivet, Daniel Prieur, Patrick ForterreAbstract: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.
Tadayuki Imanaka - One of the best experts on this subject based on the ideXlab platform.
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complete genome sequence of the hyperthermophilic archaeon thermococcus kodakaraensis kod1 and comparison with Pyrococcus genomes
Genome Research, 2005Co-Authors: Rie Matsumi, Shinsuke Fujiwara, Tamotsu Kanai, Haruyuki Atomi, Toshiaki Fukui, Tadayuki ImanakaAbstract: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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complete genome sequence of the hyperthermophilic archaeon thermococcus kodakaraensis kod1 and comparison with Pyrococcus genomes
Genome Research, 2005Co-Authors: Toshiaki Fukui, Rie Matsumi, Shinsuke Fujiwara, Tamotsu Kanai, Haruyuki Atomi, Tadayuki ImanakaAbstract:The euryarchaeal order Thermococcales, composed of two major genera Thermococcus and Pyrococcus (Huber and Stetter 2001; Itoh 2003), may be the best-studied hyperthermophiles. They are strictly anaerobic obligate heterotrophs growing on complex proteinaceous substrates, and their growth is strongly associated with the reduction of elemental sulfur. Alternatively, with a few exceptions, they are capable of gaining energy by fermentation of peptides, amino acids, and sugars, forming acids, CO2, and H2 in the absence of elemental sulfur (Amend and Shock 2001; Huber and Stetter 2001). The genus Pyrococcus, with a higher optimum growth temperature (95°C-103°C) than Thermococcus (75°C-93°C), has fascinated many microbiological researchers and has often been used as the source organism for both fundamental and application-based aspects of research. Therefore, although within the same genus, the complete genome analyses of three species—Pyrococcus horikoshii (Kawarabayasi et al. 1998), Pyrococcus furiosus (Robb et al. 2001), and Pyrococcus abyssi (Cohen et al. 2003)—have been performed. In contrast to Pyrococcus, the genus Thermococcus contains the highest number of characterized isolates (Itoh 2003). Recent culture-dependent and culture-independent studies have indicated that the members of Thermococcus are more ubiquitously present in various deep-sea hydrothermal vent systems than those of Pyrococcus (Orphan et al. 2000; Holden et al. 2001). Consequently, Thermococcus strains, with their larger population, are considered to play a major role in the ecology and metabolic activity of microbial consortia within marine hot-water ecosystems. However, despite the importance of this genus, no complete genome sequence has been determined for Thermococcus. The Thermococcus genome can be expected to encode genes responsible for various cellular functions that provide an advantage for these strains in natural high-temperature habitats. Thermococcus kodakaraensis KOD1 was isolated from a solfatara on the shore of Kodakara Island, Kagoshima, Japan (Morikawa et al. 1994; Atomi et al. 2004). Since the isolation, an abundant number of genes and their protein products from this archaeon have been examined (Imanaka and Atomi 2002), such as DNA polymerase, commercially available as an excellent enzyme for PCR amplification (Nishioka et al. 2001). Moreover, as we recently developed the first gene disruption system for a hyperthermophile with this archaeon (Sato et al. 2003), T. kodakaraensis can be regarded as one of the most useful model organisms in the research on hyperthermophiles. Here we describe the complete genome analysis of T. kodakaraensis KOD1 as well as a comparison with Pyrococcus genomes, to gain further insight into the intriguing order Thermococcales.
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characterization of dna polymerase from Pyrococcus sp strain kod1 and its application to pcr
Applied and Environmental Microbiology, 1997Co-Authors: Masahiro Takagi, Motomu Nishioka, Hirofumi Kakihara, Masao Kitabayashi, Hiroaki Inoue, Bunsei Tsurugashi Kawakami, Masanori Oka, Tadayuki ImanakaAbstract:The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conserved among eukaryotic and archaeal alpha-like DNA polymerases. The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. 3'-5' exonuclease activity was confirmed, and although KOD DNA polymerase's optimum temperature (75 degrees C) and mutation frequency (3.5 x 10(-3)) were similar to those of a DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (persistence of sequential nucleotide polymerization) 10 to 15 times higher than those of Pfu DNA polymerase. These characteristics enabled the KOD DNA polymerase to perform a more accurate PCR in a shorter reaction time.
