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Günter Schäfer - One of the best experts on this subject based on the ideXlab platform.
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Crystallization and preliminary crystallographic analysis of Rieske iron-sulfur protein II (soxF) from Sulfolobus acidocaldarius.
Acta Crystallographica Section D Biological Crystallography, 2000Co-Authors: Heiko Bönisch, Günter Schäfer, Christian L. Schmidt, Rudolf LadensteinAbstract:An archaeal Rieske iron–sulfur protein has been crystallized for the first time. The genetically constructed soluble form of the soxF protein was expressed in E. coli. It contains a correctly inserted [2Fe–2S] cluster. The authentic soxF protein is part of a terminal oxidase complex in the respiratory chain of the hyperthermoacidophilic crenarchaeon Sulfolobus acidocaldarius (DSM 639). The enzyme crystallizes in the space group P61 or P65, with unit-cell parameters a = b = 80.19, c = 75.69 A. A complete data set has been collected to 1.64 A resolution at 100 K.
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Isolation, characterization and crystallization of an iron-superoxide dismutase from the crenarchaeon Sulfolobus acidocaldarius
FEMS microbiology letters, 1996Co-Authors: S. Kardinahl, C.l. Schmidt, A. Petersen, Günter SchäferAbstract:An iron containing superoxide dismutase from the cytosol of the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius (DSM 639) has been purified to electrophoretic homogeneity. It comprises at least 11% of the cytosolic protein. The isolated protein consists of two identical subunits with an apparent molecular mass of 22.4 kDa. It contains one iron atom per dimer. The protein shows the typical EPR spectrum of a S = 3/2, rhombic high-spin iron center. It is extremely resistant against thermal and chemical denaturation. Simultaneous treatment with heat and detergent resulted in the conversion into a more active tetrameric form. Similar enzymes appear to be present in the cytosol of other members of the Sulfolobaceae. The dimeric form of the protein from S. acidocaldarius has been crystallized.
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A secY homologous gene in the crenarchaeon Sulfolobus acidocaldarius
Biochimica et biophysica acta, 1995Co-Authors: Thomas Kath, Günter SchäferAbstract:The nucleotide sequence of an open reading frame, located upstream of the gene for adenylate kinase, was determined in the thermoacidophile crenarchaeon Sulfolobus acidocaldarius. Data bank searches identified the sequence as a secY homologous gene. The DNA derived protein sequence of total 463 amino acids contains 10 hydrophobic domains. A sequence alignment with other prokaryotic and eukaryotic secY sequences reveals significant homology, but the secY primary sequence of S. acidocaldarius shows only a low degree of similarity with the secY counterparts of the euryarchaea Methanococcus vannielii and Haloarcula marismortui. A transcription analysis indicates, that the secY gene is cotranscribed with the gene coding for adenylate kinase.
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Nucleotide sequence of a gene cluster encoding ribosomal proteins in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius.
Biochimica et biophysica acta, 1995Co-Authors: Ralf Moll, Silke Schmidtke, Günter SchäferAbstract:Abstract A 1.6 kb genomic DNA fragment derived from the extremely thermoacidophilic archaeon Sulfolobus acidocaldarius (DSM 639) comprises four open reading frames. The sequence contains three genes encoding crenarchaeal ribosomal proteins with apparent molecular masses of 6.3 kDa, 15.2 kDa and 9.9 kDa, which all represent strongly basic properties. These were identified by sequence comparison as RL46, RL31 and RL33. One open reading frame encodes a new polypeptide (22.1 kDa, pI = 7.3) with no homology to known proteins. The latter is transcribed as a common mRNA with RL46 and RL31. This gene cluster immediately precedes another cluster including genes encoding the putative SRP receptor α subunit as well as the putative secEp.
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Evidence for a Rieske‐type FeS center in the thermoacidophilic archaebacterium Sulfolobus acidocaldarius
FEBS letters, 1993Co-Authors: Stefan Anemüller, Günter Schäfer, Christian L. Schmidt, Miguel TeixeiraAbstract:Abstract A high-potential iron-sulfur cluster with characteristics similar to a Rieske-type center was detected in the plasma membrane of Sulfolobus acidocaldarius by EPR spectroscopy. In the reduced form this center has g-values of gz = 2.031, gy = 1.890 and gx = 1.725 (gav = 1.88, rhombicity = 0.37) and its reduction potential at pH 7.4 was determined to be +325 ± 10 mV. The archaebacterial cluster exhibits some unique properties, in comparison to eubacterial and eukaryotic Rieske-type centers. First, the reduction potential is pH-dependent in the range from pH 6.7 to 8.2. Second, the typical inhibitor of Rieske FeS centers, DBMIB, had no effect on the g-values of this cluster. The center is reducible by both NADH and succinate in the presence of cyanide, an inhibitor of the terminal oxidases. The possible role of a Rieske-type center in an organism lacking any c-type cytochromes is discussed.
