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Karl O Stetter - One of the best experts on this subject based on the ideXlab platform.
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Complete sequential assignment and secondary structure prediction of the cannulae forming protein CanA from the hyperthermophilic archaeon Pyrodictium abyssi.
Biomolecular NMR assignments, 2020Co-Authors: Raphael Kreitner, Karl O Stetter, Claudia Elisabeth Munte, Katrin Singer, Gudrun Horn, Werner Kremer, Hans Robert KalbitzerAbstract:CanA from Pyrodictium abyssi forms a heat-resistant organic hollow-fiber network together with CanB and CanC. An N-terminally truncated construct of CanA (K1-CanA) gave NMR spectra of good quality that could be assigned by three-dimensional NMR methods on 15N and 13C-15N enriched protein. We assigned the chemical shifts of 96% of all backbone 1HN atoms, 98% of all backbone 15N atoms, 100% of all 13Cα atoms, 100% of all 1Hα atoms, 90% of all 13C' atoms, and 100% of the 13Cβ atoms. Two short helices and 10 β-strands are estimated from an analysis of the chemical shifts leading to a secondary structure content of K1-CanA of 6% helices, 44% β-pleated sheets, and 50% coils.
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Draft Genome Sequence of Pyrodictium occultum PL19T, a Marine Hyperthermophilic Species of Archaea That Grows Optimally at 105°C.
Genome announcements, 2016Co-Authors: Sagar M. Utturkar, Harald Huber, Karl O Stetter, Sebastian Leptihn, Belinda Loh, Steven D. Brown, Mircea PodarAbstract:We report here the draft genome sequence of Pyrodictium occultum PL19T, a marine hyperthermophilic archaeon. In addition, the genome provides insights into molecular and cellular adaptation mechanisms to life in extreme environments and the evolution of early organisms on Earth.
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draft genome sequence of Pyrodictium occultum pl19t a marine hyperthermophilic species of archaea that grows optimally at 105 c
Genome Announcements, 2016Co-Authors: Harald Huber, Karl O Stetter, Sagar M. Utturkar, Sebastian Leptihn, Belinda Loh, Steven D. Brown, Mircea PodarAbstract:We report here the draft genome sequence of Pyrodictium occultum PL19T, a marine hyperthermophilic archaeon. In addition, the genome provides insights into molecular and cellular adaptation mechanisms to life in extreme environments and the evolution of early organisms on Earth.
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Autotrophic CO2 fixation pathways in archaea (Crenarchaeota).
Archives of microbiology, 2003Co-Authors: Michael Hügler, Harald Huber, Karl O Stetter, Georg FuchsAbstract:Representative autotrophic and thermophilic archaeal species of different families of Crenarchaeota were examined for key enzymes of the known autotrophic CO(2) fixation pathways. Pyrobaculum islandicum ( Thermoproteaceae) contained key enzymes of the reductive citric acid cycle. This finding is consistent with the operation of this pathway in the related Thermoproteus neutrophilus. Pyrodictium abyssi and Pyrodictium occultum ( Pyrodictiaceae) contained ribulose 1,5-bisphosphate carboxylase, which was active in boiling water. Yet, phosphoribulokinase activity was not detectable. Operation of the Calvin cycle remains to be demonstrated. Ignicoccus islandicus and Ignicoccus pacificus ( Desulfurococcaceae) contained pyruvate oxidoreductase as potential carboxylating enzyme, but apparently lacked key enzymes of known pathways; their mode of autotrophic CO(2) fixation is at issue. Metallosphaera sedula, Acidianus ambivalens and Sulfolobus sp. strain VE6 ( Sulfolobaceae) contained key enzymes of a 3-hydroxypropionate cycle. This finding is in line with the demonstration of acetyl-coenzyme A (CoA) and propionyl-CoA carboxylase activities in the related Acidianus brierleyi and Sulfolobus metallicus. Enzymes of central carbon metabolism in Metallosphaera sedula were studied in more detail. Enzyme activities of the 3-hydroxypropionate cycle were strongly up-regulated during autotrophic growth, supporting their role in CO(2) fixation. However, formation of acetyl-CoA from succinyl-CoA could not be demonstrated, suggesting a modified pathway of acetyl-CoA regeneration. We conclude that Crenarchaeota exhibit a mosaic of three or possibly four autotrophic pathways. The distribution of the pathways so far correlates with the 16S-rRNA-based taxa of the Crenarchaeota.
