WorldWideScience

Sample records for archaeon methanococcoides burtonii

  1. 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

  2. Draft Genome Sequence of an Obligately Methylotrophic Methanogen, Methanococcoides methylutens, Isolated from Marine Sediment

    KAUST Repository

    Guan, Yue

    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%.

  3. Methanococcoides vulcani sp. nov., a marine methylotrophic methanogen that uses betaine, choline and N,N-dimethylethanolamine for methanogenesis, isolated from a mud volcano, and emended description of the genus Methanococcoides.

    Science.gov (United States)

    L'Haridon, Stéphane; Chalopin, Morgane; Colombo, Delphine; Toffin, Laurent

    2014-06-01

    A novel, strictly anaerobic, methylotrophic marine methanogen, strain SLH33(T), was isolated from deep sediment samples covered by an orange microbial mat collected from the Napoli Mud Volcano. Cells of strain SLH33(T) were Gram-stain-negative, motile, irregular cocci that occurred singly. Cells utilized trimethylamine, dimethylamine, monomethylamine, methanol, betaine, N,N-dimethylethanolamine and choline (N,N,N-trimethylethanolamine) as substrates for growth and methanogenesis. The optimal growth temperature was 30 °C; maximum growth rate was obtained at pH 7.0 in the presence of 0.5 M Na(+). The DNA G+C content of strain SLH33(T) was 43.4 mol%. Phylogenetic analyses based on 16S rRNA gene sequences placed strain SLH33(T) within the genus Methanococcoides. The novel isolate was related most closely to Methanococcoides methylutens TMA-10(T) (98.8% 16S rRNA gene sequence similarity) but distantly related to Methanococcoides burtonii DSM 6242(T) (97.6%) and Methanococcoides alaskense AK-5(T) (97.6%). DNA-DNA hybridization studies indicated that strain SLH33(T) represents a novel species, given that it shared less than 16% DNA-DNA relatedness with Methanococcoides methylutens TMA-10(T). The name Methanococcoides vulcani sp. nov. is proposed for this novel species, with strain SLH33(T) ( = DSM 26966(T) = JCM 19278(T)) as the type strain. An emended description of the genus Methanococcoides is also proposed.

  4. Glycine betaine as a direct substrate for methanogens (Methanococcoides spp.).

    Science.gov (United States)

    Watkins, Andrew J; Roussel, Erwan G; Parkes, R John; Sass, Henrik

    2014-01-01

    Nine marine methanogenic Methanococcoides strains, including the type strains of Methanococcoides methylutens, M. burtonii, and M. alaskense, were tested for the utilization of N-methylated glycines. Three strains (NM1, PM2, and MKM1) used glycine betaine (N,N,N-trimethylglycine) as a substrate for methanogenesis, partially demethylating it to N,N-dimethylglycine, whereas none of the strains used N,N-dimethylglycine or sarcosine (N-methylglycine). Growth rates and growth yields per mole of substrate with glycine betaine (3.96 g [dry weight] per mol) were similar to those with trimethylamine (4.11 g [dry weight] per mol). However, as glycine betaine is only partially demethylated, the yield per methyl group was significantly higher than with trimethylamine. If glycine betaine and trimethylamine are provided together, trimethylamine is demethylated to dimethyl- and methylamine with limited glycine betaine utilization. After trimethylamine is depleted, dimethylamine and glycine betaine are consumed rapidly, before methylamine. Glycine betaine extends the range of substrates that can be directly utilized by some methanogens, allowing them to gain energy from the substrate without the need for syntrophic partners.

  5. Cutaneous mycosis in a Barbastelle bat (Barbastella barbastellus) caused by Hyphopichia burtonii.

    Science.gov (United States)

    Simpson, Victor R; Borman, Andrew M; Fox, Richard I; Mathews, Fiona

    2013-07-01

    A rare barbastelle bat (Barbastella barbastellus) died shortly after being found in emaciated condition in Devon, England. The skin over the muzzle and face was grossly thickened, crusty, and in places was sloughing and ulcerated. There were numerous nodules up to 3 mm in diameter on both wings and ear pinnae. Histologically, multiple foci of epidermal hyperplasia, hyperkeratosis, and crateriform erosions containing masses of fungal spores and septate hyphae were found in the wing. Epidermal hyperplasia and follicular hyperkeratosis, with fungal masses within keratinized follicles and also in fissured stratum corneum, were found in the pinna. Hyphae did not invade the dermis, and there was no inflammation, but there was multifocal serous exudation and crusting. No parasites or other significant organisms were identified. Microscopic and multiple cultural analyses of face and wing lesions demonstrated (10/10) a fine, septate fungus bearing laterally oval to clavate conidia; morphologically and culturally this was entirely consistent with Hyphopichia burtonii, and polymerase chain reaction analysis and sequencing gave 100% identity with the type strain. The organism isolated was morphologically consistent with that repeatedly seen in histology sections and demonstrates that although H. burtonii has not previously been recognized as a dermatophyte, it clearly has the ability to invade the skin of live bats. Although not identical, the lesions in this case show similarity with those of white nose syndrome and therefore H. burtonii should be considered as a potential pathogen of bats.

  6. Isolation and cultivation of Walsby's square archaeon

    NARCIS (Netherlands)

    Bolhuis, H; Poele, EMT; Rodriguez-Valera, F

    2004-01-01

    In 1980, A. E. Walsby described a square halophilic archaeon. This archaeon is of specific interest because of its unique shape and its abundance in hypersaline ecosystems, which suggests an important ecophysiological role. Ever since its discovery, the isolation and cultivation of 'Walsby's square

  7. NCBI nr-aa BLAST: CBRC-TTRU-01-0582 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0582 ref|YP_566336.1| cation transporter, P-type ATPase [Methanococcoides burton...ii DSM 6242] gb|ABE52586.1| Cation-transporter, P-type ATPase [Methanococcoides burtonii DSM 6242] YP_566336.1 0.033 24% ...

  8. NCBI nr-aa BLAST: CBRC-BTAU-01-2823 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-BTAU-01-2823 ref|YP_565442.1| polysaccharide biosynthesis protein [Methanococcoides burton...ii DSM 6242] gb|ABE51692.1| polysaccharide biosynthesis protein [Methanococcoides burtonii DSM 6242] YP_565442.1 2.5 21% ...

  9. NCBI nr-aa BLAST: CBRC-CINT-01-0121 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CINT-01-0121 ref|YP_565930.1| hypothetical protein Mbur_1259 [Methanococcoides burton...ii DSM 6242] gb|ABE52180.1| conserved hypothetical protein [Methanococcoides burtonii DSM 6242] YP_565930.1 2.8 20% ...

  10. Sugar transport in the thermoacidophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Albers, Sonja-Verena

    2001-01-01

    Summary and concluding remarks Introduction The archaeon Sulfolobus solfataricus is a thermoacidophile preferring growth at around 80oC and a pH of 2.5 to 3.5. As a thermoacidophile S. solfataricus faces two major problems: firstly, the proton permeability of membranes increases with temperature res

  11. Response of Haloalkaliphilic Archaeon Natronococcus Jeotgali RR17 to Hypergravity

    Science.gov (United States)

    Thombre, Rebecca S.; Bhalerao, Aniruddha R.; Shinde, Vinaya D.; Dhar, Sunil Kumar; Shouche, Yogesh S.

    2017-01-01

    The survival of archaeabacteria in extreme inhabitable environments on earth that challenge organismic survival is ubiquitously known. However, the studies related to the effect of hypergravity on the growth and proliferation of archaea are unprecedented. The survival of organisms in hypergravity and rocks in addition to resistance to cosmic radiations, pressure and other extremities is imperative to study the possibilities of microbial travel between planets and endurance in hyperaccelerative forces faced during ejection of rocks from planets. The current investigation highlights the growth of an extremophilic archaeon isolated from a rocky substrate in hypergravity environment. The haloalkaliphilic archaeon, Natronococcus jeotgali RR17 was isolated from an Indian laterite rock, submerged in the Arabian sea lining Coastal Maharashtra, India. The endolithic haloarchaeon was subjected to hypergravity from 56 - 893 X gusing acceleration generated by centrifugal rotation. The cells of N. jeotgali RR17 proliferated and demonstrated good growth in hypergravity (223 X g). This is the first report on isolation of endolithic haloarchaeon N. jeotgali RR17 from an Indian laterite rock and its ability to proliferate in hypergravity. The present study demonstrates the ability of microbial life to survive and proliferate in hypergravity. Thus the inability of organismic growth in hypergravity may no longer be a limitation for astrobiology studies related to habitability of substellar objects, brown dwarfs and other planetary bodies in the universe besides planet earth.

  12. 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.

  13. Characterization of malate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum.

    Science.gov (United States)

    Yennaco, Lynda J; Hu, Yajing; Holden, James F

    2007-09-01

    Native and recombinant malate dehydrogenase (MDH) was characterized from the hyperthermophilic, facultatively autotrophic archaeon Pyrobaculum islandicum. The enzyme is a homotetramer with a subunit mass of 33 kDa. The activity kinetics of the native and recombinant proteins are the same. The apparent K ( m ) values of the recombinant protein for oxaloacetate (OAA) and NADH (at 80 degrees C and pH 8.0) were 15 and 86 microM, respectively, with specific activity as high as 470 U mg(-1). Activity decreased more than 90% when NADPH was used. The catalytic efficiency of OAA reduction by P. islandicum MDH using NADH was significantly higher than that reported for any other archaeal MDH. Unlike other archaeal MDHs, specific activity of the P. islandicum MDH back-reaction also decreased more than 90% when malate and NAD(+) were used as substrates and was not detected with NADP(+). A phylogenetic tree of 31 archaeal MDHs shows that they fall into 5 distinct groups separated largely along taxonomic lines suggesting minimal lateral mdh transfer between Archaea.

  14. 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.

  15. 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.

  16. 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......-xylosidase activity was optimal at 65degreesC. SDS-PAGE and zymogram techniques revealed the presence of two xylan-degrading proteins of approximately 45 and 67 kDa in culture supernatants. To our knowledge, this paper is the first report on hemicellulose-degrading enzymes produced by an extremely halophilic archaeon....

  17. An x-ray absorption spectroscopy study of Cd binding onto a halophilic archaeon

    Science.gov (United States)

    Showalter, Allison R.; Szymanowski, Jennifer E. S.; Fein, Jeremy B.; Bunker, Bruce A.

    2016-05-01

    X-ray absorption spectroscopy (XAS) and cadmium (Cd) isotherm experiments determine how Cd adsorbs to the surface of halophilic archaeon Halobacterium noricense. This archaeon, isolated from the Waste Isolation Pilot Plant (WIPP) near Carlsbad, New Mexico could be involved with the transport of toxic metals stored in the transuranic waste in the salt mine. The isotherm experiments show that adsorption is relatively constant across the tolerable pH range for H. noricense. The XAS results indicate that Cd adsorption occurs predominately via a sulfur site, most likely sulfhydryl, with the same site dominating all measured pH values.

  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. Production and characterization of a thermostable L-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    Machielsen, M.P.; Oost, van der J.

    2006-01-01

    The gene encoding a threonine dehydrogenase (TDH) has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The Pf-TDH protein has been functionally produced in Escherichia coli and purified to homogeneity. The enzyme has a tetrameric conformation with a molecular mass of ¿ 155 kDa.

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

    NARCIS (Netherlands)

    Swarts, D.C.; Hegge, J.W.; Hinojo, Ismael; Shiimori, Masami; Ellis, Michael A.; Dumrongkulraksa, Justin; Terns, Rebecca M.; Terns, Michael P.; Oost, Van Der 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

  1. UV-inducible cellular aggregation of the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by pili formation

    NARCIS (Netherlands)

    Froels, Sabrina; Ajon, Malgorzata; Wagner, Michaela; Teichmann, Daniela; Zolghadr, Behnam; Folea, Mihaela; Boekema, Egbert J.; Driessen, Arnold J. M.; Schleper, Christa; Albers, Sonja-Verena

    2008-01-01

    The hyperthermophilic archaeon Sulfolobus solfataricus has been shown to exhibit a complex transcriptional response to UV irradiation involving 55 genes. Among the strongest UV-induced genes was a putative pili biogenesis operon encoding a potential secretion ATPase, two pre-pilins, a putative trans

  2. Genome Sequence of "Candidatus Methanomassiliicoccus intestinalis" Issoire-Mx1, a Third Thermoplasmatales-Related Methanogenic Archaeon from Human Feces.

    Science.gov (United States)

    Borrel, Guillaume; Harris, Hugh M B; Parisot, Nicolas; Gaci, Nadia; Tottey, William; Mihajlovski, Agnès; Deane, Jennifer; Gribaldo, Simonetta; Bardot, Olivier; Peyretaillade, Eric; Peyret, Pierre; O'Toole, Paul W; Brugère, Jean-François

    2013-07-11

    "Candidatus Methanomassiliicoccus intestinalis" Issoire-Mx1 is a methanogenic archaeon found in the human gut and is a representative of the novel order of methanogens related to Thermoplasmatales. Its complete genome sequence is presented here.

  3. Membrane homeoviscous adaptation in the piezo-hyperthermophilic archaeon Thermococcus barophilus

    Directory of Open Access Journals (Sweden)

    Anaïs eCario

    2015-10-01

    Full Text Available The archaeon Thermococcus barophilus, one of the most extreme members of hyperthermophilic piezophiles known thus far, is able to grow at temperatures up to 103°C and pressures up to 80MPa. We analyzed the membrane lipids of T. barophilus by HPLC-MS as a function of pressure and temperature. In contrast to previous reports, we show that under optimal growth conditions (40 MPa, 85°C the membrane spanning tetraether lipid GDGT-0 (sometimes called caldarchaeol is a major membrane lipid of T. barophilus together with archaeol. Increasing pressure and decreasing temperature lead to an increase of the proportion of archaeol and, reversely, a higher proportion of GDGT-0 is observed under low pressure and high temperature conditions. Noticeably, pressure and temperature fluctuations also impact the level of unsaturation of non-polar lipids with an irregular polyisoprenoid carbon skeleton (polyunsaturated lycopane derivatives, suggesting a structural role for these neutral lipids in the membrane of T. barophilus. Whether these apolar lipids insert in the membrane or not remains to be addressed. However, our results raise questions about the structure of the membrane in this archaeon and other archaeon harboring a mixture of di- and tetraether lipids.

  4. Dimethyl sulfoxide reduction by a hyperhermophilic archaeon Thermococcus onnurineus NA1 via a cysteine-cystine redox shuttle.

    Science.gov (United States)

    Choi, Ae Ran; Kim, Min-Sik; Kang, Sung Gyun; Lee, Hyun Sook

    2016-01-01

    A variety of microbes grow by respiration with dimethyl sulfoxide (DMSO) as an electron acceptor, and several distinct DMSO respiratory systems, consisting of electron carriers and a terminal DMSO reductase, have been characterized. The heterotrophic growth of a hyperthermophilic archaeon Thermococcus onnurineus NA1 was enhanced by the addition of DMSO, but the archaeon was not capable of reducing DMSO to DMS directly using a DMSO reductase. Instead, the archaeon reduced DMSO via a cysteine-cystine redox shuttle through a mechanism whereby cystine is microbially reduced to cysteine, which is then reoxidized by DMSO reduction. A thioredoxin reductase-protein disulfide oxidoreductase redox couple was identified to have intracellular cystine-reducing activity, permitting recycle of cysteine. This study presents the first example of DMSO reduction via an electron shuttle. Several Thermococcales species also exhibited enhanced growth coupled with DMSO reduction, probably by disposing of excess reducing power rather than conserving energy.

  5. Biochemical evidence for the presence of two α-glucoside ABC-transport systems in the hyperthermophilic archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    Koning, Sonja M.; Konings, Wil N.; Driessen, Arnold J.M.

    2002-01-01

    The hyperthermophilic archaeon Pyrococcus furiosus can utilize different carbohydrates, such as starch, maltose and trehalose. Uptake of α-glucosides is mediated by two different, binding protein-dependent, ATP-binding cassette (ABC)-type transport systems. The maltose transporter also transports tr

  6. Membrane homeoviscous adaptation in the piezo-hyperthermophilic archaeon Thermococcus barophilus.

    Science.gov (United States)

    Cario, Anaïs; Grossi, Vincent; Schaeffer, Philippe; Oger, Philippe M

    2015-01-01

    The archaeon Thermococcus barophilus, one of the most extreme members of hyperthermophilic piezophiles known thus far, is able to grow at temperatures up to 103°C and pressures up to 80 MPa. We analyzed the membrane lipids of T. barophilus by high performance liquid chromatography-mass spectrometry as a function of pressure and temperature. In contrast to previous reports, we show that under optimal growth conditions (40 MPa, 85°C) the membrane spanning tetraether lipid GDGT-0 (sometimes called caldarchaeol) is a major membrane lipid of T. barophilus together with archaeol. Increasing pressure and decreasing temperature lead to an increase of the proportion of archaeol. Reversely, a higher proportion of GDGT-0 is observed under low pressure and high temperature conditions. Noticeably, pressure and temperature fluctuations also impact the level of unsaturation of apolar lipids having an irregular polyisoprenoid carbon skeleton (unsaturated lycopane derivatives), suggesting a structural role for these neutral lipids in the membrane of T. barophilus. Whether these apolar lipids insert in the membrane or not remains to be addressed. However, our results raise questions about the structure of the membrane in this archaeon and other Archaea harboring a mixture of di- and tetraether lipids.

  7. 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

  8. 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.

  9. 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.

  10. Biochemical characterization and helix stabilizing properties of HSNP-C' from the thermoacidophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Celestina, F; Suryanarayana, T

    2000-01-19

    Helix stabilizing nucleoid protein HSNP-C' from the thermophilic archaeon Sulfolobus acidocaldarius has been characterized with respect to its interactions with nucleic acids by gel retardation assay, affinities to immobilized matrices, electron microscopy, and fluorescence titration. The amino acids implicated in the DNA binding site of the protein have been shown by selectively modifying specific amino acyl functional groups and looking at their effects on the DNA binding properties of the protein. Lysine, arginine, tryptophan, and tyrosine residues of the protein HSNP-C' were modified with pyridoxal-5-phosphate; 2,3-butanedione; BNPS-skatole; and tetranitromethane, respectively. The modification of residues was assessed according to standard procedures. The effect of the chemical modification on the function of the protein HSNP-C' with respect to DNA protein interactions was studied and the results indicate the definite involvement of tyrosines and also the significant involvement of the flanking tryptophan residues in the DNA binding domain on the protein.

  11. Genome-scale analysis of gene function in the hydrogenotrophic methanogenic archaeon Methanococcus maripaludis.

    Science.gov (United States)

    Sarmiento, Felipe; Mrázek, Jan; Whitman, William B

    2013-03-19

    A comprehensive whole-genome analysis of gene function by transposon mutagenesis and deep sequencing methodology has been implemented successfully in a representative of the Archaea domain. Libraries of transposon mutants were generated for the hydrogenotrophic, methanogenic archaeon Methanococcus maripaludis S2 using a derivative of the Tn5 transposon. About 89,000 unique insertions were mapped to the genome, which allowed for the classification of 526 genes or about 30% of the genome as possibly essential or strongly advantageous for growth in rich medium. Many of these genes were homologous to eukaryotic genes that encode fundamental processes in replication, transcription, and translation, providing direct evidence for their importance in Archaea. Some genes classified as possibly essential were unique to the archaeal or methanococcal lineages, such as that encoding DNA polymerase PolD. In contrast, the archaeal homolog to the gene encoding DNA polymerase B was not essential for growth, a conclusion confirmed by construction of an independent deletion mutation. Thus PolD, and not PolB, likely plays a fundamental role in DNA replication in methanococci. Similarly, 121 hypothetical ORFs were classified as possibly essential and likely play fundamental roles in methanococcal information processing or metabolism that are not established outside this group of prokaryotes.

  12. Preliminary characterization of two different crystal forms of acylphosphatase from the hyperthermophile archaeon Sulfolobus solfataricus

    Energy Technology Data Exchange (ETDEWEB)

    Zuccotti, Simone [Department of Physics-INFM and Center of Excellence for Biomedical Research, University of Genova, Via Dodecaneso 33, 16132 Genova (Italy); Rosano, Camillo [National Institute for Cancer Research (IST), X-ray Structural Biology Unit, Largo R. Benzi 10, 16132 Genova (Italy); Bemporad, Francesco [Department of Biochemical Sciences, University of Firenze, Viale Morgagni 50, 50134 Florence (Italy); Stefani, Massimo [Department of Biochemical Sciences, University of Firenze, Viale Morgagni 50, 50134 Florence (Italy); Centro di Ricerca, Trasferimento e Alta Formazione MCIDNENT, University of Firenze, Viale Morgagni 50, 50134 Florence (Italy); Bolognesi, Martino, E-mail: bolognes@fisica.unige.it [Department of Physics-INFM and Center of Excellence for Biomedical Research, University of Genova, Via Dodecaneso 33, 16132 Genova (Italy)

    2005-01-01

    S. solfataricus acylphosphatase has been expressed, purified and crystallized in two different crystal forms. Preliminary characterization of a triclinic and a monoclinic crystal form is reported and data were collected to 1.27 and 1.90 Å, respectively. Acylphosphatase is a ubiquitous small enzyme that was first characterized in mammals. It is involved in the hydrolysis of carboxyl-phosphate bonds in several acylphosphate substrates, such as carbamoylphosphate and 1,3-biphosphoglycerate; however, a consensus on acylphosphatase action in vivo has not yet been reached. Recent investigations have focused on acylphosphatases from lower phyla, such as Drosophila melanogaster and Escherichia coli, in view of the application of these small proteins as models in the study of folding, misfolding and aggregation processes. An acylphosphatase from the hyperthermophilic archaeon Sulfolobus solfataricus has been cloned, expressed and purified. Here, the growth and characterization of a triclinic and a monoclinic crystal form of the hyperthermophilic enzyme are reported; X-ray diffraction data have been collected to 1.27 and 1.90 Å resolution, respectively.

  13. 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.

  14. 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.

  15. Utilization of banana peel as a novel substrate for biosurfactant production by Halobacteriaceae archaeon AS65.

    Science.gov (United States)

    Chooklin, Chanika Saenge; Maneerat, Suppasil; Saimmai, Atipan

    2014-05-01

    In this study, biosurfactant-producing bacteria was evaluated for biosurfactant production by using banana peel as a sole carbon source. From the 71 strains screened, Halobacteriaceae archaeon AS65 produced the highest biosurfactant activity. The highest biosurfactant production (5.30 g/l) was obtained when the cells were grown on a minimal salt medium containing 35 % (w/v) banana peel and 1 g/l commercial monosodium glutamate at 30 °C and 200 rpm after 54 h of cultivation. The biosurfactant obtained by extraction with ethyl acetate showed high surface tension reduction (25.5 mN/m), a small critical micelle concentration value (10 mg/l), thermal and pH stability with respect to surface tension reduction and emulsification activity, and a high level of salt tolerance. The biosurfactant obtained was confirmed as a lipopeptide by using a biochemical test FT-IR, NMR, and mass spectrometry. The crude biosurfactant showed a broad spectrum of antimicrobial activity and had the ability to emulsify oil, enhance PAHs solubility, and oil bioremediation.

  16. Characterization of a trehalose-degrading enzyme from the hyperthermophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Moon, Jeong Hyun; Lee, Whiso; Park, Jihee; Choi, Kyoung-Hwa; Cha, Jaeho

    2016-07-01

    We purified a cytosolic trehalase (TreH) from a thermoacidophilic archaeon Sulfolobus acidocaldarius. Enzyme activity in cell-free extracts indicated that trehalose degradation in the cell occurred via the hydrolytic activity of TreH, and not via TreP (phosphorolytic activity) or TreT (transfer activity). TreH was purified to near-homogeneity by DEAE anion-exchange chromatography, followed by size exclusion and HiTrap Q anion-exchange chromatography, and its molecular mass was estimated as 40 kDa. Maximum activity was observed at 85°C and pH 4.5. The half-life of TreH was 53 and 41 min at 90°C and 95°C, respectively. TreH was highly specific for trehalose and was inhibited by glucose with a Ki of 0.05 mM. Compared with TreH from other trehalases, TreH from S. acidocaldarius is the most thermostable trehalase reported so far. Furthermore, this is the first trehalase characterized in the Archaea domain.

  17. Molecular characteristics of spontaneous deletions in the hyperthermophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Grogan, Dennis W; Hansen, Josh E

    2003-02-01

    Prokaryotic genomes acquire and eliminate blocks of DNA sequence by lateral gene transfer and spontaneous deletion, respectively. The basic parameters of spontaneous deletion, which are expected to influence the course of genome evolution, have not been determined for any hyperthermophilic archaeon. We therefore screened a number of independent pyrimidine auxotrophs of Sulfolobus acidocaldarius for deletions and sequenced those detected. Deletions accounted for only 0.4% of spontaneous pyrE mutations, corresponding to a frequency of about 10(-8) per cell. Nucleotide sequence analysis of five independent deletions showed no significant association of the endpoints with short direct repeats, despite the fact that several such repeats occur within the pyrE gene and that duplication mutations in pyrE reverted at high frequencies. Endpoints of the spontaneous deletions did not coincide with short inverted repeats or potential stem-loop structures. No consensus sequence common to all the deletions could be identified, although two deletions showed the potential of being stabilized by octanucleotide sequences elsewhere in pyrE, and another pair of deletions shared an octanucleotide at their 3' ends. The unusually low frequency and low sequence dependence of spontaneous deletions in the S. acidocaldarius pyrE gene compared to other genetic systems could not be explained in terms of possible constraints imposed by the 5-fluoroorotate selection.

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

    Science.gov (United States)

    Ehlers, Claudia; Veit, Katharina; Gottschalk, Gerhard; Schmitz, Ruth A

    2002-09-01

    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.

  19. Minimal sulfur requirement for growth and sulfur-dependent metabolism of the hyperthermophilic archaeon Staphylothermus marinus

    Directory of Open Access Journals (Sweden)

    Xiaolei Hao

    2003-01-01

    Full Text Available Staphylothermus marinus is an anaerobic hyperthermophilic archaeon that uses peptides as carbon and energy sources. Elemental sulfur (S° is obligately required for its growth and is reduced to H2S. The metabolic functions and mechanisms of S° reduction were explored by examining S°-dependent growth and activities of key enzymes present in this organism. All three forms of S° tested—sublimed S°, colloidal S° and polysulfide—were used by S. marinus, and no other sulfur-containing compounds could replace S°. Elemental sulfur did not serve as physical support but appeared to function as an electron acceptor. The minimal S° concentration required for optimal growth was 0.05% (w/v. At this concentration, there appeared to be a metabolic transition from H2 production to S° reduction. Some enzymatic activities related to S°-dependent metabolism, including sulfur reductase, hydrogenase, glutamate dehydrogenase and electron transfer activities, were detected in cell-free extracts of S. marinus. These results indicate that S° plays an essential role in the heterotrophic metabolism of S. marinus. Reducing equivalents generated by the oxidation of amino acids from peptidolysis may be transferred to sulfur reductase and hydrogenase, which then catalyze the production of H2S and H2, respectively.

  20. Functional screening of hydrolytic activities reveals an extremely thermostable cellulase from a deep-sea archaeon

    Directory of Open Access Journals (Sweden)

    Benedikt eLeis

    2015-07-01

    Full Text Available Extreme habitats serve as a source of enzymes which are active under extreme conditions and are candidates for industrial applications. In this work, six large-insert mixed genomic libraries were screened for hydrolase activities in a broad temperature range (8 to 70 °C. Among a variety of hydrolytic activities, one fosmid clone, derived from a library of pooled isolates of hyperthermophilic archaea from deep sea vents, displayed hydrolytic activity on carboxymethyl cellulose substrate plates at 70 °C but not at lower temperatures. Sequence analysis of the fosmid insert revealed a gene encoding a novel glycoside hydrolase family 12 (GHF12 endo-1,4-β-glucanase, termed Cel12E. The enzyme shares 45 % sequence identity with a protein from the archaeon Thermococcus sp. AM4 and displays a unique multidomain architecture. Biochemical characterization of Cel12E revealed a remarkably thermostable protein, which appears to be of archaeal origin. The enzyme displayed maximum activity at 92 °C and was active on a variety of linear 1,4-β-glucans like carboxymethyl cellulose, β-glucan, lichenan, and phosphoric acid swollen cellulose. The protein is able to bind to various insoluble β-glucans. Product pattern analysis indicated that Cel12E is an endo-cleaving β-glucanase. Cel12E expands the toolbox of hyperthermostable archaeal cellulases with biotechnological potential.

  1. The thermoacidophilic archaeon Sulfolobus acidocaldarius contains an unusually short, highly reduced dolichyl phosphate.

    Science.gov (United States)

    Guan, Ziqiang; Meyer, Benjamin H; Albers, Sonja-Verena; Eichler, Jerry

    2011-10-01

    Polyprenoids, polymers containing varied numbers of isoprene subunits, serve numerous roles in biology. In Eukarya, dolichyl phosphate, a phosphorylated polyprenol bearing a saturated α-end isoprene subunit, serves as the glycan carrier during N-glycosylation, namely that post-translational modification whereby glycans are covalently linked to select asparagine residues of a target protein. As in Eukarya, N-glycosylation in Archaea also relies on phosphorylated dolichol. In this report, LC-ESI/MS/MS was employed to identify a novel dolichyl phosphate (DolP) in the thermoacidophilic archaeon, Sulfolobus acidocaldarius. The unusually short S. acidocaldarius DolP presents a degree of saturation not previously reported. S. acidocaldarius DolP contains not only the saturated α- and ω-end isoprene subunits observed in other archaeal DolPs, but also up to five saturated intra-chain isoprene subunits. The corresponding dolichol and hexose-charged DolP species were also detected. The results of the present study offer valuable information on the biogenesis and potential properties of this unique DolP. Furthermore, elucidation of the mechanism of α-isoprene unit reduction in S. acidocaldarius dolichol may facilitate the identification of the alternative, as yet unknown polyprenol reductase in Eukarya.

  2. Natronorubrum texcoconense sp. nov., a haloalkaliphilic archaeon isolated from soil of the former lake Texcoco (Mexico).

    Science.gov (United States)

    Ruiz-Romero, Erick; Valenzuela-Encinas, César; López-Ramírez, María Patricia; de los Angeles Coutiño-Coutiño, María; Marsch, Rodolfo; Dendooven, Luc

    2013-02-01

    A new haloalkaliphilic archaeon, strain B4(T), was isolated from the former lake Texcoco in Mexico. The cells were Gram-negative, pleomorphic-shaped, pink to red pigmented and aerobic. Strain B4(T) required at least 2.5 M NaCl for growth, with optimum growth at 3.4 M NaCl. It was able to grow over a pH range of 7.5-10.0 and temperature of 25-50 °C, with optimal growth at pH 9 and 37 °C. Cells are lysed in hypotonic treatment with less than 1.3 M NaCl. The major polar lipids of strain B4(T) were phosphatidylglycerol and methyl-phosphatidylglycerophosphate. Phospholipids were detected, but not glycolipids. The nucleotide sequence of the 16S rRNA gene revealed that the strain B4(T) was phylogenetically related to members of the genus Natronorubrum. Sequence similarity with Natronorubrum tibetense was 96.28 %, with Natronorubrum sulfidifaciens 95.06 % and Natronorubrum sediminis 94.98 %. The G+C content of the DNA was 63.3 mol%. The name of Natronorubrum texcoconense sp. nov. is proposed. The type strain is B4(T) (=CECT 8067(T) = JCM 17497(T)).

  3. Natronobacterium texcoconense sp. nov., a haloalkaliphilic archaeon isolated from soil of a former lake.

    Science.gov (United States)

    Ruiz-Romero, Erick; Sánchez-López, Katia Berenice; de los Angeles Coutiño-Coutiño, María; González-Pozos, Sirenia; Bello-López, Juan Manuel; López-Ramírez, María Patricia; Ramírez-Villanueva, Daniel Alejandro; Dendooven, Luc

    2013-11-01

    A novel haloalkaliphilic archaeon, strain B23(T) was isolated from the former lake Texcoco in Mexico. The strain was Gram-stain-negative, the cells coccoid to ovoid rods, red pigmented and aerobic. Strain B23(T) grew in 1.7-4.3 M NaCl, at pH 6.5-9.5 and at 25-45 °C with optimal growth at 2.6-3.4 M NaCl, pH 7.5-8.5 and 37 °C. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain B23(T) was most closely related to Natronobacterium gregoryi SP2(T) with 97.3 % sequence similarity. The polar lipids of strain B23(T) were phosphatidylglycerol and several unidentified phospholipids. The G+C content of the DNA of the strain was 62.5 mol%. Levels of DNA-DNA relatedness between strain B23(T) and Natronobacterium gregoryi DSM 3393(T) was 32.3 %. The name Natronobacterium texcoconense sp. nov. is proposed. The type strain is B23(T) ( = CECT 8068(T) = JCM 17655(T)).

  4. 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.

  5. High hydrostatic pressure increases amino acid requirements in the piezo-hyperthermophilic archaeon Thermococcus barophilus.

    Science.gov (United States)

    Cario, Anaïs; Lormières, Florence; Xiang, Xiao; Oger, Philippe

    2015-11-01

    We have established a defined growth medium for the piezophilic hyperthermophilic archaeon Thermococcus barophilus, which allows growth yields of ca. 10(8) cells/ml under both atmospheric and high hydrostatic pressure. Our results demonstrate a major impact of hydrostatic pressure on amino acid metabolism, with increases from 3 amino acids required at atmospheric pressure to 17 at 40 MPa. We observe in T. barophilus and other Thermococcales a similar discrepancy between the presence/absence of amino acid synthesis pathways and amino acid requirements, which supports the existence of alternate, but yet unknown, amino acid synthesis pathways, and may explain the low number of essential amino acids observed in T. barophilus and other Thermococcales. T. barophilus displays a strong metabolic preference for organic polymers such as polypeptides and chitin, which may constitute a more readily available resource of carbon and energy in situ in deep-sea hydrothermal vents. We hypothesize that the low energy yields of fermentation of organic polymers, together with energetic constraints imposed by high hydrostatic pressure, may render de novo synthesis of amino acids ecologically unfavorable. Induction of this metabolic switch to amino acid recycling can explain the requirement for non-essential amino acids by Thermococcales for efficient growth in defined medium.

  6. Dynamic Metabolite Profiling in an Archaeon Connects Transcriptional Regulation to Metabolic Consequences.

    Directory of Open Access Journals (Sweden)

    Horia Todor

    Full Text Available Previous work demonstrated that the TrmB transcription factor is responsible for regulating the expression of many enzyme-coding genes in the hypersaline-adapted archaeon Halobacterium salinarum via a direct interaction with a cis-regulatory sequence in their promoters. This interaction is abolished in the presence of glucose. Although much is known about the effects of TrmB at the transcriptional level, it remains unclear whether and to what extent changes in mRNA levels directly affect metabolite levels. In order to address this question, here we performed a high-resolution metabolite profiling time course during a change in nutrients using a combination of targeted and untargeted methods in wild-type and ΔtrmB strain backgrounds. We found that TrmB-mediated transcriptional changes resulted in widespread and significant changes to metabolite levels across the metabolic network. Additionally, the pattern of growth complementation using various purines suggests that the mis-regulation of gluconeogenesis in the ΔtrmB mutant strain in the absence of glucose results in low phosphoribosylpyrophosphate (PRPP levels. We confirmed these low PRPP levels using a quantitative mass spectrometric technique and found that they are associated with a metabolic block in de novo purine synthesis, which is partially responsible for the growth defect of the ΔtrmB mutant strain in the absence of glucose. In conclusion, we show how transcriptional regulation of metabolism affects metabolite levels and ultimately, phenotypes.

  7. 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.

  8. Identification of several intracellular carbohydrate-degrading activities from the halophilic archaeon Haloferax mediterranei.

    Science.gov (United States)

    Pérez-Pomares, F; Díaz, S; Bautista, V; Pire, C; Bravo, G; Esclapez, J; Zafrilla, B; Bonete, María-José

    2009-07-01

    Three different amylolytic activities, designated AMY1, AMY2, and AMY3 were detected in the cytoplasm of the extreme halophilic archaeon Haloferax mediterranei grown in a starch containing medium. This organism had also been reported to excrete an alpha-amylase into the external medium in such conditions. The presence of these different enzymes which are also able to degrade starch may be related to the use of the available carbohydrates and maltodextrins, including the products obtained by the action of the extracellular amylase on starch that may be transported to the cytoplasm of the organism. The behavior of these intracellular hydrolytic enzymes on starch is reported here and compared with their extracellular counterpart. Two of these glycosidic activities (AMY1, AMY3) have also been purified and further characterized. As with other halophilic enzymes, they were salt dependent and displayed maximal activity at 3 M NaCl, and 50 degrees C. The purification steps and molecular masses have also been reported. The other activity (AMY2) was also detected in extracts from cells grown in media with glycerol instead of starch and in a yeast extract medium. This enzyme was able to degrade starch yielding small oligosaccharides and displayed similar halophilic behavior with salt requirement in the range 1.5-3 M NaCl.

  9. 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.

  10. Genome-wide primary transcriptome analysis of H2-producing archaeon Thermococcus onnurineus NA1

    Science.gov (United States)

    Cho, Suhyung; Kim, Min-Sik; Jeong, Yujin; Lee, Bo-Rahm; Lee, Jung-Hyun; Kang, Sung Gyun; Cho, Byung-Kwan

    2017-01-01

    In spite of their pivotal roles in transcriptional and post-transcriptional processes, the regulatory elements of archaeal genomes are not yet fully understood. Here, we determine the primary transcriptome of the H2-producing archaeon Thermococcus onnurineus NA1. We identified 1,082 purine-rich transcription initiation sites along with well-conserved TATA box, A-rich B recognition element (BRE), and promoter proximal element (PPE) motif in promoter regions, a high pyrimidine nucleotide content (T/C) at the −1 position, and Shine-Dalgarno (SD) motifs (GGDGRD) in 5′ untranslated regions (5′ UTRs). Along with differential transcript levels, 117 leaderless genes and 86 non-coding RNAs (ncRNAs) were identified, representing diverse cellular functions and potential regulatory functions under the different growth conditions. Interestingly, we observed low GC content in ncRNAs for RNA-based regulation via unstructured forms or interaction with other cellular components. Further comparative analysis of T. onnurineus upstream regulatory sequences with those of closely related archaeal genomes demonstrated that transcription of orthologous genes are initiated by highly conserved promoter sequences, however their upstream sequences for transcriptional and translational regulation are largely diverse. These results provide the genetic information of T. onnurineus for its future application in metabolic engineering. PMID:28216628

  11. Dynamic Metabolite Profiling in an Archaeon Connects Transcriptional Regulation to Metabolic Consequences.

    Science.gov (United States)

    Todor, Horia; Gooding, Jessica; Ilkayeva, Olga R; Schmid, Amy K

    2015-01-01

    Previous work demonstrated that the TrmB transcription factor is responsible for regulating the expression of many enzyme-coding genes in the hypersaline-adapted archaeon Halobacterium salinarum via a direct interaction with a cis-regulatory sequence in their promoters. This interaction is abolished in the presence of glucose. Although much is known about the effects of TrmB at the transcriptional level, it remains unclear whether and to what extent changes in mRNA levels directly affect metabolite levels. In order to address this question, here we performed a high-resolution metabolite profiling time course during a change in nutrients using a combination of targeted and untargeted methods in wild-type and ΔtrmB strain backgrounds. We found that TrmB-mediated transcriptional changes resulted in widespread and significant changes to metabolite levels across the metabolic network. Additionally, the pattern of growth complementation using various purines suggests that the mis-regulation of gluconeogenesis in the ΔtrmB mutant strain in the absence of glucose results in low phosphoribosylpyrophosphate (PRPP) levels. We confirmed these low PRPP levels using a quantitative mass spectrometric technique and found that they are associated with a metabolic block in de novo purine synthesis, which is partially responsible for the growth defect of the ΔtrmB mutant strain in the absence of glucose. In conclusion, we show how transcriptional regulation of metabolism affects metabolite levels and ultimately, phenotypes.

  12. A transcription factor links growth rate and metabolism in the hypersaline adapted archaeon Halobacterium salinarum.

    Science.gov (United States)

    Todor, Horia; Dulmage, Keely; Gillum, Nicholas; Bain, James R; Muehlbauer, Michael J; Schmid, Amy K

    2014-09-01

    Co-ordinating metabolism and growth is a key challenge for all organisms. Despite fluctuating environments, cells must produce the same metabolic outputs to thrive. The mechanisms underlying this 'growth homeostasis' are known in bacteria and eukaryotes, but remain unexplored in archaea. In the model archaeon Halobacterium salinarum, the transcription factor TrmB regulates enzyme-coding genes in diverse metabolic pathways in response to glucose. However, H. salinarum is thought not to catabolize glucose. To resolve this discrepancy, we demonstrate that TrmB regulates the gluconeogenic production of sugars incorporated into the cell surface S-layer glycoprotein. Additionally, we show that TrmB-DNA binding correlates with instantaneous growth rate, likely because S-layer glycosylation is proportional to growth. This suggests that TrmB transduces a growth rate signal to co-regulated metabolic pathways including amino acid, purine, and cobalamin biosynthesis. Remarkably, the topology and function of this growth homeostatic network appear conserved across domains despite extensive alterations in protein components.

  13. 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

    Science.gov (United States)

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

    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 complete genome sequence analysis results of Pyrococcus sp. ST04 and report the major findings from the genome annotation, with a focus on its saccharolytic and metabolite production potential. PMID:22843576

  14. Coupled TLC and MALDI-TOF/MS Analyses of the Lipid Extract of the Hyperthermophilic Archaeon Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Simona Lobasso

    2012-01-01

    Full Text Available The lipidome of the marine hyperthermophilic archaeon Pyrococcus furiosus was studied by means of combined thin-layer chromatography and MALDI-TOF/MS analyses of the total lipid extract. 80–90% of the major polar lipids were represented by archaeol lipids (diethers and the remaining part by caldarchaeol lipids (tetraethers. The direct analysis of lipids on chromatography plate showed the presence of the diphytanylglycerol analogues of phosphatidylinositol and phosphatidylglycerol, the N-acetylglucosamine-diphytanylglycerol phosphate plus some caldarchaeol lipids different from those previously described. In addition, evidence for the presence of the dimeric ether lipid cardiolipin is reported, suggesting that cardiolipins are ubiquitous in archaea.

  15. Identification and characterization of small RNAs in the hyperthermophilic archaeon Sulfolobus solfataricus.

    Directory of Open Access Journals (Sweden)

    Ning Xu

    Full Text Available The term RNA silencing (RNA interference, RNAi describes a set of mechanisms that regulate gene expression in eukaryotes. Small interfering RNAs (siRNA and microRNAs (miRNAs are two major types of RNAi-associated small RNAs (smRNAs found in most eukaryotic organisms. Despite the presence of a plethora of non-coding RNAs longer than 50-nucleotide (nt in length in various species of Archaea, little is known about smRNAs in archaea that resemble the 20-24-nt long smRNAs found in eukaryotes, which have been implicated in the post-transcriptional control of gene expression. Here, we report the finding of a large number of smRNAs approximatelly 20-nt in length, including phased smRNAs and potential miRNAs, from the hyperthermophilic archaeon Sulfolobus solfataricus p2 (Ssp2 based on deep sequencing. The expression of some of the miRNA candidates in Ssp2 was confirmed. Consistent with the Ssp2 hyperthermophilic properties, we found that higher temperatures more efficiently induced the production of the miRNA candidates in an in vitro system using the putative foldback precursor transcripts incubated with Ssp2 extract. Although we initially predicted putative target genes of some miRNA candidates, further analysis mapped the cleavage sites downstream of the miRNA candidate complementary regions, similar to those involved in plant miRNA-mediated TAS transcript cleavage. We also identified smRNAs from clustered, regularly interspaced, short palindromic repeat (CRISPR loci, which play important roles in prokaryotic microbial defense systems. Archaea represent a unique life form next to Bacteria and Eukarya, and our results may provide a useful resource for further in-depth study on the regulation and evolution of smRNAs in this special organism.

  16. 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.

  17. Grappling archaea: ultrastructural analyses of an uncultivated, cold-loving archaeon and its biofilm

    Directory of Open Access Journals (Sweden)

    Alexandra ePerras

    2014-08-01

    Full Text Available Similarly to Bacteria, Archaea are microorganisms that interact with their surrounding environment in a versatile manner. To date, interactions based on cellular structure and surface appendages have mainly been documented using model systems of cultivable archaea under laboratory conditions. Here, we report on the microbial interactions and ultrastructural features of the uncultivated SM1 Euryarchaeon, which is highly dominant in its biotope. Therefore, biofilm samples taken from the Sippenauer Moor, Germany, were investigated via transmission electron microscopy (TEM; negative staining, thin-sectioning and scanning electron microscopy (SEM in order to elucidate the fine structures of the microbial cells and the biofilm itself. The biofilm consisted of small archaeal cocci (0.6 µm diameter, arranged in a regular pattern (1.2-2.0 µm distance from cell to cell, whereas each archaeon was connected to 6 other archaea on average. Extracellular polymeric substances (EPS were limited to the close vicinity of the archaeal cells, and specific cell surface appendages (hami, Moissl et al., 2005 protruded beyond the EPS matrix enabling microbial interaction by cell-cell contacts among the archaea and between archaea and bacteria. All analyzed hami revealed their previously described architecture of nano-grappling hooks and barb-wire basal structures. Considering the archaeal cell walls, the SM1 Euryarchaea exhibited a double-membrane, which has rarely been reported for members of this phylogenetic domain. Based on these findings, the current generalized picture on archaeal cell walls needs to be revisited, as archaeal cell structures are more complex and sophisticated than previously assumed, particularly when looking into the uncultivated majority.

  18. Crystal structure of T state aspartate carbamoyltransferase of the hyperthermophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    De Vos, Dirk; Van Petegem, Filip; Remaut, Han; Legrain, Christianne; Glansdorff, Nicolas; Van Beeumen, Jozef J

    2004-06-11

    Aspartate carbamoyltransferase (ATCase) is a model enzyme for understanding allosteric effects. The dodecameric complex exists in two main states (T and R) that differ substantially in their quaternary structure and their affinity for various ligands. Many hypotheses have resulted from the structure of the Escherichia coli ATCase, but so far other crystal structures to test these have been lacking. Here, we present the tertiary and quaternary structure of the T state ATCase of the hyperthermophilic archaeon Sulfolobus acidocaldarius (SaATC(T)), determined by X-ray crystallography to 2.6A resolution. The quaternary structure differs from the E.coli ATCase, by having altered interfaces between the catalytic (C) and regulatory (R) subunits, and the presence of a novel C1-R2 type interface. Conformational differences in the 240 s loop region of the C chain and the C-terminal region of the R chain affect intersubunit and interdomain interfaces implicated previously in the allosteric behavior of E.coli ATCase. The allosteric-zinc binding domain interface is strengthened at the expense of a weakened R1-C4 type interface. The increased hydrophobicity of the C1-R1 type interface may stabilize the quaternary structure. Catalytic trimers of the S.acidocaldarius ATCase are unstable due to a drastic weakening of the C1-C2 interface. The hyperthermophilic ATCase presents an interesting example of how an allosteric enzyme can adapt to higher temperatures. The structural rearrangement of this thermophilic ATCase may well promote its thermal stability at the expense of changes in the allosteric behavior.

  19. Calcium-induced aggregation of archaeal bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Roby Kanichay

    2003-01-01

    Full Text Available Previously, we showed that the proton permeability of small unilamellar vesicles (SUVs composed of polar lipid fraction E (PLFE from the thermoacidophilic archaeon Sulfolobus acidocaldarius was remarkably low and insensitive to temperature (Komatsu and Chong 1998. In this study, we used photon correlation spectroscopy to investigate the time dependence of PLFE SUV size as a function of Ca2+ concentration. In the absence of Ca2+, vesicle diameter changed little over 6 months. Addition of Ca2+, however, immediately induced formation of vesicle aggregates with an irregular shape, as revealed by confocal fluorescence microscopy. Aggregation was reversible upon addition of EDTA; however, the reversibility varied with temperature as well as incubation time with Ca2+. Freeze-fracture electron microscopy showed that, after a long period of incubation (2 weeks with Ca2+, the PLFE vesicles had not just aggregated, but had fused or coalesced. The initial rate of vesicle aggregation varied sigmoidally with Ca2+ concentration. At pH 6.6, the threshold calcium concentration (Cr for vesicle aggregation at 25 and 40 °C was 11 and 17 mM, respectively. At pH 3.0, the Cr at 25 °C increased to 25 mM. The temperature dependence of Cr may be attributable to changes in membrane surface potential, which was –22.0 and –13.2 mV at 25 and 40 °C, respectively, at pH 6.6, as determined by 2-(p-toluidinylnaphthalene-6-sulfonic acid fluorescence. The variation in surface potential with temperature is discussed in terms of changes in lipid conformation and membrane organization.

  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.

