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Sample records for archaeon sulfolobus solfataricus

  1. "Hot standards" for the thermoacidophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Zaparty, Melanie; Esser, Dominik; Gertig, Susanne; Haferkamp, Patrick; Kouril, Theresa; Manica, Andrea; Pham, Trong K.; Reimann, Julia; Schreiber, Kerstin; Sierocinski, Pawel; Teichmann, Daniela; van Wolferen, Marleen; von Jan, Mathias; Wieloch, Patricia; Albers, Sonja V.; Driessen, Arnold J. M.; Klenk, Hans-Peter; Schleper, Christa; Schomburg, Dietmar; van der Oost, John; Wright, Phillip C.; Siebers, Bettina

    2010-01-01

    Within the archaea, the thermoacidophilic crenarchaeote Sulfolobus solfataricus has become an important model organism for physiology and biochemistry, comparative and functional genomics, as well as, more recently also for systems biology approaches. Within the Sulfolobus Systems Biology ("SulfoSYS

  2. Proteomic mapping of the hyperthermophilic and acidophilic archaeon Sulfolobus solfataricus P2

    Energy Technology Data Exchange (ETDEWEB)

    Barry, Richard C.; Young, Mark J.; Stedman, Kenneth M.; Dratz, Edward A.

    2006-07-14

    A proteomic map of Sulfolobus solfataricus P2, an archaeon that grows optimally at 80 C and pH 3.2, was developed using high resolution two-dimensional gel electrophoresis and peptide mass fingerprinting. A total of 867 protein spots (659 aqueous tris-soluble spots and 208 aqueous tris-insoluble) were mapped over IPG 3-10, 4-7, and 6-11, with second dimension gels made of 8-18% polyacrylamide. 324 different gene products were represented by the 867 spots, with 274 gene products being identified in the tris-soluble fractions and 100 gene products in the tris-insoluble portion. Fifty gene products were found on gels from both fractions. Additionally, an average of 1.50 + 0.12 isoforms/per protein were identified. This mapping study confirmed the expression of proteins involved in numerous metabolic, transport, energy production, nucleic acid replication, translation, and transcription pathways. Of particular interest, phosphoenolpyruvate carboxykinase (SSO2537) was detected even though the pathway for gluconeogenesis is unknown for this archaeon. Tris-soluble fractions contained many cytosolic proteins while tris-insoluble fractions contained many membrane-associated proteins, including ABC transporters and an ATP synthase. This study provides an optimized 2-DE approach for investigating the biochemical pathways and post-translational modifications employed by Sulfolobus to survive in its extreme environment.

  3. A new type of didhydroorotate dehydrogenase, type 1S, from the thermoacidophilic archaeon Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Sørensen, P. G.; Dandanell, Gert

    2002-01-01

    Dihydroorotate dehydrogenase (DHOD) (EC 1.3.3.1) from the thermoacidophilic archaeon Sulfolobus solfataricus P2 (DSM 1617) was partially purified 3,158-fold, characterized, and the encoding genes identified. Based on enzymological as well as phylogenetic methods, dihydroorotate dehydrogenase from S....... solfataricus (DHODS) represents a new type of DHOD, type 1S. Furthermore, it is unable to use any of the (type-specific) natural electron acceptors employed by all other presently known DHODs. DHODS shows optimal activity at 70°C in the pH range 7-8.5. It is capable of using ferricyanide, 2...

  4. A Phosphohexomutase from the Archaeon Sulfolobus solfataricus Is Covalently Modified by Phosphorylation on Serine

    OpenAIRE

    Ray, W. Keith; Keith, Sabrina M.; DeSantis, Andrea M.; Hunt, Jeremy P.; Larson, Timothy J.; Helm, Richard F.; Kennelly, Peter J.

    2005-01-01

    A phosphoserine-containing peptide was identified from tryptic digests from Sulfolobus solfataricus P1 by liquid chromatography-tandem mass spectrometry. Its amino acid sequence closely matched that bracketing Ser-309 in the predicted protein product of open reading frame sso0207, a putative phosphohexomutase, in the genome of S. solfataricus P2. Open reading frame sso0207 was cloned, and its protein product expressed in Escherichia coli. The recombinant protein proved capable of interconvert...

  5. Mercury Inactivates Transcription and the Generalized Transcription Factor TFB in the Archaeon Sulfolobus solfataricus

    OpenAIRE

    Dixit, Vidula; Bini, Elisabetta; Drozda, Melissa; Blum, Paul

    2004-01-01

    Mercury has a long history as an antimicrobial agent effective against eukaryotic and prokaryotic organisms. Despite its prolonged use, the basis for mercury toxicity in prokaryotes is not well understood. Archaea, like bacteria, are prokaryotes but they use a simplified version of the eukaryotic transcription apparatus. This study examined the mechanism of mercury toxicity to the archaeal prokaryote Sulfolobus solfataricus. In vivo challenge with mercuric chloride instantaneously blocked cel...

  6. Genome-scale reconstruction and analysis of the metabolic network in the hyperthermophilic archaeon Sulfolobus solfataricus.

    Directory of Open Access Journals (Sweden)

    Thomas Ulas

    Full Text Available We describe the reconstruction of a genome-scale metabolic model of the crenarchaeon Sulfolobus solfataricus, a hyperthermoacidophilic microorganism. It grows in terrestrial volcanic hot springs with growth occurring at pH 2-4 (optimum 3.5 and a temperature of 75-80°C (optimum 80°C. The genome of Sulfolobus solfataricus P2 contains 2,992,245 bp on a single circular chromosome and encodes 2,977 proteins and a number of RNAs. The network comprises 718 metabolic and 58 transport/exchange reactions and 705 unique metabolites, based on the annotated genome and available biochemical data. Using the model in conjunction with constraint-based methods, we simulated the metabolic fluxes induced by different environmental and genetic conditions. The predictions were compared to experimental measurements and phenotypes of S. solfataricus. Furthermore, the performance of the network for 35 different carbon sources known for S. solfataricus from the literature was simulated. Comparing the growth on different carbon sources revealed that glycerol is the carbon source with the highest biomass flux per imported carbon atom (75% higher than glucose. Experimental data was also used to fit the model to phenotypic observations. In addition to the commonly known heterotrophic growth of S. solfataricus, the crenarchaeon is also able to grow autotrophically using the hydroxypropionate-hydroxybutyrate cycle for bicarbonate fixation. We integrated this pathway into our model and compared bicarbonate fixation with growth on glucose as sole carbon source. Finally, we tested the robustness of the metabolism with respect to gene deletions using the method of Minimization of Metabolic Adjustment (MOMA, which predicted that 18% of all possible single gene deletions would be lethal for the organism.

  7. Allosteric regulation of the GTP activated and CTP inhibited uracil phosphoribosyltransferase from the thermophilic archaeon Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Arent, Susan; Larsen, Sine;

    2005-01-01

    The upp gene, encoding uracil phosphoribosyltransferase (UPRTase) from the thermoacidophilic archaeon Sulfolobus solfataricus, was cloned and expressed in Escherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH 5.5 when...... assayed at 60 °C. Enzyme activity was strongly stimulated by GTP and inhibited by CTP. GTP caused an approximately 20-fold increase in the turnover number kcat and raised the Km values for 5-phosphoribosyl-1-diphosphate (PRPP) and uracil by two- and >10-fold, respectively. The inhibition by CTP was...

  8. Gene content and organization of a 281-kbp contig from the genome of the extremely thermophilic archaeon, Sulfolobus solfataricus P2

    NARCIS (Netherlands)

    Charlebois, R.; Confalonieri, F.; Curtis, B.; Doolittle, W.F.; Duguet, M.; Erauso, G.; Faguy, D.; Gaasterland, T.; Garrett, R.A.; Gordon, P.; Kozera, C.; Medina, N.; Oost, van der J.; Peng, X.; Ragan, M.; She, Q.; Singh, R.K.

    2000-01-01

    The sequence of a 281-kbp contig from the crenarchaeote Sulfolobus solfataricus P2 was determined and analysed. Notable features in this region include 29 ribosomal protein genes, 12 tRNA genes (four of which contain archaeal-type introns), operons encoding enzymes of histidine biosynthesis, pyrimid

  9. Specificities and pH profiles of adenine and hypoxanthine-guanine-xanthine phosphoribosyltransferases (nucleotide synthases) of the thermoacidophile archaeon Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Hansen, Michael Riis; Jensen, Kristine Steen; Rasmussen, Mads Skytte;

    2014-01-01

    Two open reading frames in the genome of Sulfolobus solfataricus (SSO2341 and SSO2424) were cloned and expressed in E. coli. The protein products were purified and their enzymatic activity characterized. Although SSO2341 was annotated as a gene (gpT-1) encoding a 6-oxopurine phosphoribosyltransfe......Two open reading frames in the genome of Sulfolobus solfataricus (SSO2341 and SSO2424) were cloned and expressed in E. coli. The protein products were purified and their enzymatic activity characterized. Although SSO2341 was annotated as a gene (gpT-1) encoding a 6-oxopurine...... phosphoribosyltransferase (PRTase), the protein product turned out to be a PRTase highly specific for adenine and we suggest that the reading frame should be renamed apT. The other reading frame SSO2424 (gpT-2) proved to be a true 6-oxopurine PRTase active with hypoxanthine, xanthine and guanine as substrates, and we.......5, while maximal activity with xanthine was observed at pH 7.5. We discuss likely reasons why SSO2341 in S. solfataricus and similar open reading frames in other Crenarchaeota could not be identified as genes encoding APRTase....

  10. Crystallization and preliminary X-ray diffraction analysis of PAT, an acetyltransferase from Sulfolobus solfataricus

    International Nuclear Information System (INIS)

    PAT, an acetyltransferase from the archaeon S. solfataricus that specifically acetylates the chromatin protein Alba, was expressed, purified and crystallized. PAT is an acetyltransferase from the archaeon Sulfolobus solfataricus that specifically acetylates the chromatin protein Alba. The enzyme was expressed, purified and subsequently crystallized using the sitting-drop vapour-diffusion technique. Native diffraction data were collected to 1.70 Å resolution on the BL13C1 beamline of NSRRC from a flash-frozen crystal at 100 K. The crystals belonged to space group P212121, with unit-cell parameters a = 44.30, b = 46.59, c = 68.39 Å

  11. Crystallization and preliminary X-ray characterization of a PaaX-like protein from Sulfolobus solfataricus P2

    International Nuclear Information System (INIS)

    In this study, the PaaX-like protein from the hyperthermophilic archaeon Sulfolobus solfataricus P2 was successfully crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. PaaX is a global regulator of the phenylacetyl-coenzyme A catabolon that adjusts the expression of different operons to that of the paa-encoded central pathway. In this study, the PaaX-like protein from the hyperthermophilic archaeon Sulfolobus solfataricus P2 was successfully crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. Diffraction data were obtained to a resolution of 3.0 Å using synchrotron radiation at the Photon Factory. The crystal belonged to space group P321, with unit-cell parameters a = 86.4, b = 86.4, c = 105.5 Å

  12. Post-genomic characterization of metabolic pathways in Sulfolobus solfataricus

    NARCIS (Netherlands)

    Walther, J.

    2012-01-01

    The physiological functions and mode of actions of different biomolecules are of continuous interest and a prerequisite to fully understand and appreciate the potential of Archaea and their molecules. We chose to study Sulfolobus solfataricus for its stable (heat-resistant) enzymes and specific meta

  13. Structure and Biochemical Characterization of Protein Acetyltransferase from Sulfolobus solfataricus

    Energy Technology Data Exchange (ETDEWEB)

    Brent, Michael M.; Iwata, Ayaka; Carten, Juliana; Zhao, Kehao; Marmorstein, Ronen; (UPENN)

    2009-09-02

    The Sulfolobus solfataricus protein acetyltransferase (PAT) acetylates ALBA, an abundant nonspecific DNA-binding protein, on Lys{sup 16} to reduce its DNA affinity, and the Sir2 deacetylase reverses the modification to cause transcriptional repression. This represents a 'primitive' model for chromatin regulation analogous to histone modification in eukaryotes. We report the 1.84-{angstrom} crystal structure of PAT in complex with coenzyme A. The structure reveals homology to both prokaryotic GNAT acetyltransferases and eukaryotic histone acetyltransferases (HATs), with an additional 'bent helix' proximal to the substrate binding site that might play an autoregulatory function. Investigation of active site mutants suggests that PAT does not use a single general base or acid residue for substrate deprotonation and product reprotonation, respectively, and that a diffusional step, such as substrate binding, may be rate-limiting. The catalytic efficiency of PAT toward ALBA is low relative to other acetyltransferases, suggesting that there may be better, unidentified substrates for PAT. The structural similarity of PAT to eukaryotic HATs combined with its conserved role in chromatin regulation suggests that PAT is evolutionarily related to the eukaryotic HATs.

  14. Discovery and characterization of a second extremely thermostable (+)-γ-lactamase from Sulfolobus solfataricus P2.

    Science.gov (United States)

    Zhu, Shaozhou; Huang, Rong; Gao, Shuaihua; Li, Xinxin; Zheng, Guojun

    2016-05-01

    A thermostable formamidase from the hyperthermophilic archaeon Sulfolobus solfataricus P2 was revealed to be a novel, thermostable (+)-γ-lactamase. This (+)-γ-lactamase (Sso2810) is composed of only 318 amino acid residues, in contrast to a previously reported (+)-γ-lactamase (Sso2122) with 504 amino acid residues from the same strain. Herein, we demonstrate that a single strain may contain diverse (+)-γ-lactamases. The gene of this thermostable (+)-γ-lactamase was cloned, functionally expressed in Escherichia coli BL21 and purified by a simple yet effective heat treatment method. Sso2810 was biochemically characterized and compared to Sso2122, with phylogenetic analysis indicating different evolutionary histories for the two encoding genes. This newly found thermostable enzyme shows promising properties for industrial applications; specifically, it could be used for the production of chirally pure (-)-γ-lactam for the synthesis of well-known carbocyclic nucleoside antiretroviral agents like Abacavir and Peramivir. The chiral product of the enzyme was purified to >99% enantiomeric excess. PMID:26685014

  15. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Andersen, Rune W.; Lo Leggio, Leila; Hove-Jensen, Bjarne;

    2015-01-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg2+-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP...

  16. Crystallization and preliminary X-ray crystallographic analysis of the Sulfolobus solfataricus nucleotide-exchange factor 1β

    Energy Technology Data Exchange (ETDEWEB)

    Ruggiero, Alessia [Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, I-80134 Napoli (Italy); Masullo, Mariorosario [Dipartimento di Scienze Farmacobiologiche, Università degli Studi Magna Graecia, Roccelletta di Borgia, I-88021 Catanzaro (Italy); Arcari, Paolo [Dipartimento di Biochimica e Biotecnologie Mediche, Università degli Studi Federico II, I-80131 Napoli (Italy); Raimo, Gennaro [Dipartimento di Biochimica e Biotecnologie Mediche, Università degli Studi Federico II, I-80131 Napoli (Italy); Dipartimento di Scienze e Tecnologie per l’Ambiente e il Territorio, Università degli Studi del Molise, I-86170 Isernia (Italy); Vitagliano, Luigi [Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, I-80134 Napoli (Italy); Centro Interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPEB), I-80134 Napoli (Italy); Zagari, Adriana, E-mail: zagari@unina.it [Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, I-80134 Napoli (Italy); Dipartimento delle Scienze Biologiche, Sezione di Biostrutture, Università degli Studi Federico II, I-80134 Napoli (Italy)

    2005-11-01

    Nucleotide-exchange factor from S. solfataricus (SsEF-1β) has been successfully crystallized. X-ray diffraction data have been collected from the native enzyme and from the selenomethionine derivative of SsEF-1β to 1.97 and 1.83 Å resolution, respectively. The nucleotide-exchange factor isolated from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-1β) consists of 90 residues and differs from eukaryal EF-1βs. The protein has been successfully crystallized using either microbatch-under-oil or vapour-diffusion methods. Crystals of native SsEF-1β diffract to 1.97 Å resolution and belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 106.46, b = 54.87, c = 44.03 Å. Diffraction data have also been collected from a selenomethionine derivative of SsEF-1β at 1.83 Å resolution. Model building using the phases derived from the MAD experiment is in progress.

  17. Crystallization and preliminary X-ray crystallographic analysis of two dimeric hyperthermostable thioredoxins isolated from Sulfolobus solfataricus

    International Nuclear Information System (INIS)

    Two thioredoxins (SsTrxA2 and SsTrxA1) isolated from S. solfataricus have been crystallized. Diffraction data have been collected from SsTrxA2 and SsTrxA1 to 1.83 and 1.90 Å resolution, respectively. The structures of both enzymes have been solved by molecular replacement. The thioredoxin system of the archaeon Sulfolobus solfataricus involves a number of different proteins: two thioredoxin reductases (SsTrxRB2 and SsTrxRB3), two distinct thioredoxins (SsTrxA1 and SsTrxA2) and a disulfide oxidoreductase (SsPDO). Here, the crystallization and preliminary crystallographic analyses of SsTrxA1 and SsTrxA2, two dimeric proteins endowed with extraordinary thermal stability, are reported. In addition to the functional thioredoxin domain, both SsTrxA1 and SsTrxA2 present an extra N-terminal fragment of approximately 30 residues. Although crystallization trials have been conducted on both forms of the proteins, crystals that were suitable for X-ray crystallographic analyses have only been obtained for their truncated variants. The crystals of SsTrxA2 belonged to space group P2, with unit-cell parameters a = 28.27, b = 27.88, c = 62.06 Å, β = 92.34°, and diffracted to 1.83 Å resolution, whereas the crystals of SsTrxA1 belonged to space group P21, with unit-cell parameters a = 51.76, b = 75.09, c = 55.35 Å, β = 112.64°, and diffracted to 1.90 Å resolution. The structures of the two proteins have been solved by molecular replacement

  18. Neuartige Tryptophan-Synthasen aus Hyperthermophilen: Charakterisierung der Enzyme aus Sulfolobus solfataricus

    OpenAIRE

    Leopoldseder, Sonja

    2006-01-01

    In der vorliegenden Arbeit wurde die Tryptophan Synthase aus Sulfolobus solfataricus strukturell und funktionell untersucht. Zu diesem Zweck wurden die Operon-ständigen Gene strpA und strpB2a, sowie das nicht Operon-ständige strpB2b getrennt in Escherichia coli exprimiert, die Genprodukte gereinigt und charakterisiert. sTrpB2a und sTrpB2b katalysieren mit vergleichbar hoher Effizienz die Synthese von Tryptophan aus Serin und Indol. Während sTrpB2b nicht mit sTrpA interagiert, bilden sTrpB2a ...

  19. Mutational analysis of divalent metal ion binding in the active site of class II α-mannosidase from sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Hansen, Dennis K.; Webb, Helen; Nielsen, Jonas Willum; Winther, Jakob R.; Willemoes, Martin; Harris, Pernille

    2015-01-01

    Mutational analysis of Sulfolobus solfataricus class II α-mannosidase was focused on side chains that interact with the hydroxyls of the-1 mannosyl of the substrate (Asp-534) or form ligands to the active site divalent metal ion (His-228 and His-533) judged from crystal structures of homologous...

  20. Functional curation of the Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 complete genome sequences

    NARCIS (Netherlands)

    Esser, D.; Kouril, T.; Zaparty, M.; Sierocinski, P.; Oost, van der J.; Makarova, K.S.; Siebers, B.

    2011-01-01

    The thermoacidophiles Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 are considered key model organisms representing a major phylum of the Crenarchaeota. Because maintaining current, accurate genome information is indispensable for modern biology, we have updated gene function annotation usin

  1. Identification of a GDP-mannose pyrophosphorylase gene from Sulfolobus solfataricus.

    Science.gov (United States)

    Sacchetti, Silvana; Bartolucci, Simonetta; Rossi, Mosè; Cannio, Raffaele

    2004-05-12

    An open reading frame (ORF) encoding a putative GDP-mannose pyrophosphorylase (SsoGMPP) was identified on the genome sequence of Sulfolobus solfataricus P2, the predicted gene product showing high amino acid sequence homology to several archaeal, bacterial, and eukaryal GDP-mannose pyrophosphorylases such as guanidine diphosphomannose pyrophosphorylases (GMPPs) from Saccharomyces cerevisiae and Arabidopsis thaliana. The sequence was PCR amplified from genomic DNA of S. solfataricus P2 and heterologous gene expression obtained as a fusion to glutathione S-transferase in Escherichia coli, under conditions suitable to reduce the formation of inclusion bodies. Specific assays performed at 60 degrees C revealed the presence of the archaeal synthesizing GDP-mannose enzyme activity in the cell extracts of the transformed E. coli. As a positive control, the same assays were performed at the mesophilic enzyme optimum temperature on the already characterized yeast recombinant GMPP. The recombinant protein was purified to homogeneity by glutathione sepharose affinity chromatography and its thermophilic nature could be verified. The enzyme was definitively identified by demonstrating its capability to catalyze also the reverse reaction of pyrophosphorolysis and, most interestingly, its high specificity for synthesizing GDP-mannose. PMID:15145064

  2. Membrane Association and Catabolite Repression of the Sulfolobus solfataricus α-Amylase

    Directory of Open Access Journals (Sweden)

    Edith Soo

    2015-09-01

    Full Text Available Sulfolobus solfataricus is a thermoacidophilic member of the archaea whose envelope consists of an ether-linked lipid monolayer surrounded by a protein S-layer. Protein translocation across this envelope must accommodate a steep proton gradient that is subject to temperature extremes. To better understand this process in vivo, studies were conducted on the S. solfataricus glycosyl hydrolyase family 57 α-Amylase (AmyA. Cell lines harboring site specific modifications of the amyA promoter and AmyA structural domains were created by gene replacement using markerless exchange and characterized by Western blot, enzyme assay and culture-based analysis. Fusion of amyA to the malAp promoter overcame amyAp-mediated regulatory responses to media composition including glucose and amino acid repression implicating action act at the level of transcription. Deletion of the AmyA Class II N-terminal signal peptide blocked protein secretion and intracellular protein accumulation. Deletion analysis of a conserved bipartite C-terminal motif consisting of a hydrophobic region followed by several charged residues indicated the charged residues played an essential role in membrane-association but not protein secretion. Mutants lacking the C-terminal bipartite motif exhibited reduced growth rates on starch as the sole carbon and energy source; therefore, association of AmyA with the membrane improves carbohydrate utilization. Widespread occurrence of this motif in other secreted proteins of S. solfataricus and of related Crenarchaeota suggests protein association with membranes is a general trait used by these organisms to influence external processes.

  3. Isolation of Extracellular Polymeric Substances from Biofilms of the Thermoacidophilic Archaeon Sulfolobus acidocaldarius

    OpenAIRE

    Jachlewski, Silke; Jachlewski, Witold D.; Linne, Uwe; Bräsen, Christopher; Wingender, Jost; Siebers, Bettina

    2015-01-01

    Extracellular polymeric substances (EPS) are the major structural and functional components of microbial biofilms. The aim of this study was to establish a method for EPS isolation from biofilms of the thermoacidophilic archaeon, Sulfolobus acidocaldarius, as a basis for EPS analysis. Biofilms of S. acidocaldarius were cultivated on the surface of gellan gum-solidified Brock medium at 78°C for 4 days. Five EPS extraction methods were compared, including shaking of biofilm suspensions in phosp...

  4. Copper tolerance of the thermoacidophilic archaeon Sulfolobus metallicus: possible role of polyphosphate metabolism.

    Science.gov (United States)

    Remonsellez, Francisco; Orell, Alvaro; Jerez, Carlos A

    2006-01-01

    It has been postulated that inorganic polyphosphate (polyP) and transport of metal-phosphate complexes could participate in heavy metal tolerance in some bacteria. To study if such a system exists in archaea, the presence of polyP was determined by the electron energy loss spectroscopy (EELS) procedure and quantified by using specific enzymic methods in Sulfolobus acidocaldarius, Sulfolobus metallicus and Sulfolobus solfataricus. All three micro-organisms synthesized polyP during growth, but only S. metallicus greatly accumulated polyP granules. The differences in the capacity to accumulate polyP between these archaea may reflect adaptive responses to their natural environment. Thus, S. metallicus could grow in and tolerate up to 200 mM copper sulfate, with a concomitant decrease in its polyP levels with increasing copper concentrations. On the other hand, S. solfataricus could not grow in or tolerate more than 1-5 mM copper sulfate, most likely due to its low levels of polyP. Shifting S. metallicus cells to copper sulfate concentrations up to 100 mM led to a rapid increase in their exopolyphosphatase (PPX) activity which was concomitant in time with a decrease in their polyP levels and a stimulation of phosphate efflux. Furthermore, copper in the range of 10 microM greatly stimulated PPX activity in cell-free extracts from S. metallicus. The results strongly suggest that a metal tolerance mechanism mediated through polyP is functional in members of the genus Sulfolobus. This ability to accumulate and hydrolyse polyP may play an important role not only in the survival of these micro-organisms in sulfidic mineral environments containing high toxic metals concentrations, but also in their applications in biomining. PMID:16385115

  5. SMV1 virus-induced CRISPR spacer acquisition from the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2

    DEFF Research Database (Denmark)

    Erdmann, Susanne; Shah, Shiraz Ali; Garrett, Roger Antony

    2013-01-01

    complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2...

  6. Biochemical, transcriptional and translational evidences of the phenol-meta-degradation pathway by the hyperthermophilic Sulfolobus solfataricus 98/2.

    Directory of Open Access Journals (Sweden)

    Alexia Comte

    Full Text Available Phenol is a widespread pollutant and a model molecule to study the biodegradation of monoaromatic compounds. After a first oxidation step leading to catechol in mesophilic and thermophilic microorganisms, two main routes have been identified depending on the cleavage of the aromatic ring: ortho involving a catechol 1,2 dioxygenase (C12D and meta involving a catechol 2,3 dioxygenase (C23D. Our work aimed at elucidating the phenol-degradation pathway in the hyperthermophilic archaea Sulfolobus solfataricus 98/2. For this purpose, the strain was cultivated in a fermentor under different substrate and oxygenation conditions. Indeed, reducing dissolved-oxygen concentration allowed slowing down phenol catabolism (specific growth and phenol-consumption rates dropped 55% and 39%, respectively and thus, evidencing intermediate accumulations in the broth. HPLC/Diode Array Detector and LC-MS analyses on culture samples at low dissolved-oxygen concentration (DOC  =  0.06 mg x L(-1 suggested, apart for catechol, the presence of 2-hydroxymuconic acid, 4-oxalocrotonate and 4-hydroxy-2-oxovalerate, three intermediates of the meta route. RT-PCR analysis on oxygenase-coding genes of S. solfataricus 98/2 showed that the gene coding for the C23D was expressed only on phenol. In 2D-DIGE/MALDI-TOF analysis, the C23D was found and identified only on phenol. This set of results allowed us concluding that S. solfataricus 98/2 degrade phenol through the meta route.

  7. A New Pepstatin-Insensitive Thermopsin-Like Protease Overproduced in Peptide-Rich Cultures of Sulfolobus solfataricus

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    Marta Gogliettino

    2014-02-01

    Full Text Available In this study, we gain insight into the extracellular proteolytic system of Sulfolobus solfataricus grown on proteinaceous substrates, providing further evidence that acidic proteases were specifically produced in response to peptide-rich media. The main proteolytic component was the previously isolated SsMTP (Sulfolobus solfataricus multi-domain thermopsin-like protease, while the less abundant (named SsMTP-1 one was purified, characterized and identified as the sso1175 gene-product. The protein revealed a multi-domain organization shared with the cognate SsMTP with a catalytic domain followed by several tandemly-repeated motifs. Moreover, both enzymes were found spread across the Crenarchaeota phylum and belonging to the thermopsin family, although segregated into diverse phylogenetic clusters. SsMTP-1 showed a 75-kDa molecular mass and was stable in the temperature range 50–90 °C, with optimal activity at 70 °C and pH 2.0. Serine, metallo and aspartic protease inhibitors did not affect the enzyme activity, designating SsMTP-1 as a new member of the pepstatin-insensitive aspartic protease family. The peptide-bond-specificity of SsMTP-1 in the cleavage of the oxidized insulin B chain was uncommon amongst thermopsins, suggesting that it could play a distinct, but cooperative role in the protein degradation machinery. Interestingly, predictions of the transmembrane protein topology of SsMTP and SsMTP-1 strongly suggest a possible contribution in signal-transduction pathways.

  8. Identification and molecular characterization of the first a-xylosidase from an Archaeon

    NARCIS (Netherlands)

    Moracci, M.; Cobucci-Ponzano, B.; Trincone, A.; Fusco, S.; Rosa, de M.; Oost, van der J.; Sensen, C.W.; Charlebois, R.L.; Rossi, M.

    2000-01-01

    We here report the first molecular characterization of an -xylosidase (XylS) from an Archaeon. Sulfolobus solfataricus is able to grow at temperatures higher than 80 °C on several carbohydrates at acidic pH. The isolated xylS gene encodes a monomeric enzyme homologous to -glucosidases, -xylosidases,

  9. Bypass of Aflatoxin B[subscript 1] Adducts by the Sulfolobus solfataricus DNA Polymerase IV

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Surajit; Brown, Kyle L.; Egli, Martin; Stone, Michael P. (Vanderbilt)

    2012-07-18

    Aflatoxin B{sub 1} (AFB{sub 1}) is oxidized to an epoxide in vivo, which forms an N7-dG DNA adduct (AFB{sub 1}-N7-dG). The AFB{sub 1}-N7-dG can rearrange to a formamidopyrimidine (AFB{sub 1}-FAPY) derivative. Both AFB{sub 1}-N7-dG and the {beta}-anomer of the AFB{sub 1}-FAPY adduct yield G {yields} T transversions in Escherichia coli, but the latter is more mutagenic. We show that the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) bypasses AFB{sub 1}-N7-dG in an error-free manner but conducts error-prone replication past the AFB{sub 1}-FAPY adduct, including misinsertion of dATP, consistent with the G {yields} T mutations observed in E. coli. Three ternary (Dpo4-DNA-dNTP) structures with AFB{sub 1}-N7-dG adducted template:primers have been solved. These demonstrate insertion of dCTP opposite the AFB{sub 1}-N7-dG adduct, and correct vs incorrect insertion of dATP vs dTTP opposite the 5'-template neighbor dT from a primed AFB{sub 1}-N7-dG:dC pair. The insertion of dTTP reveals hydrogen bonding between the template N3 imino proton and the O{sup 2} oxygen of dTTP, and between the template T O{sup 4} oxygen and the N3 imino proton of dTTP, perhaps explaining why this polymerase does not efficiently catalyze phosphodiester bond formation from this mispair. The AFB{sub 1}-N7-dG maintains the 5'-intercalation of the AFB{sub 1} moiety observed in DNA. The bond between N7-dG and C8 of the AFB{sub 1} moiety remains in plane with the alkylated guanine, creating a 16{sup o} inclination of the AFB{sub 1} moiety with respect to the guanine. A binary (Dpo4-DNA) structure with an AFB{sub 1}-FAPY adducted template:primer also maintains 5'-intercalation of the AFB{sub 1} moiety. The {beta}-deoxyribose anomer is observed. Rotation about the FAPY C5-N{sup 5} bond orients the bond between N{sup 5} and C8 of the AFB{sub 1} moiety out of plane in the 5'-direction, with respect to the FAPY base. The formamide group extends in the 3'-direction. This improves

  10. Effect of DNA binding protein Ssh12 from hyperthermophilic archaeon Sulfolobus shibatae on DNA supercoiling

    Institute of Scientific and Technical Information of China (English)

    楼慧强; 黄力; VietQ.Mai

    1999-01-01

    An 11.5-ku DNA binding protein, designated as Sshl2, was purified from the hyperthermophilic archaeon Sulfolobus shibatae by column chromatography in SP Sepharose, DNA cellulose and phosphocellulose. Sshl2 accounts for about 4 % of the total cellular protein. The protein is capable of binding to both negatively supercoiled and relaxed DNAs. Nick closure analysis revealed that Sshl2 constrains negative supercoils upon binding to DNA. While the ability of the protein to constrain supercoils is weak at 22℃ , it is enhanced substantially at temperatures higher than 37℃ . Both the cellular content and supercoil-constraining ability of Sshl2 suggest that the protein may play an important role in the organization and stabilization of the chromosome of S. shibatae.

  11. EXPRESSION, PURIFICATION, AND SMALL ANGLE X-RAY SCATTERING OF DNA REPLICATION AND REPAIR PROTEINS FROM THE HYPERTHERMOPHILE SULFOLOBUS SOLFATARICUS

    Energy Technology Data Exchange (ETDEWEB)

    Patterson, S.M.; Hatherill, J.R.; Hammel, M.; Hura, G.L.; Tainer, J.A.; Yannone, S.M.

    2008-01-01

    Vital molecular processes such as DNA replication, transcription, translation, and maintenance occur through transient protein interactions. Elucidating the mechanisms by which these protein complexes and interactions function could lead to treatments for diseases related to DNA damage and cell division control. In the recent decades since its introduction as a third domain, Archaea have shown to be simpler models for complicated eukaryotic processes such as DNA replication, repair, transcription, and translation. Sulfolobus solfataricus is one such model organism. A hyperthermophile with an optimal growth temperature of 80°C, Sulfolobus protein-protein complexes and transient protein interactions should be more stable at moderate temperatures, providing a means to isolate and study their structure and function. Here we provide the initial steps towards characterizing three DNA-related Sulfolobus proteins with small angle X-ray scattering (SAXS): Sso0257, a cell division control and origin recognition complex homolog, Sso0768, the small subunit of the replication factor C, and Sso3167, a Mut-T like protein. SAXS analysis was performed at multiple concentrations for both short and long exposure times. The Sso0257 sample was determined to be either a mixture of monomeric and dimeric states or a population of dynamic monomers in various conformational states in solution, consistent with a fl exible winged helix domain. Sso0768 was found to be a complex mixture of multimeric states in solution. Finally, molecular envelope reconstruction from SAXS data for Sso3167 revealed a novel structural component which may function as a disordered to ordered region in the presence of its substrates and/or protein partners.

  12. Molecular Characterization of Copper and Cadmium Resistance Determinants in the Biomining Thermoacidophilic Archaeon Sulfolobus metallicus

    Directory of Open Access Journals (Sweden)

    Alvaro Orell

    2013-01-01

    Full Text Available Sulfolobus metallicus is a thermoacidophilic crenarchaeon used in high-temperature bioleaching processes that is able to grow under stressing conditions such as high concentrations of heavy metals. Nevertheless, the genetic and biochemical mechanisms responsible for heavy metal resistance in S. metallicus remain uncharacterized. Proteomic analysis of S. metallicus cells exposed to 100 mM Cu revealed that 18 out of 30 upregulated proteins are related to the production and conversion of energy, amino acids biosynthesis, and stress responses. Ten of these last proteins were also up-regulated in S. metallicus treated in the presence of 1 mM Cd suggesting that at least in part, a common general response to these two heavy metals. The S. metallicus genome contained two complete cop gene clusters, each encoding a metallochaperone (CopM, a Cu-exporting ATPase (CopA, and a transcriptional regulator (CopT. Transcriptional expression analysis revealed that copM and copA from each cop gene cluster were cotranscribed and their transcript levels increased when S. metallicus was grown either in the presence of Cu or using chalcopyrite (CuFeS2 as oxidizable substrate. This study shows for the first time the presence of a duplicated version of the cop gene cluster in Archaea and characterizes some of the Cu and Cd resistance determinants in a thermophilic archaeon employed for industrial biomining.

  13. Molecular characterization of copper and cadmium resistance determinants in the biomining thermoacidophilic archaeon Sulfolobus metallicus.

    Science.gov (United States)

    Orell, Alvaro; Remonsellez, Francisco; Arancibia, Rafaela; Jerez, Carlos A

    2013-01-01

    Sulfolobus metallicus is a thermoacidophilic crenarchaeon used in high-temperature bioleaching processes that is able to grow under stressing conditions such as high concentrations of heavy metals. Nevertheless, the genetic and biochemical mechanisms responsible for heavy metal resistance in S. metallicus remain uncharacterized. Proteomic analysis of S. metallicus cells exposed to 100 mM Cu revealed that 18 out of 30 upregulated proteins are related to the production and conversion of energy, amino acids biosynthesis, and stress responses. Ten of these last proteins were also up-regulated in S. metallicus treated in the presence of 1 mM Cd suggesting that at least in part, a common general response to these two heavy metals. The S. metallicus genome contained two complete cop gene clusters, each encoding a metallochaperone (CopM), a Cu-exporting ATPase (CopA), and a transcriptional regulator (CopT). Transcriptional expression analysis revealed that copM and copA from each cop gene cluster were cotranscribed and their transcript levels increased when S. metallicus was grown either in the presence of Cu or using chalcopyrite (CuFeS2) as oxidizable substrate. This study shows for the first time the presence of a duplicated version of the cop gene cluster in Archaea and characterizes some of the Cu and Cd resistance determinants in a thermophilic archaeon employed for industrial biomining. PMID:23509422

  14. Ring-Opening of the γ-OH-PdG Adduct Promotes Error-Free Bypass by the Sulfolobus solfataricus DNA Polymerase Dpo4

    OpenAIRE

    Shanmugam, Ganesh; Minko, Irina G.; Banerjee, Surajit; Christov, Plamen P.; Kozekov, Ivan D.; Rizzo, Carmelo J.; Lloyd, R. Stephen; Egli, Martin; Michael P. Stone

    2013-01-01

    Acrolein, a mutagenic aldehyde, reacts with deoxyguanosine (dG) to form 3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a] purin-10(3H)-one (γ-OH-PdG). When placed opposite deoxycytosine (dC) in DNA, γ-OH-PdG undergoes ring-opening to the N 2-(3-oxopropyl)-dG. Ring-opening of the adduct has been hypothesized to facilitate nonmutagenic bypass, particularly by DNA polymerases of the Y family. This study examined the bypass of γ-OH-PdG by Sulfolobus solfataricus ...

  15. Purification and characterization of a protein phosphatase (PP1-Arch) from the archaebacterium Sulfolobus solfataricus, isolation and expression of its gene

    OpenAIRE

    Leng, Jie

    1994-01-01

    PP1-Arch was verified as a protein phosphatase by both acid molybdate extraction and thin layer electrophoresis. Soluble fraction was prepared from Sulfolobus solfataricus, from which PP1-Arch was purified over 1OOO-fold by DE-52 ion-exchange, hydroxyapatite, gel filtration (G- 100), and Mono Q FPLC chromatography. PP1-Arch was identified from the fmal purified sample by renaturation on an SDS-polyacrylamide gel. The molecular size of PP1-Arch was determined by both gel filtrat...

  16. Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    tang, T. H.; Polacek, N.; Zywicki, M.; Huber, Harald; Brügger, Kim; Garrett, Roger Antony; Bachellerie, J. P.; Hüttenhofer, Alexander

    2005-01-01

    -box RNAs, where the latter only exhibit partial complementarity to RNA targets. The most prominent group of antisense RNAs is transcribed in the opposite orientation to the transposase genes, encoded by insertion elements (transposons). Thus, these antisense RNAs may regulate transposition of insertion...... first report of stably expressed antisense RNAs in an archaeal species and it raises the prospect that antisense-based mechanisms are also used widely in Archaea to regulate gene expression....

  17. Analysis of ATPases of putative secretion operons in the thermoacidophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Albers, SV; Driessen, AJM

    2005-01-01

    Gram-negative bacteria use a wide variety of complex mechanisms to secrete proteins across their membranes or to assemble secreted proteins into surface structures. As most archaea only possess a cytoplasmic membrane surrounded by a membrane-anchored S-layer, the organization of such complexes might

  18. Adenine phosphoribosyltransferase from Sulfolobus solfataricus is an enzyme with unusual kinetic properties and a crystal structure that suggests it evolved from a 6-oxopurine phosphoribosyltransferase

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Hansen, Michael Riis; Jensen, Kristine Steen;

    2015-01-01

    The adenine phosphoribosyltransferase (APRTase) encoded by the open reading frame SSO2342 of Sulfolobus solfataricus P2, was subjected to crystallographic, kinetic and ligand binding analyses. The enzyme forms dimers in solution and in the crystals, and binds one molecule of the reactants 5...

  19. Isolation of extracellular polymeric substances from biofilms of the thermoacidophilic archaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Silke eJachlewski

    2015-08-01

    Full Text Available Extracellular polymeric substances (EPS are the major structural and functional components of microbial biofilms. The aim of this study was to establish a method for EPS isolation from biofilms of the thermoacidophilic archaeon Sulfolobus acidocaldarius as a basis for EPS analysis. Biofilms of S. acidocaldarius were cultivated on the surface of gellan gum-solidified Brock medium at 78 °C for 4 days. Five EPS extraction methods were compared, including shaking of biofilm suspensions in phosphate buffer, cation-exchange resin (CER extraction and stirring with addition of EDTA, crown ether or NaOH. With respect to EPS yield, impact on cell viability and compatibility with subsequent biochemical analysis, the CER extraction method was found to be the best suited isolation procedure resulting in the detection of carbohydrates and proteins as the major constituents and DNA as a minor component of the EPS. Culturability of CER-treated cells was not impaired. Analysis of the extracellular proteome using two-dimensional gel electrophoresis resulted in the detection of several hundredshundred of protein spots, mainly with molecular masses of 25 kDa to 116 kDa and pI values of 5 to 8. Identification of proteins suggested a cytoplasmic origin for many of these proteins, possibly released via membrane vesicles or biofilm-inherent cell lysis during biofilm maturation. Functional analysis of EPS proteins, using fluorogenic substrates as well as zymography, demonstrated the activity of diverse groups of enzymes such as proteases, lipases, esterases, phosphatases and glucosidases. In conclusion, the CER extraction method, as previously applied to bacterial biofilms, also represents a suitable method for isolation of water soluble EPS from the archaeal biofilms of S. acidocaldarius, allowing the investigation of composition and function of EPS components in these types of biofilms.

  20. Isolation of Extracellular Polymeric Substances from Biofilms of the Thermoacidophilic Archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Jachlewski, Silke; Jachlewski, Witold D; Linne, Uwe; Bräsen, Christopher; Wingender, Jost; Siebers, Bettina

    2015-01-01

    Extracellular polymeric substances (EPS) are the major structural and functional components of microbial biofilms. The aim of this study was to establish a method for EPS isolation from biofilms of the thermoacidophilic archaeon, Sulfolobus acidocaldarius, as a basis for EPS analysis. Biofilms of S. acidocaldarius were cultivated on the surface of gellan gum-solidified Brock medium at 78°C for 4 days. Five EPS extraction methods were compared, including shaking of biofilm suspensions in phosphate buffer, cation-exchange resin (CER) extraction, and stirring with addition of EDTA, crown ether, or NaOH. With respect to EPS yield, impact on cell viability, and compatibility with subsequent biochemical analysis, the CER extraction method was found to be the best suited isolation procedure resulting in the detection of carbohydrates and proteins as the major constituents and DNA as a minor component of the EPS. Culturability of CER-treated cells was not impaired. Analysis of the extracellular proteome using two-dimensional gel electrophoresis resulted in the detection of several hundreds of protein spots, mainly with molecular masses of 25-116 kDa and pI values of 5-8. Identification of proteins suggested a cytoplasmic origin for many of these proteins, possibly released via membrane vesicles or biofilm-inherent cell lysis during biofilm maturation. Functional analysis of EPS proteins, using fluorogenic substrates as well as zymography, demonstrated the activity of diverse enzyme classes, such as proteases, lipases, esterases, phosphatases, and glucosidases. In conclusion, the CER extraction method, as previously applied to bacterial biofilms, also represents a suitable method for isolation of water soluble EPS from the archaeal biofilms of S. acidocaldarius, allowing the investigation of composition and function of EPS components in these types of biofilms. PMID:26380258

  1. Structural characterization of ether lipids from the archaeon Sulfolobus islandicus by high-resolution shotgun lipidomics.

    Science.gov (United States)

    Jensen, Sara Munk; Brandl, Martin; Treusch, Alexander H; Ejsing, Christer S

    2015-03-01

    The molecular structures, biosynthetic pathways and physiological functions of membrane lipids produced by organisms in the domain Archaea are poorly characterized as compared with that of counterparts in Bacteria and Eukaryota. Here we report on the use of high-resolution shotgun lipidomics to characterize, for the first time, the lipid complement of the archaeon Sulfolobus islandicus. To support the identification of lipids in S. islandicus, we first compiled a database of ether lipid species previously ascribed to Archaea. Next, we analyzed the lipid complement of S. islandicus by high-resolution Fourier transform mass spectrometry using an ion trap-orbitrap mass spectrometer. This analysis identified five clusters of molecular ions that matched ether lipids in the database with sub-ppm mass accuracy. To structurally characterize and validate the identities of the potential lipid species, we performed structural analysis using multistage activation on the ion trap-orbitrap instrument as well as tandem mass analysis using a quadrupole time-of-flight machine. Our analysis identified four ether lipid species previously reported in Archaea, and one ether lipid species that had not been described before. This uncharacterized lipid species features two head group structures composed of a trisaccharide residue carrying an uncommon sulfono group (-SO3) and an inositol phosphate group. Both head groups are linked to a glycerol dialkyl glycerol tetraether core structure having isoprenoid chains with a total of 80 carbon atoms and 4 cyclopentane moieties. The shotgun lipidomics approach deployed here defines a novel workflow for exploratory lipid profiling of Archaea. PMID:25800184

  2. Transcriptome analysis of Sulfolobus solfataricus infected with two related fuselloviruses reveals novel insights into the regulation of CRISPR-Cas system

    DEFF Research Database (Denmark)

    Fusco, Salvatore; Liguori, Rossana; Limauro, Danila;

    2015-01-01

    Fuselloviruses SSV1 and SSV2 are model systems to investigate virus-host relationships in stably infected cells thanks to their temperate nature. Although they are very similar in morphology, genome organization and gene synteny, their replication is induced by different stimuli, i.e.: by UV......-light exposure (for SSV1) and by the growth progression of the host (for SSV2). In this study, we have analysed global gene expression in SSV1- and SSV2-lysogens of Sulfolobus solfataricus P2 in the absence of any stimuli. Additionally, the interplay among SSV1, SSV2 and the host has been investigated in a...... double-infected strain to explore both virus-host and virus-virus interactions. Whereas SSV1 did not induce major changes of the host gene expression, SSV2 elicited a strong host response, which includes the transcriptional activation of CRISPR loci and cas genes. As a consequence, a significant decrease...

  3. Structural characterization of ether lipids from the archaeon Sulfolobus islandicus by high-resolution shotgun lipidomics

    DEFF Research Database (Denmark)

    Jensen, Sara Munk; Brandl, Martin; Treusch, Alexander H; Ejsing, Christer S

    2015-01-01

    characterize, for the first time, the lipid complement of the archaeon Sulfolobus islandicus. To support the identification of lipids in S. islandicus, we first compiled a database of ether lipid species previously ascribed to Archaea. Next, we analyzed the lipid complement of S. islandicus by high......-resolution Fourier transform mass spectrometry using an ion trap-orbitrap mass spectrometer. This analysis identified five clusters of molecular ions that matched ether lipids in the database with sub-ppm mass accuracy. To structurally characterize and validate the identities of the potential lipid species, we...... performed structural analysis using multistage activation on the ion trap-orbitrap instrument as well as tandem mass analysis using a quadrupole time-of-flight machine. Our analysis identified four ether lipid species previously reported in Archaea, and one ether lipid species that had not been described...

  4. Evidence that the xylanase activity from Sulfolobus solfataricus Oalpha is encoded by the endoglucanase precursor gene (sso1354) and characterization of the associated cellulase activity.

    Science.gov (United States)

    Maurelli, Luisa; Giovane, Alfonso; Esposito, Alessandra; Moracci, Marco; Fiume, Immacolata; Rossi, Mosè; Morana, Alessandra

    2008-09-01

    Sulfolobus solfataricus strain Oalpha was previously isolated for its ability to grow on minimal medium supplemented with xylan as a carbon source. The strain exhibited thermostable xylanase activity but several attempts to identify the gene encoding for the activity failed. Further studies showed that the xylanase displayed activity on carboxymethylcellulose (CMC) and the new activity was characterized. It exhibited an optimal temperature and pH of 95 degrees C and 3.5, respectively, and a half-life of 53 min at 95 degrees C. The enzyme, which was demonstrated to be glycosylated, hydrolyzed CMC in an endo-manner releasing cellobiose and other cello-oligomers. Analysis of the tryptic fragments by tandem mass spectrometry led to identification of the endoglucanase precursor, encoded by the sso1354 gene, as the protein possessing dual activity. The efficiency of the SSO1354 protein in degrading cellulosic and hemicellulosic fractions contained in agronomic residues was tested at low pH and high temperature. Cellulose and xylan were degraded to glucose and xylose at 90 degrees C, pH 4 by an enzyme mix consisting of SSO1354 and additional glycosyl hydrolases from S. solfataricus Oalpha. Given its role in saccharification processes requiring high temperatures and acidic environments, SSO1354 represents an interesting candidate for the utilization of agro-industrial waste for fuel production. PMID:18568289

  5. Crystallization and preliminary X-ray diffraction studies of hyperthermophilic archaeal Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus P1

    International Nuclear Information System (INIS)

    A hyperthermophilic archaeal Rieske-type [2Fe–2S] ferredoxin (ARF) from S. solfataricus P1 has been crystallized as a recombinant protein with a vector-derived long N-terminal extension region. The P43212 crystals of recombinant ARF diffracted to 1.85 Å resolution using synchrotron radiation. The hyperthermophilic archaeal Rieske-type [2Fe–2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 contains a low-potential Rieske-type [2Fe–2S] cluster that has served as a tractable model for ligand-substitution studies on this protein family. Recombinant ARF harbouring a pET30a vector-derived N-terminal extension region plus a hexahistidine tag has been heterologously overproduced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using 0.05 M sodium acetate, 0.05 M HEPES, 2 M ammonium sulfate pH 5.5. The crystals diffracted to 1.85 Å resolution and belonged to the tetragonal space group P43212, with unit-cell parameters a = 60.72, c = 83.31 Å. The asymmetric unit contains one protein molecule

  6. Frameshift Deletion by Sulfolobus solfataricus P2 DNA Polymerase Dpo4 T239W Is Selective for Purines and Involves Normal Conformational Change Followed by Slow Phosphodiester Bond Formation*

    OpenAIRE

    Zhang, Huidong; Beckman, Jeff W.; Guengerich, F. Peter

    2009-01-01

    The human DNA polymerase κ homolog Sulfolobus solfataricus DNA polymerase IV (Dpo4) produces “−1” frameshift deletions while copying unmodified DNA and, more frequently, when bypassing DNA adducts. As judged by steady-state kinetics and mass spectrometry, bypass of purine template bases to produce these deletions occurred rarely but with 10-fold higher frequency than with pyrimidines. The DNA adduct 1,N2-etheno-2′-deoxyguanosine, with a larger stacking surface than canonical purines, showed t...

  7. Molecular Characterization of Copper and Cadmium Resistance Determinants in the Biomining Thermoacidophilic Archaeon Sulfolobus metallicus

    OpenAIRE

    Alvaro Orell; Francisco Remonsellez; Rafaela Arancibia; Jerez, Carlos A.

    2013-01-01

    Sulfolobus metallicus is a thermoacidophilic crenarchaeon used in high-temperature bioleaching processes that is able to grow under stressing conditions such as high concentrations of heavy metals. Nevertheless, the genetic and biochemical mechanisms responsible for heavy metal resistance in S. metallicus remain uncharacterized. Proteomic analysis of S. metallicus cells exposed to 100 mM Cu revealed that 18 out of 30 upregulated proteins are related to the production and conversion of energy,...

  8. Lesion-Induced Mutation in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius and Its Avoidance by the Y-Family DNA Polymerase Dbh.

    Science.gov (United States)

    Sakofsky, Cynthia J; Grogan, Dennis W

    2015-10-01

    Hyperthermophilic archaea offer certain advantages as models of genome replication, and Sulfolobus Y-family polymerases Dpo4 (S. solfataricus) and Dbh (S. acidocaldarius) have been studied intensively in vitro as biochemical and structural models of trans-lesion DNA synthesis (TLS). However, the genetic functions of these enzymes have not been determined in the native context of living cells. We developed the first quantitative genetic assays of replication past defined DNA lesions and error-prone motifs in Sulfolobus chromosomes and used them to measure the efficiency and accuracy of bypass in normal and dbh(-) strains of Sulfolobus acidocaldarius. Oligonucleotide-mediated transformation allowed low levels of abasic-site bypass to be observed in S. acidocaldarius and demonstrated that the local sequence context affected bypass specificity; in addition, most erroneous TLS did not require Dbh function. Applying the technique to another common lesion, 7,8-dihydro-8-oxo-deoxyguanosine (8-oxo-dG), revealed an antimutagenic role of Dbh. The efficiency and accuracy of replication past 8-oxo-dG was higher in the presence of Dbh, and up to 90% of the Dbh-dependent events inserted dC. A third set of assays, based on phenotypic reversion, showed no effect of Dbh function on spontaneous -1 frameshifts in mononucleotide tracts in vivo, despite the extremely frequent slippage at these motifs documented in vitro. Taken together, the results indicate that a primary genetic role of Dbh is to avoid mutations at 8-oxo-dG that occur when other Sulfolobus enzymes replicate past this lesion. The genetic evidence that Dbh is recruited to 8-oxo-dG raises questions regarding the mechanism of recruitment, since Sulfolobus spp. have eukaryotic-like replisomes but no ubiquitin. PMID:26224736

  9. A Bac Library and Paired-PCR Approach to Mapping and Completing the Genome Sequence of Sulfolobus Solfataricus P2

    DEFF Research Database (Denmark)

    She, Qunxin; Confalonieri, F.; Zivanovic, Y.;

    2000-01-01

    The original strategy used in the Sulfolobus solfatnricus genome project was to sequence non overlapping, or minimally overlapping, cosmid or lambda inserts without constructing a physical map. However, after only about two thirds of the genome sequence was completed, this approach became counter...... selected for walking over small gaps and preparing template libraries for larger ones. It is concluded that an optimal strategy for sequencing microorganism genomes involves construction of a high-resolution physical map by BAC end analyses, PCR screening and paired-PCR chromosome walking after about half...... the genome sequence has been accumulated....

  10. Heteroduplex formation, mismatch resolution, and genetic sectoring during homologous recombination in the hyperthermophilic archaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    DennisW.Grogan

    2012-06-01

    Full Text Available Hyperthermophilic archaea exhibit certain molecular-genetic features not seen in bacteria or eukaryotes, and their systems of homologous recombination (HR remain largely unexplored in vivo. We transformed a Sulfolobus acidocaldarius pyrE mutant with short DNAs that contained multiple non-selected genetic markers within the pyrE gene. From 20 to 40% of the resulting colonies were found to contain two Pyr+ clones with distinct sets of the non-selected markers. The dual-genotype colonies could not be attributed to multiple DNAs entering the cells or conjugation between transformed and non-transformed cells. These colonies thus appear to represent genetic sectoring in which stretches of heteroduplex DNA formed during HR and segregated without complete resolution of inter-strand differences. Surprisingly, sectoring was also frequent in transformation with single-stranded DNAs. Oligonucleotides, for example, produced somewhat more sectored transformants when electroporated as single strands than as a duplex, although all forms (positive-strand, negative-strand, and duplex produced a diversity of genotypes from the limited number of markers. The marker patterns in the recombinants indicate that S. acidocaldarius resolves individual mismatches through un-coordinated short-patch excision followed by re-filling of the resulting gap. These gene-conversion events exhibit little strand bias, and can occur in pre-formed heteroduplex. These properties suggest that this process does not play a central role in the fidelity of genome replication, but may generate 3’ single-strand tails, and thereby initiate the incorporation of duplex DNA into the recipient chromosome. Regardless of the molecular details of its mechanism, HR between the S. acidocaldarius chromosome and a multiply-marked DNA produces a strikingly high level of genetic diversity in a very short chromosomal interval, and suggests that HR in Sulfolobus has significant mutagenic potential if not

  11. Compound K Production from Red Ginseng Extract by β-Glycosidase from Sulfolobus solfataricus Supplemented with α-L-Arabinofuranosidase from Caldicellulosiruptor saccharolyticus.

    Directory of Open Access Journals (Sweden)

    Kyung-Chul Shin

    Full Text Available Ginsenoside compound K (C-K is attracting a lot of interest because of its biological and pharmaceutical activities, including hepatoprotective, antitumor, anti-wrinkling, and anti-skin aging activities. C-K has been used as the principal ingredient in skin care products. For the effective application of ginseng extracts to the manufacture of cosmetics, the PPD-type ginsenosides in ginseng extracts should be converted to C-K by enzymatic conversion. For increased yield of C-K from the protopanaxadiol (PPD-type ginsenosides in red-ginseng extract (RGE, the α-L-arabinofuranoside-hydrolyzing α-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus (CS-abf was used along with the β-D-glucopyranoside/α-L-arabinopyranoside-hydrolyzing β-glycosidase from Sulfolobus solfataricus (SS-bgly because SS-bgly showed very low hydrolytic activity on the α-L-arabinofuranoside linkage in ginsenosides. The optimal reaction conditions for C-K production were as follows: pH 6.0, 80°C, 2 U/mL SS-bgly, 3 U/mL CS-abf, and 7.5 g/L PPD-type ginsenosides in RGE. Under these optimized conditions, SS-bgly supplemented with CS-abf produced 4.2 g/L C-K from 7.5 g/L PPD-type ginsenosides in 12 h without other ginsenosides, with a molar yield of 100% and a productivity of 348 mg/L/h. To the best of our knowledge, this is the highest concentration and productivity of C-K from ginseng extract ever published in literature.

  12. Enzymatic synthesis of dimaltosyl-β-cyclodextrin via a transglycosylation reaction using TreX, a Sulfolobus solfataricus P2 debranching enzyme

    International Nuclear Information System (INIS)

    Di-O-α-maltosyl-β-cyclodextrin ((G2)2-β-CD) was synthesized from 6-O-α-maltosyl-β-cyclodextrin (G2-β-CD) via a transglycosylation reaction catalyzed by TreX, a debranching enzyme from Sulfolobus solfataricus P2. TreX showed no activity toward glucosyl-β-CD, but a transfer product (1) was detected when the enzyme was incubated with maltosyl-β-CD, indicating specificity for a branched glucosyl chain bigger than DP2. Analysis of the structure of the transfer product (1) using MALDI-TOF/MS and isoamylase or glucoamylase treatment revealed it to be dimaltosyl-β-CD, suggesting that TreX transferred the maltosyl residue of a G2-β-CD to another molecule of G2-β-CD by forming an α-1,6-glucosidic linkage. When [14C]-maltose and maltosyl-β-CD were reacted with the enzyme, the radiogram showed no labeled dimaltosyl-β-CD; no condensation product between the two substrates was detected, indicating that the synthesis of dimaltosyl-β-CD occurred exclusively via transglycosylation of an α-1,6-glucosidic linkage. Based on the HPLC elution profile, the transfer product (1) was identified to be isomers of 61,63- and 61,64-dimaltosyl-β-CD. Inhibition studies with β-CD on the transglycosylation activity revealed that β-CD was a mixed-type inhibitor, with a Ki value of 55.6 μmol/mL. Thus, dimaltosyl-β-CD can be more efficiently synthesized by a transglycosylation reaction with TreX in the absence of β-CD. Our findings suggest that the high yield of (G2)2-β-CD from G2-β-CD was based on both the transglycosylation action mode and elimination of the inhibitory effect of β-CD

  13. Structural and Functional Analysis of Sulfolobus solfataricus Y-Family DNA Polymerase Dpo4-Catalyzed Bypass of the Malondialdehyde−Deoxyguanosine Adduct

    Energy Technology Data Exchange (ETDEWEB)

    Eoff, Robert L.; Stafford, Jennifer B.; Szekely, Jozsef; Rizzo, Carmelo J.; Egli, Martin; Guengerich, F. Peter; Marnett, Lawrence J.; (Vanderbilt)

    2010-01-12

    Oxidative stress can induce the formation of reactive electrophiles, such as DNA peroxidation products, e.g., base propenals, and lipid peroxidation products, e.g., malondialdehyde. Base propenals and malondialdehyde react with DNA to form adducts, including 3-(2'-deoxy-{beta}-d-erythro-pentofuranosyl)pyrimido[1,2-{alpha}]purin-10(3H)-one (M{sub 1}dG). When paired opposite cytosine in duplex DNA at physiological pH, M{sub 1}dG undergoes ring opening to form N{sup 2}-(3-oxo-1-propenyl)-dG (N{sup 2}-OPdG). Previous work has shown that M{sub 1}dG is mutagenic in bacteria and mammalian cells and that its mutagenicity in Escherichia coli is dependent on induction of the SOS response, indicating a role for translesion DNA polymerases in the bypass of M{sub 1}dG. To probe the mechanism by which translesion polymerases bypass M{sub 1}dG, kinetic and structural studies were conducted with a model Y-family DNA polymerase, Dpo4 from Sulfolobus solfataricus. The level of steady-state incorporation of dNTPs opposite M{sub 1}dG was reduced 260-2900-fold and exhibited a preference for dATP incorporation. Liquid chromatography-tandem mass spectrometry analysis of the full-length extension products revealed a spectrum of products arising principally by incorporation of dC or dA opposite M{sub 1}dG followed by partial or full-length extension. A greater proportion of -1 deletions were observed when dT was positioned 5' of M{sub 1}dG. Two crystal structures were determined, including a 'type II' frameshift deletion complex and another complex with Dpo4 bound to a dC-M{sub 1}dG pair located in the postinsertion context. Importantly, M{sub 1}dG was in the ring-closed state in both structures, and in the structure with dC opposite M{sub 1}dG, the dC residue moved out of the Dpo4 active site, into the minor groove. The results are consistent with the reported mutagenicity of M{sub 1}dG and illustrate how the lesion may affect replication events.

  14. Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Huidong; Eoff, Robert L.; Kozekov, Ivan D.; Rizzo, Carmelo J.; Egli, Martin; Guengerich, F. Peter; (Vanderbilt)

    2009-08-13

    Previous work has shown that Y-family DNA polymerases tolerate large DNA adducts, but a substantial decrease in catalytic efficiency and fidelity occurs during bypass of N{sup 2},N{sup 2}-dimethyl (Me{sub 2})-substituted guanine (N{sup 2},N{sup 2}-Me{sub 2}G), in contrast to a single methyl substitution. Therefore, it is unclear why the addition of two methyl groups is so disruptive. The presence of N{sup 2},N{sup 2}-Me{sub 2}G lowered the catalytic efficiency of the model enzyme Sulfolobus solfataricus Dpo4 16,000-fold. Dpo4 inserted dNTPs almost at random during bypass of N{sup 2},N{sup 2}-Me{sub 2}G, and much of the enzyme was kinetically trapped by an inactive ternary complex when N{sup 2},N{sup 2}-Me{sub 2}G was present, as judged by a reduced burst amplitude (5% of total enzyme) and kinetic modeling. One crystal structure of Dpo4 with a primer having a 3{prime}-terminal dideoxycytosine (Cdd) opposite template N{sup 2},N{sup 2}-Me{sub 2}G in a post-insertion position showed Cdd folded back into the minor groove, as a catalytically incompetent complex. A second crystal had two unique orientations for the primer terminal Cdd as follows: (i) flipped into the minor groove and (ii) a long pairing with N{sup 2},N{sup 2}-Me{sub 2}G in which one hydrogen bond exists between the O-2 atom of Cdd and the N-1 atom of N{sup 2},N{sup 2}-Me{sub 2}G, with a second water-mediated hydrogen bond between the N-3 atom of C{sub dd} and the O-6 atom of N{sup 2},N{sup 2}-Me{sub 2}G. A crystal structure of Dpo4 with dTTP opposite template N{sup 2},N{sup 2}-Me{sub 2}G revealed a wobble orientation. Collectively, these results explain, in a detailed manner, the basis for the reduced efficiency and fidelity of Dpo4-catalyzed bypass of N{sup 2},N{sup 2}-Me{sub 2}G compared with mono-substituted N{sup 2}-alkyl G adducts.

  15. Frameshift Deletion by Sulfolobus solfataricus P2 DNA Polymerase Dpo4 T239W Is Selective for Purines and Involves Normal Conformational Change Followed by Slow Phosphodiester Bond Formation*

    Science.gov (United States)

    Zhang, Huidong; Beckman, Jeff W.; Guengerich, F. Peter

    2009-01-01

    The human DNA polymerase κ homolog Sulfolobus solfataricus DNA polymerase IV (Dpo4) produces “−1” frameshift deletions while copying unmodified DNA and, more frequently, when bypassing DNA adducts. As judged by steady-state kinetics and mass spectrometry, bypass of purine template bases to produce these deletions occurred rarely but with 10-fold higher frequency than with pyrimidines. The DNA adduct 1,N2-etheno-2′-deoxyguanosine, with a larger stacking surface than canonical purines, showed the highest frequency of formation of −1 frameshift deletions. Dpo4 T239W, a mutant we had previously shown to produce fluorescence changes attributed to conformational change following dNTP binding opposite cognate bases (Beckman, J. W., Wang, Q., and Guengerich, F. P. (2008) J. Biol. Chem. 283, 36711–36723), reported similar conformational changes when the incoming dNTP complemented the base following a templating purine base or bulky adduct (i.e. the “+1” base). However, in all mispairing cases, phosphodiester bond formation was inefficient. The frequency of −1 frameshift events and the associated conformational changes were not dependent on the context of the remainder of the sequence. Collectively, our results support a mechanism for −1 frameshift deletions by Dpo4 that involves formation of active complexes via a favorable conformational change that skips the templating base, without causing slippage or flipping out of the base, to incorporate a complementary residue opposite the +1 base, in a mechanism previously termed “dNTP-stabilized incorporation.” The driving force is attributed to be the stacking potential between the templating base and the incoming dNTP base. PMID:19837980

  16. Frameshift deletion by Sulfolobus solfataricus P2 DNA polymerase Dpo4 T239W is selective for purines and involves normal conformational change followed by slow phosphodiester bond formation.