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atp synthesis at 100 c by an atpase purified from the hyperthermophilic archaeon Pyrodictium abyssi
FEBS Letters, 2000Co-Authors: Reinhard Dirmeier, G Hauska, Karl O StetterAbstract:The chemolithoautotrophic archaeon Pyrodictium abyssi isolate TAG 11 lives close to 100°C and gains energy by sulfur respiration, with hydrogen as electron donor. From the membranes of this hyperthermophile, an ATPase complex was isolated. The purified enzyme consists of six major polypeptides, the 67, 51, 41, 26 and 22 kDa subunits composing the AF1 headpiece, and the 7 kDa proteolipid of the AF0 component. The headpiece of the enzyme restored the formation of ATP during sulfur respiration in membrane vesicles from which it had been removed by low salt treatment. Characteristics of the reconstituted activity suggest that the same enzyme is responsible for ATP formation in untreated membranes. ATP formation was neither sensitive to ionophores and uncouplers, nor to dicyclohexyl carbodiimide, but depended on closed vesicles. Both ATPase activity (up to 2 μmol per min and mg protein) as well as ATP formation (up to 0.4 μmol per min and mg membrane protein) were highest at 100°C. A P/e2 ratio of close to one can be estimated for sulfur respiration with hydrogen. In addition to ATP, autoradiographic detection revealed the formation of high quantities of 33Pi-labeled ADP and of another compound not identified so far.
Wolfgang Baumeister - One of the best experts on this subject based on the ideXlab platform.
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Pyrodictium cannulae enter the periplasmic space but do not enter the cytoplasm, as revealed by cryo-electron tomography.
Journal of structural biology, 2003Co-Authors: Stephan Nickell, Reiner Hegerl, Wolfgang Baumeister, Reinhard RachelAbstract:The hyperthermophilic archaeon Pyrodictium grows in the form of a macroscopically visible network. It consists of cells entrapped in an extracellular matrix of hollow tubules, the "cannulae." Here, we present the three-dimensional structure of a single cell in conjunction with two extracellular cannulae, as determined by cryo-electron microscopy. To achieve this, the information from two independent tilt series of the same specimen was combined, with the specimen rotated in the second series. In the three-dimensional tomographic reconstruction, we were able to trace the two cannulae in their full length, in particular, also inside the cell. One cannula enters the periplasmic space, while the other cannula contacts the surface of the cell, the S-layer. This indicates that the cannulae interconnect individual cells with each other on the level of their periplasmic space; we do not, however, have evidence that they enter the cytoplasm of the cells. The implications of these data for possible functions of the cannulae are discussed.
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Structure of a molecular chaperone from a thermophilic archaebacterium
Nature, 1993Co-Authors: Barry M. Phipps, Karl O Stetter, Reiner Hegerl, Susanne Volker, Dieter Typke, Angelika Hoffmann, Wolfgang BaumeisterAbstract:WE recently reported that a wide range of thermophilic archaebacteria have a novel ATPase complex which accumulates to high levels upon heat shock and may be a new type of molecular chaperone related to the chaperonins1. Striking similarities between the complex, referred to here as the 'thermosome', and the subsequently characterized chaperones TF55 of the thermophilic archaebacterium Sulfolobus shibatae2 and TCP-1 from eukaryotic cytosol2–5 suggest that these proteins belong to a single family. Here we determine the three-dimensional structure of the thermosome from Pyrodictium occultum by random conical tilt reconstruction from electron micrographs. The reconstruction reveals a complex consisting of two rings of eight subunits each, stacked face-to-face. The subunits are kidney-shaped and composed of at least two domains. In the centre of the thermosome is a large cavity of about 6.7 nm diameter; the opening of this cavity on each face of the complex is partially blocked by a mass which appears to be weakly connected to the eight-membered ring.