Tairo Oshima - One of the best experts on this subject based on the ideXlab platform.
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A physical map of the sulfur-dependent archaebacterium Sulfolobus acidocaldarius 7 chromosome.
Journal of bacteriology, 1993Co-Authors: S. Kondo, Akihiko Yamagishi, Tairo OshimaAbstract:A chromosomal map of the sulfur-dependent thermoacidophilic archaebacterium Sulfolobus acidocaldarius 7 was constructed with four restriction enzymes: NotI, BssHII, RsrII, and EagI. The map indicated that the chromosome is a single circular DNA of 2,760 +/- 20 kb (mean +/- standard error of the mean). rRNA genes were also mapped. They were located at one site in the genome.
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Positive selection for uracil auxotrophs of the sulfur-dependent thermophilic archaebacterium Sulfolobus acidocaldarius by use of 5-fluoroorotic acid.
Journal of Bacteriology, 1991Co-Authors: S. Kondo, Akihiko Yamagishi, Tairo OshimaAbstract:Uracil auxotrophs of Sulfolobus acidocaldarius were positively selected by using 5-fluoroorotic acid. The wild-type strain was unable to grow in medium containing 5-fluoroorotic acid, whereas the mutants grew normally. Positive selection could be done for the auxotrophs. Mutants deficient in orotidine-59-monophosphate pyrophosphorylase activity were isolated.
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Crystallization and preliminary X-ray studies on Sulfolobus acidocaldarius ferredoxin.
Journal of biochemistry, 1991Co-Authors: Tomomi Fujii, Hideaki Moriyama, Akio Takénaka, Nobuo Tanaka, Takayoshi Wakagi, Tairo OshimaAbstract:Ferredoxin from a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius, has been crystallized. The space group is P4(3)2(1)2 or P4(1)2(1)2 and the cell dimensions are a = b = 50.12 A and c = 69.52 A. The Vm value is calculated to be 1.88 A3/Da, assuming one molecule per asymmetric unit. The crystal diffracts X-rays beyond 2.0 A resolution.
Dennis W. Grogan - One of the best experts on this subject based on the ideXlab platform.
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Homologous recombination of exogenous DNA with the Sulfolobus acidocaldarius genome: Properties and uses
FEMS microbiology letters, 2005Co-Authors: Norio Kurosawa, Dennis W. GroganAbstract:In order to quantify recombination between exogenous DNA and the Sulfolobus acidocaldarius chromosome, we electroporated pyrE (uracil-auxtotrophic) recipient strains with functional pyrE sequences and counted Pyr+ transformants by direct plating. Certain culture and post-electroporation conditions increased the yield of Pyr+ recombinants from non-replicating pyrE plasmid, whereas cognate methylation of SuaI restriction sites in the plasmid decreased it. Recombination of linear DNAs with the S. acidocaldarius genome was proportional to the length of a limiting overlap, but even synthetic oligonucleotides produced reasonable numbers of recombinants with appropriate recipient strains. To investigate uses of this latter property, we electroporated an 18-bp pyrE deletion mutant with mixtures of synthetic oligonucleotides altering glycine-55 of the orotate phosphoribosyl transferase encoded by pyrE. Pyr+ transformants were recovered in which this codon was converted to each of the alternatives encoded by the oligonucleotide mixtures, thereby identifying five amino acid substitutions tolerated at this position of the thermostable enzyme.
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Conjugational Genetic Exchange in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius: Intragenic Recombination with Minimal Dependence on Marker Separation
Journal of bacteriology, 2005Co-Authors: Josh E. Hansen, Amy C. Dill, Dennis W. GroganAbstract:In Sulfolobus acidocaldarius conjugation assays, recombinant frequency was relatively constant for marker separations from 1,154 bp down to about 50 bp and readily detectable at 10 bp. Three-factor crosses revealed little, if any, genetic linkage over distances of 500 to 600 bp, and large deletion mutants were good donors but poor recipients in matings. The results indicate that most intragenic recombination events occur at one of the mutations, not in the interval between them.