    Science.gov (United States)

    Zhang, Huidong; Beckman, Jeff W; Guengerich, F Peter

    2009-12-11

    The human DNA polymerase kappa homolog Sulfolobus solfataricus DNA polymerase IV (Dpo4) produces "-1" frameshift deletions while copying unmodified DNA and, more frequently, when bypassing DNA adducts. As judged by steady-state kinetics and mass spectrometry, bypass of purine template bases to produce these deletions occurred rarely but with 10-fold higher frequency than with pyrimidines. The DNA adduct 1,N(2)-etheno-2'-deoxyguanosine, with a larger stacking surface than canonical purines, showed the highest frequency of formation of -1 frameshift deletions. Dpo4 T239W, a mutant we had previously shown to produce fluorescence changes attributed to conformational change following dNTP binding opposite cognate bases (Beckman, J. W., Wang, Q., and Guengerich, F. P. (2008) J. Biol. Chem. 283, 36711-36723), reported similar conformational changes when the incoming dNTP complemented the base following a templating purine base or bulky adduct (i.e. the "+1" base). However, in all mispairing cases, phosphodiester bond formation was inefficient. The frequency of -1 frameshift events and the associated conformational changes were not dependent on the context of the remainder of the sequence. Collectively, our results support a mechanism for -1 frameshift deletions by Dpo4 that involves formation of active complexes via a favorable conformational change that skips the templating base, without causing slippage or flipping out of the base, to incorporate a complementary residue opposite the +1 base, in a mechanism previously termed "dNTP-stabilized incorporation." The driving force is attributed to be the stacking potential between the templating base and the incoming dNTP base. PMID:19837980

  17. Deletion of the topoisomerase III gene in the hyperthermophilic archaeon Sulfolobus islandicus results in slow growth and defects in cell cycle control

    DEFF Research Database (Denmark)

    Li, Xiyang; Guo, Li; Deng, Ling;

    2011-01-01

    Topoisomerase III (topo III), a type IA topoisomerase, is widespread in hyperthermophilic archaea. In order to interrogate the in vivo role of archaeal topo III, we constructed and characterized a topo III gene deletion mutant of Sulfolobus islandicus. The mutant was viable but grew more slowly t...

  18. Replication Bypass of the trans-4-Hydroxynonenal-Derived (6S,8R,11S)-1,N[superscript 2]-Deoxyguanosine DNA Adduct by the Sulfolobus solfataricus DNA Polymerase IV

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Surajit; Christov, Plamen P.; Kozekova, Albena; Rizzo, Carmelo J.; Egli, Martin; Stone, Michael P. (Vanderbilt)

    2014-10-02

    trans-4-Hydroxynonenal (HNE) is the major peroxidation product of {omega}-6 polyunsaturated fatty acids in vivo. Michael addition of the N{sub 2}-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,N{sub 2}-dGuo (1,N{sub 2}-HNE-dGuo) adducts. The (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct was incorporated into the 18-mer templates 5'-d(TCATXGAATCCTTCCCCC)-3' and d(TCACXGAATCCTTCCCCC)-3', where X = (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct. These differed in the identity of the template 5'-neighbor base, which was either Thy or Cyt, respectively. Each of these templates was annealed with either a 13-mer primer 5'-d(GGGGGAAGGATTC)-3' or a 14-mer primer 5'-d(GGGGGAAGGATTCC)-3'. The addition of dNTPs to the 13-mer primer allowed analysis of dNTP insertion opposite to the (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6S,8R,11S)-HNE-1,N{sub 2}-dGuo:dCyd pair. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) belongs to the Y-family of error-prone polymerases. Replication bypass studies in vitro reveal that this polymerase inserted dNTPs opposite the (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct in a sequence-specific manner. If the template 5'-neighbor base was dCyt, the polymerase inserted primarily dGTP, whereas if the template 5'-neighbor base was dThy, the polymerase inserted primarily dATP. The latter event would predict low levels of Gua {yields} Thy mutations during replication bypass when the template 5'-neighbor base is dThy. When presented with a primed (6S,8R,11S)-HNE-1,N{sub 2}-dGuo:dCyd pair, the polymerase conducted full-length primer extension. Structures for ternary (Dpo4-DNA-dNTP) complexes with all four template-primers were obtained. For the 18-mer:13-mer template-primers in which the polymerase was confronted with the (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct, the (6S,8R,11S)-1,N

  19. The genome sequence of the metal-mobilizing, extremely thermoacidophilic archaeon Metallosphaera sedula provides insights into bioleaching-associated metabolism.

    Science.gov (United States)

    Auernik, Kathryne S; Maezato, Yukari; Blum, Paul H; Kelly, Robert M

    2008-02-01

    Despite their taxonomic description, not all members of the order Sulfolobales are capable of oxidizing reduced sulfur species, which, in addition to iron oxidation, is a desirable trait of biomining microorganisms. However, the complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, approximately 2,300 open reading frames [ORFs]) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins involved (directly or indirectly) with bioleaching. As expected, the M. sedula genome contains genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation, and a putative tetrathionate hydrolase, implicated in sulfur oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, was also identified. These iron- and sulfur-oxidizing components are missing from genomes of nonleaching members of the Sulfolobales, such as Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole-genome transcriptional response analysis showed that 88 ORFs were up-regulated twofold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included genes for components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized. PMID:18083856

  20. The genome of Sulfolobus acidocaldarius, a model organism of the Crenarchaeota

    DEFF Research Database (Denmark)

    Chen, L.M.; Brugger, K.; Skovgaard, Marie;

    2005-01-01

    to the Sulfolobus genus. Moreover, 82 genes for untranslated RNAs were identified and annotated. Owing to the probable absence of active autonomous and nonautonomous mobile elements, the genome stability and organization of S. acidocaldarius differ radically from those of Sulfolobus solfataricus and...... Sulfolobus tokodaii. The S. acidocaldarius genome contains an integrated, and probably encaptured, pARN-type conjugative plasmid which may facilitate intercellular chromosomal gene exchange in S. acidocaldarius. Moreover, it contains genes for a characteristic restriction modification system, a UV damage...

  1. The Genome of Sulfolobus acidocaldarius, a Model Organism of the Crenarchaeota

    DEFF Research Database (Denmark)

    Chen, Lanming; Brügger, Kim; Skovgaard, M.;

    2005-01-01

    Sulfolobus genus. Moreover, 82 genes for untranslated RNAs were identified and annotated. Owing to the probable absence of active autonomous and nonautonomous mobile elements, the genome stability and organization of S. acidocaldarius differ radically from those of Sulfolobus solfataricus and Sulfolobus...... tokodaii. The S. acidocaldarius genome contains an integrated, and probably encaptured, pARN-type conjugative plasmid which may facilitate intercellular chromosomal gene exchange in S. acidocaldarius. Moreover, it contains genes for a characteristic restriction modification system, a UV damage excision...

  2. Genus-specific protein binding to the large clusters of DNA repeats (short regularly spaced repeats) present in Sulfolobus genomes

    DEFF Research Database (Denmark)

    Peng, Xu; Brügger, Kim; Shen, Biao; Chen, Lanming; She, Qunxin; Garrett, Roger A

    2003-01-01

    structure which yields a good sequence match with a helix-turn-helix DNA-binding motif. Although this putative motif is shared by other archaeal proteins, orthologs of SSO454 were only detected in species within the Sulfolobus genus and in the closely related Acidianus genus. We infer that the genus....... For the crenarchaeon Sulfolobus solfataricus P2, the repeats in the genome fall mainly into two closely related sequence families that are arranged in seven clusters containing a total of 441 repeats which constitute ca. 1% of the genome. The Sulfolobus conjugative plasmid pNOB8 contains a small...

  3. The Mre11 protein interacts with both Rad50 and the HerA bipolar helicase and is recruited to DNA following gamma irradiation in the archaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Forterre Patrick

    2008-02-01

    Full Text Available Abstract Background The ubiquitous Rad50 and Mre11 proteins play a key role in many processes involved in the maintenance of genome integrity in Bacteria and Eucarya, but their function in the Archaea is presently unknown. We showed previously that in most hyperthermophilic archaea, rad50-mre11 genes are linked to nurA encoding both a single-strand endonuclease and a 5' to 3' exonuclease, and herA, encoding a bipolar DNA helicase which suggests the involvement of the four proteins in common molecular pathway(s. Since genetic tools for hyperthermophilic archaea are just emerging, we utilized immuno-detection approaches to get the first in vivo data on the role(s of these proteins in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius. Results We first showed that S. acidocaldarius can repair DNA damage induced by high doses of gamma rays, and we performed a time course analysis of the total levels and sub-cellular partitioning of Rad50, Mre11, HerA and NurA along with the RadA recombinase in both control and irradiated cells. We found that during the exponential phase, all proteins are synthesized and display constant levels, but that all of them exhibit a different sub-cellular partitioning. Following gamma irradiation, both Mre11 and RadA are immediately recruited to DNA and remain DNA-bound in the course of DNA repair. Furthermore, we show by immuno-precipitation assays that Rad50, Mre11 and the HerA helicase interact altogether. Conclusion Our analyses strongly support that in Sulfolobus acidocaldarius, the Mre11 protein and the RadA recombinase might play an active role in the repair of DNA damage introduced by gamma rays and/or may act as DNA damage sensors. Moreover, our results demonstrate the functional interaction between Mre11, Rad50 and the HerA helicase and suggest that each protein play different roles when acting on its own or in association with its partners. This report provides the first in vivo evidence supporting the

  4. Sulfolobus Turreted Icosahedral Virus c92 Protein Responsible for the Formation of Pyramid-Like Cellular Lysis Structures

    DEFF Research Database (Denmark)

    Snyder, Jamie C; Brumfield, Susan K; Peng, Nan;

    2011-01-01

    Host cells infected by Sulfolobus turreted icosahedral virus (STIV) have been shown to produce unusual pyramid-like structures on the cell surface. These structures represent a virus-induced lysis mechanism that is present in Archaea and appears to be distinct from the holin/endolysin system...... described for DNA bacteriophages. This study investigated the STIV gene products required for pyramid formation in its host Sulfolobus solfataricus. Overexpression of STIV open reading frame (ORF) c92 in S. solfataricus alone is sufficient to produce the pyramid-like lysis structures in cells. Gene...... disruption of c92 within STIV demonstrates that c92 is an essential protein for virus replication. Immunolocalization of c92 shows that the protein is localized to the cellular membranes forming the pyramid-like structures....

  5. Proteomic analysis of secreted membrane vesicles of archaeal Sulfolobus species reveals the presence of endosome sorting complex components

    OpenAIRE

    Ellen, Albert F.; Albers, Sonja-Verena; Huibers, Wim; Pitcher, Angela; Hobel, Cedric F. V.; Schwarz, Heinz; Folea, Mihaela; Schouten, Stefan; Boekema, Egbert J.; Poolman, Bert; Driessen, Arnold J. M.

    2009-01-01

    The crenarchaea Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii, release membrane vesicles into the medium. These membrane vesicles consist of tetraether lipids and are coated with an S-layer. A proteomic analysis reveals the presence of proteins homologous to subunits of the eukaryotic endosomal sorting complex required for transport (ESCRT). Immunodetection of one of these homologs suggest a cell surface localization in intact cells. These data suggest that the membrane vesicles ...

  6. Factor requirements for transcription in the Archaeon Sulfolobus shibatae.

    OpenAIRE

    Qureshi, S A; Bell, S.D.; Jackson, S P

    1997-01-01

    Archaea (archaebacteria) constitute a domain of life that is distinct from Bacteria (eubacteria) and Eucarya (eukaryotes). Although archaeal cells share many morphological features with eubacteria, their transcriptional apparatus is more akin to eukaryotic RNA polymerases I, II and III than it is to eubacterial transcription systems. Thus, in addition to possessing a 10 subunit RNA polymerase and a homologue of the TATA-binding protein (TBP), Archaea possess a polypeptide termed TFB that is h...

  7. Sulfolobus tengchongensis Spindle-Shaped Virus STSV1: Virus-Host Interactions and Genomic Features

    DEFF Research Database (Denmark)

    Xiang, X.; Chen, L.; Huang, X;

    2005-01-01

    A virus infecting the hyperthermophilic archaeon Sulfolobus tengchongensis has been isolated from a field sample from Tengchong, China, and characterized. The virus, denoted STSV1 (Sulfolobus tengchongensis spindle-shaped virus 1), has the morphology of a spindle (230 by 107 nm) with a tail of...... genome contains a total of 74 open reading frames (ORFs), among which 14 have a putative function. Five ORFs encode viral structural proteins, including a putative coat protein of high abundance. The products of the other nine ORFs are probably involved in polysaccharide biosynthesis, nucleotide...

  8. Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers

    DEFF Research Database (Denmark)

    Guðbergsdóttir, Sóley Ruth; Deng, Ling; Chen, Zhengjun;

    2011-01-01

    The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching...... individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different...... protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers...

  9. Tlys, a Newly Identified Sulfolobus Spindle-Shaped Virus 1 Transcript Expressed in the Lysogenic State, Encodes a DNA-Binding Protein Interacting at the Promoters of the Early Genes

    OpenAIRE

    Fusco, Salvatore; She, Qunxin; Bartolucci, Simonetta; Contursi, Patrizia

    2013-01-01

    While studying the gene expression of the Sulfolobus spindle-shaped virus 1 (SSV1) in Sulfolobus solfataricus lysogenic cells, a novel viral transcript (Tlys) was identified. Transcriptional analysis revealed that Tlys is expressed only in the absence of UV irradiation and is downregulated during the growth of the lysogenic host. The correponding gene f55 lies between two transcriptional units (T6 and Tind) that are upregulated upon UV irradiation. The open reading frame f55 encodes a 6.3-kDa...

  10. The Sulfolobus initiator element is an important contributor to promoter strength.

    Science.gov (United States)

    Ao, Xiang; Li, Yingjun; Wang, Fan; Feng, Mingxia; Lin, Yanxu; Zhao, Shumiao; Liang, Yunxiang; Peng, Nan

    2013-11-01

    Basal elements in archaeal promoters, except for putative initiator elements encompassing transcription start sites, are well characterized. Here, we employed the Sulfolobus araS promoter as a model to study the function of the initiator element (Inr) in archaea. We have provided evidence for the presence of a third core promoter element, the Sulfolobus Inr, whose action depends on a TATA box and the TFB recognition element (BRE). Substitution mutations in the araS Inr did not alter the location of the transcription start site. Using systematic mutagenesis, the most functional araS Inr was defined as +1 GAGAMK +6 (where M is A/C and K is G/T). Furthermore, WebLogo analysis of a subset of promoters with coding sequences for 5' untranslated regions (UTRs) larger than 4 nucleotides (nt) in Sulfolobus solfataricus P2 identified an Inr consensus that exactly matches the functional araS Inr sequence. Moreover, mutagenesis of 3 randomly selected promoters confirmed the Inr sequences to be important for basal promoter strength in the subgroup. Importantly, the result of the araS Inr being added to the Inr-less promoters indicates that the araS Inr, the core promoter element, is able to enhance the strength of Inr-less promoters. We infer that transcription factor B (TFB) and subunits of RNA polymerase bind the Inr to enhance promoter strength. Taken together, our data suggest that the presence or absence of an Inr on basal promoters is important for global gene regulation in Sulfolobus. PMID:24039266

  11. Crystal structure of the S. solfataricus archaeal exosome reveals conformational flexibility in the RNA-binding ring.

    Directory of Open Access Journals (Sweden)

    Changrui Lu

    Full Text Available BACKGROUND: The exosome complex is an essential RNA 3'-end processing and degradation machinery. In archaeal organisms, the exosome consists of a catalytic ring and an RNA-binding ring, both of which were previously reported to assume three-fold symmetry. METHODOLOGY/PRINCIPAL FINDINGS: Here we report an asymmetric 2.9 A Sulfolobus solfataricus archaeal exosome structure in which the three-fold symmetry is broken due to combined rigid body and thermal motions mainly within the RNA-binding ring. Since increased conformational flexibility was also observed in the RNA-binding ring of the related bacterial PNPase, we speculate that this may reflect an evolutionarily conserved mechanism to accommodate diverse RNA substrates for degradation. CONCLUSION/SIGNIFICANCE: This study clearly shows the dynamic structures within the RNA-binding domains, which provides additional insights on mechanism of asymmetric RNA binding and processing.

  12. Developing novel single molecule analyses of the single-stranded DNA binding protein from Sulfolobus solfataricus

    OpenAIRE

    Morten, Michael J.

    2015-01-01

    Single-stranded DNA binding proteins (SSB) bind to single-stranded DNA (ssDNA) that is generated by molecular machines such as helicases and polymerases. SSBs play crucial roles in DNA translation, replication and repair and their importance is demonstrated by their inclusion across all domains of life. The homotetrameric E. coli SSB and the heterotrimeric human RPA demonstrate how SSBs can vary structurally, but all fulfil their roles by employing oligonucleotide/oligosaccharide binding (OB)...

  13. Bioaccumulation of U(VI) by Sulfolobus acidocaldarius under moderate acidic conditions

    International Nuclear Information System (INIS)

    U(VI) accumulation by the acidothermophilic archaeon Sulfolobus acidocaldarius at a moderate acidic pH of 4.5 was investigated. This pH value is relevant for some heavy metal and uranium polluted environments where populations of S. acidocaldarius were found to persist. We demonstrate that U(VI) is rapidly complexed by the archaeal cells. A combination of X-ray absorption spectroscopy and time-resolved laser-induced fluorescence spectroscopy revealed that at pH 4.5 organic phosphate and carboxylic groups are involved in the U(VI) complexation. These results are in contrast to those published for most bacteria which at this pH precipitate U(VI) mainly in inorganic uranyl phosphate phases. As demonstrated by TEM only a limited part of the added U(VI) was biomineralized extracellularly in the case of the studied archaeon. Most of the U(VI) accumulates were localized in a form of intracellular deposits which were associated with the inner side of the cytoplasma membrane. Observed differences in U(VI) bioaccumulation between the studied archaeon and bacteria can be explained by the significant differences in their cell wall structures as well as by their different physiological characteristics. (orig.)

  14. A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus

    DEFF Research Database (Denmark)

    Deng, Ling; Garrett, Roger Antony; Shah, Shiraz Ali;

    2013-01-01

    Recent studies on CRISPR-based adaptive immune systems have revealed extensive structural and functional diversity of the interference complexes which often coexist intracellularly. The archaeon Sulfolobus islandicus REY15A encodes three interference modules, one of type IA and two of type IIIB...... targeting. A rationale is provided for the intracellular coexistence of the different interference systems in S.¿islandicus REY15A which cooperate functionally by sharing a single Cas6 protein for crRNA processing and utilize crRNA products from identical CRISPR spacers....

  15. Genetic manipulation in Sulfolobus islandicus and functional analysis of DNA repair genes

    DEFF Research Database (Denmark)

    Zhang, Changyi; Tian, Bin; Li, Suming;

    2013-01-01

    enzymes already impaired cell growth, highlighting their important roles in archaeal DNA repair. Systematically characterizing these mutants and generating mutants lacking two or more DNA repair genes will yield further insights into the genetic mechanisms of DNA repair in this model organism.......Recently, a novel gene-deletion method was developed for the crenarchaeal model Sulfolobus islandicus, which is a suitable tool for addressing gene essentiality in depth. Using this technique, we have investigated functions of putative DNA repair genes by constructing deletion mutants and studying...... their phenotype. We found that this archaeon may not encode a eukarya-type of NER (nucleotide excision repair) pathway because depleting each of the eukaryal NER homologues XPD, XPB and XPF did not impair the DNA repair capacity in their mutants. However, among seven homologous recombination proteins...

  16. Tlys, a newly identified Sulfolobus spindle-shaped virus 1 transcript expressed in the lysogenic state, encodes a DNA-binding protein interacting at the promoters of the early genes

    DEFF Research Database (Denmark)

    Fusco, Salvatore; She, Qunxin; Bartolucci, Simonetta;

    2013-01-01

    While studying the gene expression of the Sulfolobus spindle-shaped virus 1 (SSV1) in Sulfolobus solfataricus lysogenic cells, a novel viral transcript (Tlys) was identified. Transcriptional analysis revealed that Tlys is expressed only in the absence of UV irradiation and is downregulated during...... the growth of the lysogenic host. The correponding gene f55 lies between two transcriptional units (T6 and Tind) that are upregulated upon UV irradiation. The open reading frame f55 encodes a 6.3-kDa protein which shows sequence identity with negative regulators that fold into the ribbon...... promoter. Taking together the transcriptional analysis data and the biochemical evidences, we surmise that the protein F55 is involved in the regulation of the lysogenic state of SSV1....

  17. Diverse CRISPR-Cas responses and dramatic cellular DNA changes and cell death in pKEF9-conjugated Sulfolobus species.

    Science.gov (United States)

    Liu, Guannan; She, Qunxin; Garrett, Roger A

    2016-05-19

    The Sulfolobales host a unique family of crenarchaeal conjugative plasmids some of which undergo complex rearrangements intracellularly. Here we examined the conjugation cycle of pKEF9 in the recipient strain Sulfolobus islandicus REY15A. The plasmid conjugated and replicated rapidly generating high average copy numbers which led to strong growth retardation that was coincident with activation of CRISPR-Cas adaptation. Simultaneously, intracellular DNA was extensively degraded and this also occurred in a conjugated Δcas6 mutant lacking a CRISPR-Cas immune response. Furthermore, the integrated forms of pKEF9 in the donor Sulfolobus solfataricus P1 and recipient host were specifically corrupted by transposable orfB elements, indicative of a dual mechanism for inactivating free and integrated forms of the plasmid. In addition, the CRISPR locus of pKEF9 was progressively deleted when conjugated into the recipient strain. Factors influencing activation of CRISPR-Cas adaptation in the recipient strain are considered, including the first evidence for a possible priming effect in Sulfolobus The 3-Mbp genome sequence of the donor P1 strain is presented. PMID:27098036

  18. Genetic Studies on CRISPR-Cas Functions in Invader Defense in Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Peng, Wenfang

    Archaea and bacteria contain CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) systems that protect themselves against invasion by viruses and plasmids. There are three major types of CRISPR-Cas systems, type I, II and III, that are further divided into at...... least 11 subtypes. I employed Sulfolobus islandicus Rey15A as the model to study CRISPR mechanisms. The model archaeon encodes one subtype I-A (Cascade) and two subtype III-B (Cmr-α and Cmr-β) interference systems with no apparent redundancy in cas genes or in CRISPR systems, which is ideal for genetic...... analysis of cas gene function. Furthermore, a range of genetic tools have been developed for S. islandicus Rey15A in our laboratory and a plasmid interference assay has been successfully developed for testing CRISPR-directed DNA targeting activity, which have provided a solid basis for studying the...

  19. The Sulfolobus solfataricus Lrp-like protein LysM regulates lysine biosynthesis in response to lysine availability

    NARCIS (Netherlands)

    Brinkman, A.B.; Bell, S.D.; Lebbink, R.J.; Vos, de W.M.; Oost, van der J.

    2002-01-01

    Although the archaeal transcription apparatus resembles the eukaryal RNA polymerase II system, many bacterial-like regulators can be found in archaea. Particularly, all archaeal genomes sequenced to date contain genes encoding homologues of Lrp (leucine-responsive regulatory protein). Whereas Lrp-li

  20. Structure of the Sulfolobus solfataricus alpha-glucosidase: Implications for domain conservation and substrate recognition in GH31

    DEFF Research Database (Denmark)

    Ernst, Heidi Asschenfeldt; Lo Leggio, Leila; Willemoes, M.;

    2006-01-01

    glycogen degradation and glycoprotein processing. The structure of MalA shows clear differences from the only other structure known from GH31, a-xylosidase YicI. MalA and YicI share only 23% sequence identity. Although the two enzymes display a similar domain structure and both form hexamers, their......), whereas YicI prefers isoprimeverose (Xyl-a1,6-Glc). The structural origin of this difference occurs in the -1 subsite where MalA residues Asp251 and Trp284 could interact with OH6 of the substrate. The structure of MalA in complex with ß-octyl-glucopyranoside has been determined. It reveals Arg400, Asp87...

  1. A plasmid in the archaebacterium Sulfolobus acidocaldarius

    OpenAIRE

    Yeats, Siobhán; McWilliam, Peter; Zillig, Wolfram

    1982-01-01

    A plasmid of mol. wt. ∼9 × 106 has been isolated from the archaebacterium Sulfolobus acidocaldarius strain B12. Plasmid production is induced by u.v. radiation. A copy of the plasmid is probably carried by the chromosome, integrated at a specific site. The entire plasmid, and also restriction fragments of it, has been cloned into Escherichia coli plasmid vectors, and the cleavage sites on the plasmid DNA of three restriction endonucleases have been mapped.

  2. Two novel conjugative plasmids from a single strain of Sulfolobus

    NARCIS (Netherlands)

    Erauso, G.; Stedman, K.M.; Werken, van de H.J.G.; Zillig, W.; Oost, van der J.

    2006-01-01

    Two conjugative plasmids (CPs) were isolated and characterized from the same 'Sulfolobus islandicus' strain, SOG2/4, The plasmids were separated from each other and transferred into Sulfolobus soltataricus. One has a high copy number and is not stable (pSOG1) whereas the other has a low copy number

  3. Induction of the Sulfolobus shibatae virus SSV1 DNA replication by mitomycin C

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The temperate virus SSV1 from the hyperthermophilic archaeon Sulfolobus shibatae provides a useful model system for the study of archaeal DNA replication. Southern hybridization showed that SSV1 existed primarily as a provirus in its host that was grown without shaking. Upon UV or mitomycin C induction, the cellular level of free SSV1 DNA increased drastically whereas that of integrated viral DNA remained unchanged. The results of mitomycin C induction were more reproducible than those of UV induction. We found that, when the cells that had been grown without shaking were shaken, the replication of SSV1 DNA was also induced. Based on our results, we developed a method for the induction of SSV1 DNA replication by mitomycin C. When the S. shibatae virus production was induced using this method, the cellular level of free SSV1 DNA started to increase 10 h after induction, and peaked after 12-15 h. A fully induced S. shibatae cell contained ~50 molecules of free SSV1 DNA. The development of this induction method and the description of the process of SSV1 DNA replication following induction are valuable to the analysis of the origin and mode of replication of the virus.

  4. The Effects of Temperature and Growth Phase on the Lipidomes of Sulfolobus islandicus and Sulfolobus tokodaii

    DEFF Research Database (Denmark)

    Jensen, Sara Munk; Neesgaard, Vinnie Lund; Skjoldbjerg, Sandra Landbo Nedergaard;

    2015-01-01

    The functionality of the plasma membrane is essential for all organisms. Adaption to high growth temperatures imposes challenges and Bacteria, Eukarya, and Archaea have developed several mechanisms to cope with these. Hyperthermophilic archaea have earlier been shown to synthesize tetraether...... membrane lipids with an increased number of cyclopentane moieties at higher growth temperatures. Here we used shotgun lipidomics to study this effect as well as the influence of growth phase on the lipidomes of Sulfolobus islandicus and Sulfolobus tokodaii for the first time. Both species were cultivated...... at three different temperatures, with samples withdrawn during lag, exponential, and stationary phases. Three abundant tetraether lipid classes and one diether lipid class were monitored. Beside the expected increase in the number of cyclopentane moieties with higher temperature in both archaea, we...

  5. Studying Extrachromosomal Genetic Elements in Sulfolobus

    DEFF Research Database (Denmark)

    Guannan, Liu

    of three closely related viruses (ATV, ATV2, ATVv) revealed a conserved genome organization, but many differences in gene size and content. Comparison of the CRISPR loci in S. solfataricus P3 with those of three published S. solfataricus strains showed many shared spacers, as well as different...... studied to gain a better understanding of the interactions between conjugative plasmids and hosts. The result also demonstrated why certain archaeal conjugative plasmids are gradually lost during continuous growth. Whereas loss of pKEF9 in S. islandicus was due to interference from the host CRISPR...

  6. T(lys), a newly identified Sulfolobus spindle-shaped virus 1 transcript expressed in the lysogenic state, encodes a DNA-binding protein interacting at the promoters of the early genes.

    Science.gov (United States)

    Fusco, Salvatore; She, Qunxin; Bartolucci, Simonetta; Contursi, Patrizia

    2013-05-01

    While studying the gene expression of the Sulfolobus spindle-shaped virus 1 (SSV1) in Sulfolobus solfataricus lysogenic cells, a novel viral transcript (T(lys)) was identified. Transcriptional analysis revealed that T(lys) is expressed only in the absence of UV irradiation and is downregulated during the growth of the lysogenic host. The correponding gene f55 lies between two transcriptional units (T6 and T(ind)) that are upregulated upon UV irradiation. The open reading frame f55 encodes a 6.3-kDa protein which shows sequence identity with negative regulators that fold into the ribbon-helix-helix DNA-binding motif. DNA-binding assays demonstrated that the recombinant F55, purified from Escherichia coli, is indeed a putative transcription factor able to recognize site specifically target sequences in the promoters of the early induced T5, T6, and T(ind) transcripts, as well as of its own promoter. Binding sites of F55 are included within a tandem-repeated sequence overlapping the transcription start sites and/or the B recognition element of the pertinent genes. The strongest binding was observed with the promoters of T5 and T6, and an apparent cooperativity in binding was observed with the T(ind) promoter. Taking together the transcriptional analysis data and the biochemical evidences, we surmise that the protein F55 is involved in the regulation of the lysogenic state of SSV1. PMID:23514883

  7. The Effects of Temperature and Growth Phase on the Lipidomes of Sulfolobus islandicus and Sulfolobus tokodaii

    Directory of Open Access Journals (Sweden)

    Sara Munk Jensen

    2015-08-01

    Full Text Available The functionality of the plasma membrane is essential for all organisms. Adaption to high growth temperatures imposes challenges and Bacteria, Eukarya, and Archaea have developed several mechanisms to cope with these. Hyperthermophilic archaea have earlier been shown to synthesize tetraether membrane lipids with an increased number of cyclopentane moieties at higher growth temperatures. Here we used shotgun lipidomics to study this effect as well as the influence of growth phase on the lipidomes of Sulfolobus islandicus and Sulfolobus tokodaii for the first time. Both species were cultivated at three different temperatures, with samples withdrawn during lag, exponential, and stationary phases. Three abundant tetraether lipid classes and one diether lipid class were monitored. Beside the expected increase in the number of cyclopentane moieties with higher temperature in both archaea, we observed previously unreported changes in the average cyclization of the membrane lipids throughout growth. The average number of cyclopentane moieties showed a significant dip in exponential phase, an observation that might help to resolve the currently debated biosynthesis pathway of tetraether lipids.

  8. Characterization of the Sulfolobus host-SSV2 virus interaction

    DEFF Research Database (Denmark)

    Contursi, P.; Jensen, S.; Aucelli, T.;

    2006-01-01

    The Sulfolobus spindle virus, SSV2, encodes a tyrosine integrase which furthers provirus formation in host chromosomes. Consistently with the prediction made during sequence analysis, integration was found to occur in the downstream half of the tRNAGly (CCC) gene. In this paper we report the find...

  9. Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon

    DEFF Research Database (Denmark)

    Jaubert, Carole; Danioux, Chloë; Oberto, Jacques;

    2013-01-01

    common core genome of approximately 2 Mb and a long hyperplastic region containing most of the strain-specific genes. In LAL14/1, the latter region is enriched in insertion sequences, CRISPR (clustered regularly interspaced short palindromic repeats), glycosyl transferase genes, toxin-antitoxin genes and...... MITE (miniature inverted-repeat transposable elements). The tRNA genes of LAL14/1 are preferential targets for the integration of mobile elements but clusters of atypical genes (CAG) are also integrated elsewhere in the genome. LAL14/1 carries five CRISPR loci with 10 per cent of spacers matching...... perfectly or imperfectly the genomes of archaeal viruses and plasmids found in the Icelandic hot springs. Strikingly, the CRISPR_2 region of LAL14/1 carries an unusually long 1.9 kb spacer interspersed between two repeat regions and displays a high similarity to pING1-like conjugative plasmids. Finally, we...

  10. Genomic evidence of rapid, global-scale gene flow in a Sulfolobus species

    OpenAIRE

    Mao, Dominic; Grogan, Dennis

    2012-01-01

    Local populations of Sulfolobus islandicus diverge genetically with geographical separation, and this has been attributed to restricted transfer of propagules imposed by the unfavorable spatial distribution of acidic geothermal habitat. We tested the generality of genetic divergence with distance in Sulfolobus species by analyzing genomes of Sulfolobus acidocaldarius drawn from three populations separated by more than 8000 km. In sharp contrast to S. islandicus, the geographically diverse S. ...

  11. Promiscuità enzimatica ed evoluzione molecolare:studio di una lattonasi da Sulfolobus solfataricus in grado di degradare agenti nervini

    OpenAIRE

    Merone, Luigia

    2010-01-01

    Si definisce promiscuità catalitica la capacità di un enzima di catalizzare reazioni aggiuntive al ruolo principale, che possono essere più o meno chimicamente differenti tra loro. Questa definizione si riferisce sia alla possibilità di catalizzare la trasformazione di substrati di natura diversa che di utilizzare differenti meccanismi di reazione e/o diversi siti catalitici1,2. L'enorme importanza legata allo studio della promiscuità enzimatica è sostanzialmente duplice. Da un punto di vista...

  12. Compound K Production from Red Ginseng Extract by β-Glycosidase from Sulfolobus solfataricus Supplemented with α-L-Arabinofuranosidase from Caldicellulosiruptor saccharolyticus

    OpenAIRE

    Shin, Kyung-Chul; Choi, Hye-Yeon; Seo, Min-Ju; Oh, Deok-Kun

    2015-01-01

    Ginsenoside compound K (C-K) is attracting a lot of interest because of its biological and pharmaceutical activities, including hepatoprotective, antitumor, anti-wrinkling, and anti-skin aging activities. C-K has been used as the principal ingredient in skin care products. For the effective application of ginseng extracts to the manufacture of cosmetics, the PPD-type ginsenosides in ginseng extracts should be converted to C-K by enzymatic conversion. For increased yield of C-K from the protop...

  13. Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries

    DEFF Research Database (Denmark)

    Becker, F.; Schnorr, K.; Wilting, R.; Tolstrup, N.; Bendtsen, Jannick Dyrløv; Olsen, P.B.

    2004-01-01

    To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less beta-lactamase ('bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The 'bla gene was cloned into a bacteriophage MU...

  14. Co-expression with RadA and the characterization of stRad55B, a RadA paralog from the hyperthermophilic crenarchaea Sulfolobus tokodaii

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    ST0838 (designed stRad55B) is one of the four RadA paralogs (or Rad55 homologues) in the genome of the hyperthermophilic crenarchaeon Sulfolobus tokodaii. The gene is induced by UV irradiation, sug-gesting that it is involved in DNA recombinational repair in this organism. However, this protein could not be expressed normally in vitro. In this study, thermostable and soluble stRad55B was obtained by co-expression with S. tokodaii RadA (stRadA) in E. coli, and the enzymatic properties were examined. It was found that stRad55B bound ssDNA preferentially and had a very weak ATPase activity that was not stimulated by DNA. The recombinant protein inhibited the strand exchange activity promoted by stRadA, indicating that stRad55B might be an inhibitor to the homologous recombination in this ar-chaeon. The results will be helpful for further functional and interaction analysis of RadA paralogs and for the understanding of the mechanism of recombinational repair in archaea.

  15. Co-expression with RadA and the characterization of stRad55B, a RadA paralog from the hyperthermophilic crenarchaea Sulfolobus tokodaii

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    ST0838 (designed stRad55B) is one of the four RadA paralogs (or Rad55 homologues) in the genome of the hyperthermophilic crenarchaeon Sulfolobus tokodaii. The gene is induced by UV irradiation, suggesting that it is involved in DNA recombinational repair in this organism. However, this protein could not be expressed normally in vitro. In this study, thermostable and soluble stRad55B was obtained by co-expression with S. tokodaii RadA (stRadA) in E. coli, and the enzymatic properties were examined. It was found that stRad55B bound ssDNA preferentially and had a very weak ATPase activity that was not stimulated by DNA. The recombinant protein inhibited the strand exchange activity promoted by stRadA, indicating that stRad55B might be an inhibitor to the homologous recombination in this archaeon. The results will be helpful for further functional and interaction analysis of RadA paralogs and for the understanding of the mechanism of recombinational repair in archaea.

  16. Inter-viral conflicts that exploit host CRISPR immune systems of Sulfolobus

    OpenAIRE

    Erdmann, Susanne; Le Moine Bauer, Sven; Garrett, Roger A.

    2014-01-01

    Infection of Sulfolobus islandicus REY15A with mixtures of different Sulfolobus viruses, including STSV2, did not induce spacer acquisition by the host CRISPR immune system. However, coinfection with the tailed fusiform viruses SMV1 and STSV2 generated hyperactive spacer acquisition in both CRISPR loci, exclusively from STSV2, with the resultant loss of STSV2 but not SMV1. SMV1 was shown to activate adaptation while itself being resistant to CRISPR-mediated adaptation and DNA interference. Ex...

  17. The Identification of Sulfolobus Species from a Biocorrosion of Wastewater Pipes

    OpenAIRE

    Shakila Motamedi; Reza Marandi; Seyedeh Flour Mazhar; Kambiz Tahvildari

    2011-01-01

    One of the species which has a prominent role in the biocorrosion of wastewater pipes is Sulfolobus bacterium. This bacterium is among the iron oxidizing bacteria. Sulfolobus is from the archae bacteria genus, which provides its needed energy by oxidation of sulfur in warm and acidic environments. In this research, the water sample exist in the water supply pipelines at Sarcheshmeh copper mine was studied, sulfur corrosive bacteria were separated and identified and their biochemical activity ...

  18. The composition, structure and stability of a group II chaperonin are temperature regulated in a hyperthermophilic archaeon.

    Science.gov (United States)

    Kagawa, Hiromi K; Yaoi, Takuro; Brocchieri, Luciano; McMillan, R Andrew; Alton, Thomas; Trent, Jonathan D

    2003-04-01

    The hyperthermoacidophilic archaeon Sulfolobus shibatae contains group II chaperonins, known as rosettasomes, which are two nine-membered rings composed of three different 60 kDa subunits (TF55 alpha, beta and gamma). We sequenced the gene for the gamma subunit and studied the temperature-dependent changes in alpha, beta and gamma expression, their association into rosettasomes and their phylogenetic relationships. Alpha and beta gene expression was increased by heat shock (30 min, 86 degrees C) and decreased by cold shock (30 min, 60 degrees C). Gamma expression was undetectable at heat shock temperatures and low at normal temperatures (75-79 degrees C), but induced by cold shock. Polyacrylamide gel electrophoresis indicated that in vitro alpha and beta subunits form homo-oligomeric rosettasomes, and mixtures of alpha, beta and gamma form hetero-oligomeric rosettasomes. Transmission electron microscopy revealed that beta homo-oligomeric rosettasomes and all hetero-oligomeric rosettasomes associate into filaments. In vivo rosettasomes were hetero-oligomeric with an average subunit ratio of 1alpha:1beta:0.1gamma in cultures grown at 75 degrees C, a ratio of 1alpha:3beta:1gamma in cultures grown at 60 degrees C and a ratio of 2alpha:3beta:0gamma after 86 degrees C heat shock. Using differential scanning calorimetry, we determined denaturation temperatures (Tm) for alpha, beta and gamma subunits of 95.7 degrees C, 96.7 degrees C and 80.5 degrees C, respectively, and observed that rosettasomes containing gamma were relatively less stable than those with alpha and/or beta only. We propose that, in vivo, the rosettasome structure is determined by the relative abundance of subunits and not by a fixed geometry. Furthermore, phylogenetic analyses indicate that archaeal chaperonin subunits underwent multiple duplication events within species (paralogy). The independent evolution of these paralogues raises the possibility that chaperonins have functionally diversified between

  19. Formate hydrogenlyase in the hyperthermophilic archaeon, Thermococcus litoralis

    OpenAIRE

    Rákhely Gábor; Varga András; Bogos Balázs; Tóth András; Takács Mária; Kovács Kornél L

    2008-01-01

    Abstract Background Thermococcus litoralis is a heterotrophic facultative sulfur dependent hyperthermophilic Archaeon, which was isolated from a shallow submarine thermal spring. It has been successfully used in a two-stage fermentation system, where various keratinaceous wastes of animal origin were converted to biohydrogen. In this system T. litoralis performed better than its close relative, P. furiosus. Therefore, new alternative enzymes involved in peptide and hydrogen metabolism were as...

  20. Sugar metabolism and regulation in the hyperthermophilic archaeon Thermococcus litoralis

    OpenAIRE

    Qu, Qiuhao

    2004-01-01

    A novel trehalose glycosyl transferring synthase (TreT) from the hyperthermophilic archaeon T. litoralis was identified. This enzyme produces trehalose by the transfer of glucose from ADP-glucose (or, less effectively, UDP-glucose and GDP-glucose) onto the 1-position of glucose to form trehalose and ADP. Such an enzymatic activity in the synthesis of trehalose has not yet been described for any other organisms. It catalyzes a reversible reaction for trehalose synthesis and degradation by conv...

  1. Factors affecting Archaeal Lipid Compositions of the Sulfolobus Species

    Science.gov (United States)

    He, L.; Han, J.; Wei, Y.; Lin, L.; Wei, Y.; Zhang, C.

    2010-12-01

    Temperature is the best known variable affecting the distribution of the archaeal glycerol dibiphytanyl glycerol tetraethers (GDGTs) in marine and freshwater systems. Other variables such as pH, ionic strength, or bicarbonate concentration may also affect archaeal GDGTs in terrestrial systems. Studies of pure cultures can help us pinpoint the specific effects these variables may have on archaeal lipid distribution in natural environments. In this study, three Sulfolobus species (HG4, HB5-2, HB9-6) isolated from Tengchong hot springs (pH 2-3, temperature 73-90°C) in China were used to investigate the effects of temperature, pH, substrate, and type of strain on the composition of GDGTs. Results showed that increase in temperature had negative effects on the relative contents of GDGT-0 (no cyclopentyl rings), GDGT-1 (one cyclopentyl ring), GDGT-2 and GDGT-3 but positive effects on GDGT-4, GDGT-4', GDGT-5 and GDGT-5'. Increase in pH, on the other hand, had negative effects on GDGT-0, GDGT-1, GDGT-4', GDGT-5 and GDGT-5', and positive effects on GDGT-3 and GDGT-4. GDGT-2 remained relatively constant with changing pH. When the HG4 was grown on different substrates, GDGT-5 was five time more abundant in sucrose-grown cultures than in yeast extract- or sulfur- grown cultures, suggesting that carbohydrates may stimulate the production of GDGT-5. For all three species, the ring index (average number of rings) of GDGTs correlated positively with incubation temperature. In HG4, ring index was much lower at optimal pH (3.5) than at other pH values. Ring index of HB5-2 or HB9-6 is higher than that of HG4, suggesting that speciation may affect the degree of cyclization of GDGT of the Sulfolobus. These results indicate that individual archaeal lipids respond differently to changes in environmental variables, which may be also species specific.

  2. CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties

    DEFF Research Database (Denmark)

    Lillestøl, Reidun K; Shah, Shiraz Ali; Brügger, Kim;

    2009-01-01

    Summary CRISPRs of Sulfolobus fall into three main families based on their repeats, leader regions, associated cas genes, and putative recognition sequences on viruses and plasmids. Spacer sequence matches to different viruses and plasmids of the Sulfolobales revealed some bias particularly for...... family III CRISPRs. Transcription occurs on both strands of the five repeat-clusters of Sulfolobus acidocaldarius and a repeat-cluster of the conjugative plasmid pKEF9. Leader strand transcripts cover whole repeat-clusters and are processed mainly from the 3'-end, within repeats, yielding heterogeneous...

  3. Identification of carotenoids from the extremely halophilic archaeon Haloarcula japonica

    Directory of Open Access Journals (Sweden)

    Rie eYatsunami

    2014-03-01

    Full Text Available The carotenoids produced by extremely halophilic archaeon Haloarcula japonica were extracted and identified by their chemical, chromatographic, and spectroscopic characteristics (UV-Vis and mass spectrometry. The composition (mol% was 68.1% bacterioruberin, 22.5% monoanhydrobacterioruberin, 9.3% bisanhydrobacterioruberin, < 0.1% isopentenyldehydrorhodopin, and trace amounts of lycopene and phytoene. The in vitro scavenging capacity of a carotenoid, bacterioruberin, extracted from Ha. japonica cells against 1,1-diphenyl-2-picrylhydrazyl (DPPH radicals was evaluated. The antioxidant capacity of bacterioruberin was much higher than that of β-carotene.

  4. Functional Reconstitution of Membrane Proteins in Monolayer Liposomes from Bipolar Lipids of Sulfolobus acidocaldarius

    NARCIS (Netherlands)

    Elferink, Maria; Wit, Janny G. de; Demel, Rudy; Driessen, Arnold J.M.; Konings, Wilhelmus

    1992-01-01

    Membranes of Sulfolobus acidocaldarius, an extreme thermophilic archaebacterium, are composed of unusual bipolar lipids. They consist of macrocyclic tetraethers with two polar heads linked by two hydrophobic C40 phytanyl chains which are thought to be arranged as a monolayer in the cytoplasmic membr

  5. Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Deng, Ling; Zhu, Haojun; Chen, Zhengjun;

    2009-01-01

    Sulfolobus islandicus is being used as a model for studying archaeal biology, geo-biology and evolution. However, no genetic system is available for this organism. To produce an S. islandicus mutant suitable for genetic analyses, we screened for colonies with a spontaneous pyrEF mutation. One mut...

  6. Transcription termination in the plasmid/virus hybrid pSSVx from Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Contursi, Patrizia; Cannio, Raffaele; She, Qunxin

    2010-01-01

    The pSSVx from Sulfolobus islandicus, strain REY15/4, is a hybrid between a plasmid and a fusellovirus. A systematic study previously performed revealed the presence of nine major transcripts, the expression of which was differentially and temporally regulated over the growth cycle of S. islandic...

  7. Microarray analysis in the archaeon Halobacterium salinarum strain R1.

    Directory of Open Access Journals (Sweden)

    Jens Twellmeyer

    Full Text Available BACKGROUND: Phototrophy of the extremely halophilic archaeon Halobacterium salinarum was explored for decades. The research was mainly focused on the expression of bacteriorhodopsin and its functional properties. In contrast, less is known about genome wide transcriptional changes and their impact on the physiological adaptation to phototrophy. The tool of choice to record transcriptional profiles is the DNA microarray technique. However, the technique is still rarely used for transcriptome analysis in archaea. METHODOLOGY/PRINCIPAL FINDINGS: We developed a whole-genome DNA microarray based on our sequence data of the Hbt. salinarum strain R1 genome. The potential of our tool is exemplified by the comparison of cells growing under aerobic and phototrophic conditions, respectively. We processed the raw fluorescence data by several stringent filtering steps and a subsequent MAANOVA analysis. The study revealed a lot of transcriptional differences between the two cell states. We found that the transcriptional changes were relatively weak, though significant. Finally, the DNA microarray data were independently verified by a real-time PCR analysis. CONCLUSION/SIGNIFICANCE: This is the first DNA microarray analysis of Hbt. salinarum cells that were actually grown under phototrophic conditions. By comparing the transcriptomics data with current knowledge we could show that our DNA microarray tool is well applicable for transcriptome analysis in the extremely halophilic archaeon Hbt. salinarum. The reliability of our tool is based on both the high-quality array of DNA probes and the stringent data handling including MAANOVA analysis. Among the regulated genes more than 50% had unknown functions. This underlines the fact that haloarchaeal phototrophy is still far away from being completely understood. Hence, the data recorded in this study will be subject to future systems biology analysis.

  8. Sulfolobus Replication Factor C stimulates the activity of DNA Polymerase B1

    DEFF Research Database (Denmark)

    Xing, Xuanxuan; Zhang, Likui; Guo, Li;

    2014-01-01

    Replication factor C (RFC) is known to function in loading proliferating cell nuclear antigen (PCNA) onto primed DNA, allowing PCNA to tether DNA polymerase for highly processive DNA synthesis in eukaryotic and archaeal replication. In this report, we show that an RFC complex from the...... with the ability of RFC to facilitate DNA binding by PolB1 through protein-protein interaction. These results suggest that Sulfolobus RFC may play a role in recruiting DNA polymerase for efficient primer extension, in addition to clamp loading, during DNA replication....... hyperthermophilic archaea of the genus Sulfolobus physically interacts with DNA polymerase B1 (PolB1) and enhances both the polymerase and 3'-5' exonuclease activities of PolB1 in an ATP-independent manner. Stimulation of the PolB1 activity by RFC is independent of the ability of RFC to bind DNA but is consistent...

  9. Structure of hypothetical Mo-cofactor biosynthesis protein B (ST2315) from Sulfolobus tokodaii

    International Nuclear Information System (INIS)

    The structure of a protein involved in the molybdopterin and molybdenum co-factor biosynthesis pathways of Sulfolobus tokodaii has been solved to a resolution of 1.9 Å. The structure of a probable Mo-cofactor biosynthesis protein B from Sulfolobus tokodaii, belonging to space group P6422 with unit-cell parameters a = b = 136.68, c = 210.52 Å, was solved by molecular replacement to a resolution of 1.9 Å and refined to an R factor and Rfree of 16.8% and 18.5%, respectively. The asymmetric unit contains a trimer, while the biologically significant oligomer is predicted to be a hexamer by size-exclusion chromatography. The subunit structure and fold of ST2315 are similar to those of other enzymes that are known to be involved in the molybdopterin- and molybdenum cofactor-biosynthesis pathways

  10. Molecular cloning of the transcription factor TFIIB homolog from Sulfolobus shibatae.

    OpenAIRE

    Qureshi, S A; Khoo, B; Baumann, P; Jackson, S P

    1995-01-01

    The Archaea (archaebacteria) constitute a group of prokaryotes that are phylogenetically distinct from Eucarya (eukaryotes) and Bacteria (eubacteria). Although Archaea possess only one RNA polymerase, evidence suggests that their transcriptional apparatus is similar to that of Eucarya. For example, Archaea contain a homolog of the TATA-binding protein which interacts with the TATA-box like A-box sequence upstream of many archaeal genes. Here, we report the cloning of a Sulfolobus shibatae gen...

  11. The Potent In Vitro Skin Permeation of Archaeosome Made from Lipids Extracted of Sulfolobus acidocaldarius

    OpenAIRE

    Eskandar Moghimipour; Mohammad Kargar; Zahra Ramezani; Somayeh Handali

    2013-01-01

    Archaeosomes are a new generation of liposomes that exhibit higher stabilities under different conditions, such as high temperatures, alkaline or acidic pH, and presence of bile salts in comparison with liposomes, and can be used in biotechnology including drug, gene, and vaccine delivery. The objective of this study was to prepare archaeosomes using lipid extracted from Sulfolobus acidocaldarius and evaluate their physicochemical properties. The lipids were extracted from S. acidocaldarius a...

  12. The Identification of Sulfolobus Species from a Biocorrosion of Wastewater Pipes

    Directory of Open Access Journals (Sweden)

    Shakila Motamedi

    2011-01-01

    Full Text Available One of the species which has a prominent role in the biocorrosion of wastewater pipes is Sulfolobus bacterium. This bacterium is among the iron oxidizing bacteria. Sulfolobus is from the archae bacteria genus, which provides its needed energy by oxidation of sulfur in warm and acidic environments. In this research, the water sample exist in the water supply pipelines at Sarcheshmeh copper mine was studied, sulfur corrosive bacteria were separated and identified and their biochemical activity was researched and studied and it was found that the target species are in fact Sulfolobus archae bacteria which have different enzymatic activities. Due to these enzymatic activities, a considerable amount of sulfur is accumulated on the cell surface. Based on various incubation environments, as a result of sulfur accumulation, Sulfolubos archae bacteria were identified, and after doing isolation and purification methods, number of colonies have been decrease from 10-1 dilution to 10-10 dilution and in sample with 10-1 dilution sample 200 colonies has been reported and in plate with 10-10 dilution no colonies has been observed.

  13. Isolation and Phylogenetic Analysis of Halophilic Archaeon AJ6

    Institute of Scientific and Technical Information of China (English)

    Xu Xiaohong; Wu Min; Cao Yi; Wu Yuehong; Zhang Ting

    2006-01-01

    Halophilic archaeon A J6 was isolated and purified from the Altun Mountain National Nature Reserve of the Xinjiang Uygur Autonomous Region.Strain AJ6 is a Gram-negative rod whose size is 0.2-0.6 by 1.6-4.2 μm,wherein a few cells are globular.The optimum salt concentration for its growth is 20% NaC1 and 0.6% Mg2+,and the optimum pH is 6.0-7.0.Morphological,physiological,and biochemical characteristics of strain AJ6 were observed.The 16S rRNA encoding gene (16S rDNA)sequence of strain A J6 was amplified by PCR,and its nucteotide sequence was determined subsequently."Clustalw"and"PHYLIP"software bags were used to analyze the 16S rDNA sequence;the homology was compared,and then the phylogenetic tree was established.The results indicate that strain AJ6 is a novel species of the genus Natrinema.The GenBank accession number of the 16S rDNA sequences of strain AJ6 is AY277584.

  14. Single gene insertion drives bioalcohol production by a thermophilic archaeon

    Energy Technology Data Exchange (ETDEWEB)

    Basen, M; Schut, GJ; Nguyen, DM; Lipscomb, GL; Benn, RA; Prybol, CJ; Vaccaro, BJ; Poole, FL; Kelly, RM; Adams, MWW

    2014-12-09

    Bioethanol production is achieved by only two metabolic pathways and only at moderate temperatures. Herein a fundamentally different synthetic pathway for bioalcohol production at 70 degrees C was constructed by insertion of the gene for bacterial alcohol dehydrogenase (AdhA) into the archaeon Pyrococcus furiosus. The engineered strain converted glucose to ethanol via acetate and acetaldehyde, catalyzed by the host-encoded aldehyde ferredoxin oxidoreductase (AOR) and heterologously expressed AdhA, in an energy-conserving, redox-balanced pathway. Furthermore, the AOR/AdhA pathway also converted exogenously added aliphatic and aromatic carboxylic acids to the corresponding alcohol using glucose, pyruvate, and/or hydrogen as the source of reductant. By heterologous coexpression of a membrane-bound carbon monoxide dehydrogenase, CO was used as a reductant for converting carboxylic acids to alcohols. Redirecting the fermentative metabolism of P. furiosus through strategic insertion of foreign genes creates unprecedented opportunities for thermophilic bioalcohol production. Moreover, the AOR/AdhA pathway is a potentially game-changing strategy for syngas fermentation, especially in combination with carbon chain elongation pathways.

  15. Metabolic reconstruction of the archaeon methanogen Methanosarcina Acetivorans

    Directory of Open Access Journals (Sweden)

    Maranas Costas D

    2011-02-01

    Full Text Available Abstract Background Methanogens are ancient organisms that are key players in the carbon cycle accounting for about one billion tones of biological methane produced annually. Methanosarcina acetivorans, with a genome size of ~5.7 mb, is the largest sequenced archaeon methanogen and unique amongst the methanogens in its biochemical characteristics. By following a systematic workflow we reconstruct a genome-scale metabolic model for M. acetivorans. This process relies on previously developed computational tools developed in our group to correct growth prediction inconsistencies with in vivo data sets and rectify topological inconsistencies in the model. Results The generated model iVS941 accounts for 941 genes, 705 reactions and 708 metabolites. The model achieves 93.3% prediction agreement with in vivo growth data across different substrates and multiple gene deletions. The model also correctly recapitulates metabolic pathway usage patterns of M. acetivorans such as the indispensability of flux through methanogenesis for growth on acetate and methanol and the unique biochemical characteristics under growth on carbon monoxide. Conclusions Based on the size of the genome-scale metabolic reconstruction and extent of validated predictions this model represents the most comprehensive up-to-date effort to catalogue methanogenic metabolism. The reconstructed model is available in spreadsheet and SBML formats to enable dissemination.

  16. Structure of A197 from Sulfolobus Turreted Icosahedral Virus: a Crenarchaeal Viral Glycosyltransferase Exhibiting the GT-A Fold

    OpenAIRE

    Larson, Eric T.; Reiter, Dirk; Young, Mark; Lawrence, C. Martin

    2006-01-01

    Sulfolobus turreted icosahedral virus (STIV) was the first icosahedral virus characterized from an archaeal host. It infects Sulfolobus species that thrive in the acidic hot springs (pH 2.9 to 3.9 and 72 to 92°C) of Yellowstone National Park. The overall capsid architecture and the structure of its major capsid protein are very similar to those of the bacteriophage PRD1 and eukaryotic viruses Paramecium bursaria Chlorella virus 1 and adenovirus, suggesting a viral lineage that predates the th...

  17. Removal of Sulfur Compounds from Coal by the Thermophilic Organism Sulfolobus acidocaldarius

    OpenAIRE

    KARGI, Fikret; Robinson, James M.

    1982-01-01

    The thermophilic, reduced-sulfur, iron-oxidizing bacterium Sulfolobus acidocaldarius was used for the removal of sulfur compounds from coal. The inclusion of complex nutrients such as yeast extract and peptone, and chemical oxidizing agents, 0.01 M FeCl3 into leaching medium, reduced the rate and the extent of sulfur removal from coal. The rate of sulfur removal by S. acidocaldarius was strongly dependent on the sulfur content of the coal and on the total external surface area of coal particl...

  18. Formate hydrogenlyase in the hyperthermophilic archaeon, Thermococcus litoralis

    Directory of Open Access Journals (Sweden)

    Rákhely Gábor

    2008-06-01

    Full Text Available Abstract Background Thermococcus litoralis is a heterotrophic facultative sulfur dependent hyperthermophilic Archaeon, which was isolated from a shallow submarine thermal spring. It has been successfully used in a two-stage fermentation system, where various keratinaceous wastes of animal origin were converted to biohydrogen. In this system T. litoralis performed better than its close relative, P. furiosus. Therefore, new alternative enzymes involved in peptide and hydrogen metabolism were assumed in T. litoralis. Results An about 10.5 kb long genomic region was isolated and sequenced from Thermococcus litoralis. In silico analysis revealed that the region contained a putative operon consisting of eight genes: the fdhAB genes coding for a formate dehydrogenase and the mhyCDEFGH genes encoding a [NiFe] hydrogenase belonging to the group of the H2-evolving, energy-conserving, membrane-bound hydrogenases. Reverse transcription linked quantitative Real-Time PCR and Western blotting experiments showed that the expression of the fdh-mhy operon was up-regulated during fermentative growth on peptides and down-regulated in cells cultivated in the presence of sulfur. Immunoblotting and protein separation experiments performed on cell fractions indicated that the formate dehydrogenase part of the complex is associated to the membrane-bound [NiFe] hydrogenase. Conclusion The formate dehydrogenase together with the membrane-bound [NiFe] hydrogenase formed a formate hydrogenlyase (formate dehydrogenase coupled hydrogenase, FDH-MHY complex. The expression data suggested that its physiological role is linked to the removal of formate likely generated during anaerobic peptide fermentation.

  19. The genome of the square archaeon Haloquadratum walsbyi : life at the limits of water activity

    NARCIS (Netherlands)

    Bolhuis, Henk; Palm, Peter; Wende, Andy; Falb, Michaela; Rampp, Markus; Rodriguez-Valera, Francisco; Pfeiffer, Friedhelm; Oesterhelt, Dieter

    2006-01-01

    Background: The square halophilic archaeon Haloquadratum walsbyi dominates NaCl- saturated and MgCl2 enriched aquatic ecosystems, which imposes a serious desiccation stress, caused by the extremely low water activity. The genome sequence was analyzed and physiological and physical experiments were c

  20. Impact of Molecular Hydrogen on Chalcopyrite Bioleaching by the Extremely Thermoacidophilic Archaeon Metallosphaera sedula▿

    OpenAIRE

    Auernik, Kathryne S.; Kelly, Robert M.

    2010-01-01

    Hydrogen served as a competitive inorganic energy source, impacting the CuFeS2 bioleaching efficiency of the extremely thermoacidophilic archaeon Metallosphaera sedula. Open reading frames encoding key terminal oxidase and electron transport chain components were triggered by CuFeS2. Evidence of heterotrophic metabolism was noted after extended periods of bioleaching, presumably related to cell lysis.

  1. Lipids of the ultra-thin square halophilic archaeon Haloquadratum walsbyi

    OpenAIRE

    Simona LoBasso; Patrizia LoPalco; Giuseppe Mascolo; Angela Corcelli

    2008-01-01

    The lipid composition of the extremely halophilic archaeon Haloquadratum walsbyi was investigated by thin-layer chromatography and electrospray ionization-mass spectrometry. The analysis of neutral lipids showed the presence of vitamin MK-8, squalene, carotene, bacterioruberin and several retinal isomers. The major polar lipids were phosphatidylglycerophosphate methyl ester, phosphatidylglycerosulfate, phosphatidylg...

  2. Production of beta-xylanase and beta-xylosidase by the extremely halophilic archaeon Halorhabdus utahensis

    DEFF Research Database (Denmark)

    Wainø, M.; Ingvorsen, K.

    2003-01-01

    The extremely halophilic archaeon, Halorhabdus utahensis, isolated from the Great Salt Lake, Utah, produced beta-xylanase and beta-xylosidase activities. Both enzymes were active over a broad NaCl range from near zero to 30% NaCl when tested with culture broth. A broad NaCl optimum was observed f...

  3. First Insights into the Genome Sequence of the Halophilic Archaeon Halalkalicoccus paucihalophilus (DSM 24557).

    Science.gov (United States)

    Poehlein, Anja; Mucek, Katharina; Enders, Marieke; Pankok, Frederik; Daniel, Rolf

    2016-01-01

    Halalkalicoccus paucihalophilus is an extremely halophilic, Gram-negative, and nonmotile coccus-like archaeon, which was originally isolated from the Lop Nur region in the northwest of China. The genome consists of a single replicon (3.98 Mbp). H. paucihalophilus is able to utilize mannose, which is unique for members of this genus. PMID:27198019

  4. NCBI nr-aa BLAST: CBRC-XTRO-01-3737 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-3737 ref|NP_343011.1| Transporter (proton symporter) [Sulfolobus solfa...taricus P2] gb|AAK41801.1| Transporter (proton symporter) [Sulfolobus solfataricus P2] NP_343011.1 0.010 25% ...

  5. Genetic analysis of the Holliday junction resolvases Hje and Hjc in Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Huang, Qihong; Li, Yansheng; Zeng, Chaoning;

    2015-01-01

    The in vivo functions of Hje and Hjc, two Holliday junction resolvases in Sulfolobus islandicus were investigated. We found that deletion of either hje or hjc had no effect on normal cell growth, while deletion of both hje and hjc is lethal. Although Hjc is the conserved resolvase in all archaea,...

  6. The cobY Gene of the Archaeon Halobacterium sp. Strain NRC-1 Is Required for De Novo Cobamide Synthesis

    OpenAIRE

    Woodson, J. D.; Peck, R. F.; Krebs, M P; Escalante-Semerena, J C

    2003-01-01

    Genetic and nutritional analyses of mutants of the extremely halophilic archaeon Halobacterium sp. strain NRC-1 showed that open reading frame (ORF) Vng1581C encodes a protein with nucleoside triphosphate:adenosylcobinamide-phosphate nucleotidyltransferase enzyme activity. This activity was previously associated with the cobY gene of the methanogenic archaeon Methanobacterium thermoautotrophicum strain ΔH, but no evidence was obtained to demonstrate the direct involvement of this protein in c...

  7. Novel RepA-MCM proteins encoded in plasmids pTAU4, pORA1 and pTIK4 from Sulfolobus neozealandicus

    DEFF Research Database (Denmark)

    Greve, Bo Bjørn

    2005-01-01

    . Plasmids pORA1 and pTIK4 encode RepA proteins, only the former of which carries the novel polymerase–primase domain of other known Sulfolobus plasmids. Plasmid pTAU4 encodes a mini-chromosome maintenance protein homolog and no RepA protein; the implications for DNA replication are considered. Plasmid pORA1......Three plasmids isolated from the crenarchaeal thermoacidophile Sulfolobus neozealandicus were characterized. Plasmids pTAU4 (7,192 bp), pORA1 (9,689 bp) and pTIK4 (13,638 bp) show unusual properties that distinguish them from previously characterized cryptic plasmids of the genus Sulfolobus...... is the first Sulfolobus plasmid to be characterized that does not encode the otherwise highly conserved DNA-binding PlrA protein. Another encoded protein appears to be specific for the New Zealand plasmids. The three plasmids should provide useful model systems for functional studies of these...

  8. Transcriptome changes in STSV2-infected Sulfolobus islandicus REY15A undergoing continuous CRISPR spacer acquisition

    DEFF Research Database (Denmark)

    León-Sobrino, Carlos; Kot, Witold P; Garrett, Roger A

    2016-01-01

    A transcriptome study was performed on Sulfolobus islandicus REY15A actively undergoing CRISPR spacer acquisition from the crenarchaeal monocaudavirus STSV2 in rich and basal media over a 6 day period. Spacer acquisition preceded strong host growth retardation, altered transcriptional activity of...... four different CRISPR-Cas modules and changes in viral copy numbers, and with significant differences in the two media. Transcript levels of proteins involved in the cell cycle were reduced, while those of DNA replication, DNA repair, transcriptional regulation, and some antitoxin-toxin pairs and...... transposases were unchanged or enhanced. Antisense RNAs were implicated in the transcriptional regulation of adaptation and interference modules of the type I-A CRISPR-Cas system and evidence was found for the occurrence of functional coordination between the single CRISPR-Cas adaptation module and the...