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a novel atpase complex selectively accumulated upon heat shock is a major cellular component of thermophilic archaebacteria
The EMBO Journal, 1991Co-Authors: B. M. Phipps, Karl Otto Stetter, Anke Hoffmann, Wolfgang BaumeisterAbstract:We have discovered a large cylindrical protein complex which is an abundant component of the cytoplasm of extremely thermophilic archaebacteria. Structural analysis by image processing of electron micrographs suggests that the complex is composed of two stacked rings of eight subunits each; the rings enclose a central channel. The complex purified from the hyperthermophile Pyrodictium occultum is composed of equal quantities of two polypeptides of Mr 56,000 and 59,000. It exhibits an extremely thermostable ATPase activity with a temperature optimum of 100 degrees C. The basal level of the ATPase complex in the cell is high, and it becomes highly enriched as a result of heat shock (shift from 102 degrees C to 108 degrees C) or balanced growth at temperatures near the physiological upper limit. Immunoblotting results indicate that a related protein is present in most thermophilic archaebacteria and in Escherichia coli. This protein complex may play an important role in the adaptation of thermophilic archaebacteria to life at high temperature.
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Three-dimensional reconstruction of the surface protein of Pyrodictium brockii : comparing two image processing strategies
Journal of Structural Biology, 1991Co-Authors: Roland Dürr, Reiner Hegerl, Susanne Volker, Ute Santarius, Wolfgang BaumeisterAbstract:Abstract The three-dimensional structure of the surface layer protein of the hyperthermophilic archaebacterium Pyrodictium brockii was determined by electron crystallographic techniques to a resolution of approximately 1.6 nm. Two image processing strategies—correlation averaging and lattice unbending—were explored with regard to their potential to compensate for lattice distortions. Reconstructions performed via these two routes did not show any significant difference although the unbending approach might be expected to give superior results since, in principle, it incorporates a correction for unit cell displacement, rotation, and deformation while conventional correlation averaging compensates for displacements only. The discussion furthermore addresses some hitherto ignored problems associated with the correction of lattice disorder at higher tilts.
James F. Holden - One of the best experts on this subject based on the ideXlab platform.
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Microbe-Mineral Interaction and Novel Proteins for Iron Oxide Mineral Reduction in the Hyperthermophilic Crenarchaeon Pyrodictium delaneyi.
Applied and environmental microbiology, 2021Co-Authors: S. Kashyap, James F. HoldenAbstract:Dissimilatory iron reduction by hyperthermophilic archaea occurs in many geothermal environments and generally relies on microbe-mineral interactions that transform various iron oxide minerals. In this study, the physiology of dissimilatory iron and nitrate reduction was examined in the hyperthermophilic crenarchaeon Pyrodictium delaneyi Su06T Iron barrier experiments showed that P. delaneyi required direct contact with the Fe(III) oxide mineral ferrihydrite for reduction. The separate addition of an exogenous electron shuttle (anthraquinone-2,6-disulfonate), a metal chelator (nitrilotriacetic acid), and 75% spent cell-free supernatant did not stimulate growth with or without the barrier. Protein electrophoresis showed that the c-type cytochrome and general protein compositions of P. delaneyi changed when grown on ferrihydrite relative to nitrate. Differential proteomic analyses using tandem mass tagged protein fragments and mass spectrometry detected 660 proteins and differential production of 127 proteins. Among these, two putative membrane-bound molybdopterin-dependent oxidoreductase complexes increased in relative abundance 60- to 3,000-fold and 50-100-fold in cells grown on iron oxide. A putative 8-heme c-type cytochrome was 60-fold more abundant in iron grown cells and was unique to the Pyrodictiaceae There was also a >14,700-fold increase in a membrane transport protein in iron grown cells. There were no changes in the abundances of flagellin proteins nor a putative nitrate reductase, but a membrane nitric oxide reductase was more abundant on nitrate. These data help to elucidate the mechanisms by which hyperthermophilic crenarchaea generate energy and transfer electrons across the membrane to iron oxide minerals.IMPORTANCE Understanding iron reduction in the hyperthermophilic crenarchaeon Pyrodictium delaneyi provides insight into the diversity of mechanisms used for this process and its potential impact in geothermal environments. The ability of P. delaneyi to reduce Fe(III) oxide minerals through direct contact potentially using a novel cytochrome respiratory complex and a membrane-bound molybdopterin respiratory complex sets iron reduction in this organism apart from previously described iron reduction processes. A model is presented where obligatory H2 oxidation on the membrane coupled with electron transport and either Fe(III) oxide or nitrate reduction leads to the generation of a proton motive force and energy generation by oxidative phosphorylation. However, P. delaneyi cannot fix CO2 and relies on organic compounds from its environment for biosynthesis.