  1. Calcium-induced aggregation of archaeal bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Kanichay, Roby; Boni, Lawrence T; Cooke, Peter H; Khan, Tapan K; Chong, Parkson Lee-Gau

    2003-10-01

    Previously, we showed that the proton permeability of small unilamellar vesicles (SUVs) composed of polar lipid fraction E (PLFE) from the thermoacidophilic archaeon Sulfolobus acidocaldarius was remarkably low and insensitive to temperature (Komatsu and Chong 1998). In this study, we used photon correlation spectroscopy to investigate the time dependence of PLFE SUV size as a function of Ca2+ concentration. In the absence of Ca2+, vesicle diameter changed little over 6 months. Addition of Ca2+, however, immediately induced formation of vesicle aggregates with an irregular shape, as revealed by confocal fluorescence microscopy. Aggregation was reversible upon addition of EDTA; however, the reversibility varied with temperature as well as incubation time with Ca2+. Freeze-fracture electron microscopy showed that, after a long period of incubation (2 weeks) with Ca2+, the PLFE vesicles had not just aggregated, but had fused or coalesced. The initial rate of vesicle aggregation varied sigmoidally with Ca2+ concentration. At pH 6.6, the threshold calcium concentration (Cr) for vesicle aggregation at 25 and 40 degrees C was 11 and 17 mM, respectively. At pH 3.0, the Cr at 25 degrees C increased to 25 mM. The temperature dependence of Cr may be attributable to changes in membrane surface potential, which was -22.0 and -13.2 mV at 25 and 40 degrees C, respectively, at pH 6.6, as determined by 2-(p-toluidinyl)naphthalene-6-sulfonic acid fluorescence. The variation in surface potential with temperature is discussed in terms of changes in lipid conformation and membrane organization.

  2. 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.

  3. Citric acid cycle in the hyperthermophilic archaeon Pyrobaculum islandicum grown autotrophically, heterotrophically, and mixotrophically with acetate.

    Science.gov (United States)

    Hu, Yajing; Holden, James F

    2006-06-01

    The hyperthermophilic archaeon Pyrobaculum islandicum uses the citric acid cycle in the oxidative and reductive directions for heterotrophic and autotrophic growth, respectively, but the control of carbon flow is poorly understood. P. islandicum was grown at 95 degrees C autotrophically, heterotrophically, and mixotrophically with acetate, H2, and small amounts of yeast extract and with thiosulfate as the terminal electron acceptor. The autotrophic growth rates and maximum concentrations of cells were significantly lower than those in other media. The growth rates on H2 and 0.001% yeast extract with and without 0.05% acetate were the same, but the maximum concentration of cells was fourfold higher with acetate. There was no growth with acetate if 0.001% yeast extract was not present, and addition of H2 to acetate-containing medium greatly increased the growth rates and maximum concentrations of cells. P. islandicum cultures assimilated 14C-labeled acetate in the presence of H2 and yeast extract with an efficiency of 55%. The activities of 11 of 19 enzymes involved in the central metabolism of P. islandicum were regulated under the three different growth conditions. Pyruvate synthase and acetate:coenzyme A (CoA) ligase (ADP-forming) activities were detected only in heterotrophically grown cultures. Citrate synthase activity decreased in autotrophic and acetate-containing cultures compared to the activity in heterotrophic cultures. Acetylated citrate lyase, acetate:CoA ligase (AMP forming), and phosphoenolpyruvate carboxylase activities increased in autotrophic and acetate-containing cultures. Citrate lyase activity was higher than ATP citrate synthase activity in autotrophic cultures. These data suggest that citrate lyase and AMP-forming acetate:CoA ligase, but not ATP citrate synthase, work opposite citrate synthase to control the direction of carbon flow in the citric acid cycle.

  4. The protein interaction network of a taxis signal transduction system in a Halophilic Archaeon

    Directory of Open Access Journals (Sweden)

    Schlesner Matthias

    2012-11-01

    Full Text Available Abstract Background The taxis signaling system of the extreme halophilic archaeon Halobacterium (Hbt. salinarum differs in several aspects from its model bacterial counterparts Escherichia coli and Bacillus subtilis. We studied the protein interactions in the Hbt. salinarum taxis signaling system to gain an understanding of its structure, to gain knowledge about its known components and to search for new members. Results The interaction analysis revealed that the core signaling proteins are involved in different protein complexes and our data provide evidence for dynamic interchanges between them. Fifteen of the eighteen taxis receptors (halobacterial transducers, Htrs can be assigned to four different groups depending on their interactions with the core signaling proteins. Only one of these groups, which contains six of the eight Htrs with known signals, shows the composition expected for signaling complexes (receptor, kinase CheA, adaptor CheW, response regulator CheY. From the two Hbt. salinarum CheW proteins, only CheW1 is engaged in signaling complexes with Htrs and CheA, whereas CheW2 interacts with Htrs but not with CheA. CheY connects the core signaling structure to a subnetwork consisting of the two CheF proteins (which build a link to the flagellar apparatus, CheD (the hub of the subnetwork, two CheC complexes and the receptor methylesterase CheB. Conclusions Based on our findings, we propose two hypotheses. First, Hbt. salinarum might have the capability to dynamically adjust the impact of certain Htrs or Htr clusters depending on its current needs or environmental conditions. Secondly, we propose a hypothetical feedback loop from the response regulator to Htr methylation made from the CheC proteins, CheD and CheB, which might contribute to adaptation analogous to the CheC/CheD system of B. subtilis.

  5. Development of New Modular Genetic Tools for Engineering the Halophilic Archaeon Halobacterium salinarum.

    Directory of Open Access Journals (Sweden)

    Rafael Silva-Rocha

    Full Text Available Our ability to genetically manipulate living organisms is usually constrained by the efficiency of the genetic tools available for the system of interest. In this report, we present the design, construction and characterization of a set of four new modular vectors, the pHsal series, for engineering Halobacterium salinarum, a model halophilic archaeon widely used in systems biology studies. The pHsal shuttle vectors are organized in four modules: (i the E. coli's specific part, containing a ColE1 origin of replication and an ampicillin resistance marker, (ii the resistance marker and (iii the replication origin, which are specific to H. salinarum and (iv the cargo, which will carry a sequence of interest cloned in a multiple cloning site, flanked by universal M13 primers. Each module was constructed using only minimal functional elements that were sequence edited to eliminate redundant restriction sites useful for cloning. This optimization process allowed the construction of vectors with reduced sizes compared to currently available platforms and expanded multiple cloning sites. Additionally, the strong constitutive promoter of the fer2 gene was sequence optimized and incorporated into the platform to allow high-level expression of heterologous genes in H. salinarum. The system also includes a new minimal suicide vector for the generation of knockouts and/or the incorporation of chromosomal tags, as well as a vector for promoter probing using a GFP gene as reporter. This new set of optimized vectors should strongly facilitate the engineering of H. salinarum and similar strategies could be implemented for other archaea.

  6. Draft Genome Sequence of the Novel Thermoacidophilic Archaeon Acidianus copahuensis Strain ALE1, Isolated from the Copahue Volcanic Area in Neuquen, Argentina.

    Science.gov (United States)

    Urbieta, M Sofía; Rascovan, Nicolás; Castro, Camila; Revale, Santiago; Giaveno, M Alejandra; Vazquez, Martín; Donati, Edgardo R

    2014-05-08

    Acidianus copahuensis is a recently characterized thermoacidophilic archaeon isolated from the Copahue volcanic area in Argentina. Here, we present its draft genome sequence, in which we found genes involved in key metabolic pathways for developing under Copahue's extreme environmental conditions, such as sulfur and iron oxidation, carbon fixation, and metal tolerance.

  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...

  8. 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

  9. Thermal stability and biochemical properties of isocitrate dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum.

    Science.gov (United States)

    Stokke, Runar; Birkeland, Nils-Kåre; Steen, Ida Helene

    2007-03-01

    Isocitrate dehydrogenase [IDH; EC 1.1.1.42] from the thermoacidophilic archaeon Thermoplasma acidophilum (TaIDH) showed high thermal stability with an apparent melting temperature, T(m), of 82.2 and 84.5 degrees C at pH 7.5 and 5.8, respectively. Based on structural alignment of TaIDH with IDH from Aeropyrum pernix (ApIDH) and Archaeoglobus fulgidus (AfIDH) residues forming an aromatic cluster in the clasp-domain thought to strengthen the dimer interface in ApIDH and AfIDH were identified in the former enzyme. Moreover, TaIDH had a shortened N-terminus that may protect the enzyme from thermal denaturation. The enzyme activity of TaIDH was highest at 70 degrees C. The pH-activity profile was bell-shaped with an optimum shifted to a lower pH compared to AfIDH. The activity of TaIDH was influenced by changes in pH with a three-fold reduction in activity when the pH was shifted from the pH-optimum at 7.5 to pH 5.8. However, the specific activity at pH 5.8 was still high when compared with AfIDH. The reduction in activity at pH 5.8 was not due to instability of the enzyme as the T(m) of TaIDH was higher at pH 5.8 than at 7.5 and the enzyme retained 91% of its activity after incubation at 1 h at pH 5 and 60 degrees C. The difference in the pH-profile of TaIDH in comparison with AfIDH may thus be related to the pK(a)s of their catalytic residues involved in the initial proton abstraction and the final proton donation during the catalysis of oxidative decarboxylation of isocitrate to 2-oxoglutarate and reduced coenzyme.

  10. Involvement of a eukaryotic-like ubiquitin-related modifier in the proteasome pathway of the archaeon Sulfolobus acidocaldarius

    Science.gov (United States)

    Anjum, Rana S.; Bray, Sian M.; Blackwood, John K.; Kilkenny, Mairi L.; Coelho, Matthew A.; Foster, Benjamin M.; Li, Shurong; Howard, Julie A.; Pellegrini, Luca; Albers, Sonja-Verena; Deery, Michael J.; Robinson, Nicholas P.

    2015-09-01

    In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved.

  11. Different glycosyltransferases are involved in lipid glycosylation and protein N-glycosylation in the halophilic archaeon Haloferax volcanii.

    Science.gov (United States)

    Naparstek, Shai; Vinagradov, Evguenii; Eichler, Jerry

    2010-07-01

    Both the lipid and the protein components of biological membranes can be modified by the covalent addition of polysaccharides. Whereas eukaryal and bacterial pathways of lipid and protein glycosylation are relatively well defined, considerably less is known of the parallel processes in Archaea. Recent efforts have identified glycosyltransferases involved in N-glycosylation of the surface-layer glycoprotein of the halophilic archaeon Haloferax volcanii. In the present study, the involvement of these same glycosyltransferases in the biosynthesis of Hfx. volcanii glycolipids was considered by performing nuclear magnetic resonance analysis of the glycolipid fraction of Hfx. volcanii cells deleted of genes encoding those glycosyltransferases, as well as the oligosaccharyltransferase, AglB. The results reveal that different glycosyltransferases are involved in the biosynthesis of N-linked glycoproteins and glycolipids in Archaea.

  12. 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

    2016-03-15

    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.

  13. Restoration of the di-myo-inositol-phosphate pathway in the piezo-hyperthermophilic archaeon Thermococcus barophilus.

    Science.gov (United States)

    Cario, Anaïs; Mizgier, Alex; Thiel, Axel; Jebbar, Mohamed; Oger, Phil M

    2015-11-01

    Most Thermococcales accumulate di-myo-inositol-phosphate (DIP) as an organic solute as a response to heat stress. We have studied the accumulation of this osmolyte in the high-hydrostatic pressure adapted hyperthermophile Thermococcus barophilus. We found no accumulation of DIP under any of the stress conditions tested, although this archaeon harbors the 3 DIP synthesis genes. Lack of synthesis is due to the lack of expression of TERMP_01135 coding for the second step of DIP synthesis. In contrast to other species, the T. barophilus synthesis operon is interrupted by a four gene locus, in reverse orientation. Restoring an operon like structure at the DIP locus restored DIP synthesis, but did not have an impact on growth characteristics, suggesting that other mechanisms have evolved in this organism to cope with heat stress.

  14. Purification and biochemical properties of a cytochrome bc complex from the aerobic hyperthermophilic archaeon Aeropyrum pernix

    OpenAIRE

    Kabashima Yoshiki; Sakamoto Junshi

    2011-01-01

    Abstract Background The bioenergetics of Archaea with respect to the evolution of electron transfer systems is very interesting. In contrast to terminal oxidases, a canonical bc1 complex has not yet been isolated from Archaea. In particular, c-type cytochromes have been reported only for a limited number of species. Results Here, we isolated a c-type cytochrome-containing enzyme complex from the membranes of the hyperthermophilic archaeon, Aeropyrum pernix, grown aerobically. The redox spectr...

  15. Efficient CRISPR-Mediated Post-Transcriptional Gene Silencing in a Hyperthermophilic Archaeon Using Multiplexed crRNA Expression

    Directory of Open Access Journals (Sweden)

    Ziga Zebec

    2016-10-01

    Full Text Available CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats-mediated RNA degradation is catalyzed by a type III system in the hyperthermophilic archaeon Sulfolobus solfataricus. Earlier work demonstrated that the system can be engineered to target specifically mRNA of an endogenous host reporter gene, namely the β-galactosidase in S. solfataricus. Here, we investigated the effect of single and multiple spacers targeting the mRNA of a second reporter gene, α-amylase, at the same, and at different, locations respectively, using a minimal CRISPR (miniCR locus supplied on a viral shuttle vector. The use of increasing numbers of spacers reduced mRNA levels at progressively higher levels, with three crRNAs (CRISPR RNAs leading to ∼ 70–80% reduction, and five spacers resulting in an α-amylase gene knockdown of > 90% measured on both mRNA and protein activity levels. Our results indicate that this technology can be used to increase or modulate gene knockdown for efficient post-transcriptional gene silencing in hyperthermophilic archaea, and potentially also in other organisms.

  16. Disruption of the Operon Encoding Ehb Hydrogenase Limits AnabolicCO2 Assimilation in the Archaeon Methanococcus maripaludis

    Energy Technology Data Exchange (ETDEWEB)

    Porat, Iris; Kim, Wonduck; Hendrickson, Erik L.; Xia, Qiangwei; Zhang, Yi; Wang, Tiansong; Taub, Fred; Moore, Brian C.; Anderson, IainJ.; Hackett, Murray; Leigh, John A.; Whitman, William B.

    2006-02-01

    Methanococcus maripaludis is a mesophilic archaeon thatreduces CO2 to methane with H2 or formate as an energy source. Itcontains two membrane-bound energy-conserving hydrogenases, Eha and Ehb.To determine therole of Ehb, a deletion in the ehb operon wasconstructed to yield the mutant, strain S40. Growth of S40 was severelyimpaired in minimal medium. Both acetate and yeast extract were necessaryto restore growth to nearly wild-type levels, suggesting that Ehb wasinvolved in multiple steps in carbon assimilation. However, nodifferences in the total hydrogenase specific activities were foundbetween the wild type and mutant in either cell extracts ormembrane-purified fractions. Methanogenesis by resting cells withpyruvate as the electron donor was also reduced by 30 percent in S40,suggesting a defect in pyruvate oxidation. CO dehydrogenase/acetylcoenzyme A (CoA) synthase and pyruvate oxidoreductase had higher specificactivities in the mutant, and genes encoding these enzymes, as well asAMP-forming acetyl-CoA synthetase, were expressed at increased levels.These observations support a role for Ehb in anabolic CO2 assimilation inmethanococci.

  17. Unusual starch degradation pathway via cyclodextrins in the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324.

    Science.gov (United States)

    Labes, Antje; Schönheit, Peter

    2007-12-01

    The hyperthermophilic archaeon Archaeoglobus fulgidus strain 7324 has been shown to grow on starch and sulfate and thus represents the first sulfate reducer able to degrade polymeric sugars. The enzymes involved in starch degradation to glucose 6-phosphate were studied. In extracts of starch-grown cells the activities of the classical starch degradation enzymes, alpha-amylase and amylopullulanase, could not be detected. Instead, evidence is presented here that A. fulgidus utilizes an unusual pathway of starch degradation involving cyclodextrins as intermediates. The pathway comprises the combined action of an extracellular cyclodextrin glucanotransferase (CGTase) converting starch to cyclodextrins and the intracellular conversion of cyclodextrins to glucose 6-phosphate via cyclodextrinase (CDase), maltodextrin phosphorylase (Mal-P), and phosphoglucomutase (PGM). These enzymes, which are all induced after growth on starch, were characterized. CGTase catalyzed the conversion of starch to mainly beta-cyclodextrin. The gene encoding CGTase was cloned and sequenced and showed highest similarity to a glucanotransferase from Thermococcus litoralis. After transport of the cyclodextrins into the cell by a transport system to be defined, these molecules are linearized via a CDase, catalyzing exclusively the ring opening of the cyclodextrins to the respective maltooligodextrins. These are degraded by a Mal-P to glucose 1-phosphate. Finally, PGM catalyzes the conversion of glucose 1-phosphate to glucose 6-phosphate, which is further degraded to pyruvate via the modified Embden-Meyerhof pathway.

  18. A predicted geranylgeranyl reductase reduces the ω-position isoprene of dolichol phosphate in the halophilic archaeon, Haloferax volcanii.

    Science.gov (United States)

    Naparstek, Shai; Guan, Ziqiang; Eichler, Jerry

    2012-06-01

    In N-glycosylation in both Eukarya and Archaea, N-linked oligosaccharides are assembled on dolichol phosphate prior to transfer of the glycan to the protein target. However, whereas only the α-position isoprene subunit is saturated in eukaryal dolichol phosphate, both the α- and ω-position isoprene subunits are reduced in the archaeal lipid. The agents responsible for dolichol phosphate saturation remain largely unknown. The present study sought to identify dolichol phosphate reductases in the halophilic archaeon, Haloferax volcanii. Homology-based searches recognize HVO_1799 as a geranylgeranyl reductase. Mass spectrometry revealed that cells deleted of HVO_1799 fail to fully reduce the isoprene chains of H. volcanii membrane phospholipids and glycolipids. Likewise, the absence of HVO_1799 led to a loss of saturation of the ω-position isoprene subunit of C(55) and C(60) dolichol phosphate, with the effect of HVO_1799 deletion being more pronounced with C(60) dolichol phosphate than with C(55) dolichol phosphate. Glycosylation of dolichol phosphate in the deletion strain occurred preferentially on that version of the lipid saturated at both the α- and ω-position isoprene subunits.

  19. 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.

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

    Science.gov (United States)

    Robbins, L.L.; Van Cleave, K. A.; Ostrom, P.

    2008-01-01

    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.

  1. 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.

  2. Nucleic acid binding properties of a helix stabilising nucleoid protein from the thermoacidophilic archaeon Sulfolobus acidocaldarius that condenses DNA into compact structures.

    Science.gov (United States)

    Celestina, F; Suryanarayana, T

    1995-12-01

    Helix stabilising nucleoid protein (HSNP-C') from an acidothermophilic archaeon Sulfolobus acidocaldarius has been characterised with respect to interaction with nucleic acids by gel retardation assay, binding to nucleic acid columns, fluorescence titrations and electron microscopy. The protein exists in solution as very large multimeric aggregates as indicated by cross-linking studies. The protein binds strongly and co-operatively to double stranded DNA. Electron microscopy of the complexes of the protein with DNA shows compact structures suggesting that the protein condenses DNA.

  3. 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)).

  4. Identification of key components in the energy metabolism of the hyperthermophilic sulfate reducing archaeon Archaeoglobus fulgidus by transcriptome analyses

    Directory of Open Access Journals (Sweden)

    William Peter eHocking

    2014-03-01

    Full Text Available Energy conservation by the pathway of dissimilatory sulfate reduction is present in a diverse group of prokaryotes, but is most comprehensively studied in Deltaproteobacteria. Herein, whole-genome microarray analyses where used to provide a model of the energy me-tabolism of the sulfate reducing archaeon Archaeoglobus fulgidus, based comparative analysis litoautotrophic growth with H2/CO2 and thiosulfate, and heterotrophic growth on lactate with sulfate or thiosulfate. Only 72 genes were expressed differentially between the cultures utiliz-ing sulfate or thiosulfate whereas 269 genes were affected by a shift in energy source. We identified co-located gene cluster encoding putative lactate dehydrogenases (lldD, dld, lldEFG, also present in sulfate reducing bacteria. These enzymes may take part in energy conservation in A. fulgidus by specifically linking lactate oxidation with APS reduction via the Qmo complex. High transcriptional levels of Fqo confirm an important role of F420H2 and menaquinone mediated electron transport chain during heterotrophic growth. A putative pe-riplasmic thiosulfate reductase was identified by specific up-regulation. Also, putative genes for transport of sulfate and sulfite are discussed. We present a model for hydrogen metabo-lism, based on the probable bifurcation reaction of the Mvh:Hdl hydrogenase, that may inhibit the utilization of Fdred for energy conservation. Rather, energy conservation is probably facili-tated via menaquinone to multiple membrane bound heterodisulfide reductase complexes and the enzyme DsrC – linking periplasmic hydrogenase (Vht to the cytoplasmic reduction of sulfite. The ambiguous roles of genes corresponding to fatty acid metabolism induced during growth with H2 are discussed. Putative co-assimilation of organic acids is favored over a homologues secondary carbon fixation pathway, although both mechanisms may contribute to conserve the amount of Fdred needed during autotrophic growth

  5. Essential and non-essential DNA replication genes in the model halophilic Archaeon, Halobacterium sp. NRC-1

    Directory of Open Access Journals (Sweden)

    DasSarma Shiladitya

    2007-06-01

    Full Text Available Abstract Background Information transfer systems in Archaea, including many components of the DNA replication machinery, are similar to those found in eukaryotes. Functional assignments of archaeal DNA replication genes have been primarily based upon sequence homology and biochemical studies of replisome components, but few genetic studies have been conducted thus far. We have developed a tractable genetic system for knockout analysis of genes in the model halophilic archaeon, Halobacterium sp. NRC-1, and used it to determine which DNA replication genes are essential. Results Using a directed in-frame gene knockout method in Halobacterium sp. NRC-1, we examined nineteen genes predicted to be involved in DNA replication. Preliminary bioinformatic analysis of the large haloarchaeal Orc/Cdc6 family, related to eukaryotic Orc1 and Cdc6, showed five distinct clades of Orc/Cdc6 proteins conserved in all sequenced haloarchaea. Of ten orc/cdc6 genes in Halobacterium sp. NRC-1, only two were found to be essential, orc10, on the large chromosome, and orc2, on the minichromosome, pNRC200. Of the three replicative-type DNA polymerase genes, two were essential: the chromosomally encoded B family, polB1, and the chromosomally encoded euryarchaeal-specific D family, polD1/D2 (formerly called polA1/polA2 in the Halobacterium sp. NRC-1 genome sequence. The pNRC200-encoded B family polymerase, polB2, was non-essential. Accessory genes for DNA replication initiation and elongation factors, including the putative replicative helicase, mcm, the eukaryotic-type DNA primase, pri1/pri2, the DNA polymerase sliding clamp, pcn, and the flap endonuclease, rad2, were all essential. Targeted genes were classified as non-essential if knockouts were obtained and essential based on statistical analysis and/or by demonstrating the inability to isolate chromosomal knockouts except in the presence of a complementing plasmid copy of the gene. Conclusion The results showed that ten

  6. The structure and evolution of the ribosomal proteins encoded in the spc operon of the archaeon (Crenarchaeota) Sulfolobus acidocaldarius.

    Science.gov (United States)

    Yang, D; Kusser, I; Köpke, A K; Koop, B F; Matheson, A T

    1999-07-01

    The genes for nine ribosomal proteins, L24, L5, S14, S8, L6, L18, S5, L30, and L15, have been isolated and sequenced from the spc operon in the archaeon (Crenarchaeota) Sulfolobus acidocaldarius, and the putative amino acid sequence of the proteins coded by these genes has been determined. In addition, three other genes in the spc operon, coding for ribosomal proteins S4E, L32E, and L19E (equivalent to rat ribosomal proteins S4, L32, and L19), were sequenced and the structure of the putative proteins was determined. The order of the ribosomal protein genes in the spc operon of the Crenarchaeota kingdom of Archaea is identical to that present in the Euryarchaeota kingdom of Archaea and also identical to that found in bacteria, except for the genes for r-proteins S4E, L32E, and L19E, which are absent in bacteria. Although AUG is the initiation codon in most of the spc genes, GUG (val) and UUG (leu) are also used as initiation codons in S. acidocaldarius. Over 70% of the codons in the Sulfolobus spc operon have A or U in the third position, reflecting the low GC content of Sulfolobus DNA. Phylogenetic analysis indicated that the archaeal r-proteins are a sister group of their eucaryotic counterparts but did not resolve the question of whether the Archaea is monophyletic, as suggested by the L6P, L15P, and L18P trees, or the question of whether the Crenarchaeota is separate from the Euryarchaeota and closer to the Eucarya, as suggested by the S8P, S5P, and L24P trees. In the case of the three Sulfolobus r-proteins that do not have a counterpart in the bacterial ribosome (S4E, L32E, and L19E), the archaeal r-proteins showed substantial identity to their eucaryotic equivalents, but in all cases the archaeal proteins formed a separate group from the eucaryotic proteins.

  7. 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

  8. Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by D-amino acids.

    Science.gov (United States)

    Kawakami, Ryushi; Ohmori, Taketo; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2015-08-01

    To date, there have been few reports analyzing the amino acid requirement for growth of hyperthermophilic archaea. We here found that the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 requires Thr, Leu, Val, Phe, Tyr, Trp, His and Arg in the medium for growth, and shows slow growth in medium lacking Met or Ile. This largely corresponds to the presence, or absence, of genes related to amino acid biosynthesis in its genome, though there are exceptions. The amino acid requirements were dramatically lost by addition of D-isomers of Met, Leu, Val, allo-Ile, Phe, Tyr, Trp and Arg. Tracer analysis using (14)C-labeled D-Trp showed that D-Trp in the medium was used as a protein component in the cells, suggesting the presence of D-amino acid metabolic enzymes. Pyridoxal 5'-phosphate (PLP)-dependent racemase activity toward Met, Leu and Phe was detected in crude extract of P. horikoshii and was enhanced in cells grown in the medium supplemented with D-amino acids, especially D-allo-Ile. The gene encoding the racemase was narrowed down to one open reading frame on the basis of enzyme purification from P. horikoshii cells, and the recombinant enzyme exhibited PLP-dependent racemase activity toward several amino acids, including Met, Leu and Phe, but not Pro, Asp or Glu. This is the first report showing the presence in a hyperthermophilic archaeon of a PLP-dependent amino acid racemase with broad substrate specificity that is likely responsible for utilization of D-amino acids for growth.

  9. DNA polymerases BI and D from the hyperthermophilic archaeon Pyrococcus furiosus both bind to proliferating cell nuclear antigen with their C-terminal PIP-box motifs.

    Science.gov (United States)

    Tori, Kazuo; Kimizu, Megumi; Ishino, Sonoko; Ishino, Yoshizumi

    2007-08-01

    Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3'-5' exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex.

  10. Manual annotation, transcriptional analysis, and protein expression studies reveal novel genes in the agl cluster responsible for N glycosylation in the halophilic archaeon Haloferax volcanii.

    Science.gov (United States)

    Yurist-Doutsch, Sophie; Eichler, Jerry

    2009-05-01

    While Eukarya, Bacteria, and Archaea are all capable of protein N glycosylation, the archaeal version of this posttranslational modification is the least understood. To redress this imbalance, recent studies of the halophilic archaeon Haloferax volcanii have identified a gene cluster encoding the Agl proteins involved in the assembly and attachment of a pentasaccharide to select Asn residues of the surface layer glycoprotein in this species. However, because the automated tools used for rapid annotation of genome sequences, including that of H. volcanii, are not always accurate, a reannotation of the agl cluster was undertaken in order to discover genes not previously recognized. In the present report, reanalysis of the gene cluster that includes aglB, aglE, aglF, aglG, aglI, and aglJ, which are known components of the H. volcanii protein N-glycosylation machinery, was undertaken. Using computer-based tools or visual inspection, together with transcriptional analysis and protein expression approaches, genes encoding AglP, AglQ, and AglR are now described.

  11. Genome sequence of Methanobacterium congolense strain Buetzberg, a hydrogenotrophic, methanogenic archaeon, isolated from a mesophilic industrial-scale biogas plant utilizing bio-waste.

    Science.gov (United States)

    Tejerizo, Gonzalo Torres; Kim, Yong Sung; Maus, Irena; Wibberg, Daniel; Winkler, Anika; Off, Sandra; Pühler, Alfred; Scherer, Paul; Schlüter, Andreas

    2017-02-16

    Methanogenic Archaea are of importance at the end of the anaerobic digestion (AD) chain for biomass conversion. They finally produce methane, the end-product of AD. Among this group of microorganisms, members of the genus Methanobacterium are ubiquitously present in anaerobic habitats, such as bioreactors. The genome of a novel methanogenic archaeon, namely Methanobacterium congolense Buetzberg, originally isolated from a mesophilic biogas plant, was completely sequenced to analyze putative adaptive genome features conferring competitiveness of this isolate within the biogas reactor environment. Sequencing and assembly of the M. congolense Buetzberg genome yielded a chromosome with a size of 2,451,457bp and a mean GC-content of 38.51%. Additionally, a plasmid with a size of 18,118bp, featuring a GC content of 36.05% was identified. The M. congolense Buetzberg plasmid showed no sequence similarities with the plasmids described previously suggesting that it represents a new plasmid type. Analysis of the M. congolense Buetzberg chromosome architecture revealed a high collinearity with the Methanobacterium paludis chromosome. Furthermore, annotation of the genome and functional predictions disclosed several genes involved in cell wall and membrane biogenesis. Compilation of specific genes among Methanobacterium strains originating from AD environments revealed 474 genetic determinants that could be crucial for adaptation of these strains to specific conditions prevailing in AD habitats.

  12. The RosR transcription factor is required for gene expression dynamics in response to extreme oxidative stress in a hypersaline-adapted archaeon

    Directory of Open Access Journals (Sweden)

    Sharma Kriti

    2012-07-01

    Full Text Available Abstract Background Previous work has shown that the hypersaline-adapted archaeon, Halobacterium salinarum NRC-1, is highly resistant to oxidative stress caused by exposure to hydrogen peroxide, UV, and gamma radiation. Dynamic alteration of the gene regulatory network (GRN has been implicated in such resistance. However, the molecular functions of transcription regulatory proteins involved in this response remain unknown. Results Here we have reanalyzed several existing GRN and systems biology datasets for H. salinarum to identify and characterize a novel winged helix-turn-helix transcription factor, VNG0258H, as a regulator required for reactive oxygen species resistance in this organism. This protein appears to be unique to the haloarchaea at the primary sequence level. High throughput quantitative growth assays in a deletion mutant strain implicate VNG0258H in extreme oxidative stress resistance. According to time course gene expression analyses, this transcription factor is required for the appropriate dynamic response of nearly 300 genes to reactive oxygen species damage from paraquat and hydrogen peroxide. These genes are predicted to function in repair of oxidative damage to proteins and DNA. In vivo DNA binding assays demonstrate that VNG0258H binds DNA to mediate gene regulation. Conclusions Together these results suggest that VNG0258H is a novel archaeal transcription factor that regulates gene expression to enable adaptation to the extremely oxidative, hypersaline niche of H. salinarum. We have therefore renamed VNG0258H as RosR, for reactive oxygen species regulator.

  13. Genomic Analysis of the Extremely Halophilic Archaeon Halobacterium noricense CBA1132 Isolated from Solar Salt That Is an Essential Material for Fermented Foods.

    Science.gov (United States)

    Lim, Seul Ki; Kim, Joon Yong; Song, Hye Seon; Kwon, Min-Sung; Lee, Jieun; Oh, Young Jun; Nam, Young-Do; Seo, Myung-Ji; Lee, Dong-Gi; Choi, Jong-Soon; Yoon, Changmann; Sohn, Eunju; Rahman, Md Arif-Ur; Roh, Seong Woon; Choi, Hak-Jong

    2016-08-28

    The extremely halophilic archaeon Halobacterium noricense is a member of the genus Halobacterium. Strain CBA1132 (= KCCM 43183, JCM 31150) was isolated from solar salt. The genome of strain CBA1132 assembled with 4 contigs, including three rRNA genes, 44 tRNA genes, and 3,208 open reading frames. Strain CBA1132 had nine putative CRISPRs and the genome contained genes encoding metal resistance determinants: copper-translocating P-type ATPase (CtpA), arsenical pump-driving ATPase (ArsA), arsenate reductase (ArsC), and arsenical resistance operon repressor (ArsR). Strain CBA1132 was related to Halobacterium noricense, with 99.2% 16S rRNA gene sequence similarity. Based on the comparative genomic analysis, strain CBA1132 has distinctly evolved; moreover, essential genes related to nitrogen metabolism were only detected in the genome of strain CBA1132 among the reported genomes in the genus Halobacterium. This genome sequence of Halobacterium noricense CBA1132 may be of use in future molecular biological studies.

  14. Enhancing Heat Tolerance of the Little Dogwood Cornus canadensis L. f. with Introduction of a Superoxide Reductase Gene from the Hyperthermophilic Archaeon Pyrococcus furiosus.

    Science.gov (United States)

    Geng, Xing-Min; Liu, Xiang; Ji, Mikyoung; Hoffmann, William A; Grunden, Amy; Xiang, Qiu-Yun J

    2016-01-01

    Production of reactive oxygen species (ROS) can be accelerated under various biotic and abiotic stresses causing lipid peroxidation, protein degradation, enzyme inactivation, and DNA damage. Superoxide reductase (SOR) is a novel antioxidant enzyme from Pyrococcus furiosus and is employed by this anaerobic hyperthermophilic archaeon for efficient detoxification of ROS. In this study, SOR was introduced into a flowering plant Cornus canadensis to enhance its heat tolerance and reduce heat induced damage. A fusion construct of the SOR gene and Green Fluorescent Protein gene (GFP) was introduced into C. canadensis using Agrobacterium-mediated transformation. Heat tolerance of the GFP-SOR expressing transgenic plants was investigated by observing morphological symptoms of heat injury and by examining changes in photosynthesis, malondialdehyde (MDA), and proline levels in the plants. Our results indicate that the expression of the P. furiosus SOR gene in the transgenic plants alleviated lipid peroxidation of cell membranes and photoinhibition of PS II, and decreased the accumulation of proline at 40°C. After a series of exposures to increasing temperatures, the SOR transgenic plants remained healthy and green whereas most of the non-transgenic plants dried up and were unable to recover. While it had previously been reported that expression of SOR in Arabidopsis enhanced heat tolerance, this is the first report of the successful demonstration of improved heat tolerance in a non-model plant resulting from the introduction of P. furiosus SOR. The study demonstrates the potential of SOR for crop improvement and that inherent limitations of plant heat tolerance can be ameliorated with P. furiosus SOR.

  15. Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius has a unique Asn-linked highly branched hexasaccharide chain containing 6-sulfoquinovose.

    Science.gov (United States)

    Zähringer, U; Moll, H; Hettmann, T; Knirel, Y A; Schäfer, G

    2000-07-01

    Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639) has been described as a novel highly glycosylated membrane-bound b-type hemoprotein [Hettmann, T., Schmidt, C. L., Anemüller, S., Zähringer, U., Moll, H., Petersen, A. & Schäfer, G. (1998) J. Biol. Chem. 273, 12032-12040]. The purified cytochrome b558/566 was characterized by MALDI MS as a 64-kDa (glyco)protein expressing 17% glycosylation. Detailed chemical studies showed that it was exclusively O-mannosylated with monosaccharides and N-glycosylated with at least seven hexasaccharide units having the same unique structure. The hexasaccharide was released by cleavage with peptide:N-glycosidase (PNGase) F and found to consist of two residues each of Man and GlcNAc and one residue each of Glc and 6-deoxy-6-sulfoglucose (6-sulfoquinovose). The last sugar has been known as a component of glycolipids of plants and some prokaryotes, but has not been hitherto found in bacterial glycoproteins. Digestion with trypsin/pronase gave a mixture of glycopeptides with the same Asn-linked hexasaccharide chain, from which an N-glycosylated Tyr-Asn dipeptide was purified by gel chromatography and anion-exchange HPLC. Studies of the degradation products using methylation analysis, ESI MS, MALDI MS, and 1H and 13C NMR spectroscopy, including 1H,13C HMQC and NOESY experiments, established the structure of the unique Asn-linked hexasaccharide chain of cytochrome b558/566.

  16. Enhancing heat tolerance of the little dogwood Cornus canadensis L. f. with introduction of a superoxide reductase gene from the hyperthermophilic archaeon Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Xinmin eGeng

    2016-01-01

    Full Text Available Production of reactive oxygen species (ROS can be accelerated under various biotic and abiotic stresses causing lipid peroxidation, protein degradation, enzyme inactivation, and DNA damage. Superoxide reductase (SOR is a novel antioxidant enzyme from Pyrococcus furiosus and is employed by this anaerobic hyperthermophilic archaeon for efficient detoxification of ROS. In this study, SOR was introduced into a flowering plant Cornus canadensis to enhance its heat tolerance and reduce heat induced damage. A fusion construct of the SOR gene and Green Fluorescent Protein gene (GFP was introduced into C. canadensis using Agrobacterium-mediated transformation. Heat tolerance of the GFP-SOR expressing transgenic plants was investigated by observing morphological symptoms of heat injury and by examining changes in photosynthesis, malondialdehyde (MDA, and proline levels in the plants. Our results indicate that the expression of the P. furiosus SOR gene in the transgenic plants alleviated lipid peroxidation of cell membranes and photoinhibition of PS II, and decreased the accumulation of proline at 40°C. After a series of exposures to increasing temperatures, the SOR transgenic plants remained healthy and green whereas most of the non-transgenic plants dried up and were unable to recover. While it had previously been reported that expression of SOR in Arabidopsis enhanced heat tolerance, this is the first report of the successful demonstration of improved heat tolerance in a non-model plant resulting from the introduction of P. furiosus SOR. The study demonstrates the potential of SOR for crop improvement and that inherent limitations of plant heat tolerance can be ameliorated with P. furiosus SOR.

  17. MutS and MutL are dispensable for maintenance of the genomic mutation rate in the halophilic archaeon Halobacterium salinarum NRC-1.

    Directory of Open Access Journals (Sweden)

    Courtney R Busch

    Full Text Available BACKGROUND: The genome of the halophilic archaeon Halobacterium salinarum NRC-1 encodes for homologs of MutS and MutL, which are key proteins of a DNA mismatch repair pathway conserved in Bacteria and Eukarya. Mismatch repair is essential for retaining the fidelity of genetic information and defects in this pathway result in the deleterious accumulation of mutations and in hereditary diseases in humans. METHODOLOGY/PRINCIPAL FINDINGS: We calculated the spontaneous genomic mutation rate of H. salinarum NRC-1 using fluctuation tests targeting genes of the uracil monophosphate biosynthesis pathway. We found that H. salinarum NRC-1 has a low incidence of mutation suggesting the presence of active mechanisms to control spontaneous mutations during replication. The spectrum of mutational changes found in H. salinarum NRC-1, and in other archaea, appears to be unique to this domain of life and might be a consequence of their adaption to extreme environmental conditions. In-frame targeted gene deletions of H. salinarum NRC-1 mismatch repair genes and phenotypic characterization of the mutants demonstrated that the mutS and mutL genes are not required for maintenance of the observed mutation rate. CONCLUSIONS/SIGNIFICANCE: We established that H. salinarum NRC-1 mutS and mutL genes are redundant to an alternative system that limits spontaneous mutation in this organism. This finding leads to the puzzling question of what mechanism is responsible for maintenance of the low genomic mutation rates observed in the Archaea, which for the most part do not have MutS and MutL homologs.

  18. Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance.

    Science.gov (United States)

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

    2007-04-01

    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 (hmg(Tk)) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apu(Tk)) or a gene cluster which includes apu(Tk) and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 microM simvastatin were isolated. The transformants exhibited growth in the presence of 20 microM simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmg(Tk) locus when the endogenous hmg(Tk) gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmg(Pf)) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The Deltaapu(Tk) strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that Apu(Tk) is a major polysaccharide-degrading enzyme in T. kodakaraensis.

  19. Characterization of the TrmB-like protein, PF0124, a TGM-recognizing global transcriptional regulator of the hyperthermophilic archaeon Pyrococcus furiosus.

    Science.gov (United States)

    Lee, Sung-Jae; Surma, Melanie; Seitz, Sabine; Hausner, Winfried; Thomm, Michael; Boos, Winfried

    2007-07-01

    The characterization of the transcriptional regulator TrmBL1 of the hyperthermophilic archaeon Pyrococcus furiosus, homologous to TrmB (transcriptional regulator of the maltose system), was studied. The genome of P. furiosus contains three TrmB paralogues. One of the TrmB-like proteins (TrmBL), PF0124 (TrmBL1), was analysed in more detail. It regulated the expression of the genes encoding enzymes of the glycolytic pathway as well as the maltodextrin (MD) ABC transporter. By molecular sieve chromatography, purified TrmBL1 behaved at ambient temperature as a tetramer of 148.8 kDa. In the presence of 1 mM maltotriose or 5 mM maltose TrmBL1 formed octamers. As shown by electrophoretic mobility shift assay (EMSA) TrmBL1 was found to bind the MD (maltodextrin ABC transport genes) promoter DNA with sixfold higher binding affinity (K(d) 0.2 microM) than to the trehalose/maltose ABC transporter (TM) promoter (K(d) 1.2 microM). Maltotriose and maltose interfered in these assays indicating inducer function. In vitro transcription assays using purified transcription components corroborated the data obtained with EMSA and showed inhibition of transcription of the MD promoter by TrmBL1. Recently, van de Werken et al. (FEMS Microbiol Lett 2006; 260: 69-76) identified TGM, a conserved sequence (Thermococcales-Glycolytic-Motif) upstream of genes encoding glycolytic enzymes and the MD ABC transporter. The position of TGM is invariably located downstream of the BRE-TATA box and overlapping the transcription start site on each promoter. By footprint analysis TrmBL1 was found to recognize the TGM sequence in several TGM-containing promoter sequences. We identified the recognition helix in TrmBL1 revealing tyrosine (Y49) to be essential for target DNA binding. However, the TGM motif was not essential for TrmBL1 binding. We conclude that TrmBL1 is a global sugar-sensing transcriptional regulator controlling the genes of transport systems and of sugar-metabolizing enzymes.

  20. Evidence of active methanogen communities in shallow sediments of the sonora margin cold seeps.

    Science.gov (United States)

    Vigneron, Adrien; L'Haridon, Stéphane; Godfroy, Anne; Roussel, Erwan G; Cragg, Barry A; Parkes, R John; Toffin, Laurent

    2015-05-15

    In the Sonora Margin cold seep ecosystems (Gulf of California), sediments underlying microbial mats harbor high biogenic methane concentrations, fueling various microbial communities, such as abundant lineages of anaerobic methanotrophs (ANME). However, the biodiversity, distribution, and metabolism of the microorganisms producing this methane remain poorly understood. In this study, measurements of methanogenesis using radiolabeled dimethylamine, bicarbonate, and acetate showed that biogenic methane production in these sediments was mainly dominated by methylotrophic methanogenesis, while the proportion of autotrophic methanogenesis increased with depth. Congruently, methane production and methanogenic Archaea were detected in culture enrichments amended with trimethylamine and bicarbonate. Analyses of denaturing gradient gel electrophoresis (DGGE) fingerprinting and reverse-transcribed PCR-amplified 16S rRNA sequences retrieved from these enrichments revealed the presence of active methylotrophic Methanococcoides burtonii relatives and several new autotrophic Methanogenium lineages, confirming the cooccurrence of Methanosarcinales and Methanomicrobiales methanogens with abundant ANME populations in the sediments of the Sonora Margin cold seeps.

  1. Sugar utilization in the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324: starch degradation to acetate and CO2 via a modified Embden-Meyerhof pathway and acetyl-CoA synthetase (ADP-forming).

    Science.gov (United States)

    Labes, A; Schönheit, P

    2001-11-01

    The hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324, rather than the type strain VC16, was found to grow on starch and sulfate as energy and carbon source. Fermentation products and enzyme activities were determined in starch-grown cells and compared to those of cells grown on lactate and sulfate. During exponential growth on starch, 1 mol of glucose-equivalent was incompletely oxidized with sulfate to approximately 2 mol acetate, 2 mol CO2 and 1 mol H2S. Starch-grown cells did not contain measurable amounts of the deazaflavin factor F420 (reducer A. fulgidus strain 7324 converts starch to acetate via a modified Embden-Meyerhof pathway and acetyl-CoA synthetase (ADP-forming). This is the first report of growth of a sulfate reducer on starch, i.e. on a polymeric sugar.

  2. Mechanisms of thermal adaptation revealed from the genomes of the Antarctic

    Energy Technology Data Exchange (ETDEWEB)

    Saunders, Neil F.W.; Thomas, Torsten; Curmi, Paul M.G.; Mattick, John S.; Kuczek, Elizabeth; Slade, Rob; Davis, John; Franzmann, Peter; Boone, David; Rusterholtz, Karl; Feldman, Robert; Gates, Chris; Bench, Shellie; Sowers, Kevin; Kadner, Kristen; Aerts, Andrea; Dehal, Paramvir; Detter, Chris; Glavina, Tijana; Lucas, Susan; Richardson, Paul; Larimer, Frank; Hauser , Frank; Hauser, Loren; Land, Miriam; Cavicchioli, Richard

    2003-03-01

    We generated draft genome sequences for two cold-adapted Archaea, Methanogenium frigidum and Methanococcoides burtonii, to identify genotypic characteristics that distinguish them from Archaea with a higher optimal growth temperature (OGT). Comparative genomics revealed trends in amino acid and tRNA composition, and structural features of proteins. Proteins from the cold-adapted Archaea are characterized by a higher content of non-charged polar amino acids, particularly Gln and Thr and a lower content of hydrophobic amino acids, particularly Leu. Sequence data from nine methanogen genomes (OGT 15-98 C) was used to generate 1 111 modeled protein structures. Analysis of the models from the cold-adapted Archaea showed a strong tendency in the solvent accessible area for more Gln, Thr an hydrophobic residues and fewer charged residues. A cold shock domain (CSD) protein (CspA homolog) was identified in M. frigidum, two hypothetical proteins with CSD-folds in M. burtonii, and a unique winged helix DNA-binding domain protein in M. burtonii. This suggests that these types of nucleic acid binding proteins have a critical role in cold-adapted Archaea. Structural analysis of tRNA sequences from the Archaea indicated that GC content is the major factor influencing tRNA stability in hyperthermophiles, but not in the psychrophiles, mesophiles or moderate thermophiles. Below an OGT of 60 C, the GC content in tRNA was largely unchanged, indicating that any requirement for flexibility of tRNA in psychrophiles is mediated by other means. This is the first time that comparisons have been performed with genome data from Archaea spanning the growth temperature extremes from psychrophiles to hyperthermophiles.

  3. 新疆罗布泊地区可培养嗜盐古菌多样性及其功能酶筛选%Biodiversity and functional enzymes of cultured halophilic archaeon in Lop Nur region

    Institute of Scientific and Technical Information of China (English)

    刘冰冰; 唐蜀昆; 明红; 何松涛; 聂国兴; 关统伟; 张利莉; 李文均

    2011-01-01

    [目的]探索新疆罗布泊地区高盐环境可培养嗜盐古菌的多样性及其功能酶应用潜力.[方法]采集罗布泊地区13份土样,用纯培养并结合基于16S rRNA基因系统发育分析的方法来研究样品中嗜盐古菌的多样性.按系统进化树的聚类关系,挑选出一些菌株进行盐度耐受及淀粉酶、蛋白酶、酯酶的酶活检测.[结果]从13份土样中共分离到56株嗜盐古菌,经16S rRNA基因克隆测序,通过MEGA 4.0构建N-J树分析,56株菌分布于嗜盐古菌的10个生效发表属和5个潜在新属.运用Shannon-Wiener方法计算其多样性指数为1.820.挑选17株嗜盐古菌所测试盐浓度实验结果表明这一批嗜盐古菌的大部分生长范围在10% -35%之间,最适盐浓度在20% -25%之间.不同酶活检测结果为:淀粉酶酶活率为70.6%,蛋白酶酶活率为35.3%,酯酶酶活率为82.4%.[结论]新疆罗布泊周边地区由于气候及地理位置的独特性,蕴藏丰富的嗜盐古菌资源.本实验所设计的分离方法对嗜盐古菌的分离是极其有效的,为进一步研究新疆罗布泊及周边地区嗜盐古菌资源提供了技术基础.盐度耐受实验结果验证在低盐环境中分离嗜盐古菌新物种的可行性.同时,嗜盐古菌的酶活比率较高且活性较强为进一步开发利用嗜盐古菌资源提供了理论依据.%[ Objective ] In order to explore the diversity of cultured halophilic archaeon from hypersaline environments in Lop Nur region and their potential application. [Methods] Total 13 soil samples were collected from Lop Nur regions. Halophilic archaea strains were isolated and identified by 16S rRNA gene sequence analysis. In addition, 17 strains were selected based on different branches in pylogenetic tree, and their salt concentration tolerance and amylase, protease, esterase activities were further detected by conventional methods. [ Results ] The 16S rRNA gene sequences of 56 selected strains were

  4. 连四硫酸盐存在下利用嗜酸热古菌Acidianus copahuensis 提高锌回收率%Improving zinc recovery by thermoacidophilic archaeon Acidianus copahuensis using tetrathionate

    Institute of Scientific and Technical Information of China (English)

    Camila CASTRO; Edgardo R. DONATI

    2016-01-01

    The attachment and bioleaching experiments were conducted to evaluate the zinc recovery from Hualilan ore by the thermoacidophilic archaeon Acidianus copahuensis. Cells of this species pregrown on tetrathionate showed higher capability of attachment to the ore than cells pregrown on other energy sources and such attachment seemed to be mediated by the product of extracellular polymeric substances. A. copahuensis achieved a successful bioleaching of the ore reaching 100% of zinc recovery when tetrathionate was added. Simultaneous addition of yeast extract and tetrathionate maintained the zinc extraction at higher rate. Zinc dissolution kinetics was controlled by chemical reaction in cultures with the external addition of tetrathionate but by the diffusion through a product layer of jarosite in the other cultures.%通过吸附和生物浸出实验考察利用嗜酸热古菌 Acidianus copahuensis 从 Hualian 矿中回收锌。经过在连四硫酸盐表面预处理的菌种具有比经其他能量供给剂预处理的菌种更强的矿物吸附能力,且此吸附能力可由所产生的体外聚合物调节。当加入连四硫酸盐时,用 A. copahuensis 生物浸取 Hualian 矿中的锌,其浸出率达100%。同时添加酵母和连四硫酸盐不仅能保持较高的锌浸出率,而且能加快浸出速率。添加连四硫酸盐后,培养基中锌的溶解动力学受化学反应控制;而在未添加连四硫酸盐培养基中,锌的溶解动力学受经过黄钾铁矾反应层的扩散控制。

  5. Evidence of Molecular Adaptation to Extreme Environments and Applicability to Space Environments

    Directory of Open Access Journals (Sweden)

    Filipović, M. D.