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Biological effects of DNA damage in the hyperthermophilic archaeon Sulfolobus acidocaldarius.
FEMS microbiology letters, 2002Co-Authors: Michelle S. Reilly, Dennis W. GroganAbstract:To investigate the generality of efficient double-strand break repair and damage-induced mutagenesis in hyperthermophilic archaea, we systematically measured the effects of five DNA-damaging agents on Sulfolobus acidocaldarius and compared the results to those obtained for Escherichia coli under corresponding conditions. The observed lethality of gamma-radiation was very similar for S. acidocaldarius and E. coli, arguing against unusually efficient double-strand break repair in S. acidocaldarius. In addition, DNA-strand-breaking agents (gamma-radiation or bleomycin), as well as DNA-cross-linking agents (mechlorethamine, butadiene diepoxide or cisplatin) stimulated forward mutation, reverse mutation, and formation of recombinants via conjugation in Sulfolobus cells. Although two of the five DNA-damaging agents failed to revert the E. coli auxotrophs under these conditions, all five reverted S. acidocaldarius auxotrophs.
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Loss of genetic accuracy in mutants of the thermoacidophile Sulfolobus acidocaldarius
Archaea (Vancouver B.C.), 2002Co-Authors: Greg David Bell, Dennis W. GroganAbstract:To investigate how hyperthermophilic archaea can propagate their genomes accurately, we isolated Sulfolobus acidocaldarius mutants exhibiting abnormally high rates of spontaneous mutation. Our isolation strategy involved enrichment for mutator lineages via alternating selections, followed by screening for the production of spontaneous, 5-fluoro-orotate-resistant mutants in micro-colonies. Several candidates were evaluated and found to have high frequencies of pyrE and pyrF mutation and reversion. Neither an increased efficiency of plating of mutants on selective medium, nor the creation of a genetically unstable pyrE allele, could be implicated as the cause of these high frequencies. The strains had elevated frequencies of other mutations, and exhibited certain phenotypic differences among themselves. A large increase in sensitivity to DNA-damaging agents was not observed, however. These properties generally resemble those of bacterial mutator mutants and suggest loss of functions specific to genetic accuracy.
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Altered patterns of cellular growth, morphology, replication and division in conditional-lethal mutants of the thermophilic archaeon Sulfolobus acidocaldarius
Microbiology, 2000Co-Authors: Rolf Bernander, Andrzej Poplawski, Dennis W. GroganAbstract:As a basis for studing the essential cellular processes of hyperthermophilic archaea, thermosensitive mutants of Sulfolobus acidocaldarius were isolated and characterized. Exponential-phase liquid cultures were shifted to the non-permissive temperature and growth, viability, and distributions of cell mass and DNA content were measured as a function of time after the shift. The observed phenotypes demonstrate that chromosome replication, nucleoid organization, nucleoid partition and cell division, which normally are tightly co-ordinated during cellular growth, can be inhibited or uncoupled by mutation in this hyperthermophilic archaeon.
Sonja-verena Albers - One of the best experts on this subject based on the ideXlab platform.
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Biochemical characterization of archaeal homocitrate synthase from Sulfolobus acidocaldarius.
FEBS letters, 2019Co-Authors: Tomohiro Suzuki, Kento Takahashi, Nagisa Akiyama, Kerstin Lassak, Takeo Tomita, Sonja-verena Albers, Ayako Yoshida, Maria Florencia Haurat, Takuya Okada, Tomohisa KuzuyamaAbstract:The hyperthermophilic archaeon, Sulfolobus, synthesizes lysine via the α-aminoadipate pathway; however, the gene encoding homocitrate synthase, the enzyme responsible for the first and committed step of the pathway, has not yet been identified. In the present study, we identified saci_1304 as the gene encoding a novel type of homocitrate synthase fused with a Regulation of Amino acid Metabolism (RAM) domain at the C terminus in Sulfolobus acidocaldarius. Enzymatic characterization revealed that Sulfolobus homocitrate synthase was inhibited by lysine; however, the mutant enzyme lacking the RAM domain was insensitive to inhibition by lysine. The present results indicated that the RAM domain is responsible for enzyme inhibition.