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Reduction and Morphological Transformation of Synthetic Nanophase Iron Oxide Minerals by Hyperthermophilic Archaea.
Frontiers in microbiology, 2018Co-Authors: S. Kashyap, E. C. Sklute, M. Darby Dyar, James F. HoldenAbstract:Fe(III) (oxyhydr)oxides are electron acceptors for some hyperthermophilic archaea in mildly-reducing geothermal environments. However, the kinds of iron oxides that can be used, growth rates, extent of iron reduction, and the morphological changes that occur to minerals are poorly understood. The hyperthermophilic iron-reducing crenarchaea Pyrodictium delaneyi and Pyrobaculum islandicum were grown separately on six different synthetic nanophase Fe(III) (oxyhydr)oxides. For both organisms, growth on ferrihydrite produced the highest growth rates and the largest amounts of Fe(II), although P. delaneyi produced four times more Fe(II) (25 mM) than P. islandicum (6 mM). Both organisms grew on lepidocrocite and akaganeite and produced 2-3 mM Fe(II). Modest growth occurred for both organisms on goethite, hematite, and maghemite where ≤ 1 mM Fe(II) was produced. The diameters of the spherical mineral end-products following P. delaneyi growth increased by 30 nm for ferrihydrite and 50-150 nm for lepidocrocite relative to heated abiotic controls. For akaganeite, spherical particle sizes were the same for P. delaneyi-reacted samples and heated abiotic controls, but the spherical particles were more numerous in the P. delaneyi samples. For P. islandicum, there was no increase in grain size for the mineral end-products following growth on ferrihydrite, lepidocrocite, or akaganeite relative to the heated abiotic controls. High-resolution transmission electron microscopy (HRTEM) of lattice fringes and selected-area electron diffraction (SAED) of the minerals produced by both organisms when grown on ferrihydrite showed that magnetite and/or possibly maghemite were the end-products while the heated abiotic controls only contained ferrihydrite. These results expand the current view of bioavailable Fe(III) (oxyhydr)oxides for reduction by hyperthermophilic archaea when presented as synthetic nanophase minerals. They show that growth and reduction rates are inversely correlated with the iron (oxyhydr)oxide crystallinity and that iron (oxyhydr)oxide mineral transformation takes different forms for these two organisms.
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Reduction and Morphological Transformation of Synthetic Nanophase Iron Oxide Minerals by Hyperthermophilic Archaea
Frontiers Media S.A., 2018Co-Authors: Srishti Kashyap, E. C. Sklute, Darby M. Dyar, James F. HoldenAbstract:Fe(III) (oxyhydr)oxides are electron acceptors for some hyperthermophilic archaea in mildly reducing geothermal environments. However, the kinds of iron oxides that can be used, growth rates, extent of iron reduction, and the morphological changes that occur to minerals are poorly understood. The hyperthermophilic iron-reducing crenarchaea Pyrodictium delaneyi and Pyrobaculum islandicum were grown separately on six different synthetic nanophase Fe(III) (oxyhydr)oxides. For both organisms, growth on ferrihydrite produced the highest growth rates and the largest amounts of Fe(II), although P. delaneyi produced four times more Fe(II) (25 mM) than P. islandicum (6 mM). Both organisms grew on lepidocrocite and akaganéite and produced 2 and 3 mM Fe(II). Modest growth occurred for both organisms on goethite, hematite, and maghemite where ≤1 mM Fe(II) was produced. The diameters of the spherical mineral end-products following P. delaneyi growth increased by 30 nm for ferrihydrite and 50–150 nm for lepidocrocite relative to heated abiotic controls. For akaganéite, spherical particle sizes were the same for P. delaneyi-reacted samples and heated abiotic controls, but the spherical particles were more numerous in the P. delaneyi samples. For P. islandicum, there was no increase in grain size for the mineral end-products following growth on ferrihydrite, lepidocrocite, or akaganéite relative to the heated abiotic controls. High-resolution transmission electron microscopy of lattice fringes and selected-area electron diffraction of the minerals produced by both organisms when grown on ferrihydrite showed that magnetite and/or possibly maghemite were the end-products while the heated abiotic controls only contained ferrihydrite. These results expand the current view of bioavailable Fe(III) (oxyhydr)oxides for reduction by hyperthermophilic archaea when presented as synthetic nanophase minerals. They show that growth and reduction rates are inversely correlated with the iron (oxyhydr)oxide crystallinity and that iron (oxyhydr)oxide mineral transformation takes different forms for these two organisms