    2008-06-01

    Full Text Available This is initial investigation of gene signatures responsible for adapting microscopic life to the extreme Earth environments. We present preliminary results on identification of the clusters of orthologous groups (COGs common to several hyperthermophiles and exclusion of those common to a mesophile (non-hyperthermophile: {it Escherichia coli (E. coli K12}, will yield a group of proteins possibly involved in adaptation to life under extreme temperatures. Comparative genome analyses represent a powerful tool in discovery of novel genes responsible for adaptation to specific extreme environments. Methanogens stand out as the only group of organisms that have species capable of growth at 0D C ({it Metarhizium frigidum (M.~frigidum} and {it Methanococcoides burtonii (M.~burtonii} and 110D C ({it Methanopyrus kandleri (M.~kandleri}. Although not all the components of heat adaptation can be attributed to novel genes, the {it chaperones} known as heat shock proteins stabilize the enzymes under elevated temperature. However, highly conserved {it chaperons} found in bacteria and eukaryots are not present in hyperthermophilic Archea, rather, they have a unique {it chaperone TF55}. Our aim was to use software which we specifically developed for extremophile genome comparative analyses in order to search for additional novel genes involved in hyperthermophile adaptation. The followinghyperthermophile genomes incorporated in this software were used forthese studies: {it Methanocaldococcus jannaschii (M.~jannaschii, M.~kandleri, Archaeoglobus fulgidus (A.~fulgidus} and threespecies of {it Pyrococcus}. Common genes were annotated and groupedaccording to their roles in cellular processes where such informationwas available and proteins not previously implicated in theheat-adaptation of hyperthermophiles were identified. Additionalexperimental data are needed in order to learn more about theseproteins. To address non-gene based components of thermaladaptation

  6. Evidence of molecular adaptation to extreme environments and applicability to space environments

    Directory of Open Access Journals (Sweden)

    Filipović M.

    2008-01-01

    Full Text Available This is initial investigation of gene signatures responsible for adapting microscopic life to the extreme Earth environments. We present preliminary results on identification of the clusters of orthologous groups (COGs common to several hyperthermophiles and exclusion of those common to a mesophile (non-hyperthermophile: Escherichia coli (E. coli K12, will yield a group of proteins possibly involved in adaptation to life under extreme temperatures. Comparative genome analyses represent a powerful tool in discovery of novel genes responsible for adaptation to specific extreme environments. Methanogens stand out as the only group of organisms that have species capable of growth at 0ºC (Metarhizium frigidum (M. frigidum and Methanococcoides burtonii (M. burtonii and 110ºC (Methanopyrus kandleri (M. kandleri. Although not all the components of heat adaptation can be attributed to novel genes, the chaperones known as heat shock proteins stabilize the enzymes under elevated temperature. However, highly conserved chaperons found in bacteria and eukaryots are not present in hyperthermophilic Archea, rather, they have a unique chaperone TF55. Our aim was to use software which we specifically developed for extremophile genome comparative analyses in order to search for additional novel genes involved in hyperthermophile adaptation. The following hyperthermophile genomes incorporated in this software were used for these studies: Methanocaldococcus jannaschii (M. jannaschii, M. kandleri, Archaeoglobus fulgidus (A. fulgidus and three species of Pyrococcus. Common genes were annotated and grouped according to their roles in cellular processes where such information was available and proteins not previously implicated in the heat-adaptation of hyperthermophiles were identified. Additional experimental data are needed in order to learn more about these proteins. To address non-gene based components of thermal adaptation, all sequenced extremophiles were

  7. Hydrogenases from the Hyperthermophilic Archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    van Haaster, D.J.

    2007-01-01

    Hydrogenase is an electron-transfer protein and catalyses the simplest chemical redox reaction, the reversible two-electron oxidation of molecular hydrogen in aerobic and anaerobic microorganisms. A kinetic study of the hydrogen oxidation reaction by Fe-hydrogenase from Desulfovibrio vulgaris (Hilde

  8. Isolation,Identification and Characterization of Extremely Halophilic C50 Carotenoid-Producing Archaeon%1株产C50类胡萝卜素极端嗜盐古菌的筛选鉴定及特性分析

    Institute of Scientific and Technical Information of China (English)

    刘良森; 邓元告; 隋丽英

    2014-01-01

    An extremely halophilic C50 carotenoid-producing red archaeon was isolated from the crystalli-zer ponds in solar saltworks.The isolated strain is Gram-negative and short rod.The optimum salinity and pH for growth is 250 and 7,respectively.Phenotypic and molecular analyses of this strain indicated that it belonged to extremely halophilic archaea genus Halorubrum and named Halorubrum Sp1 (16S rRNA Genbank registration number KF697239).UV-visible scanning spectrum showed that C50 carote-noid was the major pigments presented in this strain.Pigment accumulation was maximizing at pH 8. In the salinity range of 150~300,increasing salinity resulted in declined pigment accumulation.%从日晒盐场结晶池中筛选到1株产C50类胡萝卜素的红色极端嗜盐古菌。该菌株为革兰氏阴性菌,短棒状,最适生长盐度为250,最适生长pH 为7。表型鉴定方法结合16S rDNA序列分析判定,该菌属于极端嗜盐古菌盐红菌属 Halorubrum,命名为 Halorubrum sp.Sp1(16S rRNA Genbank 登录号为KF697239)。根据紫外-可见光扫描特征光谱,确定该菌株主要色素为 C50类胡萝卜素。pH 8时单位细胞色素积累量最大,在盐度150~300范围内随盐度升高,单位细胞色素积累量逐渐降低。

  9. Distribution, structure and diversity of “bacterial” genes encoding two-component proteins in the Euryarchaeota

    Directory of Open Access Journals (Sweden)

    Mark K. Ashby

    2006-01-01

    Full Text Available The publicly available annotated archaeal genome sequences (23 complete and three partial annotations, October 2005 were searched for the presence of potential two-component open reading frames (ORFs using gene category lists and BLASTP. A total of 489 potential two-component genes were identified from the gene category lists and BLASTP. Two-component genes were found in 14 of the 21 Euryarchaeal sequences (October 2005 and in neither the Crenarchaeota nor the Nanoarchaeota. A total of 20 predicted protein domains were identified in the putative two-component ORFs that, in addition to the histidine kinase and receiver domains, also includes sensor and signalling domains. The detailed structure of these putative proteins is shown, as is the distribution of each class of two-component genes in each species. Potential members of orthologous groups have been identified, as have any potential operons containing two or more two-component genes. The number of two-component genes in those Euryarchaeal species which have them seems to be linked more to lifestyle and habitat than to genome complexity, with most examples being found in Methanospirillum hungatei, Haloarcula marismortui, Methanococcoides burtonii and the mesophilic Methanosarcinales group. The large numbers of two-component genes in these species may reflect a greater requirement for internal regulation. Phylogenetic analysis of orthologous groups of five different protein classes, three probably involved in regulating taxis, suggests that most of these ORFs have been inherited vertically from an ancestral Euryarchaeal species and point to a limited number of key horizontal gene transfer events.

  10. "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

  11. Functional genomics of the thermo-acidophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Oost, van der J.; Walther, J.; Brouns, S.J.J.; Werken, van de H.J.G.; Snijders, A.P.L.; Wright, P.C.; Andersson, A.; Bernander, R.; Vos, de W.M.

    2006-01-01

    Archaea and bacteria that optimally grow at temperatures above 60C and 80C are referred to as thermophiles and hyperthermophiles, respectively (Stetter, 1996). Since their discovery in the late 1960s (Brock and Freeze, 1969), attempts were made to reveal the secrets of the thermal resistance of thes

  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. Chromosome replication dynamics in the archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Duggin, Iain G; McCallum, Simon A; Bell, Stephen D

    2008-10-28

    The "baby machine" provides a means of generating synchronized cultures of minimally perturbed cells. We describe the use of this technique to establish the key cell-cycle parameters of hyperthermophilic archaea of the genus Sulfolobus. The 3 DNA replication origins of Sulfolobus acidocaldarius were mapped by 2D gel analysis to near 0 (oriC2), 579 (oriC1), and 1,197 kb (oriC3) on the 2,226-kb circular genome, and we present a direct demonstration of their activity within the first few minutes of a synchronous cell cycle. We also detected X-shaped DNA molecules at the origins in log-phase cells, but these were not directly associated with replication initiation or ongoing chromosome replication in synchronized cells. Whole-genome marker frequency analyses of both synchronous and log-phase cultures showed that origin utilization was close to 100% for all 3 origins per round of replication. However, oriC2 was activated slightly later on average compared with oriC1 and oriC3. The DNA replication forks moved bidirectionally away from each origin at approximately 88 bp per second in synchronous culture. Analysis of the 3 Orc1/Cdc6 initiator proteins showed a uniformity of cellular abundance and origin binding throughout the cell cycle. In contrast, although levels of the MCM helicase were constant across the cell cycle, its origin localization was regulated, because it was strongly enriched at all 3 origins in early S phase.

  14. Flagellar motility and structure in the hyperthermoacidophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Szabo, Zalan; Sani, Musa; Groeneveld, Maarten; Zolghadr, Benham; Schelert, James; Albers, Sonja-Verena; Blum, Paul; Boekema, Egbert J.; Driessen, Arnold J. M.

    2007-01-01

    Flagellation in archaea is widespread and is involved in swimming motility. Here, we demonstrate that the structural flagellin gene from the crenarchaeaon Suffolobus soffiataricus is highly expressed in stationary-phase-grown cells and under unfavorable nutritional conditions. A mutant in a flagella

  15. Sulfolobus hakonensis sp. nov., a novel species of acidothermophilic archaeon.

    Science.gov (United States)

    Takayanagi, S; Kawasaki, H; Sugimori, K; Yamada, T; Sugai, A; Ito, T; Yamasato, K; Shioda, M

    1996-04-01

    We characterized a microbial strain that was isolated from a hot spring at a geothermal area in Hakone, Japan. This isolate, whose lobed-shaped cells were about 1.0 micron in diameter, was a facultative chemolitho-autotroph that required aerobic conditions for growth. The optimum pH was 3.0 (pH range, 1.0 to 4.0), and the optimum temperature was 70 degrees C (temperature range, 50 to 80 degrees C). Lithotrophically, this strain grew on elemental sulfur and reduced sulfur compounds. The G+C content of the genomic DNA was 38.4 mol%. This organism contained calditoglycerocaldarchaeol, which is characteristic of members of the Sulfolobaceae. The levels of 16S rRNA sequence similarity between the new isolate and Sulfolobus acidocaldarius, Sulfolobus solfataricus, and Sulfolobus shibatae were less than 89.8%. Unlike S. acidocaldarius, S. solfataricus, and S. shibatae, the new isolate utilized sugars and amino acids poorly as sole carbon sources, and the levels of DNA-DNA hybridization between the new isolate and these Sulfolobus species were very low. Phenotypically, the new isolate was also distinct from the obligately lithotrophic organism Sulfolobus metallicus. We concluded that the new organism belongs to a new Sulfolobus species, for which we propose the name Sulfolobus hakonensis.

  16. Phylogenomic analysis of proteins that are distinctive of Archaea and its main subgroups and the origin of methanogenesis

    Directory of Open Access Journals (Sweden)

    Gupta Radhey S

    2007-03-01

    Full Text Available Abstract Background The Archaea are highly diverse in terms of their physiology, metabolism and ecology. Presently, very few molecular characteristics are known that are uniquely shared by either all archaea or the different main groups within archaea. The evolutionary relationships among different groups within the Euryarchaeota branch are also not clearly understood. Results We have carried out comprehensive analyses on each open reading frame (ORFs in the genomes of 11 archaea (3 Crenarchaeota – Aeropyrum pernix, Pyrobaculum aerophilum and Sulfolobus acidocaldarius; 8 Euryarchaeota – Pyrococcus abyssi, Methanococcus maripaludis, Methanopyrus kandleri, Methanococcoides burtonii, Halobacterium sp. NCR-1, Haloquadratum walsbyi, Thermoplasma acidophilum and Picrophilus torridus to search for proteins that are unique to either all Archaea or for its main subgroups. These studies have identified 1448 proteins or ORFs that are distinctive characteristics of Archaea and its various subgroups and whose homologues are not found in other organisms. Six of these proteins are unique to all Archaea, 10 others are only missing in Nanoarchaeum equitans and a large number of other proteins are specific for various main groups within the Archaea (e.g. Crenarchaeota, Euryarchaeota, Sulfolobales and Desulfurococcales, Halobacteriales, Thermococci, Thermoplasmata, all methanogenic archaea or particular groups of methanogens. Of particular importance is the observation that 31 proteins are uniquely present in virtually all methanogens (including M. kandleri and 10 additional proteins are only found in different methanogens as well as A. fulgidus. In contrast, no protein was exclusively shared by various methanogen and any of the Halobacteriales or Thermoplasmatales. These results strongly indicate that all methanogenic archaea form a monophyletic group exclusive of other archaea and that this lineage likely evolved from Archaeoglobus. In addition, 15 proteins

  17. Comparative analyses of the two proliferating cell nuclear antigens from the hyperthermophilic archaeon, Thermococcus kodakarensis.

    Science.gov (United States)

    Kuba, Yumani; Ishino, Sonoko; Yamagami, Takeshi; Tokuhara, Masahiro; Kanai, Tamotsu; Fujikane, Ryosuke; Daiyasu, Hiromi; Atomi, Haruyuki; Ishino, Yoshizumi

    2012-11-01

    The DNA sliding clamp is a multifunctional protein involved in cellular DNA transactions. In Archaea and Eukaryota, proliferating cell nuclear antigen (PCNA) is the sliding clamp. The ring-shaped PCNA encircles double-stranded DNA within its central hole and tethers other proteins on DNA. The majority of Crenarchaeota, a subdomain of Archaea, have multiple PCNA homologues, and they are capable of forming heterotrimeric rings for their functions. In contrast, most organisms in Euryarchaeota, the other major subdomain, have a single PCNA forming a homotrimeric ring structure. Among the Euryarchaeota whose genome is sequenced, Thermococcus kodakarensis is the only species with two genes encoding PCNA homologues on its genome. We cloned the two genes from the T. kodakarensis genome, and the gene products, PCNA1 and PCNA2, were characterized. PCNA1 stimulated the DNA synthesis reactions of the two DNA polymerases, PolB and PolD, from T. kodakarensis in vitro. PCNA2, however, only had an effect on PolB. We were able to disrupt the gene for PCNA2, whereas gene disruption for PCNA1 was not possible, suggesting that PCNA1 is essential for DNA replication. The sensitivities of the Δpcna2 mutant strain to ultraviolet irradiation (UV), methyl methanesulfonate (MMS) and mitomycin C (MMC) were indistinguishable from those of the wild-type strain.

  18. Domain topology of the DNA polymerase D complex from a hyperthermophilic archaeon Pyrococcus horikoshii.

    Science.gov (United States)

    Tang, Xiao-Feng; Shen, Yulong; Matsui, Eriko; Matsui, Ikuo

    2004-09-21

    Family D DNA polymerase (PolD) is a recently found DNA polymerase extensively existing in Euryarchaeota of Archaea. Here, we report the domain function of PolD in oligomerization and interaction with other proteins, which were characterized with the yeast two-hybrid (Y2H) and surface plasmon resonance (SPR) assays. A proliferating cell nuclear antigen, PhoPCNA, interacted with the N-terminus of the small subunit, DP1(1-200). Specific interaction between the remaining part of the small subunit, DP1(201-622), and the N-terminus of the large subunit, DP2(1-300), was detected by the Y2H assay. The SPR assay also indicated the intrasubunit interaction within the N-terminus, DP2(1-100), and the C-terminus, DP2(792-1163), of the large subunit. A synthetic 21 amino acid peptide corresponding to the sequence from cysteine cluster II, DP2(1290-1310), tightly interacted (a dissociation constant K(D) = 4.3 nM) with the N-terminus of the small subunit, DP1(1-200). Since the peptide could increase the 3'-5' exonuclease activity of DP1 [Shen et al. (2004) Nucleic Acids Res. 32, 158], the short region DP2(1290-1310) seems to play dual roles to form the PhoPolD complex and to regulate the 3'-5' exonuclease activity of DP1 through interaction with DP1(1-200). Furthermore, DP2(792-1163) containing the catalytic residues for DNA polymerization, Asp1122 and Asp1124, interacted with the intrasubunit domain, DP2(1-100), and the intersubunit domain, DP1(1-200). DP2(792-1163) probably forms the most important domain deeply involved in both the catalysis of DNA polymerization and stabilization of the PhoPolD complex through these multiple interactions.

  19. Physiological plasticity of the thermophilic ammonia oxidizing archaeon Nitrosocaldus yellowstonii in response to a changing environment

    Science.gov (United States)

    Jewell, T.; Johnson, A.; Gelsinger, D.; de la Torre, J. R.

    2012-12-01

    Our understanding of nitrogen biogeochemical cycling in high temperature environments underwent a dramatic revision with the discovery of ammonia oxidizing archaea (AOA). The importance of AOA to the global nitrogen cycle came to light when recent studies of marine AOA demonstrated the dominance of these organisms in the ocean microbiome and their role as producers of the greenhouse gas nitrous oxide (N2O). Understanding how AOA respond to fluctuating environments is crucial to fully comprehending their contribution to global biogeochemical cycling and climate change. In this study we use the thermophilic AOA Nitrosocaldus yellowstonii strain HL72 to explore the physiological plasticity of energy metabolism in these organisms. Previous studies have shown that HL72 grows autotrophically by aerobically oxidizing ammonia (NH3) to nitrite (NO2-). Unlike studies of marine AOA, we find that HL72 can grow over a wide ammonia concentration range (0.25 - 10 mM NH4Cl) with comparable generation times when in the presence of 0.25 to 4 mM NH4Cl. However, preliminary data indicate that amoA, the alpha subunit of ammonia monooxygenase (AMO), is upregulated at low ammonia concentrations (urea transporter. Urea ((NH2)2CO) is an organic compound ubiquitous to aquatic and soil habitats that, when hydrolyzed, forms NH3 and CO2. We examined urea as an alternate source of ammonia for the ammonia oxidation pathway. HL72 grows over a wide range of urea concentrations (0.25 - 10 mM) at rates comparable to growth on ammonia. In a substrate competition experiment HL72 preferentially consumed NH3 from NH4Cl when both substrates were provided in equal molar concentrations. However, the urease alpha subunit ureC was expressed in both the presence and absence of urea. One consequence of urea hydrolysis is consumption of intracellular protons during the reaction. As ammonia oxidation produces H+, leading to a decrease in pH, the hydrolysis of urea prior to ammonia oxidation may help alleviate metabolism-driven pH change in HL72. A survey of archaeal ureC sequences from metagenomic data covering a range of hydrothermal features revealed that ureolytic potential is common to many Nitrosocaldus-like organisms and is geographically widespread. Measurements of urea from siliceous circumneutral springs indicate that the concentrations are generally low, below 10 μM. One possible explanation for low steady state urea concentrations is high consumption rates by ureolytic organisms. This, combined with abiotic thermal degradation, may mask high fluxes of urea in microbial hot spring communities.

  20. 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.

  1. Active ammonia oxidizers in an acidic soil are phylogenetically closely related to neutrophilic archaeon.

    Science.gov (United States)

    Wang, Baozhan; Zheng, Yan; Huang, Rong; Zhou, Xue; Wang, Dongmei; He, Yuanqiu; Jia, Zhongjun

    2014-03-01

    All cultivated ammonia-oxidizing archaea (AOA) within the Nitrososphaera cluster (former soil group 1.1b) are neutrophilic. Molecular surveys also indicate the existence of Nitrososphaera-like phylotypes in acidic soil, but their ecological roles are poorly understood. In this study, we present molecular evidence for the chemolithoautotrophic growth of Nitrososphaera-like AOA in an acidic soil with pH 4.92 using DNA-based stable isotope probing (SIP). Soil microcosm incubations demonstrated that nitrification was stimulated by urea fertilization and accompanied by a significant increase in the abundance of AOA rather than ammonia-oxidizing bacteria (AOB). Real-time PCR analysis of amoA genes as a function of the buoyant density of the DNA gradient following the ultracentrifugation of the total DNA extracted from SIP microcosms indicated a substantial growth of soil AOA during nitrification. Pyrosequencing of the total 16S rRNA genes in the "heavy" DNA fractions suggested that archaeal communities were labeled to a much greater extent than soil AOB. Acetylene inhibition further showed that (13)CO2 assimilation by nitrifying communities depended solely on ammonia oxidation activity, suggesting a chemolithoautotrophic lifestyle. Phylogenetic analysis of both (13)C-labeled amoA and 16S rRNA genes revealed that most of the active AOA were phylogenetically closely related to the neutrophilic strains Nitrososphaera viennensis EN76 and JG1 within the Nitrososphaera cluster. Our results provide strong evidence for the adaptive growth of Nitrososphaera-like AOA in acidic soil, suggesting a greater metabolic versatility of soil AOA than previously appreciated.

  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. 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

  4. 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.

  5. Targeted Disruption of the α-Amylase Gene in the Hyperthermophilic Archaeon Sulfolobus solfataricus

    OpenAIRE

    Worthington, Penny; Hoang, Viet; Perez-Pomares, Francisco; Blum, Paul

    2003-01-01

    Sulfolobus solfataricus secretes an acid-resistant α-amylase (amyA) during growth on starch as the sole carbon and energy source. Synthesis of this activity is subject to catabolite repression. To better understand α-amylase function and regulation, the structural gene was identified and disrupted and the resulting mutant was characterized. Internal α-amylase peptide sequences obtained by tandem mass spectroscopy were used to identify the amyA coding sequence. Anti-α-amylase antibodies raised...

  6. Identification of a novel alpha-galatosidase from the hyperthermophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Brouns, S.J.J.; Smits, N.; Wu, H.; Wright, P.C.; Snijders, A.P.L.; Vos, de W.M.; Oost, van der J.

    2006-01-01

    Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable -galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for

  7. The ABC of ABC-transport in the hyperthermophilic archaeon Pyrococcus furiosus

    OpenAIRE

    Koning, S.

    2003-01-01

    Living organisms of our earth can be divided into two groups, the prokaryotes and the eukaryotes. Eukaryotic cells have a nucleus, a special compartment in the cell, where the genetic material, the DNA is located. The DNA in the prokaryotic cell is floating freely in the cell. The eukaryotes, that is where we belong to, together with animals, plants and fungi. Bacteria and archaea belong to the prokaryotes. Archaea resemble bacteria but in certain features they resemble more the eukaryotes. T...

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

    Science.gov (United States)

    Mao, Dominic; Grogan, Dennis W

    2012-01-01

    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 acidocaldariuspyrE 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 to conjugation between transformed and non-transformed cells. These colonies thus appear to represent genetic sectoring in which regions of heteroduplex DNA formed and then segregated after partial resolution of inter-strand differences. Surprisingly, sectoring was also frequent in cells transformed with single-stranded DNAs. Oligonucleotides produced more sectored transformants when electroporated as single strands than as a duplex, although all forms of donor DNA (positive-strand, negative-strand, and duplex) produced a diversity of genotypes, despite 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. The conversion events that occur during transformation by single-stranded DNA do not show the strand bias necessary for a system that corrects replication errors effectively; similar events also occur in pre-formed heteroduplex electroporated into the cells. Although numerous mechanistic details remain obscure, the results demonstrate that the HR system of S. acidocaldarius can generate remarkable genetic diversity from short intervals of moderately diverged DNAs.

  9. Biological effects of DNA damage in the hyperthermophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Reilly, Michelle S; Grogan, Dennis W

    2002-02-19

    To investigate the generality of efficient double-strand break repair and damage-induced mutagenesis in hyperthermophilic archaea, we systematically measured the effects of five DNA-damaging agents on Sulfolobus acidocaldarius and compared the results to those obtained for Escherichia coli under corresponding conditions. The observed lethality of gamma-radiation was very similar for S. acidocaldarius and E. coli, arguing against unusually efficient double-strand break repair in S. acidocaldarius. In addition, DNA-strand-breaking agents (gamma-radiation or bleomycin), as well as DNA-cross-linking agents (mechlorethamine, butadiene diepoxide or cisplatin) stimulated forward mutation, reverse mutation, and formation of recombinants via conjugation in Sulfolobus cells. Although two of the five DNA-damaging agents failed to revert the E. coli auxotrophs under these conditions, all five reverted S. acidocaldarius auxotrophs.

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

    Directory of Open Access Journals (Sweden)

    Dennis W. 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 controlled.

  11. Homologous recombination in the archaeon Sulfolobus acidocaldarius: effects of DNA substrates and mechanistic implications.

    Science.gov (United States)

    Rockwood, Jananie; Mao, Dominic; Grogan, Dennis W

    2013-09-01

    Although homologous recombination (HR) is known to influence the structure, stability, and evolution of microbial genomes, few of its functional properties have been measured in cells of hyperthermophilic archaea. The present study manipulated various properties of the parental DNAs in high-resolution assays of Sulfolobus acidocaldarius transformation, and measured the impact on the efficiency and pattern of marker transfer to the recipient chromosome. The relative orientation of homologous sequences, the type and position of chromosomal mutation being replaced, and the length of DNA flanking the marked region all affected the efficiency, linkage, tract continuity, and other parameters of marker transfer. Effects predicted specifically by the classical reciprocal-exchange model of HR were not observed. One analysis observed only 90 % linkage between markers defined by adjacent bases; in another series of experiments, sequence divergence up to 4 % had no detectable impact on overall efficiency of HR or on the co-transfer of a distal non-selected marker. The effects of introducing DNA via conjugation, rather than transformation, were more difficult to assess, but appeared to increase co-transfer (i.e. linkage) of relatively distant non-selected markers. The results indicate that HR events between gene-sized duplex DNAs and the S. acidocaldarius chromosome typically involve neither crossing over nor interference from a mismatch-activated anti-recombination system. Instead, the donor DNA may anneal to a transient chromosomal gap, as in the mechanism proposed for oligonucleotide-mediated transformation of Sulfolobus and other micro-organisms.

  12. 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.

  13. The Alternative Route to Heme in the Methanogenic Archaeon Methanosarcina barkeri

    Directory of Open Access Journals (Sweden)

    Melanie Kühner

    2014-01-01

    Full Text Available In living organisms heme is formed from the common precursor uroporphyrinogen III by either one of two substantially different pathways. In contrast to eukaryotes and most bacteria which employ the so-called “classical” heme biosynthesis pathway, the archaea use an alternative route. In this pathway, heme is formed from uroporphyrinogen III via the intermediates precorrin-2, sirohydrochlorin, siroheme, 12,18-didecarboxysiroheme, and iron-coproporphyrin III. In this study the heme biosynthesis proteins AhbAB, AhbC, and AhbD from Methanosarcina barkeri were functionally characterized. Using an in vivo enzyme activity assay it was shown that AhbA and AhbB (Mbar_A1459 and Mbar_A1460 together catalyze the conversion of siroheme into 12,18-didecarboxysiroheme. The two proteins form a heterodimeric complex which might be subject to feedback regulation by the pathway end-product heme. Further, AhbC (Mbar_A1793 was shown to catalyze the formation of iron-coproporphyrin III in vivo. Finally, recombinant AhbD (Mbar_A1458 was produced in E. coli and purified indicating that this protein most likely contains two [4Fe-4S] clusters. Using an in vitro enzyme activity assay it was demonstrated that AhbD catalyzes the conversion of iron-coproporphyrin III into heme.

  14. Carbon isotope fractionation by the marine ammonia-oxidizing archaeon Nitrosopumilus maritimus

    OpenAIRE

    Könneke, Martin; Lipp, Julius Sebastian; Hinrichs, Kai-Uwe

    2012-01-01

    Abstract Ammonia-oxidizing archaea (AOA) are abundant and widely distributed microorganisms in aquatic and terrestrial habitats. By catalyzing the first and rate limiting step in nitrification, these chemolithoautotrophs play a significant role in the global nitrogen cycle and contribute to primary production. Here, the carbon isotopic fractionation relative to inorganic carbon source was determined for bulk biomass, biphytanes and polar lipid bound sugars of a marine AOA pure culture. Bu...

  15. Morphological and structural aspects of the extremely halophilic archaeon Haloquadratum walsbyi.

    Directory of Open Access Journals (Sweden)

    Matilde Sublimi Saponetti

    Full Text Available Ultrathin square cell Haloquadratum walsbyi from the Archaea domain are the most abundant microorganisms in the hypersaline water of coastal salterns and continental salt lakes. In this work, we explore the cell surface of these microorganisms using amplitude-modulation atomic-force microscopy in nearly physiological conditions. We demonstrate the presence of a regular corrugation with a periodicity of 16-20 nm attributed to the surface layer (S-layer protein lattice, striped domains asymmetrically distributed on the cell faces and peculiar bulges correlated with the presence of intracellular granules. Besides, subsequent images of cell evolution during the drying process indicate the presence of an external capsule that might correspond to the giant protein halomucin, predicted by the genome but never before observed by other microscopy studies.

  16. 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.

  17. Defining the topology of the N-glycosylation pathway in the halophilic archaeon Haloferax volcanii.

    Science.gov (United States)

    Plavner, Noa; Eichler, Jerry

    2008-12-01

    In Eukarya, N glycosylation involves the actions of enzymes working on both faces of the endoplasmic reticulum membrane. The steps of bacterial N glycosylation, in contrast, transpire essentially on the cytoplasmic side of the plasma membrane, with only transfer of the assembled glycan to the target protein occurring on the external surface of the cell. For Archaea, virtually nothing is known about the topology of enzymes involved in assembling those glycans that are subsequently N linked to target proteins on the external surface of the cell. To remedy this situation, subcellular localization and topology predictive algorithms, protease accessibility, and immunoblotting, together with cysteine modification following site-directed mutagenesis, were enlisted to define the topology of Haloferax volcanii proteins experimentally proven to participate in the N-glycosylation process. AglJ and AglD, involved in the earliest and latest stages, respectively, of assembly of the pentasaccharide decorating the H. volcanii S-layer glycoprotein, were shown to present their soluble N-terminal domain, likely containing the putative catalytic site of each enzyme, to the cytosol. The same holds true for Alg5-B, Dpm1-A, and Mpg1-D, proteins putatively involved in this posttranslational event. The results thus point to the assembly of the pentasaccharide linked to certain Asn residues of the H. volcanii S-layer glycoprotein as occurring within the cell.

  18. Defining the Topology of the N-Glycosylation Pathway in the Halophilic Archaeon Haloferax volcanii▿

    OpenAIRE

    Plavner, Noa; Eichler, Jerry

    2008-01-01

    In Eukarya, N glycosylation involves the actions of enzymes working on both faces of the endoplasmic reticulum membrane. The steps of bacterial N glycosylation, in contrast, transpire essentially on the cytoplasmic side of the plasma membrane, with only transfer of the assembled glycan to the target protein occurring on the external surface of the cell. For Archaea, virtually nothing is known about the topology of enzymes involved in assembling those glycans that are subsequently N linked to ...

  19. 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.

  20. N-glycosylation in the thermoacidophilic archaeon Sulfolobus acidocaldarius involves a short dolichol pyrophosphate carrier.

    Science.gov (United States)

    Guan, Ziqiang; Delago, Antonia; Nußbaum, Phillip; Meyer, Benjamin; Albers, Sonja-Verena; Eichler, Jerry

    2016-09-01

    N-glycosylation is a post-translational modification that occurs across evolution. In the thermoacidophilic archaea Sulfolobus acidocaldarius, glycoproteins are modified by an N-linked tribranched hexasaccharide reminiscent of the N-glycans assembled in Eukarya. Previously, hexose-bearing dolichol phosphate was detected in a S. acidocaldarius Bligh-Dyer lipid extract. Here, we used a specialized protocol for extracting lipid-linked oligosaccharides to detect a dolichol pyrophosphate bearing the intact hexasaccharide, as well as its biosynthetic intermediates. Furthermore, evidence for N-glycosylation of two S. acidocaldarius proteins by the same hexasaccharide and its derivatives was collected. These findings thus provide novel insight into archaeal N-glycosylation.

  1. Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Albers, S.-V.; Jonuscheit, M.; Dinkelaker, S.; Urich, T.; Kletzin, A.; Tampé, R.; Driessen, A.J.M.; Schleper, C.

    2006-01-01

    Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archa

  2. Relationships between fuselloviruses infecting the extremely thermophilic archaeon Sulfolobus: SSV1 and SSV2

    DEFF Research Database (Denmark)

    Stedman, Kenneth M; She, Qunxin; Phan, Hien

    2003-01-01

    The fusellovirus SSV2 from an Icelandic Sulfolobus strain was isolated, characterized and its complete genomic sequence determined. SSV2 is very similar in morphology, replication, genome size and number of open reading frames (ORFs) to the type virus of the family, SSV1 from Japan, except in its...... viruses, indicating that despite this genomic dissimilarity the virus genomes are mostly homologous. Unlike SSV1, the sequence of SSV2 indicates integration into a glycyl tRNA gene and is completely missing a DNA packaging gene. There is a unique, perfectly tandemly directly repeated sequence of 62...

  3. 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.

  4. Crystallization of [Fe4S3]-ferredoxin from the hyperthermophile archaeon pyrococcus furiosus

    DEFF Research Database (Denmark)

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

    2003-01-01

    Recombinant Pyrococcus furiosus ferredoxin with a [Fe3S4]-cluster was crystallized through steps of optimization and X-ray diffraction data were collected from several crystal forms. Flat plate-like crystals were grown by hanging-drop vapour diffusion. The precipitant used was 30% PEG 400; the p...

  5. 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.;

    2005-01-01

    to target the 3'-untranslated regions of certain mRNAs. Furthermore, one of the ncRNAs that does not show antisense elements is transcribed from a repeat unit of a cluster of small regularly spaced repeats in S. solfataricus which is potentially involved in replicon partitioning. In conclusion...

  6. Pcal_1699, an extremely thermostable malate dehydrogenase from hyperthermophilic archaeon Pyrobaculum calidifontis.

    Science.gov (United States)

    Gharib, Ghazaleh; Rashid, Naeem; Bashir, Qamar; Gardner, Qura-Tul Ann Afza; Akhtar, Muhammad; Imanaka, Tadayuki

    2016-01-01

    Two malate dehydrogenase homologs, Pcal_0564 and Pcal_1699, have been found in the genome of Pyrobaculum calidifontis. The gene encoding Pcal_1699 consisted of 927 nucleotides corresponding to a polypeptide of 309 amino acids. To examine the properties of Pcal_1699, the structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Pcal_1699 was NADH specific enzyme exhibiting a high malate dehydrogenase activity (886 U/mg) at optimal pH (10) and temperature (90 °C). Unfolding studies suggested that urea could not induce complete unfolding and inactivation of Pcal_1699 even at a final concentration of 8 M; however, in the presence of 4 M guanidine hydrochloride enzyme structure was unfolded with complete loss of enzyme activity. Thermostability experiments revealed that Pcal_1699 is the most thermostable malate dehydrogenase, reported to date, retaining more than 90 % residual activity even after heating for 6 h in boiling water.

  7. The complete genome sequence of Haloferax volcanii DS2, a model archaeon.

    Directory of Open Access Journals (Sweden)

    Amber L Hartman

    Full Text Available BACKGROUND: Haloferax volcanii is an easily culturable moderate halophile that grows on simple defined media, is readily transformable, and has a relatively stable genome. This, in combination with its biochemical and genetic tractability, has made Hfx. volcanii a key model organism, not only for the study of halophilicity, but also for archaeal biology in general. METHODOLOGY/PRINCIPAL FINDINGS: We report here the sequencing and analysis of the genome of Hfx. volcanii DS2, the type strain of this species. The genome contains a main 2.848 Mb chromosome, three smaller chromosomes pHV1, 3, 4 (85, 438, 636 kb, respectively and the pHV2 plasmid (6.4 kb. CONCLUSIONS/SIGNIFICANCE: The completed genome sequence, presented here, provides an invaluable tool for further in vivo and in vitro studies of Hfx. volcanii.

  8. Identifying Potential Mechanisms Enabling Acidophily in the Ammonia-Oxidizing Archaeon

    NARCIS (Netherlands)

    Lehtovirta-Morley, L.E.; Sayavedra-Soto, L.A.; Gallois, N.; Schouten, S.; Stein, L.Y.; Prosser, J.I.; Nicol, G.W.

    2016-01-01

    Ammonia oxidation is the first and rate-limiting step in nitrification and is dominated by two distinct groups of microorganismsin soil: ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). AOA are often more abundant than AOBand dominate activity in acid soils. The mechanism of amm

  9. The ABC of ABC-transport in the hyperthermophilic archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    Koning, S

    2003-01-01

    Living organisms of our earth can be divided into two groups, the prokaryotes and the eukaryotes. Eukaryotic cells have a nucleus, a special compartment in the cell, where the genetic material, the DNA is located. The DNA in the prokaryotic cell is floating freely in the cell. The eukaryotes, that i

  10. NADP-Dependent Aldehyde Dehydrogenase from Archaeon Pyrobaculum sp.1860: Structural and Functional Features

    Directory of Open Access Journals (Sweden)

    Ekaterina Yu. Bezsudnova

    2016-01-01

    Full Text Available We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60–85°С, and the affinity of AlDHPyr1147 to the NADP+ at 60°С is comparable to that for mesophilic analogues at 25°С. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution, three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å, and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å. The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.

  11. Quantitative proteome and transcriptome analysis of the archaeon Thermoplasma acidophilum cultured under aerobic and anaerobic conditions.

    Science.gov (United States)

    Sun, Na; Pan, Cuiping; Nickell, Stephan; Mann, Matthias; Baumeister, Wolfgang; Nagy, István

    2010-09-03

    A comparative proteome and transcriptome analysis of Thermoplasma acidophilum cultured under aerobic and anaerobic conditions has been performed. One-thousand twenty-five proteins were identified covering 88% of the cytosolic proteome. Using a label-free quantitation method, we found that approximately one-quarter of the identified proteome (263 proteins) were significantly induced (>2 fold) under anaerobic conditions. Thirty-nine macromolecular complexes were identified, of which 28 were quantified and 15 were regulated under anaerobiosis. In parallel, a whole genome cDNA microarray analysis was performed showing that the expression levels of 445 genes were influenced by the absence of oxygen. Interestingly, more than 40% of the membrane protein-encoding genes (145 out of 335 ORFs) were up- or down-regulated at the mRNA level. Many of these proteins are functionally associated with extracellular protein or peptide degradation or ion and amino acid transport. Comparison of the transcriptome and proteome showed only a weak positive correlation between mRNA and protein expression changes, which is indicative of extensive post-transcriptional regulatory mechanisms in T. acidophilum. Integration of transcriptomics and proteomics data generated hypotheses for physiological adaptations of the cells to anaerobiosis, and the quantitative proteomics data together with quantitative analysis of protein complexes provide a platform for correlation of MS-based proteomics studies with cryo-electron tomography-based visual proteomics approaches.

  12. Genetic examination of initial amino acid oxidation and glutamate catabolism in the hyperthermophilic archaeon Thermococcus kodakarensis.

    Science.gov (United States)

    Yokooji, Yuusuke; Sato, Takaaki; Fujiwara, Shinsuke; Imanaka, Tadayuki; Atomi, Haruyuki

    2013-05-01

    Amino acid catabolism in Thermococcales is presumed to proceed via three steps: oxidative deamination of amino acids by glutamate dehydrogenase (GDH) or aminotransferases, oxidative decarboxylation by 2-oxoacid:ferredoxin oxidoreductases (KOR), and hydrolysis of acyl-coenzyme A (CoA) by ADP-forming acyl-CoA synthetases (ACS). Here, we performed a genetic examination of enzymes involved in Glu catabolism in Thermococcus kodakarensis. Examination of amino acid dehydrogenase activities in cell extracts of T. kodakarensis KUW1 (ΔpyrF ΔtrpE) revealed high NADP-dependent GDH activity, along with lower levels of NAD-dependent activity. NADP-dependent activities toward Gln/Ala/Val/Cys and an NAD-dependent threonine dehydrogenase activity were also detected. In KGDH1, a gene disruption strain of T. kodakarensis GDH (Tk-GDH), only threonine dehydrogenase activity was detected, indicating that all other activities were dependent on Tk-GDH. KGDH1 could not grow in a medium in which growth was dependent on amino acid catabolism, implying that Tk-GDH is the only enzyme that can discharge the electrons (to NADP(+)/NAD(+)) released from amino acids in their oxidation to 2-oxoacids. In a medium containing excess pyruvate, KGDH1 displayed normal growth, but higher degrees of amino acid catabolism were observed compared to those for KUW1, suggesting that Tk-GDH functions to suppress amino acid oxidation and plays an anabolic role under this condition. We further constructed disruption strains of 2-oxoglutarate:ferredoxin oxidoreductase and succinyl-CoA synthetase. The two strains displayed growth defects in both media compared to KUW1. Succinate generation was not observed in these strains, indicating that the two enzymes are solely responsible for Glu catabolism among the multiple KOR and ACS enzymes in T. kodakarensis.

  13. 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;

    2012-01-01

    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 INTERACTIONS...... 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......)-triphosphatase 205, thiamine monophosphate kinase 179, pyruvate formate lyase family activating protein 298, 3-hydroxy-3-methylglutaryl-CoA reductase (mevanolate), N(2), N(2)-dimethylguanosine tRNA methyltransferase 145, N2, N2-dimethylguanosine tRNA methyltransferase 170, putative 5-methylcytosine restriction...

  14. 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.

  15. 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.

  16. 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...

  17. 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.

  18. Influence of Sulfobacillus thermosulfidooxidans on Initial Attachment and Pyrite Leaching by Thermoacidophilic Archaeon Acidianus sp. DSM 29099

    Directory of Open Access Journals (Sweden)

    Jing Liu

    2016-07-01

    Full Text Available At the industrial scale, bioleaching of metal sulfides includes two main technologies, tank leaching and heap leaching. Fluctuations in temperature caused by the exothermic reactions in a heap have a pronounced effect on the growth of microbes and composition of mixed microbial populations. Currently, little is known on the influence of pre-colonized mesophiles or moderate thermophiles on the attachment and bioleaching efficiency by thermophiles. The objective of this study was to investigate the interspecies interactions of the moderate thermophile Sulfobacillus thermosulfidooxidans DSM 9293T and the thermophile Acidianus sp. DSM 29099 during initial attachment to and dissolution of pyrite. Our results showed that: (1 Acidianus sp. DSM 29099 interacted with S. thermosulfidooxidansT during initial attachment in mixed cultures. In particular, cell attachment was improved in mixed cultures compared to pure cultures alone; however, no improvement of pyrite leaching in mixed cultures compared with pure cultures was observed; (2 active or inactivated cells of S. thermosulfidooxidansT on pyrite inhibited or showed no influence on the initial attachment of Acidianus sp. DSM 29099, respectively, but both promoted its leaching efficiency; (3 S. thermosulfidooxidansT exudates did not enhance the initial attachment of Acidianus sp. DSM 29099 to pyrite, but greatly facilitated its pyrite dissolution efficiency. Our study provides insights into cell-cell interactions between moderate thermophiles and thermophiles and is helpful for understanding of the microbial interactions in a heap leaching environment.

  19. 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.

  20. Doubling Power Output of Starch Biobattery Treated by the Most Thermostable Isoamylase from an Archaeon Sulfolobus tokodaii.

    Science.gov (United States)

    Cheng, Kun; Zhang, Fei; Sun, Fangfang; Chen, Hongge; Percival Zhang, Y-H

    2015-08-20

    Biobattery, a kind of enzymatic fuel cells, can convert organic compounds (e.g., glucose, starch) to electricity in a closed system without moving parts. Inspired by natural starch metabolism catalyzed by starch phosphorylase, isoamylase is essential to debranch alpha-1,6-glycosidic bonds of starch, yielding linear amylodextrin - the best fuel for sugar-powered biobattery. However, there is no thermostable isoamylase stable enough for simultaneous starch gelatinization and enzymatic hydrolysis, different from the case of thermostable alpha-amylase. A putative isoamylase gene was mined from megagenomic database. The open reading frame ST0928 from a hyperthermophilic archaeron Sulfolobus tokodaii was cloned and expressed in E. coli. The recombinant protein was easily purified by heat precipitation at 80 (o)C for 30 min. This enzyme was characterized and required Mg(2+) as an activator. This enzyme was the most stable isoamylase reported with a half lifetime of 200 min at 90 (o)C in the presence of 0.5 mM MgCl2, suitable for simultaneous starch gelatinization and isoamylase hydrolysis. The cuvett-based air-breathing biobattery powered by isoamylase-treated starch exhibited nearly doubled power outputs than that powered by the same concentration starch solution, suggesting more glucose 1-phosphate generated.

  1. A synthetic arabinose-inducible promoter confers high levels of recombinant protein expression in hyperthermophilic archaeon Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Peng, Nan; Deng, Ling; Mei, Yuxia;

    2012-01-01

    levels of target gene expression. More strikingly, N-terminal amino acid sequencing of recombinant proteins unraveled that the protein synthesized from pEXA-N-lacS lacked the designed 6×His tag and that translation initiation did not start at the ATG codon of the fusion gene. Instead, it started...

  2. SSoNΔ and SsoNΔlong: two thermostable esterases from the same ORF in the archaeon Sulfolobus solfataricus?

    Directory of Open Access Journals (Sweden)

    Luigi Mandrich

    2006-01-01

    Full Text Available Previously, we reported from the Sulfolobus solfataricus open reading frame (ORF SSO2517 the cloning, overexpression and characterization of an esterase belonging to the hormone-sensitive lipase (HSL family and apparently having a deletion at the N-terminus, which we named SsoNΔ. Searching the recently reported Sulfolobus acidocaldarius genome by sequence alignment, using SSO2517 as a query, allowed identity of a putative esterase (ORF SAC1105 sharing high sequence similarity (82% with SSO2517. This esterase displays an N-terminus and total length similar to other known esterases of the HSL family. Analysis of the upstream DNA sequence of SS02517 revealed the possibility of expressing a longer version of the protein with an extended N-terminus; however, no clear translation signal consistent with a longer protein version was detected. This new version of SSO2517 was cloned, over-expressed, purified and characterized. The resulting protein, named SsoNΔlong, was 15-fold more active with the substrate p-nitrophenyl hexanoate than SsoNΔ. Furthermore, SsoNΔlong and SsoNΔ displayed different substrate specificities for triacylglycerols. These results and the phylogenetic relationship between S. solfataricus and S. acidocaldarius suggest a common origin of SSO2517 and SAC1105 from an ancestral gene, followed by divergent evolution. Alternatively, a yet-to-be discovered mechanism of translation that directs the expression of SsoNΔlong under specific metabolic conditions could be hypothesized.

  3. Conjugational genetic exchange in the hyperthermophilic archaeon Sulfolobus acidocaldarius: intragenic recombination with minimal dependence on marker separation.

    Science.gov (United States)

    Hansen, Josh E; Dill, Amy C; Grogan, Dennis W

    2005-01-01

    In Sulfolobus acidocaldarius conjugation assays, recombinant frequency was relatively constant for marker separations from 1,154 bp down to about 50 bp and readily detectable at 10 bp. Three-factor crosses revealed little, if any, genetic linkage over distances of 500 to 600 bp, and large deletion mutants were good donors but poor recipients in matings. The results indicate that most intragenic recombination events occur at one of the mutations, not in the interval between them.

  4. Active-site residues in the type IV prepilin peptidase homologue PibD from the archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Szabo, Z; Albers, SV; Driessen, AJM

    2006-01-01

    Archaeal preflagellin peptidases and bacterial type IV prepilin peptidases belong to a family of aspartic acid proteases that cleave the leader peptides of precursor proteins with type W prepilin signal sequences. The substrate repertoire of PibD from the crenarchaeon Sulfolobus solfataricus is unus

  5. Active-Site Residues in the Type IV Prepilin Peptidase Homologue PibD from the Archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Szabo, Zalan; Albers, Sonja-Verena; Driessen, Arnold J.M.

    2006-01-01

    Archaeal preflagellin peptidases and bacterial type IV prepilin peptidases belong to a family of aspartic acid proteases that cleave the leader peptides of precursor proteins with type IV prepilin signal sequences. The substrate repertoire of PibD from the crenarchaeon Sulfolobus solfataricus is unu

  6. 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.

  7. Crystallization and preliminary X-ray diffraction analysis of L-threonine dehydrogenase (TDH) from the hyperthermophilic archaeon Thermococcus kodakaraensis.