  9. Transcriptome changes in STSV2-infected Sulfolobus islandicus REY15A undergoing continuous CRISPR spacer acquisition

    DEFF Research Database (Denmark)

    León Sobrino, Carlos; Kot, W.P.; Garrett, Roger Antony

    2015-01-01

    transposases were unchanged or enhanced. Antisense RNAs were implicated in the transcriptional regulation of adaptation and interference modules of the type I-A CRISPR-Cas system and evidence was found for the occurrence of functional coordination between the single CRISPR-Cas adaptation module and the......A transcriptome study was performed on Sulfolobus islandicus REY15A actively undergoing CRISPR spacer acquisition from the crenarchaeal monocaudavirus STSV2 in rich and basal media over a 6 day period. Spacer acquisition preceded strong host growth retardation, altered transcriptional activity of...... four different CRISPR-Cas modules and changes in viral copy numbers, and with significant differences in the two media. Transcript levels of proteins involved in the cell cycle were reduced, while those of DNA replication, DNA repair, transcriptional regulation, and some antitoxin-toxin pairs and...

  10. Biochemical Properties of a Putative Signal Peptide Peptidase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

    OpenAIRE

    Matsumi, Rie; Atomi, Haruyuki; Imanaka, Tadayuki

    2005-01-01

    We have performed the first biochemical characterization of a putative archaeal signal peptide peptidase (SppATk) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. SppATk, comprised of 334 residues, was much smaller than its counterpart from Escherichia coli (618 residues) and harbored a single predicted transmembrane domain near its N terminus. A truncated mutant protein without the N-terminal 54 amino acid residues (ΔN54SppATk) was found to be stable against autoproteolys...

  11. Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA

    OpenAIRE

    Swarts, Daan C.; Hegge, Jorrit W.; Hinojo, Ismael; Shiimori, Masami; Ellis, Michael A.; Dumrongkulraksa, Justin; Terns, Rebecca M.; Terns, Michael P.; van der Oost, John

    2015-01-01

    Functions of prokaryotic Argonautes (pAgo) have long remained elusive. Recently, Argonautes of the bacteria Rhodobacter sphaeroides and Thermus thermophilus were demonstrated to be involved in host defense. The Argonaute of the archaeon Pyrococcus furiosus (PfAgo) belongs to a different branch in the phylogenetic tree, which is most closely related to that of RNA interference-mediating eukaryotic Argonautes. Here we describe a functional and mechanistic characterization of PfAgo. Like the bac...

  12. Acyl homoserine lactone-based quorum sensing in a methanogenic archaeon

    OpenAIRE

    Zhang, Guishan; Zhang, Fan; Ding, Gang; Li, Jie; Guo, Xiaopeng; Zhu, Jinxing; Zhou, Liguang; Cai, Shichun; Liu, Xiaoli; Luo, Yuanming; Zhang, Guifeng; Shi, Wenyuan; Dong, Xiuzhu

    2012-01-01

    Acyl homoserine lactone (AHL)-based quorum sensing commonly refers to cell density-dependent regulatory mechanisms found in bacteria. However, beyond bacteria, this cell-to-cell communication mechanism is poorly understood. Here we show that a methanogenic archaeon, Methanosaeta harundinacea 6Ac, encodes an active quorum sensing system that is used to regulate cell assembly and carbon metabolic flux. The methanogen 6Ac showed a cell density-dependent physiology transition, which was related t...

  13. Current and emerging strategies for organophosphate decontamination: special focus on hyperstable enzymes.

    Science.gov (United States)

    Jacquet, Pauline; Daudé, David; Bzdrenga, Janek; Masson, Patrick; Elias, Mikael; Chabrière, Eric

    2016-05-01

    Organophosphorus chemicals are highly toxic molecules mainly used as pesticides. Some of them are banned warfare nerve agents. These compounds are covalent inhibitors of acetylcholinesterase, a key enzyme in central and peripheral nervous systems. Numerous approaches, including chemical, physical, and biological decontamination, have been considered for developing decontamination methods against organophosphates (OPs). This work is an overview of both validated and emerging strategies for the protection against OP pollution with special attention to the use of decontaminating enzymes. Considerable efforts have been dedicated during the past decades to the development of efficient OP degrading biocatalysts. Among these, the promising biocatalyst SsoPox isolated from the archaeon Sulfolobus solfataricus is emphasized in the light of recently published results. This hyperthermostable enzyme appears to be particularly attractive for external decontamination purposes with regard to both its catalytic and stability properties. PMID:26832878

  14. A cell-free transcription system for the hyperthermophilic archaeon Pyrococcus furiosus.

    OpenAIRE

    Hethke, C; Geerling, A C; Hausner, W.; de Vos, W.M.; Thomm, M

    1996-01-01

    We describe here the establishment of a cell-free transcription system for the hyperthermophilic Archaeon Pyrococcus furiosus using the cloned glutamate dehydrogenase (gdh) gene as template. The in vitro system that operated up to a temperature of 85 degrees C initiated transcription 23 bp downstream of a TATA box located 45 bp upstream of the translational start codon of gdh mRNA, at the same site as in Pyrococcus cells. Mutational analyses revealed that this TATA box is essential for in vit...

  15. Properties of a Thermostable Nitrate Reductase from the Hyperthermophilic Archaeon Pyrobaculum aerophilum

    OpenAIRE

    Afshar, Sepideh; Johnson, Eric; de Vries, Simon; Schröder, Imke

    2001-01-01

    The nitrate reductase of the hyperthermophilic archaeon Pyrobaculum aerophilum was purified 137-fold from the cytoplasmic membrane. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme complex consists of three subunits with apparent molecular weights of 130,000, 52,000, and 32,000. The enzyme contained molybdenum (0.8-mol/mol complex), iron (15.4-mol/mol complex) and cytochrome b (0.49-mol/mol complex) as cofactors. The P. aerophilum nitrate reductase disti...

  16. Development of a Simvastatin Selection Marker for a Hyperthermophilic Acidophile, Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Zheng, Tao; Huang, Qihong; Zhang, Changyi; Ni, Jinfeng; She, Qunxin; Shen, Yulong

    2012-01-01

    A merodiploid strain of S. islandicus was constructed using pyrEF marker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (¿herA) cells generated from the herA merodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the......We report here a novel selectable marker for the hyperthermophilic crenarchaeon Sulfolobus islandicus. The marker cassette is composed of the sac7d promoter and the hmg gene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (P(sac7d)-hmg), which confers simvastatin resistance......SSRlacS indicated that the plasmid was properly maintained under selection. High-level expression of the His(6)-tagged HerA helicase was obtained with the cells harboring pSSRAherA. The establishment of two efficient selectable markers (pyrEF and hmg) was subsequently exploited for genetic analysis. A her...

  17. Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase

    Directory of Open Access Journals (Sweden)

    Suzanne Wolterink-van Loo

    2009-01-01

    Full Text Available Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA displays optimal activity at 95°C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 °C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40–60 °C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures.

  18. Membrane-bound amylopullulanase is essential for starch metabolism of Sulfolobus acidocaldarius DSM639.

    Science.gov (United States)

    Choi, Kyoung-Hwa; Cha, Jaeho

    2015-09-01

    Sulfolobus acidocaldarius DSM639 produced an acid-resistant membrane-bound amylopullulanase (Apu) during growth on starch as a sole carbon and energy source. The physiological role of Apu in starch metabolism was investigated by the growth and starch degradation pattern of apu disruption mutant as well as biochemical properties of recombinant Apu. The Δapu mutant lost the ability to grow in minimal medium in the presence of starch, and the amylolytic activity observed in the membrane fraction of the wild-type strain was not detected in the Δapu mutant when the cells were grown in YT medium. The purified membrane-bound Apu initially hydrolyzed starch, amylopectin, and pullulan into various sizes of maltooligosaccharides, and then produced glucose, maltose, and maltotriose in the end, indicating Apu is a typical endo-acting glycoside hydrolase family 57 (GH57) amylopullulanase. The maltose and maltotriose observed in the culture medium during the exponential and stationary phase growth indicates that Apu is the essential enzyme to initially hydrolyze the starch into small maltooligosaccharides to be transported into the cell. PMID:26104674

  19. Enrichment and Characterization of an Autotrophic Ammonia-Oxidizing Archaeon of Mesophilic Crenarchaeal Group I.1a from an Agricultural Soil

    NARCIS (Netherlands)

    Jung, M.Y.; Park, S.J.; Min, D.; Kim, J.S.; Rijpstra, W.I.C.; Sinninghe Damsté, J.S.; Kim, G.J.; Madsen, E.L.; Rhee, S.K.

    2011-01-01

    Soil nitrification is an important process for agricultural productivity and environmental pollution. Though one cultivated representative of ammonia-oxidizing Archaea from soil has been described, additional representatives warrant characterization. We describe an ammonia-oxidizing archaeon (strain

  20. The Potent In Vitro Skin Permeation of Archaeosome Made from Lipids Extracted of Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Eskandar Moghimipour

    2013-01-01

    Full Text Available Archaeosomes are a new generation of liposomes that exhibit higher stabilities under different conditions, such as high temperatures, alkaline or acidic pH, and presence of bile salts in comparison with liposomes, and can be used in biotechnology including drug, gene, and vaccine delivery. The objective of this study was to prepare archaeosomes using lipid extracted from Sulfolobus acidocaldarius and evaluate their physicochemical properties. The lipids were extracted from S. acidocaldarius and assayed by High Performance Thin-Layer Chromatography (HPTLC. Archaeosomes were prepared using film method and methylene blue was used as drug model. They were characterized for their vesicle size and Differential Scanning Calorimetry (DSC was used to investigate changes in their thermal behavior. The released amount of methylene blue was determined using a dialysis membrane and rat skin. HPTLC analysis of the extracted lipids showed that glycerol ether may be the major lipid with more than 78 percent probability. Results of particle size determination showed a mean size of 158.33 nm and the results of DSC indicated the possible interaction of methylene blue with lipids during the preparation of archaeosome. The addition of cholesterol significantly improved the encapsulation of methylene blue in the archaeosome so that the encapsulation efficiency was 61.66 ± 2.88%. The result of in vitro skin permeation showed that methylene blue could pass through skin model according to Peppas model and there was about 41.66% release after 6 h, whereas no release was observed through dialysis membrane. According to the results of the study, it is concluded that archaeosome may be successfully used as drug delivery system.

  1. Complete Genome Sequence of the Hyperthermophilic Archaeon Pyrococcus sp. Strain ST04, Isolated from a Deep-Sea Hydrothermal Sulfide Chimney on the Juan de Fuca Ridge

    OpenAIRE

    Jung, Jong-Hyun; Lee, Ju-Hoon; Holden, James F.; Seo, Dong-Ho; Shin, Hakdong; Kim, Hae-Yeong; Kim, Wooki; Ryu, Sangryeol; Park, Cheon-Seok

    2012-01-01

    Pyrococcus sp. strain ST04 is a hyperthermophilic, anaerobic, and heterotrophic archaeon isolated from a deep-sea hydrothermal sulfide chimney on the Endeavour Segment of the Juan de Fuca Ridge in the northeastern Pacific Ocean. To further understand the distinct characteristics of this archaeon at the genome level (polysaccharide utilization at high temperature and ATP generation by a Na+ gradient), the genome of strain ST04 was completely sequenced and analyzed. Here, we present the complet...

  2. Lipids of the ultra-thin square halophilic archaeon Haloquadratum walsbyi.

    Science.gov (United States)

    Lobasso, Simona; Lopalco, Patrizia; Mascolo, Giuseppe; Corcelli, Angela

    2008-12-01

    The lipid composition of the extremely halophilic archaeon Haloquadratum walsbyi was investigated by thin-layer chromatography and electrospray ionization-mass spectrometry. The analysis of neutral lipids showed the presence of vitamin MK-8, squalene, carotene, bacterioruberin and several retinal isomers. The major polar lipids were phosphatidylglycerophosphate methyl ester, phosphatidylglycerosulfate, phosphatidylglycerol and sulfated diglycosyl diether lipid. Among cardiolipins, the tetra-phytanyl or dimeric phospholipids, only traces of bisphosphatidylglycerol were detected. When the cells were exposed to hypotonic medium, no changes in the membrane lipid composition occurred. Distinguishing it from other extreme halophiles of the Halobacteriaceae family, the osmotic stress did not induce the neo-synthesis of cardiolipins in H. walsbyi. The difference may depend on the three-laminar structure of the cell wall, which differs significantly from that of other Haloarchaea. PMID:19054744

  3. Lipids of the ultra-thin square halophilic archaeon Haloquadratum walsbyi

    Directory of Open Access Journals (Sweden)

    Simona LoBasso

    2008-01-01

    Full Text Available The lipid composition of the extremely halophilic archaeon Haloquadratum walsbyi was investigated by thin-layer chromatography and electrospray ionization-mass spectrometry. The analysis of neutral lipids showed the presence of vitamin MK-8, squalene, carotene, bacterioruberin and several retinal isomers. The major polar lipids were phosphatidylglycerophosphate methyl ester, phosphatidylglycerosulfate, phosphatidylglycerol and sulfated diglycosyl diether lipid. Among cardiolipins, the tetra-phytanyl or dimeric phospholipids, only traces of bisphosphatidylglycerol were detected. When the cells were exposed to hypotonic medium, no changes in the membrane lipid composition occurred. Distinguishing it from other extreme halophiles of the Halobacteriaceae family, the osmotic stress did not induce the neo-synthesis of cardiolipins in H. walsbyi. The difference may depend on the three-laminar structure of the cell wall, which differs significantly from that of other Haloarchaea.

  4. Association of a multi-synthetase complex with translating ribosomes in the archaeon Thermococcus kodakarensis

    DEFF Research Database (Denmark)

    Raina, Medha; Elgamal, Sara; Santangelo, Thomas J; Ibba, Michael

    2012-01-01

    In archaea and eukaryotes aminoacyl-tRNA synthetases (aaRSs) associate in multi-synthetase complexes (MSCs), however the role of such MSCs in translation is unknown. MSC function was investigated in vivo in the archaeon Thermococcus kodakarensis, wherein six aaRSs were affinity co-purified together...... with several other factors involved in protein synthesis, suggesting that MSCs may interact directly with translating ribosomes. In support of this hypothesis, the aminoacyl-tRNA synthetase (aaRS) activities of the MSC were enriched in isolated T. kodakarensis polysome fractions. These data indicate...... that components of the archaeal protein synthesis machinery associate into macromolecular assemblies in vivo and provide the potential to increase translation efficiency by limiting substrate diffusion away from the ribosome, thus facilitating rapid recycling of tRNAs. STRUCTURED SUMMARY OF PROTEIN...

  5. Maltose Metabolism in the Hyperthermophilic Archaeon Thermococcus litoralis: Purification and Characterization of Key Enzymes

    Science.gov (United States)

    Xavier, Karina B.; Peist, Ralf; Kossmann, Marina; Boos, Winfried; Santos, Helena

    1999-01-01

    Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus litoralis. Maltose was degraded by the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected in T. litoralis cell extracts. Glucose derived from the action of 4-α-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an α-glucosidase, a p-nitrophenyl-α-d-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis, but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in T. litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98°C), of 66 U/mg. A Km of 0.46 mM was determined with heptaose as the substrate at 60°C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacterium Thermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E. coli (13%). The consensus binding site for pyridoxal 5′-phosphate is conserved in the T. litoralis

  6. Novel RepA-MCM proteins encoded in plasmids pTAU4, pORA1 and pTIK4 from Sulfolobus neozealandicus

    DEFF Research Database (Denmark)

    Greve, B.; Jensen, S.; Phan, H.;

    2005-01-01

    . Plasmids pORA1 and pTIK4 encode RepA proteins, only the former of which carries the novel polymerase-primase domain of other known Sulfolobus plasmids. Plasmid pTAU4 encodes a mini-chromosome maintenance protein homolog and no RepA protein; the implications for DNA replication are considered. Plasmid pORA1......Three plasmids isolated from the crenarchaeal thermoacidophile Sulfolobus neozealandicus were characterized. Plasmids pTAU4 (7,192 bp), pORA1 (9,689 bp) and pTIK4 (13,638 bp) show unusual properties that distinguish them from previously characterized cryptic plasmids of the genus Sulfolobus...... is the first Sulfolobus plasmid to be characterized that does not encode the otherwise highly conserved DNA-binding PlrA protein. Another encoded protein appears to be specific for the New Zealand plasmids. The three plasmids should provide useful model systems for functional studies of these...

  7. Energy conservation by oxidation of formate to carbon dioxide and hydrogen via a sodium ion current in a hyperthermophilic archaeon

    OpenAIRE

    Lim, Jae Kyu; Mayer, Florian; Kang, Sung Gyun; Müller, Volker

    2014-01-01

    We report here that oxidation of formate to CO2 and H2 that operates close to thermodynamic equilibrium is coupled to vectorial H+ and Na+ transport across the cytoplasmic membrane of the hyperthermophilic archaeon Thermococcus onnurineus NA1. The ion gradient established then drives ATP synthesis via a Na+-ATP synthase. The energy-converting enzyme complex involves a formate dehydrogenase, a membrane-bound hydrogenase with similarity to complex I of the aerobic electron transport chain and a...

  8. Molecular Cloning and Functional Expression of a Protein-Serine/Threonine Phosphatase from the Hyperthermophilic Archaeon Pyrodictium abyssi TAG11

    Science.gov (United States)

    Mai, Bianca; Frey, Gerhard; Swanson, Ronald V.; Mathur, Eric J.; Stetter, K. O.

    1998-01-01

    An open reading frame coding for a putative protein-serine/threonine phosphatase was identified in the hyperthermophilic archaeon Pyrodictium abyssi TAG11 and named Py-PP1. Py-PP1 was expressed in Escherichia coli, purified from inclusion bodies, and biochemically characterized. The phosphatase gene is part of an operon which may provide, for the first time, insight into a physiological role for archaeal protein phosphatases in vivo. PMID:9696747

  9. The cobY gene of the archaeon Halobacterium sp. strain NRC-1 is required for de novo cobamide synthesis.

    Science.gov (United States)

    Woodson, J D; Peck, R F; Krebs, M P; Escalante-Semerena, J C

    2003-01-01

    Genetic and nutritional analyses of mutants of the extremely halophilic archaeon Halobacterium sp. strain NRC-1 showed that open reading frame (ORF) Vng1581C encodes a protein with nucleoside triphosphate:adenosylcobinamide-phosphate nucleotidyltransferase enzyme activity. This activity was previously associated with the cobY gene of the methanogenic archaeon Methanobacterium thermoautotrophicum strain DeltaH, but no evidence was obtained to demonstrate the direct involvement of this protein in cobamide biosynthesis in archaea. Computer analysis of the Halobacterium sp. strain NRC-1 ORF Vng1581C gene and the cobY gene of M. thermoautotrophicum strain DeltaH showed the primary amino acid sequence of the proteins encoded by these two genes to be 35% identical and 48% similar. A strain of Halobacterium sp. strain NRC-1 carrying a null allele of the cobY gene was auxotrophic for cobinamide-GDP, a known intermediate of the late steps of cobamide biosynthesis. The auxotrophic requirement for cobinamide-GDP was corrected when a wild-type allele of cobY was introduced into the mutant strain, demonstrating that the lack of cobY function was solely responsible for the observed block in cobamide biosynthesis in this archaeon. The data also show that Halobacterium sp. strain NRC-1 possesses a high-affinity transport system for corrinoids and that this archaeon can synthesize cobamides de novo under aerobic growth conditions. To the best of our knowledge this is the first genetic and nutritional analysis of cobalamin biosynthetic mutants in archaea. PMID:12486068

  10. CrRNA-Protospacer Recognition during CRISPR- Directed DNA Interference Sulfolobus islandicus REY 15A and Structural Studies of CRISPR Binding Proteins (CBP) of Crenarchaeon Sulfolobus

    DEFF Research Database (Denmark)

    Mousaei, Marzieh

    The CRISPR-Cas (clustered regularly interspaced short palindromic repeats and associated proteins) is one of the important known immune mechanisms in archaea and bacteria. This adaptive immune system degrades invading genetic elements and protects the cell. Amongst 3 main types I, II and III of...... CRISPR system, two types (I and III) are found in archaea. However, in Sulfolobus species, subtypes IA, I-D, and III-B, III-D and rarely III-A are found. The model organism used for interference and structural studies is S. islandicus REY15A which carries subtypes I-A and III-B (α and β). Besides CRISPR...... ribonucleoprotein complex which is involved directly in defense, there are some less- known parts of the system including CPBs (CRISPR repeat-binding proteins) which are suggested to play a role in transcription. In the first part of my thesis, I provide a brief introduction to archaea and viruses that infect...

  11. Modulation of CRISPR locus transcription by the repeat-binding protein Cbp1 in Sulfolobus

    DEFF Research Database (Denmark)

    Deng, Ling; Kenchappa, Chandra Shekar; Peng, Xu;

    2012-01-01

    CRISPR loci are essential components of the adaptive immune system of archaea and bacteria. They consist of long arrays of repeats separated by DNA spacers encoding guide RNAs (crRNA), which target foreign genetic elements. Cbp1 (CRISPR DNA repeat binding protein) binds specifically to the multiple...... direct repeats of CRISPR loci of members of the acidothermophilic, crenarchaeal order Sulfolobales. cbp1 gene deletion from Sulfolobus islandicus REY15A produced a strong reduction in pre-crRNA yields from CRISPR loci but did not inhibit the foreign DNA targeting capacity of the CRISPR/Cas system....... Conversely, overexpression of Cbp1 in S. islandicus generated an increase in pre-crRNA yields while the level of reverse strand transcripts from CRISPR loci remained unchanged. It is proposed that Cbp1 modulates production of longer pre-crRNA transcripts from CRISPR loci. A possible mechanism is that it...

  12. Uracil phosphoribosyltransferase from the extreme thermoacidophilic archaebacterium Sulfolobus shibatae is an allosteric enzyme, activated by GTP and inhibited by CTP

    DEFF Research Database (Denmark)

    Linde, Lise; Jensen, Kaj Frank

    1996-01-01

    Uracil phosphoribosyltransferase, which catalyses the formation of UMP and pyrophosphate from uracil and 5-phosphoribosyl a-1-pyrophosphate (PRPP), was partly purified from the extreme thermophilic archaebacterium Sulfolobus shibatae. The enzyme required divalent metal ions for activity and it...... showed the highest activity at pH 6.4. The specific activity of the enzyme was 50-times higher at 95°C than at 37°C, but the functional half-life was short at 95°C. The activity of uracil phosphoribosyltransferase was strongly activated by GTP, which increased Vmax of the reaction by approximately 20......-fold without much effect on Km for the substrates. The concentration of GTP required for half-maximal activation was about 80 µM. CTP was a strong inhibitor and acted by raising the concentration of GTP needed for half-maximal activation of the enzyme. We conclude that uracil phosphoribosyltransferase...

  13. Microbial community profiling of the Chinoike Jigoku ("Blood Pond Hell") hot spring in Beppu, Japan: isolation and characterization of Fe(III)-reducing Sulfolobus sp. strain GA1.

    Science.gov (United States)

    Masaki, Yusei; Tsutsumi, Katsutoshi; Hirano, Shin-Ichi; Okibe, Naoko

    2016-09-01

    Chinoike Jigoku ("Blood Pond Hell") is located in the hot spring town of Beppu on the southern island of Kyushu in Japan, and is the site of a red-colored acidic geothermal pond. This study aimed to investigate the microbial population composition in this extremely acidic environment and to isolate/characterize acidophilic microorganism with metal-reducing ability. Initially, PCR (using bacteria- and archaea-specific primers) of environmental DNA samples detected the presence of bacteria, but not archaea. This was followed by random sequencing analysis, confirming the presence of wide bacterial diversity at the site (123 clones derived from 18 bacterial and 1 archaeal genera), including those closely related to known autotrophic and heterotrophic acidophiles (Acidithiobacillus sp., Sulfobacillus sp., Alicyclobacillus sp.). Nevertheless, successive culture enrichment with Fe(III) under micro-aerobic conditions led to isolation of an unknown archaeal organism, Sulfolobus sp. GA1 (with 99.7% 16S rRNA gene sequence identity with Sulfolobus shibatae). Unlike many other known Sulfolobus spp., strain GA1 was shown to lack sulfur oxidation ability. Strain GA1 possessed only minor Fe(II) oxidation ability, but readily reduced Fe(III) during heterotrophic growth under micro-aerobic conditions. Strain GA1 was capable of reducing highly toxic Cr(VI) to less toxic/soluble Cr(III), demonstrating its potential utility in bioremediation of toxic metal species. PMID:27208660

  14. Identification and characterization of arsenite methyltransferase from an archaeon, Methanosarcina acetivorans C2A.

    Science.gov (United States)

    Wang, Pei-Pei; Sun, Guo-Xin; Zhu, Yong-Guan

    2014-11-01

    Arsenic is a ubiquitous toxic contaminant in the environment. The methylation of arsenic can affect its toxicity and is primarily mediated by biological processes. Few studies have focused on the mechanism of arsenic methylation in archaea although archaea are widespread in the environment. Here, an arsenite [As(III)] methyltransferase (ArsM) was identified and characterized from an archaeon Methanosarcina acetivorans C2A. Heterologous expression of MaarsM was shown to confer As(III) resistance to an arsenic-sensitive strain of E. coli through arsenic methylation and subsequent volatilization. Purified MaArsM protein was further identified the function in catalyzing the formation of various methylated products from As(III) in vitro. Methylation of As(III) by MaArsM is highly dependent on the characteristics of the thiol cofactors used, with some of them (coenzyme M, homocysteine, and dithiothreitol) more efficient than GSH. Site-directed mutagenesis demonstrated that three conserved cysteine (Cys) residues (Cys62, Cys150, and Cys200) in MaArsM were necessary for As(III) methylation, of which only Cys150 and Cys200 were required for the methylation of monomethylarsenic. These results present a molecular pathway for arsenic methylation in archaea and provide some insight into the role of archaea in As biogeochemistry. PMID:25295694

  15. Production and properties of an exopolysaccharide synthesized by the extreme halophilic archaeon Haloterrigena turkmenica.

    Science.gov (United States)

    Squillaci, Giuseppe; Finamore, Rosario; Diana, Paola; Restaino, Odile Francesca; Schiraldi, Chiara; Arbucci, Salvatore; Ionata, Elena; La Cara, Francesco; Morana, Alessandra

    2016-01-01

    We have isolated a novel exopolysaccharide (EPS) produced by the extreme halophilic archaeon Haloterrigena turkmenica. Some features, remarkable from an industrial point of view, such as emulsifying and antioxidant properties, were investigated. H. turkmenica excreted 20.68 mg of EPS per 100 ml of culture medium when grown in usual medium supplemented with glucose. The microorganism excreted the biopolymer mainly in the middle exponential growth phase and reached the maximal production in the stationary phase. Analyses by anion exchange chromatography and SEC-TDA Viscotek indicated that the EPS was composed of two main fractions of 801.7 and 206.0 kDa. It was a sulfated heteropolysaccharide containing glucose, galactose, glucosamine, galactosamine, and glucuronic acid. Studies performed utilizing the mixture of EPS anionic fractions showed that the biopolymer had emulsifying activity towards vegetable oils comparable or superior to that exhibited by the controls, moderate antioxidant power when tested with 2,2'-diphenyl-1-picrylhydrazyl (DPPH(·)), and moisture-retention ability higher than hyaluronic acid (HA). The EPS from H. turkmenica is the first exopolysaccharide produced by an archaea to be characterized in terms of properties that can have potential biotechnological applications. PMID:26403921

  16. ATP synthesis at 100 degrees C by an ATPase purified from the hyperthermophilic archaeon Pyrodictium abyssi.

    Science.gov (United States)

    Dirmeier, R; Hauska, G; Stetter, K O

    2000-02-01

    The chemolithoautotrophic archaeon Pyrodictium abyssi isolate TAG 11 lives close to 100 degrees 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 AF(1) headpiece, and the 7 kDa proteolipid of the AF(0) 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 micromol per min and mg protein) as well as ATP formation (up to 0.4 micromol per min and mg membrane protein) were highest at 100 degrees 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 (33)P(i)-labeled ADP and of another compound not identified so far. PMID:10664465

  17. Methanosarcina subterranea sp. nov., a methanogenic archaeon isolated from a deep subsurface diatomaceous shale formation.

    Science.gov (United States)

    Shimizu, Satoru; Ueno, Akio; Naganuma, Takeshi; Kaneko, Katsuhiko

    2015-04-01

    A methanogenic archaeon, strain HC-2(T), was isolated from a deep diatomaceous shale formation. The strain grew on methanol, monomethylamine, dimethylamine, trimethylamine and dimethylsulphide, but not on acetate, H2/CO2, formate, 2-propanol, 2-butanol or cyclopentanol. Cells were Gram-stain-negative, non-motile, and coccus-like, 0.9-1.4 µm in diameter, and occurred singly, in pairs, or as aggregates. The strain grew at 10-40 °C (optimum 35 °C), pH 5.9-7.4 (optimum pH 6.6-6.8) and in 0-0.6 M NaCl (optimum 0.1-0.2 M). The genomic DNA G+C content was 41.5 mol% and the 16S rRNA gene sequence was closely related to those of Methanosarcina lacustris DSM 13486(T) (99.1%) and Methanosarcina siciliae DSM 3028(T) (98.3%). Values for DNA-DNA hybridization with these strains were less than 30%. The phenotypic and phylogenetic features of HC-2(T) indicate that it represents a novel species of the genus Methanosarcina , for which the name Methanosarcina subterranea sp. nov. is proposed. The type strain is HC-2(T) ( = DSM 22503(T) = JCM 15540(T) = NBRC 102578(T)). PMID:25604340

  18. Characterization of the proteasome from the extremely halophilic archaeon Haloarcula marismortui

    Directory of Open Access Journals (Sweden)

    B. Franzetti

    2002-01-01

    Full Text Available A 20S proteasome, comprising two subunits α and β, was purified from the extreme halophilic archaeon Haloarcula marismortui, which grows only in saturated salt conditions. The three-dimensional reconstruction of the H. marismortui proteasome (Hm proteasome, obtained from negatively stained electron micrographs, is virtually identical to the structure of a thermophilic proteasome filtered to the same resolution. The stability of the Hm proteasome was found to be less salt-dependent than that of other halophilic enzymes previously described. The proteolytic activity of the Hm proteasome was investigated using the malate dehydrogenase from H. marismortui (HmMalDH as a model substrate. The HmMalDH denatures when the salt concentration is decreased below 2 M. Under these conditions, the proteasome efficiently cleaves HmMalDH during its denaturation process, but the fully denatured HmMalDH is poorly degraded. These in vitro experiments show that, at low salt concentrations, the 20S proteasome from halophilic archaea eliminates a misfolded protein.

  19. Proteomic Insights into Sulfur Metabolism in the Hydrogen-Producing Hyperthermophilic Archaeon Thermococcus onnurineus NA1

    Directory of Open Access Journals (Sweden)

    Yoon-Jung Moon

    2015-04-01

    Full Text Available The hyperthermophilic archaeon Thermococcus onnurineus NA1 has been shown to produce H2 when using CO, formate, or starch as a growth substrate. This strain can also utilize elemental sulfur as a terminal electron acceptor for heterotrophic growth. To gain insight into sulfur metabolism, the proteome of T. onnurineus NA1 cells grown under sulfur culture conditions was quantified and compared with those grown under H2-evolving substrate culture conditions. Using label-free nano-UPLC-MSE-based comparative proteomic analysis, approximately 38.4% of the total identified proteome (589 proteins was found to be significantly up-regulated (≥1.5-fold under sulfur culture conditions. Many of these proteins were functionally associated with carbon fixation, Fe–S cluster biogenesis, ATP synthesis, sulfur reduction, protein glycosylation, protein translocation, and formate oxidation. Based on the abundances of the identified proteins in this and other genomic studies, the pathways associated with reductive sulfur metabolism, H2-metabolism, and oxidative stress defense were proposed. The results also revealed markedly lower expression levels of enzymes involved in the sulfur assimilation pathway, as well as cysteine desulfurase, under sulfur culture condition. The present results provide the first global atlas of proteome changes triggered by sulfur, and may facilitate an understanding of how hyperthermophilic archaea adapt to sulfur-rich, extreme environments.

  20. Pyrobaculum calidifontis sp. nov., a novel hyperthermophilic archaeon that grows in atmospheric air

    Directory of Open Access Journals (Sweden)

    Taku Amo

    2002-01-01

    Full Text Available A novel, facultatively aerobic, heterotrophic hyperthermophilic archaeon was isolated from a terrestrial hot spring in the Philippines. Cells of the new isolate, strain VA1, were rod-shaped with a length of 1.5 to 10 μm and a width of 0.5 to 1.0 μm. Isolate VA1 grew optimally at 90 to 95 °C and pH 7.0 under atmospheric air. Oxygen served as a final electron acceptor under aerobic growth conditions, and vigorous shaking of the medium significantly enhanced growth. Elemental sulfur inhibited cell growth under aerobic growth conditions, whereas thiosulfate stimulated cell growth. Under anaerobic growth conditions, nitrate served as a final electron acceptor, but nitrite or sulfur-containing compounds such as elemental sulfur, thiosulfate, sulfate and sulfite could not act as final electron acceptors. The G+C content of the genomic DNA was 51 mol%. Phylogenetic analysis based on 16S rRNA sequences indicated that strain VA1 exhibited close relationships to species of the genus Pyrobaculum. A DNA–DNA hybridization study revealed a low level of similarity (≤ 18% between strain VA1 and previously described members of the genus Pyrobaculum. Physiological characteristics also indicated that strain VA1 was distinct from these Pyrobaculum species. Our results indicate that isolate VA1 represents a novel species, named Pyrobaculum calidifontis.

  1. Cloning and Characterization of an Alpha-amylase Gene from the Hyperthermophilic Archaeon Thermococcus Thioreducens

    Science.gov (United States)

    Bernhardsdotter, Eva C. M. J.; Pusey, Marc L.; Ng, Joseph D.; Garriott, Owen K.

    2004-01-01

    The gene encoding an extracellular a-amylase, TTA, from the hyperthermophilic archaeon Thermococcus thioreducens was cloned and expressed in Escherichia coli. Primary structural analysis revealed high similarity with other a-amylases from the Thermococcus and Pyrococcus genera, as well as the four highly conserved regions typical for a-amylases. The 1374 bp gene encodes a protein of 457 amino acids, of which 435 constitute the mature protein preceded by a 22 amino acid signal peptide. The molecular weight of the purified recombinant enzyme was estimated to be 43 kDa by denaturing gel electrophoresis. Maximal enzymatic activity of recombinant TTA was observed at 90 C and pH 5.5 in the absence of exogenous Ca(2+), and the enzyme was considerably stable even after incubation at 90 C for 2 hours. The thermostability at 90 and 102 C was enhanced in the presence of 5 mM Ca(2+). The extraordinarily high specific activity (about 7.4 x 10(exp 3) U/mg protein at 90 C, pH 5.5 with soluble starch as substrate) together with its low pH optimum makes this enzyme an interesting candidate for starch processing applications.

  2. Cysteine desulphurase plays an important role in environmental adaptation of the hyperthermophilic archaeon Thermococcus kodakarensis.

    Science.gov (United States)

    Hidese, Ryota; Inoue, Takahiro; Imanaka, Tadayuki; Fujiwara, Shinsuke

    2014-07-01

    The sulphur atoms of sulphur-containing cofactors that are essential for numerous cellular functions in living organisms originate from L-cysteine via cysteine desulphurase (CSD) activity. However, many (hyper)thermophilic archaea, which thrive in solfataric fields and are positioned near the root of the evolutionary tree of life, lack CSD orthologues. The existence of CSD orthologues in a subset of (hyper)thermophilic archaea is of interest with respect to the evolution of sulphur-trafficking systems for the cofactors. This study demonstrates that the disruption of the csd gene of Thermococcus kodakarensis, a facultative elemental sulphur (S(0))-reducing hyperthermophilic archaeon, encoding Tk-CSD, conferred a growth defect evident only in the absence of S(0), and that growth can be restored by the addition of S(0), but not sulphide. We show that the csd gene is not required for biosynthesis of thiamine pyrophosphate or molybdopterin, irrespective of the presence or absence of S(0), but is necessary for iron-sulphur cluster biosynthesis in the absence of S(0). Recombinant form of Tk-CSD expressed in Escherichia coli was obtained and it was found to catalyse the desulphuration of L-cysteine. The obtained data suggest that hyperthermophiles might benefit from a capacity for CSD-dependent iron-sulphur cluster biogenesis, which allows them to thrive outside solfataric environments. PMID:24893566

  3. Functional organization of a single nif cluster in the mesophilic archaeon Methanosarcina mazei strain Gö1

    Directory of Open Access Journals (Sweden)

    Claudia Ehlers

    2002-01-01

    Full Text Available The mesophilic methanogenic archaeon Methanosarcina mazei strain Gö1 is able to utilize molecular nitrogen (N2 as its sole nitrogen source. We have identified and characterized a single nitrogen fixation (nif gene cluster in M. mazei Gö1 with an approximate length of 9 kbp. Sequence analysis revealed seven genes with sequence similarities to nifH, nifI1, nifI2, nifD, nifK, nifE and nifN, similar to other diazotrophic methanogens and certain bacteria such as Clostridium acetobutylicum, with the two glnB-like genes (nifI1 and nifI2 located between nifH and nifD. Phylogenetic analysis of deduced amino acid sequences for the nitrogenase structural genes of M. mazei Gö1 showed that they are most closely related to Methanosarcina barkeri nif2 genes, and also closely resemble those for the corresponding nif products of the gram-positive bacterium C. acetobutylicum. Northern blot analysis and reverse transcription PCR analysis demonstrated that the M. mazei nif genes constitute an operon transcribed only under nitrogen starvation as a single 8 kb transcript. Sequence analysis revealed a palindromic sequence at the transcriptional start site in front of the M. mazei nifH gene, which may have a function in transcriptional regulation of the nif operon.

  4. Different roles of two transcription factor B proteins in the hyperthermophilic archaeon Thermococcus kodakarensis.

    Science.gov (United States)

    Hidese, Ryota; Nishikawa, Ryo; Gao, Le; Katano, Masahiro; Imai, Tomohiro; Kato, Satoru; Kanai, Tamotsu; Atomi, Haruyuki; Imanaka, Tadayuki; Fujiwara, Shinsuke

    2014-05-01

    Two genes, TK1280 and TK2287, encode orthologous transcription factor B proteins (TFB1 and TFB2, respectively) in the hyperthermophilic archaeon Thermococcus kodakarensis. The functional difference between their TFBs remains unknown. While TFB1 and TFB2 displayed equivalent thermostability, mRNA levels of tfb1 at 93 °C were eightfold higher than those at 60 or 85 °C, and were 4- to 10-fold greater than those of tfb2 at all temperatures. This suggests that TFB1 is the abundant TFB in T. kodakarensis and is heat-inducible. By contrast, the mRNA level of tfb2 increased at 93 °C, but the levels were less than twofold of those at 60 or 85 °C. No significant differences in growth were observed among the DTF1 (∆tfb1, ∆pyrF), DTF2 (∆tfb2 ∆pyrF), and parental host strain KU216 (∆pyrF) at 60 °C. However, DTF2 showed a decrease in cell yield at 85 °C, and both DTF1 and DTF2 showed growth defects at 93 °C. Comparative transcriptome analysis between KU216 and DTF1 or DTF2 indicated that TFB1 apparently controls the expression of genes essential for motility/adhesion, whereas TFB2 regulates genes involved in mevalonate/lipid biosynthesis. In DTF1, the ratio of cells with flagella decreased at 85 and 93 °C, and reporter studies indicated that flaB1 transcription is dependent on TFB1 at 85 °C but not at 60 °C. PMID:24627188

  5. Apo and ligand-bound structures of ModA from the archaeon Methanosarcina acetivorans.

    Science.gov (United States)

    Chan, Sum; Giuroiu, Iulia; Chernishof, Irina; Sawaya, Michael R; Chiang, Janet; Gunsalus, Robert P; Arbing, Mark A; Perry, L Jeanne

    2010-03-01

    The trace-element oxyanion molybdate, which is required for the growth of many bacterial and archaeal species, is transported into the cell by an ATP-binding cassette (ABC) transporter superfamily uptake system called ModABC. ModABC consists of the ModA periplasmic solute-binding protein, the integral membrane-transport protein ModB and the ATP-binding and hydrolysis cassette protein ModC. In this study, X-ray crystal structures of ModA from the archaeon Methanosarcina acetivorans (MaModA) have been determined in the apoprotein conformation at 1.95 and 1.69 A resolution and in the molybdate-bound conformation at 2.25 and 2.45 A resolution. The overall domain structure of MaModA is similar to other ModA proteins in that it has a bilobal structure in which two mixed alpha/beta domains are linked by a hinge region. The apo MaModA is the first unliganded archaeal ModA structure to be determined: it exhibits a deep cleft between the two domains and confirms that upon binding ligand one domain is rotated towards the other by a hinge-bending motion, which is consistent with the 'Venus flytrap' model seen for bacterial-type periplasmic binding proteins. In contrast to the bacterial ModA structures, which have tetrahedral coordination of their metal substrates, molybdate-bound MaModA employs octahedral coordination of its substrate like other archaeal ModA proteins. PMID:20208152

  6. Characterization of an archaeal malic enzyme from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

    Directory of Open Access Journals (Sweden)

    Wakao Fukuda

    2005-01-01

    Full Text Available Although the interconversion between C4 and C3 compounds has an important role in overall metabolism, limited information is available on the properties and regulation of enzymes acting on these metabolites in hyperthermophilic archaea. Malic enzyme is one of the enzymes involved in this interconversion, catalyzing the oxidative decarboxylation of malate to pyruvate as well as the reductive carboxylation coupled with NAD(PH. This study focused on the enzymatic properties and expression profile of an uncharacterized homolog of malic enzyme identified in the genome of a heterotrophic, hyperthermophilic archaeon T hermococcus kodakaraensis KOD1 (Tk-Mae. The amino acid sequence of Tk-Mae was 52–58% identical to those of malic enzymes from bacteria, whereas the similarities to the eukaryotic homologs were lower. Several catalytically important regions and residues were conserved in the primary structure of Tk-Mae. The recombinant protein, which formed a homodimer, exhibited thermostable malic enzyme activity with strict divalent cation dependency. The enzyme preferred NADP+ rather than NAD+, but did not catalyze the decarboxylation of oxaloacetate, unlike the usual NADP-dependent malic enzymes. The apparent Michaelis constant (Km of Tk-Mae for malate (16.9 mM was much larger than those of known enzymes, leading to no strong preference for the reaction direction. Transcription of the gene encoding Tk-Mae and intracellular malic enzyme activity in T. kodakaraensis were constitutively weak, regardless of the growth substrates. Possible roles of Tk-Mae are discussed based on these results and the metabolic pathways of T. kodakaraensis deduced from the genome sequence.

  7. Archaeal signal transduction: impact of protein phosphatase deletions on cell size, motility, and energy metabolism in Sulfolobus acidocaldarius.

    Science.gov (United States)

    Reimann, Julia; Esser, Dominik; Orell, Alvaro; Amman, Fabian; Pham, Trong Khoa; Noirel, Josselin; Lindås, Ann-Christin; Bernander, Rolf; Wright, Phillip C; Siebers, Bettina; Albers, Sonja-Verena

    2013-12-01

    In this study, the in vitro and in vivo functions of the only two identified protein phosphatases, Saci-PTP and Saci-PP2A, in the crenarchaeal model organism Sulfolobus acidocaldarius were investigated. Biochemical characterization revealed that Saci-PTP is a dual-specific phosphatase (against pSer/pThr and pTyr), whereas Saci-PP2A exhibited specific pSer/pThr activity and inhibition by okadaic acid. Deletion of saci_pp2a resulted in pronounced alterations in growth, cell shape and cell size, which could be partially complemented. Transcriptome analysis of the three strains (Δsaci_ptp, Δsaci_pp2a and the MW001 parental strain) revealed 155 genes that were differentially expressed in the deletion mutants, and showed significant changes in expression of genes encoding the archaella (archaeal motility structure), components of the respiratory chain and transcriptional regulators. Phosphoproteome studies revealed 801 unique phosphoproteins in total, with an increase in identified phosphopeptides in the deletion mutants. Proteins from most functional categories were affected by phosphorylation, including components of the motility system, the respiratory chain, and regulatory proteins. In the saci_pp2a deletion mutant the up-regulation at the transcript level, as well as the observed phosphorylation pattern, resembled starvation stress responses. Hypermotility was also observed in the saci_pp2a deletion mutant. The results highlight the importance of protein phosphorylation in regulating essential cellular processes in the crenarchaeon S. acidocaldarius. PMID:24078887

  8. Knockout and functional analysis of two DExD/H-box family helicase genes in Sulfolobus islandicus REY15A.

    Science.gov (United States)

    Song, Xueguo; Huang, Qihong; Ni, Jinfeng; Yu, Yang; Shen, Yulong

    2016-07-01

    DExD/H-box helicases represent the largest family of helicases. They belong to superfamily 2 helicases and participate in nucleotide metabolism, ribosome biogenesis, and nucleocytoplasmic transport. The biochemical properties and structures of some DExD/H-box helicases in the archaea have been documented, but many of them have not been characterized; and reports on in vivo functional analyses are limited. In this study, we attempted gene knockout of 8 putative DExD/H-box helicases in Sulfolobus islandicus REY15A and obtained two deletion mutants, SiRe_0681 and SiRe_1605. We determined that ΔSiRe_0681 grew faster than wild type cells in the presence of methyl methanesulfonate (MMS). Flow cytometry analysis showed that this strain had fewer G1/S phase cells than the wild type, and the genes coding for cell division proteins were up-regulated. The stain ΔSiRe_1605 was more sensitive to MMS than the wild type cell, and many nucleotide metabolism and DNA repair enzymes were found to be down-regulated. Intriguingly, deletion of either gene led to silencing simultaneously of over 80 genes located at a specific region. This study provides a novel insight into the in vivo functions of predicted DExD/H-box family helicases in the archaea. PMID:27290726

  9. Recombinant production, crystallization and preliminary X-ray analysis of PCNA from the psychrophilic archaeon Methanococcoides burtonii DSM 6242

    International Nuclear Information System (INIS)

    The proliferating cell nuclear antigen (PCNA) from the eurypsychrophilic archaeon M. burtonii DSM 6242 has been cloned, overproduced, purified and crystallized. Crystals were deemed to be suitable for X-ray analysis and structure determination to 2.40 Å resolution. Proliferating cell nuclear antigen (PCNA) is a DNA-clamping protein that is responsible for increasing the processivity of the replicative polymerases during DNA replication and repair. The PCNA from the eurypsychrophilic archaeon Methanococcoides burtonii DSM 6242 (MbPCNA) has been targeted for protein structural studies. A recombinant expression system has been created that overproduces MbPCNA with an N-terminal hexahistidine affinity tag in Escherichia coli. As a result, recombinant MbPCNA with a molecular mass of 28.3 kDa has been purified to at least 95% homogeneity and crystallized by vapor-diffusion equilibration. Preliminary X-ray analysis revealed a trigonal hexagonal R3 space group, with unit-cell parameters a = b = 102.5, c = 97.5 Å. A single MbPCNA crystal was subjected to complete diffraction data-set collection using synchrotron radiation and reflections were measured to 2.40 Å resolution. The diffraction data were of suitable quality for indexing and scaling and an unrefined molecular-replacement solution has been obtained

  10. Expression, purification and preliminary X-ray analysis of proliferating cell nuclear antigen from the archaeon Thermococcus thioreducens

    International Nuclear Information System (INIS)

    The proliferating cell nuclear antigen (PCNA) from a novel hyperthermophilic archaeon Thermococcus thioreducens has been crystallized, and diffraction data have been collected to 1.86 Å. Proliferating cell nuclear antigen (PCNA) is a DNA sliding clamp which confers processivity on replicative DNA polymerases. PCNA also acts as a sliding platform that enables the association of many DNA-processing proteins with DNA in a non-sequence-specific manner. In this investigation, the PCNA from the hyperthermophilic archaeon Thermococcus thioreducens (TtPCNA) was cloned, overexpressed in Escherichia coli and purified to greater than 90% homogeneity. TtPCNA crystals were obtained by sitting-drop vapor-diffusion methods and the best ordered crystal diffracted to 1.86 Å resolution using synchrotron radiation. The crystals belonged to the hexagonal space group P63, with unit-cell parameters a = b = 89.0, c = 62.8 Å. Crystals of TtPCNA proved to be amenable to complete X-ray analysis and future structure determination

  11. SAV 1, a temperate u.v.-inducible DNA virus-like particle from the archaebacterium Sulfolobus acidocaldarius isolate B12

    OpenAIRE

    Martin, Andrea; Yeats, Siobhán; Janekovic, Davorin; Reiter, Wolf-Dieter; Aicher, Wilhelm; Zillig, Wolfram

    1984-01-01

    Sulfolobus acidocaldarius, strain B12, which harbours a double-stranded DNA species both as a plasmid and in a linear form, which is integrated at a specific site of the chromosome, produces virus-like particles upon u.v. irradiation. These particles contain the same circular DNA and a number of coat proteins and are probably surrounded by a lipid membrane. They are lemon shaped, 100 x 60 nm in size and carry tail structures at one pole. The host cell recovers and remains lysogenic after viru...

  12. Genome-wide transcriptional response of the archaeon Thermococcus gammatolerans to cadmium.

    Directory of Open Access Journals (Sweden)

    Arnaud Lagorce

    Full Text Available Thermococcus gammatolerans, the most radioresistant archaeon known to date, is an anaerobic and hyperthermophilic sulfur-reducing organism living in deep-sea hydrothermal vents. Knowledge of mechanisms underlying archaeal metal tolerance in such metal-rich ecosystem is still poorly documented. We showed that T. gammatolerans exhibits high resistance to cadmium (Cd, cobalt (Co and zinc (Zn, a weaker tolerance to nickel (Ni, copper (Cu and arsenate (AsO(4 and that cells exposed to 1 mM Cd exhibit a cellular Cd concentration of 67 µM. A time-dependent transcriptomic analysis using microarrays was performed at a non-toxic (100 µM and a toxic (1 mM Cd dose. The reliability of microarray data was strengthened by real time RT-PCR validations. Altogether, 114 Cd responsive genes were revealed and a substantial subset of genes is related to metal homeostasis, drug detoxification, re-oxidization of cofactors and ATP production. This first genome-wide expression profiling study of archaeal cells challenged with Cd showed that T. gammatolerans withstands induced stress through pathways observed in both prokaryotes and eukaryotes but also through new and original strategies. T. gammatolerans cells challenged with 1 mM Cd basically promote: 1 the induction of several transporter/permease encoding genes, probably to detoxify the cell; 2 the upregulation of Fe transporters encoding genes to likely compensate Cd damages in iron-containing proteins; 3 the induction of membrane-bound hydrogenase (Mbh and membrane-bound hydrogenlyase (Mhy2 subunits encoding genes involved in recycling reduced cofactors and/or in proton translocation for energy production. By contrast to other organisms, redox homeostasis genes appear constitutively expressed and only a few genes encoding DNA repair proteins are regulated. We compared the expression of 27 Cd responsive genes in other stress conditions (Zn, Ni, heat shock, γ-rays, and showed that the Cd transcriptional pattern is

  13. Anaerobic desulfurization of ground rubber with the thermophilic archaeon Pyrococcus furiosus--a new method for rubber recycling.

    Science.gov (United States)

    Bredberg, K; Persson, J; Christiansson, M; Stenberg, B; Holst, O

    2001-01-01

    The anaerobic sulfur-reducing archaeon Pyrococcus furiosus was investigated regarding its capacity to desulfurize rubber material. The microorganism's sensitivity towards common rubber elastomers and additives was tested and several were shown to be toxic to P. furiosus. The microorganism was shown to utilize sulfur in vulcanized natural rubber and an increase in cell density was obtained when cultivated in the presence of spent tire rubber. Ethanol-leached cryo-ground tire rubber treated with P. furiosus for 10 days was vulcanized together with virgin rubber material (15% w/w) and the mechanical properties of the resulting material were determined. The increase in the stress at break value and the decrease in swell ratio and stress relaxation rate obtained for material containing microbially treated rubber (compared to untreated material) show the positive effects of microbial desulfurization on rubber. PMID:11234957

  14. Adaptive engineering of a hyperthermophilic archaeon on CO and discovering the underlying mechanism by multi-omics analysis

    Science.gov (United States)

    Lee, Seong Hyuk; Kim, Min-Sik; Lee, Jae-Hak; Kim, Tae Wan; Bae, Seung Seob; Lee, Sung-Mok; Jung, Hae Chang; Yang, Tae-Jun; Choi, Ae Ran; Cho, Yong-Jun; Lee, Jung-Hyun; Kwon, Kae Kyoung; Lee, Hyun Sook; Kang, Sung Gyun

    2016-01-01

    The hyperthermophilic archaeon Thermococcus onnurineus NA1 can grow and produce H2 on carbon monoxide (CO) and its H2 production rates have been improved through metabolic engineering. In this study, we applied adaptive evolution to enhance H2 productivity. After over 150 serial transfers onto CO medium, cell density, CO consumption rate and H2 production rate increased. The underlying mechanism for those physiological changes could be explained by using multi-omics approaches including genomic, transcriptomic and epigenomic analyses. A putative transcriptional regulator was newly identified to regulate the expression levels of genes related to CO oxidation. Transcriptome analysis revealed significant changes in the transcript levels of genes belonging to the categories of transcription, translation and energy metabolism. Our study presents the first genome-scale methylation pattern of hyperthermophilic archaea. Adaptive evolution led to highly enhanced H2 productivity at high CO flow rates using synthesis gas produced from coal gasification. PMID:26975345

  15. The Genome Sequence of the psychrophilic archaeon, Methanococcoides burtonii: the Role of Genome Evolution in Cold-adaptation

    Energy Technology Data Exchange (ETDEWEB)

    Allen, Michelle A.; Lauro, Federico M.; Williams, Timothy J.; Burg, Dominic; Siddiqui, Khawar S.; De Francisci, David; Chong, Kevin W.Y.; Pilak, Oliver; Chew, Hwee H.; De Maere, Matthew Z.; Ting, Lily; Katrib, Marilyn; Ng, Charmaine; Sowers, Kevin R.; Galperin, Michael Y.; Anderson, Iain J.; Ivanova, Natalia; Dalin, Eileen; Martinez, Michelle; Lapidus, Alla; Hauser, Loren; Land, Miriam; Thomas, Torsten; Cavicchioli, Ricardo

    2009-04-01

    Psychrophilic archaea are abundant and perform critical roles throughout the Earth's expansive cold biosphere. Here we report the first complete genome sequence for a psychrophilic methanogenic archaeon, Methanococcoides burtonii. The genome sequence was manually annotated including the use of a five tiered Evidence Rating system that ranked annotations from Evidence Rating (ER) 1 (gene product experimentally characterized from the parent organism) to ER5 (hypothetical gene product) to provide a rapid means of assessing the certainty of gene function predictions. The genome is characterized by a higher level of aberrant sequence composition (51%) than any other archaeon. In comparison to hyper/thermophilic archaea which are subject to selection of synonymous codon usage, M. burtonii has evolved cold adaptation through a genomic capacity to accommodate highly skewed amino acid content, while retaining codon usage in common with its mesophilic Methanosarcina cousins. Polysaccharide biosynthesis genes comprise at least 3.3% of protein coding genes in the genome, and Cell wall/membrane/envelope biogenesis COG genes are over-represented. Likewise, signal transduction (COG category T) genes are over-represented and M. burtonii has a high 'IQ' (a measure of adaptive potential) compared to many methanogens. Numerous genes in these two over-represented COG categories appear to have been acquired from {var_epsilon}- and {delta}-proteobacteria, as do specific genes involved in central metabolism such as a novel B form of aconitase. Transposases also distinguish M. burtonii from other archaea, and their genomic characteristics indicate they play an important role in evolving the M. burtonii genome. Our study reveals a capacity for this model psychrophile to evolve through genome plasticity (including nucleotide skew, horizontal gene transfer and transposase activity) that enables adaptation to the cold, and to the biological and physical changes that have

  16. Crystallization and preliminary X-ray analysis of a cohesin-like module from AF2375 of the archaeon Archaeoglobus fulgidus

    International Nuclear Information System (INIS)

    A cohesin-like module from the hyperthermophilic archaeon A. fulgidus was cloned, expressed, purified and crystallized. X-ray diffraction data were collected to 1.82 Å resolution. A cohesin-like module of 160 amino-acid residues from the hypothetical protein AF2375 of the noncellulolytic, hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus was cloned, expressed, purified, crystallized and subjected to X-ray structural study in order to compare its structure with those of cellulolytic cohesins. The crystals had cubic symmetry, with unit-cell parameters a = b = c = 101.75 Å in space group P4332, and diffracted to 1.82 Å resolution. The asymmetric unit contained a single cohesin molecule. A model assembled from six cohesin structures of very low sequence identity to the cohesin-like module was used in molecular-replacement attempts, producing a marginal solution

  17. Crystallization and preliminary X-ray analysis of a novel dye-linked l-proline dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix

    International Nuclear Information System (INIS)

    A novel dye-linked l-proline dehydrogenase from a hyperthermophilic archaeon was successfully isolated and crystallized. A novel dye-linked l-proline dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 8000 as the precipitant. The crystals belonged to the tetragonal space group P41212 or its enantiomorph P43212, with unit-cell parameters a = b = 61.1, c = 276.3 Å, and diffracted to 2.87 Å resolution using a Cu Kα rotating-anode generator with an R-AXIS VII detector. The asymmetric unit contained one protein molecule, giving a crystal volume per enzyme mass (VM) of 2.75 Å3 Da−1 and a solvent content of 55.3%

  18. Crystallization and preliminary X-ray analysis of a dye-linked d-lactate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix

    International Nuclear Information System (INIS)

    A dye-linked d-lactate dehydrogenase from a hyperthermophilic archaeon was successfully isolated and crystallized. A dye-linked d-lactate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol 8000 as the precipitant. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 63.4, b = 119.4, c = 70.2 Å, β = 112.0°, and diffracted to 2.0 Å resolution on the BL26B1 beamline at SPring-8. The overall Rmerge was 4.5% and the completeness was 99.8%

  19. Characterization of an Exo-β-d-Glucosaminidase Involved in a Novel Chitinolytic Pathway from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

    OpenAIRE

    Tanaka, Takeshi; Fukui, Toshiaki; Atomi, Haruyuki; Imanaka, Tadayuki

    2003-01-01

    We previously clarified that the chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 produces diacetylchitobiose (GlcNAc2) as an end product from chitin. Here we sought to identify enzymes in T. kodakaraensis that were involved in the further degradation of GlcNAc2. Through a search of the T. kodakaraensis genome, one candidate gene identified as a putative β-glycosyl hydrolase was found in the near vicinity of the chitinase gene. The primary structure of the candida...

  20. The Genome Sequence of the Metal-Mobilizing, Extremely Thermoacidophilic Archaeon Metallosphaera sedula Provides Insights into Bioleaching-Associated Metabolism▿ †

    OpenAIRE

    Auernik, Kathryne S.; Maezato, Yukari; Paul H. Blum; Kelly, Robert M.

    2007-01-01

    Despite their taxonomic description, not all members of the order Sulfolobales are capable of oxidizing reduced sulfur species, which, in addition to iron oxidation, is a desirable trait of biomining microorganisms. However, the complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, ∼2,300 open reading frames [ORFs]) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways an...

  1. Assessment of the Carbon Monoxide Metabolism of the Hyperthermophilic Sulfate-Reducing Archaeon Archaeoglobus fulgidus VC-16 by Comparative Transcriptome Analyses

    OpenAIRE

    Hocking, William P.; Irene Roalkvam; Carina Magnussen; Runar Stokke; Steen, Ida H.

    2015-01-01

    The hyperthermophilic, sulfate-reducing archaeon, Archaeoglobus fulgidus, utilizes CO as an energy source and it is resistant to the toxic effects of high CO concentrations. Herein, transcription profiles were obtained from A. fulgidus during growth with CO and sulfate or thiosulfate, or without an electron acceptor. This provided a basis for a model of the CO metabolism of A. fulgidus. The model suggests proton translocation by “Mitchell-type” loops facilitated by Fqo catalyzing a Fdred:mena...

  2. Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance▿

    OpenAIRE

    Matsumi, Rie; Manabe, Kenji; Fukui, Toshiaki; Atomi, Haruyuki; Imanaka, Tadayuki

    2007-01-01

    We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmgTk) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apuTk) or a gene cluster which includes apuTk and genes encoding components of a putative sugar transporter. Disruption plasm...

  3. Sequence, expression in Escherichia coli, and analysis of the gene encoding a novel intracellular protease (PfpI) from the hyperthermophilic archaeon Pyrococcus furiosus.

    OpenAIRE

    Halio, S B; Blumentals, I. I.; Short, S. A.; Merrill, B M; Kelly, R.M.

    1996-01-01

    A previously identified intracellular proteolytic activity in the hyperthermophilic archaeon Pyrococcus furiosus (I. I. Blumentals, A. S. Robinson, and R. M. Kelly, Appl. Environ. Microbiol. 56:1992-1998, 1990) was found to be a homomultimer consisting of 18.8-kDa subunits. Dissociation of this native P. furiosus protease I (PfpI) into a single subunit was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but only after trichloroacetic acid precipitation; heating to...

  4. The carbamate kinase-like carbamoyl phosphate synthetase of the hyperthermophilic archaeon Pyrococcus furiosus, a missing link in the evolution of carbamoyl phosphate biosynthesis

    OpenAIRE

    Durbecq, Virginie; Legrain, Christianne; Roovers, Martine; Piérard, André; Glansdorff, Nicolas

    1997-01-01

    Microbial carbamoyl phosphate synthetases (CPS) use glutamine as nitrogen donor and are composed of two subunits (or domains), one exhibiting glutaminase activity, the other able to synthesize carbamoyl phosphate (CP) from bicarbonate, ATP, and ammonia. The pseudodimeric organization of this synthetase suggested that it has evolved by duplication of a smaller kinase, possibly a carbamate kinase (CK). In contrast to other prokaryotes the hyperthermophilic archaeon Pyrococcus furiosus was found...

  5. Crystallization and preliminary X-ray analysis of the YjgF/YER057c/UK114-family protein ST0811 from Sulfolobus tokodaii strain 7

    International Nuclear Information System (INIS)

    Overexpression, purification and crystallization of the YjgF/YER057c/UK114-family protein from S. tokodaii strain 7 allowed the collection of a complete data set to 2.0 Å resolution. ST0811 from Sulfolobus tokodaii strain 7, a member of the YjgF/YER057c/UK114 protein family, was crystallized by the sitting-drop vapour-diffusion method using PEG 10 000 as precipitant. The crystals diffracted X-rays to beyond 2.0 Å resolution using an in-house rotating-anode generator. The crystals belonged to the rhombohedral space group R3, with hexagonal unit-cell parameters a = b = 55.0, c = 223.2 Å. The crystals contained two molecules in the asymmetric unit (VM = 2.3 Å3 Da−1) and had a solvent content of 47%

  6. Genetic determinants of PAM-dependent DNA targeting and pre-crRNA processing in Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Peng, Wenfang; Li, Huan; Hallstrøm, Søren;

    2013-01-01

    Bacteria and Archaea encode clustered, regularly interspaced, short palindromic repeat (CRISPR) systems to confer adaptive immunity to invasive viruses and plasmids. Recent studies of CRISPR systems revealed that diverse CRISPR-associated (Cas) interference modules often coexist in different...... organisms but functions of cas genes have not been dissected in any of these systems. The crenarchaeon Sulfolobus islandicus encodes three distinct CRISPR interference modules, including a type IA system and two type IIIB systems: Cmr-a and Cmr-ß. To study the genetic determinants of protospacer......-adjacent motif (PAM)-dependent DNA targeting activity and mature CRISPR RNA (crRNA) production in this organism, mutants deleting individual genes of the type IA system or removing each of other Cas modules were constructed. Characterization of these mutants revealed that Cas7, Cas5, Cas6, Cas3' and Cas3" are...

  7. Improving the Thermostability and Optimal Temperature of a Lipase from the Hyperthermophilic Archaeon Pyrococcus furiosus by Covalent Immobilization

    Directory of Open Access Journals (Sweden)

    Roberta V. Branco

    2015-01-01

    Full Text Available A recombinant thermostable lipase (Pf2001Δ60 from the hyperthermophilic Archaeon Pyrococcus furiosus (PFUL was immobilized by hydrophobic interaction on octyl-agarose (octyl PFUL and by covalent bond on aldehyde activated-agarose in the presence of DTT at pH = 7.0 (one-point covalent attachment (glyoxyl-DTT PFUL and on glyoxyl-agarose at pH 10.2 (multipoint covalent attachment (glyoxyl PFUL. The enzyme’s properties, such as optimal temperature and pH, thermostability, and selectivity, were improved by covalent immobilization. The highest enzyme stability at 70°C for 48 h incubation was achieved for glyoxyl PFUL (around 82% of residual activity, whereas glyoxyl-DTT PFUL maintained around 69% activity, followed by octyl PFUL (27% remaining activity. Immobilization on glyoxyl-agarose improved the optimal temperature to 90°C, while the optimal temperature of octyl PFUL was 70°C. Also, very significant changes in activity with different substrates were found. In general, the covalent bond derivatives were more active than octyl PFUL. The E value also depended substantially on the derivative and the conditions used. It was observed that the reaction of glyoxyl-DTT PFUL using methyl mandelate as a substrate at pH 7 presented the best results for enantioselectivity E=22 and enantiomeric excess (ee (% = 91.