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Image_1_Reduction and Morphological Transformation of Synthetic Nanophase Iron Oxide Minerals by Hyperthermophilic Archaea.PDF
2018Co-Authors: Srishti Kashyap, E. C. Sklute, Darby M. Dyar, James F. HoldenAbstract:Fe(III) (oxyhydr)oxides are electron acceptors for some hyperthermophilic archaea in mildly reducing geothermal environments. However, the kinds of iron oxides that can be used, growth rates, extent of iron reduction, and the morphological changes that occur to minerals are poorly understood. The hyperthermophilic iron-reducing crenarchaea Pyrodictium delaneyi and Pyrobaculum islandicum were grown separately on six different synthetic nanophase Fe(III) (oxyhydr)oxides. For both organisms, growth on ferrihydrite produced the highest growth rates and the largest amounts of Fe(II), although P. delaneyi produced four times more Fe(II) (25 mM) than P. islandicum (6 mM). Both organisms grew on lepidocrocite and akaganéite and produced 2 and 3 mM Fe(II). Modest growth occurred for both organisms on goethite, hematite, and maghemite where ≤1 mM Fe(II) was produced. The diameters of the spherical mineral end-products following P. delaneyi growth increased by 30 nm for ferrihydrite and 50–150 nm for lepidocrocite relative to heated abiotic controls. For akaganéite, spherical particle sizes were the same for P. delaneyi-reacted samples and heated abiotic controls, but the spherical particles were more numerous in the P. delaneyi samples. For P. islandicum, there was no increase in grain size for the mineral end-products following growth on ferrihydrite, lepidocrocite, or akaganéite relative to the heated abiotic controls. High-resolution transmission electron microscopy of lattice fringes and selected-area electron diffraction of the minerals produced by both organisms when grown on ferrihydrite showed that magnetite and/or possibly maghemite were the end-products while the heated abiotic controls only contained ferrihydrite. These results expand the current view of bioavailable Fe(III) (oxyhydr)oxides for reduction by hyperthermophilic archaea when presented as synthetic nanophase minerals. They show that growth and reduction rates are inversely correlated with the iron (oxyhydr)oxide crystallinity and that iron (oxyhydr)oxide mineral transformation takes different forms for these two organisms.
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Pyrodictium delaneyi sp. nov., a hyperthermophilic autotrophic archaeon that reduces Fe(III) oxide and nitrate.
International journal of systematic and evolutionary microbiology, 2016Co-Authors: T. Jennifer Lin, Gabriel K. El Sebae, Jong-hyun Jung, Dong-hyun Jung, Cheon-seok Park, James F. HoldenAbstract:A hyperthermophilic, autotrophic iron and nitrate reducer, strain Su06T, was isolated from an active deep-sea hydrothermal vent chimney on the Endeavour Segment in the north-eastern Pacific Ocean. It was obligately anaerobic, hydrogenotrophic and reduced Fe(III) oxide to magnetite and NO3 − to N2. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain was more than 97 % similar to other species of the genera Pyrodictium and Hyperthermus . Therefore, overall genome relatedness index analyses were performed to establish whether strain Su06T represents a novel species. For each analysis, strain Su06T was most similar to Pyrodictium occultum PL-19T. Relative to this strain, the average nucleotide identity score for strain Su06T was 72 %, the genome-to-genome direct comparison score was 13–19 % and the species identification score at the protein level was 89 %. For each analysis, strain Su06T was below the species delineation cutoff. Based on its whole genome sequence and its unique phenotypic characteristics, strain Su06T is suggested to represent a novel species of the genus Pyrodictium , for which the name Pyrodictium delaneyi is proposed. The type strain is Su06T (=DSM 28599T=ATCC BAA-2559T).
James A Mccloskey - One of the best experts on this subject based on the ideXlab platform.