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crystal structure of an lrs14 like archaeal biofilm regulator from Sulfolobus acidocaldarius
Acta Crystallographica Section D-biological Crystallography, 2018Co-Authors: S L Volpel, Sonja-verena Albers, Larsoliver Essen, Ankan BanerjeeAbstract:The small winged helix-turn-helix (wHTH) proteins of the Lrs14 family are major transcriptional regulators and act as archaeal biofilm regulators (AbfRs) in the crenarchaeote Sulfolobus acidocaldarius. Here, the first crystal structure of an AbfR ortholog, AbfR2, the deletion of which is known to impair biofilm formation, is presented. Like most other wHTH orthologs, AbfR2 is dimeric in solution as well as in its 2.45 A resolution crystal structure. Given the presence of three independent AbfR2 dimers in the asymmetric unit, the crystal structure shows a considerable degree of conformational variation within the dimer, the antiparallel orientations of which are stabilized by coiled-coil interaction between H4 helices. Conserved anchor interactions between helices H0 and H4 of AbfR2 further contribute to dimer stabilization. The combined structural and bioinformatic analysis reveals cluster-specific structural differences between different members of the Lrs14 protein family.
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Expression, Purification, and Assembly of Archaellum Subcomplexes of Sulfolobus acidocaldarius.
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Paushali Chaudhury, Patrick Tripp, Sonja-verena AlbersAbstract:The archaellum assembly machinery and its filament consist of seven proteins in the crenarchaeon Sulfolobus acidocaldarius. We have so far expressed, purified, and biochemically characterized four of these archaellum subunits, namely, FlaX, FlaH, FlaI, and FlaF. FlaX, FlaH, and FlaI tightly interact and form the archaellum motor complex important for archaellum assembly and rotation. We have previously shown that FlaH forms an inner ring within a very stable FlaX ring, and therefore FlaX is believed to provide the scaffold for the assembly of the archaellum motor complex. Here we describe how to express and purify FlaX and FlaH and how the double ring structure both form can be obtained.
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Characterization of two β-decarboxylating dehydrogenases from Sulfolobus acidocaldarius
Extremophiles, 2016Co-Authors: Kento Takahashi, Fumika Nakanishi, Nagisa Akiyama, Kerstin Lassak, Takeo Tomita, Tomohisa Kuzuyama, Sonja-verena Albers, Makoto NishiyamaAbstract:Sulfolobus acidocaldarius , a hyperthermoacidophilic archaeon, possesses two β-decarboxylating dehydrogenase genes, saci_0600 and saci_2375 , in its genome, which suggests that it uses these enzymes for three similar reactions in lysine biosynthesis through 2-aminoadipate, leucine biosynthesis, and the tricarboxylic acid cycle. To elucidate their roles, these two genes were expressed in Escherichia coli in the present study and their gene products were characterized. Saci_0600 recognized 3-isopropylmalate as a substrate, but exhibited slight and no activity for homoisocitrate and isocitrate, respectively. Saci_2375 exhibited distinct and similar activities for isocitrate and homoisocitrate, but no detectable activity for 3-isopropylmalate. These results suggest that Saci_0600 is a 3-isopropylmalate dehydrogenase for leucine biosynthesis and Saci_2375 is a dual function enzyme serving as isocitrate-homoisocitrate dehydrogenase. The crystal structure of Saci_0600 was determined as a closed-form complex that binds 3-isopropylmalate and Mg^2+, thereby revealing the structural basis for the extreme thermostability and novel-type recognition of the 3-isopropyl moiety of the substrate.
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Alterations of the Transcriptome of Sulfolobus acidocaldarius by Exoribonuclease aCPSF2
PloS one, 2013Co-Authors: Birgit Märtens, Sonja-verena Albers, Lukas Zeichen, Fabian Amman, Salim Manoharadas, Alvaro Orell, Ivo L. Hofacker, Udo BläsiAbstract:Recent studies identified a 5´ to 3´ exoribonuclease termed Sso-RNase J in the crenarchaeon Sulfolobus solfataricus (Sso), which has been reclassified to the aCPSF2 (archaeal cleavage and polyadenylation specificity factor 2) group of β-CASP proteins. In this study, the Sso-aCPSF2 orthologue of Sulfolobus acidocaldarius (Saci-aCPSF2) was functionally characterized. Like Sso-aCPSF2, Saci-aCPSF2 degrades RNA with 5´ to 3´ directionality in vitro. To address the biological significance of Saci-aCPSF2, a deletion mutant was constructed, and the influence of Saci-aCPSF2 on the transcriptome profile was assessed employing high throughput RNA sequencing. This analysis revealed 560 genes with differential transcript abundance, suggesting a considerable role of this enzyme in RNA metabolism. In addition, bioinformatic analyses revealed several transcripts that are preferentially degraded at the 5´ end. This was exemplarily verified for two transcripts by Northern-blot analyses, showing for the first time that aCPSF2 proteins play a role in 5' to 3' directional mRNA decay in the crenarchaeal clade of Archaea.