  8. Crystallization and preliminary X-ray analysis of a RecB-family nuclease from the archaeon Pyrococcus abyssi

    International Nuclear Information System (INIS)

    A RecB-like nuclease from the archaeon Pyrococcus abyssi was expressed, purified and crystallized. The crystals belong to the orthorhombic space group C2221 with a = 81.5, b = 159.8, c = 100.8 Å, and a native data set was collected to 2.65 Å resolution. Nucleases are required to process and repair DNA damage in living cells. One of the best studied nucleases is the RecB protein, which functions in Escherichia coli as a component of the RecBCD enzyme complex that amends double-strand breaks in DNA. Although archaea do not contain the RecBCD complex, a RecB-like nuclease from Pyrococcus abyssi has been cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant. The crystals belong to the orthorhombic space group C2221, with unit-cell parameters a = 81.5, b = 159.8, c = 100.8 Å. Self-rotation function and native Patterson map calculations revealed that there is a dimer in the asymmetric unit with its local twofold axis running parallel to the crystallographic twofold screw axis. The crystals diffracted to about 2 Å and a complete native data set was collected to 2.65 Å resolution

  9. Highly thermostable RadA protein from the archaeon Pyrococcus woesei enhances specificity of simplex and multiplex PCR assays.

    Science.gov (United States)

    Stefanska, Aleksandra; Gaffke, Lidia; Kaczorowska, Anna-Karina; Plotka, Magdalena; Dabrowski, Slawomir; Kaczorowski, Tadeusz

    2016-05-01

    The radA gene of the hyperthermophilic archaeon Pyrococcus woesei (Thermococcales) was cloned and overexpressed in Escherichia coli. The 1050-bp gene codes for a 349-amino-acid polypeptide with an M r of 38,397 which shows 100 % positional amino acid identity to Pyrococcus furiosus RadA and 27.1 % to the E. coli RecA protein. Recombinant RadA was overproduced in Escherichia coli as a His-tagged fusion protein and purified to electrophoretic homogeneity using a simple procedure consisting of ammonium sulfate precipitation and metal-affinity chromatography. In solution RadA exists as an undecamer (11-mer). The protein binds both to ssDNA and dsDNA. RadA has been found to be highly thermostable, it remains almost unaffected by a 4-h incubation at 94 °C. The addition of the RadA protein to either simplex or multiplex PCR assays, significantly improves the specificity of DNA amplification by eliminating non-specific products. Among applications tested the RadA protein proved to be useful in allelic discrimination assay of HADHA gene associated with long-chain 3-hydroxylacyl-CoA dehydrogenase deficiency that in infancy may lead to hypotonia, serious heart and liver problems and even sudden death. PMID:26337425

  10. A novel acidophilic, thermophilic iron and sulfur-oxidizing archaeon isolated from a hot spring of tengchong, yunnan, China

    Directory of Open Access Journals (Sweden)

    Jiannan Ding

    2011-06-01

    Full Text Available A novel thermoacidophilic iron and sulfur-oxidizing archaeon, strain YN25, was isolated from an in situ enriched acid hot spring sample collected in Yunnan, China. Cells were irregular cocci, about 0.9-1.02 µm×1.0-1.31 µm in the medium containing elemental sulfur and 1.5-2.22 µm×1.8-2.54 µm in ferrous sulfate medium. The ranges of growth and pH were 50-85 (optimum 65 and pH 1.0-6.0 (optimum 1.5-2.5. The acidophile was able to grow heterotrophically on several organic substrates, including various monosaccharides, alcohols and amino acids, though the growth on single substrate required yeast extract as growth factor. Growth occurred under aerobic conditions or via anaerobic respiration using elemental sulfur as terminal electron acceptor. Results of morphology, physiology, fatty acid analysis and analysis based on 16S rRNA gene sequence indicated that the strain YN25 should be grouped in the species Acidianus manzaensis. Bioleaching experiments indicated that this strain had excellent leaching capacity, with a copper yielding ratio up to 79.16% in 24 d. The type strain YN25 was deposited in China Center for Type Culture Collection (=CCTCCZNDX0050.

  11. Crystallization of leucyl-tRNA synthetase complexed with tRNALeu from the archaeon Pyrococcus horikoshii

    International Nuclear Information System (INIS)

    The leucyl-tRNA synthetase (LeuRS) from P. horikoshii has been overexpressed in Escherichia coli and purified, and cocrystallizations with each of the tRNALeu isoacceptors have been attempted. Cocrystals were obtained by the hanging-drop vapour-diffusion method, but only when the tRNALeu isoacceptor with the anticodon CAA was used. All five tRNALeu isoacceptors from the archaeon Pyrococcus horikoshii have been transcribed in vitro and purified. The leucyl-tRNA synthetase (LeuRS) from P. horikoshii was overexpressed in Escherichia coli and purified, and cocrystallizations with each of the tRNALeu isoacceptors were attempted. Cocrystals were obtained by the hanging-drop vapour-diffusion method, but only when the tRNALeu isoacceptor with the anticodon CAA was used. Electrophoretic analyses revealed that the crystals contain both LeuRS and tRNALeu, suggesting that they are LeuRS–tRNALeu complex crystals. A data set diffracting to 3.3 Å resolution was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic space group P21212, with unit-cell parameters a = 118.18, b = 120.55, c = 231.13 Å. The asymmetric unit is expected to contain two complexes of LeuRS–tRNALeu, with a corresponding crystal volume per protein weight of 2.9 Å3 Da−1 and a solvent content of 57.3%

  12. Molecular chaperone accumulation as a function of stress evidences adaptation to high hydrostatic pressure in the piezophilic archaeon Thermococcus barophilus

    Science.gov (United States)

    Cario, Anaïs; Jebbar, Mohamed; Thiel, Axel; Kervarec, Nelly; Oger, Phil M.

    2016-01-01

    The accumulation of mannosyl-glycerate (MG), the salinity stress response osmolyte of Thermococcales, was investigated as a function of hydrostatic pressure in Thermococcus barophilus strain MP, a hyperthermophilic, piezophilic archaeon isolated from the Snake Pit site (MAR), which grows optimally at 40 MPa. Strain MP accumulated MG primarily in response to salinity stress, but in contrast to other Thermococcales, MG was also accumulated in response to thermal stress. MG accumulation peaked for combined stresses. The accumulation of MG was drastically increased under sub-optimal hydrostatic pressure conditions, demonstrating that low pressure is perceived as a stress in this piezophile, and that the proteome of T. barophilus is low-pressure sensitive. MG accumulation was strongly reduced under supra-optimal pressure conditions clearly demonstrating the structural adaptation of this proteome to high hydrostatic pressure. The lack of MG synthesis only slightly altered the growth characteristics of two different MG synthesis deletion mutants. No shift to other osmolytes was observed. Altogether our observations suggest that the salinity stress response in T. barophilus is not essential and may be under negative selective pressure, similarly to what has been observed for its thermal stress response. PMID:27378270

  13. In vivo characterization of the homing endonuclease within the polB gene in the halophilic archaeon Haloferax volcanii.

    Directory of Open Access Journals (Sweden)

    Adit Naor

    Full Text Available Inteins are parasitic genetic elements, analogous to introns that excise themselves at the protein level by self-splicing, allowing the formation of functional non-disrupted proteins. Many inteins contain a homing endonuclease (HEN gene, and rely on its activity for horizontal propagation. In the halophilic archaeon, Haloferax volcanii, the gene encoding DNA polymerase B (polB contains an intein with an annotated but uncharacterized HEN. Here we examine the activity of the polB HEN in vivo, within its natural archaeal host. We show that this HEN is highly active, and able to insert the intein into both a chromosomal target and an extra-chromosomal plasmid target, by gene conversion. We also demonstrate that the frequency of its incorporation depends on the length of the flanking homologous sequences around the target site, reflecting its dependence on the homologous recombination machinery. Although several evolutionary models predict that the presence of an intein involves a change in the fitness of the host organism, our results show that a strain deleted for the intein sequence shows no significant changes in growth rate compared to the wild type.

  14. In vivo characterization of the homing endonuclease within the polB gene in the halophilic archaeon Haloferax volcanii.

    Science.gov (United States)

    Naor, Adit; Lazary, Rona; Barzel, Adi; Papke, R Thane; Gophna, Uri

    2011-01-01

    Inteins are parasitic genetic elements, analogous to introns that excise themselves at the protein level by self-splicing, allowing the formation of functional non-disrupted proteins. Many inteins contain a homing endonuclease (HEN) gene, and rely on its activity for horizontal propagation. In the halophilic archaeon, Haloferax volcanii, the gene encoding DNA polymerase B (polB) contains an intein with an annotated but uncharacterized HEN. Here we examine the activity of the polB HEN in vivo, within its natural archaeal host. We show that this HEN is highly active, and able to insert the intein into both a chromosomal target and an extra-chromosomal plasmid target, by gene conversion. We also demonstrate that the frequency of its incorporation depends on the length of the flanking homologous sequences around the target site, reflecting its dependence on the homologous recombination machinery. Although several evolutionary models predict that the presence of an intein involves a change in the fitness of the host organism, our results show that a strain deleted for the intein sequence shows no significant changes in growth rate compared to the wild type. PMID:21283796

  15. The aerobic respiratory chain of the acidophilic archaeon Ferroplasma acidiphilum: A membrane-bound complex oxidizing ferrous iron.

    Science.gov (United States)

    Castelle, Cindy J; Roger, Magali; Bauzan, Marielle; Brugna, Myriam; Lignon, Sabrina; Nimtz, Manfred; Golyshina, Olga V; Giudici-Orticoni, Marie-Thérèse; Guiral, Marianne

    2015-08-01

    The extremely acidophilic archaeon Ferroplasma acidiphilum is found in iron-rich biomining environments and is an important micro-organism in naturally occurring microbial communities in acid mine drainage. F. acidiphilum is an iron oxidizer that belongs to the order Thermoplasmatales (Euryarchaeota), which harbors the most extremely acidophilic micro-organisms known so far. At present, little is known about the nature or the structural and functional organization of the proteins in F. acidiphilum that impact the iron biogeochemical cycle. We combine here biochemical and biophysical techniques such as enzyme purification, activity measurements, proteomics and spectroscopy to characterize the iron oxidation pathway(s) in F. acidiphilum. We isolated two respiratory membrane protein complexes: a 850 kDa complex containing an aa3-type cytochrome oxidase and a blue copper protein, which directly oxidizes ferrous iron and reduces molecular oxygen, and a 150 kDa cytochrome ba complex likely composed of a di-heme cytochrome and a Rieske protein. We tentatively propose that both of these complexes are involved in iron oxidation respiratory chains, functioning in the so-called uphill and downhill electron flow pathways, consistent with autotrophic life. The cytochrome ba complex could possibly play a role in regenerating reducing equivalents by a reverse ('uphill') electron flow. This study constitutes the first detailed biochemical investigation of the metalloproteins that are potentially directly involved in iron-mediated energy conservation in a member of the acidophilic archaea of the genus Ferroplasma. PMID:25896560

  16. Carbonate precipitation by the thermophilic archaeon Archaeoglobus fulgidus: a model of carbon flow for an ancient microorganism

    Directory of Open Access Journals (Sweden)

    P. Ostrom

    2008-08-01

    Full Text Available Microbial carbonate precipitation experiments were conducted using the archaeon bacteria Archaeoglobus fulgidus to determine chemical and isotopic fractionation of organic and inorganic carbon into mineral phases. Carbonate precipitation was induced in two different experiments using A. fulgidus to determine the relative abundance of organically derived carbon incorporated into carbonate minerals as well as to define any distinct phases or patterns that could be attributed to the precipitation process. One experiment used a medium containing 13C-depleted organic carbon and 13C-enriched inorganic carbon, and the other used a 14C-labeled organic carbon source. Results indicated that 0.9–24.8% organic carbon was incorporated into carbonates precipitated by A. fulgidus and that this process was mediated primarily by pH and CO2 emission from cells. Data showed that the carbon in the CO2 produced from this microorganism is incorporated into carbonates and that the rate at which precipitation occurs and the dynamics of the carbonate precipitation process are strongly mediated by the specific steps involved in the biochemical process for lactate oxidation by A. fulgidus.

  17. Evolution of multi-enzyme complexes: the case of tryptophan synthase.

    Science.gov (United States)

    Leopoldseder, Sonja; Hettwer, Stefan; Sterner, Reinhard

    2006-11-28

    The prototypical tryptophan synthase is a stable heterotetrameric alpha-betabeta-alpha complex. The constituting TrpA and TrpB1 subunits, which are encoded by neighboring genes in the trp operon, activate each other in a bi-directional manner. Recently, a novel class of TrpB2 proteins has been identified, whose members contain additional amino acids that might sterically prevent complex formation with TrpA. To test this hypothesis, we characterized the TrpA and TrpB proteins from Sulfolobus solfataricus. This hyperthermophilic archaeon does not contain a TrpB1 protein but instead contains two TrpB2 homologues that are encoded within (TrpB2i) and outside (TrpB2o) the trp operon. We find that TrpB2i and TrpA form a weak and transient complex during catalysis, with a uni-directional activation of TrpA by TrpB2i. In contrast, TrpB2o and TrpA do not form a detectable complex. These results suggest a model for the evolution of the tryptophan synthase in which TrpB2o, TrpB2i, and TrpB1 reflect the stepwise increase of TrpB affinity for TrpA and the refinement of functional subunit interaction, concomitant with the co-localization of the encoding genes in the trp operon. PMID:17115706

  18. Hyperthermophilic endoglucanase for in planta lignocellulose conversion

    Directory of Open Access Journals (Sweden)

    Klose Holger

    2012-08-01

    Full Text Available Abstract Background The enzymatic conversion of lignocellulosic plant biomass into fermentable sugars is a crucial step in the sustainable and environmentally friendly production of biofuels. However, a major drawback of enzymes from mesophilic sources is their suboptimal activity under established pretreatment conditions, e.g. high temperatures, extreme pH values and high salt concentrations. Enzymes from extremophiles are better adapted to these conditions and could be produced by heterologous expression in microbes, or even directly in the plant biomass. Results Here we show that a cellulase gene (sso1354 isolated from the hyperthermophilic archaeon Sulfolobus solfataricus can be expressed in plants, and that the recombinant enzyme is biologically active and exhibits the same properties as the wild type form. Since the enzyme is inactive under normal plant growth conditions, this potentially allows its expression in plants without negative effects on growth and development, and subsequent heat-inducible activation. Furthermore we demonstrate that the recombinant enzyme acts in high concentrations of ionic liquids and can therefore degrade α-cellulose or even complex cell wall preparations under those pretreatment conditions. Conclusion The hyperthermophilic endoglucanase SSO1354 with its unique features is an excellent tool for advanced biomass conversion. Here we demonstrate its expression in planta and the possibility for post harvest activation. Moreover the enzyme is suitable for combined pretreatment and hydrolysis applications.

  19. Impact of Substrate Glycoside Linkage and Elemental Sulfur on Bioenergetics of and Hydrogen Production by the Hyperthermophilic Archaeon Pyrococcus furiosus▿ †

    OpenAIRE

    Chou, Chung-Jung; Shockley, Keith R.; Conners, Shannon B.; Lewis, Derrick L.; Comfort, Donald A.; Adams, Michael W. W.; Kelly, Robert M.

    2007-01-01

    Glycoside linkage (cellobiose versus maltose) dramatically influenced bioenergetics to different extents and by different mechanisms in the hyperthermophilic archaeon Pyrococcus furiosus when it was grown in continuous culture at a dilution rate of 0.45 h−1 at 90°C. In the absence of S0, cellobiose-grown cells generated twice as much protein and had 50%-higher specific H2 generation rates than maltose-grown cultures. Addition of S0 to maltose-grown cultures boosted cell protein production fou...

  20. A dual role of divalent metal ions in catalysis and folding of RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1

    OpenAIRE

    Tannous, Elias; Yokoyama, Koji; You, Dong-Ju; Koga, Yuichi; Kanaya, Shigenori

    2012-01-01

    RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1 (Halo-RNH1) consists of an N-terminal domain with unknown function and a C-terminal RNase H domain. It is characterized by the high content of acidic residues on the protein surface. The far- and near-UV CD spectra of Halo-RNH1 suggested that Halo-RNH1 assumes a partially folded structure in the absence of salt and divalent metal ions. It requires either salt or divalent metal ions for folding. However, thermal denaturation of ...

  1. Dicty_cDB: Contig-U15070-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available us M.14.25, ... 53 3e-05 AE006641_2942( AE006641 |pid:none) Sulfolobus solfataricus P2, com... 52 8e-05 AF15...1059( AE006641 |pid:none) Sulfolobus solfataricus P2, com... 41 0.18 AF224509_3( AF224509 |pid:none) Bactero...0153_1364( CP000153 |pid:none) Sulfurimonas denitrificans DSM ... 35 7.4 CP000745_318( CP000745 |pid:none) Methanococcus mari...kn*kkkik*nscfykikrldfhtlffikfnmtpvitafls faflcfaswcvgeggeilgkkydasiiggliiawlntapeaiff...itdcssvdvaftsrpv rkcyfcskscgiprallaywektnvysininiil*ylnlfvtlllflllenyf*knkiif *fffffskikkkk*nkilvfik*kgwtftpff

  2. Dicty_cDB: Contig-U15077-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available *idiykkityqinptkty*ytnkyiiff*fnliflyfffyyfs ffyfffylhttliiyiyl*issicyhsriirr**s**...) Zebrafish DNA sequence from clone CH211-72M2 in l... 52 0.053 1 ( Y18930 ) Sulfolobus solfataricus 281 kb ...genomic DNA fragment... 52 0.053 1 ( AE006697 ) Sulfolobus solfataricus P2 section 56 of 272 of t... 52 0.05...*n*kiiqkk*nkik*n kik*nkik*knnqnkndygirpkt*nekm*vin*lsngd*fiyfili*ffffffffsll* k*ckkr*mcliidssqviqiie*ifg...k*nkik*knnqnkndygirpkt*nekm*vin*lsngd*fiyfili*ffffffffsll* k*ckkr*mcliidssqviqiie*if

  3. Dicty_cDB: Contig-U11403-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available eyqapaiytkysnshmhylnnvlkinnkk*iktnkskk*ikkk Frame B: llaywxilphpktynfiiffvfffssnk...DNA,... 62 3e-17 AE006641_870( AE006641 |pid:none) Sulfolobus solfataricus P2, co.... 52 6e-05 AE006641_430( AE006641 |pid:none) Sulfolobus solfataricus P2, comp... 52 6e-05 CP000875_100( CP00...est48= CHH790Z ,1102,1204 est49= SSL567Z ,1159,1312 Translated Amino Acid sequence llaywxilphpktynfiiff...xxyqk*NKIKKK--- ---ww*tfsrivkdye*nyq*gmgfgiigrgtcstssnv*kgfnsdkspl*igfnsnivt *r*kdsrfefpywsktl*gklvgftksrfpckcfmfrslvsndsgil*rif

  4. Thermococcus thioreducens sp. nov., a Novel Hyperthermophilic, Obligately Sulfur-Reducing Archaeon from a Deep-Sea Hydrothermal Vent

    Science.gov (United States)

    Pikuta, Elena V.; Marsic, Damien; Itoh, Takashi; Bej, Asim K.; Tang, Jane; Whitman, William B.; Ng, Joseph D.; Garriott, Owen K.; Hoover, Richard B.

    2007-01-01

    A hyperthermophilic, sulfur-reducing, organo-heterotrophic archaeon, strain OGL-20P(sup T), was isolated from 'black smoker' chimney material from the Rainbow hydrothermal vent site on the Mid-Atlantic Ridge (36.2degN, 33.9degW). The cells of strain OGL-20P(T) have an irregular coccoid shape and are motile with a single flagellum. Growth was observed within a pH range of 5.0-8.5 (optimum pH 7.0), an NaCl concentration range of 1-5%(w/v) (optimum 3%)and a temperature range of 55-94 C (optimum 83-85 C). The novel isolate is strictly anaerobic and obligately dependent upon elemental sulfur as an electron acceptor, but it does not reduce sulfate, sulfite, thiosulfate, Fe(III) or nitrate. Proteolysis products (peptone, bacto-tryptone, Casamino acids and yeast extract) are utilized as substrates during sulfur reduction. Strain OGL-20P(sup T) is resistant to ampicillin, chloram phenicol, kanamycin and gentamicin, but sensitive to tetracycline and rifampicin. The G + C content of the DNA is 52.9 mol% The 16S rRNA gene sequence analysis revealed that strain OGL-20P(sup T) is closely related to Thermococcus coalescens and related species, but no significant homology by DNA-DNA hybridization was observed between those species and the new isolate. On the basis of physiological and molecular properties of the new isolate, we conclude that strain OGL-20P(sup T) represents a new separate species within the genus Thermococcus, for which we propose the name Thermococcus thioreducens sp. nov. The type strain is OGL-20P(sup T) (=JCM 12859(exp T) = DSM 14981(exp T)=ATCC BAA-394(exp T)).

  5. Archaeal promoter architecture and mechanism of gene activation

    DEFF Research Database (Denmark)

    Peng, Nan; Ao, Xiang; Liang, Yun Xiang;

    2011-01-01

    Sulfolobus solfataricus and Sulfolobus islandicus contain several genes exhibiting D-arabinose-inducible expression and these systems are ideal for studying mechanisms of archaeal gene expression. At sequence level, only two highly conserved cis elements are present on the promoters: a regulatory...... mechanisms include TFB (transcription factor B) recruitment by the ara-box-binding factor to activate gene expression and modulation of TFB recruitment efficiency to yield differential gene expression....

  6. Dicty_cDB: VFK405 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available icswss*w**ycslyssvgmlysfrykk*krwiisittts*kamfnftiiiid tiiliigwi*cflwyfrnnllfnihwifiis*itrrrekrrs*y*ng*kkewck*tndsi nnsiinri...rrrrhr*rr*t*rtii*rcilfifrwsfnlll**tih*lnciisiilgn*sn xisilfssncfxna*nxxinfii... DSM 62... 39 0.31 AE017285_2604( AE017285 |pid:none) Desulfovibrio vulgaris subsp. v....xkrsfsky*ysffkfnwwynnqnnfingyflflwsc* ri*mgityifimldiidnlcscsiwywlfceqdktttrfnvirxflisrnntiliky Ho...id:none) Sulfolobus solfataricus P2, com... 49 2e-04 CP001399_1018( CP001399 |pid:none) Sulfolobus islandicu

  7. Structure and Cell Biology of Archaeal Virus STIV

    OpenAIRE

    Fu, Chi-yu; Johnson, John E.

    2012-01-01

    Recent investigations of archaeal viruses have revealed novel features of their structures and life cycles when compared to eukaryotic and bacterial viruses, yet there are structure-based unifying themes suggesting common ancestral relationships among dsDNA viruses in the three kingdoms of life. Sulfolobus solfataricus and the infecting virus Sulfolobus turreted icosahedral virus (STIV) is one of the well-established model systems to study archaeal virus replication and viral-host interaction...

  8. Activities of methionine-γ-lyase in the acidophilic archaeon “Ferroplasma acidarmanus” strain fer1

    Directory of Open Access Journals (Sweden)

    Khan MA

    2013-04-01

    Full Text Available M A Khan,1 Madeline M López-Muñoz,2 Charles W Kaspar,3 Kai F Hung1 1Department of Biological Sciences, Eastern Illinois University, Charleston, IL, USA; 2Department of Biology, Universidad de Puerto Rico, Mayaguez, Puerto Rico; 3Bacteriology Department, University of Wisconsin, Madison, WI, USA Abstract: Biogeochemical processes on exposed pyrite ores result in extremely high levels of sulfuric acid at these locations. Acidophiles that thrive in these conditions must overcome significant challenges, including an environment with proton concentrations at pH 3 or below. The role of sulfur metabolism in the archaeon “Ferroplasma acidarmanus” strain fer1's ability to thrive in this environment was investigated due to its growth-dependent production of methanethiol, a volatile organic sulfur compound. Two putative sequences for methionine-γ-lyase (EC 4.4.1.11, an enzyme known to carry out α, γ-elimination on L-methionine to produce methanethiol, were identified in fer1. Bioinformatic analyses identified a conserved pyridoxal-5'-phosphate (PLP binding domain and a partially conserved catalytic domain in both putative sequences. Detection of PLP-dependent and L-methionine-dependent production of α-keto compounds and thiol groups in fer1 confirmed the presence of methionine-γ-lyase activity. Further, fer1 lysate was capable of processing related substrates, including D-methionine, L-cysteine, L-cystathionine, and L/D-homocysteine. When the two putative fer1 methionine-γ-lyase gene-coded proteins were expressed in Escherichia coli cells, one sequence demonstrated an ability to carry out α, γ-elimination activity, while the other exhibited γ-replacement activity. These fer1 methionine-γ-lyases also exhibited optimum pH, substrate specificity, and catalytic preferences that are different from methionine-γ-lyases from other organisms. These differences are discussed in the context of molecular phylogeny constructed using a maximum

  9. Cloning, over expression and characterization of a new thermophilic and acidic a-amylase from Sulfolobus tokodaii strain 7 in Escherichia coil%Sulfolobus tokodaii strain 7高温酸性α-淀粉酶基因在大肠杆菌中克隆表达及其酶学性质

    Institute of Scientific and Technical Information of China (English)

    魏涛; 孙浩; 申玉龙; 毛多斌

    2013-01-01

    A gene (ST0817) encoding a putative thermophilic and acidic α-amylase from the thermophilic crenarchaeota Sulfolobus tokodaii strain 7 was cloned and functionally overexpressed in Escherichia coli.The recombinant enzyme was purified to homogeneity after heat treatment,Ni-NTA affinity and superdex-200 gel filtration.The relative molecular mass of α-amylase was 53.0 kDa on SDS-PAGE.The purified enzyme displayed optimal activity at 70 ℃ and pH 5.5.It had a half-life of 8 h at 85℃.The enzyme was found to have high pH stability and maintained about 50% of activity even after 120 min of treatment at pH 5.2.The enzymatic activities of the purified enzyme towards different substrates were listed from high to low as follows:amylose > soluble starch > amylopectin > β-cyclodextrin > glycogen > cyclodextrin > pullulan.The enzyme was found to have high tolerance to organic solvents,metal ions and detergents.%以超嗜热古菌Sulfolobus tokodaii strain 7基因组DNA为模板,通过PCR扩增高温酸性α-淀粉酶基因ST0817,将此基因克隆至表达载体pET15b,并转化大肠杆菌Escherichia coli BL21-CodenPlus(DE3)-RIL,获得重组大肠杆菌工程菌.通过热处理、镍柱亲和层析和分子筛层析,得到纯化重组酶,SDS-PAGE分析表明,该酶分子量为53.0 kDa.酶学性质研究表明,该酶最适温度和pH分别为75℃和5.5;具有较强的热稳定性和pH稳定性,在85℃处理8h保持50%左右活力,在pH 5.2处理120 min仍保持50%活力.此酶对不同底物水解活性不同,直连淀粉>可溶性淀粉>支链淀粉>β-极限糊精>糖原>环糊精>普鲁兰糖;该酶对有机溶剂、变性剂和金属离子具有一定抗性.

  10. Dicty_cDB: VSC123 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available one) Sulfolobus solfataricus P2, com... 33 4.1 X61658_1( X61658 |pid:none) B.brevis grsB gene for gramicid...in S sy... 32 9.2 (P0C063) RecName: Full=Gramicidin S synthetase 2; AltName: Full=.

  11. Archaeal viruses of the sulfolobales

    DEFF Research Database (Denmark)

    Erdmann, Susanne; Garrett, Roger Antony

    2015-01-01

    Infection of archaea with phylogenetically diverse single viruses, performed in different laboratories, has failed to activate spacer acquisition into host CRISPR loci. The first successful uptake of archaeal de novo spacers was observed on infection of Sulfolobus solfataricus P2 with an environm......Infection of archaea with phylogenetically diverse single viruses, performed in different laboratories, has failed to activate spacer acquisition into host CRISPR loci. The first successful uptake of archaeal de novo spacers was observed on infection of Sulfolobus solfataricus P2 with an...... CRISPR loci of Sulfolobus species from a second coinfecting conjugative plasmid or virus (Erdmann and Garrett, Mol Microbiol 85:1044-1056, 2012; Erdmann et al. Mol Microbiol 91:900-917, 2014). Here we describe, firstly, the isolation of archaeal virus mixtures from terrestrial hot springs and the...

  12. CLONING AND EXPRESSION OF THE GENE ENCODING NOVEL a-AMYLASE FROM SULFOLOBUS SHIBATAE IN E. COLI%芝田硫化叶菌新型α-淀粉酶基因在大肠杆菌的克隆和表达

    Institute of Scientific and Technical Information of China (English)

    刘莉; 陈炜; 金城

    2000-01-01

    A novel α-amylase gene was amplified from Sulfolobus shibatae by using PCR technique.The amplified 1.7kb DNA fragment was inserted into an expression vector pBV220 to yield the recombinant plasmid pSBAM. The novel α-amylase gene in pSBAM was expressed in E. coli. The production of the novel α-amylase activity reached over 8 units/100mL of the culture. The molecular weight of this enzyme was about 61kD by SDS-PAGE. The expressed novel α-amylase protein in E.coli DHSα accounted for about 20 % of the total protein in the recombinant cell. The cooperative action of the novel α-amylase and the maltooligosyltrehalose synthase from Sulfolobus shibatae was investigated and trehalose was detected by using HPLC analysis when using amylose and partial starch hydrolysates as substrates.

  13. Ligand-induced formation of a transient tryptophan synthase complex with αββ subunit stoichiometry.

    Science.gov (United States)

    Ehrmann, Alexander; Richter, Klaus; Busch, Florian; Reimann, Julia; Albers, Sonja-Verena; Sterner, Reinhard

    2010-12-28

    The prototypical tryptophan synthases form a stable heterotetrameric αββα complex in which the constituting TrpA and TrpB1 subunits activate each other in a bidirectional manner. The hyperthermophilic archaeon Sulfolobus solfataricus does not contain a TrpB1 protein but instead two members of the phylogenetically distinct family of TrpB2 proteins, which are encoded within (sTrpB2i) and outside (sTrpB2a) the tryptophan operon. It has previously been shown that sTrpB2a does not functionally or structurally interact with sTrpA, whereas sTrpB2i substantially activates sTrpA in a unidirectional manner. However, in the absence of catalysis, no physical complex between sTrpB2i and sTrpA could be detected. In order to elucidate the structural requirements for complex formation, we have analyzed the interaction between sTrpA (α-monomer) and sTrpB2i (ββ-dimer) by means of spectroscopy, analytical gel filtration, and analytical ultracentrifugation, as well as isothermal titration calorimetry. In the presence of the TrpA ligand glycerol 3-phosphate (GP) and the TrpB substrate l-serine, sTrpA and sTrpB2i formed a physical complex with a thermodynamic dissociation constant of about 1 μM, indicating that the affinity between the α- and ββ-subunits is weaker by at least 1 order of magnitude than the affinity between the corresponding subunits of prototypical tryptophan synthases. The observed stoichiometry of the complex was 1 subunit of sTrpA per 2 subunits of sTrpB2i, which corresponds to a αββ quaternary structure and testifies to a strong negative cooperativity for the binding of the α-monomers to the ββ-dimer. The analysis of the interaction between sTrpB2i and sTrpA in the presence of several substrate, transition state, and product analogues suggests that the αββ complex remains stable during the whole catalytic cycle and disintegrates into α- and ββ-subunits upon the release of the reaction product tryptophan. The formation of a transient tryptophan

  14. Mutation of the gene encoding the ribonuclease P RNA in the hyperthermophilic archaeon Thermococcus kodakarensis causes decreased growth rate and impaired processing of tRNA precursors.

    Science.gov (United States)

    Ueda, Toshifumi; Ishino, Sonoko; Suematsu, Kotaro; Nakashima, Takashi; Kakuta, Yoshimitsu; Kawarabayasi, Yutaka; Ishino, Yoshizumi; Kimura, Makoto

    2015-12-25

    Ribonuclease P (RNase P) catalyzes the processing of 5' leader sequences of tRNA precursors in all three phylogenetic domains. RNase P also plays an essential role in non-tRNA biogenesis in bacterial and eukaryotic cells. For archaeal RNase Ps, additional functions, however, remain poorly understood. To gain insight into the biological function of archaeal RNase Ps in vivo, we prepared archaeal mutants KUWΔP3, KUWΔP8, and KUWΔP16, in which the gene segments encoding stem-loops containing helices, respectively, P3, P8 and P16 in RNase P RNA (TkopRNA) of the hyperthermophilic archaeon Thermococcus kodakarensis were deleted. Phenotypic analysis showed that KUWΔP3 and KUWΔP16 grew slowly compared with wild-type T. kodakarensis KUW1, while KUWΔP8 displayed no difference from T. kodakarensis KUW1. RNase P isolated using an affinity-tag from KUWΔP3 had reduced pre-tRNA cleavage activity compared with that from T. kodakarensis KUW1. Moreover, quantitative RT-PCR (qRT-PCR) and Northern blots analyses of KUWΔP3 showed greater accumulation of unprocessed transcripts for pre-tRNAs than that of T. kodakarensis KUW1. The current study represents the first attempt to prepare mutant T. kodakarensis with impaired RNase P for functional investigation. Comparative whole-transcriptome analysis of T. kodakarensis KUW1 and KUWΔP3 should allow for the comprehensive identification of RNA substrates for archaeal RNase Ps. PMID:26551464

  15. Biogenic inorganic crystalline phase formation as a result of biogeochemical interactions in between the chemolithotrophic archaeon Metallosphaera sedula and meteorite: implications for potential microbial biosignatures

    Science.gov (United States)

    Milojevic, Tetyana; Blazevic, Amir; Kutlucinar, Kaan Georg

    2016-04-01

    Chemolithotrophy has been indicated as the most primordial form of microbial metabolism on the early Earth and proposed as a possible metabolic form for other iron-mineral-rich planets like Mars. Rock-eating extremophiles represent an exciting field of research for the study of microbe-mineral interactions in order to find the unique biosignatures of life in the extreme conditions. Metallosphaera sedula is the chemolithotrophic archaeon, which thrives at 73°C and pH 2, using energy derived from metal oxidation at the edge of living limits. When given an access to extraterrestrial material (a stony meteorite H5 ordinary chondrite NWA1172), M. sedula releases soluble metal ions into the solution from NWA1172 due to its metal oxidizing metabolic activity. Here we report the formation of inorganic crystalline phase as a result of biogeochemical interactions in between M. sedula and extraterrestrial material. Inorganic ions released from meteorite as a result of M. sedula mediated leaching were trapped into crystalline material by solvent evaporation technique. Scanning Electron Microscopy observations and EDX analysis revealed that this crystalline phase is mainly composed of Ni, S, Mg and O elements. Biogenicity of this inorganic crystalline material was evaluated by comparing to abiotic conditions. Biological nature of Ni-, S-, Mg- and O -containing crystalline phase was established, since it was not mimicked in abiotic experimental conditions, allowing clearly to exclude abiogenic origin. Further investigations of exact mineralogical nature of biogenic of Ni-, S-, Mg- and O -crystalline material and its implication as a biosignature for detection of life are going to be investigated.

  16. Engineering thermoacidophilic archaea using linear DNA recombination.

    Science.gov (United States)

    Maezato, Yukari; Dana, Karl; Blum, Paul

    2011-01-01

    Thermoacidophilic archaea comprise one of the major classes of extremophiles. Most belong to the family Sulfolobales within the phylum Crenarchaeota. They are of applied interest as sources of hyperstable enzymes, for biomining of base and precious metals, and for evolutionary studies because of their use of eukaryotic-like subcellular mechanisms. Genetic methods are available for several species particularly Sulfolobus solfataricus. This organism has a considerable number of methods available for the construction of novel cell lines with unique functions. This chapter presents recent developments in the use of homologous recombination and linear DNA for the engineering of site-specific changes in the genome of S. solfataricus. PMID:21815108

  17. Evidence for the horizontal transfer of an integrase gene from a fusellovirus to a pRN-like plasmid within a single strain of Sulfolobus and the implications for plasmid survival

    DEFF Research Database (Denmark)

    Peng, Xu

    2008-01-01

    A fusellovirus SSV4 and a pRN-like plasmid pXZ1 were co-isolated from a single strain of Sulfolobus. In contrast to the previously characterized virus-plasmid hybrids pSSVx and pSSVi, which can coexist intracellulary with a fusellovirus, pXZ1 is not packaged into viral particles and shows no viral...... infectivity. The virus and plasmid carry genomes of 15 135 and 6970 bp, respectively. For SSV4, 33 predicted ORFs are compactly organized with a strong preference for UGA stop codons, three-quarters of which overlap with either the Shine-Dalgarno motif or the start codon of the following gene. pXZ1 carries...... seven ORFs, three of which encode an atypical RepA, a PlrA and a CopG protein. A fourth ORF exhibits a high nucleotide sequence identity to the SSV4 integrase gene, which suggests that it has been transferred to the plasmid from SSV4. A single point mutation within an otherwise identical 500 bp region...

  18. An Immunological Assay for Detection and Enumeration of Thermophilic Biomining Microorganisms

    OpenAIRE

    Amaro, Ana M.; Hallberg, Kevin B.; Lindström, E. Börje; Jerez, Carlos A.

    1994-01-01

    A specific, fast, and sensitive nonradioactive immunobinding assay for the detection and enumeration of the moderate thermophile Thiobacillus caldus and the thermophilic archaeon Sulfolobus acidocaldarius was developed. It employs enhanced chemiluminescence or peroxidase-conjugated immunoglobulins in a dot or slot blotting system and is very convenient for monitoring thermophilic bioleaching microorganisms in effluents from industrial bioleaching processes.

  19. An immunological assay for detection and enumeration of thermophilic biomining microorganisms.

    Science.gov (United States)

    Amaro, A M; Hallberg, K B; Lindström, E B; Jerez, C A

    1994-09-01

    A specific, fast, and sensitive nonradioactive immunobinding assay for the detection and enumeration of the moderate thermophile Thiobacillus caldus and the thermophilic archaeon Sulfolobus acidocaldarius was developed. It employs enhanced chemiluminescence or peroxidase-conjugated immunoglobulins in a dot or slot blotting system and is very convenient for monitoring thermophilic bioleaching microorganisms in effluents from industrial bioleaching processes. PMID:16349398

  20. Dicty_cDB: Contig-U09371-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available AANGDFAN KIGTYSVAVLANHHNIPFHCVAPMSTVDIKCLTGKDIPIEERSQTEVQGASGAFGQVRWA PKNSKTFNPAFDVTPMTLVTSIILDTGILTKNDLNKLSE*nkiffffs...VAPMSTVDIKCLTGKDIPIEERSQTEVQGASGAFGQVRWA PKNSKTFNPAFDVTPMTLVTSIILDTGILTKNDLNKLSE*nkiffffsekkkkk Frame C: llaywklifkiiinifff...ne) Sulfolobus islandicus Y.G.57.14... 99 1e-41 AE006641_2733( AE006641 |pid:none) Sulfolobus solfataricus P...-52 CP000867_1041( CP000867 |pid:none) Methanococcus maripaludis C6, c... 131 2e-52 (Q9UZ16) RecName: Full=Puta...AY596297 |pid:none) Haloarcula marismortui ATCC 430... 35 5.1 CR954205_430( CR954205 |pid:none) Ostreococcus tauri

  1. Dicty_cDB: Contig-U12238-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available yfyfyyfqslfcfknkikk*kktmte ttpttnetkeqvitpweveaapggsvdymklvdqfgstviseeliarfekvtgkrahhfl rrgiffshrdlkeildhhe...rcnw*iyistsskqhhvfqihshifsh*miqrlrifvv*lpca idqdpyfrmtrdiahrighqkpalihskffpalqghntkmsasdtnsavylsdtpdqvkd kikkhafs...rcnw*iyistsskqhhvfqihshifsh*miqrlrifvv*lpca idqdpyfrmtrdiahrighqkpalihskff...|pid:none) Sulfolobus solfataricus P2, comp... 170 2e-53 (Q97ZX0) RecName: Full=T...31 CP000505_582( CP000505 |pid:none) Thermofilum pendens Hrk 5, compl... 137 2e-30 AF542017_1( AF542017 |pid:none) Griff

  2. Extremophiles Survival to Simulated Space Conditions: An Astrobiology Model Study

    OpenAIRE

    Mastascusa, V.; Romano, I.; Di Donato, P; A. Poli; Della Corte, V.; Rotundi, A.; Bussoletti, E.; Quarto, M.; Pugliese, M.; Nicolaus, B

    2015-01-01

    In this work we investigated the ability of four extremophilic bacteria from Archaea and Bacteria domains to resist to space environment by exposing them to extreme conditions of temperature, UV radiation, desiccation coupled to low pressure generated in a Mars’ conditions simulator. All the investigated extremophilic strains (namely Sulfolobus solfataricus, Haloterrigena hispanica, Thermotoga neapolitana and Geobacillus thermantarcticus) showed a good resistance to the simulation of the temp...

  3. Application of direct PCR in rapid rDNA ITS haplotype determination of the hyperparasitic fungus Sphaeropsis visci (Botryosphaeriaceae)

    OpenAIRE

    Varga, Ildiko; Poczai, Péter; Cernák, István; Hyvönen, Jaakko

    2014-01-01

    Abstract Background The plant pathogenic fungus, Sphaeropsis visci a dark-spored species of Botryosphaeriaceae, which causes the leaf spot disease of the European mistletoe (Viscum album). This species seems to have potential as a tool for biological control of the hemiparasite. For the rapid detection of S. visci haplotypes we tested a direct PCR assay without prior DNA purification. This approach was based on a polymerase enzyme from the crenarchaeon Sulfolobus solfataricus engineered by fu...

  4. Structural and Functional Characterization of an Archaeal Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Complex for Antiviral Defense (CASCADE)

    DEFF Research Database (Denmark)

    Lintner, Nathanael G; Kerou, Melina; Brumfield, Susan K;

    2011-01-01

    In response to viral infection, many prokaryotes incorporate fragments of virus-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA. The...... Escherichia coli "CRISPR-associated complex for antiviral defense" (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5......a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2...

  5. Extremophiles Survival to Simulated Space Conditions: An Astrobiology Model Study

    Science.gov (United States)

    Mastascusa, V.; Romano, I.; Di Donato, P.; Poli, A.; Della Corte, V.; Rotundi, A.; Bussoletti, E.; Quarto, M.; Pugliese, M.; Nicolaus, B.

    2014-09-01

    In this work we investigated the ability of four extremophilic bacteria from Archaea and Bacteria domains to resist to space environment by exposing them to extreme conditions of temperature, UV radiation, desiccation coupled to low pressure generated in a Mars' conditions simulator. All the investigated extremophilic strains (namely Sulfolobus solfataricus, Haloterrigena hispanica, Thermotoga neapolitana and Geobacillus thermantarcticus) showed a good resistance to the simulation of the temperature variation in the space; on the other hand irradiation with UV at 254 nm affected only slightly the growth of H. hispanica, G. thermantarcticus and S. solfataricus; finally exposition to Mars simulated condition showed that H. hispanica and G. thermantarcticus were resistant to desiccation and low pressure.

  6. Steady state kinetic analysis of substrate specificity of glycoside hydrolases from families 13 and 38

    DEFF Research Database (Denmark)

    Nielsen, Jonas Willum

    Glycosidases are widespread in nature, where they perform a diverse range of functions. The glycoside hydrolase (GH) family 38, α-mannosidase II enzymes play a crucial role in mammalian cells, in the maturation of N-glycosylated proteins in the Golgi apparatus and in catabolism in cytosol and...... lysosomes of glycans originating from e.g. terminally misfolded proteins. A divalent metal ion typically resides in the active site, and is essential for enzyme activity. Recently, the α-mannosidase II from Sulfolobus solfataricus (ManA) was purified and characterized as a Zn2+-containing enzyme, but also...

  7. Archaeal promoter architecture and mechanism of gene activation.

    Science.gov (United States)

    Peng, Nan; Ao, Xiang; Liang, Yun Xiang; She, Qunxin

    2011-01-01

    Sulfolobus solfataricus and Sulfolobus islandicus contain several genes exhibiting D-arabinose-inducible expression and these systems are ideal for studying mechanisms of archaeal gene expression. At sequence level, only two highly conserved cis elements are present on the promoters: a regulatory element named ara box directing arabinose-inducible expression and the basal promoter element TATA, serving as the binding site for the TATA-binding protein. Strikingly, these promoters possess a modular structure that allows an essentially inactive basal promoter to be strongly activated. The invoked mechanisms include TFB (transcription factor B) recruitment by the ara-box-binding factor to activate gene expression and modulation of TFB recruitment efficiency to yield differential gene expression. PMID:21265754

  8. The Y-Family DNA Polymerase Dpo4 Uses a Template Slippage Mechanism To Create Single-Base Deletions

    Energy Technology Data Exchange (ETDEWEB)

    Y Wu; R Wilson; J Pata

    2011-12-31

    The Y-family polymerases help cells tolerate DNA damage by performing translesion synthesis, yet they also can be highly error prone. One distinctive feature of the DinB class of Y-family polymerases is that they make single-base deletion errors at high frequencies in repetitive sequences, especially those that contain two or more identical pyrimidines with a 5? flanking guanosine. Intriguingly, different deletion mechanisms have been proposed, even for two archaeal DinB polymerases that share 54% sequence identity and originate from two strains of Sulfolobus. To reconcile these apparent differences, we have characterized Dpo4 from Sulfolobus solfataricus using the same biochemical and crystallographic approaches that we have used previously to characterize Dbh from Sulfolobus acidocaldarius. In contrast to previous suggestions that Dpo4 uses a deoxynucleoside triphosphate (dNTP)-stabilized misalignment mechanism when creating single-base deletions, we find that Dpo4 predominantly uses a template slippage deletion mechanism when replicating repetitive DNA sequences, as was previously shown for Dbh. Dpo4 stabilizes the skipped template base in an extrahelical conformation between the polymerase and the little-finger domains of the enzyme. This contrasts with Dbh, in which the extrahelical base is stabilized against the surface of the little-finger domain alone. Thus, despite sharing a common deletion mechanism, these closely related polymerases use different contacts with the substrate to accomplish the same result.

  9. Dicty_cDB: Contig-U01609-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 06641_1560( AE006641 |pid:none) Sulfolobus solfataricus P2, com... 40 0.23 CT573071_1389( CT573071 |pid:none) Kuenenia stuttgartien...5_1109( AE016795 |pid:none) Vibrio vulnificus CMCP6 chromos... 39 0.39 CT573071_1444( CT573071 |pid:none) Kuenenia stuttgartien...veyi ATCC BAA-1116 chr... 37 1.9 CT573073_670( CT573073 |pid:none) Kuenenia stuttgartiensis gen...2_978( CT573072 |pid:none) Kuenenia stuttgartiensis genome ... 35 4.3 CT573073_346( CT573073 |pid:none) Kuenenia stuttgartiensis gen...A, c... 35 4.3 CT573071_2297( CT573071 |pid:none) Kuenenia stuttgartiensis genome... 35 4.3 CT573072_395( CT573072 |pid:none) Kuenen

  10. Monitoring Acidophilic Microbes with Real-Time Polymerase Chain Reaction (PCR) Assays

    Energy Technology Data Exchange (ETDEWEB)

    Frank F. Roberto

    2008-08-01

    Many techniques that are used to characterize and monitor microbial populations associated with sulfide mineral bioleaching require the cultivation of the organisms on solid or liquid media. Chemolithotrophic species, such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, or thermophilic chemolithotrophs, such as Acidianus brierleyi and Sulfolobus solfataricus can grow quite slowly, requiring weeks to complete efforts to identify and quantify these microbes associated with bioleach samples. Real-time PCR (polymerase chain reaction) assays in which DNA targets are amplified in the presence of fluorescent oligonucleotide primers, allowing the monitoring and quantification of the amplification reactions as they progress, provide a means of rapidly detecting the presence of microbial species of interest, and their relative abundance in a sample. This presentation will describe the design and use of such assays to monitor acidophilic microbes in the environment and in bioleaching operations. These assays provide results within 2-3 hours, and can detect less than 100 individual microbial cells.

  11. Structure and function of the translesion DNA polymerases and interactions with damaged DNA

    Directory of Open Access Journals (Sweden)

    F. Peter Guengerich

    2015-03-01

    Pre-steady-state kinetic analysis has been used to develop minimum kinetic models with rate constants of (the eight individual reaction steps in the catalytic cycle. The use of single-tryptophan mutants of Sulfolobus solfataricus Dpo4 and human (h pol κ has led to discernment of the steps for the conformation change (associated with dNTP binding and relocation and nucleotidyl transfer. X-ray crystal structures have been obtained for a number of the DNA adduct/DNA polymerase pairs in both binary and ternary complexes. Two isomeric etheno guanine adducts differ considerably in their interactions with DNA polymerases, explaining the base preferences. Further, even when several DNA polymerases cause the same mispairs with a single DNA adduct, the structural bases for this can differ considerably.

  12. Alpha-amylase from the Hyperthermophilic Archaeon Thermococcus thioreducens

    Science.gov (United States)

    Bernhardsdotter, E. C. M. J.; Pusey, M. L.; Ng, M. L.; Garriott, O. K.

    2003-01-01

    Extremophiles are microorganisms that thrive in, from an anthropocentric view, extreme environments such as hot springs. The ability of survival at extreme conditions has rendered enzymes from extremophiles to be of interest in industrial applications. One approach to producing these extremozymes entails the expression of the enzyme-encoding gene in a mesophilic host such as E.coli. This method has been employed in the effort to produce an alpha-amylase from a hyperthermophile (an organism that displays optimal growth above 80 C) isolated from a hydrothermal vent at the Rainbow vent site in the Atlantic Ocean. alpha-amylases catalyze the hydrolysis of starch to produce smaller sugars and constitute a class of industrial enzymes having approximately 25% of the enzyme market. One application for thermostable alpha-amylases is the starch liquefaction process in which starch is converted into fructose and glucose syrups. The a-amylase encoding gene from the hyperthermophile Thermococcus thioreducens was cloned and sequenced, revealing high similarity with other archaeal hyperthermophilic a-amylases. The gene encoding the mature protein was expressed in E.coli. Initial characterization of this enzyme has revealed an optimal amylolytic activity between 85-90 C and around pH 5.3-6.0.

  13. Components of calcium homeostasis in Archaeon Methanobacterium thermoautotrophicum

    International Nuclear Information System (INIS)

    The cells of Archaea are interesting from several points of view. Among others there are: (a) the evolutionary relationship to procaryotes and eucaryotes and (b) the involvement of Na+ and H+ gradient in archaeal bio-energetics. The observations are presented which are devoted to the description of components of Ca2+ homeostasis, an apparatus is vital for both procaryotic and eukaryotic organisms, in obligate anaerobe Methanobacterium thermoautotrophicum. This is, after the demonstration of the ATP-dependent Ca2+ transport in Halobacterium halobium membrane vesicles, the first complex description of processes of Ca2+ homeostasis in Archaea. The Ca2+ influx and efflux was measured using radionuclide 45Ca2+. The experiment were performed under strictly anaerobic conditions. The measurement of the membrane potential by means of 3H-tetraphenyl phosphonium chloride showed that the presence of Na+ depolarized the membrane from -110 to -60 mV. The growth of M. thermoautotrophicum and methanogenesis was suppressed but nor arrested by the presence EGTA suggesting that the Ca2+ homeostasis may be involved in controlling these cellular functions. The results indicate the presence of three components involved in establishing the Ca2+ homeostasis in cell of M. thermoautotrophicum. The first is the Ca2+-carrier mediating the CA2+ influx driven by the proton motive force or the membrane potential. The Ca2+ efflux is mediated by two transport systems, Na+/Ca2+ and H+/Ca2+ anti-porters. The evidence for the presence of the Ca2+-transporting ATPase was not obtained so far. (authors)

  14. Regulation of tryptophan operon expression in the archaeon Methanothermobacter thermautotrophicus.

    Science.gov (United States)

    Xie, Yunwei; Reeve, John N

    2005-09-01

    Conserved trp genes encode enzymes that catalyze tryptophan biosynthesis in all three biological domains, and studies of their expression in Bacteria and eukaryotes have revealed a variety of different regulatory mechanisms. The results reported here provide the first detailed description of an archaeal trp gene regulatory system. We have established that the trpEGCFBAD operon in Methanothermobacter thermautotrophicus is transcribed divergently from a gene (designated trpY) that encodes a tryptophan-sensitive transcription regulator. TrpY binds to TRP box sequences (consensus, TGTACA) located in the overlapping promoter regions between trpY and trpE, inhibiting trpY transcription in the absence of tryptophan and both trpY and trpEGCFBAD transcription in the presence of tryptophan. TrpY apparently inhibits trpY transcription by blocking RNA polymerase access to the site of trpY transcription initiation and represses trpEGCFBAD transcription by preventing TATA box binding protein (TBP) binding to the TATA box sequence. Given that residue 2 (W2) is the only tryptophan in TrpY and in TrpY homologues in other Euryarchaea and that there is only one tryptophan codon in the entire trpEGCFBAD operon (trpB encodes W175), expression of the trp operon may also be regulated in vivo by the supply of charged tRNA(Trp) available to translate the second codon of the trpY mRNA. PMID:16159776

  15. Regulation of Tryptophan Operon Expression in the Archaeon Methanothermobacter thermautotrophicus

    OpenAIRE

    Xie, Yunwei; Reeve, John N.

    2005-01-01

    Conserved trp genes encode enzymes that catalyze tryptophan biosynthesis in all three biological domains, and studies of their expression in Bacteria and eukaryotes have revealed a variety of different regulatory mechanisms. The results reported here provide the first detailed description of an archaeal trp gene regulatory system. We have established that the trpEGCFBAD operon in Methanothermobacter thermautotrophicus is transcribed divergently from a gene (designated trpY) that encodes a try...

  16. Elucidating the transcription cycle of the UV-inducible hyperthermophilic archaeal virus SSV1 by DNA microarrays.

    Science.gov (United States)

    Fröls, Sabrina; Gordon, Paul M K; Panlilio, Mayi Arcellana; Schleper, Christa; Sensen, Christoph W

    2007-08-15

    The spindle-shaped Sulfolobus virus SSV1 was the first of a series of unusual and uniquely shaped viruses isolated from hyperthermophilic Archaea. Using whole-genome microarrays we show here that the circular 15.5 kb DNA genome of SSV1 exhibits a chronological regulation of its transcription upon UV irradiation, reminiscent to the life cycles of bacteriophages and eukaryotic viruses. The transcriptional cycle starts with a small UV-specific transcript and continues with early transcripts on both its flanks. The late transcripts appear after the onset of viral replication and are extended to their full lengths towards the end of the approximately 8.5 h cycle. While we detected only small differences in genome-wide analysis of the host Sulfolobus solfataricus comparing infected versus uninfected strains, we found a marked difference with respect to the strength and speed of the general UV response of the host. Models for the regulation of the virus cycle, and putative functions of genes in SSV1 are presented. PMID:17467765

  17. Structure and cell biology of archaeal virus STIV.

    Science.gov (United States)

    Fu, Chi-yu; Johnson, Johnson E

    2012-04-01

    Recent investigations of archaeal viruses have revealed novel features of their structures and life cycles when compared to eukaryotic and bacterial viruses, yet there are structure-based unifying themes suggesting common ancestral relationships among dsDNA viruses in the three kingdoms of life. Sulfolobus solfataricus and the infecting virus Sulfolobus turreted icosahedral virus (STIV) is one of the well-established model systems to study archaeal virus replication and viral-host interactions. Reliable laboratory conditions to propagate STIV and available genetic tools allowed structural characterization of the virus and viral components that lead to the proposal of common capsid ancestry with PRD1 (bacteriophage), Adenovirus (eukaryotic virus) and PBCV (chlorellavirus). Microarray and proteomics approaches systematically analyzed viral replication and the corresponding host responses. Cellular cryo-electron tomography and thin-section EM studies uncovered the assembly and maturation pathway of STIV and revealed dramatic cellular ultra-structure changes upon infection. The viral-induced pyramid-like protrusions on cell surfaces represent a novel viral release mechanism and previously uncharacterized functions in viral replication. PMID:22482708

  18. Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs.

    Directory of Open Access Journals (Sweden)

    Sarit Edelheit

    2013-06-01

    Full Text Available The presence of 5-methylcytidine (m(5C in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, and comprehensive transcriptome-wide overview of m(5C across the three domains of life, we used bisulfite treatment on total RNA from both gram positive (B. subtilis and gram negative (E. coli bacteria, an archaeon (S. solfataricus and a eukaryote (S. cerevisiae, followed by massively parallel sequencing. We were able to recover most previously documented m(5C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m(5C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m(5C was absent were also discovered. Intriguingly, we detected m(5C sites within archaeal mRNAs, and identified a consensus motif of AUCGANGU that directs methylation in S. solfataricus. Our results, which were validated using m(5C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea, suggesting that this mode of post-transcriptional regulation extends beyond the eukaryotic domain.

  19. Harnessing type I and type III CRISPR-Cas systems for genome editing

    DEFF Research Database (Denmark)

    Li, Yingjun; Pan, Saifu; Zhang, Yan;

    2016-01-01

    -CRISPR array and a donor DNA containing a non-target sequence. Transformation of a pGE plasmid would yield two alternative fates to transformed cells: wild-type cells are to be targeted for chromosomal DNA degradation, leading to cell death, whereas those carrying the mutant gene would survive the cell killing...... and selectively retained as transformants. Using this strategy, different types of mutation were generated, including deletion, insertion and point mutations. We envision this method is readily applicable to different bacteria and archaea that carry an active CRISPR-Cas system of DNA interference provided...... report on harnessing a Type I or Type III system for genome editing. Here, a method was developed to repurpose both CRISPR-Cas systems for genetic manipulation in Sulfolobus islandicus, a thermophilic archaeon. A novel type of genome-editing plasmid (pGE) was constructed, carrying an artificial mini...

  20. Geranylgeranyl reductase and ferredoxin from Methanosarcina acetivorans are required for the synthesis of fully reduced archaeal membrane lipid in Escherichia coli cells.

    Science.gov (United States)

    Isobe, Keisuke; Ogawa, Takuya; Hirose, Kana; Yokoi, Takeru; Yoshimura, Tohru; Hemmi, Hisashi

    2014-01-01

    Archaea produce membrane lipids that typically possess fully saturated isoprenoid hydrocarbon chains attached to the glycerol moiety via ether bonds. They are functionally similar to, but structurally and biosynthetically distinct from, the fatty acid-based membrane lipids of bacteria and eukaryotes. It is believed that the characteristic lipid structure helps archaea survive under severe conditions such as extremely low or high pH, high salt concentrations, and/or high temperatures. We detail here the first successful production of an intact archaeal membrane lipid, which has fully saturated isoprenoid chains, in bacterial cells. The introduction of six phospholipid biosynthetic genes from a methanogenic archaeon, Methanosarcina acetivorans, in Escherichia coli enabled the host bacterium to synthesize the archaeal lipid, i.e., diphytanylglyceryl phosphoglycerol, while a glycerol modification of the phosphate group was probably catalyzed by endogenous E. coli enzymes. Reduction of the isoprenoid chains occurred only when archaeal ferredoxin was expressed with geranylgeranyl reductase, suggesting the role of ferredoxin as a specific electron donor for the reductase. This report is the first identification of a physiological reducer for archaeal geranylgeranyl reductase. On the other hand, geranylgeranyl reductase from the thermoacidophilic archaeon Sulfolobus acidocaldarius could, by itself, replace both its orthologue and ferredoxin from M. acetivorans, which indicated that an endogenous redox system of E. coli reduced the enzyme. PMID:24214941

  1. Biomining Microorganisms: Molecular Aspects and Applications in Biotechnology and Bioremediation

    Science.gov (United States)

    Jerez, Carlos A.

    The microbial solubilization of metals using chemolithoautotrophic microorganisms has successfully been used in industrial processes called biomining to extract metals such as copper, gold, uranium and others. The most studied leaching bacteria are from the genus Acidithiobacillus belonging to the Gram-negative γ-proteobacteria. Acidithiobacillus spp. obtain their energy from the oxidation of ferrous iron, elemental sulfur, or partially oxidized sulfur compounds. Other thermophilic archaeons capable of oxidizing sulfur and iron (II) have also been known for many years, and they are mainly from the genera Sulfolobus, Acidianus, Metallosphaera and Sulfurisphaera. Recently, some mesophilic iron (II)-oxidizing archaeons such as Ferroplasma acidiphilium and F. acidarmanus belonging to the Thermoplasmales have also been isolated and characterized. Recent studies of microorganisms consider them in their consortia, integrating fundamental biological knowledge with metagenomics, metaproteomics, and other data to obtain a global picture of how a microbial community functions. The understanding of microbial growth and activities in oxidizing metal ions will be useful for improving applied microbial biotechnologies such as biomining, bioshrouding, biomonitoring and bioremediation of metals in acidic environments.

  2. Studies on DNA Damage Response in Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Han, Wenyuan

    methyl methanesulfonate (MMS), and hydroxyurea (HU) that may not introduce DNA lesions directly. Comparison of the effects of the three drugs on S. islandicus cells showed that NQO and MMS led to DNA-less cell formation, while HU did not. In addition, the DNA-less cells were featured with increased side...... scattered light, damaged cell membrane and electron-dense area. During NQO and MMS treatment, degradation of chromatin proteins was coincided with DNA-less cell formation, suggesting their roles in protecting genomic DNA from massive degradation. Further, HU inhibited NQO-induced DSB formation and DNA...

  3. Global analysis of viral infection in an archaeal model system

    Directory of Open Access Journals (Sweden)

    JosephSteffens

    2012-12-01

    Full Text Available The origin and evolutionary relationship of viruses is poorly understood. This makes archaeal virus-host of particular interest because the hosts generally root near the base of phylogenetic trees, while some of the viruses have clear structural similarities to those that infect prokaryotic and eukaryotic cells. Despite the advantageous position for use in evolutionary studies, little is known about archaeal viruses or how they interact with their hosts, compared to viruses of bacteria and eukaryotes. In addition, many archaeal viruses have been isolated from extreme environments and present a unique opportunity for elucidating factors that are important for existence at the extremes.. In this article we focus on virus-host interactions using a proteomics approach to study Sulfolobus Turreted Icosahedral Virus (STIV infection of Sulfolobus solfataricus P2. Using cultures grown from the ATCC cell stock, a single cycle of STIV infection was sampled 6 times over a 72 hr period. More than 700 proteins were identified throughout the course of the experiments. Seventy one host proteins were found to change by nearly two-fold (p<0.05 with 40 becoming more abundant and 31 less abundant. The modulated proteins represent 30 different cell pathways and 14 COG groups. 2D gel analysis showed that changes in post translational modifications were a common feature of the affected proteins. The results from these studies showed that the prokaryotic antiviral adaptive immune system CRISPR associated proteins (CAS proteins were regulated in response to the virus infection. It was found that regulated proteins come from mRNAs with a shorter than average half-life. In addition, activity-based protein profiling (ABPP profiling on 2D gels showed caspase, hydrolase and tyrosine phosphatase enzyme activity labeling at the protein isoform level. Together, this data provides a more detailed global view of archaeal cellular responses to viral infection, demonstrates the

  4. The X-ray Crystal Structure of RNA Polymerase from Archaea

    Energy Technology Data Exchange (ETDEWEB)

    Hirata,A.; Klein, B.; Murakami, K.

    2008-01-01

    The transcription apparatus in Archaea can be described as a simplified version of its eukaryotic RNA polymerase (RNAP) II counterpart, comprising an RNAPII-like enzyme as well as two general transcription factors, the TATA-binding protein (TBP) and the eukaryotic TFIIB orthologue TFB. It has been widely understood that precise comparisons of cellular RNAP crystal structures could reveal structural elements common to all enzymes and that these insights would be useful in analysing components of each enzyme that enable it to perform domain-specific gene expression. However, the structure of archaeal RNAP has been limited to individual subunits3, 4. Here we report the first crystal structure of the archaeal RNAP from Sulfolobus solfataricus at 3.4 Angstroms resolution, completing the suite of multi-subunit RNAP structures from all three domains of life. We also report the high-resolution (at 1.76 Angstroms ) crystal structure of the D/L subcomplex of archaeal RNAP and provide the first experimental evidence of any RNAP possessing an iron-sulphur (Fe-S) cluster, which may play a structural role in a key subunit of RNAP assembly. The striking structural similarity between archaeal RNAP and eukaryotic RNAPII highlights the simpler archaeal RNAP as an ideal model system for dissecting the molecular basis of eukaryotic transcription.

  5. Multiple nucleic acid cleavage modes in divergent type III CRISPR systems

    Science.gov (United States)

    Zhang, Jing; Graham, Shirley; Tello, Agnes; Liu, Huanting; White, Malcolm F.