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STRUCTURAL FEATURE OF THE INITIATOR TRNA GENE FROM Pyrodictium OCCULTUM AND THE THERMAL STABILITY OF ITS GENE PRODUCT, TRNAIMET
Biochimie, 1996Co-Authors: C. Ushida, Karl O Stetter, Pamela F Crain, James A Mccloskey, T. Muramatsu, H. Mizushima, Takuya Ueda, Kimitsuna Watanabe, Yoshiyuki KuchinoAbstract:Abstract Pyrodictium occultum is a hyperthermophilic archaeum that grows optimally at 105°C. To study how tRNA molecules in P occultum are thermally stabilized, we isolated the initiator tRNA gene from the organism using a synthetic DNA probe of 74 bp containing the known nucleotide sequences that are conserved in archaeal initiator tRNAs. A HindIII fragment of 700 bp containing the Pyrodictium initiator tRNA gene was cloned and sequenced by cycle sequencing. The nucleotide sequence revealed that the Pyrodictium initiator tRNA gene has no introns, and that the 3′ CCA terminus is encoded. The tRNA gene also contained a unique TATA-like sequence, AAGCTTATAA, which is likely the promoter proposed for archaeal tRNA genes, −50 bp upstream of the 5′ end of the tRNA coding region. In the region adjacent to the 3′ end of the tRNA coding region, there was a six G-C base pair inverted repeat followed by a C-rich sequence like the ϱ-independent transcription termination signal of bacteriol genes. The Pyrodictium initiator tRNA sequence predicted from the gene sequence contained all of the nucleotide residues A1, A37, U54, A57, U60, and U72, in addition to three G-C base pairs in the anticodon stem region, which are characteristic of archaeal initiator tRNAs. The melting temperature (Tm) of the unmodified initiator tRNA synthesized in vitro using the cloned tRNA gene as a template was 80°C, which is only two degrees lower than that calculated from the G-C content in the stem regions of the tRNA. In contrast, the Tm of the natural initiator tRNA isolated from P occultum was over 100°C. Analysis of digests of purified Pyrodictium initiator tRNA by means of HPLC-mass spectrometry and [32P] post-labeling, indicated that the tRNA contains a variety of modified nucleosides. These results suggest that the extraordinarily high melting temperature of P occultum tRNAiMet is due to posttranscriptional modification.
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The role of posttranscriptional modification in stabilization of transfer RNA from hyperthermophiles.
Biochemistry, 1994Co-Authors: Jeffrey A Kowalak, James A Mccloskey, Joseph J. Dalluge, Karl O StetterAbstract:The influence of posttranscriptional modification on structural stabilization of tRNA from hyperthermophilic archaea was studied, using Pyrococcus furiosus (growth optimum 100 degrees C) as a primary model. Optical melting temperatures (Tm) of unfractionated tRNA in 20 mM Mg2+ are 97 degrees C for P. furiosus and 101.5 degrees C for Pyrodictium occultum (growth optimum, 105 degrees C). These values are approximately 20 degrees C higher than predicted solely from G-C content and are attributed primarily to posttranscriptional modification. Twenty-three modified nucleosides were determined in total digests of P. furiosus tRNA by combined HPLC-mass spectrometry. From cells cultured at 70, 85, and 100 degrees C, progressively increased levels of modification were observed within three families of nucleosides, the most highly modified forms of which were N4-acetyl-2'-O-methylcytidine (ac4Cm), N2,N2,2'-O-trimethylguanosine (m2(2)Gm), and 5-methyl-2-thiouridine (m5s2U). Nucleosides ac4Cm and m2(2)Gm, which are unique to the archaeal hyperthermophiles, were shown in earlier NMR studies to exhibit unusually high conformational stabilities that favor the C3'-endo form [Kawai, G., et al. (1991) Nucleic Acids Symp. Ser. 21, 49-50; (1992) Nucleosides Nucleotides 11, 759-771]. The sequence location of m5s2U was determined by mass spectrometry to be primarily at tRNA position 54, a site of known thermal stabilization in the bacterial thermophile Thermus thermophilus [Horie, N., et al. (1985) Biochemistry 24, 5711-5715]. It is concluded that selected posttranscriptional modifications in archaeal thermophiles play major stabilizing roles beyond the effects of Mg2+ binding and G-C content, and are proportionally more important and have evolved with greater structural diversity at the nucleoside level in the bacterial thermophiles.