    Science.gov (United States)

    Bowyer, A; Mikolajek, H; Wright, J N; Coker, A; Erskine, P T; Cooper, J B; Bashir, Q; Rashid, N; Jamil, F; Akhtar, M

    2008-09-01

    The enzyme L-threonine dehydrogenase catalyses the NAD(+)-dependent conversion of L-threonine to 2-amino-3-ketobutyrate, which is the first reaction of a two-step biochemical pathway involved in the metabolism of threonine to glycine. Here, the crystallization and preliminary crystallographic analysis of L-threonine dehydrogenase (Tk-TDH) from the hyperthermophilic organism Thermococcus kodakaraensis KOD1 is reported. This threonine dehydrogenase consists of 350 amino acids, with a molecular weight of 38 kDa, and was prepared using an Escherichia coli expression system. The purified native protein was crystallized using the hanging-drop vapour-diffusion method and crystals grew in the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 124.5, c = 271.1 A. Diffraction data were collected to 2.6 A resolution and preliminary analysis indicates that there are four molecules in the asymmetric unit of the crystal.

  8. Identification of the S-layer glycoproteins and their covalently linked glycans in the halophilic archaeon Haloarcula hispanica.

    Science.gov (United States)

    Lu, Hua; Lü, Yang; Ren, Jinwei; Wang, Zhongfu; Wang, Qian; Luo, Yuanming; Han, Jing; Xiang, Hua; Du, Yuguo; Jin, Cheng

    2015-11-01

    Haloarcula hispanica is one of members of the Halobacteriaceae, which displays particularly low restriction activity and is therefore important as one of the most tractable haloarchaea for archaeal genetic research. Although the Har. hispanica S-layer protein has been reported glycosylated, the S-layer glycoprotein and its glycosylation have not been investigated yet. In this study, the S-layer proteins of Har. hispanica were extracted and characterized. The S-layer was found containing two different glycoproteins which shared highly similar amino acid sequences. The genes coding for these two S-layer glycoproteins were found next to each other in the genome. Moreover, the N- and O-linked glycans were released from these two S-layer glycoproteins for structural determination. Based on the mass spectrometry and nuclear magnetic resonance, the N-glycan was determined as a branched trisaccharide containing a 225 Da residue corresponded to a 2-amino-6-sulfo-2, 6-dideoxy-quinovose, which was the first time that a naturally occurring form of sulfoquinovosamine was identified. Besides, the O-glycan was characterized as a Glcα-1,4-Gal disaccharide by mass spectrometry combined with monosaccharide composition analysis and glycosidase treatment. The determination of the N- and O-glycan structure will be helpful for studying the diverse protein glycosylation pathways in archaea utilizing H. hispanica as a new model.

  9. Dynamic Metabolic Adjustments and Genome Plasticity Are Implicated in the Heat Shock Response of the Extremely Thermoacidophilic Archaeon Sulfolobus solfataricus†

    Science.gov (United States)

    Tachdjian, Sabrina; Kelly, Robert M.

    2006-01-01

    Approximately one-third of the open reading frames encoded in the Sulfolobus solfataricus genome were differentially expressed within 5 min following an 80 to 90°C temperature shift at pH 4.0. This included many toxin-antitoxin loci and insertion elements, implicating a connection between genome plasticity and metabolic regulation in the early stages of stress response. PMID:16740961

  10. Dynamic metabolic adjustments and genome plasticity are implicated in the heat shock response of the extremely thermoacidophilic archaeon Sulfolobus solfataricus.

    Science.gov (United States)

    Tachdjian, Sabrina; Kelly, Robert M

    2006-06-01

    Approximately one-third of the open reading frames encoded in the Sulfolobus solfataricus genome were differentially expressed within 5 min following an 80 to 90 degrees C temperature shift at pH 4.0. This included many toxin-antitoxin loci and insertion elements, implicating a connection between genome plasticity and metabolic regulation in the early stages of stress response.

  11. 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

    have developed a genetic system for S. islandicus LAL14/1 and created ¿pyrEF and ¿CRISPR_1 mutants using double cross-over and pop-in/pop-out approaches, respectively. Thus, LAL14/1 is a promising model to study virus-host interactions and the CRISPR/Cas defence mechanism in Archaea....... 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...

  12. 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;

    2015-01-01

    -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...

  13. A Mesophilic, Autotrophic, Ammonia-Oxidizing Archaeon of Thaumarchaeal Group I.1a Cultivated from a Deep Oligotrophic Soil Horizon

    NARCIS (Netherlands)

    Jung, M.Y.; Park, S.J.; Kim, S.J.; Kim, J.G.; Sinninghe Damsté, J.S.; Jeon, C.O.; Rhee, S.K.

    2014-01-01

    Soil nitrification plays an important role in the reduction of soil fertility and in nitrate enrichment of groundwater. Various ammonia- oxidizing archaea (AOA) are considered to be members of the pool of ammonia-oxidizing microorganisms in soil. This study reports the discovery of a chemolithoautot

  14. Ser/Thr/Tyr protein phosphorylation in the archaeon Halobacterium salinarum--a representative of the third domain of life.

    Directory of Open Access Journals (Sweden)

    Michalis Aivaliotis

    Full Text Available In the quest for the origin and evolution of protein phosphorylation, the major regulatory post-translational modification in eukaryotes, the members of archaea, the "third domain of life", play a protagonistic role. A plethora of studies have demonstrated that archaeal proteins are subject to post-translational modification by covalent phosphorylation, but little is known concerning the identities of the proteins affected, the impact on their functionality, the physiological roles of archaeal protein phosphorylation/dephosphorylation, and the protein kinases/phosphatases involved. These limited studies led to the initial hypothesis that archaea, similarly to other prokaryotes, use mainly histidine/aspartate phosphorylation, in their two-component systems representing a paradigm of prokaryotic signal transduction, while eukaryotes mostly use Ser/Thr/Tyr phosphorylation for creating highly sophisticated regulatory networks. In antithesis to the above hypothesis, several studies showed that Ser/Thr/Tyr phosphorylation is also common in the bacterial cell, and here we present the first genome-wide phosphoproteomic analysis of the model organism of archaea, Halobacterium salinarum, proving the existence/conservation of Ser/Thr/Tyr phosphorylation in the "third domain" of life, allowing a better understanding of the origin and evolution of the so-called "Nature's premier" mechanism for regulating the functional properties of proteins.

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

    Science.gov (United States)

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

    2014-08-05

    Thermococcus onnurineus NA1 is known to grow by the anaerobic oxidation of formate to CO2 and H2, a reaction that operates near thermodynamic equilibrium. Here we demonstrate that this reaction is coupled to ATP synthesis by a transmembrane ion current. Formate oxidation leads to H(+) translocation across the cytoplasmic membrane that then drives Na(+) translocation. The ion-translocating electron transfer system is rather simple, consisting of only a formate dehydrogenase module, a membrane-bound hydrogenase module, and a multisubunit Na(+)/H(+) antiporter module. The electrochemical Na(+) gradient established then drives ATP synthesis. These data give a mechanistic explanation for chemiosmotic energy conservation coupled to formate oxidation to CO2 and H2. Because it is discussed that the membrane-bound hydrogenase with the Na(+)/H(+) antiporter module are ancestors of complex I of mitochondrial and bacterial electron transport these data also shed light on the evolution of ion transport in complex I-like electron transport chains.

  16. ATP- and NAD+-dependent DNA ligases share an essential function in the halophilic archaeon Haloferax volcanii

    DEFF Research Database (Denmark)

    Zhao, A.; Gray, F. C; MacNeill, S. A.

    2006-01-01

    DNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP-dependent ligases are found predominantly in the eukarya and archaea whereas NAD+-dependent DNA ligases are found only in the eubacteria and in entomopoxviruses. Using the genetically tractable halophile...

  17. 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;

    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...

  18. Histone and TK0471/TrmBL2 form a novel heterogeneous genome architecture in the hyperthermophilic archaeon Thermococcus kodakarensis.

    Science.gov (United States)

    Maruyama, Hugo; Shin, Minsang; Oda, Toshiyuki; Matsumi, Rie; Ohniwa, Ryosuke L; Itoh, Takehiko; Shirahige, Katsuhiko; Imanaka, Tadayuki; Atomi, Haruyuki; Yoshimura, Shige H; Takeyasu, Kunio

    2011-02-01

    Being distinct from bacteria and eukaryotes, Archaea constitute a third domain of living things. The DNA replication, transcription, and translation machineries of Archaea are more similar to those of eukaryotes, whereas the genes involved in metabolic processes show more similarity to their bacterial counterparts. We report here that TK0471/TrmB-like 2 (TrmBL2), in addition to histone, is a novel type of abundant chromosomal protein in the model euryarchaeon Thermococcus kodakarensis . The chromosome of T. kodakarensis can be separated into regions enriched either with histone, in which the genetic material takes on a “beads-on-a-string” appearance, or with TK0471/TrmBL2, in which it assumes a thick fibrous structure. TK0471/TrmBL2 binds to both coding and intergenic regions and represses transcription when bound to the promoter region. These results show that the archaeal chromosome is organized into heterogeneous structures and that TK0471/TrmBL2 acts as a general chromosomal protein as well as a global transcriptional repressor.

  19. The apt/6-Methylpurine Counterselection System and Its Applications in Genetic Studies of the Hyperthermophilic Archaeon Sulfolobus islandicus

    Science.gov (United States)

    Bi, Hongkai; Whitaker, Rachel J.

    2016-01-01

    ABSTRACT Sulfolobus islandicus serves as a model for studying archaeal biology as well as linking novel biology to evolutionary ecology using functional population genomics. In the present study, we developed a new counterselectable genetic marker in S. islandicus to expand the genetic toolbox for this species. We show that resistance to the purine analog 6-methylpurine (6-MP) in S. islandicus M.16.4 is due to the inactivation of a putative adenine phosphoribosyltransferase encoded by M164_0158 (apt). The application of the apt gene as a novel counterselectable marker was first illustrated by constructing an unmarked α-amylase deletion mutant. Furthermore, the 6-MP counterselection feature was employed in a forward (loss-of-function) mutation assay to reveal the profile of spontaneous mutations in S. islandicus M.16.4 at the apt locus. Moreover, the general conservation of apt genes in the crenarchaea suggests that the same strategy can be broadly applied to other crenarchaeal model organisms. These results demonstrate that the apt locus represents a new tool for genetic manipulation and sequence analysis of the hyperthermophilic crenarchaeon S. islandicus. IMPORTANCE Currently, the pyrEF/5-fluoroorotic acid (5-FOA) counterselection system remains the sole counterselection marker in crenarchaeal genetics. Since most Sulfolobus mutants constructed by the research community were derived from genetic hosts lacking the pyrEF genes, the pyrEF/5-FOA system is no longer available for use in forward mutation assays. Demonstration of the apt/6-MP counterselection system for the Sulfolobus model renders it possible to again study the mutation profiles in mutants that have already been constructed by the use of strains with a pyrEF-deficient background. Furthermore, additional counterselectable markers will allow us to conduct more sophisticated genetic studies, i.e., investigate mechanisms of chromosomal DNA transfer and quantify recombination frequencies among S. islandicus strains. PMID:26969706

  20. Cloning and expression of the catalase-peroxidase gene from the hyperthermophilic archaeon Archaeoglobus fulgidus and characterization of the enzyme

    NARCIS (Netherlands)

    Kengen, S.W.M.; Bikker, F.; Vos, de W.M.; Oost, van der J.

    2001-01-01

    A putative perA gene from Archaeoglobus fulgidus was cloned and expressed in Escherichia coli BL21(DE3), and the recombinant catalase-peroxidase was purified to homogeneity. The enzyme is a homodimer with a subunit molecular mass of 85 kDa. UV-visible spectroscopic analysis indicated the presence of

  1. Uncovering Mechanisms for Repair and Protection in Cold Environments Through Studies of Cold Adapted Archaea

    Science.gov (United States)

    2009-12-18

    Contract Number: FA9550-07-1-0085 Rick Cavicchioli, Tim Williams, Oliver Pilak University of New South Wales, Sydney, Australia Introduction...adapted microorganism . Objective 1: Progress All three chaperonin genes from M. burtonii have been cloned and the gene products have been...2ºC, 1ºC, 4ºC, 10ºC, 16ºC, 23ºC, 28ºC), which encompasses the lower to upper growth limits of M. burtonii. We also examined the effect of

  2. Draft genome of Haloarcula rubripromontorii strain SL3, a novel halophilic archaeon isolated from the solar salterns of Cabo Rojo, Puerto Rico

    Directory of Open Access Journals (Sweden)

    Rubén Sánchez-Nieves

    2016-03-01

    Full Text Available The genus Haloarcula belongs to the family Halobacteriaceae which currently has 10 valid species. Here we report the draft genome sequence of strain SL3, a new species within this genus, isolated from the Solar Salterns of Cabo Rojo, Puerto Rico. Genome assembly performed using NGEN Assembler resulted in 18 contigs (N50 = 601,911 bp, the largest of which contains 1,023,775 bp. The genome consists of 3.97 MB and has a GC content of 61.97%. Like all species of Haloarcula, the genome encodes heterogeneous copies of the small subunit ribosomal RNA. In addition, the genome includes 6 rRNAs, 48 tRNAs, and 3797 protein coding sequences. Several carbohydrate-active enzymes genes were found, as well as enzymes involved in the dihydroxyacetone processing pathway which are not found in other Haloarcula species. The NCBI accession number for this genome is LIUF00000000 and the strain deposit number is CECT9001.

  3. Draft genome sequence of Halorubrum tropicale strain V5, a novel halophilic archaeon isolated from the solar salterns of Cabo Rojo, Puerto Rico

    Directory of Open Access Journals (Sweden)

    Rubén Sánchez-Nieves

    2016-03-01

    Full Text Available The genus Halorubrum is a member of the family Halobacteriaceae which currently has the highest number of described species (31 of all the haloarchaea. Here we report the draft genome sequence of strain V5, a new species within this genus that was isolated from the solar salterns of Cabo Rojo, Puerto Rico. Assembly was performed and rendered the genome into 17 contigs (N50 = 515,834 bp, the largest of which contains 1,031,026 bp. The genome consists of 3.57 MB in length with G + C content of 67.6%. In general, the genome includes 4 rRNAs, 52 tRNAs, and 3246 protein-coding sequences. The NCBI accession number for this genome is LIST00000000 and the strain deposit number is CECT9000.

  4. SulfoSYS (Sulfolobus Systems Biology) : towards a silicon cell model for the central carbohydrate metabolism of the archaeon Sulfolobus solfataricus under temperature variation

    NARCIS (Netherlands)

    Albers, Sonja-Verena; Birkeland, Nils-Kare; Driessen, Arnold J. M.; Gertig, Susanne; Haferkamp, Patrick; Klenk, Hans-Peter; Kouril, Theresa; Manica, Andrea; Pham, Trong K.; Ruoff, Peter; Schleper, Christa; Schomburg, Dietmar; Sharkey, Kieran J.; Siebers, Bettina; Sierocinski, Pawel; Steuer, Ralf; van der Oost, John; Westerhoff, Hans V.; Wieloch, Patricia; Wright, Phillip C.; Zaparty, Melanie; Birkeland, Nils-Kåre

    2009-01-01

    SulfoSYS (Sulfolobus Systems Biology) focuses on the study of the CCM (central carbohydrate metabolism) of Sulfolobus solfataricus and its regulation under temperature variation at the systems level. in Archaea, carbohydrates are metabolized by modifications of the classical pathways known from Bact

  5. SulfoSYS (Sulfolobus Systems Biology): towards a silicon cell model for the central carbohydrate metabolism of the archaeon Sulfolobus solfataricus under temperature variation.

    NARCIS (Netherlands)

    Albers, S.V.; Birkeland, N.K.; Driessen, A.J.; Gertig, S.; Haferkamp, P.; Klenk, H.P.; Kouril, T.; Manica, A.; Pham, T.K.; Ruoff, P.; Schleper, C.; Schomburg, D.; Sharkey, K.J.; Siebers, A.G.; Sierocinski, P.; Steuer, R.; Oost, J. van der; Westerhoff, H.V.; Wieloch, P.; Wright, P.C.; Zaparty, M.

    2009-01-01

    SulfoSYS (Sulfolobus Systems Biology) focuses on the study of the CCM (central carbohydrate metabolism) of Sulfolobus solfataricus and its regulation under temperature variation at the systems level. In Archaea, carbohydrates are metabolized by modifications of the classical pathways known from Bact

  6. SulfoSYS (Sulfolobus Systems Biology): towards a silicon cell model for the central carbohydrate metabolism of the archaeon Sulfolobus solfataricus under temperature variation

    NARCIS (Netherlands)

    Albers, S.V.; Birkeland, N.K.; Driessen, A.J.M.; Gertig, S.; Haferkamp, P.; Klenk, H.P.; Kouril, T.; Manica, A.; Pham, T.K.; Ruoff, P.; Schleper, C.; Schomburg, D.; Sharkey, K.; Siebers, B.; Sierocinski, P.; Steur, R.; Oost, van der J.; Westerhoff, H.V.; Wieloch, P.; Wright, P.C.; Zaparty, M.

    2009-01-01

    SulfoSYS (Sulfolobus Systems Biology) focuses on the study of the CCM (central carbohydrate metabolism) of Sulfolobus solfataricus and its regulation under temperature variation at the systems level. In Archaea, carbohydrates are metabolized by modifications of the classical pathways known from Bact

  7. Tetrahydrofolate-specific enzymes in Methanosarcina barkeri and growth dependence of this methanogenic archaeon on folic acid or p-aminobenzoic acid.

    Science.gov (United States)

    Buchenau, Bärbel; Thauer, Rudolf K

    2004-10-01

    Methanogenic archaea are generally thought to use tetrahydromethanopterin or tetrahydrosarcinapterin (H4SPT) rather than tetrahydrofolate (H4F) as a pterin C1 carrier. However, the genome sequence of Methanosarcina species recently revealed a cluster of genes, purN, folD, glyA and metF, that are predicted to encode for H4F-specific enzymes. We show here for folD and glyA from M. barkeri that this prediction is correct: FolD (bifunctional N5,N10-methylene-H4F dehydrogenase/N5,N10-methenyl-H4F cyclohydrolase) and GlyA (serine:H4F hydroxymethyltransferase) were heterologously overproduced in Escherichia coli, purified and found to be specific for methylene-H4F and H4F, respectively (apparent Km below 5 microM). Western blot analyses and enzyme activity measurements revealed that both enzymes were synthesized in M. barkeri. The results thus indicate that M. barkeri should contain H4F, which was supported by the finding that growth of M. barkeri was dependent on folic acid and that the vitamin could be substituted by p-aminobenzoic acid, a biosynthetic precursor of H4F. From the p-aminobenzoic acid requirement, an intracellular H4F concentration of approximately 5 M was estimated. Evidence is presented that the p-aminobenzoic acid taken up by the growing cells was not required for the biosynthesis of H4SPT, which was found to be present in the cells at a concentration above 3 mM. The presence of both H4SPT and H4F in M. barkeri is in agreement with earlier isotope labeling studies indicating that there are two separate C1 pools in these methanogens.

  8. Complete Genome Sequence of the Hyperthermophilic Piezophilic Archaeon Pyrococcus kukulkanii NCB100 Isolated from the Rebecca's Roost Hydrothermal Vent in the Guaymas Basin.

    Science.gov (United States)

    Oger, Philippe M; Callac, Nolwenn; Oger-Desfeux, Christine; Hughes, Sandrine; Gillet, Benjamin; Jebbar, Mohamed; Godfroy, Anne

    2017-02-16

    Members of the order Thermococcales are common inhabitants of high-temperature hydrothermal vent systems (black smokers) that are represented in clone libraries mostly by isolates from the Thermococcus genus. We report the complete sequence of a novel species from the Pyrococcus genus, P. kukulkanii strain NCB100, which has been isolated from a flange fragment of the Rebecca's Roost hydrothermal vent system in the Guaymas Basin.

  9. Bipolar tetraether lipids derived from thermoacidophilic archaeon Sulfolobus acidocaldarius for membrane stabilization of chlorin e6 based liposomes for photodynamic therapy.

    Science.gov (United States)

    Mahmoud, Gihan; Jedelská, Jarmila; Strehlow, Boris; Bakowsky, Udo

    2015-09-01

    The initial burst release of water-soluble photosensitizers is one of the major problems encountered the development of controlled release formulations. In this study, the freely water soluble chlorin e6 (Ce6) was assembled with cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) to improve its loading efficiency in the liposomal bilayer. Tetraether lipids (TELs) derived from Sulfolobus acidocaldarius were added to DOTAP:Ce6 assembly in a concentration range of 2.5×10(-4)-1.6×10(-3)M to stabilize the membrane rigidity of the liposomes and to provide controlled release system. From the comparative spectroscopic experiments, it has been shown that the assembled DOTAP:Ce6 along with addition of TELs have improved the loading efficiency of Ce6 in TELs-liposomes and obviously modified the release profile of Ce6. The in vitro cell viability of Ce6 in mouse neuro-blastoma (Neuro-2a) and ovarian cell carcinoma (SK-OV-3) confirmed neglected dark cytotoxicity and presented potential photo-induced cytotoxicity with the effect was being more pronounced in Neuro 2a than in SK-OV-3. In-situ IV-injection of chick chorioallantoic membrane (CAM) showed hemorrhage and necrosis 30 min post irradiation at 1.8 mol% TELs (19.9J/cm(2)). Higher TELs of 2.2 and 3.7 mol% in particular demonstrated localized vascular destruction within the irradiated area. Our results suggest that TELs favored slower release rates of Ce6. This, in turn, tetraether lipids can be considered as a versatile class of lipids for photodynamic modality for destruction of cancer cells and tumor vasculature while sparing the quiescent ones.

  10. Enrichment and genome sequence of the group I.1a ammonia-oxidizing Archaeon "Ca. Nitrosotenuis uzonensis" representing a clade globally distributed in thermal habitats.

    Directory of Open Access Journals (Sweden)

    Elena V Lebedeva

    Full Text Available The discovery of ammonia-oxidizing archaea (AOA of the phylum Thaumarchaeota and the high abundance of archaeal ammonia monooxygenase subunit A encoding gene sequences in many environments have extended our perception of nitrifying microbial communities. Moreover, AOA are the only aerobic ammonia oxidizers known to be active in geothermal environments. Molecular data indicate that in many globally distributed terrestrial high-temperature habits a thaumarchaeotal lineage within the Nitrosopumilus cluster (also called "marine" group I.1a thrives, but these microbes have neither been isolated from these systems nor functionally characterized in situ yet. In this study, we report on the enrichment and genomic characterization of a representative of this lineage from a thermal spring in Kamchatka. This thaumarchaeote, provisionally classified as "Candidatus Nitrosotenuis uzonensis", is a moderately thermophilic, non-halophilic, chemolithoautotrophic ammonia oxidizer. The nearly complete genome sequence (assembled into a single scaffold of this AOA confirmed the presence of the typical thaumarchaeotal pathways for ammonia oxidation and carbon fixation, and indicated its ability to produce coenzyme F420 and to chemotactically react to its environment. Interestingly, like members of the genus Nitrosoarchaeum, "Candidatus N. uzonensis" also possesses a putative artubulin-encoding gene. Genome comparisons to related AOA with available genome sequences confirmed that the newly cultured AOA has an average nucleotide identity far below the species threshold and revealed a substantial degree of genomic plasticity with unique genomic regions in "Ca. N. uzonensis", which potentially include genetic determinants of ecological niche differentiation.

  11. 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.

  12. The membrane-extrinsic domain of cytochrome b(558/566) from the archaeon Sulfolobus acidocaldarius performs pivoting movements with respect to the membrane surface.

    Science.gov (United States)

    Schoepp-Cothenet, B; Schütz, M; Baymann, F; Brugna, M; Nitschke, W; Myllykallio, H; Schmidt, C

    2001-01-05

    The orientation of the membrane-attached cytochrome b(558/566)-haem with respect to the membrane was determined by electron paramagnetic resonance spectroscopy on two-dimensionally ordered oxidised membrane fragments from Sulfolobus acidocaldarius. Unlike the other redox centres in the membrane, the cytochrome b(558/566)-haem was found to cover a range of orientations between 25 degrees and 90 degrees. The described results are reminiscent of those obtained on the Rieske cluster of bc complexes and indicate that the membrane-extrinsic domain of cytochrome b(558/566) can perform pivoting motion between two extreme positions. Such a conformational flexibility is likely to play a role in electron transfer with its redox partners.

  13. Disruption of the gene encoding restriction endonuclease SuaI and development of a host-vector system for the thermoacidophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Suzuki, Shoji; Kurosawa, Norio

    2016-03-01

    Sulfolobus acidocaldarius is a useful model organism for the genetic study of thermophilic archaea due to its ease of cultivation. Here we describe the development of a host-vector system for S. acidocaldarius consisting of SuaI restriction system-deficient strain SK-1 and shuttle vector pSAV2. The new host strain SK-1 was constructed by pop-out recombination based on the pyrE marker gene. Plasmid pSAV2 was constructed from the S. islandicus native plasmid pRN1, in which selectable markers and functional genes were inserted in suitable locations and orientations followed by the deletion of non-essential open reading frames. SK-1 allowed direct transformation without N(4)-methylation at SuaI restriction sites, so unmethylated vector pSAV2 could be introduced directly into SK-1 by electroporation. The transformants were selected by pyrEF complementation on xyrose-tryptone solid medium without prior liquid culturing. The transformation efficiency was approximately 1.0 × 10(3)/μg DNA. After replication in S. acidocaldarius, pSAV2 was successfully recovered from transformant cultures by the standard alkaline lysis method. Plasmid yield was approximately 40-50 ng/ml from late-log through stationary phase cultures. In addition, pSAV2 was maintained stably and at relatively high copy number in S. acidocaldarius.

  14. Specific partial reduction of geranylgeranyl diphosphate by an enzyme from the thermoacidophilic archaeon Sulfolobus acidocaldarius yields a reactive prenyl donor, not a dead-end product.

    Science.gov (United States)

    Sato, Sho; Murakami, Motomichi; Yoshimura, Tohru; Hemmi, Hisashi

    2008-06-01

    Geranylgeranyl reductase from Sulfolobus acidocaldarius was shown to catalyze the reduction of geranylgeranyl groups in the precursors of archaeal membrane lipids, generally reducing all four double bonds. However, when geranylgeranyl diphosphate was subjected to the reductase reaction, only three of the four double bonds were reduced. Mass spectrometry and acid hydrolysis indicated that the allylic double bond was preserved in the partially reduced product derived from geranylgeranyl diphosphate. Thus, the reaction product was shown to be phytyl diphosphate, which is a substrate for archaeal prenyltransferases, unlike the completely reduced compound phytanyl diphosphate.

  15. 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...... suggest that the gene should be renamed gpT. Both enzymes exhibited unusual profiles of activity versus pH. The adenine PRTase showed the highest activity at pH 7.5-8.5, but had a distinct peak of activity also at pH 4.5. The 6-oxo PRTase showed maximal activity with hypoxanthine and guanine around pH 4...

  16. 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...

  17. The Geoglobus acetivorans genome: Fe(III) reduction, acetate utilization, autotrophic growth, and degradation of aromatic compounds in a hyperthermophilic archaeon.

    Science.gov (United States)

    Mardanov, Andrey V; Slododkina, Galina B; Slobodkin, Alexander I; Beletsky, Alexey V; Gavrilov, Sergey N; Kublanov, Ilya V; Bonch-Osmolovskaya, Elizaveta A; Skryabin, Konstantin G; Ravin, Nikolai V

    2015-02-01

    Geoglobus acetivorans is a hyperthermophilic anaerobic euryarchaeon of the order Archaeoglobales isolated from deep-sea hydrothermal vents. A unique physiological feature of the members of the genus Geoglobus is their obligate dependence on Fe(III) reduction, which plays an important role in the geochemistry of hydrothermal systems. The features of this organism and its complete 1,860,815-bp genome sequence are described in this report. Genome analysis revealed pathways enabling oxidation of molecular hydrogen, proteinaceous substrates, fatty acids, aromatic compounds, n-alkanes, and organic acids, including acetate, through anaerobic respiration linked to Fe(III) reduction. Consistent with the inability of G. acetivorans to grow on carbohydrates, the modified Embden-Meyerhof pathway encoded by the genome is incomplete. Autotrophic CO2 fixation is enabled by the Wood-Ljungdahl pathway. Reduction of insoluble poorly crystalline Fe(III) oxide depends on the transfer of electrons from the quinone pool to multiheme c-type cytochromes exposed on the cell surface. Direct contact of the cells and Fe(III) oxide particles could be facilitated by pilus-like appendages. Genome analysis indicated the presence of metabolic pathways for anaerobic degradation of aromatic compounds and n-alkanes, although an ability of G. acetivorans to grow on these substrates was not observed in laboratory experiments. Overall, our results suggest that Geoglobus species could play an important role in microbial communities of deep-sea hydrothermal vents as lithoautotrophic producers. An additional role as decomposers would close the biogeochemical cycle of carbon through complete mineralization of various organic compounds via Fe(III) respiration.

  18. A zinc-dependent protease AMZ-tk from a thermophilic archaeon is a new member of the archaemetzincin protein family

    Directory of Open Access Journals (Sweden)

    Baolei eJia

    2015-12-01

    Full Text Available A putative zinc-dependent protease (TK0512 in Thermococcus kodakarensis KOD1 shares a conserved motif with archaemetzincins, which are metalloproteases found in archaea, bacteria, and eukarya. Phylogenetic and sequence analyses showed that TK0512 and its homologues in Thermococcaceae represent new members in the archaemetzincins family, which we named AMZ-tk. We further confirmed its proteolytic activity biochemically by overexpression of the recombinant AMZ-tk in E. coli and characterization of the purified enzyme. In the presence of zinc, the purified enzyme degraded casein, while adding EDTA strongly inhibited the enzyme activity. AMZ-tk also exhibited self-cleavage activity that required Zn2+. These results demonstrated that AMZ-tk is a zinc-dependent protease within the archaemetzincin family. The enzyme displayed activity at alkaline pHs ranging from 7.0-10.0, with the optimal pH being 8.0. The optimum temperature for the catalytic activity of AMZ-tk was 55ºC. Quantitative reverse transcription-PCR revealed that transcription of AMZ-tk was also up-regulated after exposing the cells to 55 ºC and 65ºC. Mutant analysis suggests that Zn2+ binding histidine and catalytic glutamate plays key roles in proteolysis. AMZ-tk was thermostable on incubation for 4 h at 70°C in the presence of EDTA. AMZ-tk also retained >50% of its original activity in the presence of both laboratory surfactants and commercial laundry detergents. AMZ-tk further showed antibacterial activity against several bacteria. Therefore, AMZ-tk is of considerable interest for many purposes in view of its activity at alkaline pH, detergents, and thermostability.

  19. Replication factor C from the hyperthermophilic archaeon Pyrococcus abyssi does not need ATP hydrolysis for clamp-loading and contains a functionally conserved RFC PCNA-binding domain.

    Science.gov (United States)

    Henneke, Ghislaine; Gueguen, Yannick; Flament, Didier; Azam, Philippe; Querellou, Joël; Dietrich, Jacques; Hübscher, Ulrich; Raffin, Jean-Paul

    2002-11-08

    The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in Pyrococcus abyssi (Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in Escherichia coli. Two inteins present in the gene encoding the small subunit (PabRFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the PabRFC-small subunit could be purified, while the large subunit (PabRFC-large) alone was completely insoluble. The highly purified PabRFC complex possessed an ATPase activity, which was not enhanced by DNA. The Pab proliferating cell nuclear antigen (PCNA) activated the PabRFC complex in a DNA-dependent manner, but the PabRFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The PabRFC complex was able to stimulate PabPCNA-dependent DNA synthesis by the Pabfamily D heterodimeric DNA polymerase. Finally, (i) the PabRFC-large fraction cross-reacted with anti-human-RFC PCNA-binding domain antibody, corroborating the conservation of the protein sequence, (ii) the human PCNA stimulated the PabRFC complex ATPase activity in a DNA-dependent way and (iii) the PabRFC complex could load human PCNA onto primed single-stranded circular DNA, suggesting that the PCNA-binding domain of RFC has been functionally conserved during evolution. In addition, ATP hydrolysis was not required either for DNA polymerase stimulation or PCNA-loading in vitro.

  20. High-throughput Screening of the Enantioselectivity of Hyperthermophilic Mutant Esterases from Archaeon Aeropyrum pernix K1 for Resolution of (R,S)-2-Octanol Acetate

    Institute of Scientific and Technical Information of China (English)

    ZHANG Gui-rong; GAO Ren-jun; ZHANG Ai-jun; RAO Lang; CAO Shu-gui

    2007-01-01

    To identify the desired hyperthermophilic variants within a mutant esterase library for the resolution of (R,S)-2-octanol acetate, a simple, reliable, and versatile method was developed in this study. We built a screening strategy including two steps, first we selected agar plate with substrate to screen the enzymatic activity; secondly we used a pH indicator to screen the enantioselectivity. This method could rapidly detect favorable mutants with high activity and enantioselectivity. A total of 96.2% of tedious screening work can be precluded using this screening strategy. It is an effective screening for alkyl ester and can be applied to relative screening researches. The four improved mutants were screened from the mutant esterase library. Their enantioselectivities, activities, and structures were investigated at different temperatures.

  1. TrmB, a sugar-specific transcriptional regulator of the trehalose/maltose ABC transporter from the hyperthermophilic archaeon Thermococcus litoralis.

    Science.gov (United States)

    Lee, Sung-Jae; Engelmann, Afra; Horlacher, Reinhold; Qu, Qiuhao; Vierke, Gudrun; Hebbeln, Carina; Thomm, Michael; Boos, Winfried

    2003-01-10

    We report the characterization of TrmB, a protein of 38,800 apparent molecular weight, that is involved in the maltose-specific regulation of a gene cluster in Thermococcus litoralis, malE malF malG orf trmB malK, encoding a binding protein-dependent ABC transporter for trehalose and maltose. TrmB binds maltose and trehalose half-maximally at 20 microm and 0.5 mm sugar concentration, respectively. Binding of maltose but not of trehalose showed indications of sigmoidality and quenched the intrinsic tryptophan fluorescence by 15%, indicating a conformational change on maltose binding. TrmB causes a shift in electrophoretic mobility of DNA fragments harboring the promoter and upstream regulatory motif identified by footprinting. Band shifting by TrmB can be prevented by maltose. In vitro transcription assays with purified components from Pyrococcus furiosus have been established to show pmalE promoter-dependent transcription at 80 degrees C. TrmB specifically inhibits transcription, and this inhibition is counteracted by maltose and trehalose. These data characterize TrmB as a maltose-specific repressor for the trehalose/maltose transport operon of Thermococcus litoralis.

  2. The three-dimensional structure of TrmB, a transcriptional regulator of dual function in the hyperthermophilic archaeon Pyrococcus furiosus in complex with sucrose.

    Science.gov (United States)

    Krug, Michael; Lee, Sung-Jae; Boos, Winfried; Diederichs, Kay; Welte, Wolfram

    2013-06-01

    TrmB is a repressor that binds maltose, maltotriose, and sucrose, as well as other α-glucosides. It recognizes two different operator sequences controlling the TM (Trehalose/Maltose) and the MD (Maltodextrin) operon encoding the respective ABC transporters and sugar-degrading enzymes. Binding of maltose to TrmB abrogates repression of the TM operon but maintains the repression of the MD operon. On the other hand, binding of sucrose abrogates repression of the MD operon but maintains repression of the TM operon. The three-dimensional structure of TrmB in complex with sucrose was solved and refined to a resolution of 3.0 Å. The structure shows the N-terminal DNA binding domain containing a winged-helix-turn-helix (wHTH) domain followed by an amphipathic helix with a coiled-coil motif. The latter promotes dimerization and places the symmetry mates of the putative recognition helix in the wHTH motif about 30 Å apart suggesting a canonical binding to two successive major grooves of duplex palindromic DNA. This suggests that the structure resembles the conformation of TrmB recognizing the pseudopalindromic TM promoter but not the conformation recognizing the nonpalindromic MD promoter.

  3. The role of TrmB and TrmB-like transcriptional regulators for sugar transport and metabolism in the hyperthermophilic archaeon Pyrococcus furiosus.

    Science.gov (United States)

    Lee, Sung-Jae; Surma, Melanie; Hausner, Winfried; Thomm, Michael; Boos, Winfried

    2008-09-01

    TrmB of Pyrococcus furiosus was discovered as the trehalose/maltose-specific repressor for the genes encoding the trehalose/maltose high-affinity ABC transporter (the TM system). TrmB also represses the genes encoding the high affinity maltodextrin-specific ABC transporter (the MD system) with maltodextrin and sucrose as inducers. In addition, TrmB binds glucose leading to an increased repression of both, the TM and the MD system. Thus, TrmB recognizes different promoters and depending on the promoter it will be activated or inactivated for promoter binding by different sugar effectors. The TrmB-like protein TrmBL1 of P. furiosus is a global regulator and recognizes preferentially, but not exclusively, the TGM (for Thermococcales-glycolytic motif) sequence that is found upstream of the MD system as well as of genes encoding enzymes involved in the glycolytic and the gluconeogenic pathway. It responds to maltose and maltotriose as inducers and functions as repressor for the genes encoding the MD system and glycolytic enzymes, but as activator for genes encoding gluconeogenic enzymes. The TrmB-like protein TrmBL2 of P. furiosus lacks the sugar-binding domain that has been determined in TrmB. It recognizes the MD promoter, but not all TGM harboring promoters. It is evolutionary the most conserved among the Thermococcales. The regulatory range of TrmBL2 remains unclear.

  4. The magic spot ppGpp influences in vitro the molecular and functional properties of the elongation factor 1α from the archaeon Sulfolobus solfataricus.

    Science.gov (United States)

    Martucci, Nicola M; Lamberti, Anna; Vitagliano, Luigi; Cantiello, Piergiuseppe; Ruggiero, Immacolata; Arcari, Paolo; Masullo, Mariorosario

    2012-09-01

    Guanosine tetra-phosphate (ppGpp), also known as "magic spot I", is a key molecule in the stringent control of most eubacteria and some eukarya. Here, we show that ppGpp affects the functional and molecular properties of the archaeal elongation factor 1α from Sulfolobus solfataricus (SsEF-1α). Indeed, ppGpp inhibited archaeal protein synthesis in vitro, even though the concentration required to get inhibition was higher than that required for the eubacterial and eukaryal systems. Regarding the partial reactions catalysed by SsEF-1α the effect produced by ppGpp on the affinity for aa-tRNA was lower than that measured in the presence of GTP but higher than that for GDP. Magic spot I was also able to bind SsEF-1α with an intermediate affinity in comparison to that displayed by GDP and GTP. Furthermore, ppGpp inhibited the intrinsic GTPase of SsEF-1α with a competitive behaviour. Finally, the binding of ppGpp to SsEF-1α rendered the elongation factor more resistant to heat treatment and the analysis of the molecular model of the complex between SsEF-1α and ppGpp suggests that this stabilisation arises from the charge optimisation on the surface of the protein.

  5. Characteristics Of Phytases From Soybeans And Microorganisms Involved In The Tempe Production

    OpenAIRE

    K A Buckle2); Sutardi*

    2004-01-01

    Tempe was prepared from Forest variety of soybeans inoculated with pure culture of the mould Rhizopus oligosporus strain CT11K2. Partially purified phytases from soybeans, mould of the R. oligosporus strain CT11K2, yeasts (Endomycopsis burtonii, Candida diddensiae and Candida tropicalis) and bacteria (Streptococcus faecium and Streptococcus dysgalactiae) involved during tempe production were prepared according to procedure developed by Sutardi (1988). The crude phytases were characterized and...

  6. The protein ORF80 from the acidophilic and thermophilic archaeon Sulfolobus islandicus binds highly site-specifically to double-stranded DNA and represents a novel type of basic leucine zipper protein

    Science.gov (United States)

    Lipps, Georg; Ibanez, Pablo; Stroessenreuther, Thomas; Hekimian, Katya; Krauss, Gerhard

    2001-01-01

    The cryptic high copy number plasmid pRN1 from the thermophilic and acidophilic crenarchaeote Sulfolobus islandicus shares three conserved open reading frames with other S.islandicus plasmids. One of the open reading frames, namely orf80, encodes a 9.5 kDa protein that has no homology to any characterised protein. Recombinant ORF80 purified from Escherichia coli binds to double-stranded DNA in a sequence-specific manner as suggested by EMSA experiments and DNase I footprints. Two highly symmetrical binding sites separated by ∼60 bp were found upstream of the orf80 gene. Both binding sites contain two TTAA motifs as well as other conserved bases. Fluorescence measurements show that short duplex DNAs derived from a single binding site sequence are bound with submicromolar affinity and moderate cooperativity by ORF80. On DNA fragments carrying both binding sites, a rather large protein–DNA complex is formed in a highly cooperative manner. ORF80 contains an N-terminal leucine zipper motif and a highly basic domain at its C-terminus. Compared to all known basic leucine zipper proteins the order of the domains is reversed in ORF80. ORF80 may therefore constitute a new subclass of basic leucine zipper DNA-binding proteins. PMID:11812827

  7. Genome sequence of the mud-dwelling archaeon Methanoplanus limicola type strain (DSM 2279(T)), reclassification of Methanoplanus petrolearius as Methanolacinia petrolearia and emended descriptions of the genera Methanoplanus and Methanolacinia.

    Science.gov (United States)

    Göker, Markus; Lu, Megan; Fiebig, Anne; Nolan, Matt; Lapidus, Alla; Tice, Hope; Del Rio, Tijana Glavina; Cheng, Jan-Fang; Han, Cliff; Tapia, Roxanne; Goodwin, Lynne A; Pitluck, Sam; Liolios, Konstantinos; Mavromatis, Konstantinos; Pagani, Ioanna; Ivanova, Natalia; Mikhailova, Natalia; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam; Mayilraj, Shanmugam; Rohde, Manfred; Detter, John C; Bunk, Boyke; Spring, Stefan; Wirth, Reinhard; Woyke, Tanja; Bristow, James; Eisen, Jonathan A; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C; Klenk, Hans-Peter

    2014-06-15

    Methanoplanus limicola Wildgruber et al. 1984 is a mesophilic methanogen that was isolated from a swamp composed of drilling waste near Naples, Italy, shortly after the Archaea were recognized as a separate domain of life. Methanoplanus is the type genus in the family Methanoplanaceae, a taxon that felt into disuse since modern 16S rRNA gene sequences-based taxonomy was established. Methanoplanus is now placed within the Methanomicrobiaceae, a family that is so far poorly characterized at the genome level. The only other type strain of the genus with a sequenced genome, Methanoplanus petrolearius SEBR 4847(T), turned out to be misclassified and required reclassification to Methanolacinia. Both, Methanoplanus and Methanolacinia, needed taxonomic emendations due to a significant deviation of the G+C content of their genomes from previously published (pre-genome-sequence era) values. Until now genome sequences were published for only four of the 33 species with validly published names in the Methanomicrobiaceae. Here we describe the features of M. limicola, together with the improved-high-quality draft genome sequence and annotation of the type strain, M3(T). The 3,200,946 bp long chromosome (permanent draft sequence) with its 3,064 protein-coding and 65 RNA genes is a part of the G enomic E ncyclopedia of B acteria and Archaea project.

  8. 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 implication of the Mre11 protein in DNA repair processes in the Archaea and showing its interaction with both Rad50 and the HerA bipolar helicase. Further studies on the functional interactions between these proteins, the NurA nuclease and the RadA recombinase, will allow us to define their roles and mechanism of action.

  9. Inhibiting NAD+-dependent DNA ligase activity with 2-(cyclopentyloxy)-5'-deoxyadenosine (CPOdA) offers a new tool for DNA replication and repair studies in the model archaeon Haloferax volcanii.

    Science.gov (United States)

    Giroux, Xavier; MacNeill, Stuart A

    2015-11-01

    DNA ligases play an essential role in many aspects of DNA metabolism in all three domains of life. The haloarchaeal organism Haloferax volcanii encodes both ATP- and NAD(+)-dependent DNA ligase enzymes designated LigA and LigN, respectively. Neither LigA nor LigN alone is required for cell viability but they share an essential function, most likely the ligation of Okazaki fragments during chromosome replication. Here we show that 2-(cyclopentyloxy)-5'-deoxyadenosine (referred to as CPOdA), originally developed as a inhibitor of bacterial NAD(+)-dependent DNA ligases, is a potent inhibitor of the growth of Hfx. volcanii cells expressing LigN alone, causing chromosome fragmentation and cell death, while cells expressing LigA are unaffected. Growth inhibition occurs at significantly lower CPOdA concentrations (MIC ≤ 50 ng ml(-1)) than those required for inhibition of bacterial growth (≥2 μg ml(-1)). CPOdA has the potential to become a vital tool in DNA replication and repair studies in this important model organism.

  10. Biochemical Properties of DNA Ligase From The Hyperthermophilic Archaeon Sulfolobus shibatae%极端嗜热古菌--芝田硫化叶菌DNA连接酶的生化性质

    Institute of Scientific and Technical Information of China (English)

    赖小勤; 黄力

    2005-01-01

    极端嗜热古菌--芝田硫化叶菌DNA连接酶(Ssh连接酶)的最适辅因子为ATP,在dATP存在时,该酶也能表现出较弱的连接活性.ATP或dATP都能够使该酶发生腺苷化,腺苷化的Ssh连接酶能够将腺苷基团转移至含切刻的DNA上.电泳迁移率改变实验表明,Ssh连接酶能够结合双链DNA,且与含切刻及不含切刻的DNA结合的亲和力相同,但不结合单链DNA.酵母双杂交实验显示,硫磺矿硫化叶菌(与芝田硫化叶菌亲缘关系很近)的DNA连接酶,与该菌所含的3个增殖细胞核抗原(PCNA)同源蛋白中的一个(PCNA-1)有相互作用,而与另外2个同源蛋白(PCNA-like和PCNA-2)则无相互作用.在古菌中高度保守的Sac10b蛋白家族成员Ssh10b能够激活Ssh连接酶的活性,而硫化叶菌中的主要染色体蛋白--7kuDNA结合蛋白(Ssh7)则对该酶活性没有影响.

  11. NCBI nr-aa BLAST: CBRC-MDOM-11-0005 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MDOM-11-0005 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 1e-07 25% ...

  12. NCBI nr-aa BLAST: CBRC-OSAT-03-0013 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OSAT-03-0013 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 2e-21 31% ...

  13. NCBI nr-aa BLAST: CBRC-MMUS-02-0423 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MMUS-02-0423 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 2e-43 40% ...

  14. NCBI nr-aa BLAST: CBRC-GACU-11-0006 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-GACU-11-0006 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 3e-33 35% ...

  15. NCBI nr-aa BLAST: CBRC-DMEL-06-0001 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DMEL-06-0001 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 5e-18 41% ...

  16. NCBI nr-aa BLAST: CBRC-RNOR-12-0025 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-RNOR-12-0025 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 2e-12 42% ...

  17. NCBI nr-aa BLAST: CBRC-TGUT-37-0114 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TGUT-37-0114 ref|YP_685385.1| hypothetical protein RCIX660 [uncultured methanogen...ic archaeon RC-I] emb|CAJ36059.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_685385.1 3e-16 39% ...

  18. NCBI nr-aa BLAST: CBRC-DMEL-06-0001 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DMEL-06-0001 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 4e-20 50% ...

  19. NCBI nr-aa BLAST: CBRC-BTAU-01-2840 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-BTAU-01-2840 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 8e-14 33% ...

  20. NCBI nr-aa BLAST: CBRC-PABE-10-0012 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PABE-10-0012 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 8e-09 29% ...

  1. NCBI nr-aa BLAST: CBRC-MMUR-01-1504 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MMUR-01-1504 ref|YP_685385.1| hypothetical protein RCIX660 [uncultured methanogen...ic archaeon RC-I] emb|CAJ36059.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_685385.1 3e-24 31% ...

  2. NCBI nr-aa BLAST: CBRC-BTAU-01-1360 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-BTAU-01-1360 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 6e-09 30% ...

  3. NCBI nr-aa BLAST: CBRC-OLAT-26-0110 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OLAT-26-0110 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 1e-52 42% ...

  4. NCBI nr-aa BLAST: CBRC-AGAM-07-0025 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-07-0025 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 2e-31 49% ...

  5. NCBI nr-aa BLAST: CBRC-PHAM-01-0645 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PHAM-01-0645 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 2e-21 33% ...

  6. NCBI nr-aa BLAST: CBRC-DYAK-06-0001 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DYAK-06-0001 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 4e-21 50% ...

  7. NCBI nr-aa BLAST: CBRC-MEUG-01-2690 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MEUG-01-2690 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 1e-50 50% ...

  8. NCBI nr-aa BLAST: CBRC-ETEL-01-0356 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ETEL-01-0356 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 3e-30 38% ...

  9. NCBI nr-aa BLAST: CBRC-OGAR-01-0238 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OGAR-01-0238 ref|YP_685385.1| hypothetical protein RCIX660 [uncultured methanogen...ic archaeon RC-I] emb|CAJ36059.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_685385.1 6e-12 27% ...

  10. NCBI nr-aa BLAST: CBRC-OSAT-03-0017 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OSAT-03-0017 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 1e-06 28% ...

  11. NCBI nr-aa BLAST: CBRC-AGAM-07-0025 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-07-0025 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 7e-31 50% ...

  12. NCBI nr-aa BLAST: CBRC-LAFR-01-2518 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-2518 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 1e-11 29% ...

  13. NCBI nr-aa BLAST: CBRC-LAFR-01-2518 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-2518 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 4e-08 28% ...

  14. NCBI nr-aa BLAST: CBRC-EEUR-01-0548 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-EEUR-01-0548 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 0.016 24% ...