    2016-01-01

    CRISPR-Cas is an RNA-guided adaptive immune system that protects bacteria and archaea from invading nucleic acids. Type III systems (Cmr, Csm) have been shown to cleave RNA targets in vitro and some are capable of transcription-dependent DNA targeting. The crenarchaeon Sulfolobus solfataricus has two divergent subtypes of the type III system (Sso-IIID and a Cmr7-containing variant of Sso-IIIB). Here, we report that both the Sso-IIID and Sso-IIIB complexes cleave cognate RNA targets with a ruler mechanism and 6 or 12 nt spacing that relates to the organization of the Cas7 backbone. This backbone-mediated cleavage activity thus appears universal for the type III systems. The Sso-IIIB complex is also known to possess a distinct ‘UA’ cleavage mode. The predominant activity observed in vitro depends on the relative molar concentration of protein and target RNA. The Sso-IIID complex can cleave plasmid DNA targets in vitro, generating linear DNA products with an activity that is dependent on both the cyclase and HD nuclease domains of the Cas10 subunit, suggesting a role for both nuclease active sites in the degradation of double-stranded DNA targets. PMID:26801642

  6. Characterization of Fatty Acids in Crenarchaeota by GC-MS and NMR.

    Science.gov (United States)

    Hamerly, Timothy; Tripet, Brian; Wurch, Louie; Hettich, Robert L; Podar, Mircea; Bothner, Brian; Copié, Valérie

    2015-01-01

    Lipids composed of condensed isoprenyl units connected to glycerol backbones by ether linkages are a distinguishing feature of Archaea. Data suggesting that fatty acids with linear hydrocarbon chains are present in some Archaea have been available for decades. However, lack of genomic and biochemical evidence for the metabolic machinery required to synthesize and degrade fatty acids has left the field unclear on this potentially significant biochemical aspect. Because lipids are energy currency and cell signaling molecules, their presence in Archaea is significant for understanding archaeal biology. A recent large-scale bioinformatics analysis reignited the debate as to the importance of fatty acids in Archaea by presenting genetic evidence for the presence of enzymes required for anabolic and catabolic fatty acid metabolism across the archaeal domain. Here, we present direct biochemical evidence from gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy for the presence of fatty acids in two members of the Crenarchaeota, Sulfolobus solfataricus and Ignicoccus hospitalis. This is the first report providing biochemical data for the existence of fatty acids in these Crenarchaeota, opening new discussions on energy balance and the potential for the discovery of new thermostable enzymes for industry. PMID:26880868

  7. Characterization of Fatty Acids in Crenarchaeota by GC-MS and NMR

    Directory of Open Access Journals (Sweden)

    Timothy Hamerly

    2015-01-01

    Full Text Available Lipids composed of condensed isoprenyl units connected to glycerol backbones by ether linkages are a distinguishing feature of Archaea. Data suggesting that fatty acids with linear hydrocarbon chains are present in some Archaea have been available for decades. However, lack of genomic and biochemical evidence for the metabolic machinery required to synthesize and degrade fatty acids has left the field unclear on this potentially significant biochemical aspect. Because lipids are energy currency and cell signaling molecules, their presence in Archaea is significant for understanding archaeal biology. A recent large-scale bioinformatics analysis reignited the debate as to the importance of fatty acids in Archaea by presenting genetic evidence for the presence of enzymes required for anabolic and catabolic fatty acid metabolism across the archaeal domain. Here, we present direct biochemical evidence from gas chromatography-mass spectrometry (GC-MS and nuclear magnetic resonance (NMR spectroscopy for the presence of fatty acids in two members of the Crenarchaeota, Sulfolobus solfataricus and Ignicoccus hospitalis. This is the first report providing biochemical data for the existence of fatty acids in these Crenarchaeota, opening new discussions on energy balance and the potential for the discovery of new thermostable enzymes for industry.

  8. CRISPRTarget: bioinformatic prediction and analysis of crRNA targets.

    Science.gov (United States)

    Biswas, Ambarish; Gagnon, Joshua N; Brouns, Stan J J; Fineran, Peter C; Brown, Chris M

    2013-05-01

    The bacterial and archaeal CRISPR/Cas adaptive immune system targets specific protospacer nucleotide sequences in invading organisms. This requires base pairing between processed CRISPR RNA and the target protospacer. For type I and II CRISPR/Cas systems, protospacer adjacent motifs (PAM) are essential for target recognition, and for type III, mismatches in the flanking sequences are important in the antiviral response. In this study, we examine the properties of each class of CRISPR. We use this information to provide a tool (CRISPRTarget) that predicts the most likely targets of CRISPR RNAs (http://bioanalysis.otago.ac.nz/CRISPRTarget). This can be used to discover targets in newly sequenced genomic or metagenomic data. To test its utility, we discover features and targets of well-characterized Streptococcus thermophilus and Sulfolobus solfataricus type II and III CRISPR/Cas systems. Finally, in Pectobacterium species, we identify new CRISPR targets and propose a model of temperate phage exposure and subsequent inhibition by the type I CRISPR/Cas systems. PMID:23492433

  9. Expression and Characterization of the RKOD DNA Polymerase in Pichia pastoris.

    Directory of Open Access Journals (Sweden)

    Fei Wang

    Full Text Available The present study assessed high-level expression of the KOD DNA polymerase in Pichia pastoris. Thermococcus kodakaraensis KOD1 is a DNA polymerase that is widely used in PCR. The DNA coding sequence of KOD was optimized based on the codon usage bias of P. pastoris and synthesized by overlapping PCR, and the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus was fused to the C-terminus of KOD. The resulting novel gene was cloned into a pHBM905A vector and introduced into P. pastoris GS115 for secretory expression. The yield of the target protein reached approximately 250 mg/l after a 6-d induction with 1% (v/v methanol in shake flasks. This yield is much higher than those of other DNA polymerases expressed heterologously in Escherichia coli. The recombinant enzyme was purified, and its enzymatic features were studied. Its specific activity was 19,384 U/mg. The recombinant KOD expressed in P. pastoris exhibited excellent thermostability, extension rate and fidelity. Thus, this report provides a simple, efficient and economic approach to realize the production of a high-performance thermostable DNA polymerase on a large scale. This is the first report of the expression in yeast of a DNA polymerase for use in PCR.

  10. Structural Basis for Conserved Regulation and Adaptation of the Signal Recognition Particle Targeting Complex.

    Science.gov (United States)

    Wild, Klemens; Bange, Gert; Motiejunas, Domantas; Kribelbauer, Judith; Hendricks, Astrid; Segnitz, Bernd; Wade, Rebecca C; Sinning, Irmgard

    2016-07-17

    The signal recognition particle (SRP) is a ribonucleoprotein complex with a key role in targeting and insertion of membrane proteins. The two SRP GTPases, SRP54 (Ffh in bacteria) and FtsY (SRα in eukaryotes), form the core of the targeting complex (TC) regulating the SRP cycle. The architecture of the TC and its stimulation by RNA has been described for the bacterial SRP system while this information is lacking for other domains of life. Here, we present the crystal structures of the GTPase heterodimers of archaeal (Sulfolobus solfataricus), eukaryotic (Homo sapiens), and chloroplast (Arabidopsis thaliana) SRP systems. The comprehensive structural comparison combined with Brownian dynamics simulations of TC formation allows for the description of the general blueprint and of specific adaptations of the quasi-symmetric heterodimer. Our work defines conserved external nucleotide-binding sites for SRP GTPase activation by RNA. Structural analyses of the GDP-bound, post-hydrolysis states reveal a conserved, magnesium-sensitive switch within the I-box. Overall, we provide a general model for SRP cycle regulation by RNA. PMID:27241309

  11. Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Yingang, E-mail: fengyg@qibebt.ac.cn [Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101 (China); Song, Xiaxia [Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Lin, Jinzhong [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Xuan, Jinsong [Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Cui, Qiu [Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101 (China); Wang, Jinfeng [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-07-18

    Highlights: • The archaea-specific ribosomal protein L46a has no homology to known proteins. • Three dimensional structure and backbone dynamics of L46a were determined by NMR. • The structure of L46a represents a novel protein fold. • A potential rRNA-binding surface on L46a was identified. • The potential position of L46a on the ribosome was proposed. - Abstract: Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae.

  12. Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

    International Nuclear Information System (INIS)

    Highlights: • The archaea-specific ribosomal protein L46a has no homology to known proteins. • Three dimensional structure and backbone dynamics of L46a were determined by NMR. • The structure of L46a represents a novel protein fold. • A potential rRNA-binding surface on L46a was identified. • The potential position of L46a on the ribosome was proposed. - Abstract: Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae

  13. NurA Is Endowed with Endo- and Exonuclease Activities that Are Modulated by HerA: New Insight into Their Role in DNA-End Processing.

    Science.gov (United States)

    De Falco, Mariarosaria; Catalano, Federico; Rossi, Mosè; Ciaramella, Maria; De Felice, Mariarita

    2015-01-01

    The nuclease NurA and the ATPase HerA are present in all known thermophilic archaea and cooperate with the highly conserved MRE11/RAD50 proteins to facilitate efficient DNA double-strand break end processing during homologous recombinational repair. However, contradictory results have been reported on the exact activities and mutual dependence of these two enzymes. To understand the functional relationship between these two enzymes we deeply characterized Sulfolobus solfataricus NurA and HerA proteins. We found that NurA is endowed with exo- and endonuclease activities on various DNA substrates, including linear (single-stranded and double stranded) as well as circular molecules (single stranded and supercoiled double-stranded). All these activities are not strictly dependent on the presence of HerA, require divalent ions (preferably Mn2+), and are inhibited by the presence of ATP. The endo- and exonculease activities have distinct requirements: whereas the exonuclease activity on linear DNA fragments is stimulated by HerA and depends on the catalytic D58 residue, the endonuclease activity on circular double-stranded DNA is HerA-independent and is not affected by the D58A mutation. On the basis of our results we propose a mechanism of action of NurA/HerA complex during DNA end processing. PMID:26560692

  14. NurA Is Endowed with Endo- and Exonuclease Activities that Are Modulated by HerA: New Insight into Their Role in DNA-End Processing.

    Directory of Open Access Journals (Sweden)

    Mariarosaria De Falco

    Full Text Available The nuclease NurA and the ATPase HerA are present in all known thermophilic archaea and cooperate with the highly conserved MRE11/RAD50 proteins to facilitate efficient DNA double-strand break end processing during homologous recombinational repair. However, contradictory results have been reported on the exact activities and mutual dependence of these two enzymes. To understand the functional relationship between these two enzymes we deeply characterized Sulfolobus solfataricus NurA and HerA proteins. We found that NurA is endowed with exo- and endonuclease activities on various DNA substrates, including linear (single-stranded and double stranded as well as circular molecules (single stranded and supercoiled double-stranded. All these activities are not strictly dependent on the presence of HerA, require divalent ions (preferably Mn2+, and are inhibited by the presence of ATP. The endo- and exonculease activities have distinct requirements: whereas the exonuclease activity on linear DNA fragments is stimulated by HerA and depends on the catalytic D58 residue, the endonuclease activity on circular double-stranded DNA is HerA-independent and is not affected by the D58A mutation. On the basis of our results we propose a mechanism of action of NurA/HerA complex during DNA end processing.

  15. An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination.

    Science.gov (United States)

    Del Giudice, Immacolata; Coppolecchia, Rossella; Merone, Luigia; Porzio, Elena; Carusone, Teresa Maria; Mandrich, Luigi; Worek, Franz; Manco, Giuseppe

    2016-04-01

    In vitro evolution of enzymes represents a powerful device to evolve new or to improve weak enzymatic functions. In the present work a semi-rational engineering approach has been used to design an efficient and thermostable organophosphate hydrolase, starting from a lactonase scaffold (SsoPox from Sulfolobus solfataricus). In particular, by in vitro evolution of the SsoPox ancillary promiscuous activity, the triple mutant C258L/I261F/W263A has been obtained which, retaining its inherent stability, showed an enhancement of its hydrolytic activity on paraoxon up to 300-fold, achieving absolute values of catalytic efficiency up to 10(5) M(-1) s(-1) . The kinetics and structural determinants of this enhanced activity were thoroughly investigated and, in order to evaluate its potential biotechnological applications, the mutant was tested in formulations of different solvents (methanol or ethanol) or detergents (SDS or a commercial soap) for the cleaning of pesticide-contaminated surfaces. Biotechnol. Bioeng. 2016;113: 724-734. © 2015 Wiley Periodicals, Inc. PMID:26416557

  16. Solution Structure of Archaeoglobus fulgidis Peptidyl-tRNA Hydrolase(Pth2) Provides Evidence for an Extensive Conserved Family of Pth2 Enzymes in Archaea, Bacteria and Eukaryotes.

    Energy Technology Data Exchange (ETDEWEB)

    Powers, Robert; Mirkovic, Nebojsa; Goldsmith-Fischman, Sharon; Acton, Thomas; Chiang, Yiwen; Huang, Yuanpeng; Ma, LiChung; Rajan, Paranji K.; Cort, John R.; Kennedy, Michael A.; Liu, Jinfeng; Rost, Burkhard; Honig, Barry; Murray, Diana; Montelione, Gaetano

    2005-11-01

    The solution structure of protein AF2095 from the thermophilic archaea Archaeglobus fulgidis, a 123-residue (13.6 kDa) protein, has been determined by NMR methods. The structure of AF2095 is comprised of four a-helices and a mixed b-sheet consisting of four parallel and anti-parallel b-strands, where the a-helices sandwich the b-sheet. Sequence and structural comparison of AF2095 with proteins from Homo sapiens, Methanocaldococcus jannaschii and Sulfolobus solfataricus, reveals that AF2095 is a peptidyl-tRNA hydrolase (Pth2). This structural comparison also identifies putative catalytic residues and a tRNA interaction region for AF2095. The structure of AF2095 is also similar to the structure of protein TA0108 from archaea Thermoplasma acidophilum, which is deposited in the Protein Database but not functionally annotated. The NMR structure of AF2095 has been further leveraged to obtain good quality structural models for 55 other proteins. Although earlier studies have proposed that the Pth2 protein family is restricted to archeal and eukaryotic organisms, the similarity of the AF2095 structure to human Pth2, the conservation of key active-site residues, and the good quality of the resulting homology models demonstrate a large family of homologous Pth2 proteins that are conserved in eukaryotic, archaeal and bacterial organisms, providing novel insights in the evolution of the Pth and Pth2 enzyme families.

  17. Conservation of chromosomal arrangement among three strains of the genetically unstable archaeon Halobacterium salinarium.

    Science.gov (United States)

    Hackett, N R; Bobovnikova, Y; Heyrovska, N

    1994-12-01

    Phenotypic variants of Halobacterium salinarium NRC-1 arise at a frequency of 10(-2). These result from transpositions of halobacterial insertion sequences and rearrangements mediated by halobacterial insertion sequences. We have tested the hypothesis that such mutations are confined to only a portion of the genome by comparing the chromosomal restriction map of H. salinarium NRC-1 and that of the derivative S9, which was made in 1969. The two chromosomes were mapped by using two-dimensional pulsed-field gel electrophoresis and the restriction enzymes AflII, AseI, and DraI. A comparison of the two deduced maps showed a domain of about 210 kbp to be subject to many rearrangements, including an inversion in S9 relative to NRC-1. However, the rest of the chromosome was conserved among NRC-1, S9, and an independent Halobacterium isolate, GRB, previously mapped by St. Jean et al. (A. St. Jean, B. A. Trieselmann, and R. L. Charlebois, Nucleic Acids Res. 22:1476-1483, 1994). This concurs with data from eubacteria suggesting strong selective forces maintaining gene order even in the face of rearrangement events occurring at a high frequency. PMID:8002597

  18. Conservation of chromosomal arrangement among three strains of the genetically unstable archaeon Halobacterium salinarium.

    OpenAIRE

    Hackett, N R; Bobovnikova, Y; Heyrovska, N

    1994-01-01

    Phenotypic variants of Halobacterium salinarium NRC-1 arise at a frequency of 10(-2). These result from transpositions of halobacterial insertion sequences and rearrangements mediated by halobacterial insertion sequences. We have tested the hypothesis that such mutations are confined to only a portion of the genome by comparing the chromosomal restriction map of H. salinarium NRC-1 and that of the derivative S9, which was made in 1969. The two chromosomes were mapped by using two-dimensional ...

  19. Identifying Potential Mechanisms Enabling Acidophily in the Ammonia-Oxidizing Archaeon “Candidatus Nitrosotalea devanaterra”

    Science.gov (United States)

    Sayavedra-Soto, Luis A.; Gallois, Nicolas; Schouten, Stefan; Stein, Lisa Y.; Prosser, James I.; Nicol, Graeme W.

    2016-01-01

    Ammonia oxidation is the first and rate-limiting step in nitrification and is dominated by two distinct groups of microorganisms in soil: ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). AOA are often more abundant than AOB and dominate activity in acid soils. The mechanism of ammonia oxidation under acidic conditions has been a long-standing paradox. While high rates of ammonia oxidation are frequently measured in acid soils, cultivated ammonia oxidizers grew only at near-neutral pH when grown in standard laboratory culture. Although a number of mechanisms have been demonstrated to enable neutrophilic AOB growth at low pH in the laboratory, these have not been demonstrated in soil, and the recent cultivation of the obligately acidophilic ammonia oxidizer “Candidatus Nitrosotalea devanaterra” provides a more parsimonious explanation for the observed high rates of activity. Analysis of the sequenced genome, transcriptional activity, and lipid content of “Ca. Nitrosotalea devanaterra” reveals that previously proposed mechanisms used by AOB for growth at low pH are not essential for archaeal ammonia oxidation in acidic environments. Instead, the genome indicates that “Ca. Nitrosotalea devanaterra” contains genes encoding both a predicted high-affinity substrate acquisition system and potential pH homeostasis mechanisms absent in neutrophilic AOA. Analysis of mRNA revealed that candidate genes encoding the proposed homeostasis mechanisms were all expressed during acidophilic growth, and lipid profiling by high-performance liquid chromatography–mass spectrometry (HPLC-MS) demonstrated that the membrane lipids of “Ca. Nitrosotalea devanaterra” were not dominated by crenarchaeol, as found in neutrophilic AOA. This study for the first time describes a genome of an obligately acidophilic ammonia oxidizer and identifies potential mechanisms enabling this unique phenotype for future biochemical characterization. PMID:26896134

  20. Identifying Potential Mechanisms Enabling Acidophily in the Ammonia-Oxidizing Archaeon "Candidatus Nitrosotalea devanaterra".

    Science.gov (United States)

    Lehtovirta-Morley, Laura E; Sayavedra-Soto, Luis A; Gallois, Nicolas; Schouten, Stefan; Stein, Lisa Y; Prosser, James I; Nicol, Graeme W

    2016-05-01

    Ammonia oxidation is the first and rate-limiting step in nitrification and is dominated by two distinct groups of microorganisms in soil: ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). AOA are often more abundant than AOB and dominate activity in acid soils. The mechanism of ammonia oxidation under acidic conditions has been a long-standing paradox. While high rates of ammonia oxidation are frequently measured in acid soils, cultivated ammonia oxidizers grew only at near-neutral pH when grown in standard laboratory culture. Although a number of mechanisms have been demonstrated to enable neutrophilic AOB growth at low pH in the laboratory, these have not been demonstrated in soil, and the recent cultivation of the obligately acidophilic ammonia oxidizer "Candidatus Nitrosotalea devanaterra" provides a more parsimonious explanation for the observed high rates of activity. Analysis of the sequenced genome, transcriptional activity, and lipid content of "Ca Nitrosotalea devanaterra" reveals that previously proposed mechanisms used by AOB for growth at low pH are not essential for archaeal ammonia oxidation in acidic environments. Instead, the genome indicates that "Ca Nitrosotalea devanaterra" contains genes encoding both a predicted high-affinity substrate acquisition system and potential pH homeostasis mechanisms absent in neutrophilic AOA. Analysis of mRNA revealed that candidate genes encoding the proposed homeostasis mechanisms were all expressed during acidophilic growth, and lipid profiling by high-performance liquid chromatography-mass spectrometry (HPLC-MS) demonstrated that the membrane lipids of "Ca Nitrosotalea devanaterra" were not dominated by crenarchaeol, as found in neutrophilic AOA. This study for the first time describes a genome of an obligately acidophilic ammonia oxidizer and identifies potential mechanisms enabling this unique phenotype for future biochemical characterization. PMID:26896134

  1. LUCA, or just a conserved Archaeon?: Comments on Xue et al. (2003)

    Czech Academy of Sciences Publication Activity Database

    Čejchan, Petr

    2004-01-01

    Roč. 333, Supplement (2004), s. 47-50. ISSN 0378-1119 R&D Projects: GA ČR(CZ) GA205/03/1124 Keywords : Last universal common ancestor * universal tree * phylogeny Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.705, year: 2004

  2. Variation of the virus-related elements within syntenic genomes of the hyperthermophilic archaeon aeropyrum

    DEFF Research Database (Denmark)

    Daifuku, Takashi; Yoshida, Takashi; Kitamura, Takayuki;

    2013-01-01

    having stable genomes, interference of synteny occurred with two proviruses, A. pernix spindle-shaped virus 1 (APSV1) and A. pernix ovoid virus 1 (APOV1), and clustered regularly interspaced short palindromic repeat (CRISPR) elements. Spacer sequences derived from the A. camini CRISPR showed significant...

  3. A phytoene desaturase homolog gene from the methanogenic archaeon Methanosarcina acetivorans is responsible for hydroxyarchaeol biosynthesis.

    Science.gov (United States)

    Mori, Takeshi; Isobe, Keisuke; Ogawa, Takuya; Yoshimura, Tohru; Hemmi, Hisashi

    2015-10-16

    Hydroxyarchaeols are the typical core structures of archaeal membrane lipids uniquely produced by a limited number of methanogenic lineages, which are mainly classified in orders Methanosarcinales and Methanococcales. However, the biosynthetic machinery that is used for the biosynthesis of hydroxyarcheol core lipids has not been discovered. In this study, the ma0127 gene from Methanosarcina acetivorans, which encodes a phytoene desaturase-like protein, was found to be responsible for the hydration of a geranylgeranyl group in an archaeal-lipid precursor, sn-2,3-O-digeranylgeranylglyceryl phosphoglycerol, produced in Escherichia coli cells expressing several archaeal enzymes. LC-ESI-tandem-MS analyses proved that hydration occurs at the 2',3'-double bond of the geranylgeranyl group, yielding a 3'-hydroxylated lipid precursor. This result suggests that the encoded protein MA0127 is a hydratase involved in hydroxyarchaeol biosynthesis, because M. acetivorans is known to produce hydroxyarchaeol core lipids with a 3'-hydroxyphytanyl group. Furthermore, the distribution of the putative orthologs of ma0127 among methanogens is generally in good agreement with that of hydroxyarchaeol producers, including anaerobic methanotrophs (ANMEs). PMID:26361140

  4. Protein modification in archaeon%古菌蛋白质修饰研究进展

    Institute of Scientific and Technical Information of China (English)

    卢化; 金城

    2014-01-01

    20世纪50年代中期,在古菌的表层(S-层)首次发现了糖蛋白;21世纪初又在空肠弯曲菌(Campylobacter jejuni)中发现了蛋白质N-糖基化修饰.由此,同行开始认识到,蛋白质的糖基化修饰广泛存在于古菌、细菌及真核生物三域中.近十年来,古菌蛋白质糖基化修饰的研究取得了进展,特别是古菌蛋白质N-糖基化修饰研究进展快速.但对古菌糖蛋白O-糖基化修饰和脂修饰的了解甚少.本文综述了古菌蛋白质糖基化修饰的研究进展.

  5. Thermococcus acidaminovorans sp. nov., a new hyperthermophilic alkalophilic archaeon growing on amino acids.

    Science.gov (United States)

    Dirmeier, R; Keller, M; Hafenbradl, D; Braun, F J; Rachel, R; Burggraf, S; Stetter, K O

    1998-05-01

    From a shallow marine hydrothermal system at Vulcano (Italy), a new hyperthermophilic member of the Archaea was isolated. The cells are coccoid - shaped and possess up to five flagella. They grow between 56 degrees and 93 degrees C (optimum 85 degrees C) and pH 5.0-9.5 (optimum 9.0). The organism is strictly anaerobic and grows heterotrophically on defined amino acids and complex organic substrates such as casamino acids, yeast extract, peptone, meat extract, tryptone, and casein. Polysulfide and elemental sulfur are reduced to H2S. In the absence of polysulfide or elemental sulfur, the isolate grows at a significantly reduced rate. Growth is not influenced by the presence of H2. DNA- DNA hybridization and 16S rRNA partial sequences indicated that the new isolate belongs to the genus Thermococcus, and represents a new species, Thermococcus acidaminovorans. The type stain is isolate AEDII10 (DSM 11906). PMID:9672685

  6. Halorhabdus rudnickae sp. nov., a halophilic archaeon isolated from a salt mine borehole in Poland.

    Science.gov (United States)

    Albuquerque, Luciana; Kowalewicz-Kulbat, Magdalena; Drzewiecka, Dominika; Stączek, Paweł; d'Auria, Giuseppe; Rosselló-Móra, Ramon; da Costa, Milton S

    2016-03-01

    Two halophilic archaea, designated strains WSM-64(T) and WSM-66, were isolated from a sample taken from a borehole in the currently unexploited Barycz mining area belonging to the "Wieliczka" Salt Mine Company, in Poland. Strains are red pigmented and form non-motile cocci that stain Gram-negative. Strains WSM-64(T) and WSM-66 showed optimum growth at 40°C, in 20% NaCl and at pH 6.5-7.5. The strains were facultative anaerobes. The major polar lipids of the two strains were phosphatidylglycerol (PG2), phosphatidylglycerol phosphate methyl ester (PGP-Me) and sulfated diglycosyl diether (S-DGD). Menaquinone MK-8 was the major respiratory quinone. The DNA G+C content of strain WSM-64(T) was 61.2mol% by HPLC method; 61.0mol% by genome sequencing. Analysis of the almost complete 16S rRNA gene sequence indicated that the strains WSM-64(T) and WSM-66 (99.7% identity) represented a member of the genus Halorhabdus in the family Halobacteriaceae. Both strains formed a distinct cluster and were most closely related to Halorhabdus tiamatea SARL4B(T) and Halorhabdus utahensis AX-2(T) (DSM 12940(T)) (95.4% and 95.6%, respectively). ANI values of WSM-64(T) with the closest relative type strains were <78.5%. Based on 16S rRNA gene sequence and whole genome analyses, physiological and biochemical characteristics we describe a new species represented by strain WSM-64(T) (=DSM 29498(T) =CECT 8673(T)) for which we propose the name Halorhabdus rudnickae sp. nov. PMID:26749115

  7. Differential transport properties of D-leucine and L-leucine in the archaeon, Halobacterium salinarum.

    Science.gov (United States)

    Tanaka, M; Mukohata, Y; Yuasa, S

    2000-04-01

    The transport of D-leucine was compared with that of L-leucine in Halobacterium salinarum. When a high-outside/low-inside Na+ gradient was imposed, D-leucine as well as L-leucine accumulated in envelope vesicles, supporting the hypothesis that D-leucine is transported via a symport system along with Na+. Kinetic analyses, including inhibition experiments, indicated that both enantiomers are transported via a common carrier. However, a Hill plot indicated a single binding site for Na+ during L-leucine transport, but dual binding sites for Na+ during D-leucine transport. Furthermore, D-leucine transport was dependent on electrical membrane potential, suggesting that a transporter bound with D-leucine is positively charged. L-leucine transport was slightly, if at all, dependent on membrane potential, suggesting that a transporter bound with L-leucine is electrically neutral. These results indicate that the leucine carrier in Halobacterium salinarum translocates two moles of Na+ per mole of D-leucine, and one mole of Na+ per mole of L-leucine. PMID:10779875

  8. Inhibitory Effect of Maillard Reaction Products on Growth of the Aerobic Marine Hyperthermophilic Archaeon Aeropyrum pernix

    OpenAIRE

    Kim, Kee Woung; Lee, Sun Bok

    2003-01-01

    It was found that the growth of Aeropyrum pernix was severely inhibited in a medium containing reducing sugars and tryptone due to the formation of Maillard reaction products. The rate of the Maillard browning reaction was markedly enhanced under aerobic conditions, and the addition of Maillard reaction products to the culture medium caused fatal growth inhibition.

  9. Genomic Analysis of the Archaeon Ferroplasma acidarmanus: New Insights Into the Evolution of Arsenic Resistance

    Science.gov (United States)

    Gihring, T. M.; Banfield, J. F.

    2001-12-01

    Arsenic resistance allows microbes to live in As-rich solutions and in proximity to As-bearing minerals and thus, to impact dissolution and precipitation reactions. As-resistance is a characteristic of both Bacteria and Archaea. In some cases, it is conferred by the acquisition of a plasmid, in other instances the genes are located on the chromosome. Through analysis of newly acquired genomic data for the highly arsenic-resistant, iron-oxidizing extreme acidophile Ferroplasma acidarmanus and genomic data for other organisms we can gain insight into the mechanisms, origin, and evolution of genes that confer arsenic resistance. The deduced protein sequences of all known arsenic resistance genes were compiled from genomic and protein databases. Sequence alignments and phylogenic analyses were performed. Comparisons of arsenite efflux pump (ArsB) and 16S rDNA phylogenies indicated a parallel evolutionary history for the two genes. Results indicate that the As(III) efflux pump was present in the ancestor common to the Bacterial and Archaeal domains. This pump, conferring resistance to arsenite, was probably important to early life living in metal-rich environments. Previous work has shown that the gene encoding for ArsA, an ATPase that increases the efficiency of arsenic efflux, clearly arose via gene duplication. Ferroplasma and related Thermoplasmales group organisms contain a pre-duplication subunit of uncertain function. ArsA phylogeny shows two separate and distinct lines of evolution for the chromosomal- and plasmid-based proteins. This study provides the first evidence of metal resistance developing early in the evolution of life and gives new insights into the evolutionary history of the arsenic resistance proteins.

  10. A novel ammonia-oxidizing archaeon from wastewater treatment plant: Its enrichment, physiological and genomic characteristics

    Science.gov (United States)

    Li, Yuyang; Ding, Kun; Wen, Xianghua; Zhang, Bing; Shen, Bo; Yang, Yunfeng

    2016-03-01

    Ammonia-oxidizing archaea (AOA) are recently found to participate in the ammonia removal processes in wastewater treatment plants (WWTPs), similar to their bacterial counterparts. However, due to lack of cultivated AOA strains from WWTPs, their functions and contributions in these systems remain unclear. Here we report a novel AOA strain SAT1 enriched from activated sludge, with its physiological and genomic characteristics investigated. The maximal 16S rRNA gene similarity between SAT1 and other reported AOA strain is 96% (with “Ca. Nitrosotenuis chungbukensis”), and it is affiliated with Wastewater Cluster B (WWC-B) based on amoA gene phylogeny, a cluster within group I.1a and specific for activated sludge. Our strain is autotrophic, mesophilic (25 °C–33 °C) and neutrophilic (pH 5.0–7.0). Its genome size is 1.62 Mb, with a large fragment inversion (accounted for 68% genomic size) inside. The strain could not utilize urea due to truncation of the urea transporter gene. The lack of the pathways to synthesize usual compatible solutes makes it intolerant to high salinity (>0.03%), but could adapt to low salinity (0.005%) environments. This adaptation, together with possibly enhanced cell-biofilm attachment ability, makes it suitable for WWTPs environment. We propose the name “Candidatus Nitrosotenuis cloacae” for the strain SAT1.

  11. Activation of methanogenesis by cadmium in the marine archaeon Methanosarcina acetivorans.

    Directory of Open Access Journals (Sweden)

    Elizabeth Lira-Silva

    Full Text Available Methanosarcina acetivorans was cultured in the presence of CdCl(2 to determine the metal effect on cell growth and biogas production. With methanol as substrate, cell growth and methane synthesis were not altered by cadmium, whereas with acetate, cadmium slightly increased both, growth and methane rate synthesis. In cultures metabolically active, incubations for short-term (minutes with 10 µM total cadmium increased the methanogenesis rate by 6 and 9 folds in methanol- and acetate-grown cells, respectively. Cobalt and zinc but not copper or iron also activated the methane production rate. Methanogenic carbonic anhydrase and acetate kinase were directly activated by cadmium. Indeed, cells cultured in 100 µM total cadmium removed 41-69% of the heavy metal from the culture and accumulated 231-539 nmol Cd/mg cell protein. This is the first report showing that (i Cd(2+ has an activating effect on methanogenesis, a biotechnological relevant process in the bio-fuels field; and (ii a methanogenic archaea is able to remove a heavy metal from aquatic environments.

  12. Did group II intron proliferation in an endosymbiont-bearing archaeon create eukaryotes?

    Directory of Open Access Journals (Sweden)

    Poole Anthony M

    2006-12-01

    Full Text Available Abstract Martin & Koonin recently proposed that the eukaryote nucleus evolved as a quality control mechanism to prevent ribosome readthrough into introns. In their scenario, the bacterial ancestor of mitochondria was resident in an archaeal cell, and group II introns (carried by the fledgling mitochondrion inserted into coding regions in the archaeal host genome. They suggest that if transcription and translation were coupled, and because splicing is expected to have been slower than translation, the effect of insertion would have been ribosome readthrough into introns, resulting in production of aberrant proteins. The emergence of the nuclear compartment would thus have served to separate transcription and splicing from translation, thereby alleviating this problem. In this article, I argue that Martin & Koonin's model is not compatible with current knowledge. The model requires that group II introns would spread aggressively through an archaeal genome. It is well known that selfish elements can spread through an outbreeding sexual population despite a substantial fitness cost to the host. The same is not true for asexual lineages however, where both theory and observation argue that such elements will be under pressure to reduce proliferation, and may be lost completely. The recent introduction of group II introns into archaea by horizontal transfer provides a natural test case with which to evaluate Martin & Koonin's model. The distribution and behaviour of these introns fits prior theoretical expectations, not the scenario of aggressive proliferation advocated by Martin & Koonin. I therefore conclude that the mitochondrial seed hypothesis for the origin of eukaryote introns, on which their model is based, better explains the early expansion of introns in eukaryotes. The mitochondrial seed hypothesis has the capacity to separate the origin of eukaryotes from the origin of introns, leaving open the possibility that the cell that engulfed the ancestor of mitochondria was a sexually outcrossing eukaryote cell.

  13. Structural characterization of the N-linked pentasaccharide decorating glycoproteins of the halophilic archaeon Haloferax volcanii.

    Science.gov (United States)

    Kandiba, Lina; Lin, Chia-Wei; Aebi, Markus; Eichler, Jerry; Guerardel, Yann

    2016-07-01

    N-Glycosylation is a post-translational modification performed in all three domains of life. In the halophilic archaea Haloferax volcanii, glycoproteins such as the S-layer glycoprotein are modified by an N-linked pentasaccharide assembled by a series of Agl (archaeal glycosylation) proteins. In the present study, mass spectrometry (MS) and nuclear magnetic resonance spectroscopy were used to define the structure of this glycan attached to at least four of the seven putative S-layer glycoprotein N-glycosylation sites, namely Asn-13, Asn-83, Asn-274 and Asn-279. Such approaches detected a trisaccharide corresponding to glucuronic acid (GlcA)-β1,4-GlcA-β1,4-glucose-β1-Asn, a tetrasaccharide corresponding to methyl-O-4-GlcA-β-1,4-galacturonic acid-α1,4-GlcA-β1,4-glucose-β1-Asn, and a pentasaccharide corresponding to hexose-1,2-[methyl-O-4-]GlcA-β-1,4-galacturonic acid-α1,4-GlcA-β1,4-glucose-β1-Asn, with previous MS and radiolabeling experiments showing the hexose at the non-reducing end of the pentasaccharide to be mannose. The present analysis thus corrects the earlier assignment of the penultimate sugar as a methyl ester of a hexuronic acid, instead revealing this sugar to be a methylated GlcA. The assignments made here are in good agreement with what was already known of the Hfx. volcanii N-glycosylation pathway from previous genetic and biochemical efforts while providing new insight into the process. PMID:26863921

  14. Molecular and biochemical characterization of the ADP-dependent phosphofructokinase from the hyperthermophilic archaeon Pyrococcus furiosus.

    Science.gov (United States)

    Tuininga, J E; Verhees, C H; van der Oost, J; Kengen, S W; Stams, A J; de Vos, W M

    1999-07-23

    Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two ADP-dependent kinases. Using the N-terminal amino acid sequence of the previously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome of P. furiosus. Both genes were successfully cloned and expressed in Escherichia coli, yielding highly thermoactive ADP-dependent glucokinase and phosphofructokinase. The deduced amino acid sequences of both kinases were 21.1% identical but did not reveal significant homology with those of other known sugar kinases. The ADP-dependent phosphofructokinase was purified and characterized. The oxygen-stable protein had a native molecular mass of approximately 180 kDa and was composed of four identical 52-kDa subunits. It had a specific activity of 88 units/mg at 50 degrees C and a pH optimum of 6.5. As phosphoryl group donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent. The K(m) values for fructose 6-phosphate and ADP were 2.3 and 0.11 mM, respectively. The phosphofructokinase did not catalyze the reverse reaction, nor was it regulated by any of the known allosteric modulators of ATP-dependent phosphofructokinases. ATP and AMP were identified as competitive inhibitors of the phosphofructokinase, raising the K(m) for ADP to 0.34 and 0.41 mM, respectively. PMID:10409652

  15. TreT, a novel trehalose glycosyltransferring synthase of the hyperthermophilic archaeon Thermococcus litoralis.

    Science.gov (United States)

    Qu, Qiuhao; Lee, Sung-Jae; Boos, Winfried

    2004-11-12

    The gene cluster in Thermococcus litoralis encoding a multicomponent and binding protein-dependent ABC transporter for trehalose and maltose contains an open reading frame of unknown function. We cloned this gene (now called treT), expressed it in Escherichia coli, purified the encoded protein, and identified it as an enzyme forming trehalose and ADP from ADP-glucose and glucose. The enzyme can also use UDP- and GDP-glucose but with less efficiency. The reaction is reversible, and ADP-glucose plus glucose can also be formed from trehalose and ADP. The rate of reaction and the equilibrium favor the formation of trehalose. At 90 degrees C, the optimal temperature for the enzymatic reaction, the half-maximal concentration of ADP-glucose at saturating glucose concentrations is 1.14 mm and the V(max) is 160 units/mg protein. In the reverse reaction, the half-maximal concentration of trehalose at saturating ADP concentrations is 11.5 mm and the V(max) was estimated to be 17 units/mg protein. Under non-denaturating in vitro conditions the enzyme behaves as a dimer of identical subunits of 48 kDa. As the transporter encoded in the same gene cluster, TreT is induced by trehalose and maltose in the growth medium. PMID:15364950

  16. Cloning and characterization of ftsZ and pyrF from the archaeon Thermoplasma acidophilum.

    Science.gov (United States)

    Yaoi, T; Laksanalamai, P; Jiemjit, A; Kagawa, H K; Alton, T; Trent, J D

    2000-09-01

    To characterize cytoskeletal components of archaea, the ftsZ gene from Thermoplasma acidophilum was cloned and sequenced. In T. acidophilum ftsZ, which is involved in cell division, was found to be in an operon with the pyrF gene, which encodes orotidine-5'-monophosphate decarboxylase (ODC), an essential enzyme in pyrimidine biosynthesis. Both ftsZ and pyrF from T. acidophilum were expressed in Escherichia coli and formed functional proteins. FtsZ expression in wild-type E. coli resulted in the filamentous phenotype characteristic of ftsZ mutants. T. acidophilum pyrF expression in an E. coli mutant lacking pyrF complemented the mutation and rescued the strain. Sequence alignments of ODCs from archaea, bacteria, and eukarya reveal five conserved regions, two of which have homology to 3-hexulose-6-phosphate synthase (HPS), suggesting a common substrate recognition and binding motif. PMID:10973825

  17. Purification and biochemical characterization of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease

    DEFF Research Database (Denmark)

    Studdert, C A; Herrera Seitz, M K; Plasencia, I; Sanchez, J J; de Castro, R E

    2001-01-01

    other halobacteria nor with commercial proteases except subtilisin. The amino acid sequences of three tryptic peptides obtained from Natronococcus occultus protease did not show significant similarity to other known proteolytic enzymes. This fact, in addition to its high molecular mass suggests that...

  18. An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum

    Science.gov (United States)

    Burggraf, S.; Larsen, N.; Woese, C. R.; Stetter, K. O.

    1993-01-01

    The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly. The two organisms are closely related by this measure (over 98% similar). However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum. To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote. Upon excision the intron is circularized. A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea. The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence.

  19. Biotransformation of Two Pharmaceuticals by the Ammonia-Oxidizing Archaeon Nitrososphaera gargensis.

    Science.gov (United States)

    Men, Yujie; Han, Ping; Helbling, Damian E; Jehmlich, Nico; Herbold, Craig; Gulde, Rebekka; Onnis-Hayden, Annalisa; Gu, April Z; Johnson, David R; Wagner, Michael; Fenner, Kathrin

    2016-05-01

    The biotransformation of some micropollutants has previously been observed to be positively associated with ammonia oxidation activities and the transcript abundance of the archaeal ammonia monooxygenase gene (amoA) in nitrifying activated sludge. Given the increasing interest in and potential importance of ammonia-oxidizing archaea (AOA), we investigated the capabilities of an AOA pure culture, Nitrososphaera gargensis, to biotransform ten micropollutants belonging to three structurally similar groups (i.e., phenylureas, tertiary amides, and tertiary amines). N. gargensis was able to biotransform two of the tertiary amines, mianserin (MIA) and ranitidine (RAN), exhibiting similar compound specificity as two ammonia-oxidizing bacteria (AOB) strains that were tested for comparison. The same MIA and RAN biotransformation reactions were carried out by both the AOA and AOB strains. The major transformation product (TP) of MIA, α-oxo MIA was likely formed via a two-step oxidation reaction. The first hydroxylation step is typically catalyzed by monooxygenases. Three RAN TP candidates were identified from nontarget analysis. Their tentative structures and possible biotransformation pathways were proposed. The biotransformation of MIA and RAN only occurred when ammonia oxidation was active, suggesting cometabolic transformations. Consistently, a comparative proteomic analysis revealed no significant differential expression of any protein-encoding gene in N. gargensis grown on ammonium with MIA or RAN compared with standard cultivation on ammonium only. Taken together, this study provides first important insights regarding the roles played by AOA in micropollutant biotransformation. PMID:27046099

  20. Activation of Methanogenesis by Cadmium in the Marine Archaeon Methanosarcina acetivorans

    OpenAIRE

    Elizabeth Lira-Silva; M Geovanni Santiago-Martínez; Viridiana Hernández-Juárez; Rodolfo García-Contreras; Rafael Moreno-Sánchez; Ricardo Jasso-Chávez

    2012-01-01

    Methanosarcina acetivorans was cultured in the presence of CdCl(2) to determine the metal effect on cell growth and biogas production. With methanol as substrate, cell growth and methane synthesis were not altered by cadmium, whereas with acetate, cadmium slightly increased both, growth and methane rate synthesis. In cultures metabolically active, incubations for short-term (minutes) with 10 µM total cadmium increased the methanogenesis rate by 6 and 9 folds in methanol- and acetate-grown cel...

  1. Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins

    Directory of Open Access Journals (Sweden)

    Koga Yuichi

    2010-07-01

    Full Text Available Abstract Background The unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI. Results To clarify whether the difference in the unfolding rate is due to differences in the types of RNase H or differences in proteins from archaea and bacteria, we examined the equilibrium stability and unfolding reaction of RNases HII from the hyperthermophilic bacteria Thermotoga maritima (Tm-RNase HII and Aquifex aeolicus (Aa-RNase HII and RNase HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI. These proteins from hyperthermophiles are more stable than Ec-RNase HI over all the temperature ranges examined. The observed unfolding speeds of all hyperstable proteins at the different denaturant concentrations studied are much lower than those of Ec-RNase HI, which is in accordance with the familiar slow unfolding of hyperstable proteins. However, the unfolding rate constants of these RNases H in water are dispersed, and the unfolding rate constant of thermophilic archaeal proteins is lower than that of thermophilic bacterial proteins. Conclusions These results suggest that the nature of slow unfolding of thermophilic proteins is determined by the evolutionary history of the organisms involved. The unfolding rate constants in water are related to the amount of buried hydrophobic residues in the tertiary structure.

  2. Genome Analyses of Icelandic Strains of Sulfolobus islandicus, Model Organisms for Genetic and Virus-Host Interaction Studies

    DEFF Research Database (Denmark)

    Guo, Li; Brügger, Kim; Liu, Chao;

    2011-01-01

    (IS) elements and high proportions of the orphan orfB elements and SMN1 miniature inverted-repeat transposable elements (MITEs), as well as the clustered regular interspaced short palindromic repeat (CRISPR)-based immune systems, which are complex and diverse in both strains, consistent with them...

  3. The saci_2123 gene of the hyperthermoacidophile Sulfolobus acidocaldarius encodes an ATP-binding cassette multidrug transporter

    NARCIS (Netherlands)

    Yang, Nuan; Driessen, Arnold J. M.

    2015-01-01

    Multidrug resistance (MDR) transporters are capable of secreting structurally and functionally unrelated toxic compounds from the cell. Among this group are ATP-binding cassette (ABC) transporters. These membrane proteins are typically arranged as either hetero- or homo-dimers of ABC half-transporte

  4. Chaperonin Polymers in Archaea: The Cytoskeleton of Prokaryotes?

    Science.gov (United States)

    Trent, J. D.; Kagawa, H. K.; Zaluzec, N. J.

    1997-07-01

    Chaperonins are protein complexes that play a critical role in folding nascent polypeptides under normal conditions and refolding damaged proteins under stress conditions. In all organisms these complexes are composed of evolutionarily conserved 60-kDa proteins arranged in double-ring structures with between 7 and 9 protein subunits per ring. These double ring structures are assumed to be the functional units in vivo, although they have never been observed inside cells. Here the authors show that the purified chaperonin from the hyperthermophilic archaeon Sulfolobus shibatae, which is closely related to chaperonins in eukaryotes, has a double ring structure at low concentrations (0.1 mg/ml), but at more physiological concentrations, the rings stack end to end to form polymers. The polymers are stable at physiological temperatures (75 C) and closely resemble structures observed inside unfixed S. shibatae cells. The authors suggest that in vivo chaperonin activity may be regulated by polymerization and that chaperonin polymers may act as a cytoskeleton-like structure in archaea and bacteria.

  5. Magnetic Au Nanoparticles on Archaeal S-Layer Ghosts as Templates

    Directory of Open Access Journals (Sweden)

    Sonja Selenska-Pobell

    2011-10-01

    Full Text Available Cell‐ghosts representing empty cells of the archaeon Sulfolobus acidocaldarius, consisting only of their highly ordered and unusually stable outermost proteinaceous surface layer (S‐layer, were used as templates for Au nanoparticles fabrication. The properties of these archaeal Au nanoparticles differ significantly from those produced earlier by us onto bacterial S‐layer sheets. The archaeal Au nanoparticles, with a size of about 2.5 nm, consist exclusively of metallic Au(0, while those produced on the bacterial S‐layer had a size of about 4 nm and represented a mixture of Au(0 and Au(III in the ratio of 40 to 60 %. The most impressive feature of the archaeal Au nanoparticles is that they are strongly paramagnetic, in contrast to the bacterial ones and also to bulk gold. SQUID magnetometry and XMCD measurements demonstrated that the archaeal Au nanoparticles possess a rather large magnetic moment of about 0.1 µB/atom. HR‐ TEM‐EDX analysis revealed that the archaeal Au nanoparticles are linked to the sulfur atoms of the thiol groups of the amino acid cysteine, characteristic only for archaeal S‐layers. This is the first study demonstrating the formation of such unusually strong magnetic Au nanoparticles on a non‐modified archaeal S‐layer.

  6. Chaperonin filaments : their formation and an evaluation of methods for studying them.

    Energy Technology Data Exchange (ETDEWEB)

    Yaoi, T.; Kagawa, K. H.; Trent, J. D.; Center for Mechanistic Biology and Biotechnology

    1998-08-01

    Chaperonins are multisubunit protein complexes that can be isolated from cells as high-molecular-weight structures that appear as double rings in the electron microscope. We recently discovered that chaperonin double rings isolated from the hyperthermophilic archaeon Sulfolobus shibatae, when incubated at physiological temperatures in the presence of ATP and Mg{sup 2+}, stacked into filaments; we hypothesized that these filaments are related to filaments seen inside S. shibatae cells and that chaperonins exist as filaments in vivo. This paper elucidates the conditions under which we have observed S. shibatae chaperonins to form filaments and evaluates native polyacrylamide gel electrophoresis (PAGE), TEM, spectrophotometry, and centrifugation as methods for studying these filaments. We observed that in the presence of Mg{sup 2+} combined with ATP, ADP, ATP{gamma}S, or GTP, native PAGE indicated that chaperonin subunits assembled into double rings and that the conformation of these double rings was effected by nucleotide binding, but we saw no indication of chaperonin filament formation. Under these same conditions, however, TEM, spectroscopy, and centrifugation methods indicated that chaperonin subunits and double rings had assembled into filaments. We determined that this discrepancy in the representation of the chaperonin structure was due to the native PAGE method itself. When we exposed chaperonin filaments to the electrophoretic field used in native PAGE, the filaments dissociated into double rings. This suggests that TEM, spectrophotometry, and centrifugation are the preferred methods for studying the higher-order structures of chaperonins, which are likely to be of biological significance.

  7. Chaperonin filaments: The archaeal cytoskeleton?

    Science.gov (United States)

    Trent, Jonathan D.; Kagawa, Hiromi K.; Yaoi, Takuro; Olle, Eric; Zaluzec, Nestor J.

    1997-01-01

    Chaperonins are high molecular mass double-ring structures composed of 60-kDa protein subunits. In the hyperthermophilic archaeon Sulfolobus shibatae the two chaperonin proteins represent ≈4% of its total protein and have a combined intracellular concentration of >30 mg/ml. At concentrations ≥ 0.5 mg/ml purified chaperonins form filaments in the presence of Mg2+ and nucleotides. Filament formation requires nucleotide binding (not hydrolysis), and occurs at physiological temperatures in biologically relevant buffers, including a buffer made from cell extracts. These observations suggest that chaperonin filaments may exist in vivo and the estimated 4600 chaperonins per cell suggest that such filaments could form an extensive cytostructure. We observed filamentous structures in unfixed, uranyl-acetate-stained S. shibatae cells, which resemble the chaperonin filaments in size and appearance. ImmunoGold (Janssen) labeling using chaperonin antibodies indicated that many chaperonins are associated with insoluble cellular structures and these structures appear to be filamentous in some areas, although they could not be uranyl-acetate-stained. The existence of chaperonin filaments in vivo suggests a mechanism whereby their protein-folding activities can be regulated. More generally, the filaments themselves may play a cytoskeletal role in Archaea. PMID:9144246

  8. Chaperonin filaments: The archael cytoskeleton

    Energy Technology Data Exchange (ETDEWEB)

    Trent, J.D.; Kagawa, H.K.; Yaoi, Takuro; Olle, E.; Zaluzec, N.J.

    1997-08-01

    Chaperonins are multi-subunit double-ring complexed composed of 60-kDa proteins that are believed to mediate protein folding in vivo. The chaperonins in the hyperthermophilic archaeon Sulfolobus shibatae are composed of the organism`s two most abundant proteins, which represent 4% of its total protein and have an intracellular concentration of {ge} 3.0 mg/ml. At concentrations of 1.0 mg/ml, purified chaperonin proteins aggregate to form ordered filaments. Filament formation, which requires Mg{sup ++} and nucleotide binding (not hydrolysis), occurs at physiological temperatures under conditions suggesting filaments may exist in vivo. If the estimated 4,600 chaperonins per cell, formed filaments in vivo, they could create a matrix of filaments that would span the diameter of an average S. shibatae cell 100 times. Direct observations of unfixed, minimally treated cells by intermediate voltage electron microscopy (300 kV) revealed an intracellular network of filaments that resembles chaperonin filaments produced in vitro. The hypothesis that the intracellular network contains chaperonins is supported by immunogold analyses. The authors propose that chaperonin activity may be regulated in vivo by filament formation and that chaperonin filaments may serve a cytoskeleton-like function in archaea and perhaps in other prokaryotes.

  9. Evaluation of elemental sulphur in biodesulphurized low rank coals

    Energy Technology Data Exchange (ETDEWEB)

    L. Gonsalvesh; S.P. Marinov; M. Stefanova; R. Carleer; J. Yperman [Bulgarian Academy of Sciences, Sofia (Bulgaria). Institute of Organic Chemistry

    2011-09-15

    A new procedure for elemental sulphur (S{sup el}) determination in coal and its fractions is offered. It includes exhaustive CHCl{sub 3} extraction and subsequent quantitative analysis of the extracts by HPLC using C{sub 18} reversed phase column. Its application gives ground to achieve better sulphur balance and to specify the changes in the organic and elemental sulphur as a result of biotreatments. Two Bulgarian high sulphur containing coal samples, i.e. subbituminious (Pirin) and lignite (Maritza East), and one Turkish lignite (Cayirhan-Beypazari) are used. Prior to biotreatments, the samples are demineralized and depyritized. In the biodesulphurization processes, the applied microorganisms are: the white rot fungi 'Phanerochaeta Chrysosporium' - ME446 and the thermophilic and acidophilic archae 'Sulfolobus Solfataricus' - ATCC 35091. In the preliminary demineralized and depyritized coals, the highest presence of S{sup el} is registered, which is explained by their natural weathering. As a result of the implemented biotreatments, the amount of S{sup el} could be reduced in the range of 16.1-53.8%. The content of S{sub el} is also assessed as part of the total sulphur and organic sulphur. The following range of S{sup el} content is measured: 0.01-0.16 wt.% or 0.3-4.6% of total sulphur and 0.3-5.1% of organic sulphur. In this way, more precise information is obtained concerning the content of organic sulphur presence. 31 refs., 4 figs., 6 tabs.

  10. Microbial metalloproteomes are largely uncharacterized.

    Science.gov (United States)

    Cvetkovic, Aleksandar; Menon, Angeli Lal; Thorgersen, Michael P; Scott, Joseph W; Poole, Farris L; Jenney, Francis E; Lancaster, W Andrew; Praissman, Jeremy L; Shanmukh, Saratchandra; Vaccaro, Brian J; Trauger, Sunia A; Kalisiak, Ewa; Apon, Junefredo V; Siuzdak, Gary; Yannone, Steven M; Tainer, John A; Adams, Michael W W

    2010-08-01

    Metal ion cofactors afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and have a great impact on protein stability. Consequently, metalloproteins have key roles in most biological processes, including respiration (iron and copper), photosynthesis (manganese) and drug metabolism (iron). Yet, predicting from genome sequence the numbers and types of metal an organism assimilates from its environment or uses in its metalloproteome is currently impossible because metal coordination sites are diverse and poorly recognized. We present here a robust, metal-based approach to determine all metals an organism assimilates and identify its metalloproteins on a genome-wide scale. This shifts the focus from classical protein-based purification to metal-based identification and purification by liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) to characterize cytoplasmic metalloproteins from an exemplary microorganism (Pyrococcus furiosus). Of 343 metal peaks in chromatography fractions, 158 did not match any predicted metalloprotein. Unassigned peaks included metals known to be used (cobalt, iron, nickel, tungsten and zinc; 83 peaks) plus metals the organism was not thought to assimilate (lead, manganese, molybdenum, uranium and vanadium; 75 peaks). Purification of eight of 158 unexpected metal peaks yielded four novel nickel- and molybdenum-containing proteins, whereas four purified proteins contained sub-stoichiometric amounts of misincorporated lead and uranium. Analyses of two additional microorganisms (Escherichia coli and Sulfolobus solfataricus) revealed species-specific assimilation of yet more unexpected metals. Metalloproteomes are therefore much more extensive and diverse than previously recognized, and promise to provide key insights for cell biology, microbial growth and toxicity mechanisms. PMID:20639861

  11. NrpRII mediates contacts between NrpRI and general transcription factors in the archaeon Methanosarcina mazei Gö1.

    Science.gov (United States)

    Weidenbach, Katrin; Ehlers, Claudia; Kock, Jutta; Schmitz, Ruth A

    2010-11-01

    We report here on the formation of a complex between the two NrpR homologs present in Methanosarcina mazei Gö1 and their binding properties to the nifH and glnK(1) promoters. Reciprocal co-chromatography demonstrated that NrpRI forms stable complexes with NrpRII (at an NrpRI : NrpRII molar ratio of ∼ 1 : 3), which are not affected by 2-oxoglutarate. Promoter-binding, analyses using DNA-affinity chromatography and electrophoretic gel mobility shift assays, verified that NrpRII is not able to bind to either the nifH promoter or the glnK(1) promoter except when in complex with NrpRI. Specific binding of NrpRI to the nifH and glnK(1) promoters was shown to be highly sensitive to 2-oxoglutarate, regardless of whether only NrpRI, or NrpRI in complex with NrpRII, bound to the promoter. Finally, strong interactions between NrpRII and the general transcription factors TATA-binding proteins (TBP) 1-3 and the general transcription factor TFIIB (TFB) were demonstrated, interactions which are also sensitive to 2-oxoglutarate. On the basis of these findings we propose the following: under nitrogen sufficiency NrpRII binds from solution to either the nifH promoter or the glnK(1) promoter by simultaneously contacting NrpRI and TBP plus TFB, resulting in full repression of transcription; whereas, under nitrogen limitation, increasing 2-oxoglutarate concentrations significantly decrease the binding of NrpRI to the operator as well as the binding of NrpRII to TBP and TFB, ultimately allowing recruitment of RNA polymerase to the promoter. PMID:20875081

  12. The origin of a derived superkingdom: how a gram-positive bacterium crossed the desert to become an archaeon

    Directory of Open Access Journals (Sweden)

    Bourne Philip E

    2011-02-01

    Full Text Available Abstract Background The tree of life is usually rooted between archaea and bacteria. We have previously presented three arguments that support placing the root of the tree of life in bacteria. The data have been dismissed because those who support the canonical rooting between the prokaryotic superkingdoms cannot imagine how the vast divide between the prokaryotic superkingdoms could be crossed. Results We review the evidence that archaea are derived, as well as their biggest differences with bacteria. We argue that using novel data the gap between the superkingdoms is not insurmountable. We consider whether archaea are holophyletic or paraphyletic; essential to understanding their origin. Finally, we review several hypotheses on the origins of archaea and, where possible, evaluate each hypothesis using bioinformatics tools. As a result we argue for a firmicute ancestry for archaea over proposals for an actinobacterial ancestry. Conclusion We believe a synthesis of the hypotheses of Lake, Gupta, and Cavalier-Smith is possible where a combination of antibiotic warfare and viral endosymbiosis in the bacilli led to dramatic changes in a bacterium that resulted in the birth of archaea and eukaryotes. Reviewers This article was reviewed by Patrick Forterre, Eugene Koonin, and Gáspár Jékely

  13. The ultrastructure of Ignicoccus: Evidence for a novel outer membrane and for intracellular vesicle budding in an archaeon

    Directory of Open Access Journals (Sweden)

    Reinhard Rachel

    2002-01-01

    Full Text Available A novel genus of hyperthermophilic, strictly chemolithotrophic archaea, Ignicoccus, has been described recently, with (so far three isolates in pure culture. Cells were prepared for ultrastructural investigation by cultivation in cellulose capillaries and processing by high-pressure freezing, freeze-substitution and embedding in Epon. Cells prepared in accordance with this protocol consistently showed a novel cell envelope structure previously unknown among the Archaea: a cytoplasmic membrane; a periplasmic space with a variable width of 20 to 400 nm, containing membrane-bound vesicles; and an outer sheath, approximately 10 nm wide, resembling the outer membrane of gram-negative bacteria. This sheath contained three types of particles: numerous tightly, irregularly packed single particles, about 8 nm in diameter; pores with a diameter of 24 nm, surrounded by tiny particles, arranged in a ring with a diameter of 130 nm; and clusters of up to eight particles, each particle 12 nm in diameter. Freeze-etched cells exhibited a smooth surface, without a regular pattern, with frequent fracture planes through the outer sheath, indicating the presence of an outer membrane and the absence of an S-layer. The study illustrates the novel complex architecture of the cell envelope of Ignicoccus as well as the importance of elaborate preparation procedures for ultrastructural investigations.

  14. Mutational analyses of the enzymes involved in the metabolism of hydrogen by the hyperthermophilic archaeon Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Gerrit J Schut

    2012-05-01

    Full Text Available Pyrococcus furiosus grows optimally near 100°C by fermenting carbohydrates to produce hydrogen (H2 or, if elemental sulfur (S0, is present hydrogen sulfide instead. It contains two cytoplasmic hydrogenases, SHI and SHII, that use NADP(H as an electron carrier, and a membrane bound hydrogenase (MBH, that utilizes the redox protein ferredoxin. We previously constructed deletion strains lacking SHI and/or SHII and showed that they exhibited no obvious phenotype. This study has now been extended to include biochemical analyses and growth studies using the ΔSHI and ΔSHII deletion strains together with strains lacking a functional MBH (ΔMbhL. Hydrogenase activities in cytoplasmic extracts of ΔSHII and the parent strain were similar but were much lower (<10% in the ΔSHI strain, and no activity was detected in the ΔSHIΔSHII double deletion strain, indicating that SHI is responsible for most of the cytoplasmic hydrogenase activity. In contrast, the ΔmbhL strain showed no growth in the absence of S0, confirming the hypothesis that, in the absence of S0, MBH is the only enzyme that can dispose of reductant (as H2 generated during sugar oxidation. The deletion strain devoid of all three hydrogenases also grew only in the presence of S0 and did not produce any detectable H2. When grown in the presence of limiting S0, both H2S and H2 were produced by the parent and ΔSHI/ΔSHII strains. A significant amount of H2 was also produced by the ΔmbhL strain, showing that SHI can produce H2 from NADPH in vivo, although this does not enable significant growth of ΔmbhL in the absence of S0. We propose that the physiological function of SHI is to recycle H2 and provide a link between external H2 and the intracellular pool of NADPH needed for biosynthesis. This likely has a distinct energetic advantage in the environment, but it is clearly not required for growth of the organism under the usual laboratory conditions. The function of SHII, however, remains unknown.

  15. The 1.5 resolution structure of the [Fe4S3]-ferredoxin from the hyperthermiphilic archaeon Pyrococcus furiosus

    DEFF Research Database (Denmark)

    Nielsen, Michael Ericsson Skovbo; Harris, Pernille; Ooi, Bee Lean; Christensen, Hans Erik Mølager

    2004-01-01

    contains a double-conformation disulfide bond existing in a left-handed and a right-handed spiral conformation. The crystal packing reveals a beta-sheet interaction, which supports the suggestion that P. furiosus ferredoxin is a functional dimer. The extraordinary thermostability of P. furiosus ferredoxin...

  16. Molecular cloning and enzymological characterization of pyridoxal 5'-phosphate independent aspartate racemase from hyperthermophilic archaeon Thermococcus litoralis DSM 5473.

    Science.gov (United States)

    Washio, Tsubasa; Kato, Shiro; Oikawa, Tadao

    2016-09-01

    We succeeded in expressing the aspartate racemase homolog gene from Thermococcus litoralis DSM 5473 in Escherichia coli Rosetta (DE3) and found that the gene encodes aspartate racemase. The aspartate racemase gene consisted of 687 bp and encoded 228 amino acid residues. The purified enzyme showed aspartate racemase activity with a specific activity of 1590 U/mg. The enzyme was a homodimer with a molecular mass of 56 kDa and did not require pyridoxal 5'-phosphate as a coenzyme. The enzyme showed aspartate racemase activity even at 95 °C, and the activation energy of the enzyme was calculated to be 51.8 kJ/mol. The enzyme was highly thermostable, and approximately 50 % of its initial activity remained even after incubation at 90 °C for 11 h. The enzyme showed a maximum activity at a pH of 7.5 and was stable between pH 6.0 and 7.0. The enzyme acted on L-cysteic acid and L-cysteine sulfinic acid in addition to D- and L-aspartic acids, and was strongly inhibited by iodoacetic acid. The site-directed mutagenesis of the enzyme showed that the essential cysteine residues were conserved as Cys83 and Cys194. D-Forms of aspartic acid, serine, alanine, and valine were contained in T. litoralis DSM 5473 cells. PMID:27438592

  17. FK506 Binding Protein from the Hyperthermophilic Archaeon Pyrococcus horikoshii Suppresses the Aggregation of Proteins in Escherichia coli

    OpenAIRE

    Ideno, Akira; Furutani, Masahiro; Iba, Yoshitaka; Kurosawa, Yoshikazu; Maruyama, Tadashi

    2002-01-01

    The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (kcat/Km) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15°C. Besides t...

  18. FK506 binding protein from the hyperthermophilic archaeon Pyrococcus horikoshii suppresses the aggregation of proteins in Escherichia coli.

    Science.gov (United States)

    Ideno, Akira; Furutani, Masahiro; Iba, Yoshitaka; Kurosawa, Yoshikazu; Maruyama, Tadashi

    2002-02-01

    The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (k(cat)/K(m)) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15 degrees C. Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro. In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100 degrees C. When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E. coli, whose expressed product tended to form an inactive aggregate in E. coli, it improved the yield of the soluble Fab fragments with antibody specificity. PhFKBP29 exerted protein folding and aggregation suppression in E. coli cells. PMID:11823179

  19. Draft Genome Sequence of a Highly Flagellated, Fast-Swimming Archaeon, Methanocaldococcus villosus Strain KIN24-T80 (DSM 22612)

    KAUST Repository

    Thennarasu, Sugumar

    2013-07-11

    We report the draft genome sequence of a hyperthermophilic Methanocaldococcus villosus strain, KIN24-T80. The gene associated with its heavy flagellum formation was annotated in the 1.2-Mb draft genome sequence, and this strain may be a good model system to study the extensive functional role of flagella and their fast motor activity.

  20. Genome sequence of Halorhabdus tiamatea, the first archaeon isolated from a deep-sea anoxic brine lake.

    KAUST Repository

    Antunes, Andre

    2011-09-01

    We present the draft genome of Halorhabdus tiamatea, the first member of the Archaea ever isolated from a deep-sea anoxic brine. Genome comparison with Halorhabdus utahensis revealed some striking differences, including a marked increase in genes associated with transmembrane transport and putative genes for a trehalose synthase and a lactate dehydrogenase.

  1. Functional Analysis of Homologous Recombination Repair Proteins HerA and NurA in the Thermophile Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Huang, Qihong

    cells with more than two HerA foci increased after UV-irradiation, suggesting that HerA could be involved in the repair of UV-induced DNA damage. The pattern of NurA foci was similar to that of HerA, while the numbers of RadA foci in most cells were 0 or 1, and did not increase apparently after UV...... live in harsh conditions, such as high temperature, high pressure, extreme pH, or strong radiation, which introduce more DNA damages in genomes than normal environments. But the stability of archaeal genomes is comparable with that in other two domains of life, suggesting that archaea could harbor more......-treatment, indicating that RadA may work differently from HerA-NurA. To better understand other putative functions of HerA in vivo, this protein was overexpressed in S. islandicus cells. We found that HerA overexpression reduced cell viability and produced abnormal cells with enlarged size and increased DNA contents...