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5S rRNA modification in the hyperthermophilic archaea Sulfolobus solfataricus and Pyrodictium occultum.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1993Co-Authors: Eveline Bruenger, Karl O Stetter, Jeffrey A Kowalak, James A Mccloskey, H. Mizushima, Y. Kuchino, Pamela F CrainAbstract:The 5S rRNAs from Sulfolobus solfataricus and Pyrodictium occultum were digested to nucleosides and analyzed using directly-combined HPLC/mass spectrometry. P. occultum 5S rRNA contains two modifie...
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Sulfolobus solfataricus and Pyrodictium occultum
1993Co-Authors: Eveline Bruenger, James A Mccloskey, Jeffrey A Kowalak, Yoshiyuki Kuchino, Pamela F CrainAbstract:The 5S rRNAs from Sulfolobus solfataricus and Pyrodictium occultum were digested to nucleosides and analyzed using directly-combined HPLC/mass spec- trometry. P. occultum 5S rRNA contains two modified nucleoside species, N4-acetylcytidine (ac4C) and N4-acetyl-2' -O-methylcytidine (acCm). Oligonucleotides were generated from P occultum 5S rRNA by RNase T1 hydrolysis, and their molecular weights were determined using electrospray mass spectrometry and compared with those predicted from the P. occuitum 5S RNA gene se- quence. Deviation in mass between expected and ob- served molecular weights permitted ac4Cm to be located at position 35, in the nonanucleotide CAA- CACC(ac4CmIG, and the ac4C in one or both of two (C,U)G trinucleotides. 2'-O-Methylcytidine is unambig- uously characterized in S. solfataricus 5S rRNA, confirming earlier tentative assignments at the analogous sequence position (Stahl,D. A., Luehrsen, K. R., Woese, C. R., and Pace, N. R. (1981)Nucleic Acids Res., Vol. 9,
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posttranscriptional modification of trna in thermophilic archaea archaebacteria
Journal of Bacteriology, 1991Co-Authors: Charles G Edmonds, Karl O Stetter, Pamela F Crain, Ramesh Gupta, Takeshi Hashizume, C H Hocart, Jeffrey A Kowalak, Steven C Pomerantz, James A MccloskeyAbstract:Nucleoside modification has been studied in unfractionated tRNA from 11 thermophilic archaea (archaebacteria), including phylogenetically diverse representatives of thermophilic methanogens and sulfur-metabolizing hyperthermophiles which grow optimally in the temperature range of 56 (Thermoplasma acidophilum) to 105 degrees C (Pyrodictium occultum), and for comparison from the most thermophilic bacterium (eubacterium) known, Thermotoga maritima (80 degrees C). Nine nucleosides are found to be unique to the archaea, six of which are structurally novel in being modified both in the base and by methylation in ribose and occur primarily in tRNA from the extreme thermophiles in the Crenarchaeota of the archaeal phylogenetic tree. 2-Thiothymine occurs in tRNA from Thermococcus sp., and constitutes the only known occurrence of the thymine moiety in archaeal RNA, in contrast to its near-ubiquitous presence in tRNA from bacteria and eukarya. A total of 33 modified nucleosides are rigorously characterized in archaeal tRNA in the present study, demonstrating that the structural range of posttranscriptional modifications in archaeal tRNA is more extensive than previously known. From a phylogenetic standpoint, certain tRNA modifications occur in the archaea which are otherwise unique to either the bacterial or eukaryal domain, although the overall patterns of modification are more typical of eukaryotes than bacteria.
Robertj . Maier - One of the best experts on this subject based on the ideXlab platform.
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Thermal Tolerance of Archael Membrane-Bound Enzymes.
1998Co-Authors: Robertj . MaierAbstract:Abstract : The molecular mechanisms for thermal tolerance of proteins and other components from hyperthermophilic microorganisms (organisms that grow above 100 deg C) have yet to be determined. The objective of this work was to identify an approach to understand the role of hydrophobic membrane components (i.e., lipids) in conferring thermal tolerance to a heat-stable membrane-bound enzyme. A tightly-membrane-associated enzyme (hydrogenase) that is a key component of the energy metabolism of the hyperthermophile Pyrodictium abyssi (grows at up to 110 deg C), can be assayed at temperatures up to 120 deg C. The specific aims for this project period were to reconstitute partially purified P. abyssi hydrogenase into proteoliposomes to enable assessment of the roles of lipids in conferring stability. This objective was partially achieved, although the amount of enzyme incorporation into liposomes was low, and enzyme activity loss was rapid. The P. abyssi detergent-solubilized enzyme was characterized for some biochemical and physiological properties especially for its function as a membrane-bound respiratory-type uptake hydrogenase enzyme. Another membrane-bound enzyme, sulfur reductase, was partially purified and characterized, and a S(exp 0)-reducing electron transport chain was partially identified.