  15. NCBI nr-aa BLAST: CBRC-TGUT-22-0001 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TGUT-22-0001 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 9e-06 26% ...

  16. NCBI nr-aa BLAST: CBRC-DRER-13-0009 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DRER-13-0009 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 6e-06 25% ...

  17. NCBI nr-aa BLAST: CBRC-CFAM-31-0002 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CFAM-31-0002 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 1e-24 39% ...

  18. NCBI nr-aa BLAST: CBRC-MEUG-01-2690 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MEUG-01-2690 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 6e-55 55% ...

  19. NCBI nr-aa BLAST: CBRC-ACAR-01-0632 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ACAR-01-0632 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 6e-08 32% ...

  20. NCBI nr-aa BLAST: CBRC-TTRU-01-1377 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-1377 ref|YP_686723.1| putative Na(+)/alanine symporter [uncultured methanogen...ic archaeon RC-I] emb|CAJ37397.1| putative Na(+)/alanine symporter [uncultured methanogenic archaeon RC-I] YP_686723.1 0.11 23% ...

  1. NCBI nr-aa BLAST: CBRC-AGAM-07-0037 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-07-0037 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 2e-10 30% ...

  2. NCBI nr-aa BLAST: CBRC-TNIG-22-0115 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TNIG-22-0115 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 4e-07 25% ...

  3. NCBI nr-aa BLAST: CBRC-TTRU-01-1331 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-1331 ref|YP_685247.1| hypothetical protein RCIX493 [uncultured methanogen...ic archaeon RC-I] emb|CAJ35921.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_685247.1 0.001 26% ...

  4. NCBI nr-aa BLAST: CBRC-CFAM-31-0002 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CFAM-31-0002 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 3e-27 38% ...

  5. NCBI nr-aa BLAST: CBRC-OSAT-03-0013 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OSAT-03-0013 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 3e-20 30% ...

  6. NCBI nr-aa BLAST: CBRC-AGAM-02-0057 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-02-0057 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 2e-10 33% ...

  7. NCBI nr-aa BLAST: CBRC-FRUB-02-0719 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-FRUB-02-0719 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 4e-87 56% ...

  8. NCBI nr-aa BLAST: CBRC-TTRU-01-0296 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0296 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 6e-05 31% ...

  9. NCBI nr-aa BLAST: CBRC-LAFR-01-2791 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-2791 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 1e-04 28% ...

  10. NCBI nr-aa BLAST: CBRC-GACU-16-0138 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-GACU-16-0138 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 5e-36 44% ...

  11. NCBI nr-aa BLAST: CBRC-BTAU-01-2840 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-BTAU-01-2840 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 9e-10 32% ...

  12. NCBI nr-aa BLAST: CBRC-OANA-01-2166 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OANA-01-2166 ref|YP_685385.1| hypothetical protein RCIX660 [uncultured methanogen...ic archaeon RC-I] emb|CAJ36059.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_685385.1 1e-09 38% ...

  13. NCBI nr-aa BLAST: CBRC-FRUB-02-0759 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-FRUB-02-0759 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 1e-27 53% ...

  14. NCBI nr-aa BLAST: CBRC-GACU-19-0030 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-GACU-19-0030 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 3e-24 38% ...

  15. NCBI nr-aa BLAST: CBRC-MDOM-09-0038 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MDOM-09-0038 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 2e-59 42% ...

  16. NCBI nr-aa BLAST: CBRC-OSAT-05-0027 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OSAT-05-0027 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 1e-09 30% ...

  17. NCBI nr-aa BLAST: CBRC-AGAM-02-0102 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-02-0102 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 7e-12 27% ...

  18. NCBI nr-aa BLAST: CBRC-PMAR-01-0237 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PMAR-01-0237 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 6e-19 34% ...

  19. NCBI nr-aa BLAST: CBRC-SARA-01-1960 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-SARA-01-1960 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 9e-19 40% ...

  20. NCBI nr-aa BLAST: CBRC-OSAT-05-0027 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OSAT-05-0027 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 5e-09 27% ...

  1. NCBI nr-aa BLAST: CBRC-ETEL-01-0356 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ETEL-01-0356 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 6e-35 39% ...

  2. NCBI nr-aa BLAST: CBRC-OSAT-03-0017 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OSAT-03-0017 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 6e-08 28% ...

  3. NCBI nr-aa BLAST: CBRC-GACU-01-0014 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-GACU-01-0014 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 9e-12 33% ...

  4. NCBI nr-aa BLAST: CBRC-PHAM-01-0645 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PHAM-01-0645 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 3e-24 35% ...

  5. NCBI nr-aa BLAST: CBRC-OCUN-01-0349 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OCUN-01-0349 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 3e-27 34% ...

  6. NCBI nr-aa BLAST: CBRC-RNOR-12-0025 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-RNOR-12-0025 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 2e-12 37% ...

  7. NCBI nr-aa BLAST: CBRC-DRER-03-0097 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DRER-03-0097 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 8e-44 38% ...

  8. NCBI nr-aa BLAST: CBRC-TGUT-20-0001 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TGUT-20-0001 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 2e-09 34% ...

  9. NCBI nr-aa BLAST: CBRC-DYAK-06-0001 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DYAK-06-0001 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 4e-19 47% ...

  10. NCBI nr-aa BLAST: CBRC-OLAT-26-0110 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OLAT-26-0110 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 3e-48 43% ...

  11. NCBI nr-aa BLAST: CBRC-MDOM-07-0027 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MDOM-07-0027 ref|YP_686429.1| hypothetical protein RCIX1938 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37103.1| conserved hypothetical protein [uncultured methanogenic archaeon RC-I] YP_686429.1 4.4 25% ...

  12. NCBI nr-aa BLAST: CBRC-DNOV-01-1123 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-1123 ref|YP_686048.1| hypothetical protein RCIA111 [uncultured methanogen...ic archaeon RC-I] emb|CAJ36722.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_686048.1 8.8 48% ...

  13. NCBI nr-aa BLAST: CBRC-RNOR-15-0080 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-RNOR-15-0080 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 2e-05 32% ...

  14. NCBI nr-aa BLAST: CBRC-ACAR-01-0270 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ACAR-01-0270 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 3e-06 25% ...

  15. NCBI nr-aa BLAST: CBRC-BTAU-01-1360 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-BTAU-01-1360 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 3e-09 27% ...

  16. NCBI nr-aa BLAST: CBRC-MMUS-02-0423 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MMUS-02-0423 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 1e-40 34% ...

  17. NCBI nr-aa BLAST: CBRC-GACU-01-0014 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-GACU-01-0014 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 1e-09 32% ...

  18. NCBI nr-aa BLAST: CBRC-AGAM-02-0057 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-02-0057 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 9e-11 35% ...

  19. NCBI nr-aa BLAST: CBRC-DRER-13-0011 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DRER-13-0011 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 6e-06 25% ...

  20. NCBI nr-aa BLAST: CBRC-TNIG-22-0115 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TNIG-22-0115 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 7e-08 26% ...

  1. NCBI nr-aa BLAST: CBRC-SARA-01-1960 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-SARA-01-1960 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 7e-22 39% ...

  2. NCBI nr-aa BLAST: CBRC-ACAR-01-0632 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ACAR-01-0632 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 1e-08 32% ...

  3. NCBI nr-aa BLAST: CBRC-PABE-10-0012 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PABE-10-0012 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 5e-10 26% ...

  4. NCBI nr-aa BLAST: CBRC-EEUR-01-0242 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-EEUR-01-0242 ref|YP_686482.1| hypothetical protein RCIX1999 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37156.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_686482.1 6e-11 20% ...

  5. NCBI nr-aa BLAST: CBRC-MDOM-09-0038 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MDOM-09-0038 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 2e-54 48% ...

  6. NCBI nr-aa BLAST: CBRC-DRER-03-0097 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DRER-03-0097 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 2e-40 36% ...

  7. NCBI nr-aa BLAST: CBRC-MDOM-02-0142 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MDOM-02-0142 ref|YP_687482.1| putative Na(+)/H(+) antiporter [uncultured methanogen...ic archaeon RC-I] emb|CAJ38156.1| putative Na(+)/H(+) antiporter [uncultured methanogenic archaeon RC-I] YP_687482.1 0.75 25% ...

  8. NCBI nr-aa BLAST: CBRC-AGAM-07-0037 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-07-0037 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 5e-11 28% ...

  9. NCBI nr-aa BLAST: CBRC-EEUR-01-0548 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-EEUR-01-0548 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 0.013 27% ...

  10. NCBI nr-aa BLAST: CBRC-FRUB-02-0759 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-FRUB-02-0759 ref|YP_687145.1| hypothetical protein RRC34 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37819.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687145.1 1e-24 50% ...

  11. NCBI nr-aa BLAST: CBRC-FRUB-02-0542 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-FRUB-02-0542 ref|YP_687144.1| hypothetical protein RRC32 [uncultured methanogen...ic archaeon RC-I] emb|CAJ37818.1| hypothetical protein [uncultured methanogenic archaeon RC-I] YP_687144.1 2e-68 47% ...

  12. Draft Genome Sequence of Pyrodictium occultum PL19T, a Marine Hyperthermophilic Species of Archaea That Grows Optimally at 105°C.

    Science.gov (United States)

    Utturkar, Sagar M; Huber, Harald; Leptihn, Sebastian; Loh, Belinda; Brown, Steven D; Stetter, Karl O; Podar, Mircea

    2016-02-25

    We report here the draft genome sequence of Pyrodictium occultum PL19(T), a marine hyperthermophilic archaeon. The genome provides insights into molecular and cellular adaptation mechanisms to life in extreme environments and the evolution of early organisms on Earth.

  13. Validation of a predictive model for the growth of chalk yeasts on bread.

    Science.gov (United States)

    Burgain, Anaïs; Bensoussan, Maurice; Dantigny, Philippe

    2015-07-02

    The present study focused on the effects of temperature, T, and water activity, aw, on the growth of Hyphopichia burtonii, Pichia anomala, and Saccharomycopsis fibuligera on Sabouraud Agar Medium. Cardinal values were estimated by means of cardinal models with inflection. All the yeasts were xerophilic, and they exhibited growth at 0.85 aw. The combined effects of T, aw, and pH on the growth of these species were described by the gamma-concept and validated on bread in the range of 15-25 °C, 0.91-0.97 aw, and pH 4.6-6.8. The optimum growth rates on bread were 2.88, 0.259, and 1.06 mm/day for H. burtonii, P. anomala, and S. fibuligera, respectively. The optimal growth rate of S. fibuligera on bread was about 2 fold that obtained on Sabouraud. Due to reproduction by budding, P. anomala exhibited low growth on Sabouraud and bread. However, this species is of major concern in the baker's industry because of the production of ethyl acetate in bread.

  14. The Expression, Purification of Chaperonin β Subunit from the Thermoacidophilic Archaeon,Acidianus tengchongensis and its Activity Analysis%腾冲嗜酸热两面菌S5分子伴侣β亚基的表达、纯化和活性的初步分析

    Institute of Scientific and Technical Information of China (English)

    马晴; 张渝英

    2007-01-01

    用NdeI和BamHI酶切回收腾冲嗜酸热两面菌S5的分子伴侣β亚基基因片段插入pET-23b的相应位置,并分别在BL21(DE3)和Rosetta-gamiTMB(DE3)pLysS中表达.表达的β亚基以可溶的形式存在.β亚基在Rosetta-gamiTMB(DE3)pLysS中表达较高,其占菌体总蛋白的16.2%,且以单体和聚体形式同时存在.表达的菌体经超声破碎、70℃热处理后,上清中β亚基蛋白含量达到30%,再经(NH4)2SO4沉淀、Bio-Gel A-1.5m和DEAE-Sepharose CL-6B柱层析,得到在SDS-PAGE呈电泳均一的β亚基,Native-PAGE表明其为聚体,有弱的ATPase活性.

  15. Occurrence and Diversity of Pichia spp. in Marine Environments

    Institute of Scientific and Technical Information of China (English)

    LI Jing; CHI Zhenming; WANG Xianghong; WANG Lin; SHENG Jun; GONG Fang

    2008-01-01

    A total of 328 yeast strains from seawater, sediments, mud of saltems, the guts of marine fish and marine algae were obtained. The results of routine identification and molecular methods show that five yeast strains obtained in this study belonged to Pichia spp., including Pichia guilliermondii luv-small, Pichia ohmeri YF04d, Pichiafermentans YF12b, Pichia burtonii YF11A and Pichia anomala YF07b. Further studies revealed that Pichia anomala YF07b could produce killer toxin against pathogenic yeasts in crabs while Pichia guilliermondii luv-small could produce high activity of extracellular inulinase. It is advisable to test if Pichia ohmeri YF04d obtained in this study is related to central-venous-catheter-associated infection.

  16. Genomic Evolution of the Ascomycete Yeasts

    Energy Technology Data Exchange (ETDEWEB)

    Riley, Robert; Haridas, Sajeet; Salamov, Asaf; Boundy-Mills, Kyria; Goker, Markus; Hittinger, Chris; Klenk, Hans-Peter; Lopes, Mariana; Meir-Kolthoff, Jan P.; Rokas, Antonis; Rosa, Carlos; Scheuner, Carmen; Soares, Marco; Stielow, Benjamin; Wisecaver, Jennifer H.; Wolfe, Ken; Blackwell, Meredith; Kurtzman, Cletus; Grigoriev, Igor; Jeffries, Thomas

    2015-03-16

    Yeasts are important for industrial and biotechnological processes and show remarkable metabolic and phylogenetic diversity despite morphological similarities. We have sequenced the genomes of 16 ascomycete yeasts of taxonomic and industrial importance including members of Saccharomycotina and Taphrinomycotina. Phylogenetic analysis of these and previously published yeast genomes helped resolve the placement of species including Saitoella complicata, Babjeviella inositovora, Hyphopichia burtonii, and Metschnikowia bicuspidata. Moreover, we find that alternative nuclear codon usage, where CUG encodes serine instead of leucine, are monophyletic within the Saccharomycotina. Most of the yeasts have compact genomes with a large fraction of single exon genes, and a tendency towards more introns in early-diverging species. Analysis of enzyme phylogeny gives insights into the evolution of metabolic capabilities such as methanol utilization and assimilation of alternative carbon sources.

  17. Molybdenum incorporation in tungsten aldehyde oxidoreductase enzymes from Pyrococcus furiosus

    NARCIS (Netherlands)

    Sevcenco, A.M; Bevers, L.E.; Pinkse, M.W.H.; Krijger, G.C.; Wolterbeek, H.T.; Verhaert, P.D.E.M.; Hagen, W.R.; Hagedoorn, P.L.

    2010-01-01

    The hyperthermophilic archaeon Pyrococcus furiosus expresses five aldehyde oxidoreductase (AOR) enzymes, all containing a tungsto-bispterin cofactor. The growth of this organism is fully dependent on the presence of tungsten in the growth medium. Previous studies have suggested that molybdenum is no

  18. One- and two-electron reduction of molybdate reversibly bound to the archaeal tungstate/molybdate transporter WtpA

    NARCIS (Netherlands)

    Bevers, L.E.; Hagen, W.R.

    2009-01-01

    Reversible binding of the tetrahedral oxoanions MoO4 2- and WO4 2- to two carboxylato ligands of the soluble scavenger protein WtpA from the hyperthermophilic archaeon Pyrococcus furiosus enforces a quasi-octahedral MO6 coordination in which the +VI oxidation state is destabilized.

  19. 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

  20. Complete genome sequence of Desulfurococcus fermentans, a hyperthermophilic cellulolytic crenarchaeon isolated from a freshwater hot spring in Kamchatka, Russia.

    Science.gov (United States)

    Susanti, Dwi; Johnson, Eric F; Rodriguez, Jason R; Anderson, Iain; Perevalova, Anna A; Kyrpides, Nikos; Lucas, Susan; Han, James; Lapidus, Alla; Cheng, Jan-Fang; Goodwin, Lynne; Pitluck, Sam; Mavrommatis, Konstantinos; Peters, Lin; Land, Miriam L; Hauser, Loren; Gopalan, Venkat; Chan, Patricia P; Lowe, Todd M; Atomi, Haruyuki; Bonch-Osmolovskaya, Elizaveta A; Woyke, Tanja; Mukhopadhyay, Biswarup

    2012-10-01

    Desulfurococcus fermentans is the first known cellulolytic archaeon. This hyperthermophilic and strictly anaerobic crenarchaeon produces hydrogen from fermentation of various carbohydrates and peptides without inhibition by accumulating hydrogen. The complete genome sequence reported here suggested that D. fermentans employs membrane-bound hydrogenases and novel glycohydrolases for hydrogen production from cellulose.

  1. Complete Genome Sequence of Desulfurococcus fermentans, a Hyperthermophilic Cellulolytic Crenarchaeon Isolated from a Freshwater Hot Spring in Kamchatka, Russia

    Energy Technology Data Exchange (ETDEWEB)

    Susanti, Dwi [Virginia Polytechnic Institute and State University (Virginia Tech); Johnson, Eric F [Virginia Polytechnic Institute and State University (Virginia Tech); Rodriquez, Jason [Virginia Polytechnic Institute and State University (Virginia Tech); Anderson, Iain [U.S. Department of Energy, Joint Genome Institute; Perevalova, Anna [Virginia Polytechnic Institute and State University (Virginia Tech); Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Han, James [U.S. Department of Energy, Joint Genome Institute; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Cheng, Jan-Fang [U.S. Department of Energy, Joint Genome Institute; Goodwin, Lynne A. [Los Alamos National Laboratory (LANL); Pitluck, Sam [U.S. Department of Energy, Joint Genome Institute; Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Peters, Lin [U.S. Department of Energy, Joint Genome Institute; Land, Miriam L [ORNL; Hauser, Loren John [ORNL; Gopapan, Venkay [Ohio State University; Chan, Patricia [University of California, Santa Cruz; Atomi, Haruyuki [Kyoto University, Japan; Bonch-Osmolovskaya, Elizaveta [Russian Academy of Sciences, Moscow; Woyke, Tanja [U.S. Department of Energy, Joint Genome Institute; Mukhopadhyay, Biswarup [Virginia Polytechnic Institute and State University (Virginia Tech)

    2012-01-01

    Desulfurococcus fermentans is the first known cellulolytic archaeon. This hyperthermophilic and strictly anaerobic crenarchaeon produces hydrogen from fermentation of various carbohydrates and peptides without inhibition by accumulating hydrogen. The complete genome sequence reported here suggested that D. fermentans employs membrane-bound hydrogenases and novel glycohydrolases for hydrogen production from cellulose.

  2. Iron-Sulfur Proteins Investigated by EPR-, Moessbauer- and EXAFS-Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Wegner, P.; Bever, M.; Schuenemann, V.; Trautwein, A. X. [University of Luebeck, Institute of Physics (Germany); Schmidt, C. [University of Luebeck, Institute of Biochemistry (Germany); Boenisch, H. [Center for Structural Biochemistry, Karolinska Institutet, Dept. of Biosciences at NOVUM (Sweden); Gnida, M.; Meyer-Klaucke, W. [DESY, EMBL Outstation Hamburg (Germany)

    2004-12-15

    The structural and spectroscopic properties of the biologically active [Fe-4S] site of three different mutants of the wild-type rubredoxin from the archaeon Pyrococcus abyssi were investigated and compared with each other and additionally with those of the rubredoxin from the bacterium Clostridium pasteurianum.

  3. 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 ...

  4. Puromycin-rRNA interaction sites at the peptidyl transferase center

    DEFF Research Database (Denmark)

    Rodriguez-Fonseca, Christina; Phan, Hien; Long, Katherine Sarah

    2000-01-01

    The binding site of puromycin was probed chemically in the peptidyl-transferase center of ribosomes from Escherichia coli and of puromycin-hypersensitive ribosomes from the archaeon Haloferax gibbonsii. Several nucleotides of the 23S rRNAs showed altered chemical reactivities in the presence of p...

  5. STABILITY AND PROTON-PERMEABILITY OF LIPOSOMES COMPOSED OF ARCHAEAL TETRAETHER LIPIDS

    NARCIS (Netherlands)

    Elferink, Marieke G.L.; Wit, Janny G. de; Driessen, Arnold J.M.; Konings, Wil N.

    1994-01-01

    Liposomes composed of tetraether lipids originating from the thermoacidophilic archaeon Sulfolobus acidocaldarius were analyzed for their stability and proton permeability from 20 degrees C up to 80 degrees C. At room temperature, these liposomes are considerably more stable and have a much lower pr

  6. Generation of proton-motive force by an archaeal terminal quinol oxidase from Sulfolobus acidocaldarius

    NARCIS (Netherlands)

    Gleissner, Michael; Elferink, Maria; Driessen, Arnold J.M.; Konings, Wilhelmus; Anemüller, Stefan; Schäfer, Günter

    1994-01-01

    The terminal quinol oxidase of the cytochrome aa3 type was isolated from the extreme thermo-acidophilic archaeon Sulfolobus acidocaldarius. In micellar solution, the enzyme oxidized various quinols and exerted the highest activity with the physiological substrate caldariella quinol. The enzyme was f

  7. Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site

    NARCIS (Netherlands)

    Sayer, Christopher; Finnigan, William; Isupov, Michail N.; Levisson, Mark; Kengen, Servé W.M.; Oost, van der John; Harmer, Nicholas J.; Littlechild, Jennifer A.

    2016-01-01

    A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optim

  8. 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...

  9. Identification and molecular characterization of a novel type of alpha-galactosidase from Pyrococcus furiosus

    NARCIS (Netherlands)

    Lieshout, van J.F.T.; Verhees, C.H.; Ettema, T.J.G.; Sar, van der S.; Imamura, H.; Matsuzawa, H.; Oost, van der J.; Vos, de W.M.

    2003-01-01

    An -galactosidase gene from Pyrococcus furiosus was identified, cloned and functionally expressed in Escherichia coli. It is the first -galactosidase from a hyperthermophilic archaeon described to date. The gene encodes a unique amino acid sequence compared to other -galactosidases. Highest homology

  10. 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

  11. Structural insight into substrate binding and catalysis of a novel 2-keto-3-deoxy-D-arabinonate dehydratase illustrates common mechanistic features of the FAH superfamily

    NARCIS (Netherlands)

    Brouns, S.J.J.; Barends, T.R.M.; Worm, P.; Akerboom, J.; Turnbull, A.P.; Salmon, L.; Oost, van der J.

    2008-01-01

    The archaeon Sulfolobus solfataricus converts d-arabinose to 2-oxoglutarate by an enzyme set consisting of two dehydrogenases and two dehydratases. The third step of the pathway is catalyzed by a novel 2-keto-3-deoxy-D-arabinonate dehydratase (KdaD). In this study, the crystal structure of the enzym

  12. Identification of a system required for the functional surface localization of sugar binding proteins with class III signal peptides in Sulfolobus solfataricus

    NARCIS (Netherlands)

    Zolghadr, Behnam; Weber, Stefan; Szabo, Zalan; Driessen, Arnold J. M.; Albers, Sonja-Verena

    2007-01-01

    The hyperthermophilic archaeon Sulfolobus solfataricus contains an unusual large number of sugar binding proteins that are synthesized as precursors with a class III signal peptide. Such signal peptides are commonly used to direct archaeal flagellin subunits or bacterial (pseudo)pilins into extracel

  13. Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry

    DEFF Research Database (Denmark)

    Kirpekar, F; Douthwaite, S; Roepstorff, P

    2000-01-01

    and the archaeon Sulfolobus acidocaldarius, as well as the halophile archaea Halobacterium halobium and Haloarcula marismortui. One S. acidocaldarius posttranscriptional modification was identified and was further characterized by PSD as a methylation of cytidine32. The modified C is located in a region...

  14. Evaluation of Three Automated Genome Annotations for Halorhabdus utahensis

    DEFF Research Database (Denmark)

    Bakke, Peter; Carney, Nick; DeLoache, Will

    2009-01-01

    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...

  15. 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...

  16. The tungsten metallome of Pyrococcus furiosus

    NARCIS (Netherlands)

    Sevcenco, A.M.; Pinkse, M.; Bol, E.; Krijgen, G.; Wolterbeek, H.; Verhaert, P.D.E.M.; Hagedoorn, P.L.; Hagen, W.R.

    2009-01-01

    The tungsten metallome of the hyperthermophilic archaeon Pyrococcus furiosus has been investigated using electroanalytical metal analysis and native–native 2D-PAGE with the radioactive tungsten isotope W-187 (t1/2 = 23.9 h). P. furiosus cells have an intracellular tungsten concentration of 29 mM, of

  17. The tungsten metallome of Pyrococcus furiosus

    NARCIS (Netherlands)

    Sevcenco, A.M.; Pinkse, M.W.H.; Bol, E.; Krijger, G.C.; Wolterbeek, H.T.; Verhaert, P.; Hagedoorn, P.L.; Hagen, W.R.

    2009-01-01

    The tungsten metallome of the hyperthermophilic archaeon Pyrococcus furiosus has been investigated using electroanalytical metal analysis and native-native 2D-PAGE with the radioactive tungsten isotope W-187 (t(1/2) = 23.9 h). P. furiosus cells have an intracellular tungsten concentration of 29 mu M

  18. A global transcriptional regulator in Thermococcus kodakaraensis controls the expression levels of both glycolytic and gluconeogenic enzyme-encoding genes

    NARCIS (Netherlands)

    Kanai, T.; Akerboom, A.P.; Takedomi, S.; Werken, van de H.J.G.; Blombach, F.; Oost, van der J.; Murakami, T.; Atomi, H.; Imanaka, T.

    2007-01-01

    We identified a novel regulator, Thermococcales glycolytic regulator (Tgr), functioning as both an activator and a repressor of transcription in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Tgr (TK1769) displays similarity (28% identical) to Pyrococcus furiosus TrmB (PF1743), a tr

  19. Permanent draft genome sequence of Desulfurococcus mobilis type strain DSM 2161, a thermoacidophilic sulfur-reducing crenarchaeon isolated from acidic hot springs of Hveravellir, Iceland.

    Science.gov (United States)

    Susanti, Dwi; Johnson, Eric F; Lapidus, Alla; Han, James; Reddy, T B K; Pilay, Manoj; Ivanova, Natalia N; Markowitz, Victor M; Woyke, Tanja; Kyrpides, Nikos C; Mukhopadhyay, Biswarup

    2016-01-01

    This report presents the permanent draft genome sequence of Desulfurococcus mobilis type strain DSM 2161, an obligate anaerobic hyperthermophilic crenarchaeon that was isolated from acidic hot springs in Hveravellir, Iceland. D. mobilis utilizes peptides as carbon and energy sources and reduces elemental sulfur to H2S. A metabolic construction derived from the draft genome identified putative pathways for peptide degradation and sulfur respiration in this archaeon. Existence of several hydrogenase genes in the genome supported previous findings that H2 is produced during the growth of D. mobilis in the absence of sulfur. Interestingly, genes encoding glucose transport and utilization systems also exist in the D. mobilis genome though this archaeon does not utilize carbohydrate for growth. The draft genome of D. mobilis provides an additional mean for comparative genomic analysis of desulfurococci. In addition, our analysis on the Average Nucleotide Identity between D. mobilis and Desulfurococcus mucosus suggested that these two desulfurococci are two different strains of the same species.

  20. The antibiotic thiostrepton inhibits a functional transition within protein L11 at the ribosomal GTPase centre

    DEFF Research Database (Denmark)

    Porse, B T; Leviev, I; Mankin, A S

    1998-01-01

    A newly identified class of highly thiostrepton-resistant mutants of the archaeon Halobacterium halobium carry a missense mutation at codon 18 within the gene encoding ribosomal protein L11. In the mutant proteins, a proline, conserved in archaea and bacteria, is converted to either serine or thr....... This putative inhibitory mechanism of thiostrepton is critically dependent on proline 18/22. Moreover, the absence of this proline from eukaryotic protein L11 sequences would account for the high thiostrepton resistance of eukaryotic ribosomes.......A newly identified class of highly thiostrepton-resistant mutants of the archaeon Halobacterium halobium carry a missense mutation at codon 18 within the gene encoding ribosomal protein L11. In the mutant proteins, a proline, conserved in archaea and bacteria, is converted to either serine...

  1. Effect of physical constraints on the mechanisms of membrane fusion: bolaform lipid vesicles as model systems.

    OpenAIRE

    1996-01-01

    Bolaform lipid vesicles were used to study the effect of physical constraints on membrane fusion. In these vesicles the membrane is organized in a single monolayer, because of the presence of covalent bonds in its middle plane. Therefore, the formation of fusion intermediates is subject to higher energy barriers and greater geometrical constraints than is usual in bilayer membranes. Bolaform lipids were extracted from the thermophilic archaeon Sulfolobus solfataricus. These lipids can be divi...

  2. Purple membranes from Halobacterium salinarum as building blocks for nanobiotechnology: The importance of mechanical and thermal properties for matrix and surface applications

    OpenAIRE

    Rhinow, Daniel Christopher

    2008-01-01

    Bacteriorhodopsin (BR) is a light-driven proton pump and the key protein in halobacterial photosynthesis. In its native host, the archaeon Halobacterium salinarum, BR trimers arrange into a 2-D crystalline lattice, the so-called purple membranes (PMs) which comprise BR and lipids only. Along with the PM assembly BR is astonishingly stable against thermal and chemical stress which makes it an excellent candidate for a variety of ...

  3. 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.

  4. Novel class III phosphoribosyl diphosphate synthase: structure and properties of the tetrameric, phosphate-activated, non-allosterically inhibited enzyme from Methanocaldococcus jannaschii

    DEFF Research Database (Denmark)

    Kadziola, Anders; Jepsen, Clemens H; Johansson, Eva;

    2005-01-01

    The prs gene encoding phosphoribosyl diphosphate (PRPP) synthase of the hyperthermophilic autotrophic methanogenic archaeon Methanocaldococcus jannaschii has been cloned and expressed in Escherichia coli. Subsequently, M.jannaschii PRPP synthase has been purified, characterised, crystallised, and....... The properties of M.jannaschii PRPP synthase differ widely from previously characterised PRPP synthases by its tetrameric quaternary structure and the simultaneous phosphate ion-activation and lack of allosteric inhibition, and, thus, constitute a novel class of PRPP synthases....

  5. 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...

  6. New Therapeutic Strategies for Antibiotic-Resistant Select Agents

    Science.gov (United States)

    2007-12-31

    Soriano, A., Zhao, W., Gullo, V. P., and Chan, T.-M. (2004) Two new bacterial DNA primase inhibitors from the plant Polygonum cuspidatum, Bioorg...hyperthermophilic archaeon Pyrococcus horikoshii, J. Biochem. 130, 727-730. 26. Sheaff, R. J., and Kuchta, R. D. (1993) Mechanism of calf thymus DNA primase...Misincorporation of nucleotides by calf thymus DNA primase and elongation of primers containing multiple noncognate nucleotides by DNA-polymerase-alpha, J

  7. Sequence, Structure, and Binding Analysis of Cyclodextrinase (TK1770) from T. kodakarensis (KOD1) Using an In Silico Approach

    OpenAIRE

    Ramzan Ali; Muhammad Imtiaz Shafiq

    2015-01-01

    Thermostable cyclodextrinase (Tk1770 CDase) from hyperthermophilic archaeon Thermococcus kodakarensis (KOD1) hydrolyzes cyclodextrins into linear dextrins. The sequence of Tk1770 CDase retrieved from UniProt was aligned with sequences of sixteen CD hydrolyzing enzymes and a phylogenetic tree was constructed using Bayesian inference. The homology model of Tk1770 CDase was constructed and optimized with Modeller v9.14 program. The model was validated with ProSA server and PROCHECK analysis. Fou...

  8. Paracatenula, an ancient symbiosis between thiotrophic Alphaproteobacteria and catenulid flatworms

    OpenAIRE

    Gruber-Vodicka, Harald Ronald; Dirks, Ulrich; Leisch, Nikolaus; Baranyi, Christian; Stoecker, Kilian; Bulgheresi, Silvia; Heindl, Niels Robert; Horn, Matthias; Lott, Christian; Loy, Alexander; Wagner, Michael; Ott, Jörg

    2011-01-01

    Harnessing chemosynthetic symbionts is a recurring evolutionary strategy. Eukaryotes from six phyla as well as one archaeon have acquired chemoautotrophic sulfur-oxidizing bacteria. In contrast to this broad host diversity, known bacterial partners apparently belong to two classes of bacteria—the Gamma- and Epsilonproteobacteria. Here, we characterize the intracellular endosymbionts of the mouthless catenulid flatworm genus Paracatenula as chemoautotrophic sulfur-oxidizing Alphaproteobacteria...

  9. Pivotal Enzyme in Glutamate Metabolism of Poly-γ-Glutamate-Producing Microbes

    OpenAIRE

    Tohru Kamei; Takashi Yamamoto; Makoto Ashiuchi

    2013-01-01

    The extremely halophilic archaeon Natrialba aegyptiaca secretes the L-homo type of poly-g-glutamate (PGA) as an extremolyte. We examined the enzymes involved in glutamate metabolism and verified the presence of L-glutamate dehydrogenases, L-aspartate aminotransferase, and L-glutamate synthase. However, neither glutamate racemase nor D-amino acid aminotransferase activity was detected, suggesting the absence of sources of D-glutamate. In contrast, D-glutamate-rich PGA producers mostly possess ...

  10. Effects of Nitrogen and Carbon Sources on Transcription of Soluble Methyltransferases in Methanosarcina mazei Strain Gö1†

    OpenAIRE

    Veit, Katharina; Ehlers, Claudia; Schmitz, Ruth A.

    2005-01-01

    The methanogenic archaeon Methanosarcina mazei strain Gö1 uses versatile carbon sources and is able to fix molecular nitrogen with methanol as carbon and energy sources. Here, we demonstrate that when growing on trimethylamine (TMA), nitrogen fixation does not occur, indicating that ammonium released during TMA degradation is sufficient to serve as a nitrogen source and represses nif gene induction. We further report on the transcriptional regulation of soluble methyltransferases, which catal...

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

    Energy Technology Data Exchange (ETDEWEB)

    Reitz, T.; Merroun, M.L.; Rossberg, A.; Steudtner, R.; Selenska-Pobell, S. [Helmholtz-Zentrum Dresden-Rossendorf, Dresden (Germany). Inst. of Radiochemistry

    2011-07-01

    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.)

  12. Evidence of molecular adaptation to extreme environments and applicability to space environments

    CERN Document Server

    Filipovic, M D; Ognjanovic, M

    2008-01-01

    This is initial study of a gene signatures responsible for adapting microscopic life to the life in extreme Earth environments. We present a results on ID of the clusters of COGs common to several hyperthermophiles and exclusion of those common to a mesophile: E.coli.K12, will yield a group of proteins possibly involved in adaptation to life under extreme T. Methanogens stand out as the only group of organisms that have species capable of growth at 0C (M.frigidum and M.burtonii) and 110C (M.kandleri). Not all the components of heat adaptation can be attributed to novel genes, the chaperones known as heat shock proteins stabilize the enzymes under elevated temperature. Highly conserved chaperons found in bacteria and eukaryots are not present in hyperthermophilic Archea, rather, they have a unique chaperone TF55. Our aim is to use software which we specifically developed for extremophile genome comparative analyses in order to search for additional novel genes involved in hyperthermophile adaptation. The follo...

  13. Characteristics Of Phytases From Soybeans And Microorganisms Involved In The Tempe Production

    Directory of Open Access Journals (Sweden)

    K A Buckle2

    2004-12-01

    Full Text Available Tempe was prepared from Forest variety of soybeans inoculated with pure culture of the mould Rhizopus oligosporus strain CT11K2. Partially purified phytases from soybeans, mould of the R. oligosporus strain CT11K2, yeasts (Endomycopsis burtonii, Candida diddensiae and Candida tropicalis and bacteria (Streptococcus faecium and Streptococcus dysgalactiae involved during tempe production were prepared according to procedure developed by Sutardi (1988. The crude phytases were characterized and phytic acid, inorganic and total phosphorous content of soybeans, intermediate products and tempe, were also determined.Results show that phytases, produced by microorganisms involved in tempe production, and by endogenous soybean phytase, had significantly different characteristics especially on optimum pH, temperature and substrate, Km and Vmax values, inactivation and activation energy and also temperature coefficient. All phytases contribute to the reduction in phytic acid content; and mould phytase, especially the extra-cellular enzyme, and the endogenous soybean phytase, showed the dominant effect on phytic acid degradation compared with other microbial phytases and physical treatments such as soaking, boiling and steaming.Phytic acid content of soybeans was reduced by more than 50% from about 10.4 to about 4.9 - 5.0 mg/g (DWB in fresh tempe (after 40 h fermentation at 30oC

  14. Assessment of the yeast species composition of cocoa bean fermentations in different cocoa-producing regions using denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Papalexandratou, Zoi; De Vuyst, Luc

    2011-11-01

    The yeast species composition of 12 cocoa bean fermentations carried out in Brazil, Ecuador, Ivory Coast and Malaysia was investigated culture-independently. Denaturing gradient gel electrophoresis of 26S rRNA gene fragments, obtained through polymerase chain reaction with universal eukaryotic primers, was carried out with two different commercial apparatus (the DCode and CBS systems). In general, this molecular method allowed a rapid monitoring of the yeast species prevailing during fermentation. Under similar and optimal denaturing gradient gel electrophoresis conditions, the CBS system allowed a better separated band pattern than the DCode system and an unambiguous detection of the prevailing species present in the fermentation samples. The most frequent yeast species were Hanseniaspora sp., followed by Pichia kudriavzevii and Saccharomyces cerevisiae, independent of the origin of the cocoa. This indicates a restricted yeast species composition of the cocoa bean fermentation process. Exceptionally, the Ivorian cocoa bean box fermentation samples showed a wider yeast species composition, with Hyphopichia burtonii and Meyerozyma caribbica among the main representatives. Yeasts were not detected in the samples when the temperature inside the fermenting cocoa pulp-bean mass reached values higher than 45 °C or under early acetic acid production conditions.

  15. Identification of yeast strains isolated from marcha in Sikkim, a microbial starter for amylolytic fermentation.

    Science.gov (United States)

    Tsuyoshi, Naoko; Fudou, Ryosuke; Yamanaka, Shigeru; Kozaki, Michio; Tamang, Namrata; Thapa, Saroj; Tamang, Jyoti P

    2005-03-15

    Marcha or murcha is a traditional amylolytic starter used to produce sweet-sour alcoholic drinks, commonly called jaanr in the Himalayan regions of India, Nepal, Bhutan, and Tibet (China). The aim of this study was to examine the microflora of marcha collected from Sikkim in India, focusing on yeast flora and their roles. Twenty yeast strains were isolated from six samples of marcha and identified by genetic and phenotypic methods. They were first classified into four groups (Group I, II, III, and IV) based on physiological features using an API test. Phylogenetic, morphological, and physiological characterization identified the isolates as Saccharomyces bayanus (Group I); Candida glabrata (Group II); Pichia anomala (Group III); and Saccharomycopsis fibuligera, Saccharomycopsis capsularis, and Pichia burtonii (Group IV). Among them, the Group I, II, and III strains produced ethanol. The isolates of Group IV had high amylolytic activity. Because all marcha samples tested contained both starch degraders and ethanol producers, it was hypothesized that all four groups of yeast (Group I, II, III, and IV) contribute to starch-based alcohol fermentation.

  16. Methanogenic diversity and activity in hypersaline sediments of the centre of the Napoli mud volcano, Eastern Mediterranean Sea.

    Science.gov (United States)

    Lazar, Cassandre Sara; Parkes, R John; Cragg, Barry A; L'Haridon, Stéphane; Toffin, Laurent

    2011-08-01

    Submarine mud volcanoes are a significant source of methane to the atmosphere. The Napoli mud volcano, situated in the brine-impacted Olimpi Area of the Eastern Mediterranean Sea, emits mainly biogenic methane particularly at the centre of the mud volcano. Temperature gradients support the suggestion that Napoli is a cold mud volcano with moderate fluid flow rates. Biogeochemical and molecular genetic analyses were carried out to assess the methanogenic activity rates, pathways and diversity in the hypersaline sediments of the centre of the Napoli mud volcano. Methylotrophic methanogenesis was the only significant methanogenic pathway in the shallow sediments (0-40 cm) but was also measured throughout the sediment core, confirming that methylotrophic methanogens could be well adapted to hypersaline environments. Hydrogenotrophic methanogenesis was the dominant pathway below 50 cm; however, low rates of acetoclastic methanogenesis were also present, even in sediment layers with the highest salinity, showing that these methanogens can thrive in this extreme environment. PCR-DGGE and methyl coenzyme M reductase gene libraries detected sequences affiliated with anaerobic methanotrophs (mainly ANME-1) as well as Methanococcoides methanogens. Results show that the hypersaline conditions in the centre of the Napoli mud volcano influence active biogenic methane fluxes and methanogenic/methylotrophic diversity.

  17. Archaeal populations in hypersaline sediments underlying orange microbial mats in the Napoli mud volcano.

    Science.gov (United States)

    Lazar, Cassandre Sara; L'haridon, Stéphane; Pignet, Patricia; Toffin, Laurent

    2011-05-01

    Microbial mats in marine cold seeps are known to be associated with ascending sulfide- and methane-rich fluids. Hence, they could be visible indicators of anaerobic oxidation of methane (AOM) and methane cycling processes in underlying sediments. The Napoli mud volcano is situated in the Olimpi Area that lies on saline deposits; from there, brine fluids migrate upward to the seafloor. Sediments associated with a brine pool and microbial orange mats of the Napoli mud volcano were recovered during the Medeco cruise. Based on analysis of RNA-derived sequences, the "active" archaeal community was composed of many uncultured lineages, such as rice cluster V or marine benthic group D. Function methyl coenzyme M reductase (mcrA) genes were affiliated with the anaerobic methanotrophic Archaea (ANME) of the ANME-1, ANME-2a, and ANME-2c groups, suggesting that AOM occurred in these sediment layers. Enrichment cultures showed the presence of viable marine methylotrophic Methanococcoides in shallow sediment layers. Thus, the archaeal community diversity seems to show that active methane cycling took place in the hypersaline microbial mat-associated sediments of the Napoli mud volcano.

  18. 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....

  19. AcEST: BP919260 [AcEST

    Lifescience Database Archive (English)

    Full Text Available YMU001_000123_A09 469 Adiantum capillus-veneris mRNA. clone: YMU001_000123_A09. BP919260 - Show BP919260...is mRNA. clone: YMU001_000123_A09. Accession BP919260 Tissue type prothallium Developmental stage - Contig I...earch programs, Nucleic Acids Res. 25:3389-3402. Query= BP919260|Adiantum capillu...ion of protein database search programs, Nucleic Acids Res. 25:3389-3402. Query= BP919260...ed signal transduction protein OS=Uncultured methanogenic archaeon RC-I GN=UNCMA_12260

  20. Complete genome sequence of Archaeoglobus profundus type strain (AV18T)

    Energy Technology Data Exchange (ETDEWEB)

    von Jan, Mathias [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Glavina Del Rio, Tijana [U.S. Department of Energy, Joint Genome Institute; Copeland, A [U.S. Department of Energy, Joint Genome Institute; Tice, Hope [U.S. Department of Energy, Joint Genome Institute; Cheng, Jan-Fang [U.S. Department of Energy, Joint Genome Institute; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Chen, Feng [U.S. Department of Energy, Joint Genome Institute; Nolan, Matt [U.S. Department of Energy, Joint Genome Institute; Goodwin, Lynne A. [Los Alamos National Laboratory (LANL); Han, Cliff [Los Alamos National Laboratory (LANL); Pitluck, Sam [U.S. Department of Energy, Joint Genome Institute; Liolios, Konstantinos [U.S. Department of Energy, Joint Genome Institute; Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Ovchinnikova, Galina [U.S. Department of Energy, Joint Genome Institute; Chertkov, Olga [Los Alamos National Laboratory (LANL); Pati, Amrita [U.S. Department of Energy, Joint Genome Institute; Chen, Amy [U.S. Department of Energy, Joint Genome Institute; Palaniappan, Krishna [U.S. Department of Energy, Joint Genome Institute; Land, Miriam L [ORNL; Hauser, Loren John [ORNL; Chang, Yun-Juan [ORNL; Jeffries, Cynthia [Oak Ridge National Laboratory (ORNL); Saunders, Elizabeth H [Los Alamos National Laboratory (LANL); Brettin, Thomas S [ORNL; Detter, J. Chris [U.S. Department of Energy, Joint Genome Institute; Chain, Patrick S. G. [Lawrence Livermore National Laboratory (LLNL); Eichinger, Konrad [Universitat Regensburg, Regensburg, Germany; Huber, Harald [Universitat Regensburg, Regensburg, Germany; Spring, Stefan [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Rohde, Manfred [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Goker, Markus [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Wirth, Reinhard [Universitat Regensburg, Regensburg, Germany; Woyke, Tanja [U.S. Department of Energy, Joint Genome Institute; Bristow, James [U.S. Department of Energy, Joint Genome Institute; Eisen, Jonathan [U.S. Department of Energy, Joint Genome Institute; Markowitz, Victor [U.S. Department of Energy, Joint Genome Institute; Hugenholtz, Philip [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute; Klenk, Hans-Peter [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany

    2010-01-01

    Archaeoglobus profundus (Burggraf et al. 1990) is a hyperthermophilic archaeon in the euryarchaeal class Archaeoglobi, which is currently represented by six validly named species and two taxonomically challenged 'Geoglobus' strains, all belonging to the same family Archaeoglobaceae. All members were isolated from marine hydrothermal habitats and are obligate anaerobes. Here we describe the features of the organism, together with the complete genome sequence and annotation. This is the second completed genome sequence of a member of the class Archaeoglobi. The 1,563,423 bp genome with its 1,858 protein-coding and 52 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  1. Acylation of Quercetin with a Novel Thermophilic Esterase as Biocatalyst

    Institute of Scientific and Technical Information of China (English)

    XIE Xiao-na; ZHANG Chun-li; XUN Er-na; WANG Jia-xin; ZHANG Hong; WANG Lei; WANG Zhi

    2012-01-01

    The regioselective acylation of quercetin catalyzed by a novel thermophilic esterase(APE1547)from the archaeon Aeropyrum pernix K1 was successfully conducted in organic solvents.The effects of acyl donor,substrate ratio,organic solvent,temperature,and water activity were investigated.Under the optimum conditions,a yield of 74% for its mono ester could be achieved in the reaction for about 6 h.With the reaction time extending to about 30 h,the final conversion of quercetin was about 100% and three products were synthesized.

  2. Mutations and Rearrangements in the Genome of Sulfolobus solfataricus P2

    DEFF Research Database (Denmark)

    Redder, P.; Garrett, R. A.

    2006-01-01

    The genome of Sulfolobus solfataricus P2 carries a larger number of transposable elements than any other sequenced genome from an archaeon or bacterium and, as a consequence, may be particularly susceptible to rearrangement and change. In order to gain more insight into the natures and frequencies...... were defined using a specially developed "in vitro library" strategy. Moreover, while searching for the donor mobile elements, evidence was found for two major changes that had occurred in the genome of strain P2, one constituting a single deletion of about 4% of the total genome (124 kb), while...

  3. 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-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are widespread in archaea and bacteria, and research on their molecular mechanisms has led to the development of genome-editing techniques based on a few Type II systems. However, there has not been any...... 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...

  4. Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR.

    OpenAIRE

    1997-01-01

    The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conse...

  5. Sites of interaction of streptogramin A and B antibiotics in the peptidyl transferase loop of 23 S rRNA and the synergism of their inhibitory mechanisms

    DEFF Research Database (Denmark)

    Porse, B T; Garrett, R A

    1999-01-01

    Streptogramin antibiotics contain two active A and B components that inhibit peptide elongation synergistically. Mutants resistant to the A component (virginiamycin M1 and pristinamycin IIA) were selected for the archaeon Halobacterium halobium. The mutations mapped to the universally conserved...... and within the bacterial cells. It is inferred that position 2058 and the sites of mutation, A2059 and A2503, are involved in the synergistic inhibition by the two antibiotics. A structural model is presented which links A2059 and A2503 and provides a structural rationale for the rRNA footprints....

  6. 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...... 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 metabolism...