  2. A liposomal formulation for the oral application of the investigational hepatitis B drug Myrcludex B.

    Science.gov (United States)

    Uhl, P; Helm, F; Hofhaus, G; Brings, S; Kaufman, C; Leotta, Karin; Urban, S; Haberkorn, U; Mier, W; Fricker, G

    2016-06-01

    The aim of this study was the development of a liposomal formulation containing specific tetraether lipids for the oral administration of the investigational hepatitis B peptide drug Myrcludex B. For this purpose, tetraether lipids were extracted from the extremophilic archaeon Sulfolobus acidocaldarius and purified in order to obtain the desired glycerylcaldityltetraether lipids (GCTE). Myrcludex B was synthesized by solid-phase synthesis and incorporated into liposomes containing 5mol% of GCTE. These liposomes showed a size, polydispersity index and zeta potential comparable to the standard liposomes. Cryo-EM micrographs of both liposomal formulations displayed low lamellarity, the prerequisite for high drug loading capacity. Long term storage of the GCTE-liposomes was achieved by freeze-drying using 100-500mM sucrose or trehalose as lyoprotectors. The lyophilized product showed high stability with a recovery rate of 82.7±1.6% of intact Myrcludex B observed after storage for 3months at -20°C as compared to a recovery rate of 83.3±1.3% directly after the freeze-drying process. In vivo, the GCTE-liposomal formulation led to substantial enhancement of the liver uptake of iodine-131-labeled Myrcludex B in Wistar rats. 3h after oral application, approximately 7% of the initial dose (corresponding to a 3.5-fold increase compared to the free peptide) could be detected in the liver. In summary, the GCTE-liposomes enabled efficient oral administration of Myrcludex B and provided long term storage by freeze-drying. PMID:27049970

  3. Chemistry and Biology of Aflatoxin-DNA Adducts

    Energy Technology Data Exchange (ETDEWEB)

    Stone, Michael P.; Banerjee, Surajit; Brown, Kyle L.; Egli, Martin (Vanderbilt)

    2012-03-27

    Aspergillus flavus is a fungal contaminant of stored rice, wheat, corn, and other grainstuffs, and peanuts. This is of concern to human health because it produces the mycotoxin aflatoxin B{sub 1} (AFB{sub 1}), which is genotoxic and is implicated in the etiology of liver cancer. AFB{sub 1} is oxidized in vivo by cytochrome P450 to form aflatoxin B{sub 1} epoxide, which forms an N7-dG adduct (AFB{sub 1}-N7-dG) in DNA. The latter rearranges to a formamidopyrimidine (AFB{sub 1}-FAPY) derivative that equilibrates between {alpha} and {beta} anomers of the deoxyribose. In DNA, both the AFB{sub 1}-N7-dG and AFB{sub 1}-{beta}-FAPY adducts intercalate above the 5'-face of the damaged guanine. Each produces G {yields} T transversions in Escherichia coli, but the AFB{sub 1}-{beta}-FAPY adduct is more mutagenic. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) provides a model for understanding error-prone bypass of the AFB{sub 1}-N7-dG and AFB{sub 1}-{beta}-FAPY adducts. It bypasses the AFB{sub 1}-N7-dG adduct, but it conducts error-prone replication past the AFB{sub 1}-FAPY adduct, including mis-insertion of dATP, consistent with the G {yields} T mutations characteristic of AFB{sub 1} mutagenesis in E. coli. Crystallographic analyses of a series of binary and ternary complexes with the Dpo4 polymerase revealed differing orientations of the N7-C8 bond of the AFB{sub 1}-N7-dG adduct as compared to the N{sup 5}-C8 bond in the AFB{sub 1}-{beta}-FAPY adduct, and differential accommodation of the intercalated AFB{sub 1} moieties within the active site. These may modulate AFB{sub 1} lesion bypass by this polymerase.

  4. The genes coding for the hsp70(dnaK) molecular chaperone machine occur in the moderate thermophilic archaeon Methanosarcina thermophila TM-1

    DEFF Research Database (Denmark)

    Hofman-Bang, H Jacob Peider; Lange, Marianne; Ahring, Birgitte Kiær

    1999-01-01

    response by hsp70(dnaK), and a similar response by trkA. The data suggest that the moderate thermophile TM-1 has an active Hsp70(DnaK)-chaperone machine in contrast to hyperthermophilic archaea, and that trkA is a stress gene, inasmuch as it responds like classic heat-shock genes to stressors that induce a...... typical heat-shock response. (C) 1999 Elsevier Science B.V. All rights reserved.......-negative bacteria - first described in the S-6 molecule and later found to be present in all homologs from archaea and Gram positives. The genes responded to a temperature elevation in a manner that demonstrated that they are heat-shock genes, functionally active in vivo. Ammonia also induced a heat-shock type of...

  5. Genetic and transcriptomic analysis of transcription factor genes in the model halophilic Archaeon: coordinate action of TbpD and TfbA

    Directory of Open Access Journals (Sweden)

    DasSarma Shiladitya

    2007-09-01

    Full Text Available Abstract Background Archaea are prokaryotic organisms with simplified versions of eukaryotic transcription systems. Genes coding for the general transcription factors TBP and TFB are present in multiple copies in several Archaea, including Halobacterium sp. NRC-1. Multiple TBP and TFBs have been proposed to participate in transcription of genes via recognition and recruitment of RNA polymerase to different classes of promoters. Results We attempted to knock out all six TBP and seven TFB genes in Halobacterium sp. NRC-1 using the ura3-based gene deletion system. Knockouts were obtained for six out of thirteen genes, tbpCDF and tfbACG, indicating that they are not essential for cell viability under standard conditions. Screening of a population of 1,000 candidate mutants showed that genes which did not yield mutants contained less that 0.1% knockouts, strongly suggesting that they are essential. The transcriptomes of two mutants, ΔtbpD and ΔtfbA, were compared to the parental strain and showed coordinate down regulation of many genes. Over 500 out of 2,677 total genes were regulated in the ΔtbpD and ΔtfbA mutants with 363 regulated in both, indicating that over 10% of genes in both strains require the action of both TbpD and TfbA for normal transcription. Culturing studies on the ΔtbpD and ΔtfbA mutant strains showed them to grow more slowly than the wild-type at an elevated temperature, 49°C, and they showed reduced viability at 56°C, suggesting TbpD and TfbA are involved in the heat shock response. Alignment of TBP and TFB protein sequences suggested the expansion of the TBP gene family, especially in Halobacterium sp. NRC-1, and TFB gene family in representatives of five different genera of haloarchaea in which genome sequences are available. Conclusion Six of thirteen TBP and TFB genes of Halobacterium sp. NRC-1 are non-essential under standard growth conditions. TbpD and TfbA coordinate the expression of over 10% of the genes in the NRC-1 genome. The ΔtbpD and ΔtfbA mutant strains are temperature sensitive, possibly as a result of down regulation of heat shock genes. Sequence alignments suggest the existence of several families of TBP and TFB transcription factors in Halobacterium which may function in transcription of different classes of genes.

  6. Genetic and transcriptomic analysis of transcription factor genes in the model halophilic Archaeon: coordinate action of TbpD and TfbA

    OpenAIRE

    DasSarma Shiladitya; Coker James A

    2007-01-01

    Abstract Background Archaea are prokaryotic organisms with simplified versions of eukaryotic transcription systems. Genes coding for the general transcription factors TBP and TFB are present in multiple copies in several Archaea, including Halobacterium sp. NRC-1. Multiple TBP and TFBs have been proposed to participate in transcription of genes via recognition and recruitment of RNA polymerase to different classes of promoters. Results We attempted to knock out all six TBP and seven TFB genes...

  7. Genomic Analysis of Anaerobic Respiration in the Archaeon Halobacterium sp. Strain NRC-1: Dimethyl Sulfoxide and Trimethylamine N-Oxide as Terminal Electron Acceptors†

    OpenAIRE

    Müller, Jochen A.; DasSarma, Shiladitya

    2005-01-01

    We have investigated anaerobic respiration of the archaeal model organism Halobacterium sp. strain NRC-1 by using phenotypic and genetic analysis, bioinformatics, and transcriptome analysis. NRC-1 was found to grow on either dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO) as the sole terminal electron acceptor, with a doubling time of 1 day. An operon, dmsREABCD, encoding a putative regulatory protein, DmsR, a molybdopterin oxidoreductase of the DMSO reductase family (DmsEABC), and...

  8. Identification and characterization of a thermostable bifunctional enzyme with phosphomannose isomerase and sugar-1-phosphate nucleotidylyltransferase activities from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3.

    Science.gov (United States)

    Akutsu, Jun-ichi; Zhang, Zilian; Morita, Rihito; Kawarabayasi, Yutaka

    2015-11-01

    Mannosylglycerate is known as a compatible solute, and plays important roles for salinity adaptation and high temperature stability of microorganisms. In the gene cluster for the mannosylglycerate biosynthetic pathway predicted from the genomic data of Pyrococcus horikoshii OT3, the PH0925 protein was found as a putative bifunctional enzyme with phosphomannose isomerase (PMI) and mannose-1-phosphate guanylyltransferase (Man-1-P GTase) activities, which can synthesize GDP-mannose when accompanied by a phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme (PH0923). The recombinant PH0925 protein, expressed in E. coli, exhibited both expected PMI and Man-1-P GTase activities, as well as absolute thermostability; 95 °C was the optimum reaction temperature. According to the guanylyltransferase activity (GTase) of the PH0925 protein, it was found that the protein can catalyze glucose-1-phosphate (Glc-1-P) and glucosamine-1-phosphate (GlcN-1-P) in addition to Man-1-P. The analyses of C-terminus-truncated forms of the PH0925 protein indicated that sugar-1-phosphate nucleotidylyltransferase (Sugar-1-P NTase) activity was located in the region from the N-terminus to the 345th residue, and that the C-terminal 114 residue region of the PH0925 protein inhibited the Man-1-P GTase activity. Conversely, the PMI activity was abolished by deletion of the C-terminal 14 residues. This is the first report of a thermostable enzyme with both PMI and multiple Sugar-1-P NTase activities. PMID:26290359

  9. Methanospirillum stamsii sp. nov., a psychrotolerant, hydrogenotrophic, methanogenic archaeon isolated from an anaerobic expanded granular sludge bed bioreactor operated at low temperature.

    Science.gov (United States)

    Parshina, Sofiya N; Ermakova, Anna V; Bomberg, Malin; Detkova, Ekaterina N

    2014-01-01

    A psychrotolerant hydrogenotrophic methanogen, strain Pt1, was isolated from a syntrophic propionate-oxidizing methanogenic consortium obtained from granulated biomass of a two-stage low-temperature (3-8 °C) anaerobic expanded granular sludge bed (EGSB) bioreactor, fed with a mixture of volatile fatty acids (VFAs) (acetate, propionate and butyrate). The strain was strictly anaerobic, and cells were curved rods, 0.4-0.5×7.5-25 µm, that sometimes formed wavy filaments from 25 to several hundred micrometres in length. Cells stained Gram-negative and were non-sporulating. They were gently motile by means of tufted flagella. The strain grew at 5-37 °C (optimum at 20-30 °C), at pH 6.0-10 (optimum 7.0-7.5) and with 0-0.3 M NaCl (optimum 0 M NaCl). Growth and methane production was found with H2/CO2 and very weak growth with formate. Acetate and yeast extract stimulated growth, but were not essential. The G+C content of the DNA of strain Pt1 was 40 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Pt1 was a member of the genus Methanospirillum and showed 97.5 % sequence similarity to Methanospirillum hungatei JF1(T) and 94 % sequence similarity to Methanospirillum lacunae Ki8-1(T). DNA-DNA hybridization of strain Pt1 with Methanospirillum hungatei JF1(T) revealed 39 % relatedness. On the basis of its phenotypic characteristics and phylogenetic position, strain Pt1 is a representative of a novel species of the genus Methanospirillum, for which the name Methanospirillum stamsii sp. nov. is proposed. The type strain is Pt1(T) ( = DSM 26304(T) = VKM B-2808(T)). PMID:24048867

  10. The Elemental Sulfur-Responsive Protein (SipA) from the Hyperthermophilic Archaeon Pyrococcus furiosus Is Regulated by Sulfide in an Iron-Dependent Manner ▿

    OpenAIRE

    Clarkson, Sonya M; Newcomer, Elizabeth C.; Young, Everett G.; Adams, Michael W. W.

    2010-01-01

    The gene (sipA) encoding the sulfur-induced protein A (PF2025) is highly upregulated during growth of Pyrococcus furiosus on elemental sulfur (S0). Expression of sipA is regulated by sulfide, the product of S0 reduction, but in an iron-dependent manner. SipA is proposed to play a role in intracellular iron sulfide detoxification.

  11. Cloning, functional organization, transcript studies, and phylogenetic analysis of the complete nitrogenase structural genes (nifHDK2) and associated genes in the archaeon Methanosarcina barkeri 227.

    Science.gov (United States)

    Chien, Y T; Zinder, S H

    1996-01-01

    Determination of the nucleotide sequence of the nitrogenase structural genes (nifHDK2) from Methanosarcina barkeri 227 was completed in this study by cloning and sequencing a 2.7-kb BamHI fragment containing the 3' end of nifK2 and 1,390 bp of the nifE2-homologous genes. Open reading frame nifK2 is 1,371 bp long including the stop codon TAA and encodes a polypeptide of 456 amino acids. Phylogenetic analysis of the deduced amino acid sequences of the nifK2 and nifE2 gene products from M. barkeri showed that both genes cluster most closely with the corresponding nif-1 gene products from Clostridium pasteurianum, consistent with our previous analyses of nifH2 and nifD2. The nifE gene product is known to be homologous to that of nifD, and our analysis shows that the branching pattern for the nifE proteins resembles that for the nifD product (with the exception of vnfE from Azotobacter vinelandii), suggesting that a gene duplication occurred before the divergence of nitrogenases. Primer extension showed that nifH2 had a single transcription start site located 34 nucleotides upstream of the ATG translation start site for nifH2, and a sequence resembling the archaeal consensus promoter sequence [TTTA(A/T)ATA] was found 32 nucleotides upstream from that transcription start site. A tract of four T's, previously identified as a transcription termination site in archaea, was found immediately downstream of the nifK2 gene, and a potential promoter was located upstream of the nifE2 gene. Hybridization with nifH2 and nifDK2 probes with M. barkeri RNA revealed a 4.6-kb transcript from N2-grown cells, large enough to harbor nifHDK genes and their internal open reading frames, while no transcript was detected from NH4(+)-grown cells. These results support a model in which the nitrogenase structural genes in M. barkeri are cotranscribed in a single NH4(+)-repressed operon. PMID:8550408

  12. Cloning, functional organization, transcript studies, and phylogenetic analysis of the complete nitrogenase structural genes (nifHDK2) and associated genes in the archaeon Methanosarcina barkeri 227.

    OpenAIRE

    Chien, Y T; Zinder, S. H.

    1996-01-01

    Determination of the nucleotide sequence of the nitrogenase structural genes (nifHDK2) from Methanosarcina barkeri 227 was completed in this study by cloning and sequencing a 2.7-kb BamHI fragment containing the 3' end of nifK2 and 1,390 bp of the nifE2-homologous genes. Open reading frame nifK2 is 1,371 bp long including the stop codon TAA and encodes a polypeptide of 456 amino acids. Phylogenetic analysis of the deduced amino acid sequences of the nifK2 and nifE2 gene products from M. barke...

  13. Crystallization and preliminary X-ray crystallographic analysis of the catalytic domain of pyrrolysyl-tRNA synthetase from the methanogenic archaeon Methanosarcina mazei

    International Nuclear Information System (INIS)

    Pyrrolysyl-tRNA synthetase (PylRS) from M. mazei has been overexpressed in an N-terminally truncated form PylRS(c270) in Escherichia coli, purified to homogeneity and crystallized by the hanging-drop vapour-diffusion method. Pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei was overexpressed in an N-terminally truncated form PylRS(c270) in Escherichia coli, purified to homogeneity and crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. The native PylRS(c270) crystals in complex with an ATP analogue belonged to space group P64, with unit-cell parameters a = b = 104.88, c = 70.43 Å, α = β = 90, γ = 120°, and diffracted to 1.9 Å resolution. The asymmetric unit contains one molecule of PylRS(c270). Selenomethionine-substituted protein crystals were prepared in order to solve the structure by the MAD phasing method

  14. Draft Genome Sequence of Candidatus Methanomethyllophilus sp. 1R26m -Enriched from Bovine Rumen, a Methanogenic Archaeon Belonging to the Methanomassiliicoccales Order

    DEFF Research Database (Denmark)

    Noel, Samantha Joan; Højberg, Ole; Urich, T.;

    2016-01-01

    Olsenella scatoligenes SK9K4(T) is a strictly anaerobic bacterium isolated from pig feces that produces the malodorous compounds 3-methylindole (skatole) and 4-methylphenol (p-cresol). Here, we report the 2.47 Mbp draft genome sequence of SK9K4(T), exploring pathways for the synthesis of skatole...... and p-cresol from the amino acids tryptophan and tyrosine, respectively....

  15. Functional-Genomics-Based Identification and Characterization of Open Reading Frames Encoding α-Glucoside-Processing Enzymes in the Hyperthermophilic Archaeon Pyrococcus furiosus▿ †

    OpenAIRE

    Comfort, Donald A.; Chou, Chung-Jung; Conners, Shannon B.; VanFossen, Amy L.; Kelly, Robert M.

    2007-01-01

    Bioinformatics analysis and transcriptional response information for Pyrococcus furiosus grown on α-glucans led to the identification of a novel isomaltase (PF0132) representing a new glycoside hydrolase (GH) family, a novel GH57 β-amylase (PF0870), and an extracellular starch-binding protein (1,141 amino acids; PF1109-PF1110), in addition to several other putative α-glucan-processing enzymes.

  16. Draft Genome Sequence of “Candidatus Methanomethylophilus” sp. 1R26, Enriched from Bovine Rumen, a Methanogenic Archaeon Belonging to the Methanomassiliicoccales Order

    Science.gov (United States)

    Højberg, Ole; Urich, Tim

    2016-01-01

    Here, we present the draft genome of “Candidatus Methanomethylophilus” sp. 1R26, a member of the newly described Methanomassiliicoccales order of Euryarcheaota. The enrichment culture was established from bovine rumen contents and produced methane from trimethylamine and methanol. The draft genome contains genes for methanogenesis from methylated compounds. PMID:26893425

  17. The genes coding for the hsp70(dnaK) molecular chaperone machine occur in the moderate thermophilic archaeon Methanosarcina thermophila TM-1

    DEFF Research Database (Denmark)

    Hofman-Bang, H Jacob Peider; Lange, Marianne; Ahring, Birgitte Kiær

    1999-01-01

    response by hsp70(dnaK), and a similar response by trkA. The data suggest that the moderate thermophile TM-1 has an active Hsp70(DnaK)-chaperone machine in contrast to hyperthermophilic archaea, and that trkA is a stress gene, inasmuch as it responds like classic heat-shock genes to stressors that induce a...

  18. Biology and survival of extremely halophilic archaeon Haloarcula marismortui RR12 isolated from Mumbai salterns, India in response to salinity stress.

    Science.gov (United States)

    Thombre, Rebecca S; Shinde, Vinaya D; Oke, Radhika S; Dhar, Sunil Kumar; Shouche, Yogesh S

    2016-01-01

    Haloarchaea are unique microorganism's resistant to environmental and osmotic stresses and thrive in their habitats despite extreme fluctuating salinities. In the present study, haloarchaea were isolated from hypersaline thalossohaline salterns of Bhandup, Mumbai, India and were identified as Haloferax prahovense, Haloferax alexandrines, Haloferax lucentense, Haloarcula tradensis, Haloarcula marismortui and Haloarcula argentinensis. The mechanism of adaptation to contrasting salinities (1.5 M and 4.5 M) was investigated in the extreme haloarchaeon, Hal. marismortui RR12. Hal. marismortui RR12 increased the intracellular sequestration of K(+) and Cl(-) ions in hypo salinity and hyper salinity respectively as detected by Energy-dispersive X-ray spectroscopy microanalysis (EDAX) and Inductively Coupled Plasma- atomic Emission Spectroscopy (ICP-AES) indicating the presence of 'salt-in' strategy of osmoadaptation. As a cellular response to salinity stress, it produced small heat shock like proteins (sHSP) identified using MALDI-TOF MS and increased the production of protective red carotenoid pigment. This is the first report on the study of the concomitant cellular, molecular and physiological mechanism adapted by Hal. marismortui RR12 when exposed to contrasting salinities in external environment. PMID:27231230

  19. Draft Genome Sequence of “Candidatus Methanomethylophilus” sp. 1R26, Enriched from Bovine Rumen, a Methanogenic Archaeon Belonging to the Methanomassiliicoccales Order

    OpenAIRE

    Noel, Samantha Joan; Højberg, Ole; Urich, Tim; Poulsen, Morten

    2016-01-01

    Here, we present the draft genome of “Candidatus Methanomethylophilus” sp. 1R26, a member of the newly described Methanomassiliicoccales order of Euryarcheaota. The enrichment culture was established from bovine rumen contents and produced methane from trimethylamine and methanol. The draft genome contains genes for methanogenesis from methylated compounds.

  20. Draft Genome Sequence of "Candidatus Methanomethylophilus" sp. 1R26, Enriched from Bovine Rumen, a Methanogenic Archaeon Belonging to the Methanomassiliicoccales Order.

    Science.gov (United States)

    Noel, Samantha Joan; Højberg, Ole; Urich, Tim; Poulsen, Morten

    2016-01-01

    Here, we present the draft genome of "Candidatus Methanomethylophilus" sp. 1R26, a member of the newly described Methanomassiliicoccales order of Euryarcheaota. The enrichment culture was established from bovine rumen contents and produced methane from trimethylamine and methanol. The draft genome contains genes for methanogenesis from methylated compounds. PMID:26893425

  1. Purification, crystallization and preliminary X-ray crystallographic analysis of ST1022, a putative member of the Lrp/AsnC family of transcriptional regulators isolated from Sulfolobus tokodaii strain 7

    International Nuclear Information System (INIS)

    A putative member of the Lrp/AsnC family of transcriptional regulators, ST1022 from S. tokodaii strain 7, has been purified and crystallized in the absence and presence of the effector l-glutamine. A molecular-replacement solution was found using the FL11 transcriptional regulator from Pyrococcus sp. OT3 as a model and structural refinement is under way. The Lrp/AsnC family of transcriptional regulators, also known as feast/famine transcriptional regulators, are widely distributed among bacteria and archaea. This family of proteins are likely to be involved in cellular metabolism, with exogenous amino acids functioning as effectors. Here, the crystallization and preliminary X-ray diffraction analysis of ST1022, a member of the Lrp/AsnC family of proteins, is reported with and without exogenous glutamine as the effector molecule. The crystals of native ST1022 and of the putative complex belong to the tetragonal space group I422, with unit-cell parameters a = b = 103.771, c = 73.297 Å and a = b = 103.846, c = 73.992 Å, respectively. Preliminary X-ray diffraction data analysis and molecular-replacement solution revealed the presence of one monomer per asymmetric unit

  2. Reaction Mechanism and Structural Model of ADP-forming Acetyl-CoA Synthetase from the Hyperthermophilic Archaeon Pyrococcus furiosus: EVIDENCE FOR A SECOND ACTIVE SITE HISTIDINE RESIDUE*S⃞

    OpenAIRE

    Bräsen, Christopher; Schmidt, Marcel; Grötzinger, Joachim; Schönheit, Peter

    2008-01-01

    In Archaea, acetate formation and ATP synthesis from acetyl-CoA is catalyzed by an unusual ADP-forming acetyl-CoA synthetase (ACD) (acetyl-CoA + ADP + Pi ⇆ acetate + ATP + HS-CoA) catalyzing the formation of acetate from acetyl-CoA and concomitant ATP synthesis by the mechanism of substrate level phosphorylation. ACD belongs to the protein superfamily of nucleoside diphosphate-forming acyl-CoA synthetases, which also include succinyl-CoA synthetases (SCSs). ACD differs from SCS in domain orga...

  3. Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5′-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi

    OpenAIRE

    Ishikawa, Ken; Watanabe, Miki; Kuroita, Toshihiro; Uchiyama, Ikuo; Bujnicki, Janusz M.; Kawakami, Bunsei; Tanokura, Masaru; Kobayashi, Ichizo

    2005-01-01

    To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction–modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restricti...

  4. GenBank blastx search result: AK061906 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061906 001-041-H10 AF042099.1 Sulfolobus metallicus putative carboxyl transferase (pccB), biotin ... in carboxylase (accC), and biotin ... carboxyl carrier protein (accB) genes, complete cd ...

  5. GenBank blastx search result: AK060716 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060716 001-031-D11 AF042099.1 Sulfolobus metallicus putative carboxyl transferase (pccB), biotin ... in carboxylase (accC), and biotin ... carboxyl carrier protein (accB) genes, complete cd ...

  6. NCBI nr-aa BLAST: CBRC-PCAP-01-0334 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available e component [Sulfolobus islandicus Y.G.57.14] gb|ACP44753.1| binding-protein-dependent transport systems inner membran...CBRC-PCAP-01-0334 ref|YP_002836675.1| binding-protein-dependent transport systems inner membran...e component [Sulfolobus islandicus Y.G.57.14] YP_002836675.1 1.3 34% ...

  7. Four newly isolated fuselloviruses from extreme geothermal environments reveal unusual morphologies and a possible interviral recombination mechanism

    DEFF Research Database (Denmark)

    Redder, Peter; Peng, Xu; Brügger, Kim;

    2009-01-01

    Spindle-shaped virus-like particles are abundant in extreme geothermal environments, from which five spindle-shaped viral species have been isolated to date. They infect members of the hyperthermophilic archaeal genus Sulfolobus, and constitute the Fuselloviridae, a family of double-stranded DNA ...... the spacers of the Sulfolobus CRISPR antiviral system are not biased to the highly similar regions of the fusellovirus genomes....

  8. Halobacterium Salinarum: Polyextremophile Model for Life Inside Martian Halite

    Science.gov (United States)

    Srivastava, A.

    2014-07-01

    The present work briefly reviews the recent studies on long-term survival potential of Halobacterium salinarum in ancient terrestrial halite and studies the possible survival ability of this poly-extremophilic archaeon inside martian halite.

  9. Promoter recognition in archaea is mediated by transcription factors: identification of transcription factor aTFB from Methanococcus thermolithotrophicus as archaeal TATA-binding protein.

    OpenAIRE

    Gohl, H P; Gröndahl, B; Thomm, M

    1995-01-01

    At least two transcription factors, aTFB and aTFA, are required for accurate and faithful in vitro transcription of homologous templates in cell-free extracts from the methanogenic Archaeon Methanococcus thermolithotrophicus. We have recently shown that the function of aTFB can be replaced by eucaryal TATA-binding proteins. Here we demonstrate using template commitment experiments that promoter recognition in an Archaeon is mediated by transcription factors. The archaeal TATA box was identifi...

  10. Dicty_cDB: Contig-U16269-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 1( FN320319 |pid:none) Schistosoma japonicum isolate Anhu... 325 2e-87 CR380948_179( CR380948 |pid:none) Candi...F526207_1( AF526207 |pid:none) Argopecten irradians isolate iolsa... 212 2e-53 EU558306_1( EU558306 |pid:none) Novocrania an..._1282( CP001400 |pid:none) Sulfolobus islandicus M.14.25, ... 133 1e-29 (A6UV67) RecName: Full=50S ribosomal...mal protein L4P; &BA000011_329... 134 6e-30 CP001403_1389( CP001403 |pid:none) Sulfolobus islandicus Y.G...red marine group II euryar... 132 2e-29 CP001399_1369( CP001399 |pid:none) Sulfolobus islandicus L.S.2.15,..

  11. Dicty_cDB: Contig-U06081-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available N320319_1( FN320319 |pid:none) Schistosoma japonicum isolate Anhu... 275 2e-72 CR380948_179( CR380948 |pid:none) Candi...AF526207 |pid:none) Argopecten irradians isolate iolsa... 212 2e-53 CP000080_113( CP000080 |pid:none) Leishman.... 104 6e-21 CP001399_1369( CP001399 |pid:none) Sulfolobus islandicus L.S.2.15,... 103 1e-20 (A7I5P0) RecName: Full=50S riboso...d:none) Staphylothermus marinus F1, com... 106 2e-21 CP001403_1389( CP001403 |pid:none) Sulfolobus islandicu...00_1282( CP001400 |pid:none) Sulfolobus islandicus M.14.25, ... 104 5e-21 EU68663

  12. Dicty_cDB: Contig-U05942-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available APV-2005 mRNA for ri... 181 4e-44 AF526207_1( AF526207 |pid:none) Argopecten irradians isolate iols... (Q9UXA6) RecName: Full=50S ribosomal protein L4P; &AE006641_651... 85 5e-15 CP001403_1389( CP001403 |pid:none) Sulfolobus islandi...cus Y.G.57.14... 83 2e-14 CP001399_1369( CP001399 |pid:none) Sulfolobus islandi...cus L.S.2.15,... 82 3e-14 CP001400_1282( CP001400 |pid:none) Sulfolobus islandicus M.14....yzae RIB40 genomic... 175 4e-78 FM992688_1292( FM992688 |pid:none) Candida dubliniensis

  13. Molecular Assemblies, Genes and Genomics Integrated Efficiently (MAGGIE)

    Energy Technology Data Exchange (ETDEWEB)

    Baliga, Nitin S

    2011-05-26

    Final report on MAGGIE. We set ambitious goals to model the functions of individual organisms and their community from molecular to systems scale. These scientific goals are driving the development of sophisticated algorithms to analyze large amounts of experimental measurements made using high throughput technologies to explain and predict how the environment influences biological function at multiple scales and how the microbial systems in turn modify the environment. By experimentally evaluating predictions made using these models we will test the degree to which our quantitative multiscale understanding wilt help to rationally steer individual microbes and their communities towards specific tasks. Towards this end we have made substantial progress towards understanding evolution of gene families, transcriptional structures, detailed structures of keystone molecular assemblies (proteins and complexes), protein interactions, biological networks, microbial interactions, and community structure. Using comparative analysis we have tracked the evolutionary history of gene functions to understand how novel functions evolve. One level up, we have used proteomics data, high-resolution genome tiling microarrays, and 5' RNA sequencing to revise genome annotations, discover new genes including ncRNAs, and map dynamically changing operon structures of five model organisms: For Desulfovibrio vulgaris Hildenborough, Pyrococcus furiosis, Sulfolobus solfataricus, Methanococcus maripaludis and Haiobacterium salinarum NROL We have developed machine learning algorithms to accurately identify protein interactions at a near-zero false positive rate from noisy data generated using tagfess complex purification, TAP purification, and analysis of membrane complexes. Combining other genome-scale datasets produced by ENIGMA (in particular, microarray data) and available from literature we have been able to achieve a true positive rate as high as 65% at almost zero false positives

  14. Molecular biology of fuselloviruses and their satellites

    DEFF Research Database (Denmark)

    Contursi, Patrizia; Fusco, Salvatore; Cannio, Raffaele;

    2014-01-01

    Fuselloviruses, also known as Sulfolobus Spindle-shaped viruses (SSVs), are "lemon"- or "spindle"-shaped double-stranded DNA viruses. Among them, SSV1, SSV2 and the satellite viruses pSSVx and pSSVi have been investigated at the structural, genetic, transcriptomic, proteomic and biochemical level...

  15. Exceptional thermal stability and organic solvent tolerance of an esterase expressed from a thermophilic host

    DEFF Research Database (Denmark)

    Mei, Yuxia; Peng, Nan; Zhao, Shumiao; Hu, Yongmei; Wang, Huacai; Liang, Yunxiang; She, Qunxin

    2012-01-01

    A protein expression system recently developed for the thermophilic crenarchaeon Sulfolobus islandicus was employed to produce recombinant protein for EstA, a thermophilic esterase encoded in the same organism. Large amounts of protein were readily obtained by an affinity protein purification, gi...

  16. Familial relationships in hyperthermo- and acidophilic archaeal viruses

    DEFF Research Database (Denmark)

    Happonen, Lotta Johanna; Redder, Peter; Peng, Xu;

    2010-01-01

    Archaea often live in extreme, harsh environments such as acidic hot springs and hypersaline waters. To date, only two icosahedrally symmetric, membrane-containing archaeal viruses, SH1 and Sulfolobus turreted icosahedral virus (STIV), have been described in detail. We report the sequence and thr...

  17. Four newly isolated fuselloviruses from extreme geothermal environments reveal unusual morphologies and a possible interviral recombination mechanism

    DEFF Research Database (Denmark)

    Greve, Bo Bjørn

    2009-01-01

    Spindle-shaped virus-like particles are abundant in extreme geothermal environments, from which five spindle-shaped viral species have been isolated to date. They infect members of the hyperthermophilic archaeal genus Sulfolobus, and constitute the Fuselloviridae, a family of double-stranded DNA...

  18. Exploring the reductive capacity of Pyrococcus furiosus. The reduction of carboxylic acids and pyridine nucleotides

    NARCIS (Netherlands)

    Ban, van den E.C.D.

    2001-01-01

    This Ph.D. project started in 1997 and its main goal was to obtain insight in the reductive capacity of the hyperthermophilic archaeon Pyrococcus furiosus . The research was focused on the biocatalytic reduction of carboxylic acids.Reductions of carboxylic acids are interes

  19. Tryptophan auxotrophs were obtained by random transposon insertions in the Methanococcus maripaludis tryptophan operon.

    Science.gov (United States)

    Porat, Iris; Whitman, William B

    2009-08-01

    Methanococcus maripaludis is an anaerobic, methane-producing archaeon that utilizes H(2) or formate for the reduction of CO(2) to methane. Tryptophan auxotrophs were constructed by in vitro insertions of the Tn5 transposon into the tryptophan operon, followed by transformation into M. maripaludis. This method could serve for rapid insertions into large cloned DNA regions. PMID:19566682

  20. Tryptophan auxotrophs were obtained by random transposon insertions in the Methanonococcus maripaludis tryptophan operon

    OpenAIRE

    Porat, Iris; Whitman, William B.

    2009-01-01

    Methanococcus maripaludis is an anaerobic, methane-producing archaeon that utilizes H2 or formate for the reduction of CO2 to methane. Tryptophan auxotrophs were constructed by in vitro insertions of the Tn5 transposon into the tryptophan operon, followed by transformation into M. maripaludis. This method could serve for rapid insertions into large cloned DNA regions.

  1. Amylomaltase of Pyrobaculum aerophilum IM2 produces thermoreversible starch gels

    NARCIS (Netherlands)

    Kaper, T.; Talik, B.; Ettema, T.J.; Bos, H.; Maarel, M.J.E.C. van der; Dijkhuizen, L.

    2005-01-01

    Amylomaltases are 4-α-glucanotransferases (EC 2.4.1.25) of glycoside hydrolase family 77 that transfer α-1,4-linked glucans to another acceptor, which can be the 4-OH group of an α-1,4-linked glucan or glucose. The amylomaltase-encoding gene (PAE1209) from the hyperthermophilic archaeon Pyrobaculum

  2. Amylomaltase of Pyrobaculum aerophilum IM2 produces thermoreversible starch gels

    NARCIS (Netherlands)

    Kaper, T.; Talik, B.; Ettema, T.J.G.; Bos, H.; Maarel, van der M.J.E.C.; Dijkhuizen, L.

    2005-01-01

    Amylomaltases are 4-¿-glucanotransferases (EC 2.4.1.25) of glycoside hydrolase family 77 that transfer ¿-1,4-linked glucans to another acceptor, which can be the 4-OH group of an ¿-1,4-linked glucan or glucose. The amylomaltase-encoding gene (PAE1209) from the hyperthermophilic archaeon Pyrobaculum

  3. Interdomain Conjugal Transfer of DNA from Bacteria to Archaea▿ †

    OpenAIRE

    Dodsworth, Jeremy A.; Li, Lei; Wei, Shiping; Hedlund, Brian P.; Leigh, John A.; de Figueiredo, Paul

    2010-01-01

    Escherichia coli transforms the methanogenic archaeon Methanococcus maripaludis at frequencies ranging from 0.2 × 10−6 to 2 × 10−6 per recipient cell. Transformation requires cell-to-cell contact, oriT, and tra functions, is insensitive to DNase I, and otherwise displays hallmarks of conjugation.

  4. Temperature effect on the sulfur isotope fractionation during sulfate reduction by two strains of the hyperthermophilic Archaeoglobus fulgidus

    NARCIS (Netherlands)

    Mitchell, K.; Heyer, A.; Canfield, D.E.; Hoek, J.; Habicht, K.S.

    2009-01-01

    Summary Sulfur isotope fractionation during dissimilatory sulfate reduction by two strains of the thermophilic archaeon Archaeoglobus fulgidus (strains VC-16 and Z) was explored over the entire temperature range of growth. The optimal cell-specific sulfate reduction rate (14 fmol cell-1 h -1) was fo

  5. Specific single-cell isolation and genomic amplification of uncultured microorganisms

    DEFF Research Database (Denmark)

    Kvist, Thomas; Ahring, Birgitte Kiær; Lasken, R.S.; Westermann, Peter

    2007-01-01

    We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific pri...

  6. Virology: Independent virus development outside a host

    DEFF Research Database (Denmark)

    Häring, M.; Vestergaard, Gisle Alberg; Rachel, R.;

    2005-01-01

    Viruses are thought to be functionally inactive once they are outside and independent of their host cell 1 . Here we describe an exceptional property of a newly discovered virus that infects a hyperthermophilic archaeon growing in acidic hot springs: the lemon-shaped viral particle develops a very...

  7. Regulation of the hydrogen metabolism in Methanothermobacter thermoautotrophicus

    NARCIS (Netherlands)

    Poorter, Linda Martine Isabel de

    2002-01-01

    Methanothermobacter thermoautotrophicus is an archaeon that reduces CO2 into methane with hydrogen as the electron donor. Under natural and laboratory conditions, hydrogen concentrations may vary over orders of magnitude. The organism has to adapt to these changes. In this thesis, the adaptation of

  8. Practical applications of hydrogenase I from Pyrococcus furiosus for NADPH generation and regeneration

    NARCIS (Netherlands)

    Mertens, R.; Greiner, L.; Ban, van den E.C.D.; Haaker, H.B.C.M.; Liese, A.

    2003-01-01

    The soluble hydrogenase I (H-2:NADP(+) oxidoreductase, EC 1.18.99.1) from the marine hyperthermophilic strain of the archaeon Pyrococcus furiosus was partially purified by anion-exchange chromatography. This P furiosus hydrogenase I preparation (PF H(2)ase I) has been used as biocatalyst in the enzy

  9. Draft Genome Sequence of an Obligately Methylotrophic Methanogen, Methanococcoides methylutens, Isolated from Marine Sediment

    KAUST Repository

    Guan, Y.

    2014-11-20

    Methanococcoides methylutens, the type species of the genus Methanococcoides, is a slightly halophilic methanogenic archaeon with a methylotrophic metabolism. Here, we present the annotated draft genome sequence of M. methylutens, which comprises 2,508,511 bp with 2,482 coding sequences, 51 tRNA genes, and a G+C content of 42.5%.

  10. Biological Membranes in Extreme Conditions: Simulations of Anionic Archaeal Tetraether Lipid Membranes

    Science.gov (United States)

    Pineda De Castro, Luis Felipe; Dopson, Mark

    2016-01-01

    In contrast to the majority of organisms that have cells bound by di-ester phospholipids, archaeal membranes consist of di- and tetraether phospholipids. Originating from organisms that withstand harsh conditions (e.g., low pH and a wide range of temperatures) such membranes have physical properties that make them attractive materials for biological research and biotechnological applications. We developed force-field parameters based on the widely used Generalized Amber Force Field (GAFF) to enable the study of anionic tetraether membranes of the model archaean Sulfolobus acidocaldarius by computer simulations. The simulations reveal that the physical properties of these unique membranes depend on the number of cyclopentane rings included in each lipid unit, and on the size of cations that are used to ensure charge neutrality. This suggests that the biophysical properties of Sulfolobus acidocaldarius cells depend not only on the compositions of their membranes but also on the media in which they grow. PMID:27167213

  11. Studies of archaeal virus-host systems in thermal environments

    DEFF Research Database (Denmark)

    Erdmann, Susanne

    comparisons of viral variants are presented. STSV2 (Sulfolobus tengchongensis spindle-shaped virus), a large virus exhibiting one tail, has been studied in detail with respect to its host range, the virion structure and the relationship with host cells and, in particular, the CRISPR based immune system of the...... possible functions are proposed. This virus exhibits the exceptional property of undergoing a major extracellular morphological development of two tails that occurs independently of the host cells. In addition, the response of the CRISPR (Clustered regularly interspaced short palindromic repeats) -Cas...... system of Sulfolobus species was investigated when challenged by different genetic elements. This adaptive immune system has a major impact on virus-host interactions. The adaptation mechanism, involving the uptake of fragments of genetic elements as spacer regions in CRISPR arrays was induced using an...

  12. Biological Membranes in Extreme Conditions: Simulations of Anionic Archaeal Tetraether Lipid Membranes.

    Directory of Open Access Journals (Sweden)

    Luis Felipe Pineda De Castro

    Full Text Available In contrast to the majority of organisms that have cells bound by di-ester phospholipids, archaeal membranes consist of di- and tetraether phospholipids. Originating from organisms that withstand harsh conditions (e.g., low pH and a wide range of temperatures such membranes have physical properties that make them attractive materials for biological research and biotechnological applications. We developed force-field parameters based on the widely used Generalized Amber Force Field (GAFF to enable the study of anionic tetraether membranes of the model archaean Sulfolobus acidocaldarius by computer simulations. The simulations reveal that the physical properties of these unique membranes depend on the number of cyclopentane rings included in each lipid unit, and on the size of cations that are used to ensure charge neutrality. This suggests that the biophysical properties of Sulfolobus acidocaldarius cells depend not only on the compositions of their membranes but also on the media in which they grow.

  13. Dicty_cDB: Contig-U10445-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 6 1e-66 AF526233_1( AF526233 |pid:none) Argopecten irradians isolate iolsa... 255 2e-66 ( P50880 ) RecName: Full=60S riboso...7 |pid:none) Mus musculus strain BALB/c ribosomal p... 155 2e-36 CP001400_1281( CP001400 |pid:none) Sulfolobus islandi...1(... 108 4e-22 CP001399_1368( CP001399 |pid:none) Sulfolobus islandicus L.S.2.15,... 107 6e-22 (Q3IMY8) RecName: Full=50S riboso...4 FN316316_1( FN316316 |pid:none) Schistosoma japonicum isolate Anhu... 248 4e-64 FN357411_20( FN357411 |pid:none) Schistosoma manso...382_30(A... 42 0.033 CP001277_1681( CP001277 |pid:none) Candidatus Hamiltonella defensa... 42 0.033 (Q47LJ3)

  14. Dicty_cDB: Contig-U06051-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 12 CP001400_1281( CP001400 |pid:none) Sulfolobus islandicus M.14.25, ... 75 2e-12 (B0R656) RecName: Full=50S riboso...mal protein L3P; &(Q9HPD4) Rec... 75 2e-12 CP001399_1368( CP001399 |pid:none) Sulfolobus islandicus L...22_1( EU752622 |pid:none) Populus tremula isolate swl21-002 ... 56 7e-07 AF526233_1( AF526233 |pid:none) Argopecten irradians isol...e-60 FN316317_1( FN316317 |pid:none) Schistosoma japonicum isolate Anhu... 233 4e-60 CR940347_663( CR940347 |pid:none) Theileria an...169 5e-41 DQ206525_1( DQ206525 |pid:none) Suberites fuscus isolate TOA20 rib... 157 3e-37 AM494969_341( AM494969 |pid:none) Leishman

  15. Dicty_cDB: Contig-U16272-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available muelleri putative 60S rib... 251 7e-71 AF526233_1( AF526233 |pid:none) Argopecten irradians isolate iols... &CP000477_169... 142 2e-40 CP001400_1281( CP001400 |pid:none) Sulfolobus islandicus M.14.25, ... 132 4e-40 ...CP001399_1368( CP001399 |pid:none) Sulfolobus islandicus L.S.2.15,... 130 1e-39 AK297711_1( AK297711 |pid:no...39... 42 0.046 FM992695_51( FM992695 |pid:none) Candida dubliniensis CD36 chromos... 42 0.046 (A1T4N9) RecName: Full=50S riboso...6 (B6EPS5) RecName: Full=50S ribosomal protein L3; &FM178379_320(... 42 0.046 CP001277_1681( CP001277 |pid:none) Candi

  16. Archaeal virus-host interactions

    OpenAIRE

    Quax, T.E.F.

    2013-01-01

      The work presented in this thesis provides novel insights in several aspects of the molecular biology of archaea, bacteria and their viruses. Three fundamentally different groups of viruses are associated with the three domains of life. Archaeal viruses are characterized by a particularly high morphological and genetic diversity. Some archaeal viruses, such as Sulfolobus islandicus rod-shaped virus 2 (SIRV2), have quite remarkable infection cycles. As described in Chapter 1, infection ...

  17. Dicty_cDB: Contig-U05174-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 2-4, complete s... 48 0.29 1 ( BA000023 ) Sulfolobus tokodaii str. 7 DNA, complete genome. 48 0.29 1 ( ES370710 ) EoEST2521 Oil... 1 ( EY407440 ) pOP-EAP01865_EST_C_1_pBSK_SK EA (Oil Palm Embryoi... 44 4.6 1 ( EY396582 ) pOP-CBP00122_EST_C_1_pBSK_SK CB (Oil

  18. Direct observation of defect structure in protein crystals by atomic force and transmission electron microscopy.

    OpenAIRE

    Devaud, G; Furcinitti, P S; Fleming, J. C.; Lyon, M K; Douglas, K

    1992-01-01

    We have examined the structure of S-layers isolated from Sulfolobus acidocaldarius using atomic force microscopy (AFM) and transmission electron microscopy (TEM). From the AFM images, we were able to directly observe individual dimers of the crystal, defects in the crystal structure, and twin boundaries. We have identified two types of boundaries, one defined by a mirror plane and the other by a glide plane. This work shows that twin boundaries are highly structured regions that are directly ...

  19. Identification and Characterization of a Highly Conserved Crenarchaeal Protein Lysine Methyltransferase with Broad Substrate Specificity

    OpenAIRE

    Chu, Yindi; Zhang, Zhenfeng; Wang, Qian; Luo, Yuanming; Huang, Li

    2012-01-01

    Protein lysine methylation occurs extensively in the Crenarchaeota, a major kingdom in the Archaea. However, the enzymes responsible for this type of posttranslational modification have not been found. Here we report the identification and characterization of the first crenarchaeal protein lysine methyltransferase, designated aKMT, from the hyperthermophilic crenarchaeon Sulfolobus islandicus. The enzyme was capable of transferring methyl groups to selected lysine residues in a substrate prot...

  20. Expression, purification and crystallization of the ammonium transporter Amt-1 from Archaeoglobus fulgidus

    Energy Technology Data Exchange (ETDEWEB)

    Andrade, Susana L. A., E-mail: sandrad@uni-goettingen.de; Dickmanns, Antje; Ficner, Ralf; Einsle, Oliver, E-mail: sandrad@uni-goettingen.de [Abteilung Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen (Germany)

    2005-09-01

    The ammonium transporter Amt-1 from the cytoplasmic membrane of the hyperthermophilic archaeon A. fulgidus has been purified and crystallized. Ammonium transporters (Amts) are a class of membrane-integral transport proteins found in organisms from all kingdoms of life. Their key function is the transport of nitrogen in its reduced bioavailable form, ammonia, across cellular membranes, a crucial step in nitrogen assimilation for biosynthetic purposes. The genome of the hyperthermophilic archaeon Archaeoglobus fulgidus has been annotated with three individual genes for ammonium transporters, amt1–3, the roles of which are as yet unknown. The amt1 gene product has been produced by heterologous overexpression in Escherichia coli and the resulting protein has been purified to electrophoretic homogeneity. Crystals of Amt-1 have been obtained by sitting-drop vapour diffusion and diffraction data have been collected.

  1. Events during Initiation of Archaeal Transcription: Open Complex Formation and DNA-Protein Interactions

    OpenAIRE

    Hausner, Winfried; Thomm, Michael

    2001-01-01

    Transcription in Archaea is initiated by association of a TATA box binding protein (TBP) with a TATA box. This interaction is stabilized by the binding of the transcription factor IIB (TFIIB) orthologue TFB. We show here that the RNA polymerase of the archaeon Methanococcus, in contrast to polymerase II, does not require hydrolysis of the β-γ bond of ATP for initiation of transcription and open complex formation on linearized DNA. Permanganate probing revealed that the archaeal open complex s...

  2. Archaeal Transcription: Function of an Alternative Transcription Factor B from Pyrococcus furiosus▿

    OpenAIRE

    Micorescu, Michael; Grünberg, Sebastian; Franke, Andreas; Cramer, Patrick; Thomm, Michael; Bartlett, Michael

    2007-01-01

    The genome of the hyperthermophile archaeon Pyrococcus furiosus encodes two transcription factor B (TFB) paralogs, one of which (TFB1) was previously characterized in transcription initiation. The second TFB (TFB2) is unusual in that it lacks recognizable homology to the archaeal TFB/eukaryotic TFIIB B-finger motif. TFB2 functions poorly in promoter-dependent transcription initiation, but photochemical cross-linking experiments indicated that the orientation and occupancy of transcription com...

  3. Exploring the reductive capacity of Pyrococcus furiosus. The reduction of carboxylic acids and pyridine nucleotides

    OpenAIRE

    Ban, van den, A.W.

    2001-01-01

    This Ph.D. project started in 1997 and its main goal was to obtain insight in the reductive capacity of the hyperthermophilic archaeon Pyrococcus furiosus . The research was focused on the biocatalytic reduction of carboxylic acids.Reductions of carboxylic acids are interesting reactions, since the generated products, aldehydes and alcohols, are potentially applicable in the fine-chemical industry. However, the reduction of carboxylic acids to the corresponding aldehydes is a thermodynamicall...

  4. Fructose Degradation in the Haloarchaeon Haloferax volcanii Involves a Bacterial Type Phosphoenolpyruvate-Dependent Phosphotransferase System, Fructose-1-Phosphate Kinase, and Class II Fructose-1,6-Bisphosphate Aldolase

    OpenAIRE

    Pickl, Andreas; Johnsen, Ulrike; Schönheit, Peter

    2012-01-01

    The halophilic archaeon Haloferax volcanii utilizes fructose as a sole carbon and energy source. Genes and enzymes involved in fructose uptake and degradation were identified by transcriptional analyses, deletion mutant experiments, and enzyme characterization. During growth on fructose, the gene cluster HVO_1495 to HVO_1499, encoding homologs of the five bacterial phosphotransferase system (PTS) components enzyme IIB (EIIB), enzyme I (EI), histidine protein (HPr), EIIA, and EIIC, was highly ...

  5. Enumeration and Characterization of Acidophilic Microorganisms Isolated from a Pilot Plant Stirred-Tank Bioleaching Operation

    OpenAIRE

    Okibe, Naoko; Gericke, Mariekie; Hallberg, Kevin B.; Johnson, D. Barrie

    2003-01-01

    Microorganisms were enumerated and isolated on selective solid media from a pilot-scale stirred-tank bioleaching operation in which a polymetallic sulfide concentrate was subjected to biologically accelerated oxidation at 45°C. Four distinct prokaryotes were isolated: three bacteria (an Acidithiobacillus caldus-like organism, a thermophilic Leptospirillum sp., and a Sulfobacillus sp.) and one archaeon (a Ferroplasma-like isolate). The relative numbers of these prokaryotes changed in the three...

  6. Isolation and Characterization of Methanomethylovorans hollandica gen. nov., sp. nov., Isolated from Freshwater Sediment, a Methylotrophic Methanogen Able To Grow on Dimethyl Sulfide and Methanethiol

    OpenAIRE

    Bart P. Lomans; Maas, Ronald; Luderer, Rianne; Op den Camp, Huub J.M.; Pol, Arjan; van der Drift, Chris; Vogels, Godfried D.

    1999-01-01

    A newly isolated methanogen, strain DMS1T, is the first obligately anaerobic archaeon which was directly enriched and isolated from a freshwater sediment in defined minimal medium containing dimethyl sulfide (DMS) as the sole carbon and energy source. The use of a chemostat with a continuous DMS-containing gas stream as a method of enrichment, followed by cultivation in deep agar tubes, resulted in a pure culture. Since the only substrates utilized by strain DMS1T are methanol, methylamines, ...

  7. Charakterisierung von TrmB und TrmBL1, zwei Transkriptionsregulatoren im Zentrum des Zuckerstoffwechsels von Pyrococcus furiosus

    OpenAIRE

    Surma, Melanie

    2011-01-01

    Die vorliegende Arbeit beschäftigt sich mit Untersuchungen zur Funktionsweise der beiden zuckerspezifischen Transkriptionsregulatoren TrmB und TrmBL1 innerhalb des Zuckerstoffwechsels des hyperthermophilen Archaeons Pyrococcus furiosus. Die Aufnahme von Kohlenhydraten in die Zelle wird bei P. furiosus von zwei substratspezifischen ABC–Transportern vermittelt. Der Maltose/Trehalose-Transporter ist dabei ausschließlich für den Transport von Maltose und Trehalose zuständig, während der Malto...

  8. A Simple Laser-Based Device for Simultaneous Microbial Culture and Absorbance Measurement

    CERN Document Server

    Abrevaya, X C; Areso, O; Mauas, P J D

    2012-01-01

    In this work we present a device specifically designed to study microbial growth with several applications related to environmental microbiology and other areas of research as astrobiology. The Automated Measuring and Cultivation device (AMC-d) enables semi-continuous absorbance measurements directly during cultivation. It can measure simultaneously up to 16 samples. Growth curves using low and fast growing microorganism were plotted, including: Escherichia coli, and Haloferax volcanii, an halophilic archaeon.

  9. Analysis of Nanoarchaeum equitans genome and proteome composition: indications for hyperthermophilic and parasitic adaptation

    OpenAIRE

    Bag Sumit K; Paul Sandip; Das Sabyasachi; Dutta Chitra

    2006-01-01

    Abstract Background Nanoarchaeum equitans, the only known hyperthermophilic archaeon exhibiting parasitic life style, has raised some new questions about the evolution of the Archaea and provided a model of choice to study the genome landmarks correlated with thermo-parasitic adaptation. In this context, we have analyzed the genome and proteome composition of N. equitans and compared the same with those of other mesophiles, hyperthermophiles and obligatory host-associated organisms. Results A...

  10. Thermal adaptation analyzed by comparison of protein sequences from mesophilic and extremely thermophilic Methanococcus species

    OpenAIRE

    Haney, Paul J.; Jonathan H Badger; Buldak, Gerald L.; Reich, Claudia I.; Woese, Carl R.; Olsen, Gary J.

    1999-01-01

    The genome sequence of the extremely thermophilic archaeon Methanococcus jannaschii provides a wealth of data on proteins from a thermophile. In this paper, sequences of 115 proteins from M. jannaschii are compared with their homologs from mesophilic Methanococcus species. Although the growth temperatures of the mesophiles are about 50°C below that of M. jannaschii, their genomic G+C contents are nearly identical. The properties most correlated with the proteins of the thermophile include hig...

  11. Small RNA-guided processes in the hyperthermophilic methanogen Methanopyrus kandleri

    OpenAIRE

    Su, Andreas A. H.

    2015-01-01

    In this thesis, a combination of RNAseq, computational and biochemical methods was applied to analyze processes that use small RNAs (sRNAs) as guide molecules at extreme temperatures. Here, the hyperthermophilic archaeon Methanopyrus kandleri, which grows at temperatures of up to 110°C, was used as a model organism. The genome of M. kandleri harbors two CRISPR-Cas systems that use CRISPR RNA (crRNA) as guide molecules to target fo...

  12. 食品廃棄物のメタン発酵及び水素・メタン二段発酵

    OpenAIRE

    Nakashimada, Yutaka; Nishio, Naomichi

    2003-01-01

    To treat and recover energy from food industrial solid wastes, methane fermantion is very altractive, in this article, therefore, researches on methane fermentation of solid wastes from a Japanese Sake brewery, bread manufacture and soy sauce brewery were reviewed. To solubilize and acidify the waste from a Japanese Sake brewery, hyperthemophilic archaeon Pyrococcus furiosus could be applied. A perfusion culture developed to increase cell concentration gave 0.4 g/l/h of acetate production rat...

  13. Comparative genomic and transcriptional analyses of CRISPR systems across the genus Pyrobaculum

    OpenAIRE

    Bernick, David L.; Cox, Courtney L.; Dennis, Patrick P.; Lowe, Todd M.

    2012-01-01

    Within the domain Archaea, the CRISPR immune system appears to be nearly ubiquitous based on computational genome analyses. Initial studies in bacteria demonstrated that the CRISPR system targets invading plasmid and viral DNA. Recent experiments in the model archaeon Pyrococcus furiosus have uncovered a novel RNA-targeting variant of the CRISPR system. Because our understanding of CRISPR system evolution in other archaea is limited, we have taken a comparative genomic and transcriptomic view...

  14. Comparative Genomic and Transcriptional Analyses of CRISPR Systems Across the Genus Pyrobaculum

    OpenAIRE

    Bernick, David L.; Cox, Courtney L.; Dennis, Patrick P.; Lowe, Todd M.

    2012-01-01

    Within the domain Archaea, the CRISPR immune system appears to be nearly ubiquitous based on computational genome analyses. Initial studies in bacteria demonstrated that the CRISPR system targets invading plasmid and viral DNA. Recent experiments in the model archaeon Pyrococcus furiosus uncovered a novel RNA-targeting variant of the CRISPR system potentially unique to archaea. Because our understanding of CRISPR system evolution in other archaea is limited, we have taken a comparative genom...

  15. Anaerobic Ammonium-Oxidizing Bacteria: Unique Microorganisms with Exceptional Properties

    OpenAIRE

    Niftrik, L.A.M.P. van; Jetten, M.S.M.

    2012-01-01

    Summary: Anaerobic ammonium-oxidizing (anammox) bacteria defy many microbiological concepts and share numerous properties with both eukaryotes and archaea. Among their most intriguing characteristics are their compartmentalized cell plan and archaeon-like cell wall. Here we review our current knowledge about anammox cell biology. The anammox cell is divided into three separate compartments by bilayer membranes. The anammox cell consists of (from outside to inside) the cell wall, paryphoplasm,...

  16. Biocatalytic Synthesis of Poly(δ-Valerolactone) Using a Thermophilic Esterase from Archaeoglobus fulgidus as Catalyst

    OpenAIRE

    Xuedong Fang; Lingfei Zhang; Yan Yang; Jiebing Yang; Guangquan Li; Haobo Han; Hong Cao; Quanshun Li

    2012-01-01

    The ring-opening polymerization of δ-valerolactone catalyzed by a thermophilic esterase from the archaeon Archaeoglobus fulgidus was successfully conducted in organic solvents. The effects of enzyme concentration, temperature, reaction time and reaction medium on monomer conversion and product molecular weight were systematically evaluated. Through the optimization of reaction conditions, poly(δ-valerolactone) was produced in 97% monomer conve...

  17. CbiZ, an amidohydrolase enzyme required for salvaging the coenzyme B12 precursor cobinamide in archaea

    OpenAIRE

    Woodson, Jesse D.; Escalante-Semerena, Jorge C.

    2004-01-01

    The existence of a pathway for salvaging the coenzyme B12 precursor dicyanocobinamide (Cbi) from the environment was established by genetic and biochemical means. The pathway requires the function of a previously unidentified amidohydrolase enzyme that converts adenosylcobinamide to adenosylcobyric acid, a bona fide intermediate of the de novo coenzyme B12 biosynthetic route. The cbiZ gene of the methanogenic archaeon Methanosarcina mazei strain Göl was cloned, was overproduced in Escherichia...

  18. Regulation of the hydrogen metabolism in Methanothermobacter thermoautotrophicus

    OpenAIRE

    Poorter, Linda Martine Isabel de

    2002-01-01

    Methanothermobacter thermoautotrophicus is an archaeon that reduces CO2 into methane with hydrogen as the electron donor. Under natural and laboratory conditions, hydrogen concentrations may vary over orders of magnitude. The organism has to adapt to these changes. In this thesis, the adaptation of M. thermoautotrophicus to varying hydrogen concentrations is investigated at the bioenergetic and physiological levels. The study includes the development of new methods for the determination of in...

  19. Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes

    OpenAIRE

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F.

    2015-01-01

    Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners—the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)—and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic ...

  20. Genome Update: alignment of bacterial chromosomes

    DEFF Research Database (Denmark)

    Ussery, David; Jensen, Mette; Poulsen, Tine Rugh;

    2004-01-01

    There are four new microbial genomes listed in this month's Genome Update, three belonging to Gram-positive bacteria and one belonging to an archaeon that lives at pH 0; all of these genomes are listed in Table 1⇓. The method of genome comparison this month is that of genome alignment and......, as an example, an alignment of seven Staphylococcus aureus genomes and one Staphylococcus epidermidis genome is presented....

  1. Assessing methanotrophy and carbon fixation for biofuel production by Methanosarcina acetivorans

    OpenAIRE

    Nazem-Bokaee, Hadi; Gopalakrishnan, Saratram; James G. Ferry; Thomas K. Wood; Maranas, Costas D.

    2016-01-01

    Background Methanosarcina acetivorans is a model archaeon with renewed interest due to its unique reversible methane production pathways. However, the mechanism and relevant pathways implicated in (co)utilizing novel carbon substrates in this organism are still not fully understood. This paper provides a comprehensive inventory of thermodynamically feasible routes for anaerobic methane oxidation, co-reactant utilization, and maximum carbon yields of major biofuel candidates by M. acetivorans....

  2. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analyses of threonyl-tRNA synthetase editing domain from Aeropyrum pernix

    International Nuclear Information System (INIS)

    The editing domain of threonyl-tRNA synthetase from the archaeon Aeropyrum pernix has been overexpressed, purified and crystallized. The crystal diffracted to a resolution of 1.66 Å. The proofreading function of aminoacyl-tRNA synthetases is crucial in maintaining the fidelity of protein synthesis. Most archaeal threonyl-tRNA synthetases (ThrRSs) possess a unique proofreading domain unrelated to their eukaryotic/bacterial counterpart. The crystal structure of this domain from the archaeon Pyrococcus abysii in complex with its cognate and noncognate substrate analogues had given insights into its catalytic and discriminatory mechanisms. To probe further into the mechanistic and evolutionary aspects of this domain, work has been extended to another archaeon Aeropyrum pernix. The organism possesses two proteins corresponding to threonyl-tRNA synthetase, i.e. ThrRS1 and ThrRS2, encoded by two different genes, thrS1 and thrS2, respectively. ThrRS1 is responsible for aminoacylation and ThrRS2 for proofreading activity. Here the purification, crystallization and preliminary X-ray crystallographic investigation of the N-terminal proofreading domain of ThrRS2 from A. pernix is reported. The crystals belong to either the P41212 or P43212 space group and consist of one monomer per asymmetric unit

  3. Dicty_cDB: Contig-U14889-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 23 |pid:none) Sulfolobus tokodaii str. 7 DNA,... 79 2e-13 AY123115_1( AY123115 |pid:none) Griffithsia japoni...g*nc y*islfit*chlftigc*nciqaflayiqyywhwe**slifitidchgfnniiidgfvhk ctqw*ettihq*fyit*lllc***tw**n*slf*fy*ffs... CP000112_2261( CP000112 |pid:none) Desulfovibrio desulfuricans G20... 87 4e-16 CP000489_2182( CP000489 |pid:none) Par...ective length of database: 649,847,548 Effective search space: 70833382732 Effective search space used: 70833382732 Neighbori...ntig length 699 Chromosome number (1..6, M) 5 Chromosome length 5062330 Start point 4866664 End point 486735

  4. A novel rudivirus, ARV1, of the hyperthermophilic archaeal genus Acidianus

    DEFF Research Database (Denmark)

    Vestergaard, Gisle Alberg; Häring, Monika; Peng, Xu;

    2005-01-01

    tail fibers protruding at each end which appear to be involved in adsorption onto the host cell surface. The virions contain two protein components, a major one of 14.4 kDa, which is glycosylated and a minor of about 124 kDa. The linear double-stranded DNA genome yielded 24,655 bp of sequence....... Other assigned functions include a thymidylate synthase and three DNA-binding proteins. The genome sequence and composition differ strongly from those of the Sulfolobus rudiviruses SIRV1 and SIRV2, and the genome stability is very high, with no sequence variants being detected. Although the sequences of...

  5. AcEST: DK957646 [AcEST

    Lifescience Database Archive (English)

    Full Text Available TST39A01NGRL0028_O17 589 Adiantum capillus-veneris mRNA. clone: TST39A01NGRL0028_O17. 5' end seq ... ion of protein database search programs", Nucleic Aci ds Res. 25:3389-3402. Query= DK957646|Adiantum capi ... .......................done Score E Sequences produci ng significant alignments: (bits) Value sp|Q5ZJ54|T ... |THSB_SULAC Thermosome subunit beta OS=Sulfolobus aci do... 124 3e-28 sp|O30560|THS2_HALVO Thermosome sub ... -DRETLIDVAKTSLRTKVHTELADILTEAVVDSVL 176 Query: 582 CI ... 587 + Sbjct: 177 AV 178 >sp|P80317|TCPZ_MOUSE T-co ...

  6. Dissection of the functional domains of an archaeal holliday junction helicase

    DEFF Research Database (Denmark)

    Hong, Ye; Chu, Mingzhu; Li, Yansheng;

    2012-01-01

    Helicases and nucleases form complexes that play very important roles in DNA repair pathways some of which interact with each other at Holliday junctions. In this study, we present in vitro and in vivo analysis of Hjm and its interaction with Hjc in Sulfolobus. In vitro studies employed Hjm from...... propagation assay, suggesting that Hjm/Hjc mediated resolution of stalled replication forks is of crucial importance in archaea. A tentative pathway with which Hjm/Hjc interaction could have occurred at stalled replication forks is discussed....