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Hydrogen-oxidizing electron transport components in the hyperthermophilic archaebacterium Pyrodictium brockii.
Journal of bacteriology, 1992Co-Authors: T D Pihl, Brenda A. Schulman, L. K. Black, Robertj . MaierAbstract:The hyperthermophilic archaebacterium Pyrodictium brockii grows optimally at 105 degrees C by a form of metabolism known as hydrogen-sulfur autotrophy, which is characterized by the oxidation of H2 by S0 to produce ATP and H2S. UV-irradiated membranes were not able to carry out the hydrogen-dependent reduction of sulfur. However, the activity could be restored by the addition of ubiquinone Q10 or ubiquinone Q6 to the UV-damaged membranes. A quinone with thin-layer chromatography migration properties similar to those of Q6 was purified by thin-layer chromatography from membranes of P. brockii, but nuclear magnetic resonance analysis failed to confirm its identity as a ubiquinone. P. brockii quinone was capable of restoring hydrogen-dependent sulfur reduction to UV-irradiated membranes. Hydrogen-reduced-minus-air-oxidized absorption difference spectra on membranes revealed absorption peaks characteristic of c-type cytochromes. A c-type cytochrome with alpha, beta, and gamma peaks at 553, 522, and 421 nm, respectively, was solubilized from membranes with 0.5% Triton X-100. Pyridine ferrohemochrome spectra confirmed its identity as a c-type cytochrome, and heme staining of membranes loaded on sodium dodecyl sulfate gels revealed a single heme-containing component of 13 to 14 kDa. Studies with the ubiquinone analog 2-n-heptyl-4-hydroxyquinoline-N-oxide demonstrated that the P. brockii quinone is located on the substrate side of the electron transport chain with respect to the c-type cytochrome. These first characterizations of the strictly anaerobic, presumably primitive P. brockii electron transport chain suggest that the hydrogenase operates at a relatively high redox potential and that the H2-oxidizing chain more closely resembles those of aerobic eubacterial H2-oxidizing bacteria than those of the H2-metabolizing systems of anaerobes or the hyperthermophile Pyrococcus furiosus.
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Purification and characterization of the hydrogen uptake hydrogenase from the hyperthermophilic archaebacterium Pyrodictium brockii.
Journal of bacteriology, 1991Co-Authors: T D Pihl, Robertj . MaierAbstract:Abstract Pyrodictium brockii is a hyperthermophilic archaebacterium with an optimal growth temperature of 105 degrees C. P. brockii is also a chemolithotroph, requiring H2 and CO2 for growth. We have purified the hydrogen uptake hydrogenase from membranes of P. brockii by reactive red affinity chromatography and sucrose gradient centrifugation. The molecular mass of the holoenzyme was 118,000 +/- 19,000 Da in sucrose gradients. The holoenzyme consisted of two subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large subunit had a molecular mass of 66,000 Da, and the small subunit had a molecular mass of 45,000 Da. Colorometric analysis of Fe and S content in reactive red-purified hydrogenase revealed 8.7 +/- 0.6 mol of Fe and 6.2 +/- 1.2 mol of S per mol of hydrogenase. Growth of cells in 63NiCl2 resulted in label incorporation into reactive red-purified hydrogenase. Growth of cells in 63NiCl2 resulted in label incorporation into reactive red-purified hydrogenase. Temperature stability studies indicated that the membrane-bound form of the enzyme was more stable than the solubilized purified form over a period of minutes with respect to temperature. However, the membranes were not able to protect the enzyme from thermal inactivation over a period of hours. The artificial electron acceptor specificity of the pure enzyme was similar to that of the membrane-bound form, but the purified enzyme was able to evolve H2 in the presence of reduced methyl viologen. The Km of membrane-bound hydrogenase for H2 was approximately 19 microM with methylene blue as the electron acceptor, whereas the purified enzyme had a higher Km value.