  7. Spatial variations of community structures and methane cycling across a transect of Lei-Gong-Hou mud volcanoes in eastern Taiwan

    Directory of Open Access Journals (Sweden)

    Pei-Ling eWang

    2014-03-01

    Full Text Available This study analyzed cored sediments retrieved from sites distributed across a transect of the Lei-Gong-Hou mud volcanoes in eastern Taiwan to uncover the spatial distributions of biogeochemical processes and community assemblages involved in methane cycling. The profiles of methane concentration and carbon isotopic composition revealed various orders of the predominance of specific methane-related metabolisms along depth. At a site proximal to the bubbling pool, the methanogenic zone was sandwiched by the anaerobic methanotrophic zones. For two sites distributed toward the topographic depression, the methanogenic zone overlaid the anaerobic methanotrophic zone. The predominance of anaerobic methanotrophy at specific depth intervals is supported by the enhanced copy numbers of the ANME-2a 16S rRNA gene and coincides with high dissolved Fe/Mn concentrations and copy numbers of the Desulfuromonas/Pelobacter 16S rRNA gene. Assemblages of 16S rRNA and mcrA genes revealed that methanogenesis was mediated by Methanococcoides and Methanosarcina. pmoA genes and a few 16S rRNA genes related to aerobic methanotrophs were detected in limited numbers of subsurface samples. While dissolved Fe/Mn signifies the presence of anaerobic metabolisms near the surface, the correlations between geochemical characteristics and gene abundances, and the absence of aerobic methanotrophs in top sediments suggest that anaerobic methanotrophy is potentially dependent on iron/manganese reduction and dominates over aerobic methanotrophy for the removal of methane produced in situ or from a deep source. Near-surface methanogenesis contributes to the methane emissions from mud platform. The alternating arrangements of methanogenic and methanotrophic zones at different sites suggest that the interactions between mud deposition, evaporation, oxidation and fluid transport modulate the assemblages of microbial communities and methane cycling in different compartments of terrestrial

  8. Archaeal community diversity and abundance changes along a natural salinity gradient in estuarine sediments.

    Science.gov (United States)

    Webster, Gordon; O'Sullivan, Louise A; Meng, Yiyu; Williams, Angharad S; Sass, Andrea M; Watkins, Andrew J; Parkes, R John; Weightman, Andrew J

    2015-02-01

    Archaea are widespread in marine sediments, but their occurrence and relationship with natural salinity gradients in estuarine sediments is not well understood. This study investigated the abundance and diversity of Archaea in sediments at three sites [Brightlingsea (BR), Alresford (AR) and Hythe (HY)] along the Colne Estuary, using quantitative real-time PCR (qPCR) of 16S rRNA genes, DNA hybridization, Archaea 16S rRNA and mcrA gene phylogenetic analyses. Total archaeal 16S rRNA abundance in sediments were higher in the low-salinity brackish sediments from HY (2-8 × 10(7) 16S rRNA gene copies cm(-3)) than the high-salinity marine sites from BR and AR (2 × 10(4)-2 × 10(7) and 4 × 10(6)-2 × 10(7) 16S rRNA gene copies cm(-3), respectively), although as a proportion of the total prokaryotes Archaea were higher at BR than at AR or HY. Phylogenetic analysis showed that members of the 'Bathyarchaeota' (MCG), Thaumarchaeota and methanogenic Euryarchaeota were the dominant groups of Archaea. The composition of Thaumarchaeota varied with salinity, as only 'marine' group I.1a was present in marine sediments (BR). Methanogen 16S rRNA genes from low-salinity sediments at HY were dominated by acetotrophic Methanosaeta and putatively hydrogentrophic Methanomicrobiales, whereas the marine site (BR) was dominated by mcrA genes belonging to methylotrophic Methanococcoides, versatile Methanosarcina and methanotrophic ANME-2a. Overall, the results indicate that salinity and associated factors play a role in controlling diversity and distribution of Archaea in estuarine sediments.

  9. Spatial variations of community structures and methane cycling across a transect of Lei-Gong-Hou mud volcanoes in eastern Taiwan.

    Science.gov (United States)

    Wang, Pei-Ling; Chiu, Yi-Ping; Cheng, Ting-Wen; Chang, Yung-Hsin; Tu, Wei-Xain; Lin, Li-Hung

    2014-01-01

    This study analyzed cored sediments retrieved from sites distributed across a transect of the Lei-Gong-Hou mud volcanoes in eastern Taiwan to uncover the spatial distributions of biogeochemical processes and community assemblages involved in methane cycling. The profiles of methane concentration and carbon isotopic composition revealed various orders of the predominance of specific methane-related metabolisms along depth. At a site proximal to the bubbling pool, the methanogenic zone was sandwiched by the anaerobic methanotrophic zones. For two sites distributed toward the topographic depression, the methanogenic zone overlaid the anaerobic methanotrophic zone. The predominance of anaerobic methanotrophy at specific depth intervals is supported by the enhanced copy numbers of the ANME-2a 16S rRNA gene and coincides with high dissolved Fe/Mn concentrations and copy numbers of the Desulfuromonas/Pelobacter 16S rRNA gene. Assemblages of 16S rRNA and mcrA genes revealed that methanogenesis was mediated by Methanococcoides and Methanosarcina. pmoA genes and a few 16S rRNA genes related to aerobic methanotrophs were detected in limited numbers of subsurface samples. While dissolved Fe/Mn signifies the presence of anaerobic metabolisms near the surface, the correlations between geochemical characteristics and gene abundances, and the absence of aerobic methanotrophs in top sediments suggest that anaerobic methanotrophy is potentially dependent on iron/manganese reduction and dominates over aerobic methanotrophy for the removal of methane produced in situ or from a deep source. Near-surface methanogenesis contributes to the methane emissions from mud platform. The alternating arrangements of methanogenic and methanotrophic zones at different sites suggest that the interactions between mud deposition, evaporation, oxidation and fluid transport modulate the assemblages of microbial communities and methane cycling in different compartments of terrestrial mud volcanoes.

  10. Lysine and arginine biosyntheses mediated by a common carrier protein in Sulfolobus.

    Science.gov (United States)

    Ouchi, Takuya; Tomita, Takeo; Horie, Akira; Yoshida, Ayako; Takahashi, Kento; Nishida, Hiromi; Lassak, Kerstin; Taka, Hikari; Mineki, Reiko; Fujimura, Tsutomu; Kosono, Saori; Nishiyama, Chiharu; Masui, Ryoji; Kuramitsu, Seiki; Albers, Sonja-Verena; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2013-04-01

    LysW has been identified as a carrier protein in the lysine biosynthetic pathway that is active through the conversion of α-aminoadipate (AAA) to lysine. In this study, we found that the hyperthermophilic archaeon, Sulfolobus acidocaldarius, not only biosynthesizes lysine through LysW-mediated protection of AAA but also uses LysW to protect the amino group of glutamate in arginine biosynthesis. In this archaeon, after LysW modification, AAA and glutamate are converted to lysine and ornithine, respectively, by a single set of enzymes with dual functions. The crystal structure of ArgX, the enzyme responsible for modification and protection of the amino moiety of glutamate with LysW, was determined in complex with LysW. Structural comparison and enzymatic characterization using Sulfolobus LysX, Sulfolobus ArgX and Thermus LysX identify the amino acid motif responsible for substrate discrimination between AAA and glutamate. Phylogenetic analysis reveals that gene duplication events at different stages of evolution led to ArgX and LysX.

  11. Homology modelling of two subtilisin-like proteases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus stetteri.

    Science.gov (United States)

    Voorhorst, W G; Warner, A; de Vos, W M; Siezen, R J

    1997-08-01

    The hyperthermophilic archaeon Pyrococcus furiosus produces an extracellular, glycosylated hyperthermostable subtilisin-like serine protease, termed pyrolysin (Voorhorst,W.G.B., Eggen,R.I.L., Geerling,A.C.M., Platteeuw,C., Siezen,R.J. and de Vos,W.M. (1996) J. Biol. Chem., 271, 20426-20431). Based on the pyrolysin coding sequence, a pyrolysin-like gene fragment was cloned and characterized from the extreme thermophilic archaeon Thermococcus stetteri. Like pyrolysin, the deduced sequence of this serine protease, designated stetterlysin, contains a catalytic domain with high homology with other subtilases, allowing homology modelling starting from known crystal structures. Comparison of the predicted three-dimensional models of the catalytic domain of stetterlysin and pyrolysin with the crystal structure of subtilases from mesophilic and thermophilic origin, i.e. subtilisin BPN' and thermitase, and the homology model of subtilisin S41 from psychrophilic origin, led to the identification of features that could be related to protein stabilization. Higher thermostability was found to be correlated with an increased number of residues involved in pairs and networks of charge-charge and aromatic-aromatic interactions. These highly thermostable proteases have several extra surface loops and inserts with a relatively high frequency of aromatic residues and Asn residues. The latter are often present in putative N-glycosylation sites. Results from modelling of known substrates in the substrate-binding region support the broad substrate range and the autocatalytic activation previously suggested for pyrolysin.

  12. A predictive computational model of the kinetic mechanism of stimulus-induced transducer methylation and feedback regulation through CheY in archaeal phototaxis and chemotaxis

    Directory of Open Access Journals (Sweden)

    Oesterhelt Dieter

    2010-03-01

    Full Text Available Abstract Background Photo- and chemotaxis of the archaeon Halobacterium salinarum is based on the control of flagellar motor switching through stimulus-specific methyl-accepting transducer proteins that relay the sensory input signal to a two-component system. Certain members of the transducer family function as receptor proteins by directly sensing specific chemical or physical stimuli. Others interact with specific receptor proteins like the phototaxis photoreceptors sensory rhodopsin I and II, or require specific binding proteins as for example some chemotaxis transducers. Receptor activation by light or a change in receptor occupancy by chemical stimuli results in reversible methylation of glutamate residues of the transducer proteins. Both, methylation and demethylation reactions are involved in sensory adaptation and are modulated by the response regulator CheY. Results By mathematical modeling we infer the kinetic mechanisms of stimulus-induced transducer methylation and adaptation. The model (deterministic and in the form of ordinary differential equations correctly predicts experimentally observed transducer demethylation (as detected by released methanol in response to attractant and repellent stimuli of wildtype cells, a cheY deletion mutant, and a mutant in which the stimulated transducer species is methylation-deficient. Conclusions We provide a kinetic model for signal processing in photo- and chemotaxis in the archaeon H. salinarum suggesting an essential role of receptor cooperativity, antagonistic reversible methylation, and a CheY-dependent feedback on transducer demethylation.

  13. Microbial weeds in hypersaline habitats: the enigma of the weed-like Haloferax mediterranei

    Science.gov (United States)

    Oren, Aharon; Hallsworth, John E.

    2014-10-01

    Heterotrophic prokaryotic communities that inhabit saltern crystallizer ponds are typically dominated by two species, the archaeon Haloquadratum walsbyi and the bacterium Salinibacter ruber, regardless of location. These organisms behave as 'microbial weeds' as defined by Cray et al. (Microb Biotechnol6: 453–492, 2013) that possess the biological traits required to dominate the microbiology of these open habitats. Here, we discuss the enigma of the less abundant Haloferax mediterranei, an archaeon that grows faster than any other, comparable extreme halophile. It has a wide window for salt tolerance, can grow on simple as well as on complex substrates and degrade polymeric substances, has different modes of anaerobic growth, can accumulate storage polymers, produces gas vesicles, and excretes halocins capable of killing other Archaea. Therefore, Hfx. mediterranei is apparently more qualified as a 'microbial weed' than Haloquadratum and Salinibacter. However, the former differs because it produces carotenoid pigments only in the lower salinity range and lacks energy-generating retinal-based, light-driven ion pumps such as bacteriorhodopsin and halorhodopsin. We discuss these observations in relation to microbial weed biology in, and the open-habitat ecology of, hypersaline systems.

  14. Analysis of Carotenoid Production by Halorubrum sp. TBZ126; an Extremely Halophilic Archeon from Urmia Lake

    Science.gov (United States)

    Naziri, Davood; Hamidi, Masoud; Hassanzadeh, Salar; Tarhriz, Vahideh; Maleki Zanjani, Bahram; Nazemyieh, Hossein; Hejazi, Mohammd Amin; Hejazi, Mohammad Saeid

    2014-01-01

    Purpose: Carotenoids are of great interest in many scientific disciplines because of their wide distribution, diverse functions and interesting properties. The present report describes a new natural source for carotenoid production. Methods: Halorubrum sp., TBZ126, an extremely halophilic archaeon, was isolated from Urmia Lack following culture of water sample on marine agar medium and incubation at 30 °C. Then single colonies were cultivated in broth media. After that the cells were collected and carotenoids were extracted with acetone-methanol (7:3 v/v). The identification of carotenoids was performed by UV-VIS spectroscopy and confirmed by thin layer chromatography (TLC) in the presence of antimony pentachloride (SbCl5). The production profile was analyzed using liquid-chromatography mass spectroscopy (LC-MS) techniques. Phenotypic characteristics of the isolate were carried out and the 16S rRNA gene was amplified using polymerase chain reaction (PCR). Results: LC-MS analytical results revealed that produced carotenoids are bacterioruberin, lycopene and β-carotene. Bacterioruberin was found to be the predominant produced carotenoid. 16S rRNA analysis showed that TBZ126 has 100% similarity with Halorubrum chaoviator Halo-G*T (AM048786). Conclusion: Halorubrum sp. TBZ126, isolated from Urmia Lake has high capacity in the production of carotenoids. This extremely halophilic archaeon could be considered as a prokaryotic candidate for carotenoid production source for future studies. PMID:24409411

  15. Methanothermobacter thermautotrophicus modulates its membrane lipids in response to hydrogen and nutrient availability

    Science.gov (United States)

    Yoshinaga, Marcos Y.; Gagen, Emma J.; Wörmer, Lars; Broda, Nadine K.; Meador, Travis B.; Wendt, Jenny; Thomm, Michael; Hinrichs, Kai-Uwe

    2015-01-01

    Methanothermobacter thermautotrophicus strain ΔH is a model hydrogenotrophic methanogen, for which extensive biochemical information, including the complete genome sequence, is available. Nevertheless, at the cell membrane lipid level, little is known about the responses of this archaeon to environmental stimuli. In this study, the lipid composition of M. thermautotrophicus was characterized to verify how this archaeon modulates its cell membrane components during growth phases and in response to hydrogen depletion and nutrient limitation (potassium and phosphate). As opposed to the higher abundance of phospholipids in the stationary phase of control experiments, cell membranes under nutrient, and energy stress were dominated by glycolipids that likely provided a more effective barrier against ion leakage. We also identified particular lipid regulatory mechanisms in M. thermautotrophicus, which included the accumulation of polyprenols under hydrogen-limited conditions and an increased content of sodiated adducts of lipids in nutrient-limited cells. These findings suggest that M. thermautotrophicus intensely modulates its cell membrane lipid composition to cope with energy and nutrient availability in dynamic environments. PMID:25657645

  16. Methanothermobacter thermautotrophicus modulates its membrane lipids in response to hydrogen and nutrient availability

    Directory of Open Access Journals (Sweden)

    Marcos Yukio Yoshinaga

    2015-01-01

    Full Text Available Methanothermobacter thermautotrophicus strain ∆H is a model hydrogenotrophic methanogen, for which extensive biochemical information, including the complete genome sequence, is available. Nevertheless, at the cell membrane lipid level, little is known about the responses of this archaeon to environmental stimuli. In this study, the lipid composition of M. thermautotrophicus was characterized to verify how this archaeon modulates its cell membrane components during growth phases and in response to hydrogen depletion and nutrient limitation (potassium and phosphate. As opposed to the higher abundance of phospholipids in the stationary phase of control experiments, cell membranes under nutrient and energy stress were dominated by glycolipids that likely provided a more effective barrier against ion leakage. We also identified particular lipid regulatory mechanisms in M. thermautotrophicus, which included the accumulation of polyprenols under hydrogen-limited conditions and an increased content of sodiated adducts of lipids in nutrient-limited cells. These findings suggest that M. thermautotrophicus intensely modulates its cell membrane lipid composition to cope with energy and nutrient availability in dynamic environments.

  17. Thermococcus kodakarensis modulates its polar membrane lipids and elemental composition according to growth stage and phosphate availability

    Science.gov (United States)

    Meador, Travis B.; Gagen, Emma J.; Loscar, Michael E.; Goldhammer, Tobias; Yoshinaga, Marcos Y.; Wendt, Jenny; Thomm, Michael; Hinrichs, Kai-Uwe

    2014-01-01

    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. PMID:24523718

  18. Thermococcus kodakarensis modulates its polar membrane lipids and elemental composition according to growth stage and phosphate availability

    Directory of Open Access Journals (Sweden)

    Travis B. 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.

  19. Analysis of Carotenoid Production by Halorubrum sp. TBZ126; an Extremely Halophilic Archeon from Urmia Lake

    Directory of Open Access Journals (Sweden)

    Davood Naziri

    2014-03-01

    Full Text Available Purpose: Carotenoids are of great interest in many scientific disciplines because of their wide distribution, diverse functions and interesting properties. The present report describes a new natural source for carotenoid production. Methods: Halorubrum sp., TBZ126, an extremely halophilic archaeon, was isolated from Urmia Lack following culture of water sample on marine agar medium and incubation at 30 °C. Then single colonies were cultivated in broth media. After that the cells were collected and carotenoids were extracted with acetone-methanol (7:3 v/v. The identification of carotenoids was performed by UV-VIS spectroscopy and confirmed by thin layer chromatography (TLC in the presence of antimony pentachloride (SbCl5. The production profile was analyzed using liquid-chromatography mass spectroscopy (LC-MS techniques. Phenotypic characteristics of the isolate were carried out and the 16S rRNA gene was amplified using polymerase chain reaction (PCR. Results: LC-MS analytical results revealed that produced carotenoids are bacterioruberin, lycopene and β-carotene. Bacterioruberin was found to be the predominant produced carotenoid. 16S rRNA analysis showed that TBZ126 has 100% similarity with Halorubrum chaoviator Halo-G*T (AM048786. Conclusion: Halorubrum sp. TBZ126, isolated from Urmia Lake has high capacity in the production of carotenoids. This extremely halophilic archaeon could be considered as a prokaryotic candidate for carotenoid production source for future studies.

  20. Structural insights into the adaptation of proliferating cell nuclear antigen (PCNA) from Haloferax volcanii to a high-salt environment

    Energy Technology Data Exchange (ETDEWEB)

    Morgunova, Ekaterina, E-mail: ekaterina.morgunova@ki.se [Karolinska Institutet, NOVUM, Centre of Structural Biochemistry, S-14157 Huddinge (Sweden); Gray, Fiona C. [Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen (Denmark); MacNeill, Stuart A. [Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen (Denmark); Centre for Biomolecular Sciences, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST,Scotland (United Kingdom); Ladenstein, Rudolf [Karolinska Institutet, NOVUM, Centre of Structural Biochemistry, S-14157 Huddinge (Sweden)

    2009-10-01

    The crystal structure of PCNA from the halophilic archaeon H. volcanii reveals specific features of the charge distribution on the protein surface that reflect adaptation to a high-salt environment and suggests a different type of interaction with DNA in halophilic PCNAs. The sliding clamp proliferating cell nuclear antigen (PCNA) plays vital roles in many aspects of DNA replication and repair in eukaryotic cells and in archaea. Realising the full potential of archaea as a model for PCNA function requires a combination of biochemical and genetic approaches. In order to provide a platform for subsequent reverse genetic analysis, PCNA from the halophilic archaeon Haloferax volcanii was subjected to crystallographic analysis. The gene was cloned and expressed in Escherichia coli and the protein was purified by affinity chromatography and crystallized by the vapour-diffusion technique. The structure was determined by molecular replacement and refined at 3.5 Å resolution to a final R factor of 23.7% (R{sub free} = 25%). PCNA from H. volcanii was found to be homotrimeric and to resemble other homotrimeric PCNA clamps but with several differences that appear to be associated with adaptation of the protein to the high intracellular salt concentrations found in H. volcanii cells.

  1. 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

  2. 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-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are widespread in archaea and bacteria, and research on their molecular mechanisms has led to the development of genome-editing techniques based on a few Type II systems. However, there has not been any...... 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-CRISPR...... 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...

  3. 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...

  4. First structure of archaeal branched-chain amino acid aminotransferase from Thermoproteus uzoniensis specific for L-amino acids and R-amines.

    Science.gov (United States)

    Boyko, Konstantin M; Stekhanova, Tatiana N; Nikolaeva, Alena Yu; Mardanov, Andrey V; Rakitin, Andrey L; Ravin, Nikolai V; Bezsudnova, Ekaterina Yu; Popov, Vladimir O

    2016-03-01

    The gene TUZN1299 from the genome of the hyperthermophilic archaeon Thermoproteus uzoniensis encoding a new 32.8 kDa branched-chain amino acid aminotransferase (BCAT) was expressed in Escherichia coli. The recombinant protein TUZN1299 was purified to homogeneity in the PLP-bound form. TUZN1299 was active towards branched-chain amino acids (L-Val, L-Leu, L-Ile) and showed low but detectable activity toward (R)-alpha-methylbenzylamine. The enzyme exhibits high-temperature optimum, thermal stability, and tolerance to organic solvents. The structure of an archaeal BCAT called TUZN1299 was solved for the first time (at 2.0 Å resolution). TUZN1299 has a typical BCAT type IV fold, and the organization of its active site is similar to that of bacterial BCATs. However, there are some differences in the amino acid composition of the active site.

  5. 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.

  6. Extensive inter-domain lateral gene transfer in the evolution of the human commensal Methanosphaera stadtmanae

    Directory of Open Access Journals (Sweden)

    Mor Nadia Lurie-Weinberger

    2012-09-01

    Full Text Available Methanosphaera stadtmanae is a commensal methanogenic archaeon found in the human gut. As most of its niche-neighbors are bacteria, it is expected that lateral gene transfer (LGT from bacteria might have contributed to the evolutionary history of this organism. We performed a phylogenomic survey of putative lateral gene transfer events in M. stadtmanae, using a phylogenetic pipeline. Our analysis indicates that a substantial fraction of the proteins of M. stadtmanae are inferred to have been involved in inter-domain LGT. Laterally acquired genes have had a large contribution to surface functions, by providing novel glycosyltransferase functions. In addition, several ABC transporters seem to be of bacterial origin, including the molybdate transporter. Thus, bacterial genes contributed to the adaptation of M. stadtmanae to a host dependent lifestyle by allowing a larger variation in surface structures and increasing transport efficiency in the gut niche which is diverse and competitive

  7. Biosynthetic mechanism for L-Gulose in main polar lipids of Thermoplasma acidophilum and possible resemblance to plant ascorbic acid biosynthesis.

    Science.gov (United States)

    Yamauchi, Noriaki; Nakayama, Yusuke

    2013-01-01

    L-Gulose is a very rare sugar, but appears as a sugar component of the main polar lipids characteristic in such a thermophilic archaeon as Thermoplasma acidophilum that lives without cell walls in a highly acidic environment. The biosynthesis of L-gulose in this thermophilic organism was investigated with deuterium-labeling experiments. L-Gulose was found to be biosynthesized from D-glucose via stepwise stereochemical inversion at C-2 and C-5. The involvement of an epimerase related to GDP-mannose 3,5-epimerase, the key enzyme of plant ascorbate biosynthesis, was also suggested in this C-5 inversion. The resemblance of L-gulose biosynthesis in archaea and plants might be suggested from these results.

  8. Archaeal acylamino acid releasing enzyme/lipase: Crystallization and preliminary crystallographic analysis in a new crystal form

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A primitive orthorhombic crystal form of acylamino acid releasing enzyme/lipase (APE1547) from hyperthermophilic archaeon Aeropyrum pernix strain K1 has been obtained at 291 K. The diffraction pattern of the crystal extends to 0.27 nm resolution at 100 K using Cu Kαradiation. The crystal belongs to the space group P212121 with unit cell dimensions of a = 6.399, b = 10.439 and c = 16.953 nm. The presence of two molecules per asymmetric unit gives a crystal volume per protein mass (Vm) of 0.0022 nm3 Da-1 and a solvent content of 43% by volume. A full set of X-ray diffraction data were collected to 0.3 nm from the native crystal.

  9. Ultrasound-Assisted Enantioselective Esterification of Ibuprofen Catalyzed by a Flower-Like Nanobioreactor

    Directory of Open Access Journals (Sweden)

    Baiyi An

    2016-04-01

    Full Text Available A flower-like nanobioreactor was prepared for resolution of ibuprofen in organic solvents. Ultrasound irradiation has been used to improve the enzyme performance of APE1547 (a thermophilic esterase from the archaeon Aeropyrum pernix K1 in the enantioselective esterification. Under optimum reaction conditions (ultrasound power, 225 W; temperature, 45 °C; water activity, 0.21, the immobilized APE1547 showed an excellent catalytic performance (enzyme activity, 13.26 μmol/h/mg; E value, 147.1. After ten repeated reaction batches, the nanobioreactor retained almost 100% of its initial enzyme activity and enantioselectivity. These results indicated that the combination of the immobilization method and ultrasound irradiation can enhance the enzyme performance dramatically.

  10. Complex archaea that bridge the gap between prokaryotes and eukaryotes.

    Science.gov (United States)

    Spang, Anja; Saw, Jimmy H; Jørgensen, Steffen L; Zaremba-Niedzwiedzka, Katarzyna; Martijn, Joran; Lind, Anders E; van Eijk, Roel; Schleper, Christa; Guy, Lionel; Ettema, Thijs J G

    2015-05-14

    The origin of the eukaryotic cell remains one of the most contentious puzzles in modern biology. Recent studies have provided support for the emergence of the eukaryotic host cell from within the archaeal domain of life, but the identity and nature of the putative archaeal ancestor remain a subject of debate. Here we describe the discovery of 'Lokiarchaeota', a novel candidate archaeal phylum, which forms a monophyletic group with eukaryotes in phylogenomic analyses, and whose genomes encode an expanded repertoire of eukaryotic signature proteins that are suggestive of sophisticated membrane remodelling capabilities. Our results provide strong support for hypotheses in which the eukaryotic host evolved from a bona fide archaeon, and demonstrate that many components that underpin eukaryote-specific features were already present in that ancestor. This provided the host with a rich genomic 'starter-kit' to support the increase in the cellular and genomic complexity that is characteristic of eukaryotes.

  11. High protein flexibility and reduced hydration water dynamics are key pressure adaptive strategies in prokaryotes

    KAUST Repository

    Martinez, N.

    2016-09-06

    Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus and the closely related pressure-sensitive Thermococcus kodakarensis, at 0.1 and 40 MPa. We show that cells of the pressure sensitive organism exhibit higher intrinsic stability. Both the hydration water dynamics and the fast protein and lipid dynamics are reduced under pressure. In contrast, the proteome of T. barophilus is more pressure sensitive than that of T. kodakarensis. The diffusion coefficient of hydration water is reduced, while the fast protein and lipid dynamics are slightly enhanced with increasing pressure. These findings show that the coupling between hydration water and cellular constituents might not be simply a master-slave relationship. We propose that the high flexibility of the T. barophilus proteome associated with reduced hydration water may be the keys to the molecular adaptation of the cells to high hydrostatic pressure.

  12. 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.

  13. Crystal structure of Sulfolobus acidocaldarius aspartate carbamoyltransferase in complex with its allosteric activator CTP.

    Science.gov (United States)

    De Vos, Dirk; Xu, Ying; Aerts, Tony; Van Petegem, Filip; Van Beeumen, Jozef J

    2008-07-18

    Aspartate carbamoyltransferase (ATCase) is a paradigm for allosteric regulation of enzyme activity. B-class ATCases display very similar homotropic allosteric behaviour, but differ extensively in their heterotropic patterns. The ATCase from the thermoacidophilic archaeon Sulfolobus acidocaldarius, for example, is strongly activated by its metabolic pathway's end product CTP, in contrast with Escherichia coli ATCase which is inhibited by CTP. To investigate the structural basis of this property, we have solved the crystal structure of the S. acidocaldarius enzyme in complex with CTP. Structure comparison reveals that effector binding does not induce similar large-scale conformational changes as observed for the E. coli ATCase. However, shifts in sedimentation coefficients upon binding of the bi-substrate analogue PALA show the existence of structurally distinct allosteric states. This suggests that the so-called "Nucleotide-Perturbation model" for explaining heterotropic allosteric behaviour, which is based on global conformational strain, is not a general mechanism of B-class ATCases.

  14. Hot and sweet: protein glycosylation in Crenarchaeota.

    Science.gov (United States)

    Meyer, Benjamin H; Albers, Sonja-Verena

    2013-02-01

    Every living cell is covered with a dense and complex array of covalently attached sugars or sugar chains. The majority of these glycans are linked to proteins via the so-called glycosylation process. Protein glycosylation is found in all three domains of life: Eukarya, Bacteria and Archaea. However, on the basis of the limit in analytic tools for glycobiology and genetics in Archaea, only in the last few years has research on archaeal glycosylation pathways started mainly in the Euryarchaeota Haloferax volcanii, Methanocaldococcus maripaludis and Methanococcus voltae. Recently, major steps of the crenarchaeal glycosylation process of the thermoacidophilic archaeon Sulfolobus acidocaldarius have been described. The present review summarizes the proposed N-glycosylation pathway of S. acidocaldarius, describing the phenotypes of the mutants disrupted in N-glycan biosynthesis as well as giving insights into the archaeal O-linked and glycosylphosphatidylinositol anchor glycosylation process.

  15. Interaction of hexa-His tag with acidic amino acids results in facilitated refolding of halophilic nucleoside diphosphate kinase.

    Science.gov (United States)

    Ishibashi, Matsujiro; Ida, Keiko; Tatsuda, Shuhei; Arakawa, Tsutomu; Tokunaga, Masao

    2011-11-01

    We have previously reported that amino-terminal extension sequence containing hexa-His facilitated refolding and assembly of hexameric nucleoside diphosphate kinase from extremely halophilic archaeon Halobacterium salinarum (NDK). In this study, we made various mutations in both the tag sequence and within NDK molecule. SerNDK, in which hexa-His was replaced with hexa-Ser, showed no facilitated folding. In addition, HisD58GD63G, in which both Asp58 and Asp63 in NDK were replaced with Gly, also showed no refolding enhancement. These results suggest that hexa-His in His-tag interact cooperatively with either Asp58 or Asp63 or both. Furthermore, G114D mutant, which formed a dimer in low salt solution, was strongly stabilized by His-tag to form a stable hexamer.

  16. Ultrasound-Assisted Enantioselective Esterification of Ibuprofen Catalyzed by a Flower-Like Nanobioreactor.

    Science.gov (United States)

    An, Baiyi; Fan, Hailin; Wu, Zhuofu; Zheng, Lu; Wang, Lei; Wang, Zhi; Chen, Guang

    2016-04-28

    A flower-like nanobioreactor was prepared for resolution of ibuprofen in organic solvents. Ultrasound irradiation has been used to improve the enzyme performance of APE1547 (a thermophilic esterase from the archaeon Aeropyrum pernix K1) in the enantioselective esterification. Under optimum reaction conditions (ultrasound power, 225 W; temperature, 45 °C; water activity, 0.21), the immobilized APE1547 showed an excellent catalytic performance (enzyme activity, 13.26 μmol/h/mg; E value, 147.1). After ten repeated reaction batches, the nanobioreactor retained almost 100% of its initial enzyme activity and enantioselectivity. These results indicated that the combination of the immobilization method and ultrasound irradiation can enhance the enzyme performance dramatically.

  17. 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.

  18. Complete genome sequence of Halorhabdus utahensis type strain (AX-2T)

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Iain [U.S. Department of Energy, Joint Genome Institute; Tindall, Brian [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Pomrenke, Helge [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Goker, Markus [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Nolan, Matt [U.S. Department of Energy, Joint Genome Institute; Copeland, A [U.S. Department of Energy, Joint Genome Institute; Glavina Del Rio, Tijana [U.S. Department of Energy, Joint Genome Institute; Chen, Feng [U.S. Department of Energy, Joint Genome Institute; Tice, Hope [U.S. Department of Energy, Joint Genome Institute; Cheng, Jan-Fang [U.S. Department of Energy, Joint Genome Institute; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Chertkov, Olga [Los Alamos National Laboratory (LANL); Bruce, David [Los Alamos National Laboratory (LANL); Detter, J. Chris [U.S. Department of Energy, Joint Genome Institute; Han, Cliff [Los Alamos National Laboratory (LANL); Goodwin, Lynne A. [Los Alamos National Laboratory (LANL); Land, Miriam L [ORNL; Hauser, Loren John [ORNL; Chang, Yun-Juan [ORNL; Jeffries, Cynthia [Oak Ridge National Laboratory (ORNL); Pitluck, Sam [U.S. Department of Energy, Joint Genome Institute; Pati, Amrita [U.S. Department of Energy, Joint Genome Institute; Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Ovchinnikova, Galina [U.S. Department of Energy, Joint Genome Institute; Chen, Amy [U.S. Department of Energy, Joint Genome Institute; Palaniappan, Krishna [U.S. Department of Energy, Joint Genome Institute; Chain, Patrick S. G. [Lawrence Livermore National Laboratory (LLNL); Rohde, Manfred [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Bristow, James [U.S. Department of Energy, Joint Genome Institute; Eisen, Jonathan [U.S. Department of Energy, Joint Genome Institute; Markowitz, Victor [U.S. Department of Energy, Joint Genome Institute; Hugenholtz, Philip [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute; Klenk, Hans-Peter [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany

    2009-01-01

    Halorhabdus utahensis Wain et al. 2000 is the type species of the genus, which is of phylogenetic interest because of its location on one of the deepest branches within the very extensive euryarchaeal family Halobacteriaceae. H. utahensis is a free-living, motile, rod shaped to pleomorphic, Gram-negative archaeon, which was originally isolated from a sediment sample collected from the southern arm of Great Salt Lake, Utah, USA. When grown on appropriate media, H. utahensis can form polyhydroxybutyrate (PHB). Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the a member of halobacterial genus Halorhabdus, and the 3,116,795 bp long single replicon genome with its 3027 protein-coding and 48 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

  19. Crystallization and preliminary X-ray diffraction analysis of the hyperthermophilic Sulfolobus solfataricus phosphotriesterase

    Energy Technology Data Exchange (ETDEWEB)

    Elias, Mikael [Laboratoire de Cristallographie et Modélisation des Matériaux Minéraux et Biologiques, CNRS-Université Henri Poincaré, 54506 Vandoeuvre-lès-Nancy (France); Dupuy, Jérôme [Laboratoire de Cristallogenèse et Cristallographie des Protéines, Institut de Biologie Structurale J.-P. Ebel, 38027 Grenoble (France); Merone, Luigia [Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Napoli (Italy); Lecomte, Claude [Laboratoire de Cristallographie et Modélisation des Matériaux Minéraux et Biologiques, CNRS-Université Henri Poincaré, 54506 Vandoeuvre-lès-Nancy (France); Rossi, Mosè [Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Napoli (Italy); Masson, Patrick [Unité d’Enzymologie, Département de Toxicologie, Centre de Recherches du Service de Santé des Armées, 38702 La Tronche (France); Manco, Giuseppe [Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Napoli (Italy); Chabriere, Eric, E-mail: eric.chabriere@lcm3b.uhp-nancy.fr [Laboratoire de Cristallographie et Modélisation des Matériaux Minéraux et Biologiques, CNRS-Université Henri Poincaré, 54506 Vandoeuvre-lès-Nancy (France); Unité d’Enzymologie, Département de Toxicologie, Centre de Recherches du Service de Santé des Armées, 38702 La Tronche (France)

    2007-07-01

    A phosphotriesterase (PTE) from the hyperthermophilic archaeon S. solfataricus has been crystallized. Combined with biochemical and bioengineering studies, it is expected that the structure of this protein will provide insight into the natural function of the PTE family and provide important data for achieving an efficient organophosphate biodecontaminant. Organophosphates constitute the largest class of insecticides used worldwide and some of them are potent nerve agents. Consequently, organophosphate-degrading enzymes are of paramount interest as they could be used as bioscavengers and biodecontaminants. Phosphotriesterases (PTEs) are capable of hydrolyzing these toxic compounds with high efficiency. A distant and hyperthermophilic representative of the PTE family was cloned from the archeon Sulfolobus solfataricus MT4, overexpressed in Escherichia coli and crystallized; the crystals diffracted to 2.54 Å resolution. Owing to its exceptional thermostability, this PTE may be an excellent candidate for obtaining an efficient organophosphate biodecontaminant. Here, the crystallization conditions and data collection for the hyperthermophilic S. solfataricus PTE are reported.

  20. Complete genome sequence of Halorhabdus utahensis type strain (AX-2).

    Science.gov (United States)

    Anderson, Iain; Tindall, Brian J; Pomrenke, Helga; Göker, Markus; Lapidus, Alla; Nolan, Matt; Copeland, Alex; Glavina Del Rio, Tijana; Chen, Feng; Tice, Hope; Cheng, Jan-Fang; Lucas, Susan; Chertkov, Olga; Bruce, David; Brettin, Thomas; Detter, John C; Han, Cliff; Goodwin, Lynne; Land, Miriam; Hauser, Loren; Chang, Yun-Juan; Jeffries, Cynthia D; Pitluck, Sam; Pati, Amrita; Mavromatis, Konstantinos; Ivanova, Natalia; Ovchinnikova, Galina; Chen, Amy; Palaniappan, Krishna; Chain, Patrick; Rohde, Manfred; Bristow, Jim; Eisen, Jonathan A; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C; Klenk, Hans-Peter

    2009-11-22

    Halorhabdus utahensis Wainø et al. 2000 is the type species of the genus, which is of phylogenetic interest because of its location on one of the deepest branches within the very extensive euryarchaeal family Halobacteriaceae. H. utahensis is a free-living, motile, rod shaped to pleomorphic, Gram-negative archaeon, which was originally isolated from a sediment sample collected from the southern arm of Great Salt Lake, Utah, USA. When grown on appropriate media, H. utahensis can form polyhydroxybutyrate (PHB). Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the a member of halobacterial genus Halorhabdus, and the 3,116,795 bp long single replicon genome with its 3027 protein-coding and 48 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

  1. Methanopyrus kandleri: an archaeal methanogen unrelated to all other known methanogens

    Science.gov (United States)

    Burggraf, S.; Stetter, K. O.; Rouviere, P.; Woese, C. R.

    1991-01-01

    Analysis of its 16S rRNA sequence shows that the newly discovered hyperthermophilic methanogen, Methanopryus kandleri, is phylogenetically unrelated to any other known methanogen. The organism represents a separate lineage originating near the root of the archaeal tree. Although the 16S rRNA sequence of Mp. kandleri resembles euryarchaeal 16S rRNAs more than it does crenarchaeal, it shows more crenarchaeal signature features than any known euryarchaeal rRNA. Attempts to place it in relation to the root of the archaeal tree show that the Mp. kandleri lineage likely arises from the euryarchaeal branch of the tree. While the existence of so deeply branching a methanogenic lineage brings into question the thesis that methanogenesis evolved from an earlier metabolism similar to that seen in Thermococcus, it at the same time reinforces the notion that the aboriginal [correction of aborginal] archaeon was a thermophile.

  2. High protein flexibility and reduced hydration water dynamics are key pressure adaptive strategies in prokaryotes

    Science.gov (United States)

    Martinez, N.; Michoud, G.; Cario, A.; Ollivier, J.; Franzetti, B.; Jebbar, M.; Oger, P.; Peters, J.

    2016-09-01

    Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus and the closely related pressure-sensitive Thermococcus kodakarensis, at 0.1 and 40 MPa. We show that cells of the pressure sensitive organism exhibit higher intrinsic stability. Both the hydration water dynamics and the fast protein and lipid dynamics are reduced under pressure. In contrast, the proteome of T. barophilus is more pressure sensitive than that of T. kodakarensis. The diffusion coefficient of hydration water is reduced, while the fast protein and lipid dynamics are slightly enhanced with increasing pressure. These findings show that the coupling between hydration water and cellular constituents might not be simply a master-slave relationship. We propose that the high flexibility of the T. barophilus proteome associated with reduced hydration water may be the keys to the molecular adaptation of the cells to high hydrostatic pressure.

  3. 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.

  4. Specific single-cell isolation and genomic amplification of uncultured microorganisms

    DEFF Research Database (Denmark)

    Kvist, Thomas; Ahring, Birgitte Kiær; Lasken, R.S.

    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 primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms...... sequence analysis and shotgun-cloned for additional genomic analysis. Sequence analysis showed > 99% 16S rRNA gene homology to soil crenarchaeotal clone SCA1170 and shotgun fragments had the closest match to a crenarchaeotal BAC clone previously retrieved from a soil sample. The system was validated using...

  5. Global transcriptional regulator TrmB family members in prokaryotes.

    Science.gov (United States)

    Kim, Minwook; Park, Soyoung; Lee, Sung-Jae

    2016-10-01

    Members of the TrmB family act as global transcriptional regulators for the activation or repression of sugar ABC transporters and central sugar metabolic pathways, including glycolytic, gluconeogenic, and other metabolic pathways, and also as chromosomal stabilizers in archaea. As a relatively newly classified transcriptional regulator family, there is limited experimental evidence for their role in Thermococcales, halophilic archaeon Halobacterium salinarum NRC1, and crenarchaea Sulfolobus strains, despite being one of the extending protein families in archaea. Recently, the protein structures of Pyrococcus furiosus TrmB and TrmBL2 were solved, and the transcriptomic data uncovered by microarray and ChIP-Seq were published. In the present review, recent evidence of the functional roles of TrmB family members in archaea is explained and extended to bacteria.

  6. Enzymatic resolution of ibuprofen in an organic solvent under ultrasound irradiation.

    Science.gov (United States)

    Zhao, Dantong; Yue, Hong; Chen, Ge; Jiang, Liyan; Zhang, Hong; Wang, Zhi; Liu, Guangchun

    2014-01-01

    Ultrasound has been successfully adopted to improve the biocatalytic properties of APE1547 (a novel esterase from the archaeon Aeropyrum pernix K1) in the resolution of ibuprofen. After optimizing the conditions (ultrasound power, 200 W; temperature, 35 °C), the best biocatalytic performance of APE1547 (enzyme activity, 5.39 µmol/H/mg; E value, 130.8) was obtained. Compared with the conventional reaction in an orbital shaker, the enzyme activity was significantly enhanced about 90-fold, and the enantioselectivity was enhanced about fourfold after an ultrasound. The results of scanning electron microscopy clearly indicated that the activation effect of ultrasound on APE1547 originated mainly in the morphological change of the enzyme powder. Both lower particle size and conformational change of APE1547 under ultrasound might be helpful to enhance the enantioselectivity. In addition, APE1547 kept its best performance under the low-power ultrasound for at least five reaction cycles.

  7. 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.

  8. 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.)

  9. Metabolic capabilities of microorganisms involved in and associated with the anaerobic oxidation of methane

    Directory of Open Access Journals (Sweden)

    Gunter eWegener

    2016-02-01

    Full Text Available In marine sediments the anaerobic oxidation of methane with sulfate as electron acceptor (AOM is responsible for the removal of a major part of the greenhouse gas methane. AOM is performed by consortia of anaerobic methane-oxidizing archaea (ANME and their specific partner bacteria. The physiology of these organisms is poorly understood, which is due to their slow growth with doubling times in the order of months and the phylogenetic diversity in natural and in vitro AOM enrichments. Here we study sediment-free long-term AOM enrichments that were cultivated from seep sediments sampled off the Italian Island Elba (20°C; hereon called E20 and from hot vents of the Guaymas Basin, Gulf of California, cultivated at 37°C (G37 or at 50°C (G50. These enrichments were dominated by consortia of ANME-2 archaea and Seep-SRB2 partner bacteria (E20 or by ANME-1, forming consortia with Seep-SRB2 bacteria (G37 or with bacteria of the HotSeep-1 cluster (G50. We investigate lipid membrane compositions as possible factors for the different temperature affinities of the different ANME clades and show autotrophy as characteristic feature for both ANME clades and their partner bacteria. Although in the absence of additional substrates methane formation was not observed, methanogenesis from methylated substrates (methanol and methylamine could be quickly stimulated in the E20 and the G37 enrichment. Responsible for methanogenesis are archaea from the genus Methanohalophilus and Methanococcoides, which are minor community members during AOM (1 to 7‰ of archaeal 16S rRNA gene amplicons. In the same two cultures also sulfur disproportionation could be quickly stimulated by addition of zero-valent colloidal sulfur. The isolated partner bacteria are likewise minor community members (1 to 9‰ of bacterial 16S rRNA gene amplicons, whereas the dominant partner bacteria (Seep-SRB1a, Seep-SRB2 or HotSeep-1 did not grow on elemental sulfur. Our results support a

  10. Crystallization and preliminary X-ray diffraction analysis on the homing endonuclease I-Dmo-I in complex with its target DNA

    Energy Technology Data Exchange (ETDEWEB)

    Redondo, Pilar [Macromolecular Crystallography Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Centre (CNIO), c/Melchor Fdez. Almagro 3, 28029 Madrid (Spain); Prieto, Jesús; Ramos, Elena; Blanco, Francisco J. [NMR Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Centre (CNIO), c/Melchor Fdez. Almagro 3, 28029 Madrid (Spain); Montoya, Guillermo, E-mail: gmontoya@cnio.es [Macromolecular Crystallography Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Centre (CNIO), c/Melchor Fdez. Almagro 3, 28029 Madrid (Spain)

    2007-12-01

    I-Dmo-I is a well characterized homing endonuclease from the archaeon Desulfurococcus mobilis. The enzyme was cloned and overexpressed in Escherichia coli. Crystallization experiments of I-Dmo-I in complex with its DNA target in the presence of Ca{sup 2+} and Mg{sup 2+} yielded crystals that were suitable for X-ray diffraction analysis. Homing endonucleases are highly specific DNA-cleaving enzymes that recognize long stretches of base pairs. The availability of these enzymes has opened novel perspectives for genome engineering in a wide range of fields, including gene therapy, by taking advantage of the homologous gene-targeting enhancement induced by a double-strand break. I-Dmo-I is a well characterized homing endonuclease from the archaeon Desulfurococcus mobilis. The enzyme was cloned and overexpressed in Escherichia coli. Crystallization experiments of I-Dmo-I in complex with its DNA target in the presence of Ca{sup 2+} and Mg{sup 2+} yielded crystals that were suitable for X-ray diffraction analysis. The crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 106.75, b = 70.18, c = 106.85 Å, α = γ = 90, β = 119.93°. The self-rotation function and the Matthews coefficient suggested the presence of three protein–DNA complexes per asymmetric unit. The crystals diffracted to a resolution limit of 2.6 Å using synchrotron radiation at the Swiss Light Source (SLS) and the European Synchrotron Radiation Facility (ESRF)

  11. 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.

  12. Metabolism of pentose sugars in the hyperthermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius.

    Science.gov (United States)

    Nunn, Charlotte E M; Johnsen, Ulrike; Schönheit, Peter; Fuhrer, Tobias; Sauer, Uwe; Hough, David W; Danson, Michael J

    2010-10-29

    We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, D-xylose and L-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of D-xylose and L-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.

  13. Heterologous Production of an Energy-Conserving Carbon Monoxide Dehydrogenase Complex in the Hyperthermophile Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Gerrit Jan Schut

    2016-01-01

    Full Text Available 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 carbon monoxide 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.

  14. Anaerobic coculture of microalgae with Thermosipho globiformans and Methanocaldococcus jannaschii at 68°C enhances generation of n-alkane-rich biofuels after pyrolysis.

    Science.gov (United States)

    Yamane, Kunio; Matsuyama, Shigeru; Igarashi, Kensuke; Utsumi, Motoo; Shiraiwa, Yoshihiro; Kuwabara, Tomohiko

    2013-02-01

    We tested different alga-bacterium-archaeon consortia to investigate the production of oil-like mixtures, expecting that n-alkane-rich biofuels might be synthesized after pyrolysis. Thermosipho globiformans and Methanocaldococcus jannaschii were cocultured at 68°C with microalgae for 9 days under two anaerobic conditions, followed by pyrolysis at 300°C for 4 days. Arthrospira platensis (Cyanobacteria), Dunaliella tertiolecta (Chlorophyta), Emiliania huxleyi (Haptophyta), and Euglena gracilis (Euglenophyta) served as microalgal raw materials. D. tertiolecta, E. huxleyi, and E. gracilis cocultured with the bacterium and archaeon inhibited their growth and CH(4) production. E. huxleyi had the strongest inhibitory effect. Biofuel generation was enhanced by reducing impurities containing alkanenitriles during pyrolysis. The composition and amounts of n-alkanes produced by pyrolysis were closely related to the lipid contents and composition of the microalgae. Pyrolysis of A. platensis and D. tertiolecta containing mainly phospholipids and glycolipids generated short-carbon-chain n-alkanes (n-tridecane to n-nonadecane) and considerable amounts of isoprenoids. E. gracilis also produced mainly short n-alkanes. In contrast, E. huxleyi containing long-chain (31 and 33 carbon atoms) alkenes and very long-chain (37 to 39 carbon atoms) alkenones, in addition to phospholipids and glycolipids, generated a high yield of n-alkanes of various lengths (n-tridecane to n-pentatriacontane). The gas chromatography-mass spectrometry (GC-MS) profiles of these n-alkanes were similar to those of native petroleum crude oils despite containing a considerable amount of n-hentriacontane. The ratio of phytane to n-octadecane was also similar to that of native crude oils.