  7. Dicty_cDB: Contig-U16443-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available AP008937 |pid:none) Lactobacillus fermentum IFO 395... 44 0.021 CP001399_72( CP001399 |pid:none) Sulfolobus...gth of database: 101,790,757,118 Length adjustment: 24 Effective length of query: 1509 Effective len...successfully gapped: 326 Length of query: 511 Length of database: 1,061,185,681 Length adjustmen...gth of query: 1533 length of database: 8,075,542 effective HSP length: 16 effe...ctive length of query: 1517 effective length of database: 7,941,110 effective search space: 12046663870 effe

  8. Crystallization and improvement of crystal quality for x-ray diffraction of maltooligosyl trehalose synthase by reductive methylation of lysine residues.

    Science.gov (United States)

    Kobayashi, M; Kubota, M; Matsuura, Y

    1999-04-01

    Maltooligosyl trehalose synthase, one of the two enzymes in the coupled trehalose biosynthesis system in Sulfolobus acidocaldarius, has been purified and crystallized. The chemical modification of this enzyme by reductive methylation of lysine residues significantly improved the crystal quality for X-ray diffraction experiments. The crystals of the modified enzyme belong to orthorhombic space group P212121, with unit-cell parameters a = 56.70, b = 140.1, c = 205.2 A measured at cryo-temperature, and are found to contain two enzyme molecules per asymmetric unit. PMID:10089339

  9. Dicty_cDB: Contig-U09816-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available M97514 ) Saccharomyces douglasii mitochondrial cytochrome c ... 32 0.45 6 ( AE014824 ) Plasmodium falciparum 3D7 chromo...ne) Mus musculus 10 days neonate skin ... 35 4.1 AE016879_2470( AE016879 |pid:none) Bacillus anthracis str. ...44( AE016853 |pid:none) Pseudomonas syringae pv. tomato... 34 9.2 CP001399_90( CP001399 |pid:none) Sulfolobus islan... malaccensis strain MP75 mitochondrion... 42 3e-04 5 ( AF063866 ) Melanoplus sanguinipes entomo.... 36 0.071 10 ( AL929354 ) Plasmodium falciparum strain 3D7, chromosome 5, s... 32 0.071 16 ( AP009180 ) Candidat

  10. Dicty_cDB: Contig-U16361-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available n... 45 0.021 EU423621_1( EU423621 |pid:none) Echinacea pallida isolate 315A-112....:none) Variovorax paradoxus S110 chrom... 42 3e-04 DQ014506_1( DQ014506 |pid:none) Eucalyptu...nthase ... 45 0.007 CP000077_888( CP000077 |pid:none) Sulfolobus acidocaldarius DSM 63... 40 0.008 DQ898153_1( DQ898153 |pid...:none) Pinus radiata cellulose synthase (... 44 0.009 AF458083_1( AF458083 |pid:none) Arabidopsis thali... letters Score E Sequences producing significant alignments: (bits) Value Contig-U16361-1 (C

  11. Dicty_cDB: Contig-U16454-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available :none) Enterococcus faecalis V583, com... 45 0.006 CP001146_1816( CP001146 |pid:none) D............................done Score E Sequences producing significant alignments: (bits) Value N ( BJ376046 ) Dictyostelium discoid...:none) Proteus mirabilis strain HI4320... 213 4e-95 CP000644_2108( CP000644 |pid:none) Aeromonas salmonicida...nus TCF52B, co... 44 0.014 CP000077_598( CP000077 |pid:none) Sulfolobus acidocalda...tus Cloacamonas acidamin... 39 0.45 CP000886_1029( CP000886 |pid:none) Salmonella enterica

  12. Dicty_cDB: Contig-U16123-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available region ... 48 1.3 1 ( AY881806 ) Anas americana isolate USWS-4634579 control...J302282 ) Homo sapiens satellite DNA, isolate D8S2622. 34 6.1 3 ( EI711217 ) 2B42..._1333( CP001399 |pid:none) Sulfolobus islandicus L.S.2.15,... 38 1.6 CR380958_183...5( FM992693 |pid:none) Candida dubliniensis CD36 chromos... 35 8.0 FN357384_26( FN357384 |pid:none) Schistosoma manso..., clone FCL-AC02. 1289 0.0 3 ( AC116920 ) Dictyostelium discoideum chromosome 2 m

  13. Archaeal Life on Tangkuban Perahu- Sampling and Culture Growth in Indonesian Laboratories

    Directory of Open Access Journals (Sweden)

    SRI HANDAYANI

    2012-09-01

    Full Text Available The aim of the expedition to Tangkuban Perahu, West Java was to obtain archaeal samples from the solfatara fields located in Domas crater. This was one of the places, where scientists from the University of Regensburg Germany had formerly isolated Indonesian archaea, especially Thermoplasma and Sulfolobus species but not fully characterized. We collected five samples from mud holes with temperatures from 57 to 88 oC and pH of 1.5-2. A portion of each sample was grown at the University of Regensburg in modified Allen’s medium at 80 oC. From four out of five samples enrichment cultures were obtained, autotrophically on elemental sulphur and heterotrophically on sulfur and yeast extract; electron micrographs are presented. In the laboratories of Universitas Indonesia the isolates were cultured at 55-60 oC in order to grow tetraetherlipid synthesizing archaea, both Thermoplasmatales and Sulfolobales. Here, we succeeded to culture the same type of archaeal cells, which had been cultured in Regensburg, probably a Sulfolobus species and in Freundt’s medium, Thermoplasma species. The harvested cells are documented by phase contrast microscope equipped with a digital camera. Our next steps will be to further characterize genetically the cultured cells from Tangkuban Perahu isolates.

  14. Bacterial catalytic processes for transformation of metals.

    Science.gov (United States)

    Paknikar, K M

    1993-01-01

    Microorganisms actively participate in the transformation of metals and metalloids by various processes including adsorption, absorption, alkylation, oxidation and reduction reactions. Bacteria of the genera Thiobacillus and Sulfolobus have a chemolithotrophic mode of metabolism and catalyze various metal transformations. These bacteria are primarily involved in oxidation-reduction reactions of metals. Metal sulfides can be either directly oxidized by the microbes or oxidized by ferric iron, an end-product of microbial metabolism. These processes result in solubilization of iron, copper, molybdenum, uranium, and many other metals as well. Bacteria capable of these reactions are found, and they function in a variety of environments. Conditions which favor their growth are acidic pH (1.5 to 3.5) and temperatures which can be as high as 75 degrees C for Sulfolobus. The microbes have potential for mobilization of pollutants, or toxic elements, during resource extraction processes. In the absence of oxygen, iron, molybdenum and chromium may be reduced by microbes. PMID:8181951

  15. A proton shelter inspired by the sugar coating of acidophilic archaea

    OpenAIRE

    Xiumei Wang; Bei’er Lv; Guixin Cai; Long Fu; Yuanzi Wu; Xiang Wang; Bin Ren; Hongwei Ma

    2012-01-01

    The acidophilic archaeons are a group of single-celled microorganisms that flourish in hot acid springs (usually pH < 3) but maintain their internal pH near neutral. Although there is a lack of direct evidence, the abundance of sugar modifications on the cell surface has been suggested to provide the acidophiles with protection against proton invasion. In this study, a hydroxyl (OH)-rich polymer brush layer was prepared to mimic the OH-rich sugar coating. Using a novel pH-sensitive dithioacet...

  16. Comparison of four phaC genes from Haloferax mediterranei and their function in different PHBV copolymer biosyntheses in Haloarcula hispanica

    DEFF Research Database (Denmark)

    Han, Jing; Li, Ming; Hou, Jing;

    2010-01-01

    BACKGROUND: The halophilic archaeon Haloferax mediterranei is able to accumulate large amounts of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with high molar fraction of 3-hydroxyvalerate (3HV) from unrelated carbon sources. A Polyhydroxyalkanoate (PHA) synthase composed of two subunits, ...... might meet various application requirements. CONCLUSION: We discover three cryptic phaC genes in Hfx. mediterranei, and demonstrate that genetic engineering of these newly identified phaC genes has biotechnological potential for PHBV production with tailor-made material properties....

  17. A Single-Culture Bioprocess of Methanothermobacter thermautotrophicus to Upgrade Digester Biogas by CO 2 -to-CH 4 Conversion with H 2

    OpenAIRE

    Martin, Matthew R.; Fornero, Jeffrey J.; Rebecca Stark; Laurens Mets; Largus T. Angenent

    2013-01-01

    We optimized and tested a postbioprocessing step with a single-culture archaeon to upgrade biogas (i.e., increase methane content) from anaerobic digesters via conversion of CO2 into CH4 by feeding H2 gas. We optimized a culture of the thermophilic methanogen Methanothermobacter thermautotrophicus using: (1) a synthetic H2/CO2 mixture; (2) the same mixture with pressurization; (3) a synthetic biogas with different CH4 contents and H2; and (4) an industrial, untreated biogas and H2. A laborato...

  18. A New Thermoactive Pullulanase from Desulfurococcus mucosus: Cloning, Sequencing, Purification, and Characterization of the Recombinant Enzyme after Expression in Bacillus subtilis

    OpenAIRE

    Duffner, Fiona; Bertoldo, Costanzo; Andersen, Jens T.; Wagner, Karen; Antranikian, Garabed

    2000-01-01

    The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosus showed 45.4% pairwise amino acid identity with the pullulanase from Thermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuA gene...

  19. Utilization of vinasse for production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) by Haloferax mediterranei

    OpenAIRE

    Bhattacharyya, Anirban; Pramanik, Arnab; Maji, Sudipta Kumar; Haldar, Saubhik; Mukhopadhyay, Ujjal Kumar; Mukherjee, Joydeep

    2012-01-01

    Vinasse, a highly polluting waste of the ethanol industry was utilized for the production of polyhydroxyalkanoate (PHA) by the extremely halophilic archaeon, Haloferax mediterranei in shake-flasks. Following pre-treatment through adsorption on activated carbon, 25%-50% (v/v) pre-treated vinasse was utilized leading to 70% maximum accumulation of PHA. Maximum PHA concentration of 19.7 g/l, product yield coefficient (based on total carbohydrates) of 0.87 and 0.21 g/l h volumetric productivity w...

  20. Insights into archaeal evolution and symbiosis from the genomes of a Nanoarchaeon and its crenarchaeal host from Yellowstone National Park

    Energy Technology Data Exchange (ETDEWEB)

    Podar, Mircea [ORNL; Graham, David E [ORNL; Reysenbach, Anna-Louise [Portland State University; Koonin, Eugene [National Center for Biotechnology Information; Wolf, Yuri [National Center for Biotechnology Information; Makarova, Kira S. [National Center for Biotechnology Information

    2013-01-01

    A hyperthemophilic member of the Nanoarchaeota from Obsidian Pool, a thermal feature in Yellowstone National Park was characterized using single cell isolation and sequencing, together with its putative host, a Sulfolobales archaeon. This first representative of a non-marine Nanoarchaeota (Nst1) resembles Nanoarchaeum equitans by lacking most biosynthetic capabilities, the two forming a deep-branching archaeal lineage. However, the Nst1 genome is over 20% larger, encodes a complete gluconeogenesis pathway and a full complement of archaeal flagellum proteins. Comparison of the two genomes suggests that the marine and terrestrial Nanoarchaeota lineages share a common ancestor that was already a symbiont of another archaeon. With a larger genome, a smaller repertoire of split protein encoding genes and no split non-contiguous tRNAs, Nst1 appears to have experienced less severe genome reduction than N. equitans. The inferred host of Nst1 is potentially autotrophic, with a streamlined genome and simplified central and energetic metabolism as compared to other Sulfolobales. The two distinct Nanoarchaeota-host genomic data sets offer insights into the evolution of archaeal symbiosis and parasitism and will further enable studies of the cellular and molecular mechanisms of these relationships.

  1. High CO2 subsurface environment enriches for novel microbial lineages capable of autotrophic carbon fixation

    Science.gov (United States)

    Probst, A. J.; Jerett, J.; Castelle, C. J.; Thomas, B. C.; Sharon, I.; Brown, C. T.; Anantharaman, K.; Emerson, J. B.; Hernsdorf, A. W.; Amano, Y.; Suzuki, Y.; Tringe, S. G.; Woyke, T.; Banfield, J. F.

    2015-12-01

    Subsurface environments span the planet but remain little understood from the perspective of the capacity of the resident organisms to fix CO2. Here we investigated the autotrophic capacity of microbial communities in range of a high-CO2 subsurface environments via analysis of 250 near-complete microbial genomes (151 of them from distinct species) that represent the most abundant organisms over a subsurface depth transect. More than one third of the genomes belonged to the so-called candidate phyla radiation (CPR), which have limited metabolic capabilities. Approximately 30% of the community members are autotrophs that comprise 70% of the microbiome with metabolism likely supported by sulfur and nitrogen respiration. Of the carbon fixation pathways, the Calvin Benson Basham Cycle was most common, but the Wood-Ljungdhal pathway was present in the greatest phylogenetic diversity of organisms. Unexpectedly, one organism from a novel phylum sibling to the CPR is predicted to fix carbon by the reverse TCA cycle. The genome of the most abundant organism, an archaeon designated "Candidatus Altiarchaeum hamiconexum", was also found in subsurface samples from other continents including Europe and Asia. The archaeon was proven to be a carbon fixer using a novel reductive acetyl-CoA pathway. These results provide evidence that carbon dioxide is the major carbon source in these environments and suggest that autotrophy in the subsurface represents a substantial carbon dioxide sink affecting the global carbon cycle.

  2. Thermococcus kodakarensis modulates its polar membrane lipids and elemental composition according to growth stage and phosphate availability

    Directory of Open Access Journals (Sweden)

    TravisB.Meador

    2014-01-01

    Full Text Available We observed significant changes in the elemental and intact polar lipid (IPL composition of the archaeon Thermococcus kodakarensis (KOD1 in response to growth stage and phosphorus supply. Reducing the amount of organic supplements and phosphate in growth media resulted in significant decreases in cell size and cellular quotas of carbon (C, nitrogen (N, and phosphorus (P, which coincided with significant increases in cellular IPL quota and IPLs comprising multiple P atoms and hexose moieties. Relatively more cellular P was stored as IPLs in P-limited cells (2-8% compared to control cells (< 0.8%. We also identified a specific IPL biomarker containing a phosphatidyl-N-acetylhexoseamine headgroup that was relatively enriched during rapid cell division. These observations serve as empirical evidence of IPL adaptations in Archaea that will help to interpret the distribution of these biomarkers in natural systems. The reported cell quotas of C, N, and P represent the first such data for a specific archaeon and suggest that thermophiles are C-rich compared to the cell carbon-to-volume relationship reported for planktonic bacteria.

  3. Circular Permutation of a Chaperonin Protein: Biophysics and Application to Nanotechnology

    Science.gov (United States)

    Paavola, Chad; Chan, Suzanne; Li, Yi-Fen; McMillan, R. Andrew; Trent, Jonathan

    2004-01-01

    We have designed five circular permutants of a chaperonin protein derived from the hyperthermophilic organism Sulfolobus shibatae. These permuted proteins were expressed in E. coli and are well-folded. Furthermore, all the permutants assemble into 18-mer double rings of the same form as the wild-type protein. We characterized the thermodynamics of folding for each permutant by both guanidine denaturation and differential scanning calorimetry. We also examined the assembly of chaperonin rings into higher order structures that may be used as nanoscale templates. The results show that circular permutation can be used to tune the thermodynamic properties of a protein template as well as facilitating the fusion of peptides, binding proteins or enzymes onto nanostructured templates.

  4. Dicty_cDB: Contig-U10033-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available =Replication factor C small subunit; ... 219 1e-55 EU016664_18( EU016664 |pid:none) Uncultured marine microorga...0910_3306( CP000910 |pid:none) Renibacterium salmoninarum ATCC... 88 7e-16 AP009552_5393( AP009552 |pid:none) Microcystis aerugin...CP001404 |pid:none) Sulfolobus islandicus Y.N.15.51... 237 5e-61 (A4WGV2) RecName: Full=Replication factor C small subuni... GB-M1 of ... 126 1e-27 DQ149023_152( DQ149023 |pid:none) Synechococcus cyanophage syn9, c... AG1412 ) DNA polymerase III (gamma and tau chains) homolog... 94 1e-17 (Q12TX1) RecName: Full=Replication factor C large subuni

  5. The bioleaching of different sulfide concentrates using thermophilic bacteria

    Science.gov (United States)

    Torres, F.; Blázquez, M. L.; González, F.; Ballester, A.; Mier, J. L.

    1995-05-01

    The bioleaching of different mineral sulfide concentrates with thermophilic bacteria (genus Sulfolobus @#@) was studied. Since the use of this type of bacteria in leaching systems involves stirring and the control of temperature, the influence of the type of stirring and the pulp density on dissolution rates was studied in order to ascertain the optimum conditions for metal recovery. At low pulp densities, the dissolution kinetic was favored by pneumatic stirring, but for higher pulp densities, orbital stirring produced the best results. A comparative study of three differential concentrates, one mixed concentrate, and one global concentrate was made. Copper and iron extraction is directly influenced by bacterial activity, while zinc dissolution is basically due to an indirect mechanism that is activated in the presence of copper ions. Galvanic interactions between the different sulfides favors the selective bioleaching of some phases (sphalerite and chalcopyrite) and leads to high metal recovery rates. However, the formation of galvanic couples depends on the type of concentrate.

  6. Dicty_cDB: Contig-U15680-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available s) Database: CSM 8402 sequences; 8,075,542 total letters Score E Sequences producing significant alignments: (bits) Value Cont...AM270075_67( AM270075 |pid:none) Aspergillus niger contig An04c014... 209 e-104 T29637( T29637 ) hypothetical protein F49E8.3 - Caen...3e-79 BC059334_1( BC059334 |pid:none) Xenopus laevis hypothetical protei... 174 7e-79 CP000927_3( CP000927 |pid:none) Caulobacter...Sulfolobus islandicus Y.G.57.14... 132 6e-62 CP000414_448( CP000414 |pid:none) Leuconostoc mesenteroides sub... BT010001 |pid:none) Drosophila melanogaster RE31064 fu... 80 4e-15 T29564( T29564 )hypothetical protein T12E12.6 - Caen

  7. Dicty_cDB: Contig-U16379-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 189_2123( CP001189 |pid:none) Gluconacetobacter diazotrophicu... 132 4e-40 T23883( T23883 ) hypothetical protein R03G8.4 - Caen...65( AP007281 |pid:none) Lactobacillus reuteri JCM 1112 ... 157 3e-49 CP000077_358( CP000077 |pid:none) Sulfolobus acidocal...remor... 155 1e-46 CP000414_448( CP000414 |pid:none) Leuconostoc mesenteroides su...ig-U16379-1Q.Seq.d (5131 letters) Database: CSM 8402 sequences; 8,075,542 total letter...s Score E Sequences producing significant alignments: (bits) Value Contig-U16379-1 (Contig-U16379-1Q) /CSM_Contig/Cont

  8. CRISPR-based immune systems of the Sulfolobales: complexity and diversity

    DEFF Research Database (Denmark)

    Garrett, Roger Antony; Shah, Shiraz Ali; Vestergaard, Gisle Alberg;

    2011-01-01

    CRISPR (cluster of regularly interspaced palindromic repeats)/Cas and CRISPR/Cmr systems of Sulfolobus, targeting DNA and RNA respectively of invading viruses or plasmids are complex and diverse. We address their classification and functional diversity, and the wide sequence diversity of RAMP...... (repeat-associated mysterious protein)-motif containing proteins encoded in Cmr modules. Factors influencing maintenance of partially impaired CRISPR-based systems are discussed. The capacity for whole CRISPR transcripts to be generated despite the uptake of transcription signals within spacer sequences...... is considered. Targeting of protospacer regions of invading elements by Cas protein-crRNA (CRISPR RNA) complexes exhibit relatively low sequence stringency, but the integrity of protospacer-associated motifs appears to be important. Different mechanisms for circumventing or inactivating the immune...

  9. STSV2 as a Model Crenarchaeal Virus for Studying Virus-Host Interactions and CRISPR-Cas Adaptive Immunity

    DEFF Research Database (Denmark)

    León Sobrino, Carlos

    archaea harbour their own viruses, which constitute an extraordinarily diverse group with exotic morphologies and unique features. Prokaryotes possess a variety of defence mechanisms. The CRISPR-Cas adaptive immune system is of great importance for archaea –84% of them possess it, compared to 45% for...... generate immune memory by inserting in its own genome short invader-derived DNA fragments forming a database –the CRISPR locus. Little was known about this system until recent years, and the generation of immune memory has been the most elusive step. In this work, the interactions of the spindle......-shaped monocaudavirus STSV2 and its host Sulfolobus islandicus REY15A were studied. This interaction produced, after several days, de novo CRISPR adaptation – that is, without any previous memory that can act as a trigger. We employed transcriptome sequencing to characterise the long-term progression of this...

  10. An archaeal CRISPR type III-B system exhibiting distinctive RNA targeting features and mediating dual RNA and DNA interference

    DEFF Research Database (Denmark)

    Peng, Wenfang; Feng, Mingxia; Feng, Xu;

    2015-01-01

    CRISPR-Cas systems provide a small RNA-based mechanism to defend against invasive genetic elements in archaea and bacteria. To investigate the in vivo mechanism of RNA interference by two type III-B systems (Cmr-α and Cmr-β) in Sulfolobus islandicus, a genetic assay was developed using plasmids...... carrying an artificial mini-CRISPR (AC) locus with a single spacer. After pAC plasmids were introduced into different strains, Northern analyses confirmed that mature crRNAs were produced from the plasmid-borne CRISPR loci, which then guided gene silencing to target gene expression. Spacer mutagenesis....... islandicus Cmr-α mediated transcription-dependent DNA interference, the Cmr-α constitutes the first CRISPR system exhibiting dual targeting of RNA and DNA....

  11. Ab initio single and multideterminant methods used in the determination of reduction potentials and magnetic properties of Rieske ferredoxins

    Science.gov (United States)

    Powers, Nathan Lee

    2008-10-01

    The [Fe2S2]2+/[Fe2S 2]+ electronic structure of seven Rieske protein active sites (bovine mitochondrial cytochrome bc1 complex, spinach chloroplast cytochrome b6f complex, Rieske-type ferredoxin associated with biphenyl dioxygenase from Burkholderia cepacia, yeast cytochrome bcl complex from Saccharomyces cerevisiae, Rieske subunit of arsenite oxidase from Alcaligenes faecalis, respiratory-type Rieske protein from Thermus thermophilus, and Rieske protein II (soxF) from Sulfolobus acidocaldarius), which lie in a reduction potential range from -150 mV to 375 mV, have been studied by both single and multi-determinant quantum mechanical methods. Calculated reduction potentials and magnetic properties are found comparable to experimental values.

  12. Dicty_cDB: Contig-U16486-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 770( CP001025 |pid:none) Burkholderia ambifaria MC40-6 ch... 76 5e-12 CP001472_2469( CP001472 |pid:none) Acidobacterium cap...J2315 c... 77 3e-12 CP001472_1731( CP001472 |pid:none) Acidobacterium capsulatum ATCC ... 76 4e-12 CP001025_...01472 |pid:none) Acidobacterium capsulatum ATCC ... 49 9e-04 CP001399_2737( CP001399 |pid:none) Sulfolobus i...6840_2270( AP006840 |pid:none) Symbiobacterium thermophilum IA... 40 0.31 AY826921_1( AY826921 |pid:none) Heterocap..... 37 2.0 BA000011_1521( BA000011 |pid:none) Thermoplasma volcanium GSS1 DNA... 37 2.6 BX842637_13( BX842637 |pid:none) Neur

  13. Dicty_cDB: Contig-U10929-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 3( AE008692 |pid:none) Zymomonas mobilis subsp. mobilis... 77 3e-12 EF676622_1( EF676622 |pid:none) Picea sit...monas mobilis fosmid clone 42D... 72 5e-11 AE016822_1177( AE016822 |pid:none) Leifsonia xyli subsp. xyli...11 A37855( A37855 ) glucose-facilitated diffusion protein - Zymomonas... 74 2e-11..._4096( CP001127 |pid:none) Salmonella enterica subsp. ente... 69 5e-10 DQ152824_1( DQ152824 |pid:none) Danio rerio facilit...( CP001403 |pid:none) Sulfolobus islandicus Y.G.57.14... 65 8e-09 AF518411_1( AF518411 |pid:none) Toxoplasma gondii facilit

  14. Dicty_cDB: Contig-U02831-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ngipalpis EST clone NSFM-153d06, sequ... 44 9.7 1 ( AI999603 ) 701556638 A. thaliana, Columbia Col...ne LE_H... 46 2.4 1 ( AR514840 ) Sequence 19800 from patent US 6703491. 46 2.4 1 ( AR499558 ) Sequence 4518 from... patent US 6703491. 46 2.4 1 ( AY456633 ) Martelia tanganyicensis voucher 220.041a 16S....001 CP001402_1231( CP001402 |pid:none) Sulfolobus islandicus M.16.4, c... 47 0.001 FJ897525_1( FJ897525 |pid:none) Bombyx man...9887_345( AM999887 |pid:none) Wolbachia endosymbiont of Culex ... 45 0.005 EF030714_1( EF030714 |pid:none) Danio rerio slo

  15. Structure of the acidianus filamentous virus 3 and comparative genomics of related archaeal lipothrixviruses

    DEFF Research Database (Denmark)

    Vestergaard, Gisle Alberg; Aramayo, Ricardo; Basta, Tamara;

    2008-01-01

    Four novel filamentous viruses with double-stranded DNA genomes, namely, Acidianus filamentous virus 3 (AFV3), AFV6, AFV7, and AFV8, have been characterized from the hyperthermophilic archaeal genus Acidianus, and they are assigned to the Betalipothrixvirus genus of the family Lipothrixviridae. The...... structures of the approximately 2-mum-long virions are similar, and one of them, AFV3, was studied in detail. It consists of a cylindrical envelope containing globular subunits arranged in a helical formation that is unique for any known double-stranded DNA virus. The envelope is 3.1 nm thick and encases an...... high level of conservation in both gene content and gene order over large regions, with this similarity extending partly to the earlier described betalipothrixvirus Sulfolobus islandicus filamentous virus. A few predicted gene products of each virus, in addition to the structural proteins, could be...

  16. Dicty_cDB: Contig-U14936-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ma cDNA, c... 306 9e-82 CP001140_1265( CP001140 |pid:none) Desulfurococcus kamcha...:none) Guillardia theta DNA for complet... 292 2e-77 AC006081_7( AC006081 |pid:none) Arabidopsis thaliana ch...CP000481 |pid:none) Acidothermus cellulolyticus 11B ... 214 5e-54 EF558547_15( EF558547 |pid:none) Uncultured haloarcha...1404 |pid:none) Sulfolobus islandicus Y.N.15.51... 213 1e-53 CP001140_1067( CP001140 |pid:none) Desulfurococcus kamcha...7298 ) CAXY4611.rev CAXY Lottia gigantea from male gonad... 94 2e-46 6 ( ES731865 ) Nad03b_35_F03_C005.g1 Nupha

  17. AcEST: BP918083 [AcEST

    Lifescience Database Archive (English)

    Full Text Available )-5-[(5- phosphoribosylamino)methylideneamino] imidazole-4-carboxamide isomerase OS=Staphylococcus epidermid...Q8CQ93|HIS4_STAES 1-(5-phosphoribosyl)-5-[(5- phosphoribosylamino)methylideneamino] imidazole-4-carboxamide isomerase OS=Staphyl...|HIS4_STAEQ 1-(5-phosphoribosyl)-5-[(5-phosphoribosylam... 30 4.9 sp|Q5VKN9|VGLG_...oc... 30 6.5 sp|Q8CQ93|HIS4_STAES 1-(5-phosphoribosyl)-5-[(5-phosphoribosylam... 30 6.5 sp|A0A...WCBV Glycoprotein G OS=West Caucasian bat virus G... 30 6.4 sp|Q4J904|SYR_SULAC Arginyl-tRNA synthetase OS=Sulfolobus acid

  18. Dicty_cDB: Contig-U12139-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available eus AH187, complete... 130 1e-28 CP000511_1585( CP000511 |pid:none) Mycobacterium vanbaalen...i partial pgm gene f... 49 8e-04 AE003852_599( AE003852 |pid:none) Vibrio cholerae O1 biovar el...0627_131( CP000627 |pid:none) Vibrio cholerae O395 chromosome ... 47 0.002 AJ619013_1( AJ619013 |pid:none) Oenococcus oeni partial...8 1.4 CP001399_1929( CP001399 |pid:none) Sulfolobus islandicus L.S.2.15,... 38 1.4 AE017220_718( AE017220 |pid:none) Salmonella ent...is subsp. ... 35 6.9 CP000885_2302( CP000885 |pid:none) Clostridium phytofermentans ISD... 35 9

  19. Dicty_cDB: Contig-U05085-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ca cDNA clon... 34 2.5 2 ( CO216404 ) WS0044.B21_O22 SS-R-A-5 Picea sitchensis cDNA clo... 34 2.5 2 ( CX4133...um tetani E88, complete... 34 1.8 CP001400_1957( CP001400 |pid:none) Sulfolobus islandicus M.14.25, ... 33 2.4 CP000554_2470( CP000...H250760 ) BOGAH67TF BOGA Brassica oleracea genomic clone BO... 32 9.3 2 ( DX412140 ) SOYF232TH LargeInsertSo...3 4 ( CU469464 ) Candidatus Phytoplasma mali strain AT complete ch... 36 0.034 9 ( FG321702 ) gmnlem_0005a14...l-Ocean-Sampling_GS-30-02-01-1... 38 1.3 2 ( CD326164 ) AGENCOURT_14147097 NICHD_XGC_Eye1 Xenopus laevis ... 38 1.3 2 ( CD36214

  20. Dicty_cDB: Contig-U15098-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available mal protein L32; AltName: Full=... 118 2e-32 AF526230_1( AF526230 |pid:none) Argopecten irradians isolate iols...mal protein L32e; &AP006878_15... 57 4e-07 CP001399_1385( CP001399 |pid:none) Sulfolobus islandi...9 6e-32 (Q8WRF3) RecName: Full=60S ribosomal protein L32; AltName: Full=... 119 6e-32 AB244986_1( AB244986 |pid:none) Polypedi...-21 FM992693_149( FM992693 |pid:none) Candida dubliniensis CD36 chromo... 96 6e-21 CR382125_297( CR382125 |p... |pid:none) Lutzomyia intermedia isolate IAFrp... 77 3e-13 AJ965641_1( AJ965641 |pid:none) Nyctotherus ovalis

  1. Dicty_cDB: Contig-U15146-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available AF526198_1( AF526198 |pid:none) Argopecten irradians isolate iolsa... 135 3e-31 DQ440259_1( DQ440259 |pid:no...P001399 |pid:none) Sulfolobus islandicus L.S.2.15,... 66 3e-10 CP001398_1924( CP001398 |pid:none) Thermococcus gammatoleran...53 ) Mus Musculus Strain C57BL6/J chromosome 3 BAC, RP... 48 0.25 1 ( EF134343 ) Oxyrrhis marina isol...ate Om-5p-21-2 ribosomal prot... 48 0.25 1 ( EF134342 ) Oxyrrhis marina isolate Om-5p-21-1 riboso.... 124 8e-28 EF134343_1( EF134343 |pid:none) Oxyrrhis marina isolate Om-5p-21-2... 124 8e-28 BT046434_1( BT04

  2. Application of microorganisms in coal cleaning processes

    International Nuclear Information System (INIS)

    A secure energy supply is one of the basic pre-requisites for a sound economic system, sustained standard and quality of life and eventually for the social well-being of each individual. For a progressive country like Pakistan, it is obligatory that all energy options must be pursued vigorously including coal utilization, which given the relatively large resources available, is considered to be one of the major options for the next few hundred years. Bioprocessing of coal in an emerging technology which has started to receive considerable research attention. Recent research activities involving coal cleaning, direct coal conversion, and indirect conversion of coal-derived materials have generated a plethora of facts regarding biochemistry, chemistry, and thermodynamic behavior of coal, in that its bioprocessing is on the verge of becoming and acceptable means to great coals. In this research report, investigations pertaining to the various aspects of coal bio processing, including desulfurization and depyritization are discussed. Bituminous coals varying in total sulfur contents of 3-6% were depyritized more than 90% by mesophilic acidophiles like Thiobacillus ferroxidans and Thiobacillus thio oxidans and thermophilic Sulfolobus brierleyi. The archaebacterium, Sulfolobus brierleyi was found to desulfurize inorganic and organic sulfur components of the coal. Conditions were established under which it can remove more than 30% of the organic sulfur present in the coals. Heterotrophic microorganisms including oxenic and soil isolates were also employed for studying sulfurization. A soil isolate, Oil-2, was found to remove more than 70% dibenzothiophenic sulfur present in an oil-water emulsion (1:20 ratio). Pseudomonas putida and the bacterium oil-2 also remove 60-70% organic sulfur present in the shale-oil. Preliminary results indicate the presence of putatively known Kodama's pathway in the oil-2. The mass balance for sulfate indicated the possibility of the presence

  3. Biosynthesis of membrane lipids of thermophilic archaebacteria and its implication to early evolution of life

    International Nuclear Information System (INIS)

    The unit lipid of cell membranes of archaebacteria is unique ether lipids, O-dialkylated glycerol with a polar head group at sn-1 position. The chirality of glycerol moiety of the lipids is opposite to that of other kingdoms. The hydrophobic potion consists of saturated C20 isoprenoid hydrocarbon backbone and is connected to glycerol by an ether linkage. In addition, cell membrane of some of thermophilic archaebacteria are monolayer (in stead of bilayer) of tetraether lipids in which both tails of hydrocarbon chains of two diether lipids are covalently connected in a tail-to-tail fashion. Although the host cell from which contemporary eukaryotes have been derived by endosymbiosis, is speculated to be an archaebacterium, the unique ether lipids raised a serious question to the idea of archabacterial origin of eukaryote cells; why the unique ether lipids are not used to construct cytoplasmic membranes of eukaryotes? The author and his colleagues have studied biosynthesis of membrane liquids of two thermo-acidophilic archaebacteria, Thermoplasma and Sulfolobus. It was found that origins of stereospecificity of glycerol moiety of archaebacterial ether lipids differs form species to species. In Sulfolobus sn-glycerol-1-phosphate (the abnormal isomer of glycerol phosphate) seems to be directly synthesized from glycerol, whereas in Halobacterium stereospecificity of glycerol phosphate is inverted during the lipid synthesis. Recently we found that specific inhibitors for eukaryotes squalene epoxidase inhibit the condensation of diether lipids to tetraether lipids in cell-free extracts of these thermophilic archaebacteria. The results suggest evolutionary implication of archaebacterial tetraether condensing enzyme to eukaryote sterol biosynthesis. Relationships between chemical structures of membrane lipids and early evolution of life will be discussed. (author). Abstract only

  4. Microbial Community Structure and Arsenic Biogeochemistry in an Acid Vapor-Formed Spring in Tengchong Geothermal Area, China.

    Directory of Open Access Journals (Sweden)

    Zhou Jiang

    Full Text Available Arsenic biogeochemistry has been studied extensively in acid sulfate-chloride hot springs, but not in acid sulfate hot springs with low chloride. In this study, Zhenzhuquan in Tengchong geothermal area, a representative acid sulfate hot spring with low chloride, was chosen to study arsenic geochemistry and microbial community structure using Illumina MiSeq sequencing. Over 0.3 million 16S rRNA sequence reads were obtained from 6-paired parallel water and sediment samples along its outflow channel. Arsenic oxidation occurred in the Zhenxhuquan pool, with distinctly high ratios of arsenate to total dissolved arsenic (0.73-0.86. Coupled with iron and sulfur oxidation along the outflow channel, arsenic accumulated in downstream sediments with concentrations up to 16.44 g/kg and appeared to significantly constrain their microbial community diversity. These oxidations might be correlated with the appearance of some putative functional microbial populations, such as Aquificae and Pseudomonas (arsenic oxidation, Sulfolobus (sulfur and iron oxidation, Metallosphaera and Acidicaldus (iron oxidation. Temperature, total organic carbon and dissolved oxygen significantly shaped the microbial community structure of upstream and downstream samples. In the upstream outflow channel region, most microbial populations were microaerophilic/anaerobic thermophiles and hyperthermophiles, such as Sulfolobus, Nocardia, Fervidicoccus, Delftia, and Ralstonia. In the downstream region, aerobic heterotrophic mesophiles and thermophiles were identified, including Ktedonobacteria, Acidicaldus, Chthonomonas and Sphingobacteria. A total of 72.41-95.91% unassigned-genus sequences were derived from the downstream high arsenic sediments 16S rRNA clone libraries. This study could enable us to achieve an integrated understanding on arsenic biogeochemistry in acid hot springs.

  5. Microbial Community Structure and Arsenic Biogeochemistry in an Acid Vapor-Formed Spring in Tengchong Geothermal Area, China.

    Science.gov (United States)

    Jiang, Zhou; Li, Ping; Jiang, Dawei; Dai, Xinyue; Zhang, Rui; Wang, Yanhong; Wang, Yanxin

    2016-01-01

    Arsenic biogeochemistry has been studied extensively in acid sulfate-chloride hot springs, but not in acid sulfate hot springs with low chloride. In this study, Zhenzhuquan in Tengchong geothermal area, a representative acid sulfate hot spring with low chloride, was chosen to study arsenic geochemistry and microbial community structure using Illumina MiSeq sequencing. Over 0.3 million 16S rRNA sequence reads were obtained from 6-paired parallel water and sediment samples along its outflow channel. Arsenic oxidation occurred in the Zhenxhuquan pool, with distinctly high ratios of arsenate to total dissolved arsenic (0.73-0.86). Coupled with iron and sulfur oxidation along the outflow channel, arsenic accumulated in downstream sediments with concentrations up to 16.44 g/kg and appeared to significantly constrain their microbial community diversity. These oxidations might be correlated with the appearance of some putative functional microbial populations, such as Aquificae and Pseudomonas (arsenic oxidation), Sulfolobus (sulfur and iron oxidation), Metallosphaera and Acidicaldus (iron oxidation). Temperature, total organic carbon and dissolved oxygen significantly shaped the microbial community structure of upstream and downstream samples. In the upstream outflow channel region, most microbial populations were microaerophilic/anaerobic thermophiles and hyperthermophiles, such as Sulfolobus, Nocardia, Fervidicoccus, Delftia, and Ralstonia. In the downstream region, aerobic heterotrophic mesophiles and thermophiles were identified, including Ktedonobacteria, Acidicaldus, Chthonomonas and Sphingobacteria. A total of 72.41-95.91% unassigned-genus sequences were derived from the downstream high arsenic sediments 16S rRNA clone libraries. This study could enable us to achieve an integrated understanding on arsenic biogeochemistry in acid hot springs. PMID:26761709

  6. Transcriptome changes and cAMP oscillations in an archaeal cell cycle

    Directory of Open Access Journals (Sweden)

    Soppa Jörg

    2007-06-01

    Full Text Available Abstract Background The cell cycle of all organisms includes mass increase by a factor of two, replication of the genetic material, segregation of the genome to different parts of the cell, and cell division into two daughter cells. It is tightly regulated and typically includes cell cycle-specific oscillations of the levels of transcripts, proteins, protein modifications, and signaling molecules. Until now cell cycle-specific transcriptome changes have been described for four eukaryotic species ranging from yeast to human, but only for two prokaryotic species. Similarly, oscillations of small signaling molecules have been identified in very few eukaryotic species, but not in any prokaryote. Results A synchronization procedure for the archaeon Halobacterium salinarum was optimized, so that nearly 100% of all cells divide in a time interval that is 1/4th of the generation time of exponentially growing cells. The method was used to characterize cell cycle-dependent transcriptome changes using a genome-wide DNA microarray. The transcript levels of 87 genes were found to be cell cycle-regulated, corresponding to 3% of all genes. They could be clustered into seven groups with different transcript level profiles. Cluster-specific sequence motifs were detected around the start of the genes that are predicted to be involved in cell cycle-specific transcriptional regulation. Notably, many cell cycle genes that have oscillating transcript levels in eukaryotes are not regulated on the transcriptional level in H. salinarum. Synchronized cultures were also used to identify putative small signaling molecules. H. salinarum was found to contain a basal cAMP concentration of 200 μM, considerably higher than that of yeast. The cAMP concentration is shortly induced directly prior to and after cell division, and thus cAMP probably is an important signal for cell cycle progression. Conclusion The analysis of cell cycle-specific transcriptome changes of H. salinarum

  7. ANME-2D Archaea Catalyze Methane Oxidation in Deep Subsurface Sediments Independent of Nitrate Reduction

    Science.gov (United States)

    Hernsdorf, A. W.; Amano, Y.; Suzuki, Y.; Ise, K.; Thomas, B. C.; Banfield, J. F.

    2015-12-01

    Terrestrial sediments are an important global reservoir for methane. Microorganisms in the deep subsurface play a critical role in the methane cycle, yet much remains to be learned about their diversity and metabolisms. To provide more comprehensive insight into the microbiology of the methane cycle in the deep subsurface, we conducted a genome-resolved study of samples collected from the Horonobe Underground Research Laboratory (HURL), Japan. Groundwater samples were obtained from three boreholes from a depth range of between 140 m and 250 m in two consecutive years. Groundwater was filtered and metagenomic DNA extracted and sequenced, and the sequence data assembled. Based on the sequences of phylogenetically informative genes on the assembled fragments, we detected a high degree of overlap in community composition across a vertical transect within one borehole at the two sampling times. However, there was comparatively little similarity observed among communities across boreholes. Spatial and temporal abundance patterns were used in combination with tetranucleotide signatures of assembled genome fragments to bin the data and reconstruct over 200 unique draft genomes, of which 137 are considered to be of high quality (>90% complete). The deepest samples from one borehole were highly dominated by an archaeon identified as ANME-2D; this organism was also present at lower abundance in all other samples from that borehole. Also abundant in these microbial communities were novel members of the Gammaproteobacteria, Saccharibacteria (TM7) and Tenericute phyla. Notably, a ~2 Mbp draft genome for the ANME-2D archaeon was reconstructed. As expected, the genome encodes all of the genes predicted to be involved in the reverse methanogenesis pathway. In contrast with the previously reported ANME2-D genome, the HURL ANME-2D genome lacks the capacity to reduce nitrate. However, we identified many multiheme cytochromes with closest similarity to those of the known Fe

  8. Immobilization and Characterization of a Recombinant Thermostable Lipase (Pf2001 from Pyrococcus furiosus on Supports with Different Degrees of Hydrophobicity

    Directory of Open Access Journals (Sweden)

    Roberta Vieira Branco

    2010-01-01

    Full Text Available We studied the immobilization of a recombinant thermostable lipase (Pf2001Δ60 from the hyperthermophilic archaeon Pyrococcus furiosus on supports with different degrees of hydrophobicity: butyl Sepabeads and octadecyl Sepabeads. The enzyme was strongly adsorbed in both supports. When it was adsorbed on these supports, the enzyme showed 140 and 237% hyperactivation, respectively. The assessment of storage stability showed that the octadecyl Sepabeads immobilized enzyme showed 100% of residual activity after 30 days of storage. However, the greatest stability at 70∘C was obtained in butyl Sepabeads immobilized enzyme, which retained 77% activity after 1 hour incubation. The maximum activity of the immobilized preparations was obtained with the pH between 6 and 7, at 70∘C. Thus, this study achieved a new extremophilic biocatalyst with greater stability, for use in several biotechnological processes.

  9. A proton shelter inspired by the sugar coating of acidophilic archaea

    Science.gov (United States)

    Wang, Xiumei; Lv, Bei'er; Cai, Guixin; Fu, Long; Wu, Yuanzi; Wang, Xiang; Ren, Bin; Ma, Hongwei

    2012-11-01

    The acidophilic archaeons are a group of single-celled microorganisms that flourish in hot acid springs (usually pH polymer brush layer was prepared to mimic the OH-rich sugar coating. Using a novel pH-sensitive dithioacetal molecule as a probe, we studied the proton-resisting property and found that a 10-nm-thick polymer layer was able to raise the pH from 1.0 to > 5.0, indicating that the densely packed OH-rich layer is a proton shelter. As strong evidence for the role of sugar coatings as proton barriers, this biomimetic study provides insight into evolutionary biology, and the results also could be expanded for the development of biocompatible anti-acid materials.

  10. Crystallization and preliminary X-ray analysis of PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins

    International Nuclear Information System (INIS)

    PH1010, a DUF54-family protein from the hyperthermophilic archaeon P. horikoshii OT3, was crystallized and X-ray diffraction data were collected to 1.90 Å resolution. PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins, was expressed, purified and crystallized. Crystallization was performed by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.90 Å resolution using a synchrotron-radiation source. The space group of the crystal was determined to be P212121, with unit-cell parameters a = 46.9, b = 49.5, c = 132.7 Å. The crystal contained two PH1010 molecules in the asymmetric unit (VM = 2.4 Å3 Da−1) and had a solvent content of 48%

  11. Microbial metalloproteomes explored using MIRAGE.

    Science.gov (United States)

    Sevcenco, Ana-Maria; Hagen, Wilfred R; Hagedoorn, Peter-Leon

    2012-09-01

    Metalloproteomics is a rapidly developing field of science that involves the comprehensive analysis of all metal-containing or metal-binding proteins in a biological sample. The purpose of this review is to offer an overview of the research involving Metal Isotope native RadioAutography in Gel Electrophoresis (MIRAGE), a powerful new method to visualize and study the proteome of a particular metal ion. MIRAGE involves four steps: i) labelling of target proteins with a radioisotope; ii) separation of intact holo-proteins using native isoelectric focusing (1D) combined with Blue Native PAGE (2D); iii) spot visualization and quantification using autoradiography; and iv) protein identification by tandem mass spectrometry. MIRAGE Investigations of the soluble Cu, Zn, and Fe metalloproteomes of Escherichia coli, and of the soluble Mo and W proteomes of the hyperthermophilic archaeon Pyrococcus furiosus are reviewed. PMID:22976984

  12. Diversity and similarity of microbial communities in petroleum crude oils produced in Asia.

    Science.gov (United States)

    Yamane, Kunio; Maki, Hideaki; Nakayama, Tsuyoshi; Nakajima, Toshiaki; Nomura, Nobuhiko; Uchiyama, Hiroo; Kitaoka, Motomitsu

    2008-11-01

    To understand microbial communities in petroleum crude oils, we precipitated DNA using high concentrations of 2,2,4-trimethylpentane (isooctane) and purified. Samples of DNA from five crude oils, (Middle East, 3; China, 1; and Japan, 1) were characterized based upon their 16S rRNA gene sequences after PCR amplification and the construction of clone libraries. We detected 48 eubacterial species, one cyanobacterium, and one archaeon in total. The microbial constituents were diverse in the DNA samples. Most of the bacteria affiliated with the sequences of the three oils from the Middle East comprised similar mesophilic species. Acinetobacter, Propionibacterium, Sphingobium and a Bacillales were common. In contrast, the bacterial communities in Japanese and Chinese samples were unique. Thermophilic Petrotoga-like bacteria (11%) and several anaerobic-thermophilic Clostridia- and Synergistetes-like bacteria (20%) were detected in the Chinese sample. Different thermophiles (12%) and Clostridia (2%) were detected in the Japanese sample. PMID:18997416

  13. Effects of Oxytetracycline on Methane Production and the Microbial Communities During Anaerobic Digestion of Cow Manure

    Institute of Scientific and Technical Information of China (English)

    KE Xin; WANG Chun-yong; LI Run-dong; ZHANG Yun

    2014-01-01

    The effects of different concentrations of oxytetracycline (OTC) on the dynamics of bacterial and archaeal communities during the mesophilic anaerobic digestion (37°C) of cow manure were investigated. Before anaerobic digestion, OTC was added to digesters at concentrations of 20, 50, and 80 mg L-1, respectively. Compared with no-antibiotic control, all methane productions underwent different levels of inhibition at different concentrations of OTC. Changes in the bacterial and archaeal communities were discussed by using PCR-denaturing gradient gel electrophoresis (DGGE). Results showed that OTC affected the richness and diversity of bacterial and archaeal communities. The bacterial genus Flavobacterium and an uncultured bacterium (JN256083.1) were detected throughout the entire process of anaerobic digestion and seemed to be the functional bacteria. Methanobrevibacter boviskoreani and an uncultured archaeon (FJ230982.1) dominated the archaeal communities during anaerobic digestion. These microorganisms may have high resistance to OTC and may play vital roles in methane production.

  14. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    International Nuclear Information System (INIS)

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Å resolution.

  15. Preliminary crystallography confirms that the archaeal DNA-binding and tryptophan-sensing regulator TrpY is a dimer.

    Science.gov (United States)

    Cafasso, Jacquelyn; Manjasetty, Babu A; Karr, Elizabeth A; Sandman, Kathleen; Chance, Mark R; Reeve, John N

    2010-11-01

    TrpY regulates the transcription of the metabolically expensive tryptophan-biosynthetic operon in the thermophilic archaeon Methanothermobacter thermautotrophicus. TrpY was crystallized using the hanging-drop method with ammonium sulfate as the precipitant. The crystals belonged to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 87, c = 147 Å, and diffracted to 2.9 Å resolution. The possible packing of molecules within the cell based on the values of the Matthews coefficient (V(M)) and analysis of the self-rotation function are consistent with the asymmetric unit being a dimer. Determining the structure of TrpY in detail will provide insight into the mechanisms of DNA binding, tryptophan sensing and transcription regulation at high temperature by this novel archaeal protein. PMID:21045304

  16. Preliminary Crystallography Confirms that the Archaeal DNA-binding and Tryptophan-sensing Regulator TrpY is a Dimer

    Energy Technology Data Exchange (ETDEWEB)

    J Cafasso; B Manjasetty; E Karr; K Sandman; M Chance; J Reeve

    2011-12-31

    TrpY regulates the transcription of the metabolically expensive tryptophan-biosynthetic operon in the thermophilic archaeon Methanothermobacter thermautotrophicus. TrpY was crystallized using the hanging-drop method with ammonium sulfate as the precipitant. The crystals belonged to the tetragonal space group P4{sub 3}2{sub 1}2 or P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 87, c = 147 {angstrom}, and diffracted to 2.9 {angstrom} resolution. The possible packing of molecules within the cell based on the values of the Matthews coefficient (V{sub M}) and analysis of the self-rotation function are consistent with the asymmetric unit being a dimer. Determining the structure of TrpY in detail will provide insight into the mechanisms of DNA binding, tryptophan sensing and transcription regulation at high temperature by this novel archaeal protein.

  17. Dicty_cDB: Contig-U15152-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available fatibacillum alkenivorans ... 62 8e-08 EU874252_13( EU874252 |pid:none) Uncultured so... 4e-05 CU861906_909( CU861906 |pid:none) Ralstonia solanacearum strain Mo... 53 4...00480_4791( CP000480 |pid:none) Mycobacterium smegmatis str. MC... 49 4e-04 CP000140_1569( CP000140 |pid:none) Parabacteroid...W, com... 88 1e-15 AM114193_508( AM114193 |pid:none) Uncultured methanogenic archaeon... 87 1e-15 (Q12YW2) RecName: Full=Digera...eptomyces antibioticus acetyltr... 84 1e-14 AP009153_2587( AP009153 |pid:none) Gemmatimonas auran

  18. Dicty_cDB: Contig-U10475-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available e) Rickettsia peacockii str. Rustic... 49 7e-05 CP000140_1264( CP000140 |pid:none) Parabacteroides distasoni...genomic c... 40 0.39 2 ( AL445564 ) Mycoplasma pulmonis (strain UAB CTIP) complete ge... 36 0.42 9 ( AC223854 ) Bos taurus clon...e-05 CP000103_1930( CP000103 |pid:none) Nitrosospira multiformis ATCC 2... 52 1e-... 33 5.2 AM910994_300( AM910994 |pid:none) Plasmodium knowlesi strain H chr... 33 5.2 FN357596_1( FN357596 |pid:none) Schistoso...complete... 33 5.2 AY714825_20( AY714825 |pid:none) Uncultured archaeon GZfos18B6 clo... 33 6.8 S07558( S07558 ) translation elon

  19. Evaluation of Three Automated Genome Annotations for Halorhabdus utahensis

    DEFF Research Database (Denmark)

    Bakke, Peter; Carney, Nick; DeLoache, Will;

    2009-01-01

    Genome annotations are accumulating rapidly and depend heavily on automated annotation systems. Many genome centers offer annotation systems but no one has compared their output in a systematic way to determine accuracy and inherent errors. Errors in the annotations are routinely deposited in...... databases such as NCBI and used to validate subsequent annotation errors. We submitted the genome sequence of halophilic archaeon Halorhabdus utahensis to be analyzed by three genome annotation services. We have examined the output from each service in a variety of ways in order to compare the methodology...... and effectiveness of the annotations, as well as to explore the genes, pathways, and physiology of the previously unannotated genome. The annotation services differ considerably in gene calls, features, and ease of use. We had to manually identify the origin of replication and the species...

  20. Over-expression of carboxypeptidase of extreme thermophile pyrococcus furiosus in escherichia coli

    International Nuclear Information System (INIS)

    Thermophiles and extreme thermophiles are potential source of thermostable proteases for economical application. This study deals with cloning and over-expression of a carboxypeptidase (CBP) from the extreme thermophile archaeon Pyrococcus furiosus in E. coli. Using the forward and the reverse primers designed according to the putative CBP gene sequence analysed from the published genome sequence of P. furiosus, 1.5 kb fragment of CBP gene was PCR amplified. After TA-cloning in pTZ57R/T vector, the gene was ligated into pET-22b(+) and the recombinant plasmid thus obtained was used to transform E. coli BL21 (DE3)RIPL. On induction with IPTG for 6-8 hours CBP was expressed up to 30% of the total cell proteins. The enzyme, however, was expressed in an insoluble form which was refolded to an active state by treatment with urea. (author)

  1. Birth of the eukaryotes by a set of reactive innovations: New insights force us to relinquish gradual models.

    Science.gov (United States)

    Speijer, Dave

    2015-12-01

    Of two contending models for eukaryotic evolution the "archezoan" has an amitochondriate eukaryote take up an endosymbiont, while "symbiogenesis" states that an Archaeon became a eukaryote as the result of this uptake. If so, organelle formation resulting from new engulfments is simplified by the primordial symbiogenesis, and less informative regarding the bacterium-to-mitochondrion conversion. Gradualist archezoan visions still permeate evolutionary thinking, but are much less likely than symbiogenesis. Genuine amitochondriate eukaryotes have never been found and rapid, explosive adaptive periods characteristic of symbiogenetic models explain this. Mitochondrial proteomes, encoded by genes of "eukaryotic origin" not easily linked to host or endosymbiont, can be understood in light of rapid adjustments to new evolutionary pressures. Symbiogenesis allows "expensive" eukaryotic inventions via efficient ATP generation by nascent mitochondria. However, efficient ATP production equals enhanced toxic internal ROS formation. The synergistic combination of these two driving forces gave rise to the rapid evolution of eukaryotes. Also watch the Video Abstract. PMID:26577075

  2. Gas Vesicle Nanoparticles for Antigen Display

    Directory of Open Access Journals (Sweden)

    Shiladitya DasSarma

    2015-09-01

    Full Text Available Microorganisms like the halophilic archaeon Halobacterium sp. NRC-1 produce gas-filled buoyant organelles, which are easily purified as protein nanoparticles (called gas vesicles or GVNPs. GVNPs are non-toxic, exceptionally stable, bioengineerable, and self-adjuvanting. A large gene cluster encoding more than a dozen proteins has been implicated in their biogenesis. One protein, GvpC, found on the exterior surface of the nanoparticles, can accommodate insertions near the C-terminal region and results in GVNPs displaying the inserted sequences on the surface of the nanoparticles. Here, we review the current state of knowledge on GVNP structure and biogenesis as well as available studies on immunogenicity of pathogenic viral, bacterial, and eukaryotic proteins and peptides displayed on the nanoparticles. Recent improvements in genetic tools for bioengineering of GVNPs are discussed, along with future opportunities and challenges for development of vaccines and other applications.

  3. Electrochemical behavior of dye-linked L-proline dehydrogenase on glassy carbon electrodes modified by multi-walled carbon nanotubes

    Directory of Open Access Journals (Sweden)

    Haitao Zheng

    2010-12-01

    Full Text Available A glassy carbon electrode (GC was modified by multi-walled carbon nanotubes (MWCNTs. The modified electrode showed a pair of redox peaks that resulted from the oxygen-containing functional groups on the nanotube surface. A recombinant thermostable dye-linked L-proline dehydrogenase (L-proDH from hyperthermophilic archaeon (Thermococcus profundus was further immobilized by physical adsorption. The modified electrode (GC/MWCNTs/L-proDH exhibited an electrocatalytic signal for L-proline compared to bare GC, GC/L-proDH and GC/MWCNTs electrodes, which suggested that the presence of MWCNTs efficiently enhances electron transfer between the active site of enzyme and electrode surface. The immobilized L-proDH showed a typical Michaelis–Menten catalytic response with lower apparent constant.

  4. Dicty_cDB: Contig-U00318-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 001140 |pid:none) Desulfurococcus kamchatkensis 1... 111 7e-23 T31596( T31596 )hypothetical protein Y48C3A.n - Caenorhabditis ele...U0) RecName: Full=Putative tRNA pseudouridine synthase Pus1... 213 1e-53 AK029866_1( AK029866 |pid:none) Mus musculus adult male... AM114193_809( AM114193 |pid:none) Uncultured methanogenic archaeon... 103 1e-20 CP000562_1953( CP000562 |pid:none) Methanoculle...10 sequences; 101,790,757,118 total letters Searching..................................................done ...ized gastrula pE... 62 5e-06 2 ( AK017063 ) Mus musculus adult male testis cDNA, RIKEN full-

  5. Utilization of keratin-containing biowaste to produce biohydrogen

    Energy Technology Data Exchange (ETDEWEB)

    Balint, B.; Rakhely, G.; Kovacs, K.L. [Szeged Univ. (Hungary). Dept. of Biotechnology; Hungarian Academy of Sciences, Szeged (Hungary). Inst. of Biophysics; Bagi, Z.; Perei, K. [Szeged Univ. (Hungary). Dept. of Biotechnology; Toth, A. [Hungarian Academy of Sciences, Szeged (Hungary). Inst. of Biophysics

    2005-12-01

    A two-stage fermentation system was constructed to test and demonstrate the feasibility of biohydrogen generation from keratin-rich biowaste. We isolated a novel aerobic Bacillus strain (Bacillus licheniformis KK1) that displays outstanding keratinolytic activity. The isolated strain was employed to convert keratin-containing biowaste into a fermentation product that is rich in amino acids and peptides. The process was optimized for the second fermentation step, in which the product of keratin fermentation-supplemented with essential minerals-was metabolized by Thermococcus litoralis, an anaerobic hyperthermophilic archaeon. T. litoralis grew on the keratin hydrolysate and produced hydrogen gas as a physiological fermentation byproduct. Hyperthermophilic cells utilized the keratin hydrolysate in a similar way as their standard nutrient, i.e., bacto-peptone. The generalization of the findings to protein-rich waste treatment and production of biohydrogen is discussed and possible means of further improvements are listed. (orig.)

  6. Haloarchaeal Protein Translocation via the Twin Arginine Translocation Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Pohlschroder Mechthild

    2009-02-03

    Protein transport across hydrophobic membranes that partition cellular compartments is essential in all cells. The twin arginine translocation (Tat) pathway transports proteins across the prokaryotic cytoplasmic membranes. Distinct from the universally conserved Sec pathway, which secretes unfolded proteins, the Tat machinery is unique in that it secretes proteins in a folded conformation, making it an attractive pathway for the transport and secretion of heterologously expressed proteins that are Sec-incompatible. During the past 7 years, the DOE-supported project has focused on the characterization of the diversity of bacterial and archaeal Tat substrates as well as on the characterization of the Tat pathway of a model archaeon, Haloferax volcanii, a member of the haloarchaea. We have demonstrated that H. volcanii uses this pathway to transport most of its secretome.

  7. A conserved chloramphenicol binding site at the entrance to the ribosomal peptide exit tunnel

    DEFF Research Database (Denmark)

    Long, Katherine S; Porse, Bo T

    2003-01-01

    The antibiotic chloramphenicol produces modifications in 23S rRNA when bound to ribosomes from the bacterium Escherichia coli and the archaeon Halobacterium halobium and irradiated with 365 nm light. The modifications map to nucleotides m(5)U747 and C2611/C2612, in domains II and V, respectively......, of E.coli 23S rRNA and G2084 (2058 in E.coli numbering) in domain V of H.halobium 23S rRNA. The modification sites overlap with a portion of the macrolide binding site and cluster at the entrance to the peptide exit tunnel. The data correlate with the recently reported chloramphenicol binding site on...

  8. Model Construction and Analysis of Respiration in Halobacterium salinarum.

    Directory of Open Access Journals (Sweden)

    Cherryl O Talaue

    Full Text Available The archaeon Halobacterium salinarum can produce energy using three different processes, namely photosynthesis, oxidative phosphorylation and fermentation of arginine, and is thus a model organism in bioenergetics. Compared to its bacteriorhodopsin-driven photosynthesis, less attention has been devoted to modeling its respiratory pathway. We created a system of ordinary differential equations that models its oxidative phosphorylation. The model consists of the electron transport chain, the ATP synthase, the potassium uniport and the sodium-proton antiport. By fitting the model parameters to experimental data, we show that the model can explain data on proton motive force generation, ATP production, and the charge balancing of ions between the sodium-proton antiporter and the potassium uniport. We performed sensitivity analysis of the model parameters to determine how the model will respond to perturbations in parameter values. The model and the parameters we derived provide a resource that can be used for analytical studies of the bioenergetics of H. salinarum.

  9. POLYPEPTIDE AND POLYSACCHARIDE PROCESSING IN HYPERTHERMOPHILIC MICROORGANISMS

    Energy Technology Data Exchange (ETDEWEB)

    KELLY, ROBERT M.

    2008-12-22

    This project focused on the microbial physiology and biochemistry of heterotrophic hyperthermophiles with respect to mechanisms by which these organisms process polypeptides and polysaccharides under normal and stressed conditions. Emphasis is on two model organisms, for which completed genome sequences are available: Pyrococcus furiosus (growth Topt of 98°C), an archaeon, and Thermotoga maritima (growth Topt of 80°C), a bacterium. Both organisms are obligately anaerobic heterotrophs that reduce sulfur facultatively. Whole genome cDNA spotted microarrays were used to follow transcriptional response to a variety of environmental conditions in order to identify genes encoding proteins involved in the acquisition, synthesis, processing and utilization of polypeptides and polysaccharides. This project provided new insights into the physiological aspects of hyperthermophiles as these relate to microbial biochemistry and biological function in high temperature habitats. The capacity of these microorganisms to produce biohydrogen from renewable feedstocks makes them important for future efforts to develop biofuels.

  10. Biocatalytic Synthesis of Poly(δ-Valerolactone Using a Thermophilic Esterase from Archaeoglobus fulgidus as Catalyst

    Directory of Open Access Journals (Sweden)

    Hong Cao

    2012-09-01

    Full Text Available The ring-opening polymerization of δ-valerolactone catalyzed by a thermophilic esterase from the archaeon Archaeoglobus fulgidus was successfully conducted in organic solvents. The effects of enzyme concentration, temperature, reaction time and reaction medium on monomer conversion and product molecular weight were systematically evaluated. Through the optimization of reaction conditions, poly(δ-valerolactone was produced in 97% monomer conversion, with a number-average molecular weight of 2225 g/mol, in toluene at 70 °C for 72 h. This paper has produced a new biocatalyst for the synthesis of poly(δ-valerolactone, and also deeper insight has been gained into the mechanism of thermophilic esterase-catalyzed ring-opening polymerization.

  11. Biocatalytic synthesis of poly(δ-valerolactone) using a thermophilic esterase from archaeoglobus fulgidus as catalyst.

    Science.gov (United States)

    Cao, Hong; Han, Haobo; Li, Guangquan; Yang, Jiebing; Zhang, Lingfei; Yang, Yan; Fang, Xuedong; Li, Quanshun

    2012-01-01

    The ring-opening polymerization of δ-valerolactone catalyzed by a thermophilic esterase from the archaeon Archaeoglobus fulgidus was successfully conducted in organic solvents. The effects of enzyme concentration, temperature, reaction time and reaction medium on monomer conversion and product molecular weight were systematically evaluated. Through the optimization of reaction conditions, poly(δ-valerolactone) was produced in 97% monomer conversion, with a number-average molecular weight of 2225 g/mol, in toluene at 70 °C for 72 h. This paper has produced a new biocatalyst for the synthesis of poly(δ-valerolactone), and also deeper insight has been gained into the mechanism of thermophilic esterase-catalyzed ring-opening polymerization. PMID:23202895

  12. Purification, crystallization and preliminary crystallographic analysis of the non-Pfam protein AF1514 from Archeoglobus fulgidus DSM 4304

    International Nuclear Information System (INIS)

    A non-Pfam protein, AF1514, from A. fulgidus has been crystallized. A 10.5 kDa non-Pfam hypothetical protein, AF1514, from the hyperthermophilic archaeon Archeoglobus fulgidus has been overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted X-rays to 2.09 Å resolution and a data set was collected at 100 K using Cu Kα radiation from a rotating-anode X-ray source. The crystals belong to space group P41212 or P43212, with unit-cell parameters a = b = 49.27, c = 106.61 Å. The calculated Matthews coefficient was 3.16 Å3 Da−1, suggesting the presence of one molecule in the asymmetric unit

  13. Heterologous Production of an Energy-Conserving Carbon Monoxide Dehydrogenase Complex in the Hyperthermophile Pyrococcus furiosus.