Masashi Kurimoto - One of the best experts on this subject based on the ideXlab platform.
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Production of Trehalose from Starch by Thermostable Enzymes from Sulfolobus acidocaldarius
Starch - Stärke, 1997Co-Authors: Kazuhisa Mukai, Tetsuya Nakada, Hiroto Chaen, Shigeharu Fukuda, Masashi Kurimoto, Akihiko Tabuchi, Takashi Shibuya, Yoshio TsujisakaAbstract:The optimum conditions for the production of trehalose from starch were investigated using two thermostable enzymes, maltooligosyl trehalose synthase (MTSase) and maltooligosyl trehalose trehalohydrolase (MTHase), from Sulfolobus acidocaldarius ATCC 33909. The optimum pH was 5.5 and the optimum temperature was 55—57°C using isoamylase from Pseudomonas amyloderamosa as a debranching enzyme. The addition of CGTase to the reaction mixture during the saccharification process caused an increase in trehalose and a decrease in maltose and maltotriose. Isoamylase was better than pullulanase as a debranching enzyme. The yield of trehalose was independent of the type of starch used. Under optimum conditions, the yield of trehalose from corn starch at 30% concentration was more than 82%.
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Cloning and sequencing of a cluster of genes encoding novel enzymes of trehalose biosynthesis from thermophilic archaebacterium Sulfolobus acidocaldarius.
Biochimica et biophysica acta, 1996Co-Authors: Kazuhiko Maruta, Hitoshi Mitsuzumi, Tetsuya Nakada, Michio Kubota, Hiroto Chaen, Shigeharu Fukuda, Toshiyuki Sugimoto, Masashi KurimotoAbstract:Abstract Trehalose biosynthesis genes, treZ, treX and treY, encoding maltooligosyltrehalose trehalohydrolase (TreZ), glycogen debranching enzyme (TreX), and maltooligosyltrehalose synthase (TreY) have been cloned from the thermophilic archaebacterium Sulfolobus acidocaldarius ATCC33909. The amino-acid sequences deduced from treZ, treX and treY are composed of 556, 713 and 720 amino-acid residues, respectively. TreZ and TreY are 33–40% homologous to the corresponding enzymes from Arthrobacter sp. Q36. We have proposed that the biosynthesis of trehalose in Sulfolobus occurs via the actions of the three enzymes encoded by treZXY.
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purification and characterization of thermostable maltooligosyl trehalose trehalohydrolase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius
Bioscience Biotechnology and Biochemistry, 1996Co-Authors: Tetsuya Nakada, Michio Kubota, Hiroto Chaen, Shigeharu Fukuda, Toshiyuki Sugimoto, Masashi Kurimoto, Shoji Ikegami, Yoshio TsujisakaAbstract:A thermostable maltooligosyl trehalose trehalohydrolase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius ATCC 33909 was purified from a cell-free extract to an electrophoretically pure state by successive column chromatographies on Sepabeads FP-DA13, Butyl-Toyopearl 650M, DEAE-Toyopearl 650S, Toyopearl HW-55S and Ultrogel AcA44. The enzyme had a molecular mass of 59,000 by SDS-polyacrylamide gel electrophoresis and a pI of 6.1 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The enzyme showed the highest activity from pH 5.5 to 6.0 and at 75°C, and was stable from pH 5.5 to 9.5 and up to 85°C. The activity was inhibited by Hg2 +, Cu2 +, Fe2 +, Pb2 +, and Zn2 +. The Km values of the enzyme for maltosyl trehalose, maltotriosyl trehalose, maltotetraosyl trehalose, and maltopentaosyl trehalose were 16.7 mM, 2.7 mm, 3.7 mM, and 4.9 mM, respectively.