  15. Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification Is Controlled by the Transcription Regulator NarO

    Science.gov (United States)

    Hattori, Tatsuya; Shiba, Hiromichi; Ashiki, Ken-ichi; Araki, Takuma; Nagashima, Yoh-kow; Yoshimatsu, Katsuhiko

    2016-01-01

    ABSTRACT The extremely halophilic archaeon Haloferax volcanii grows anaerobically by denitrification. A putative DNA-binding protein, NarO, is encoded upstream of the respiratory nitrate reductase gene of H. volcanii. Disruption of the narO gene resulted in a loss of denitrifying growth of H. volcanii, and the expression of the recombinant NarO recovered the denitrification capacity. A novel CXnCXCX7C motif showing no remarkable similarities with known sequences was conserved in the N terminus of the NarO homologous proteins found in the haloarchaea. Restoration of the denitrifying growth was not achieved by expression of any mutant NarO in which any one of the four conserved cysteines was individually replaced by serine. A promoter assay experiment indicated that the narO gene was usually transcribed, regardless of whether it was cultivated under aerobic or anaerobic conditions. Transcription of the genes encoding the denitrifying enzymes nitrate reductase and nitrite reductase was activated under anaerobic conditions. A putative cis element was identified in the promoter sequence of haloarchaeal denitrifying genes. These results demonstrated a significant effect of NarO, probably due to its oxygen-sensing function, on the transcriptional activation of haloarchaeal denitrifying genes. IMPORTANCE H. volcanii is an extremely halophilic archaeon capable of anaerobic growth by denitrification. The regulatory mechanism of denitrification has been well understood in bacteria but remains unknown in archaea. In this work, we show that the helix-turn-helix (HTH)-type regulator NarO activates transcription of the denitrifying genes of H. volcanii under anaerobic conditions. A novel cysteine-rich motif, which is critical for transcriptional regulation, is present in NarO. A putative cis element was also identified in the promoter sequence of the haloarchaeal denitrifying genes. PMID:26787768

  16. A role for programmed cell death in the microbial loop.

    Directory of Open Access Journals (Sweden)

    Mónica V Orellana

    Full Text Available The microbial loop is the conventional model by which nutrients and minerals are recycled in aquatic eco-systems. Biochemical pathways in different organisms become metabolically inter-connected such that nutrients are utilized, processed, released and re-utilized by others. The result is that unrelated individuals end up impacting each others' fitness directly through their metabolic activities. This study focused on the impact of programmed cell death (PCD on a population's growth as well as its role in the exchange of carbon between two naturally co-occurring halophilic organisms. Flow cytometric, biochemical, ¹⁴C radioisotope tracing assays, and global transcriptomic analyses show that organic algal photosynthate released by Dunalliela salina cells undergoing PCD complements the nutritional needs of other non-PCD D. salina cells. This occurs in vitro in a carbon limited environment and enhances the growth of the population. In addition, a co-occurring heterotroph Halobacterium salinarum re-mineralizes the carbon providing elemental nutrients for the mixoheterotrophic chlorophyte. The significance of this is uncertain and the archaeon can also subsist entirely on the lysate of apoptotic algae. PCD is now well established in unicellular organisms; however its ecological relevance has been difficult to decipher. In this study we found that PCD in D. salina causes the release of organic nutrients such as glycerol, which can be used by others in the population as well as a co-occurring halophilic archaeon. H. salinarum also re-mineralizes the dissolved material promoting algal growth. PCD in D. salina was the mechanism for the flow of dissolved photosynthate between unrelated organisms. Ironically, programmed death plays a central role in an organism's own population growth and in the exchange of nutrients in the microbial loop.

  17. 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.

  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. Viruses in the Oceanic Basement

    Science.gov (United States)

    Jungbluth, Sean P.; Lin, Huei-Ting; Hsieh, Chih-Chiang; Miranda, Jaclyn A.; Schvarcz, Christopher R.; Rappé, Michael S.

    2017-01-01

    ABSTRACT Microbial life has been detected well into the igneous crust of the seafloor (i.e., the oceanic basement), but there have been no reports confirming the presence of viruses in this habitat. To detect and characterize an ocean basement virome, geothermally heated fluid samples (ca. 60 to 65°C) were collected from 117 to 292 m deep into the ocean basement using seafloor observatories installed in two boreholes (Integrated Ocean Drilling Program [IODP] U1362A and U1362B) drilled in the eastern sediment-covered flank of the Juan de Fuca Ridge. Concentrations of virus-like particles in the fluid samples were on the order of 0.2 × 105 to 2 × 105 ml−1 (n = 8), higher than prokaryote-like cells in the same samples by a factor of 9 on average (range, 1.5 to 27). Electron microscopy revealed diverse viral morphotypes similar to those of viruses known to infect bacteria and thermophilic archaea. An analysis of virus-like sequences in basement microbial metagenomes suggests that those from archaeon-infecting viruses were the most common (63 to 80%). Complete genomes of a putative archaeon-infecting virus and a prophage within an archaeal scaffold were identified among the assembled sequences, and sequence analysis suggests that they represent lineages divergent from known thermophilic viruses. Of the clustered regularly interspaced short palindromic repeat (CRISPR)-containing scaffolds in the metagenomes for which a taxonomy could be inferred (163 out of 737), 51 to 55% appeared to be archaeal and 45 to 49% appeared to be bacterial. These results imply that the warmed, highly altered fluids in deeply buried ocean basement harbor a distinct assemblage of novel viruses, including many that infect archaea, and that these viruses are active participants in the ecology of the basement microbiome. PMID:28270584

  20. 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

  1. Genomewide and biochemical analyses of DNA-binding activity of Cdc6/Orc1 and Mcm proteins in Pyrococcus sp.

    Science.gov (United States)

    Matsunaga, Fujihiko; Glatigny, Annie; Mucchielli-Giorgi, Marie-Hélène; Agier, Nicolas; Delacroix, Hervé; Marisa, Laetitia; Durosay, Patrice; Ishino, Yoshizumi; Aggerbeck, Lawrence; Forterre, Patrick

    2007-01-01

    The origin of DNA replication (oriC) of the hyperthermophilic archaeon Pyrococcus abyssi contains multiple ORB and mini-ORB repeats that show sequence similarities to other archaeal ORB (origin recognition box). We report here that the binding of Cdc6/Orc1 to a 5 kb region containing oriC in vivo was highly specific both in exponential and stationary phases, by means of chromatin immunoprecipitation coupled with hybridization on a whole genome microarray (ChIP-chip). The oriC region is practically the sole binding site for the Cdc6/Orc1, thereby distinguishing oriC in the 1.8 M bp genome. We found that the 5 kb region contains a previously unnoticed cluster of ORB and mini-ORB repeats in the gene encoding the small subunit (dp1) for DNA polymerase II (PolD). ChIP and the gel retardation analyses further revealed that Cdc6/Orc1 specifically binds both of the ORB clusters in oriC and dp1. The organization of the ORB clusters in the dp1 and oriC is conserved during evolution in the order Thermococcales, suggesting a role in the initiation of DNA replication. Our ChIP-chip analysis also revealed that Mcm alters the binding specificity to the oriC region according to the growth phase, consistent with its role as a licensing factor.

  2. Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System.

    Science.gov (United States)

    Stachler, Aris-Edda; Marchfelder, Anita

    2016-07-15

    The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon.

  3. 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.

  4. 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.

  5. Modeling DNA Repair: Approaching In Vivo Techniques in the Hyperthermophile Sulfolobus Solfataricus

    Energy Technology Data Exchange (ETDEWEB)

    Blanton, J.; Fuss, J.; Yannone, S.M.; Tainer, J.A.; Cooper, P.K.

    2005-01-01

    Archaea are found in some of the most extreme environments on earth and represent a third domain of life distinct from Eukarya and Eubacteria. The hyperthermophilic archaeon Sulfolobus solfataricus, isolated from acidic hot springs (80oC, pH 3) in Yellowstone National Park, has emerged as a potential model system for studying human DNA repair processes. Archaea are more closely related to Eukarya than to Eubacteria, suggesting that archaeal DNA repair machinery may model the complex human system much more closely than that of other prokaryotes. DNA repair requires coordinated protein-protein interactions that are frequently transient. Protein complexes that are transient at extreme temperatures where archaea thrive may be more stable at room temperature, allowing for the characterization of otherwise short-lived complexes. However, characterization of these systems in archaea has been limited by the absence of a stable in vivo transformation and expression system. The work presented here is a pilot study in gene cloning and recombinant protein expression in S. solfataricus. Three genes associated with DNA repair were selected for expression: MRE11, PCNA1, and a putative CSB homologue. Though preparation of these recombinant genes followed standard methods, preparation of a suitable vector proved more challenging. The shuttle vector pSSV64, derived from the SSV1 virus and the E. coli vector pBSSK+, was most successfully isolated from the DH5α E. coli strain. Currently, alternative vectors are being designed for more efficient genetic manipulations in S. solfataricus.

  6. Development of β-Lactamase as a Tool for Monitoring Conditional Gene Expression by a Tetracycline-Riboswitch in Methanosarcina acetivorans

    Directory of Open Access Journals (Sweden)

    Shemsi Demolli

    2014-01-01

    Full Text Available The use of reporter gene fusions to assess cellular processes such as protein targeting and regulation of transcription or translation is established technology in archaeal, bacterial, and eukaryal genetics. Fluorescent proteins or enzymes resulting in chromogenic substrate turnover, like β-galactosidase, have been particularly useful for microscopic and screening purposes. However, application of such methodology is of limited use for strictly anaerobic organisms due to the requirement of molecular oxygen for chromophore formation or color development. We have developed β-lactamase from Escherichia coli (encoded by bla in conjunction with the chromogenic substrate nitrocefin into a reporter system usable under anaerobic conditions for the methanogenic archaeon Methanosarcina acetivorans. By using a signal peptide of a putative flagellin from M. acetivorans and different catabolic promoters, we could demonstrate growth substrate-dependent secretion of β-lactamase, facilitating its use in colony screening on agar plates. Furthermore, a series of fusions comprised of a constitutive promoter and sequences encoding variants of the synthetic tetracycline-responsive riboswitch (tc-RS was created to characterize its influence on translation initiation in M. acetivorans. One tc-RS variant resulted in more than 11-fold tetracycline-dependent regulation of bla expression, which is in the range of regulation by naturally occurring riboswitches. Thus, tc-RS fusions represent the first solely cis-active, that is, factor-independent system for controlled gene expression in Archaea.

  7. Hyperthermophilic endoglucanase for in planta lignocellulose conversion

    Science.gov (United States)

    2012-01-01

    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. PMID:22928996

  8. DNA binding properties of the small cascade subunit Csa5.

    Directory of Open Access Journals (Sweden)

    Michael Daume

    Full Text Available CRISPR-Cas systems provide immunity against viral attacks in archaeal and bacterial cells. Type I systems employ a Cas protein complex termed Cascade, which utilizes small CRISPR RNAs to detect and degrade the exogenic DNA. A small sequence motif, the PAM, marks the foreign substrates. Previously, a recombinant type I-A Cascade complex from the archaeon Thermoproteus tenax was shown to target and degrade DNA in vitro, dependent on a native PAM sequence. Here, we present the biochemical analysis of the small subunit, Csa5, of this Cascade complex. T. tenax Csa5 preferentially bound ssDNA and mutants that showed decreased ssDNA-binding and reduced Cascade-mediated DNA cleavage were identified. Csa5 oligomerization prevented DNA binding. Specific recognition of the PAM sequence was not observed. Phylogenetic analyses identified Csa5 as a universal member of type I-A systems and revealed three distinct groups. A potential role of Csa5 in R-loop stabilization is discussed.

  9. Bioenergetic studies of coal sulfur oxidation by extremely thermophilic bacteria. Final report, September 15, 1992--August 31, 1997

    Energy Technology Data Exchange (ETDEWEB)

    Kelly, R.M.; Han, C.J.

    1997-12-31

    Thermoacidophilic microorganisms have been considered for inorganic sulfur removal from coal because of expected improvements in rates of both biotic and abiotic sulfur oxidation reactions with increasing temperature. In this study, the bioenergetic response of the extremely thermoacidophilic archaeon, Metallosphaera sedula, to environmental changes have been examined in relation to its capacity to catalyze pyrite oxidation in coal. Given an appropriate bioenergetic challenge, the metabolic response was to utilize additional amounts of energy sources (i.e., pyrite) to survive. Of particular interest were the consequences of exposing the organism to various forms of stress (chemical, nutritional, thermal, pH) in the presence of coal pyrite. Several approaches to take advantage of stress response to accelerate pyrite oxidation by this organism were examined, including attempts to promote acquired thermal tolerance to extend its functional range, exposure to chemical uncouplers and decouplers, and manipulation of heterotrophic and chemolithotrophic tendencies to optimize biomass concentration and biocatalytic activity. Promising strategies were investigated in a continuous culture system. This study identified environmental conditions that promote better coupling of biotic and abiotic oxidation reactions to improve biosulfurization rates of thermoacidophilic microorganisms.

  10. Role of Mn2+ and Compatible Solutes in the Radiation Resistance of Thermophilic Bacteria and Archaea

    Directory of Open Access Journals (Sweden)

    Kimberly M. Webb

    2012-01-01

    Full Text Available Radiation-resistant bacteria have garnered a great deal of attention from scientists seeking to expose the mechanisms underlying their incredible survival abilities. Recent analyses showed that the resistance to ionizing radiation (IR in the archaeon Halobacterium salinarum is dependent upon Mn-antioxidant complexes responsible for the scavenging of reactive oxygen species (ROS generated by radiation. Here we examined the role of the compatible solutes trehalose, mannosylglycerate, and di-myo-inositol phosphate in the radiation resistance of aerobic and anaerobic thermophiles. We found that the IR resistance of the thermophilic bacteria Rubrobacter xylanophilus and Rubrobacter radiotolerans was highly correlated to the accumulation of high intracellular concentration of trehalose in association with Mn, supporting the model of Mn2+-dependent ROS scavenging in the aerobes. In contrast, the hyperthermophilic archaea Thermococcus gammatolerans and Pyrococcus furiosus did not contain significant amounts of intracellular Mn, and we found no significant antioxidant activity from mannosylglycerate and di-myo-inositol phosphate in vitro. We therefore propose that the low levels of IR-generated ROS under anaerobic conditions combined with highly constitutively expressed detoxification systems in these anaerobes are key to their radiation resistance and circumvent the need for the accumulation of Mn-antioxidant complexes in the cell.

  11. Without salt, the 'thermophilic' protein Mth10b is just mesophilic.

    Directory of Open Access Journals (Sweden)

    Nan Zhang

    Full Text Available Most proteins from thermophiles or hyperthermophiles are intrinsically thermostable. However, though Methanobacterium thermoautotrophicum ΔH is a thermophilic archaeon with an optimal growth temperature of 65 °C, Mth10b, an atypical member the Sac10b protein family from M. thermoautotrophicum ΔH, seems not intrinsically thermostable. In this work, to clarify the molecular mechanism of Mth10b remaining stable under its physiological conditions, the thermodynamic properties of Mth10b were studied through equilibrium unfolding experiments performed at pH 7.0 monitored by circular dichroism (CD spectra in detail. Our work demonstrated that Mth10b is not intrinsically thermostable and that due to the masking effect upon the large numbers of destabilizing electrostatic repulsions resulting from the extremely uneven distribution of charged residues over the surface of Mth10b, salt can contribute to the thermostability of Mth10b greatly. Considering that the intracellular salt concentration is high to 0.7 M, we concluded that salt is the key extrinsic factor to Mth10b remaining stable under its physiological conditions. In other word, without salt, 'thermophilic' protein Mth10b is just a mesophilic one.

  12. Crystallization and preliminary crystallographic analysis of histamine dehydrogenase from Natrinema gari BCC 24369.

    Science.gov (United States)

    Zhou, Dongwen; Visessanguan, Wonnop; Chaikaew, Siriporn; Benjakul, Soottawat; Oda, Kohei; Wlodawer, Alexander

    2014-07-01

    Histamine dehydrogenase (HADH) catalyzes the oxidative deamination of histamine, resulting in the production of imidazole acetaldehyde and an ammonium ion. The enzyme isolated from the newly identified halophilic archaeon Natrinema gari BCC 24369 is significantly different from the previously described protein from Nocardioides simplex. This newly identified HADH comprises three subunits with molecular weights of 49.0, 24.7 and 23.9 kDa, respectively, and is optimally active under high-salt conditions (3.5-5 M NaCl). As a step in the exploration of the unique properties of the protein, the HADH heterotrimer was purified and crystallized. Crystals were obtained using the sitting-drop vapor-diffusion method from a solution composed of 0.2 M calcium chloride dihydrate, 0.1 M HEPES pH 7.5, 28% PEG 400. Diffraction data were collected at -173°C to a resolution limit of 2.4 Å on the Southeast Regional Collaborative Access Team (SER-CAT) beamline 22-ID at the Advanced Photon Source, Argonne National Laboratory. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a=211.9, b=58.6, c=135.4 Å, β=103.0°. The estimated Matthews coefficient is 3.21 Å3 Da(-1), corresponding to 61.7% solvent content.

  13. Crystal structures of the all-cysteinyl-coordinated D14C variant of Pyrococcus furiosus ferredoxin: [4Fe–4S] ↔ [3Fe–4S] cluster conversion

    DEFF Research Database (Denmark)

    Løvgreen, Monika Nøhr; Martic, Maja; Windahl, Michael S.;

    2011-01-01

    The structure of the all-cysteinyl-coordinated D14C variant of [4Fe–4S] ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus has been determined to 1.7 Å resolution from a crystal belonging to space group C2221 with two types of molecules, A and B, in the asymmetric unit. A and B...... and purification are carried out at pH 5.8, only the monomer is obtained. The crystal structure of D14C [3Fe–4S] P. furiosus ferredoxin monomer was determined to 2.8 Å resolution from a crystal belonging to space group P212121 with two molecules in the asymmetric unit. The molecules resemble molecule A of D14C [4...... molecules have different crystal packing and intramolecular disulfide bond conformation. The crystal packing reveals a β-sheet interaction between A molecules in adjacent asymmetric units, whereas B molecules are packed as monomers in a less rigid position next to the A–A extended β-sheet dimers...

  14. Thermodynamics and H2 Transfer in a Methanogenic, Syntrophic Community.

    Directory of Open Access Journals (Sweden)

    Joshua J Hamilton

    2015-07-01

    Full Text Available Microorganisms in nature do not exist in isolation but rather interact with other species in their environment. Some microbes interact via syntrophic associations, in which the metabolic by-products of one species serve as nutrients for another. These associations sustain a variety of natural communities, including those involved in methanogenesis. In anaerobic syntrophic communities, energy is transferred from one species to another, either through direct contact and exchange of electrons, or through small molecule diffusion. Thermodynamics plays an important role in governing these interactions, as the oxidation reactions carried out by the first community member are only possible because degradation products are consumed by the second community member. This work presents the development and analysis of genome-scale network reconstructions of the bacterium Syntrophobacter fumaroxidans and the methanogenic archaeon Methanospirillum hungatei. The models were used to verify proposed mechanisms of ATP production within each species. We then identified additional constraints and the cellular objective function required to match experimental observations. The thermodynamic S. fumaroxidans model could not explain why S. fumaroxidans does not produce H2 in monoculture, indicating that current methods might not adequately estimate the thermodynamics, or that other cellular processes (e.g., regulation play a role. We also developed a thermodynamic coculture model of the association between the organisms. The coculture model correctly predicted the exchange of both H2 and formate between the two species and suggested conditions under which H2 and formate produced by S. fumaroxidans would be fully consumed by M. hungatei.

  15. A novel type of replicative enzyme harbouring ATPase, primase and DNA polymerase activity

    Science.gov (United States)

    Lipps, Georg; Röther, Susanne; Hart, Christina; Krauss, Gerhard

    2003-01-01

    Although DNA replication is a process common in all domains of life, primase and replicative DNA polymerase appear to have evolved independently in the bacterial domain versus the archaeal/eukaryal branch of life. Here, we report on a new type of replication protein that constitutes the first member of the DNA polymerase family E. The protein ORF904, encoded by the plasmid pRN1 from the thermoacidophile archaeon Sulfolobus islandicus, is a highly compact multifunctional enzyme with ATPase, primase and DNA polymerase activity. Recombinant purified ORF904 hydrolyses ATP in a DNA-dependent manner. Deoxynucleotides are preferentially used for the synthesis of primers ∼8 nucleotides long. The DNA polymerase activity of ORF904 synthesizes replication products of up to several thousand nucleotides in length. The primase and DNA polymerase activity are located in the N-terminal half of the protein, which does not show homology to any known DNA polymerase or primase. ORF904 constitutes a new type of replication enzyme, which could have evolved indepen dently from the eubacterial and archaeal/eukaryal proteins of DNA replication. PMID:12743045

  16. 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.

  17. Stability against temperature and external agents of vesicles composed of archael bolaform lipids and egg PC.

    Science.gov (United States)

    Fan, Q; Relini, A; Cassinadri, D; Gambacorta, A; Gliozzi, A

    1995-11-22

    The bolaform lipid PLE extracted from the thermophilic archaeon Sulfolobus solfataricus and its mixtures with egg phosphatidyl-choline (egg PC) have been used to prepare sonicated vesicles. The leakage of entrapped calcein was continuously monitored by fluorescence dequenching. The half times of leakage have been used to compare vesicle stability under different conditions of temperature, lipid composition and presence of destabilizing agents like Ca2+ ions and poly(ethylene glycol) (PEG). It has been found that leakage is primarily modulated by the monopolar/bipolar lipid ratio. In particular, the half time of leakage for vesicles formed from a mixture of the polar lipid extract (PLE) and egg PC is characterized by a maximum at about 1:2 molar ratio. The free energy of mixing has been evaluated from pressure-area isotherms on monolayers at the air/water interface. The results indicate a non monotonous behaviour of the excess free energy of mixing as a function of the molar ratio and the occurrence of a minimum at a fixed molar ratio. The possible formation of a complex is discussed and compared with previous calorimetric measurements on similar compounds.

  18. Bipolar tetraether lipids: chain flexibility and membrane polarity gradients from spin-label electron spin resonance.

    Science.gov (United States)

    Bartucci, R; Gambacorta, A; Gliozzi, A; Marsh, D; Sportelli, L

    2005-11-15

    Membranes of thermophilic Archaea are composed of unique tetraether lipids in which C40, saturated, methyl-branched biphytanyl chains are linked at both ends to polar groups. In this paper, membranes composed of bipolar lipids P2 extracted from the acidothermophile archaeon Sulfolobus solfataricus are studied. The biophysical basis for the membrane formation and thermal stability is investigated by using electron spin resonance (ESR) of spin-labeled lipids. Spectral anisotropy and isotropic hyperfine couplings are used to determine the chain flexibility and polarity gradients, respectively. For comparison, similar measurements have been carried out on aqueous dispersions of diacyl reference lipid dipalmitoyl phosphatidylcholine and also of diphytanoyl phosphatidylcholine, which has methyl-branched chains. At a given temperature, the bolaform lipid chains are more ordered and less flexible than in normal bilayer membranes. Only at elevated temperatures (80 degrees C) does the flexibility of the chain environment in tetraether lipid assemblies approach that of fluid bilayer membranes. The height of the hydrophobic barrier formed by a monolayer of archaebacterial lipids is similar to that in conventional fluid bilayer membranes, and the permeability barrier width is comparable to that formed by a bilayer of C16 lipid chains. At a mole ratio of 1:2, the tetraether P2 lipids mix well with dipalmitoyl phosphatidylcholine lipids and stabilize conventional bilayer membranes. The biological as well as the biotechnological relevance of the results is discussed.

  19. Conserved substitution patterns around nucleosome footprints in eukaryotes and Archaea derive from frequent nucleosome repositioning through evolution.

    Directory of Open Access Journals (Sweden)

    Tobias Warnecke

    Full Text Available Nucleosomes, the basic repeat units of eukaryotic chromatin, have been suggested to influence the evolution of eukaryotic genomes, both by altering the propensity of DNA to mutate and by selection acting to maintain or exclude nucleosomes in particular locations. Contrary to the popular idea that nucleosomes are unique to eukaryotes, histone proteins have also been discovered in some archaeal genomes. Archaeal nucleosomes, however, are quite unlike their eukaryotic counterparts in many respects, including their assembly into tetramers (rather than octamers from histone proteins that lack N- and C-terminal tails. Here, we show that despite these fundamental differences the association between nucleosome footprints and sequence evolution is strikingly conserved between humans and the model archaeon Haloferax volcanii. In light of this finding we examine whether selection or mutation can explain concordant substitution patterns in the two kingdoms. Unexpectedly, we find that neither the mutation nor the selection model are sufficient to explain the observed association between nucleosomes and sequence divergence. Instead, we demonstrate that nucleosome-associated substitution patterns are more consistent with a third model where sequence divergence results in frequent repositioning of nucleosomes during evolution. Indeed, we show that nucleosome repositioning is both necessary and largely sufficient to explain the association between current nucleosome positions and biased substitution patterns. This finding highlights the importance of considering the direction of causality between genetic and epigenetic change.

  20. Ultrasensitive biotin assay of a noncompetitive format in a homogeneous solution based on resonance energy transfer induced by a protein-protein interaction.

    Science.gov (United States)

    Ikeda, Tomohiro; Miyao, Hiroki; Sueda, Shinji

    2014-06-17

    Biotin is a water-soluble vitamin serving as a cofactor for several metabolic enzymes and plays crucial roles in every living cell. In the present study, we describe a noncompetitive assay for determination of biotin in a homogeneous solution. Our assay is based on a biotinylation reaction from archaeon Sulfolobus tokodaii. S. tokodaii biotinylation has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). Determination of biotin was performed by monitoring the complexation reaction between BPL and BCCP through biotinylation, based on luminescence resonance energy transfer (LRET) from a Tb(3+) complex to fluorescein, where BPL and BCCP were labeled with a Tb(3+) complex and fluorescein, respectively. Our assay allows for ultrasensitive detection of biotin with a detection limit of approximately 1 pM (or 0.2 fmol in a 0.2 mL sample volume) by a simple procedure without use of radioactive materials or enzymatic signal amplification. In addition, owing to its noncompetitive format, our assay has a very wide measurement range of at least 3 orders of magnitude. Our assay is also beneficial as a model system for interaction analysis based on LRET.

  1. Tungsten transport protein A (WtpA) in Pyrococcus furiosus: the first member of a new class of tungstate and molybdate transporters.

    Science.gov (United States)

    Bevers, Loes E; Hagedoorn, Peter-Leon; Krijger, Gerard C; Hagen, Wilfred R

    2006-09-01

    A novel tungstate and molybdate binding protein has been discovered from the hyperthermophilic archaeon Pyrococcus furiosus. This tungstate transport protein A (WtpA) is part of a new ABC transporter system selective for tungstate and molybdate. WtpA has very low sequence similarity with the earlier-characterized transport proteins ModA for molybdate and TupA for tungstate. Its structural gene is present in the genome of numerous archaea and some bacteria. The identification of this new tungstate and molybdate binding protein clarifies the mechanism of tungstate and molybdate transport in organisms that lack the known uptake systems associated with the ModA and TupA proteins, like many archaea. The periplasmic protein of this ABC transporter, WtpA (PF0080), was cloned and expressed in Escherichia coli. Using isothermal titration calorimetry, WtpA was observed to bind tungstate (dissociation constant [K(D)] of 17 +/- 7 pM) and molybdate (K(D) of 11 +/- 5 nM) with a stoichiometry of 1.0 mol oxoanion per mole of protein. These low K(D) values indicate that WtpA has a higher affinity for tungstate than do ModA and TupA and an affinity for molybdate similar to that of ModA. A displacement titration of molybdate-saturated WtpA with tungstate showed that the tungstate effectively replaced the molybdate in the binding site of the protein.

  2. A laboratory experiment of intact polar lipid degradation in sandy sediments

    Directory of Open Access Journals (Sweden)

    J. Logemann

    2011-03-01

    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.

  3. 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.

  4. 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.

  5. 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.

  6. Mutational and Bioinformatic Analysis of Haloarchaeal Lipobox-Containing Proteins

    Directory of Open Access Journals (Sweden)

    Stefanie Storf

    2010-01-01

    Full Text Available A conserved lipid-modified cysteine found in a protein motif commonly referred to as a lipobox mediates the membrane anchoring of a subset of proteins transported across the bacterial cytoplasmic membrane via the Sec pathway. Sequenced haloarchaeal genomes encode many putative lipoproteins and recent studies have confirmed the importance of the conserved lipobox cysteine for signal peptide processing of three lipobox-containing proteins in the model archaeon Haloferax volcanii. We have extended these in vivo analyses to additional Hfx. volcanii substrates, supporting our previous in silico predictions and confirming the diversity of predicted Hfx. volcanii lipoproteins. Moreover, using extensive comparative secretome analyses, we identified genes encodining putative lipoproteins across a wide range of archaeal species. While our in silico analyses, supported by in vivo data, indicate that most haloarchaeal lipoproteins are Tat substrates, these analyses also predict that many crenarchaeal species lack lipoproteins altogether and that other archaea, such as nonhalophilic euryarchaeal species, transport lipoproteins via the Sec pathway. To facilitate the identification of genes that encode potential haloarchaeal Tat-lipoproteins, we have developed TatLipo, a bioinformatic tool designed to detect lipoboxes in haloarchaeal Tat signal peptides. Our results provide a strong foundation for future studies aimed at identifying components of the archaeal lipoprotein biogenesis pathway.

  7. Identification of amino acids related to catalytic function of Sulfolobus solfataricus P1 carboxylesterase by site-directed mutagenesis and molecular modeling

    Science.gov (United States)

    Choi, Yun-Ho; Lee, Ye-Na; Park, Young-Jun; Yoon, Sung-Jin; Lee, Hee-Bong

    2016-01-01

    The archaeon Sulfolobus solfataricus P1 carboxylesterase is a thermostable enzyme with a molecular mass of 33.5 kDa belonging to the mammalian hormone-sensitive lipase (HSL) family. In our previous study, we purified the enzyme and suggested the expected amino acids related to its catalysis by chemical modification and a sequence homology search. For further validating these amino acids in this study, we modified them using site-directed mutagenesis and examined the activity of the mutant enzymes using spectrophotometric analysis and then estimated by homology modeling and fluorescence analysis. As a result, it was identified that Ser151, Asp244, and His274 consist of a catalytic triad, and Gly80, Gly81, and Ala152 compose an oxyanion hole of the enzyme. In addition, it was also determined that the cysteine residues are located near the active site or at the positions inducing any conformational changes of the enzyme by their replacement with serine residues. [BMB Reports 2016; 49(6): 349-354] PMID:27222124

  8. “Altiarchaeales”: Uncultivated Archaea from the Subsurface

    Directory of Open Access Journals (Sweden)

    Alexander J. Probst

    2015-05-01

    Full Text Available Due to the limited cultivability of the vast majority of microorganisms, researchers have applied environmental genomics and other state-of-the-art technologies to gain insights into the biology of uncultivated Archaea and bacteria in their natural biotope. In this review, we summarize the scientific findings on a recently proposed order-level lineage of uncultivated Archaea called Altiarchaeales, which includes “Candidatus Altiarchaeum hamiconexum” as the most well-described representative. Ca. A. hamiconexum possesses a complex biology: thriving strictly anaerobically, this microorganism is capable of forming highly-pure biofilms, connecting the cells by extraordinary cell surface appendages (the “hami” and has other highly unusual traits, such as a double-membrane-based cell wall. Indicated by genomic information from different biotopes, the Altiarchaeales seem to proliferate in deep, anoxic groundwater of Earth’s crust bearing a potentially very important function: carbon fixation. Although their net carbon fixation rate has not yet been determined, they appear as highly abundant organisms in their biotopes and may thus represent an important primary producer in the subsurface. In sum, the research over more than a decade on Ca. A. hamiconexum has revealed many interesting features of its lifestyle, its genomic information, metabolism and ultrastructure, making this archaeon one of the best-studied uncultivated Archaea in the literature.

  9. Complete genome sequence Methanothermus fervidus type strain (V24ST)

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Iain [U.S. Department of Energy, Joint Genome Institute; Djao, Olivier Duplex [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Misra, Monica [Los Alamos National Laboratory (LANL); Chertkov, Olga [Los Alamos National Laboratory (LANL); Nolan, Matt [U.S. Department of Energy, Joint Genome Institute; Lucas, Susan [U.S. Department of Energy, Joint Genome Institute; Lapidus, Alla L. [U.S. Department of Energy, Joint Genome Institute; Glavina Del Rio, Tijana [U.S. Department of Energy, Joint Genome Institute; Tice, Hope [U.S. Department of Energy, Joint Genome Institute; Cheng, Jan-Fang [U.S. Department of Energy, Joint Genome Institute; Tapia, Roxanne [Los Alamos National Laboratory (LANL); Han, Cliff [Los Alamos National Laboratory (LANL); Goodwin, Lynne A. [Los Alamos National Laboratory (LANL); Pitluck, Sam [U.S. Department of Energy, Joint Genome Institute; Liolios, Konstantinos [U.S. Department of Energy, Joint Genome Institute; Ivanova, N [U.S. Department of Energy, Joint Genome Institute; Mavromatis, K [U.S. Department of Energy, Joint Genome Institute; Mikhailova, Natalia [U.S. Department of Energy, Joint Genome Institute; Pati, Amrita [U.S. Department of Energy, Joint Genome Institute; Brambilla, Evelyne-Marie [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Chen, Amy [U.S. Department of Energy, Joint Genome Institute; Palaniappan, Krishna [U.S. Department of Energy, Joint Genome Institute; Land, Miriam L [ORNL; Hauser, Loren John [ORNL; Chang, Yun-Juan [ORNL; Jeffries, Cynthia [Oak Ridge National Laboratory (ORNL); Sikorski, Johannes [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Spring, Stefan [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Rohde, Manfred [HZI - Helmholtz Centre for Infection Research, Braunschweig, Germany; Eichinger, Konrad [Universitat Regensburg, Regensburg, Germany; Huber, Harald [Universitat Regensburg, Regensburg, Germany; Wirth, Reinhard [Universitat Regensburg, Regensburg, Germany; Goker, Markus [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany; Detter, J. Chris [U.S. Department of Energy, Joint Genome Institute; Woyke, Tanja [U.S. Department of Energy, Joint Genome Institute; Bristow, James [U.S. Department of Energy, Joint Genome Institute; Eisen, Jonathan [U.S. Department of Energy, Joint Genome Institute; Markowitz, Victor [U.S. Department of Energy, Joint Genome Institute; Hugenholtz, Philip [U.S. Department of Energy, Joint Genome Institute; Kyrpides, Nikos C [U.S. Department of Energy, Joint Genome Institute; Klenk, Hans-Peter [DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany

    2010-01-01

    Methanothermus fervidus Stetter 1982 is the type strain of the genus Methanothermus. This hyperthermophilic genus is of a thought to be endemic in Icelandic hot springs. M. fervidus was not only the first characterized organism with a maximal growth temperature (97 C) close to the boiling point of water, but also the first archaeon in which a detailed functional analysis of its histone protein was reported and the first one in which the function of 2,3-cyclodiphosphoglycerate in thermoadaptation was characterized. Strain V24ST is of interest because of its very low substrate ranges, it grows only on H2 + CO2. This is the first completed genome sequence of the family Methanothermaceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,243,342 bp long genome with its 1,311 protein-coding and 50 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

  10. De Novo Sequences of Haloquadratum walsbyi from Lake Tyrrell, Australia, Reveal a Variable Genomic Landscape

    Directory of Open Access Journals (Sweden)

    Benjamin J. Tully

    2015-01-01

    Full Text Available Hypersaline systems near salt saturation levels represent an extreme environment, in which organisms grow and survive near the limits of life. One of the abundant members of the microbial communities in hypersaline systems is the square archaeon, Haloquadratum walsbyi. Utilizing a short-read metagenome from Lake Tyrrell, a hypersaline ecosystem in Victoria, Australia, we performed a comparative genomic analysis of H. walsbyi to better understand the extent of variation between strains/subspecies. Results revealed that previously isolated strains/subspecies do not fully describe the complete repertoire of the genomic landscape present in H. walsbyi. Rearrangements, insertions, and deletions were observed for the Lake Tyrrell derived Haloquadratum genomes and were supported by environmental de novo sequences, including shifts in the dominant genomic landscape of the two most abundant strains. Analysis pertaining to halomucins indicated that homologs for this large protein are not a feature common for all species of Haloquadratum. Further, we analyzed ATP-binding cassette transporters (ABC-type transporters for evidence of niche partitioning between different strains/subspecies. We were able to identify unique and variable transporter subunits from all five genomes analyzed and the de novo environmental sequences, suggesting that differences in nutrient and carbon source acquisition may play a role in maintaining distinct strains/subspecies.

  11. Construction of an Immobilized Thermophilic Esterase on Epoxy Support for Poly(ε-caprolactone Synthesis

    Directory of Open Access Journals (Sweden)

    Hui Ren

    2016-06-01

    Full Text Available Developing an efficient immobilized enzyme is of great significance for improving the operational stability of enzymes in poly(ε-caprolactone synthesis. In this paper, a thermophilic esterase AFEST from the archaeon Archaeoglobus fulgidus was successfully immobilized on the epoxy support Sepabeads EC-EP via covalent attachment, and the immobilized enzyme was then employed as a biocatalyst for poly(ε-caprolactone synthesis. The enzyme loading and recovered activity of immobilized enzyme was measured to be 72 mg/g and 10.4 U/mg using p-nitrophenyl caprylate as the substrate at 80 °C, respectively. Through the optimization of reaction conditions (enzyme concentration, temperature, reaction time and medium, poly(ε-caprolactone was obtained with 100% monomer conversion and low number-average molecular weight (Mn < 1300 g/mol. Further, the immobilized enzyme exhibited excellent reusability, with monomer conversion values exceeding 75% during 15 batch reactions. Finally, poly(ε-caprolactone was enzymatically synthesized with an isolated yield of 75% and Mn value of 3005 g/mol in a gram-scale reaction.

  12. 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.

  13. The Sso7d DNA-binding protein from Sulfolobus solfataricus has ribonuclease activity.

    Science.gov (United States)

    Shehi, E; Serina, S; Fumagalli, G; Vanoni, M; Consonni, R; Zetta, L; Dehò, G; Tortora, P; Fusi, P

    2001-05-25

    Sso7d is a small, basic, abundant protein from the thermoacidophilic archaeon Sulfolobus solfataricus. Previous research has shown that Sso7d can bind double-stranded DNA without sequence specificity by placing its triple-stranded beta-sheet across the minor groove. We previously found RNase activity both in preparations of Sso7d purified from its natural source and in recombinant, purified protein expressed in Escherichia coli. This paper provides conclusive evidence that supports the assignment of RNase activity to Sso7d, shown by the total absence of activity in the single-point mutants E35L and K12L, despite the preservation of their overall structure under the assay conditions. In keeping with our observation that the residues putatively involved in RNase activity and those playing a role in DNA binding are located on different surfaces of the molecule, the activity was not impaired in the presence of DNA. If a small synthetic RNA was used as a substrate, Sso7d attacked both predicted double- and single-stranded RNA stretches, with no evident preference for specific sequences or individual bases. Apparently, the more readily attacked bonds were those intrinsically more unstable.

  14. 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.

  15. Growth of ammonia-oxidizing archaea and bacteria in cattle manure compost under various temperatures and ammonia concentrations.

    Science.gov (United States)

    Oishi, Ryu; Tada, Chika; Asano, Ryoki; Yamamoto, Nozomi; Suyama, Yoshihisa; Nakai, Yutaka

    2012-05-01

    A recent study showed that ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) coexist in the process of cattle manure composting. To investigate their physiological characteristics, liquid cultures seeded with fermenting cattle manure compost were incubated at various temperatures (37°C, 46°C, or 60°C) and ammonium concentrations (0.5, 1, 4, or 10 mM NH (4) (+) -N). The growth rates of the AOB and AOA were monitored using real-time polymerase chain reaction analysis targeting the bacterial and archaeal ammonia monooxygenase subunit A genes. AOB grew at 37°C and 4 or 10 mM NH (4) (+) -N, whereas AOA grew at 46°C and 10 mM NH (4) (+) -N. Incubation with allylthiourea indicated that the AOB and AOA grew by oxidizing ammonia. Denaturing gradient gel electrophoresis and subsequent sequencing analyses revealed that a bacterium related to Nitrosomonas halophila and an archaeon related to Candidatus Nitrososphaera gargensis were the predominant AOB and AOA, respectively, in the seed compost and in cultures after incubation. This is the first report to demonstrate that the predominant AOA in cattle manure compost can grow and can probably oxidize ammonia under moderately thermophilic conditions.

  16. Perchlorate and halophilic prokaryotes: implications for possible halophilic life on Mars.

    Science.gov (United States)

    Oren, Aharon; Elevi Bardavid, Rahel; Mana, Lily

    2014-01-01

    In view of the finding of perchlorate among the salts detected by the Phoenix Lander on Mars, we investigated the relationships of halophilic heterotrophic microorganisms (archaea of the family Halobacteriaceae and the bacterium Halomonas elongata) toward perchlorate. All strains tested grew well in NaCl-based media containing 0.4 M perchlorate, but at the highest perchlorate concentrations, tested cells were swollen or distorted. Some species (Haloferax mediterranei, Haloferax denitrificans, Haloferax gibbonsii, Haloarcula marismortui, Haloarcula vallismortis) could use perchlorate as an electron acceptor for anaerobic growth. Although perchlorate is highly oxidizing, its presence at a concentration of 0.2 M for up to 2 weeks did not negatively affect the ability of a yeast extract-based medium to support growth of the archaeon Halobacterium salinarum. These findings show that presence of perchlorate among the salts on Mars does not preclude the possibility of halophilic life. If indeed the liquid brines that may exist on Mars are inhabited by salt-requiring or salt-tolerant microorganisms similar to the halophiles on Earth, presence of perchlorate may even be stimulatory when it can serve as an electron acceptor for respiratory activity in the anaerobic Martian environment.

  17. 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.

  18. Formaldehyde as a carbon and electron shuttle between autotroph and heterotroph populations in acidic hydrothermal vents of Norris Geyser Basin, Yellowstone National Park.

    Science.gov (United States)

    Moran, James J; Whitmore, Laura M; Isern, Nancy G; Romine, Margaret F; Riha, Krystin M; Inskeep, William P; Kreuzer, Helen W

    2016-05-01

    The Norris Geyser Basin in Yellowstone National Park contains a large number of hydrothermal systems, which host microbial populations supported by primary productivity associated with a suite of chemolithotrophic metabolisms. We demonstrate that Metallosphaera yellowstonensis MK1, a facultative autotrophic archaeon isolated from a hyperthermal acidic hydrous ferric oxide (HFO) spring in Norris Geyser Basin, excretes formaldehyde during autotrophic growth. To determine the fate of formaldehyde in this low organic carbon environment, we incubated native microbial mat (containing M. yellowstonensis) from a HFO spring with (13)C-formaldehyde. Isotopic analysis of incubation-derived CO2 and biomass showed that formaldehyde was both oxidized and assimilated by members of the community. Autotrophy, formaldehyde oxidation, and formaldehyde assimilation displayed different sensitivities to chemical inhibitors, suggesting that distinct sub-populations in the mat selectively perform these functions. Our results demonstrate that electrons originally resulting from iron oxidation can energetically fuel autotrophic carbon fixation and associated formaldehyde excretion, and that formaldehyde is both oxidized and assimilated by different organisms within the native microbial community. Thus, formaldehyde can effectively act as a carbon and electron shuttle connecting the autotrophic, iron oxidizing members with associated heterotrophic members in the HFO community.

  19. Formaldehyde as a carbon and electron shuttle between autotroph and heterotroph populations in acidic hydrothermal vents of Norris Geyser Basin, Yellowstone National Park

    Energy Technology Data Exchange (ETDEWEB)

    Moran, James J.; Whitmore, Laura M.; Isern, Nancy G.; Romine, Margaret F.; Riha, Krystin M.; Inskeep, William P.; Kreuzer, Helen W.

    2016-03-19

    The Norris Geyser Basin in Yellowstone National Park contains a large number of hydrothermal systems, which host microbial populations supported by primary productivity associated with a suite of chemolithotrophic metabolisms. We demonstrate that Metallosphaera yellowstonesis MK1, a facultative autotrophic archaeon isolated from a hyperthermal acidic hydrous ferric oxide (HFO) spring in Norris Geyser Basin, excretes formaldehyde during autotrophic growth. To determine the fate of formaldehyde in this low organic carbon environment, we incubated native microbial mat (containing M. yellowstonensis) from a HFO spring with 13C-formaldehyde. Isotopic analysis of incubation-derived CO2 and biomass showed that formaldehyde was both oxidized and assimilated by members of the community. Autotrophy, formaldehyde oxidation, and formaldehyde assimilation displayed different sensitivities to chemical inhibitors, suggesting that distinct sub-populations in the mat selectively perform these functions. Our results demonstrate that electrons originally resulting from iron oxidation can energetically fuel autotrophic carbon fixation and associated formaldehyde excretion, and that formaldehyde is both oxidized and assimilated by different organisms within the native microbial community. Thus, formaldehyde can effectively act as a carbon and electron shuttle connecting the autotrophic, iron oxidizing members with associated heterotrophic members in the HFO community.

  20. Preparation of linear maltodextrins using a hyperthermophilic amylopullulanase with cyclodextrin- and starch-hydrolysing activities.

    Science.gov (United States)

    Li, Xiaolei; Li, Dan

    2015-03-30

    A novel method for the preparation of linear maltodextrins from cyclodextrins and starch was proposed. To accomplish this process, an amylopullulanase from hyperthermophilic archaeon Caldivirga maquilingensis (CMApu) was characterized and used. CMApu with an estimated molecular mass of 62.7 kDa by SDS-PAGE had a maximal pullulan-hydrolysing activity at 100°C and pH 5.0. It could also hydrolyse amylopectin (AP), starch, β-CD and amylose (AM), in a decreasing order of relative activities from 88.96% to 57.17%. TLC and HPAEC analysis revealed that CMApu catalyzed the debranching and degrading reactions to produce linear malto-oligosaccharides (≤ G8-G1) from G8-β-CD and/or normal CDs, amylodextrins (DP6-96) from AM, and amylodextrins (DP1-76) from AP and potato starch. Our results showed that CMApu had a great potential for the industrial preparation of linear maltodextrins from normal starch instead of waxy starch, malto-oligosaccharides or sucrose. And the high optimal temperature of CMApu facilitated the simultaneous gelatinization and hydrolysis of cereal starch.

  1. Hans Georg Trüper (1936–2016) and His Contributions to Halophile Research

    Science.gov (United States)

    Oren, Aharon

    2016-01-01

    Prof. Hans Georg Trüper, one of the most important scientists in the field of halophile research, passed away on 9 March 2016 at the age of 79. I here present a brief obituary with special emphasis on Prof. Trüper’s contributions to our understanding of the halophilic prokaryotes and their adaptations to life in hypersaline environments. He has pioneered the study of the halophilic anoxygenic phototrophic sulfur bacteria of the Ectothiorhodospira—Halorhodospira group. Some of the species he and his group isolated from hypersaline and haloalkaline environments have become model organisms for the study of the mechanisms of haloadaptation: the functions of three major organic compounds – glycine betaine, ectoine, and trehalose – known to serve as “compatible solutes” in halophilic members of the Bacteria domain, were discovered during studies of these anoxygenic phototrophs. Prof. Trüper’s studies of hypersaline alkaline environments in Egypt also led to the isolation of the first known extremely halophilic archaeon (Natronomonas pharaonis). The guest editors dedicate this special volume of Life to the memory of Prof. Hans Georg Trüper. PMID:27187481

  2. Speculations on the origin of life and thermophily: Review of available information on reverse gyrase suggests that hyperthermophilic procaryotes are not so primitive

    Science.gov (United States)

    Forterre, Patrick; Confalonier, Fabrice; Charbonnier, Franck; Duguet, Michel

    1995-06-01

    All present-day hyperthermophiles studied so far (eitherBacteria orArchaea) contain a unique DNA topoisomerase, reverse gyrase, which probably helps to stabilize genomic DNA at high temperature. Herein the data relating this enzyme is reviewed and discussed from the perspective of the nature of the last detectable common ancestor and the origin of life. The sequence of the gene encoding reverse gyrase from an archaeon,Sulfolobus acidocaldarius, suggests that this enzyme contains both a helicase and a topoisomerase domains (Confalonieriet al.,Proc. Natl. Acad. Sci., 1993, 90, 4735). Accordingly, it has been proposed that reverse gyrase originated by the fusion of DNA helicase and DNA topoisomerase genes. If reverse gyrase is essential for life at high temperature, its composite structure suggests that DNA helicases and topoisomerases appeared independently and first evolved in a mesophilic world. Such scenario contradicts the hypothesis that a direct link connects present day hyperthermophiles to a hot origin of life. We discuss different patterns for the early cellular evolution in which reverse gyrase appeared either before the emergence of the last common ancestor ofArchaea, Bacteria andEucarya, or in a lineage common to the two procaryotic domains. The latter scenario could explain why all today hyperthermophiles are procaryotes.

  3. Studies on structural stability of thermophilic Sulfolobus acidocaldarius ribosomes.

    Science.gov (United States)

    Yangala, Kalavathi; Suryanarayana, Tangirala

    2007-02-01

    Structural stability of thermophilic archaeon Sulfolobus acidocaldarius ribosomes, with respect their susceptibility to pancreatic RNase A and stability to temperature (deltaTm), on treatment with various stabilizing (polyamines) and destabilizing (sulfhydryl and intercalating) agents were studied and compared with mesophilic E. coli ribosomes, to understand the structural differences between thermophilic and mesophilic ribosomes. Thermophilic archaeal ribosomes and their subunits were 10-times less susceptible to pancreatic RNase A, compared to mesophilic ribosomes, showing the presence of strong and compact structural organization in them. Thermophilic ribosomes treated with destabilizing agents, such as sulfhydryl reagents [5,5'-Dithio-bis-(2-nitrobenzoic acid), N-ethylmaleimide and p-hydroxymercurybenzoate) and intercalating agents (ethidium bromide, EtBr) showed higher stability to RNase A, compared to similarly treated mesophilic ribosomes, indicating the unavailability of thiol-reactive groups and the presence of strong solvent inaccessible inner core. Higher stability of thermophilic ribosomes compared to mesophilic ribosomes to unfolding agents like urea further supported the presence of strong inner core particle. Thermophilic ribosomes treated with intercalating agents, such as EtBr were less susceptible to RNase A, though they bound to more reagent, showing the rigidity or resilience of their macromolecular structure to alterations caused by destabilizing agents. Overall, these results indicated that factors such as presence of strong solvent inaccessible inner core and rigidity of ribosome macromolecular structure contributed stability of thermophilic ribosomes to RNase A and other destabilizing agents, when compared to mesophilic ribosomes.

  4. Electron cryotomography of ESCRT assemblies and dividing Sulfolobus cells suggests that spiraling filaments are involved in membrane scission.