    Science.gov (United States)

    Schut, Gerrit J; Lipscomb, Gina L; Nguyen, Diep M N; Kelly, Robert M; Adams, Michael W W

    2016-01-01

    Carbon monoxide (CO) is an important intermediate in anaerobic carbon fixation pathways in acetogenesis and methanogenesis. In addition, some anaerobes can utilize CO as an energy source. In the hyperthermophilic archaeon Thermococcus onnurineus, which grows optimally at 80°C, CO oxidation and energy conservation is accomplished by a respiratory complex encoded by a 16-gene cluster containing a CO dehydrogenase, a membrane-bound [NiFe]-hydrogenase and a Na(+)/H(+) antiporter module. This complex oxidizes CO, evolves CO2 and H2, and generates a Na(+) motive force that is used to conserve energy by a Na(+)-dependent ATP synthase. Herein we used a bacterial artificial chromosome to insert the 13.2 kb gene cluster encoding the CO-oxidizing respiratory complex of T. onnurineus into the genome of the heterotrophic archaeon, Pyrococcus furiosus, which grows optimally at 100°C. P. furiosus is normally unable to utilize CO, however, the recombinant strain readily oxidized CO and generated H2 at 80°C. Moreover, CO also served as an energy source and allowed the P. furiosus strain to grow with a limiting concentration of sugar or with peptides as the carbon source. Moreover, CO oxidation by P. furiosus was also coupled to the re-utilization, presumably for biosynthesis, of acetate generated by fermentation. The functional transfer of CO utilization between Thermococcus and Pyrococcus species demonstrated herein is representative of the horizontal gene transfer of an environmentally relevant metabolic capability. The transfer of CO utilizing, hydrogen-producing genetic modules also has applications for biohydrogen production and a CO-based industrial platform for various thermophilic organisms. PMID:26858706

  14. Perchlorate reduction by microbes inhabiting oil reservoirs

    Science.gov (United States)

    Liebensteiner, Martin; Stams, Alfons; Lomans, Bart

    2014-05-01

    Microbial perchlorate and chlorate reduction is a unique type of anaerobic respiration as during reduction of (per)chlorate chlorite is formed, which is then split into chloride and molecular oxygen. In recent years it was demonstrated that (per)chlorate-reducing bacteria may employ oxygenase-dependent pathways for the degradation of aromatic and aliphatic hydrocarbons. These findings suggested that (per)chlorate may be used as oxygen-releasing compound in anoxic environments that contain hydrocarbons, such as polluted soil sites and oil reservoirs. We started to study perchlorate reduction by microbes possibly inhabiting oil reservoirs. One of the organisms studied was Archaeoglobus fulgidus. This extremely thermophilic archaeon is known as a major contributor to souring in hot oil reservoirs. A. fulgidus turned out to be able to use perchlorate as terminal electron acceptor for growth with lactate (Liebensteiner et al 2013). Genome based physiological experiments indicated that A. fulgidus possesses a novel perchlorate reduction pathway. Perchlorate is first reduced to chlorite, but chlorite is not split into chloride and molecular oxygen as occurs in bacteria. Rather, chlorite reacts chemically with sulfide, forming oxidized sulfur compounds, which are reduced to sulfide in the electron transport chain by the archaeon. The dependence of perchlorate reduction on sulfur compounds could be shown. The implications of our findings as novel strategy for microbiological enhanced oil recovery and for souring mitigation are discussed. Liebensteiner MG, Pinkse MWH, Schaap PJ, Stams AJM and Lomans BP (2013) Archaeal (per)chlorate reduction at high temperature, a matter of abiotic-biotic reactions. Science 340: 85-87

  15. Cloning, purification and preliminary X-ray crystallographic analysis of a hypothetical protein, MJ0754, from Methanococcus jannaschii DSM 2661

    International Nuclear Information System (INIS)

    In this study, two forms of MJ0754 from the archaeon M. jannaschii were overexpressed and crystallized. The crystal of MJ0754 belonged to the hexagonal space group P61 and diffracted to 3.1 Å resolution, while the crystal of MJ0754t belonged to the orthogonal space group C2221 and diffracted to 1.3 Å resolution using synchrotron radiation. The protein encoded by the MJ0754 gene from the archaeon Methanococcus jannaschii DSM 2661 is an unknown hypothetical protein. Two recombinant proteins, MJ0754 (residues 1–185) and MJ0754t (a truncated form of MJ0754, residues 11–185), were cloned from MJ0754, overexpressed as His-tag fusion proteins and purified. The crystals were found to grow under two different conditions and to have two different shapes. The crystal of MJ0754 belonged to space group P61, with unit-cell parameters a = b = 127.015, c = 48.929 Å, a calculated Matthews coefficient of 2.85 Å3 Da−1 and two molecules per asymmetric unit. The crystal of MJ0754t belonged to space group C2221, with unit-cell parameters a = 51.915, b = 79.122, c = 93.869 Å, a calculated Matthews coefficient of 2.41 Å3 Da−1 and one molecule per asymmetric unit. The SeMet-labelled P61 crystal diffracted to a resolution of 3.1 Å, while the native C2221 crystal diffracted to 1.3 Å resolution

  16. Production of glycolic acid by chemolithotrophic iron- and sulfur-oxidizing bacteria and its role in delineating and sustaining acidophilic sulfide mineral-oxidizing consortia.

    Science.gov (United States)

    Nancucheo, Ivan; Johnson, D Barrie

    2010-01-01

    Glycolic acid was detected as an exudate in actively growing cultures of three chemolithotrophic acidophiles that are important in biomining operations, Leptospirillum ferriphilum, Acidithiobacillus (At.) ferrooxidans, and At. caldus. Although similar concentrations of glycolic acid were found in all cases, the concentrations corresponded to ca. 24% of the total dissolved organic carbon (DOC) in cultures of L. ferriphilum but only ca. 5% of the total DOC in cultures of the two Acidithiobacillus spp. Rapid acidification (to pH 1.0) of the culture medium of At. caldus resulted in a large increase in the level of DOC, although the concentration of glycolic acid did not change in proportion. The archaeon Ferroplasma acidiphilum grew in the cell-free spent medium of At. caldus; glycolic acid was not metabolized, although other unidentified compounds in the DOC pool were metabolized. Glycolic acid exhibited levels of toxicity with 21 strains of acidophiles screened similar to those of acetic acid. The most sensitive species were chemolithotrophs (L. ferriphilum and At. ferrivorans), while the most tolerant species were chemoorganotrophs (Acidocella, Acidobacterium, and Ferroplasma species), and the ability to metabolize glycolic acid appeared to be restricted (among acidophiles) to Firmicutes (chiefly Sulfobacillus spp.). Results of this study help explain why Sulfobacillus spp. rather than other acidophiles are the main organic carbon-degrading bacteria in continuously fed stirred tanks used to bioprocess sulfide mineral concentrates and also why temporary cessation of pH control in these systems, resulting in rapid acidification, often results in a plume of the archaeon Ferroplasma. PMID:19933342

  17. Role of the denitrifying Haloarchaea in the treatment of nitrite-brines.

    Science.gov (United States)

    Nájera-Fernández, Cindy; Zafrilla, Basilio; Bonete, María José; Martínez-Espinosa, Rosa María

    2012-09-01

    Haloferax mediterranei is a denitrifying halophilic archaeon able to reduce nitrate and nitrite under oxic and anoxic conditions. In the presence of oxygen, nitrate and nitrite are used as nitrogen sources for growth. Under oxygen scarcity, this haloarchaeon uses both ions as electron acceptors via a denitrification pathway. In the present work, the maximal nitrite concentration tolerated by this organism was determined by studying the growth of H. mediterranei in minimal medium containing 30, 40 and 50 mM nitrite as sole nitrogen source and under initial oxic conditions at 42 degrees C. The results showed the ability of H. mediterranei to withstand nitrite concentrations up to 50 mM. At the beginning of the incubation, nitrate was detected in the medium, probably due to the spontaneous oxidation of nitrite under the initial oxic conditions. The complete removal of nitrite and nitrate was accomplished in most of the tested conditions, except in culture medium containing 50 mM nitrite, suggesting that this concentration compromised the denitrification capacity of the cells. Nitrite and nitrate reductases activities were analyzed at different growth stages of H. mediterranei. In all cases, the activities of the respiratory enzymes were higher than their assimilative counterparts; this was especially the case for NirK. The denitrifying and possibly detoxifying role of this enzyme might explain the high nitrite tolerance of H. mediterranei. This archaeon was also able to remove 60% of the nitrate and 75% of the nitrite initially present in brine samples collected from a wastewater treatment facility. These results suggest that H. mediterranei, and probably other halophilic denitrifying Archaea, are suitable candidates for the bioremediation of brines with high nitrite and nitrate concentrations. PMID:23847815

  18. Genomic expansion of Domain Archaea highlights roles for organisms from new phyla in anaerobic carbon cycling

    Energy Technology Data Exchange (ETDEWEB)

    Castelle, Cindy; Wrighton, Kelly C.; Thomas, Brian C.; Hug, Laura A.; Brown, Christopher T.; Wilkins, Michael J.; Frischkorn, Kyle R.; Tringe, Susannah G.; Singh, Andrea; Markillie, Lye Meng; Taylor, Ronald C.; Williams, Kenneth H.; Banfield, Jillian F.

    2015-03-01

    Domain Archaea is currently represented by one phylum (Euryarchaeota) and two superphyla (TACK and DPANN). However, gene surveys indicate the existence of a vast diversity of uncultivated archaea for which metabolic information is lacking. We sequenced DNA from complex sediment- and groundwater-associated microbial communities sampled prior to and during an acetate biostimulation field experiment to investigate the diversity and physiology of uncultivated subsurface archaea. We sampled 15 genomes that improve resolution of a new phylum within the TACK superphylum and 119 DPANN genomes that highlight a major subdivision within the archaeal domain that separates DPANN from TACK/Euryarchaeota lineages. Within the DPANN superphylum, which lacks any isolated representatives, we defined two new phyla using sequences from 100 newly sampled genomes. The first new phylum, for which we propose the name Woesearchaeota, was defined using 54 new sequences. We reconstructed a complete (finished) genome for an archaeon from this phylum that is only 0.8 Mb in length and lacks almost all core biosynthetic pathways, but has genes encoding enzymes predicted to interact with bacterial cell walls, consistent with a symbiotic lifestyle. The second new phylum, for which we propose the name Pacearchaeota, was defined based on 46 newly sampled archaeal genomes. This phylum includes the first non-methanogen with an intermediate Type II/III RuBisCO. We also reconstructed a complete (1.24 Mb) genome for another DPANN archaeon, a member of the Diapherotrites phylum. Metabolic prediction and transcriptomic data indicate that this organism has a fermentation-based lifestyle. In fact, genomic analyses consistently indicate lack of recognizable pathways for sulfur, nitrogen, methane, oxygen, and metal cycling, and suggest that symbiotic and fermentation-based lifestyles are widespread across the DPANN superphylum. Thus, as for a recently identified superphylum of bacteria with small genomes and no

  19. XPD Helicase Structures And Activities: Insights Into the Cancer And Aging Phenotypes From XPD Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Fan, L.; Fuss, J.O.; Cheng, Q.J.; Arvai, A.S.; Hammel, M.; Roberts, V.A.; Cooper, P.K.; Tainer, J.A.

    2009-05-18

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  20. Mechanism of DNA loading by the DNA repair helicase XPD.

    Science.gov (United States)

    Constantinescu-Aruxandei, Diana; Petrovic-Stojanovska, Biljana; Penedo, J Carlos; White, Malcolm F; Naismith, James H

    2016-04-01

    The xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH complex in eukaryotes and plays an essential role in DNA repair in the nucleotide excision repair pathway. XPD is a 5' to 3' helicase with an essential iron-sulfur cluster. Structural and biochemical studies of the monomeric archaeal XPD homologues have aided a mechanistic understanding of this important class of helicase, but several important questions remain open. In particular, the mechanism for DNA loading, which is assumed to require large protein conformational change, is not fully understood. Here, DNA binding by the archaeal XPD helicase fromThermoplasma acidophilumhas been investigated using a combination of crystallography, cross-linking, modified substrates and biochemical assays. The data are consistent with an initial tight binding of ssDNA to helicase domain 2, followed by transient opening of the interface between the Arch and 4FeS domains, allowing access to a second binding site on helicase domain 1 that directs DNA through the pore. A crystal structure of XPD fromSulfolobus acidocaldiariusthat lacks helicase domain 2 has an otherwise unperturbed structure, emphasizing the stability of the interface between the Arch and 4FeS domains in XPD. PMID:26896802

  1. Horizontal transfer, not duplication, drives the expansion of protein families in prokaryotes.

    Directory of Open Access Journals (Sweden)

    Todd J Treangen

    Full Text Available Gene duplication followed by neo- or sub-functionalization deeply impacts the evolution of protein families and is regarded as the main source of adaptive functional novelty in eukaryotes. While there is ample evidence of adaptive gene duplication in prokaryotes, it is not clear whether duplication outweighs the contribution of horizontal gene transfer in the expansion of protein families. We analyzed closely related prokaryote strains or species with small genomes (Helicobacter, Neisseria, Streptococcus, Sulfolobus, average-sized genomes (Bacillus, Enterobacteriaceae, and large genomes (Pseudomonas, Bradyrhizobiaceae to untangle the effects of duplication and horizontal transfer. After removing the effects of transposable elements and phages, we show that the vast majority of expansions of protein families are due to transfer, even among large genomes. Transferred genes--xenologs--persist longer in prokaryotic lineages possibly due to a higher/longer adaptive role. On the other hand, duplicated genes--paralogs--are expressed more, and, when persistent, they evolve slower. This suggests that gene transfer and gene duplication have very different roles in shaping the evolution of biological systems: transfer allows the acquisition of new functions and duplication leads to higher gene dosage. Accordingly, we show that paralogs share most protein-protein interactions and genetic regulators, whereas xenologs share very few of them. Prokaryotes invented most of life's biochemical diversity. Therefore, the study of the evolution of biology systems should explicitly account for the predominant role of horizontal gene transfer in the diversification of protein families.

  2. Eukaryotic and archaeal TBP and TFB/TF(II)B follow different promoter DNA bending pathways.

    Science.gov (United States)

    Gietl, Andreas; Holzmeister, Phil; Blombach, Fabian; Schulz, Sarah; von Voithenberg, Lena Voith; Lamb, Don C; Werner, Finn; Tinnefeld, Philip; Grohmann, Dina

    2014-06-01

    During transcription initiation, the promoter DNA is recognized and bent by the basal transcription factor TATA-binding protein (TBP). Subsequent association of transcription factor B (TFB) with the TBP-DNA complex is followed by the recruitment of the ribonucleic acid polymerase resulting in the formation of the pre-initiation complex. TBP and TFB/TF(II)B are highly conserved in structure and function among the eukaryotic-archaeal domain but intriguingly have to operate under vastly different conditions. Employing single-pair fluorescence resonance energy transfer, we monitored DNA bending by eukaryotic and archaeal TBPs in the absence and presence of TFB in real-time. We observed that the lifetime of the TBP-DNA interaction differs significantly between the archaeal and eukaryotic system. We show that the eukaryotic DNA-TBP interaction is characterized by a linear, stepwise bending mechanism with an intermediate state distinguished by a distinct bending angle. TF(II)B specifically stabilizes the fully bent TBP-promoter DNA complex and we identify this step as a regulatory checkpoint. In contrast, the archaeal TBP-DNA interaction is extremely dynamic and TBP from the archaeal organism Sulfolobus acidocaldarius strictly requires TFB for DNA bending. Thus, we demonstrate that transcription initiation follows diverse pathways on the way to the formation of the pre-initiation complex. PMID:24744242

  3. Experimental fossilisation of viruses from extremophilic Archaea

    Science.gov (United States)

    Orange, F.; Chabin, A.; Gorlas, A.; Lucas-Staat, S.; Geslin, C.; Le Romancer, M.; Prangishvili, D.; Forterre, P.; Westall, F.

    2011-06-01

    The role of viruses at different stages of the origin of life has recently been reconsidered. It appears that viruses may have accompanied the earliest forms of life, allowing the transition from an RNA to a DNA world and possibly being involved in the shaping of tree of life in the three domains that we know presently. In addition, a large variety of viruses has been recently identified in extreme environments, hosted by extremophilic microorganisms, in ecosystems considered as analogues to those of the early Earth. Traces of life on the early Earth were preserved by the precipitation of silica on the organic structures. We present the results of the first experimental fossilisation by silica of viruses from extremophilic Archaea (SIRV2 - Sulfolobus islandicus rod-shaped virus 2, TPV1 - Thermococcus prieurii virus 1, and PAV1 - Pyrococcus abyssi virus 1). Our results confirm that viruses can be fossilised, with silica precipitating on the different viral structures (proteins, envelope) over several months in a manner similar to that of other experimentally and naturally fossilised microorganisms. This study thus suggests that viral remains or traces could be preserved in the rock record although their identification may be challenging due to the small size of the viral particles.

  4. Microbial recovery of metals from spent coal liquefaction catalysts. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Sperl, P.L.; Sperl, G.T.

    1995-07-01

    This project was initiated on October 1, 1989, for the purpose of recovering metals from spent coal liquefaction catalysts. Two catalyst types were the subject of the contract. The first was a Ni-No catalyst support on alumina (Shell 324), the catalyst used in a pilot scale coal liquefaction facility at Wilsonville, Alabama. The second material was an unsupported ammonium molybdate catalyst used in a pilot process by the Department of Energy at the Pittsburgh Energy Technology Center. This material was obtained in late February 1990 but has not been pursued since the Mo content of this particular sample was too low for the current studies and the studies at the Pittsburgh Energy Technology Center have been discontinued. The object of the contract was to treat these spent catalysts with microorganisms, especially Thiobacillus ferrooxidans , but also other Thiobacillus spp. and possibly Sulfolobus and other potential microorganisms, to leach and remove the metals (Ni and Mo) from the spent catalysts into a form which could be readily recovered by conventional techniques.

  5. Novel viral genomes identified from six metagenomes reveal wide distribution of archaeal viruses and high viral diversity in terrestrial hot springs.

    Science.gov (United States)

    Gudbergsdóttir, Sóley Ruth; Menzel, Peter; Krogh, Anders; Young, Mark; Peng, Xu

    2016-03-01

    Limited by culture-dependent methods the number of viruses identified from thermophilic Archaea and Bacteria is still very small. In this study we retrieved viral sequences from six hot spring metagenomes isolated worldwide, revealing a wide distribution of four archaeal viral families, Ampullaviridae, Bicaudaviridae, Lipothrixviridae and Rudiviridae. Importantly, we identified 10 complete or near complete viral genomes allowing, for the first time, an assessment of genome conservation and evolution of the Ampullaviridae family as well as Sulfolobus Monocaudavirus 1 (SMV1)-related viruses. Among the novel genomes, one belongs to a putative thermophilic virus infecting the bacterium Hydrogenobaculum, for which no virus has been reported in the literature. Moreover, a high viral diversity was observed in the metagenomes, especially among the Lipothrixviridae, as indicated by the large number of unique contigs and the lack of a completely assembled genome for this family. This is further supported by the large number of novel genes in the complete and partial genomes showing no sequence similarities to public databases. CRISPR analysis revealed hundreds of novel CRISPR loci and thousands of novel CRISPR spacers from each metagenome, reinforcing the notion of high viral diversity in the thermal environment. PMID:26439881

  6. Life in blue: copper resistance mechanisms of bacteria and archaea used in industrial biomining of minerals.

    Science.gov (United States)

    Orell, Alvaro; Navarro, Claudio A; Arancibia, Rafaela; Mobarec, Juan C; Jerez, Carlos A

    2010-01-01

    Industrial biomining processes to extract copper, gold and other metals involve the use of extremophiles such as the acidophilic Acidithiobacillus ferrooxidans (Bacteria), and the thermoacidophilic Sulfolobus metallicus (Archaea). Together with other extremophiles these microorganisms subsist in habitats where they are exposed to copper concentrations higher than 100mM. Herein we review the current knowledge on the Cu-resistance mechanisms found in these microorganisms. Recent information suggests that biomining extremophiles respond to extremely high Cu concentrations by using simultaneously all or most of the following key elements: 1) a wide repertoire of Cu-resistance determinants; 2) duplication of some of these Cu-resistance determinants; 3) existence of novel Cu chaperones; 4) a polyP-based Cu-resistance system, and 5) an oxidative stress defense system. Further insight of the biomining community members and their individual response to copper is highly relevant, since this could provide key information to the mining industry. In turn, this information could be used to select the more fit members of the bioleaching community to attain more efficient industrial biomining processes. PMID:20627124

  7. XPD Helicase Structures and Activities: Insights into the Cancer and Aging Phenotypes from XPD Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Tainer, John; Fan, Li; Fuss, Jill O.; Cheng, Quen J.; Arvai, Andrew S.; Hammel, Michal; Roberts, Victoria A.; Cooper, Priscilla K.; Tainer, John A.

    2008-06-02

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  8. Dicty_cDB: Contig-U15849-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 06 CP000774_3583( CP000774 |pid:none) Parvibaculum lavamentivorans DS... 45 0.006 (Q9K8Y2) RecName: Full=Tryptophanyl...te... 43 0.031 CP001399_1622( CP001399 |pid:none) Sulfolobus islandicus L.S.2.15,... 43 0.031 (P0A2P2) RecName: Full=Tryptophanyl...c; ... 137 2e-30 (Q5ZJ08) RecName: Full=Tyrosyl-tRNA synthetase, cytoplasmic; ... 136 2e-30 AK076634_1( AK076634 |pid...es 2.4.1 ch... 55 8e-06 ( P04803 ) RecName: Full=Tryptophanyl-tRNA synthetase, mitochondri... 55 8e-06 (Q83EX7) RecNam.... 54 2e-05 (Q8Y577) RecName: Full=Tryptophanyl-tRNA synthetase; EC... 53 3e-05 CP001230_176( CP001230 |pid:none) Persephonella

  9. Crystal structures of type III{sub H} NAD-dependent D-3-phosphoglycerate dehydrogenase from two thermophiles

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, S.M. [Department of Studies in Physics, University of Mysore, Mysore 570 006 (India); Pampa, K.J. [Department of Studies in Microbiology, University of Mysore, Mysore 570 006 (India); Manjula, M. [Department of Studies in Physics, University of Mysore, Mysore 570 006 (India); Hemantha Kumar, G. [Department of Studies in Computer Science, University of Mysore, Mysore 570 006 (India); Kunishima, Naoki [Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, Hyogo 679-5148 (Japan); Lokanath, N.K., E-mail: lokanath@physics.uni-mysore.ac.in [Department of Studies in Physics, University of Mysore, Mysore 570 006 (India)

    2014-08-15

    Highlights: • Determined the crystal structures of PGDH from two thermophiles. • Monomer is composed of nucleotide binding domain and substrate binding domain. • Crystal structures of type III{sub H} PGDH. - Abstract: In the L-Serine biosynthesis, D-3-phosphoglycerate dehydrogenase (PGDH) catalyzes the inter-conversion of D-3-phosphoglycerate to phosphohydroxypyruvate. PGDH belongs to 2-hydroxyacid dehydrogenases family. We have determined the crystal structures of PGDH from Sulfolobus tokodaii (StPGDH) and Pyrococcus horikoshii (PhPGDH) using X-ray diffraction to resolution of 1.77 Å and 1.95 Å, respectively. The PGDH protomer from both species exhibits identical structures, consisting of substrate binding domain and nucleotide binding domain. The residues and water molecules interacting with the NAD are identified. The catalytic triad residues Glu-His-Arg are highly conserved. The residues involved in the dimer interface and the structural features responsible for thermostability are evaluated. Overall, structures of PGDHs with two domains and histidine at the active site are categorized as type III{sub H} and such PGDHs structures having this type are reported for the first time.

  10. Novel archaeal plasmid pAH1 and its interactions with the lipothrixvirus AFV1

    DEFF Research Database (Denmark)

    Basta, Tamara; Smyth, John; Forterre, Patrick; Prangishvili, David; Peng, Xu

    2009-01-01

    At present very little is known about interactions between extrachromosomal genetic elements in Archaea. Here we describe an Acidianus strain which carries naturally a novel 28 kb conjugative plasmid-like element, pAH1, and also serves as a laboratory host for lipothrixvirus AFV1. In an attempt t...... observed in bacteria where relevant bacteriophages either are dependent on a conjugative plasmid for successful infection or are excluded by a resident plasmid.......At present very little is known about interactions between extrachromosomal genetic elements in Archaea. Here we describe an Acidianus strain which carries naturally a novel 28 kb conjugative plasmid-like element, pAH1, and also serves as a laboratory host for lipothrixvirus AFV1. In an attempt to...... establish a system for studying plasmid-virus interactions we characterized the genome of pAH1 which closely resembles those of the Sulfolobus conjugative plasmids pARN3 and pARN4. pAH1 integrates site specifically into, and excises from, the host chromosome indicating a dynamic interaction with the latter...

  11. Protein Acetylation in Archaea, Bacteria, and Eukaryotes

    Directory of Open Access Journals (Sweden)

    Jörg Soppa

    2010-01-01

    Full Text Available Proteins can be acetylated at the alpha-amino group of the N-terminal amino acid (methionine or the penultimate amino acid after methionine removal or at the epsilon-amino group of internal lysines. In eukaryotes the majority of proteins are N-terminally acetylated, while this is extremely rare in bacteria. A variety of studies about N-terminal acetylation in archaea have been reported recently, and it was revealed that a considerable fraction of proteins is N-terminally acetylated in haloarchaea and Sulfolobus, while this does not seem to apply for methanogenic archaea. Many eukaryotic proteins are modified by differential internal acetylation, which is important for a variety of processes. Until very recently, only two bacterial proteins were known to be acetylation targets, but now 125 acetylation sites are known for E. coli. Knowledge about internal acetylation in archaea is extremely limited; only two target proteins are known, only one of which—Alba—was used to study differential acetylation. However, indications accumulate that the degree of internal acetylation of archaeal proteins might be underestimated, and differential acetylation has been shown to be essential for the viability of haloarchaea. Focused proteomic approaches are needed to get an overview of the extent of internal protein acetylation in archaea.

  12. (1)H, (13)C, and (15)N backbone resonance assignments of the full-length 40 kDa S. acidocaldarius Y-family DNA polymerase, dinB homolog.

    Science.gov (United States)

    Moro, Sean L; Cocco, Melanie J

    2015-10-01

    The dinB homolog (Dbh) is a member of the Y-family of translesion DNA polymerases, which are specialized to accurately replicate DNA across from a wide variety of lesions in living cells. Lesioned bases block the progression of high-fidelity polymerases and cause detrimental replication fork stalling; Y-family polymerases can bypass these lesions. The active site of the translesion synthesis polymerase is more open than that of a replicative polymerase; consequently Dbh polymerizes with low fidelity. Bypass polymerases also have low processivity. Short extension past the lesion allows the high-fidelity polymerase to switch back onto the site of replication. Dbh and the other Y-family polymerases have been used as structural models to investigate the mechanisms of DNA polymerization and lesion bypass. Many high-resolution crystal structures of Y-family polymerases have been reported. NMR dynamics studies can complement these structures by providing a measure of protein motions. Here we report the (15)N, (1)H, and (13)C backbone resonance assignments at two temperatures (35 and 50 °C) for Sulfolobus acidocaldarius Dbh polymerase. Backbone resonance assignments have been obtained for 86 % of the residues. The polymerase active site is assigned as well as the majority of residues in each of the four domains. PMID:26154586

  13. Characterization of a phosphotriesterase-like lactonase from the hyperthermoacidophilic crenarchaeon Vulcanisaeta moutnovskia.

    Science.gov (United States)

    Kallnik, Verena; Bunescu, Alina; Sayer, Christopher; Bräsen, Christopher; Wohlgemuth, Roland; Littlechild, Jennifer; Siebers, Bettina

    2014-11-20

    The phosphotriesterase-like lactonase (PLL) encoded by Vmut_2255 in the hyperthermoacidophilic crenarchaeon Vulcanisaeta moutnovskia (VmutPLL), represents the only hyperthermophilic PLL homologue identified so far in addition to the previously characterized thermophilic PLLs from Sulfolobus spp. The Vmut_2255 gene was cloned, heterologously expressed in Escherichia coli; the resultant protein purified and characterized as a 82kDa homodimer (36kDa subunits). The VmutPLL converted lactones and acyl-homoserine lactones (AHLs) with comparable activities. Towards organophosphates (OP) VmutPLL showed a promiscuous but significantly lower activity and only minor activity was observed with carboxylesters. The catalytic activity strictly depended on bivalent cations (Cd(2+)>Ni(2+)>Co(2+)>Mn(2+)>Zn(2+)). Furthermore, VmutPLL showed a pH optimum around 8.0, a temperature optimum of 80°C, and thermostability with a half-life of 26min at 90°C. In this work, the stereoselectivity of a PLL enzyme was investigated for the first time using enantiopure lactones. The VmutPLL showed a slight preference but not an exclusive specificity for the (R)-enantiomers of capro- and valerolactone. The high thermal stability as well as the broad substrate spectrum towards lactones, AHLs and OPs underlines the high biotechnological potential of VmutPLL. PMID:24858677

  14. Experimental fossilisation of viruses from extremophilic Archaea

    Directory of Open Access Journals (Sweden)

    F. Orange

    2011-06-01

    Full Text Available The role of viruses at different stages of the origin of life has recently been reconsidered. It appears that viruses may have accompanied the earliest forms of life, allowing the transition from an RNA to a DNA world and possibly being involved in the shaping of tree of life in the three domains that we know presently. In addition, a large variety of viruses has been recently identified in extreme environments, hosted by extremophilic microorganisms, in ecosystems considered as analogues to those of the early Earth. Traces of life on the early Earth were preserved by the precipitation of silica on the organic structures. We present the results of the first experimental fossilisation by silica of viruses from extremophilic Archaea (SIRV2 – Sulfolobus islandicus rod-shaped virus 2, TPV1 – Thermococcus prieurii virus 1, and PAV1 – Pyrococcus abyssi virus 1. Our results confirm that viruses can be fossilised, with silica precipitating on the different viral structures (proteins, envelope over several months in a manner similar to that of other experimentally and naturally fossilised microorganisms. This study thus suggests that viral remains or traces could be preserved in the rock record although their identification may be challenging due to the small size of the viral particles.

  15. Structures of three members of Pfam PF02663 (FmdE) implicated in microbial methanogenesis reveal a conserved α+β core domain and an auxiliary C-terminal treble-clef zinc finger

    International Nuclear Information System (INIS)

    The first structures from the FmdE Pfam family (PF02663) reveal that some members of this family form tightly intertwined dimers consisting of two domains (N-terminal α+β core and C-terminal zinc-finger domains), whereas others contain only the core domain. The presence of the zinc-finger domain suggests that some members of this family may perform functions associated with transcriptional regulation, protein–protein interaction, RNA binding or metal-ion sensing. Examination of the genomic context for members of the FmdE Pfam family (PF02663), such as the protein encoded by the fmdE gene from the methanogenic archaeon Methanobacterium thermoautotrophicum, indicates that 13 of them are co-transcribed with genes encoding subunits of molybdenum formylmethanofuran dehydrogenase (EC 1.2.99.5), an enzyme that is involved in microbial methane production. Here, the first crystal structures from PF02663 are described, representing two bacterial and one archaeal species: B8FYU2-DESHY from the anaerobic dehalogenating bacterium Desulfitobacterium hafniense DCB-2, Q2LQ23-SYNAS from the syntrophic bacterium Syntrophus aciditrophicus SB and Q9HJ63-THEAC from the thermoacidophilic archaeon Thermoplasma acidophilum. Two of these proteins, Q9HJ63-THEAC and Q2LQ23-SYNAS, contain two domains: an N-terminal thioredoxin-like α+β core domain (NTD) consisting of a five-stranded, mixed β-sheet flanked by several α-helices and a C-terminal zinc-finger domain (CTD). B8FYU2-DESHY, on the other hand, is composed solely of the NTD. The CTD of Q9HJ63-THEAC and Q2LQ23-SYNAS is best characterized as a treble-clef zinc finger. Two significant structural differences between Q9HJ63-THEAC and Q2LQ23-SYNAS involve their metal binding. First, zinc is bound to the putative active site on the NTD of Q9HJ63-THEAC, but is absent from the NTD of Q2LQ23-SYNAS. Second, whereas the structure of the CTD of Q2LQ23-SYNAS shows four Cys side chains within coordination distance of the Zn atom, the structure

  16. Pyruvate: A key Nutrient in Hypersaline Environments?

    Directory of Open Access Journals (Sweden)

    Aharon Oren

    2015-08-01

    Full Text Available Some of the most commonly occurring but difficult to isolate halophilic prokaryotes, Archaea as well as Bacteria, require or prefer pyruvate as carbon and energy source. The most efficient media for the enumeration and isolation of heterotrophic prokaryotes from natural environments, from freshwater to hypersaline, including the widely used R2A agar medium, contain pyruvate as a key ingredient. Examples of pyruvate-loving halophiles are the square, extremely halophilic archaeon Haloquadratum walsbyi and the halophilic gammaproteobacterium Spiribacter salinus. However, surprisingly little is known about the availability of pyruvate in natural environments and about the way it enters the cell. Some halophilic Archaea (Halorubrum saccharovorum, Haloarcula spp. partially convert sugars and glycerol to pyruvate and other acids (acetate, lactate which are excreted to the medium. Pyruvate formation from glycerol was also shown during a bloom of halophilic Archaea in the Dead Sea. However, no pyruvate transporters were yet identified in the genomes of halophilic Archaea, and altogether, our understanding of pyruvate transport in the prokaryote world is very limited. Therefore, the preference for pyruvate by fastidious and often elusive halophiles and the empirically proven enhanced colony recovery on agar media containing pyruvate are still poorly understood.

  17. Flexibility of the Cytoplasmic Domain of the Phototaxis Transducer II from Natronomonas pharaonis

    Directory of Open Access Journals (Sweden)

    Ivan L. Budyak

    2008-01-01

    Full Text Available Chemo- and phototaxis systems in bacteria and archaea serve as models for more complex signal transduction mechanisms in higher eukaryotes. Previous studies of the cytoplasmic fragment of the phototaxis transducer (pHtrII-cyt from the halophilic archaeon Natronomonas pharaonis showed that it takes the shape of a monomeric or dimeric rod under low or high salt conditions, respectively. CD spectra revealed only approximately 24% helical structure, even in 4 M KCl, leaving it an open question how the rod-like shape is achieved. Here, we conducted CD, FTIR, and NMR spectroscopic studies under different conditions to address this question. We provide evidence that pHtrII-cyt is highly dynamic with strong helical propensity, which allows it to change from monomeric to dimeric helical coiled-coil states without undergoing dramatic shape changes. A statistical analysis of predicted disorder for homologous sequences suggests that structural flexibility is evolutionarily conserved within the methyl-accepting chemotaxis protein family.

  18. Phototaxis of Haloarcula marismortui revealed through a novel microbial motion analysis algorithm.

    Science.gov (United States)

    Lin, Yu-Cheng; Fu, Hsu-Yuan; Yang, Chii-Shen

    2010-01-01

    Haloarcula marismortui has been described to be nonmotile prior to the recent identification of flagellar filaments, suggesting the motile nature of H. marismortui. Here we observed the locomotion of freshly cultured H. marismortui cells and tracked the swimming trajectories via ImageJ. Trajectories of H. marismortui are intrinsically noisy, posing difficulties in motion analysis with previously established algorithms. By introducing the concept of "window vector," a Microsoft Excel-VBA-implemented microbial motion analysis algorithm reported here was able to (1) discriminate nonswimming objects from swimming cells without empirical customization by applying a power-law relationship and (2) reduce the noise caused by Brownian motion, thus enhancing the accuracy of swim reversal identification. Based on this motion analysis algorithm, two recently identified sensory rhodopsins, HmSRI and HmSRII, were shown to mediate photoattractant and photorepellent responses, respectively, revealing the phototactic activity of H. marismortui, the only archaeon showing such phenomenon other than Halobacterium salinarum. PMID:20553410

  19. Crystallization and preliminary X-ray analysis of flap endonuclease 1 (FEN1) from Desulfurococcus amylolyticus

    International Nuclear Information System (INIS)

    Flap endonuclease 1 from D. amylolyticus was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 2.00 Å resolution. Flap endonuclease 1 (FEN1) is a structure-specific nuclease that removes 5′-overhanging flaps in DNA repair and removes the RNA/DNA primer during maturation of the Okazaki fragment in lagging-strand DNA replication. FEN1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with monoammonium dihydrogen phosphate as the precipitant at pH 8.3. X-ray diffraction data were collected to 2.00 Å resolution. The space group of the crystal was determined as the primitive hexagonal space group P321, with unit-cell parameters a = b = 103.76, c = 84.58 Å. The crystal contained one molecule in the asymmetric unit

  20. Extracellular DNA metabolism in Haloferax volcanii

    Directory of Open Access Journals (Sweden)

    ScottChimileski

    2014-02-01

    Full Text Available Extracellular DNA is found in all environments and is a dynamic component of the micro-bial ecosystem. Microbial cells produce and interact with extracellular DNA through many endogenous mechanisms. Extracellular DNA is processed and internalized for use as genetic information and as a major source of macronutrients, and plays several key roles within prokaryotic biofilms. Hypersaline sites contain some of the highest extracellular DNA con-centrations measured in nature–a potential rich source of carbon, nitrogen and phosphorus for halophilic microorganisms. We conducted DNA growth studies for the halophilic archaeon Haloferax volcanii DS2 and show that this model Halobacteriales strain is capable of using exogenous double-stranded DNA as a nutrient. Further experiments with varying medium composition, DNA concentration and DNA types revealed that DNA is utilized primarily as a phosphorus source, that growth on DNA is concentration-dependent and that DNA isolated from different sources is metabolized selectively, with a bias against highly divergent methylated DNA sources. Additionally, fluorescence microscopy experiments showed that labeled DNA colocalized with Haloferax volcanii cells. The gene Hvo_1477 was also identified using a comparative genomic approach as a factor likely to be involved in extracellular DNA processing at the cell surface, and deletion of Hvo_1477 created an H. volcanii strain deficient in its ability to grow on extracellular DNA. Widespread distribution of Hvo_1477 homologs in archaea suggests metabolism of extracellular DNA may be of broad ecological and physiological relevance in this domain of life.

  1. Genome sequence of Thermofilum pendens reveals an exceptional loss of biosynthetic pathways without genome reduction

    Energy Technology Data Exchange (ETDEWEB)

    Kyrpides, Nikos; Anderson, Iain; Rodriguez, Jason; Susanti, Dwi; Porat, Iris; Reich, Claudia; Ulrich, Luke E.; Elkins, James G.; Mavromatis, Kostas; Lykidis, Athanasios; Kim, Edwin; Thompson, Linda S.; Nolan, Matt; Land, Miriam; Copeland, Alex; Lapidus, Alla; Lucas, Susan; Detter, Chris; Zhulin, Igor B.; Olsen, Gary J.; Whitman, William; Mukhopadhyay, Biswarup; Bristow, James; Kyrpides, Nikos

    2008-01-01

    We report the complete genome of Thermofilum pendens, a deep-branching, hyperthermophilic member of the order Thermoproteales within the archaeal kingdom Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It is an extracellular commensal, requiring an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. In fact T. pendens has fewer biosynthetic enzymes than obligate intracellular parasites, although it does not display other features common among obligate parasites and thus does not appear to be in the process of becoming a parasite. It appears that T. pendens has adapted to life in an environment rich in nutrients. T. pendens was known to utilize peptides as an energy source, but the genome reveals substantial ability to grow on carbohydrates. T. pendens is the first crenarchaeote and only the second archaeon found to have a transporter of the phosphotransferase system. In addition to fermentation, T. pendens may gain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogenlyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time this enzyme has been found outside of Methanosarcinales, and a presenilin-related protein. Predicted highly expressed proteins do not include housekeeping genes, and instead include ABC transporters for carbohydrates and peptides, and CRISPR-associated proteins.

  2. Solution Structure of Pfu RPP21, a Component of the Archaeal RNase P Holoenzyme, and Interactions with its RPP29 Protein Partner

    Science.gov (United States)

    Amero, Carlos D; Boomershine, William P; Xu, Yiren; Foster, Mark

    2009-01-01

    RNase P is the ubiquitous ribonucleoprotein metalloenzyme responsible for cleaving the 5′-leader sequence of precursor tRNAs during their maturation. While the RNA subunit is catalytically active on its own at high monovalent and divalent ion concentration, four proteins subunits are associated with archaeal RNase P activity in vivo: RPP21, RPP29, RPP30 and POP5. These proteins have been shown to function in pairs: RPP21-RPP29 and POP5-RPP30. We have determined the solution structure of RPP21 from the hyperthermophilic archaeon Pyrococcus furiosus (Pfu) using conventional and paramagnetic NMR techniques. Pfu RPP21 in solution consists of an unstructured N-terminus, two alpha helices, a zinc binding motif, and an unstructured C-terminus. Moreover, we have used chemical shift perturbations to characterize the interaction of RPP21 with Pfu RPP29. The data show that the primary contact with RPP29 is localized to the two helices of RPP21. This information represents a fundamental step towards understanding structure-function relationships of the archaeal RNase P holoenzyme. PMID:18922021

  3. Solution structure of Pyrococcus furiosus RPP21, a component of the archaeal RNase P holoenzyme, and interactions with its RPP29 protein partner.

    Science.gov (United States)

    Amero, Carlos D; Boomershine, William P; Xu, Yiren; Foster, Mark

    2008-11-11

    RNase P is the ubiquitous ribonucleoprotein metalloenzyme responsible for cleaving the 5'-leader sequence of precursor tRNAs during their maturation. While the RNA subunit is catalytically active on its own at high monovalent and divalent ion concentrations, four protein subunits are associated with archaeal RNase P activity in vivo: RPP21, RPP29, RPP30, and POP5. These proteins have been shown to function in pairs: RPP21-RPP29 and POP5-RPP30. We have determined the solution structure of RPP21 from the hyperthermophilic archaeon Pyrococcus furiosus ( Pfu) using conventional and paramagnetic NMR techniques. Pfu RPP21 in solution consists of an unstructured N-terminus, two alpha-helices, a zinc binding motif, and an unstructured C-terminus. Moreover, we have used chemical shift perturbations to characterize the interaction of RPP21 with RPP29. The data show that the primary contact with RPP29 is localized to the two helices of RPP21. This information represents a fundamental step toward understanding structure-function relationships of the archaeal RNase P holoenzyme. PMID:18922021

  4. Microbial redox processes in deep subsurface environments and the potential application of (perchlorate in oil reservoirs

    Directory of Open Access Journals (Sweden)

    Martin G Liebensteiner

    2014-09-01

    Full Text Available The ability of microorganisms to thrive under oxygen-free conditions in subsurface environments relies on the enzymatic reduction of oxidized elements, such as sulfate, ferric iron or CO2, coupled to the oxidation of inorganic or organic compounds. A broad phylogenetic and functional diversity of microorganisms from subsurface environments has been described using isolation-based and advanced molecular ecological techniques. The physiological groups reviewed here comprise iron-, manganese- and nitrate-reducing microorganisms. In the context of recent findings also the potential of chlorate and perchlorate [jointly termed (perchlorate] reduction in oil reservoirs will be discussed. Special attention is given to elevated temperatures that are predominant in the deep subsurface. Microbial reduction of (perchlorate is a thermodynamically favorable redox process, also at high temperature. However, knowledge about (perchlorate reduction at elevated temperatures is still scarce and restricted to members of the Firmicutes and the archaeon Archaeoglobus fulgidus. By analyzing the diversity and phylogenetic distribution of functional genes in (metagenome databases and combining this knowledge with extrapolations to earlier-made physiological observations we speculate on the potential of (perchlorate reduction in the subsurface and more precisely oil fields. In addition, the application of (perchlorate for bioremediation, souring control and microbial enhanced oil recovery are addressed.

  5. Clustering of OB-fold domains of the partner protease complexed with trimeric stomatin from Thermococcales.

    Science.gov (United States)

    Yokoyama, Hideshi; Matsui, Eriko; Hiramoto, Kana; Forterre, Patrick; Matsui, Ikuo

    2013-07-01

    The C-terminal soluble domain of stomatin operon partner protein (STOPP) of the hyperthermophilic archaeon Pyrococcus horikoshii has an oligonucleotide binding-fold (OB-fold). STOPP lacks the conserved surface residues necessary for binding to DNA/RNA. A tryptophan (W) residue is conserved instead at the molecular surface. Solvent-accessible W residues are often found at interfaces of protein-protein complexes, which suggested the possibility of self-assembling of STOPP. Protein-protein interactions among the C-terminal soluble domains of STOPP PH1510 (1510-C) were then analyzed by chemical linking and blue native polyacrylamide gel electrophoresis (BN-PAGE) methods. These results suggest that the soluble domains of STOPP could assemble into homo-oligomers. Since hexameric subcomplex I from archaeal proteasome consists of coiled-coil segments and OB-fold domains, molecular modeling of 1510-C was performed using hexameric subcomplex I as a template. Although 1510-C is a comparatively small polypeptide consisting of approximately 60 residues, numerous salt bridges and hydrophobic interactions were observed in the predicted hexamer of 1510-C, suggesting the stability of the homo-oligomeric structure. This oligomeric property of STOPP may be favorable for triplicate proteolysis of the trimer of prokaryotic stomatin. PMID:23587725

  6. Comparative Genomic and Transcriptional Analyses of CRISPR Systems Across the Genus Pyrobaculum

    Directory of Open Access Journals (Sweden)

    David L Bernick

    2012-07-01

    Full Text Available Within the domain Archaea, the CRISPR immune system appears to be nearly ubiquitous based on computational genome analyses. Initial studies in bacteria demonstrated that the CRISPR system targets invading plasmid and viral DNA. Recent experiments in the model archaeon Pyrococcus furiosus uncovered a novel RNA-targeting variant of the CRISPR system potentially unique to archaea. Because our understanding of CRISPR system evolution in other archaea is limited, we have taken a comparative genomic and transcriptomic view of the CRISPR arrays across six diverse species within the crenarchaeal genus Pyrobaculum. We present transcriptional data from each of four species in the genus (P. aerophilum, P. islandicum, P. calidifontis, P. arsenaticum, analyzing mature CRISPR-associated small RNA abundance from over 20 arrays. Within the genus, there is remarkable conservation of CRISPR array structure, as well as unique features that are have not been studied in other archaeal systems. These unique features include: a nearly invariant CRISPR promoter, conservation of direct repeat families, the 5' polarity of CRISPR-associated small RNA abundance, and a novel CRISPR-specific association with homologues of nurA and herA. These analyses provide a genus-level evolutionary perspective on archaeal CRISPR systems, broadening our understanding beyond existing non-comparative model systems.

  7. MTH187 from Methanobacterium thermoautotrophicum has three HEAT-like Repeats

    Energy Technology Data Exchange (ETDEWEB)

    Julien, Olivier; Gignac, Isabelle [Universite Laval, Departement de biochimie et de microbiologie and CREFSIP (Canada); Hutton, Anna [University of Alberta, Institute for Biomolecular Design (Canada); Yee, Adelinda; Arrowsmith, Cheryl H. [University of Toronto, Division of Molecular and Structural Biology, Department of Medical Biophysics, Ontario Cancer Institute (Canada); Gagne, Stephane M. [Universite Laval, Departement de biochimie et de microbiologie and CREFSIP (Canada)], E-mail: sgagne@rsvs.ulaval.ca

    2006-06-15

    With the completion of genome sequencing projects, there are a large number of proteins for which we have little or no functional information. Since protein function is closely related to three-dimensional conformation, structural proteomics is one avenue where the role of proteins with unknown function can be investigated. In the present structural project, the structure of MTH187 has been determined by solution-state NMR spectroscopy. This protein of 12.4 kDa is one of the 424 non-membrane proteins that were cloned and purified for the structural proteomic project of Methanobacterium thermoautotrophicum [Christendat, D., Yee, A., Dharamsi, A., Kluger, Y., Gerstein, M., Arrowsmith, C.H. and Edwards, A.M. (2000) Prog. Biophys. Mol. Biol., 73, 339-345]. Methanobacterium thermoautotrophicum is a thermophilic archaeon that grows optimally at 65 deg. C. A particular characteristic of this microorganism is its ability to generate methane from carbon dioxide and hydrogen [Smith, D.R., Doucette-Stamm, L.A., Deloughery, C., Lee, H., Dubois, J., Aldredge, T., Bashirzadeh, R., Blakely, D., Cook, R., Gilbert, K., Harrison, D., Hoang, L., Keagle, P., Lumm, W., Pothier, B., Qiu, D., Spadafora, R., Vicaire, R., Wang, Y., Wierzbowski, J., Gibson, R., Jiwani, N., Caruso, A., Bush, D., Reeve, J. N. et al. (1997) J. Bacteriol., 179, 7135-7155].

  8. Air-adapted Methanosarcina acetivorans shows high methane production and develops resistance against oxygen stress.

    Directory of Open Access Journals (Sweden)

    Ricardo Jasso-Chávez

    Full Text Available Methanosarcina acetivorans, considered a strict anaerobic archaeon, was cultured in the presence of 0.4-1% O2 (atmospheric for at least 6 months to generate air-adapted cells; further, the biochemical mechanisms developed to deal with O2 were characterized. Methane production and protein content, as indicators of cell growth, did not change in air-adapted cells respect to cells cultured under anoxia (control cells. In contrast, growth and methane production significantly decreased in control cells exposed for the first time to O2. Production of reactive oxygen species was 50 times lower in air-adapted cells versus control cells, suggesting enhanced anti-oxidant mechanisms that attenuated the O2 toxicity. In this regard, (i the transcripts and activities of superoxide dismutase, catalase and peroxidase significantly increased; and (ii the thiol-molecules (cysteine + coenzyme M-SH + sulfide and polyphosphate contents were respectively 2 and 5 times higher in air-adapted cells versus anaerobic-control cells. Long-term cultures (18 days of air-adapted cells exposed to 2% O2 exhibited the ability to form biofilms. These data indicate that M. acetivorans develops multiple mechanisms to contend with O2 and the associated oxidative stress, as also suggested by genome analyses for some methanogens.

  9. Anion binding in biological systems

    Energy Technology Data Exchange (ETDEWEB)

    Feiters, Martin C [Department of Organic Chemistry, Institute for Molecules and Materials, Faculty of Science, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen (Netherlands); Meyer-Klaucke, Wolfram [EMBL Hamburg Outstation at DESY, Notkestrasse 85, D-22607 Hamburg (Germany); Kostenko, Alexander V; Soldatov, Alexander V [Faculty of Physics, Southern Federal University, Sorge 5, Rostov-na-Donu, 344090 (Russian Federation); Leblanc, Catherine; Michel, Gurvan; Potin, Philippe [Centre National de la Recherche Scientifique and Universite Pierre et Marie Curie Paris-VI, Station Biologique de Roscoff, Place Georges Teissier, BP 74, F-29682 Roscoff cedex, Bretagne (France); Kuepper, Frithjof C [Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, Argyll PA37 1QA, Scotland (United Kingdom); Hollenstein, Kaspar; Locher, Kaspar P [Institute of Molecular Biology and Biophysics, ETH Zuerich, Schafmattstrasse 20, Zuerich, 8093 (Switzerland); Bevers, Loes E; Hagedoorn, Peter-Leon; Hagen, Wilfred R, E-mail: m.feiters@science.ru.n [Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft (Netherlands)

    2009-11-15

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L{sub 3} (2p{sub 3/2}) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  10. Anion binding in biological systems

    Science.gov (United States)

    Feiters, Martin C.; Meyer-Klaucke, Wolfram; Kostenko, Alexander V.; Soldatov, Alexander V.; Leblanc, Catherine; Michel, Gurvan; Potin, Philippe; Küpper, Frithjof C.; Hollenstein, Kaspar; Locher, Kaspar P.; Bevers, Loes E.; Hagedoorn, Peter-Leon; Hagen, Wilfred R.

    2009-11-01

    We compare aspects of biological X-ray absorption spectroscopy (XAS) studies of cations and anions, and report on some examples of anion binding in biological systems. Brown algae such as Laminaria digitata (oarweed) are effective accumulators of I from seawater, with tissue concentrations exceeding 50 mM, and the vanadate-containing enzyme haloperoxidase is implicated in halide accumulation. We have studied the chemical state of iodine and its biological role in Laminaria at the I K edge, and bromoperoxidase from Ascophyllum nodosum (knotted wrack) at the Br K edge. Mo is essential for many forms of life; W only for certain archaea, such as Archaeoglobus fulgidus and the hyperthermophilic archaeon Pyrococcus furiosus, and some bacteria. The metals are bound and transported as their oxo-anions, molybdate and tungstate, which are similar in size. The transport protein WtpA from P. furiosus binds tungstate more strongly than molybdate, and is related in sequence to Archaeoglobus fulgidus ModA, of which a crystal structure is known. We have measured A. fulgidus ModA with tungstate at the W L3 (2p3/2) edge, and compared the results with the refined crystal structure. XAS studies of anion binding are feasible even if only weak interactions are present, are biologically relevant, and give new insights in the spectroscopy.

  11. Crystallization and preliminary X-ray diffraction analysis of a flavoenzyme amine dehydrogenase/oxidase from Pyrococcus furiosus DSM 3638

    International Nuclear Information System (INIS)

    This report describes the crystallization of a recombinant flavoprotein amine dehydrogenase/oxidase with specificity for l-proline from the hyperthermophile P. furiosus DSM 3638 and X-ray diffraction data collection. Crystals belonged to space group P1 and diffracted to a resolution of 3.3 Å. A flavoprotein amine dehydrogenase/oxidase with subunit molecular weights of 54.8 kDa (α-subunit) and 42.4 kDa (β-subunit) and specificity for l-proline was cloned from the genomic DNA of the hyperthermophilic marine archaeon Pyrococcus furiosus DSM 3638. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The enzyme was crystallized using the sitting-drop vapour-diffusion technique. Diffraction data from two different crystal forms were collected to 3.3 and 3.6 Å, respectively, using synchrotron radiation. Both crystals belonged to space group P1, with unit-cell parameters a = 91.3, b = 136.3, c = 203.8 Å, α = 94.5, β = 99.4, γ = 102.7° and a = 93.7, b = 116.3, c = 126.9 Å, α = 97.3, β = 99.9, γ = 104.6°

  12. A Gateway platform for functional genomics in Haloferax volcanii: deletion of three tRNA modification genes

    Directory of Open Access Journals (Sweden)

    Basma El Yacoubi

    2009-01-01

    Full Text Available In part due to the existence of simple methods for its cultivation and genetic manipulation, Haloferax volcanii is a major archaeal model organism. It is the only archaeon for which the whole set of post-transcriptionally modified tRNAs has been sequenced, allowing for an in silico prediction of all RNA modification genes present in the organism. One approach to check these predictions experimentally is via the construction of targeted gene deletion mutants. Toward this goal, an integrative “Gateway vector” that allows gene deletion in H. volcanii uracil auxotrophs was constructed. The vector was used to delete three predicted tRNA modification genes: HVO_2001 (encoding an archaeal transglycosyl tranferase or arcTGT, which is involved in archeosine biosynthesis; HVO_2348 (encoding a newly discovered GTP cyclohydrolase I, which catalyzes the first step common to archaeosine and folate biosynthesis; and HVO_2736 (encoding a member of the COG1444 family, which is involved in N4-acetylcytidine (ac4C formation. Preliminary phenotypic analysis of the deletion mutants was conducted, and confirmed all three predictions.

  13. Evidence for surfactant production by the haloarchaeon Haloferax sp. MSNC14 in hydrocarbon-containing media.

    Science.gov (United States)

    Djeridi, Ikram; Militon, Cécile; Grossi, Vincent; Cuny, Philippe

    2013-07-01

    The potential for surfactant production by the extreme halophilic archaeon Haloferax sp. MSNC14 in the presence of individual hydrocarbon substrates was studied. This strain was selected for its ability to grow on different types of hydrocarbons at high NaCl concentrations. Linear (n-heptadecane or C17) and isoprenoid (pristane) alkanes, a polyaromatic hydrocarbon (phenanthrene) and ammonium acetate (highly water-soluble control compound) were used as growth substrates. The adherence potential was demonstrated by the ability of the cells to adhere to liquid or solid hydrocarbons. The biosurfactant production was indicated by the reduction of the surface tension (ST) and by the emulsification activity (EA) of cell-free supernatants. Growth on acetate was accompanied by a low EA (lower than 0.1) and a high ST (~70 mN/m), whereas an important EA (up to 0.68 ± 0.08) and a reduction of ST (down to 32 ± 2.3 mN/m) were observed during growth on the different hydrocarbons. Both ST and EA varied with the growth phase. The adhesion to hydrocarbons was higher when cells were grown on C17 (by 60-70 %) and pristane (by 30-50 %) than on phenanthrene (~25 %). The results demonstrated that strain MNSC14 was able to increase the bioavailability of insoluble hydrocarbons, thus facilitating their uptake and their biodegradation even at high salt concentration. PMID:23748377

  14. A laboratory experiment of intact polar lipid degradation in sandy sediments

    Directory of Open Access Journals (Sweden)

    J. Logemann

    2011-09-01

    Full Text Available Intact polar lipids (IPLs are considered biomarkers for living biomass. Their degradation in marine sediments, however, is poorly understood and complicates interpretation of their occurrence in geological samples. To investigate the turnover of IPLs, a degradation experiment with anoxic sandy sediments from the North Sea was conducted. Intact cells of two organisms that do not naturally occur in North Sea sediments were chosen as IPL sources: (i Saccharomyces cerevisiae, representative for ester-bound acyl lipids that also occur in Bacteria, and (ii the archaeon Haloferax volcanii, representative for ether-bound isoprenoid lipids. Surprisingly, IPLs with phosphoester-bound head groups showed approximately the same degradation rate as IPLs with glycosidic head groups. Furthermore, the results indicate a relatively fast degradation of S. cerevisiae IPLs with ester-bound moieties (analogs of bacterial membrane lipids and no significant degradation of archaeal IPLs with ether-bound moieties. Pore water and 16S rRNA-based DGGE analysis showed only a minor influence of the IPL source on microbial metabolism and community profiles. Due to our results, the IPL-based quantification of Archaea and Bacteria should be interpreted with caution.

  15. A laboratory experiment of intact polar lipid degradation in sandy sediments

    Science.gov (United States)

    Logemann, J.; Graue, J.; Köster, J.; Engelen, B.; Rullkötter, J.; Cypionka, H.

    2011-09-01

    Intact polar lipids (IPLs) are considered biomarkers for living biomass. Their degradation in marine sediments, however, is poorly understood and complicates interpretation of their occurrence in geological samples. To investigate the turnover of IPLs, a degradation experiment with anoxic sandy sediments from the North Sea was conducted. Intact cells of two organisms that do not naturally occur in North Sea sediments were chosen as IPL sources: (i) Saccharomyces cerevisiae, representative for ester-bound acyl lipids that also occur in Bacteria, and (ii) the archaeon Haloferax volcanii, representative for ether-bound isoprenoid lipids. Surprisingly, IPLs with phosphoester-bound head groups showed approximately the same degradation rate as IPLs with glycosidic head groups. Furthermore, the results indicate a relatively fast degradation of S. cerevisiae IPLs with ester-bound moieties (analogs of bacterial membrane lipids) and no significant degradation of archaeal IPLs with ether-bound moieties. Pore water and 16S rRNA-based DGGE analysis showed only a minor influence of the IPL source on microbial metabolism and community profiles. Due to our results, the IPL-based quantification of Archaea and Bacteria should be interpreted with caution.

  16. Purification and properties of an extremely thermostable membrane-bound sulfur-reducing complex from the hyperthermophilic Pyrodictium abyssi.

    Science.gov (United States)

    Dirmeier, R; Keller, M; Frey, G; Huber, H; Stetter, K O

    1998-03-15

    The chemolithoautotrophic archaeon Pyrodictium abyssi isolate TAG 11 gains energy by reducing sulfur with H2 to H2S. From this hyperthermophile, a sulfur-reducing complex catalyzing this reaction was purified 13.5-fold. The native complex exhibited a brownish-yellow colour and showed an apparent molecular mass of 520 kDa. SDS/PAGE revealed the presence of nine different major polypeptides with apparent molecular masses of 82, 72, 65, 50, 47, 42, 40, 30 and 24 kDa. The native complex contained 50-55 mol acid-labile sulfur, 50-55 mol iron, 1.6 mol nickel, 1.2 mol copper, 2.8 mol cytochrome b and 0.3 mol cytochrome c (all per mol native complex). The temperature optimum of the H2:sulfur oxidoreductase complex was 100 degrees C, which is consistent with the physiological growth optimum of the native organism. The complex is extremely heat stable. During 5 h incubation at 100 degrees C, no decrease in H2S-forming activity could be observed. PMID:9546664

  17. Temperature and pH dependence of DNA ejection from archaeal lemon-shaped virus His1.

    Science.gov (United States)

    Hanhijärvi, K J; Ziedaite, G; Hæggström, E; Bamford, D H

    2016-07-01

    The archaeal virus His1 isolated from a hypersaline environment infects an extremely halophilic archaeon Haloarcula hispanica. His1 features a lemon-shaped capsid, which is so far found only in archaeal viruses. This unique capsid can withstand high salt concentrations, and can transform into a helical tube, which in turn is resistant to extremely harsh conditions. Hypersaline environments exhibit a wide range of temperatures and pH conditions, which present an extra challenge to their inhabitants. We investigated the influence of pH and temperature on DNA ejection from His1 virus using single-molecule fluorescence experiments. The observed number of ejecting viruses is constant in pH 5 to 9, while the ejection process is suppressed at pH below 5. Similarly, the number of ejections within 15-42 °C shows only a minor increase around 25-37 °C. The maximum velocity of single ejected DNA increases with temperature, in qualitative agreement with the continuum model of dsDNA ejection. PMID:26820561

  18. S-layers at second glance? Altiarchaeal grappling hooks (hami resemble archaeal S-layer proteins in structure and sequence

    Directory of Open Access Journals (Sweden)

    Alexandra Kristin Perras

    2015-06-01

    Full Text Available The uncultivated Ca. Altiarchaeum hamiconexum (formerly known as SM1 Euryarchaeon carries highly specialized nano-grappling hooks (hami on its cell surface. Until now little is known about the major protein forming these structured fibrous cell surface appendages, the genes involved or membrane anchoring of these filaments. These aspects were analyzed in depth in this study using environmental transcriptomics combined with imaging methods. Since a laboratory culture of this archaeon is not yet available, natural biofilm samples with high Ca. A. hamiconexum abundance were used for the entire analyses. The filamentous surface appendages spanned both membranes of the cell, which are composed of glycosyl-archaeol. The hami consisted of multiple copies of the same protein, the corresponding gene of which was identified via metagenome-mapped transcriptome analysis. The hamus subunit proteins, which are likely to self-assemble due to their predicted beta sheet topology, revealed no similiarity to known microbial flagella-, archaella-, fimbriae- or pili-proteins, but a high similarity to known S-layer proteins of the archaeal phylum at their N-terminal region (47-44% identity. Our results provide new insights into the structure of the unique hami and their major protein and indicate their divergent evolution with S-layer proteins.

  19. Molecular genetic and physical analysis of gas vesicles in buoyant enterobacteria.

    Science.gov (United States)

    Tashiro, Yosuke; Monson, Rita E; Ramsay, Joshua P; Salmond, George P C

    2016-04-01

    Different modes of bacterial taxis play important roles in environmental adaptation, survival, colonization and dissemination of disease. One mode of taxis is flotation due to the production of gas vesicles. Gas vesicles are proteinaceous intracellular organelles, permeable only to gas, that enable flotation in aquatic niches. Gene clusters for gas vesicle biosynthesis are partially conserved in various archaea, cyanobacteria, and some proteobacteria, such as the enterobacterium, Serratia sp. ATCC 39006 (S39006). Here we present the first systematic analysis of the genes required to produce gas vesicles in S39006, identifying how this differs from the archaeon Halobacterium salinarum. We define 11 proteins essential for gas vesicle production. Mutation of gvpN or gvpV produced small bicone gas vesicles, suggesting that the cognate proteins are involved in the morphogenetic assembly pathway from bicones to mature cylindrical forms. Using volumetric compression, gas vesicles were shown to comprise 17% of S39006 cells, whereas in Escherichia coli heterologously expressing the gas vesicle cluster in a deregulated environment, gas vesicles can occupy around half of cellular volume. Gas vesicle production in S39006 and E. coli was exploited to calculate the instantaneous turgor pressure within cultured bacterial cells; the first time this has been performed in either strain. PMID:26743231

  20. Methanohalophilus levihalophilus sp. nov., a slightly halophilic, methylotrophic methanogen isolated from natural gas-bearing deep aquifers, and emended description of the genus Methanohalophilus.

    Science.gov (United States)

    Katayama, Taiki; Yoshioka, Hideyoshi; Mochimaru, Hanako; Meng, Xian-Ying; Muramoto, Yoshiyuki; Usami, Jun; Ikeda, Hidefumi; Kamagata, Yoichi; Sakata, Susumu

    2014-06-01

    A mesophilic, slightly halophilic, obligately methylotrophic, methanogenic archaeon, designated strain GTA13(T), was isolated from natural gas-bearing confined aquifers in the Minami-Kanto gas field, Japan. The cells were non-motile, slightly irregular cocci, 0.7-1.0 µm in diameter and occurred singly, in pairs or as small aggregates. The cells grew with tri- or dimethylamine but not with H2/CO2, formate, acetate, methanol or dimethyl sulphide. Vitamins, sodium and magnesium were required for growth. Optimal growth occurred at pH 7.0-7.5, 35 °C, 0.35-0.40 M NaCl and 15-50 mM MgCl2. The NaCl range for growth was 0.2-1.3 M. The DNA G+C content was 43.7 mol%. Strain GTA13(T) showed highest levels of 16S rRNA gene sequence similarity with Methanohalophilus portucalensis FDF-1(T) (96.4% sequence similarity) and Methanohalophilus halophilus DSM 3094(T) (96.0%). On the basis of physiological and phylogenetic features, strain GTA13(T) is considered to represent a novel species of the genus Methanohalophilus, for which the name Methanohalophilus levihalophilus sp. nov. is proposed. The type strain is GTA13(T) ( = NBRC 110099(T) = DSM 28452(T)). An emended description of the genus Methanohalophilus is also proposed. PMID:24670897