    Science.gov (United States)

    Dobro, Megan J; Samson, Rachel Y; Yu, Zhiheng; McCullough, John; Ding, H Jane; Chong, Parkson Lee-Gau; Bell, Stephen D; Jensen, Grant J

    2013-08-01

    The endosomal-sorting complex required for transport (ESCRT) is evolutionarily conserved from Archaea to eukaryotes. The complex drives membrane scission events in a range of processes, including cytokinesis in Metazoa and some Archaea. CdvA is the protein in Archaea that recruits ESCRT-III to the membrane. Using electron cryotomography (ECT), we find that CdvA polymerizes into helical filaments wrapped around liposomes. ESCRT-III proteins are responsible for the cinching of membranes and have been shown to assemble into helical tubes in vitro, but here we show that they also can form nested tubes and nested cones, which reveal surprisingly numerous and versatile contacts. To observe the ESCRT-CdvA complex in a physiological context, we used ECT to image the archaeon Sulfolobus acidocaldarius and observed a distinct protein belt at the leading edge of constriction furrows in dividing cells. The known dimensions of ESCRT-III proteins constrain their possible orientations within each of these structures and point to the involvement of spiraling filaments in membrane scission.

  5. Microbial desulfurization of coal by thermophilic archaea

    Energy Technology Data Exchange (ETDEWEB)

    Olsson, Gunnel

    1994-04-01

    The investigation was focused on the removal of pyrite as well as organic sulfur. One major objective was to identify and outline difficulties associated with microbial desulfurization of coal. The work has particularly been concentrated on the desulfurization environment of the microorganisms, the reprecipitation of dissolved sulfate as jarosite, the effect of microbial treatment on the properties of the coal and the comparison of different thermophilic archaea suggested for coal desulfurization. The investigated microorganisms were the thermophilic archaea Acidianus brierleyi, Sulfolobus acidocaldarius and Sulfolobus solfataricus and for comparison the mesophilic bacterium Thiobacillus ferrooxidans. The major part of the work has been done with Acidianus brierleyi. Compounds leached from coal may seriously affect the growth of microorganisms suggested for coal desulfurization. Degradation of pyritic sulfur with the used strains of S. solfataricus and S. acidocaldarius, was observed to be impossible. However, both the thermophilic archaeon Acidianus brierleyi and the mesophilic bacterium Thiobacillus ferrooxidans, were capable of degrading pure pyrite as well as pyrite from low-sulfur coals. Up to 85% removal of pyritic sulfur was obtained for coals when staring with a pyrite sulfur content of 0.5-0.7%. From kinetic studies, it was shown that A. brierleyi and T. ferrooxidans remove sulfur from coal at roughly the same rate, at least for the coals investigated in this study. However, the rate for microbial oxidation of pure pyrite was seen to be much higher for A. brierleyi than for T. ferroxidans. 62 refs, 16 figs, 5 tabs

  6. Recombination of synthetic oligonucleotides with prokaryotic chromosomes: substrate requirements of the Escherichia coli/lambdaRed and Sulfolobus acidocaldarius recombination systems.

    Science.gov (United States)

    Grogan, Dennis W; Stengel, Kristy R

    2008-09-01

    In order to reveal functional properties of recombination involving short ssDNAs in hyperthermophilic archaea, we evaluated oligonucleotide-mediated transformation (OMT) in Sulfolobus acidocaldarius and Escherichia coli as a function of the molecular properties of the ssDNA substrates. Unmodified ssDNAs as short as 20-22 nt yielded recombinants in both organisms, as did longer DNAs forming as few as 2-5 base pairs on one side of the genomic mutation. The two OMT systems showed similar responses to certain end modifications of the oligonucleotides, but E. coli was found to require a 5' phosphate on 5'-limited ssDNA whereas this requirement was not evident in S. acidocaldarius. The ability of both E. coli and S. acidocaldarius to incorporate short, mismatched ssDNAs into their genomes raises questions about the biological significance of this capability, including its phylogenetic distribution among microorganisms and its impact on genome stability. These questions seem particularly relevant for S. acidocaldarius, as this archaeon has natural competence for OMT, encodes no MutSL homologues and thrives under environmental conditions that accelerate DNA decomposition.

  7. Interactions of Sulfolobus acidocaldarius with uranium

    Energy Technology Data Exchange (ETDEWEB)

    Reitz, T.; Merroun, M.L.; Rossberg, A.; Selenska-Pobell, S. [Inst. of Radiochemistry, Forschungszentrum Rossendorf, Dresden (Germany)

    2010-07-01

    Interactions of the acidothermophilic archaeon Sulfolobus acidocaldarius DSM 639 with U(VI) were studied by using a combination of batch experiments, X-ray absorption spectroscopy (XAS), and time-resolved laser-induced fluorescence spectroscopy (TRLFS). We demonstrated that at pH 2 this archaeal strain possesses a low tolerance to U(VI) and that its growth is limited to a uranium concentration below 1.1 mM. At similarly high acidic conditions (pH 1.5 and 3.0), covering the physiological pH growth optimum of S. acidocaldarius, at which U(VI) is soluble and highly toxic, rapid accumulation of the radionuclide by the cells of the strain occurred. About half of the uranium binding capacity was reached by the strain after an incubation of five minutes and nearly total saturation of the binding sites was achieved after 30 min. Both, EXAFS- and TRLF-spectroscopic analyses showed that the accumulated U(VI) was complexed mainly through organic phosphate groups. The EXAFS measurements revealed that U(VI) is coordinated to the organic phosphate ligands of the archaeal cells in a monodentate binding mode with an average U-P bond distance of 3.60 {+-} 0.02 A (orig.)

  8. Inducible and constitutive promoters for genetic systems in Sulfolobus acidocaldarius.

    Science.gov (United States)

    Berkner, Silvia; Wlodkowski, Alexander; Albers, Sonja-Verena; Lipps, Georg

    2010-05-01

    Central to genetic work in any organism are the availability of a range of inducible and constitutive promoters. In this work we studied several promoters for use in the hyperthermophilic archaeon Sulfolobus acidocaldarius. The promoters were tested with the aid of an E. coli-Sulfolobus shuttle vector in reporter gene experiments. As the most suitable inducible promoter a maltose inducible promoter was identified. It comprises 266 bp of the sequence upstream of the gene coding for the maltose/maltotriose binding protein (mbp, Saci_1165). Induction is feasible with either maltose or dextrin at concentrations of 0.2-0.4%. The highest increase in expression (up to 17-fold) was observed in late exponential and stationary phase around 30-50 h after addition of dextrin. Whereas in the presence of glucose and xylose higher basal activity and reduced inducibility with maltose is observed, sucrose can be used in the growth medium additionally without affecting the basal activity or the inducibility. The minimal promoter region necessary could be narrowed down to 169 bp of the upstream sequence. The ABCE1 protein from S. solfataricus was successfully expressed under control of the inducible promoter with the shuttle vector pC and purified from the S. acidocaldarius culture with a yield of about 1 mg L(-1) culture. In addition we also determined the promoter strength of several constitutive promoters.

  9. Characterization of two β-decarboxylating dehydrogenases from Sulfolobus acidocaldarius.

    Science.gov (United States)

    Takahashi, Kento; Nakanishi, Fumika; Tomita, Takeo; Akiyama, Nagisa; Lassak, Kerstin; Albers, Sonja-Verena; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2016-11-01

    Sulfolobus acidocaldarius, a hyperthermoacidophilic archaeon, possesses two β-decarboxylating dehydrogenase genes, saci_0600 and saci_2375, in its genome, which suggests that it uses these enzymes for three similar reactions in lysine biosynthesis through 2-aminoadipate, leucine biosynthesis, and the tricarboxylic acid cycle. To elucidate their roles, these two genes were expressed in Escherichia coli in the present study and their gene products were characterized. Saci_0600 recognized 3-isopropylmalate as a substrate, but exhibited slight and no activity for homoisocitrate and isocitrate, respectively. Saci_2375 exhibited distinct and similar activities for isocitrate and homoisocitrate, but no detectable activity for 3-isopropylmalate. These results suggest that Saci_0600 is a 3-isopropylmalate dehydrogenase for leucine biosynthesis and Saci_2375 is a dual function enzyme serving as isocitrate-homoisocitrate dehydrogenase. The crystal structure of Saci_0600 was determined as a closed-form complex that binds 3-isopropylmalate and Mg(2+), thereby revealing the structural basis for the extreme thermostability and novel-type recognition of the 3-isopropyl moiety of the substrate.

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

    Science.gov (United States)

    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-transporters with each subunit consisting of a membrane domain fused at the C-terminus to an ATP-binding domain, or as full transporters in which the two subunits are fused into a single polypeptide. The saci_2123 gene of the thermoacidophilic archaeon Sulfolobus acidocaldarius is the only gene in the genome that encodes an ATP-binding cassette half-transporter, while a homologous gene is present in the genomes of S. solfataricus, S. tokodaii and S islandicus. Saci_2123 shares homology with well-characterized bacterial and mammalian MDR transporters. The saci_2132 gene is up-regulated when cells are exposed to drugs. A deletion mutant of saci_2132 was found to be more vulnerable to a set of toxic compounds, including detergents, antibiotics and uncouplers as compared to the wild-type strain, while the drug resistance could be restored through the plasmid-based expression of saci_2132. These data demonstrate that Saci_2132 is an archaeal ABC-MDR transporter and therefore it was termed Smr1 (Sulfolobus multidrug resistance transporter 1).

  11. Thermoacidophilic archaea for pyrite oxidation in coal desulphurization

    Energy Technology Data Exchange (ETDEWEB)

    Larsson, Liselotte

    1995-10-01

    The desulfurization of low-sulfur coals has been demonstrated with the thermoacidophilic archaeon Acidianus brierleyi. A. brierleyi facilitates the removal of inorganic sulfur from coal and oxidizes mineral pyrite. The results imply that the mechanism behind microbial coal desulfurization and pyrite oxidation is a combination of biotic and abiotic leaching of pyrite. The extent of sulfur removal is dependent on the type of coal and is closely related to he amount of pyritic sulfur in the coal. Studies have shown that neither ash content nor heating value were dramatically affected by the microbial treatment. The use of the archaea Sulfolobus acidocaldarius and Sulfolobus solfataricus, as well as the mesophilic bacteria Thiobacillus ferrooxidans and several Pseudomonas species, has also been studied for coal desulfurization and mineral pyrite oxidation. The archaea and Pseudomonas species did not grow autotrophically on mineral pyrite neither did they oxidize pyrite in coal. The oxidation rate was, however, 5-10 times less than with A. brierleyi on mineral pyrite. The rate of sulfur removal from coal was in the same range as for A. brierleyi which indicates that different reactions are rate limiting in coal depyritization than in mineral pyrite oxidation. 133 refs, 18 figs, 3 tabs

  12. Insights into Hydrocarbon-rich Environments from Studies of Protein Dynamics, Thermodynamics, and Spectroscopy

    Science.gov (United States)

    Magyar, J. S.; Asous, N. K.; Barth, S. J.; Benzik, E.; Chou, J.; Dalchand, N.; Gallagher, G. E.; Montero, K. S.; Lone, S. K.; Salerno, G. J.

    2015-12-01

    Extraordinary amounts of information are now available from genomic and metagenomic analyses of a wide variety of environments of geological and biological interest. Using such genomic information as a starting point, we are interested in looking at microbial systems at the molecular level, using the tools and approaches of inorganic chemistry, physical chemistry, and molecular biology. From these studies, spanning the molecular to the global, we gain insights into relationships between microbial life and the geochemical environment in which it lives. In our work to date, we have focused on hydrocarbon-rich environments, including the La Brea Tar Pits and the Gulf of Mexico. Starting from genomic information, we have identified proteins of interest, cloned synthetic genes into E. coli, overexpressed and purified the proteins, and characterized them by UV-visible absorption, circular dichroism, and NMR spectroscopies; X-ray crystallography; and electrochemistry. Using as examples our recent studies of a metal-uptake protein from a methanogenic archaeon native to the La Brea Tar Pits, and of electron-transfer and hydrocarbon-degrading proteins from cold marine ecosystems, we describe how new combinations of genomics, molecular biology, and bioinorganic chemistry can provide novel insights into geobiological processes.

  13. 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.

  14. Preservation of Archaeal Surface Layer Structure During Mineralization

    Science.gov (United States)

    Kish, Adrienne; Miot, Jennyfer; Lombard, Carine; Guigner, Jean-Michel; Bernard, Sylvain; Zirah, Séverine; Guyot, François

    2016-05-01

    Proteinaceous surface layers (S-layers) are highly ordered, crystalline structures commonly found in prokaryotic cell envelopes that augment their structural stability and modify interactions with metals in the environment. While mineral formation associated with S-layers has previously been noted, the mechanisms were unconstrained. Using Sulfolobus acidocaldarius a hyperthermophilic archaeon native to metal-enriched environments and possessing a cell envelope composed only of a S-layer and a lipid cell membrane, we describe a passive process of iron phosphate nucleation and growth within the S-layer of cells and cell-free S-layer “ghosts” during incubation in a Fe-rich medium, independently of metabolic activity. This process followed five steps: (1) initial formation of mineral patches associated with S-layer; (2) patch expansion; (3) patch connection; (4) formation of a continuous mineral encrusted layer at the cell surface; (5) early stages of S-layer fossilization via growth of the extracellular mineralized layer and the mineralization of cytosolic face of the cell membrane. At more advanced stages of encrustation, encrusted outer membrane vesicles are formed, likely in an attempt to remove damaged S-layer proteins. The S-layer structure remains strikingly well preserved even upon the final step of encrustation, offering potential biosignatures to be looked for in the fossil record.

  15. S-layers at second glance? Altiarchaeal grappling hooks (hami) resemble archaeal S-layer proteins in structure and sequence.

    Science.gov (United States)

    Perras, Alexandra K; Daum, Bertram; Ziegler, Christine; Takahashi, Lynelle K; Ahmed, Musahid; Wanner, Gerhard; Klingl, Andreas; Leitinger, Gerd; Kolb-Lenz, Dagmar; Gribaldo, Simonetta; Auerbach, Anna; Mora, Maximilian; Probst, Alexander J; Bellack, Annett; Moissl-Eichinger, Christine

    2015-01-01

    The uncultivated "Candidatus 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 domain at their N-terminal region (44-47% 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.

  16. The cytochrome ba complex from the thermoacidophilic crenarchaeote Acidianus ambivalens is an analog of bc(1) complexes.

    Science.gov (United States)

    Bandeiras, Tiago M; Refojo, Patricia N; Todorovic, Smilja; Murgida, Daniel H; Hildebrandt, Peter; Bauer, Christian; Pereira, Manuela M; Kletzin, Arnulf; Teixeira, Miguel

    2009-01-01

    A novel cytochrome ba complex was isolated from aerobically grown cells of the thermoacidophilic archaeon Acidianus ambivalens. The complex was purified with two subunits, which are encoded by the cbsA and soxN genes. These genes are part of the pentacistronic cbsAB-soxLN-odsN locus. The spectroscopic characterization revealed the presence of three low-spin hemes, two of the b and one of the a(s)-type with reduction potentials of +200, +400 and +160 mV, respectively. The SoxN protein is proposed to harbor the heme b of lower reduction potential and the heme a(s), and CbsA the other heme b. The soxL gene encodes a Rieske protein, which was expressed in E. coli; its reduction potential was determined to be +320 mV. Topology predictions showed that SoxN, CbsB and CbsA should contain 12, 9 and one transmembrane alpha-helices, respectively, with SoxN having a predicted fold very similar to those of the cytochromes b in bc(1) complexes. The presence of two quinol binding motifs was also predicted in SoxN. Based on these findings, we propose that the A. ambivalens cytochrome ba complex is analogous to the bc(1) complexes of bacteria and mitochondria, however with distinct subunits and heme types.

  17. Anaerobic degradation of homocyclic aromatic compounds via arylcarboxyl-coenzyme A esters: organisms, strategies and key enzymes.

    Science.gov (United States)

    Boll, Matthias; Löffler, Claudia; Morris, Brandon E L; Kung, Johannes W

    2014-03-01

    Next to carbohydrates, aromatic compounds are the second most abundant class of natural organic molecules in living organic matter but also make up a significant proportion of fossil carbon sources. Only microorganisms are capable of fully mineralizing aromatic compounds. While aerobic microbes use well-studied oxygenases for the activation and cleavage of aromatic rings, anaerobic bacteria follow completely different strategies to initiate catabolism. The key enzymes related to aromatic compound degradation in anaerobic bacteria are comprised of metal- and/or flavin-containing cofactors, of which many use unprecedented radical mechanisms for C-H bond cleavage or dearomatization. Over the past decade, the increasing number of completed genomes has helped to reveal a large variety of anaerobic degradation pathways in Proteobacteria, Gram-positive microbes and in one archaeon. This review aims to update our understanding of the occurrence of aromatic degradation capabilities in anaerobic microorganisms and serves to highlight characteristic enzymatic reactions involved in (i) the anoxic oxidation of alkyl side chains attached to aromatic rings, (ii) the carboxylation of aromatic rings and (iii) the reductive dearomatization of central arylcarboxyl-coenzyme A intermediates. Depending on the redox potential of the electron acceptors used and the metabolic efficiency of the cell, different strategies may be employed for identical overall reactions.

  18. Rapid evolution of stability and productivity at the origin of a microbial mutualism

    Energy Technology Data Exchange (ETDEWEB)

    Hillesland, Kristina L.; Stahl, David A.

    2009-12-01

    Mutualistic interactions are taxonomically and functionally diverse. Despite their ubiquity, the basic ecological and evolutionary processes underlying their origin and maintenance are poorly understood. A major reason for this has been the lack of an experimentally tractable model system. We examine the evolution of an experimentally imposed obligate mutualism between sulfate-reducing and methanogenic microorganisms that have no known history of prior interaction. Twenty-four independent pairings (cocultures) of the bacterium Desulfovibrio vulgaris and the archaeon Methanococcus maripaludis were established and followed for 300 community doublings in two environments, one allowing for the development of a heterogeneous distribution of resources and the other not. Evolved cocultures grew up to 80percent faster and were up to 30percent more productive (biomass yield per mole substrate) than the ancestors. The evolutionary process was marked by periods of significant instability leading to extinction of two of the cocultures, but resulted in more stable, efficient, and productive mutualisms for most replicated pairings. Comparisons of evolved cocultures with those assembled from one evolved and one ancestral mutualist showed that evolution of both species contributed to improved productivity. Surprisingly, however, overall improvements in growth rate and yield were less than the sum of individual contributions, suggesting antagonistic interactions between mutations from the coevolved populations. Physical constraints on the transfer of metabolites in the evolution environment affected the evolution of M. maripaludis but not D. vulgaris. Together, these results show that challenges can imperil nascent obligate mutualisms and demonstrate the evolutionary responses that enable their persistence and future evolution.

  19. High prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae detected in the human gut using an improved DNA detection protocol.

    Directory of Open Access Journals (Sweden)

    Bédis Dridi

    Full Text Available BACKGROUND: The low and variable prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae DNA in human stool contrasts with the paramount role of these methanogenic Archaea in digestion processes. We hypothesized that this contrast is a consequence of the inefficiencies of current protocols for archaeon DNA extraction. We developed a new protocol for the extraction and PCR-based detection of M. smithii and M. stadtmanae DNA in human stool. METHODOLOGY/PRINCIPAL FINDINGS: Stool specimens collected from 700 individuals were filtered, mechanically lysed twice, and incubated overnight with proteinase K prior to DNA extraction using a commercial DNA extraction kit. Total DNA was used as a template for quantitative real-time PCR targeting M. smithii and M. stadtmanae 16S rRNA and rpoB genes. Amplification of 16S rRNA and rpoB yielded positive detection of M. smithii in 95.7% and M. stadtmanae in 29.4% of specimens. Sequencing of 16S rRNA gene PCR products from 30 randomly selected specimens (15 for M. smithii and 15 for M. stadtmanae yielded a sequence similarity of 99-100% using the reference M. smithii ATCC 35061 and M. stadtmanae DSM 3091 sequences. CONCLUSIONS/SIGNIFICANCE: In contrast to previous reports, these data indicate a high prevalence of the methanogens M. smithii and M. stadtmanae in the human gut, with the former being an almost ubiquitous inhabitant of the intestinal microbiome.

  20. Comparative structural biology of eubacterial and archaeal oligosaccharyltransferases.

    Science.gov (United States)

    Maita, Nobuo; Nyirenda, James; Igura, Mayumi; Kamishikiryo, Jun; Kohda, Daisuke

    2010-02-12

    Oligosaccharyltransferase (OST) catalyzes the transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. In the bacterium Campylobacter jejuni, a single-subunit membrane protein, PglB, catalyzes N-glycosylation. We report the 2.8 A resolution crystal structure of the C-terminal globular domain of PglB and its comparison with the previously determined structure from the archaeon Pyrococcus AglB. The two distantly related oligosaccharyltransferases share unexpected structural similarity beyond that expected from the sequence comparison. The common architecture of the putative catalytic sites revealed a new catalytic motif in PglB. Site-directed mutagenesis analyses confirmed the contribution of this motif to the catalytic function. Bacterial PglB and archaeal AglB constitute a protein family of the catalytic subunit of OST along with STT3 from eukaryotes. A structure-aided multiple sequence alignment of the STT3/PglB/AglB protein family revealed three types of OST catalytic centers. This novel classification will provide a useful framework for understanding the enzymatic properties of the OST enzymes from Eukarya, Archaea, and Bacteria.

  1. AglP is a S-adenosyl-L-methionine-dependent methyltransferase that participates in the N-glycosylation pathway of Haloferax volcanii.

    Science.gov (United States)

    Magidovich, Hilla; Yurist-Doutsch, Sophie; Konrad, Zvia; Ventura, Valeria V; Dell, Anne; Hitchen, Paul G; Eichler, Jerry

    2010-04-01

    While pathways for N-glycosylation in Eukarya and Bacteria have been solved, considerably less is known of this post-translational modification in Archaea. In the halophilic archaeon Haloferax volcanii, proteins encoded by the agl genes are involved in the assembly and attachment of a pentasaccharide to select asparagine residues of the S-layer glycoprotein. AglP, originally identified based on the proximity of its encoding gene to other agl genes whose products were shown to participate in N-glycosylation, was proposed, based on sequence homology, to serve as a methyltransferase. In the present report, gene deletion and mass spectrometry were employed to reveal that AglP is responsible for adding a 14 Da moiety to a hexuronic acid found at position four of the pentasaccharide decorating the Hfx. volcanii S-layer glycoprotein. Subsequent purification of a tagged version of AglP and development of an in vitro assay to test the function of the protein confirmed that AglP is a S-adenosyl-L-methionine-dependent methyltransferase.

  2. N-linked glycosylation in Archaea: a structural, functional, and genetic analysis.

    Science.gov (United States)

    Jarrell, Ken F; Ding, Yan; Meyer, Benjamin H; Albers, Sonja-Verena; Kaminski, Lina; Eichler, Jerry

    2014-06-01

    N-glycosylation of proteins is one of the most prevalent posttranslational modifications in nature. Accordingly, a pathway with shared commonalities is found in all three domains of life. While excellent model systems have been developed for studying N-glycosylation in both Eukarya and Bacteria, an understanding of this process in Archaea was hampered until recently by a lack of effective molecular tools. However, within the last decade, impressive advances in the study of the archaeal version of this important pathway have been made for halophiles, methanogens, and thermoacidophiles, combining glycan structural information obtained by mass spectrometry with bioinformatic, genetic, biochemical, and enzymatic data. These studies reveal both features shared with the eukaryal and bacterial domains and novel archaeon-specific aspects. Unique features of N-glycosylation in Archaea include the presence of unusual dolichol lipid carriers, the use of a variety of linking sugars that connect the glycan to proteins, the presence of novel sugars as glycan constituents, the presence of two very different N-linked glycans attached to the same protein, and the ability to vary the N-glycan composition under different growth conditions. These advances are the focus of this review, with an emphasis on N-glycosylation pathways in Haloferax, Methanococcus, and Sulfolobus.

  3. Phylogenetic- and genome-derived insight into the evolution of N-glycosylation in Archaea.

    Science.gov (United States)

    Kaminski, Lina; Lurie-Weinberger, Mor N; Allers, Thorsten; Gophna, Uri; Eichler, Jerry

    2013-08-01

    N-glycosylation, the covalent attachment of oligosaccharides to target protein Asn residues, is a post-translational modification that occurs in all three domains of life. In Archaea, the N-linked glycans that decorate experimentally characterized glycoproteins reveal a diversity in composition and content unequaled by their bacterial or eukaryal counterparts. At the same time, relatively little is known of archaeal N-glycosylation pathways outside of a handful of model strains. To gain insight into the distribution and evolutionary history of the archaeal version of this universal protein-processing event, 168 archaeal genome sequences were scanned for the presence of aglB, encoding the known archaeal oligosaccharyltransferase, an enzyme key to N-glycosylation. Such analysis predicts the presence of AglB in 166 species, with some species seemingly containing multiple versions of the protein. Phylogenetic analysis reveals that the events leading to aglB duplication occurred at various points during archaeal evolution. In many cases, aglB is found as part of a cluster of putative N-glycosylation genes. The presence, arrangement and nucleotide composition of genes in aglB-based clusters in five species of the halophilic archaeon Haloferax points to lateral gene transfer as contributing to the evolution of archaeal N-glycosylation.

  4. New oligosaccharyltransferase assay method.

    Science.gov (United States)

    Kohda, Daisuke; Yamada, Masaki; Igura, Mayumi; Kamishikiryo, Jun; Maenaka, Katsumi

    2007-11-01

    We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.

  5. N-glycosylation in Archaea: on the coordinated actions of Haloferax volcanii AglF and AglM.

    Science.gov (United States)

    Yurist-Doutsch, Sophie; Magidovich, Hilla; Ventura, Valeria V; Hitchen, Paul G; Dell, Anne; Eichler, Jerry

    2010-02-01

    Like Eukarya and Bacteria, Archaea are also capable of performing N-glycosylation. In the halophilic archaeon Haloferax volcanii, N-glycosylation is mediated by the products of the agl gene cluster. In the present report, this gene cluster was expanded to include an additional sequence, aglM, shown to participate in the biosynthesis of hexuronic acids contained within a pentasaccharide decorating the S-layer glycoprotein, a reporter H. volcanii glycoprotein. In response to different growth conditions, changes in the transcription profile of aglM mirrored changes in the transcription profiles of aglF, aglG and aglI, genes encoding confirmed participants in the H. volcanii N-glycosylation pathway, thus offering support to the hypothesis that in H. volcanii, N-glycosylation serves an adaptive role. Following purification, biochemical analysis revealed AglM to function as a UDP-glucose dehydrogenase. In a scoupled reaction with AglF, a previously identified glucose-1-phosphate uridyltransferase, UDP-glucuronic acid was generated from glucose-1-phosphate and UTP in a NAD(+)-dependent manner. These experiments thus represent the first step towards in vitro reconstitution of the archaeal N-glycosylation process.

  6. Archaeal membrane-associated proteases: insights on Haloferax volcanii and other haloarchaea.

    Science.gov (United States)

    Giménez, María I; Cerletti, Micaela; De Castro, Rosana E

    2015-01-01

    The function of membrane proteases range from general house-keeping to regulation of cellular processes. Although the biological role of these enzymes in archaea is poorly understood, some of them are implicated in the biogenesis of the archaeal cell envelope and surface structures. The membrane-bound ATP-dependent Lon protease is essential for cell viability and affects membrane carotenoid content in Haloferax volcanii. At least two different proteases are needed in this archaeon to accomplish the posttranslational modifications of the S-layer glycoprotein. The rhomboid protease RhoII is involved in the N-glycosylation of the S-layer protein with a sulfoquinovose-containing oligosaccharide while archaeosortase ArtA mediates the proteolytic processing coupled-lipid modification of this glycoprotein facilitating its attachment to the archaeal cell surface. Interestingly, two different signal peptidase I homologs exist in H. volcanii, Sec11a and Sec11b, which likely play distinct physiological roles. Type IV prepilin peptidase PibD processes flagellin/pilin precursors, being essential for the biogenesis and function of the archaellum and other cell surface structures in H. volcanii.

  7. Archaeal membrane-associated proteases: insights on Haloferax volcanii and other haloarchaea

    Directory of Open Access Journals (Sweden)

    Maria Ines Giménez

    2015-02-01

    Full Text Available The function of membrane proteases range from general house-keeping to regulation of cellular processes. Although the biological role of these enzymes in archaea is poorly understood, some of them are implicated in the biogenesis of the archaeal cell envelope and surface structures. The membrane-bound ATP-dependent Lon protease is essential for cell viability and affects membrane carotenoid content in Haloferax volcanii. At least two different proteases are needed in this archaeon to accomplish the posttranslational modifications of the S-layer glycoprotein. The rhomboid protease RhoII is involved in the N-glycosylation of the S-layer protein with a sulfoquinovose-containing oligosaccharide while archaeosortase ArtA mediates the proteolytic processing coupled-lipid modification of this glycoprotein facilitating its attachment to the archaeal cell surface. Interestingly, two different signal peptidase I homologs exist in H. volcanii, Sec11a and Sec11b, which likely play distinct physiological roles. Type IV prepilin peptidase PibD processes flagellin/pilin precursors, being essential for the biogenesis and function of the archaellum and other cell surface structures in H. volcanii.

  8. Decarboxylation of Pyruvate to Acetaldehyde for Ethanol Production by Hyperthermophiles

    Directory of Open Access Journals (Sweden)

    Mohammad S. Eram

    2013-08-01

    Full Text Available Pyruvate decarboxylase (PDC encoded by pdc is a thiamine pyrophosphate (TPP-containing enzyme responsible for the conversion of pyruvate to acetaldehyde in many mesophilic organisms. However, no pdc/PDC homolog has yet been found in fully sequenced genomes and proteomes of hyper/thermophiles. The only PDC activity reported in hyperthermophiles was a bifunctional, TPP- and CoA-dependent pyruvate ferredoxin oxidoreductase (POR/PDC enzyme from the hyperthermophilic archaeon Pyrococcus furiosus. Another enzyme known to be involved in catalysis of acetaldehyde production from pyruvate is CoA-acetylating acetaldehyde dehydrogenase (AcDH encoded by mhpF and adhE. Pyruvate is oxidized into acetyl-CoA by either POR or pyruvate formate lyase (PFL, and AcDH catalyzes the reduction of acetyl-CoA to acetaldehyde in mesophilic organisms. AcDH is present in some mesophilic (such as clostridia and thermophilic bacteria (e.g., Geobacillus and Thermoanaerobacter. However, no AcDH gene or protein homologs could be found in the released genomes and proteomes of hyperthermophiles. Moreover, no such activity was detectable from the cell-free extracts of different hyperthermophiles under different assay conditions. In conclusion, no commonly-known PDCs was found in hyperthermophiles. Instead of the commonly-known PDC, it appears that at least one multifunctional enzyme is responsible for catalyzing the non-oxidative decarboxylation of pyruvate to acetaldehyde in hyperthermophiles.

  9. Cloning, expression and evolution of the gene encoding the elongation factor 1alpha from a low thermophilic Sulfolobus solfataricus strain.

    Science.gov (United States)

    Masullo, Mariorosario; Cantiello, Piergiuseppe; Lamberti, Annalisa; Longo, Olimpia; Fiengo, Antonio; Arcari, Paolo

    2003-01-28

    The gene encoding the elongation factor 1alpha (EF-1alpha) from the archaeon Sulfolobus solfataricus strain MT3 (optimum growth temperature 75 degrees C) was cloned, sequenced and expressed in Escherichia coli. The structural and biochemical properties of the purified enzyme were compared to those of EF-1alpha isolated from S. solfataricus strain MT4 (optimum growth temperature 87 degrees C). Only one amino acid change (Val15-->Ile) was found. Interestingly, the difference was in the first guanine nucleotide binding consensus sequence G(13)HIDHGK and was responsible for a reduced efficiency in protein synthesis, which was accompanied by an increased affinity for both guanosine diphosphate (GDP) and guanosine triphosphate (GTP), and an increased efficiency in the intrinsic GTPase activity. Despite the different thermophilicities of the two microorganisms, only very marginal effects on the thermal properties of the enzyme were observed. Molecular evolution among EF-1alpha genes from Sulfolobus species showed that the average rate of nucleotide substitution per site per year (0.0312x10(-9)) is lower than that reported for other functional genes.

  10. Discovering Antioxidant Molecules in the Archaea Domain: Peroxiredoxin Bcp1 from Sulfolobus solfataricus Protects H9c2 Cardiomyoblasts from Oxidative Stress

    Science.gov (United States)

    Sarcinelli, Carmen; Pizzo, Elio

    2016-01-01

    Peroxiredoxins (Prxs) are ubiquitous thiol peroxidases that are involved in the reduction of peroxides. It has been reported that prokaryotic Prxs generally show greater structural robustness than their eukaryotic counterparts, making them less prone to inactivation by overoxidation. This difference has inspired the search for new antioxidants from prokaryotic sources that can be used as possible therapeutic biodrugs. Bacterioferritin comigratory proteins (Bcps) of the hyperthermophilic archaeon Sulfolobus solfataricus that belong to the Prx family have recently been characterized. One of these proteins, Bcp1, was chosen to determine its antioxidant effects in H9c2 rat cardiomyoblast cells. Bcp1 activity was measured in vitro under physiological temperature and pH conditions that are typical of mammalian cells; the yeast thioredoxin reductase (yTrxR)/thioredoxin (yTrx) reducing system was used to evaluate enzyme activity. A TAT-Bcp1 fusion protein was constructed to allow its internalization and verify the effect of Bcp1 on H9c2 rat cardiomyoblasts subjected to oxidative stress. The results reveal that TAT-Bcp1 is not cytotoxic and inhibits H2O2-induced apoptosis in H9c2 cells by reducing the H2O2 content inside these cells. PMID:27752237

  11. Preservation of Archaeal Surface Layer Structure During Mineralization.

    Science.gov (United States)

    Kish, Adrienne; Miot, Jennyfer; Lombard, Carine; Guigner, Jean-Michel; Bernard, Sylvain; Zirah, Séverine; Guyot, François

    2016-05-25

    Proteinaceous surface layers (S-layers) are highly ordered, crystalline structures commonly found in prokaryotic cell envelopes that augment their structural stability and modify interactions with metals in the environment. While mineral formation associated with S-layers has previously been noted, the mechanisms were unconstrained. Using Sulfolobus acidocaldarius a hyperthermophilic archaeon native to metal-enriched environments and possessing a cell envelope composed only of a S-layer and a lipid cell membrane, we describe a passive process of iron phosphate nucleation and growth within the S-layer of cells and cell-free S-layer "ghosts" during incubation in a Fe-rich medium, independently of metabolic activity. This process followed five steps: (1) initial formation of mineral patches associated with S-layer; (2) patch expansion; (3) patch connection; (4) formation of a continuous mineral encrusted layer at the cell surface; (5) early stages of S-layer fossilization via growth of the extracellular mineralized layer and the mineralization of cytosolic face of the cell membrane. At more advanced stages of encrustation, encrusted outer membrane vesicles are formed, likely in an attempt to remove damaged S-layer proteins. The S-layer structure remains strikingly well preserved even upon the final step of encrustation, offering potential biosignatures to be looked for in the fossil record.

  12. Rescuing Those Left Behind: Recovering and Characterizing Underdigested Membrane and Hydrophobic Proteins To Enhance Proteome Measurement Depth.

    Science.gov (United States)

    Giannone, Richard J; Wurch, Louie L; Podar, Mircea; Hettich, Robert L

    2015-08-04

    The marine archaeon Nanoarchaeum equitans is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. As this interaction is thought to be membrane-associated, involving a myriad of membrane-anchored proteins, proteomic efforts to better characterize this difficult to analyze interface are paramount to uncovering the mechanism of their association. By extending multienzyme digestion strategies that use sample filtration to recover underdigested proteins for reprocessing/consecutive proteolytic digestion, we applied chymotrypsin to redigest the proteinaceous material left over after initial proteolysis with trypsin of sodium dodecyl sulfate (SDS)-extracted I. hospitalis-N. equitans proteins. Using this method, we show that proteins with increased hydrophobic character, including membrane proteins with multiple transmembrane helices, are enriched and recovered in the underdigested fraction. Chymotryptic reprocessing provided significant sequence coverage gains in both soluble and hydrophobic proteins alike, with the latter benefiting more so in terms of membrane protein representation. These gains were despite a large proportion of high-quality peptide spectra remaining unassigned in the underdigested fraction suggesting high levels of protein modification on these often surface-exposed proteins. Importantly, these gains were achieved without applying extensive fractionation strategies usually required for thorough characterization of membrane-associated proteins and were facilitated by the generation of a distinct, complementary set of peptides that aid in both the identification and quantitation of this important, under-represented class of proteins.

  13. Life on the edge: functional genomic response of Ignicoccus hospitalis to the presence of Nanoarchaeum equitans.

    Science.gov (United States)

    Giannone, Richard J; Wurch, Louie L; Heimerl, Thomas; Martin, Stanton; Yang, Zamin; Huber, Harald; Rachel, Reinhard; Hettich, Robert L; Podar, Mircea

    2015-01-01

    The marine hyperthermophilic crenarchaeon Ignicoccus hospitalis supports the propagation on its surface of Nanoarchaeum equitans, an evolutionarily enigmatic archaeon that resembles highly derived parasitic and symbiotic bacteria. The cellular and molecular mechanisms that enable this interarchaea relationship and the intimate physiologic consequences to I. hospitalis are unknown. Here, we used concerted proteomic and transcriptomic analyses to probe into the functional genomic response of I. hospitalis as N. equitans multiplies on its surface. The expression of over 97% of the genes was detected at mRNA level and over 80% of the predicted proteins were identified and their relative abundance measured by proteomics. These indicate that little, if any, of the host genomic information is silenced during growth in the laboratory. The primary response to N. equitans was at the membrane level, with increases in relative abundance of most protein complexes involved in energy generation as well as that of several transporters and proteins involved in cellular membrane stabilization. Similar upregulation was observed for genes and proteins involved in key metabolic steps controlling nitrogen and carbon metabolism, although the overall biosynthetic pathways were marginally impacted. Proliferation of N. equitans resulted, however, in selective downregulation of genes coding for transcription factors and replication and cell cycle control proteins as I. hospitalis shifted its physiology from its own cellular growth to that of its ectosymbiont/parasite. The combination of these multiomic approaches provided an unprecedented level of detail regarding the dynamics of this interspecies interaction, which is especially pertinent as these organisms are not genetically tractable.

  14. 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.

  15. Modeling and structural analysis of evolutionarily diverse S8 family serine proteases.

    Science.gov (United States)

    Laskar, Aparna; Rodger, Euan James; Chatterjee, Aniruddha; Mandal, Chhabinath

    2011-01-01

    Serine proteases are an abundant class of enzymes that are involved in a wide range of physiological processes and are classified into clans sharing structural homology. The active site of the subtilisin-like clan contains a catalytic triad in the order Asp, His, Ser (S8 family) or a catalytic tetrad in the order Glu, Asp and Ser (S53 family). The core structure and active site geometry of these proteases is of interest for many applications. The aim of this study was to investigate the structural properties of different S8 family serine proteases from a diverse range of taxa using molecular modeling techniques. In conjunction with 12 experimentally determined three-dimensional structures of S8 family members, our predicted structures from an archaeon, protozoan and a plant were used for analysis of the catalytic core. Amino acid sequences were obtained from the MEROPS database and submitted to the LOOPP server for threading based structure prediction. The predicted structures were refined and validated using PROCHECK, SCRWL and MODELYN. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. Encompassing a wide range of taxa, our structural analysis provides an evolutionary perspective on S8 family serine proteases. Focusing on the common core containing the catalytic site of the enzyme, the analysis presented here is beneficial for future molecular modeling strategies and structure-based rational drug design.

  16. Solid-state fermentation as a potential technique for esterase/lipase production by halophilic archaea.

    Science.gov (United States)

    Martin del Campo, Martha; Camacho, Rosa M; Mateos-Díaz, Juan C; Müller-Santos, Marcelo; Córdova, Jesus; Rodríguez, Jorge A

    2015-11-01

    Halophilic archaea are extremophiles, adapted to high-salt environments, showing a big biotechnological potential as enzyme, lipids and pigments producers. Four inert supports (perlite, vermiculite, polyurethane foam and glass fiber) were employed for solid-state fermentation (SSF) of the halophilic archaeon Natronococcus sp. TC6 to investigate biomass and esterase production. A very low esterase activity and high water activity were observed when perlite, vermiculite and polyurethane were used as supports. When glass fiber was employed, an important moisture loss was observed (8.6%). Moreover, moisture retention was improved by mixing polyurethane and glass fiber, resulting in maximal biomass and esterase production. Three halophilic archaea: Natronococcus sp. TC6, Halobacterium sp. NRC-1 and Haloarcula marismortui were cultured by submerged fermentation (SmF) and by SSF; an improvement of 1.3- to 6.2-fold was observed in the biomass and esterase production when SSF was used. Growth was not homogeneous in the mixture, but was predominant in the glass fiber thus was probably because the glass fiber provides a holder to the cells, while the polyurethane acts as an impregnation medium reservoir. To the best of our knowledge, this work is the first report on haloarchaea cultivation by SSF aiming biomass and esterase/lipase activity production.

  17. Hans Georg Trüper (1936–2016 and His Contributions to Halophile Research

    Directory of Open Access Journals (Sweden)

    Aharon Oren

    2016-05-01

    Full Text Available Prof. Hans Georg Trüper, one of the most important scientists in the field of halophile research, passed away on 9 March 2016 at the age of 79. I here present a brief obituary with special emphasis on Prof. Trüper’s contributions to our understanding of the halophilic prokaryotes and their adaptations to life in hypersaline environments. He has pioneered the study of the halophilic anoxygenic phototrophic sulfur bacteria of the Ectothiorhodospira—Halorhodospira group. Some of the species he and his group isolated from hypersaline and haloalkaline environments have become model organisms for the study of the mechanisms of haloadaptation: the functions of three major organic compounds – glycine betaine, ectoine, and trehalose – known to serve as “compatible solutes” in halophilic members of the Bacteria domain, were discovered during studies of these anoxygenic phototrophs. Prof. Trüper’s studies of hypersaline alkaline environments in Egypt also led to the isolation of the first known extremely halophilic archaeon (Natronomonas pharaonis. The guest editors dedicate this special volume of Life to the memory of Prof. Hans Georg Trüper.

  18. An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA.

    Science.gov (United States)

    Fischer, Susan; Maier, Lisa-Katharina; Stoll, Britta; Brendel, Jutta; Fischer, Eike; Pfeiffer, Friedhelm; Dyall-Smith, Mike; Marchfelder, Anita

    2012-09-28

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.

  19. Thermogladius calderae gen. nov., sp. nov., an anaerobic, hyperthermophilic crenarchaeote from a Kamchatka hot spring.

    Science.gov (United States)

    Kochetkova, Tatiana V; Kublanov, Ilya V; Toshchakov, Stepan V; Osburn, Magdalena R; Novikov, Andrei A; Bonch-Osmolovskaya, Elizaveta A; Perevalova, Anna A

    2016-01-21

    An obligately anaerobic, hyperthermophilic, organoheterotrophic archaeon, strain 1633T, was isolated from a terrestrial hot spring of the Uzon Caldera (Kamchatka Peninsula, Russia). Cells were regular cocci, 0.5-0.9 μm in diameter, with one flagellum. The temperature range for growth was 80-95°C, with an optimum at 84°C. Strain 1633T grew on yeast extract, beef extract, peptone, cellulose and cellobiose. No growth was detected on other sugars or carbohydrates, organic acids, or under autotrophic conditions. The only detected growth products were CO2, acetate, and H2. Growth rate was stimulated by elemental sulfur, which was reduced to hydrogen sulfide. In silico calculated G+C content of strain 1633T genomic DNA was 55.64 mol%. 16S rRNA gene sequence analysis placed the strain 1633T together with the non-validly published "Thermogladius shockii" strain WB1 in a separate genus-level cluster within the Desulfurococcaceae family. ANI results revealed 75.72% identity between 1633T and WB1. Based on these results we propose a novel genus and species, for which the name Thermogladius calderae gen. nov., sp. nov. (type strain 1633T=DSM 22663T=VKM B-2946T) is proposed.

  20. Structural basis for the transglycosylase activity of a GH57-type glycogen branching enzyme from Pyrococcus horikoshii.

    Science.gov (United States)

    Na, Soohui; Park, Minjeong; Jo, Inseong; Cha, Jaeho; Ha, Nam-Chul

    2017-03-18

    Glycogen branching enzyme (GBE) catalyzes the formation of α-1,6-branching points during glycogenesis by cleaving α-1,4 bonds and making new α-1,6 bonds. Most GBEs belong to the glycoside hydrolase 13 family (GH13), but new GBEs in the GH57 family have been isolated from Archaea. Here, we determined the crystal structure of a GH57 GBE from the hyperthermophilic archaeon Pyrococcus horikoshii (PhGBE) at a resolution of 2.3 Å. PhGBE exhibits both α-1,6-branching activity and endo-α-1,4 hydrolytic activity. PhGBE has a central (β/α)7-barrel domain that contains an embedded helix domain and an α-helix-rich C-terminal domain. The active-site cleft is located at the interface of the central and C-terminal domains. Amino acid substitution at Trp22, which is separate from the catalytic nucleophilic residue, abolished both enzymatic activities, indicating that Trp22 might be responsible for substrate recognition. We also observed that shortening of the flexible loop near the catalytic residue changed branched chain lengths of the reaction products with increased hydrolytic activity. Taken together, our findings propose a molecular mechanism for how GH57 GBEs exhibit the two activities and where the substrate binds the enzyme.

  1. Inositol lipids: from an archaeal origin to phosphatidylinositol 3,5-bisphosphate faults in human disease.

    Science.gov (United States)

    Michell, Robert H

    2013-12-01

    The last couple of decades have seen an extraordinary transformation in our knowledge and understanding of the multifarious biological roles of inositol phospholipids. Herein, I briefly consider two topics. The first is the role that recently acquired biochemical and genomic information - especially from archaeons - has played in illuminating the possible evolutionary origins of the biological employment of inositol in lipids, and some questions that these studies raise about the 'classical' biosynthetic route to phosphatidylinositol. The second is the growing recognition of the importance in eukaryotic cells of phosphatidylinositol 3,5-bisphosphate. Phosphatidylinositol 3,5-bisphosphate only entered our phosphoinositide consciousness quite recently, but it is speedily gathering a plethora of roles in diverse cellular processes and diseases thereof. These include: control of endolysosomal vesicular trafficking and of the activity of ion channels and pumps in the endolysosomal compartment; control of constitutive and stimulated protein traffic to and from plasma membrane subdomains; control of the nutrient and stress-sensing target of rapamycin complex 1 pathway (TORC1); and regulation of key genes in some central metabolic pathways.

  2. Hydrogen gas production by combined systems of Rhodobacter sphaeroides O.U.001 and Halobacterium salinarum in a photobioreactor

    Energy Technology Data Exchange (ETDEWEB)

    Zabut, Baker; El-Kahlout, Kamal [Department of Biochemistry, School of Science, IUG, Gaza (PS); Yuecel, Meral [Department of Biology, Middle East Technical University, 06531 Ankara (Turkey); Guenduez, Ufuk; Tuerker, Lemi [Department of Chemistry, Middle East Technical University, 06531 Ankara (Turkey); Eroglu, Inci [Department of Chemical Engineering, Middle East Technical University, 06531 Ankara (Turkey)

    2006-09-15

    Rhodobacter sphaeroides O.U.001 is a photosynthetic non-sulfur bacterium which produces hydrogen from organic compounds under anaerobic conditions. Halobacterium salinarum is an archaeon and lives under extremely halophilic conditions (4M NaCl). H. salinarum contains a retinal protein bacteriorhodopsin in its purple membrane which acts as a light-driven proton pump. In this study the Rhodobacter sphaeroides O.U.001 culture was combined with different amounts of packed cells of H. salinarum S9 or isolated purple membrane fragments in order to increase the photofermentative hydrogen gas production. The packed cells of H. salinarum have the ability to pump protons upon illumination due to the presence of bacteriorhodopsin. The proton gradient produced may be used for the formation of ATP or protons may be used for H{sub 2} production by R. sphaeroides. Similar to intact cells purple membrane fragments may also form vesicles around certain ions and may act like closed systems. The hydrogen production experiments were carried out using 400ml water-jacketed-glass column stirred photobioreactors. In combined systems 10-200nmol of bacteriorhodopsin was used. Hydrogen gas production was enhanced by four- to sixfold in combined systems of H. salinarum packed cells with R. sphaeroides O.U.001 cell. Stirring both increased the total gas produced and enhanced the rate of hydrogen production. The light energy conversion efficiency was increased from 0.6% to 2.25% in combined systems. (author)

  3. Diurnally entrained anticipatory behavior in archaea.

    Directory of Open Access Journals (Sweden)

    Kenia Whitehead

    Full Text Available By sensing changes in one or few environmental factors biological systems can anticipate future changes in multiple factors over a wide range of time scales (daily to seasonal. This anticipatory behavior is important to the fitness of diverse species, and in context of the diurnal cycle it is overall typical of eukaryotes and some photoautotrophic bacteria but is yet to be observed in archaea. Here, we report the first observation of light-dark (LD-entrained diurnal oscillatory transcription in up to 12% of all genes of a halophilic archaeon Halobacterium salinarum NRC-1. Significantly, the diurnally entrained transcription was observed under constant darkness after removal of the LD stimulus (free-running rhythms. The memory of diurnal entrainment was also associated with the synchronization of oxic and anoxic physiologies to the LD cycle. Our results suggest that under nutrient limited conditions halophilic archaea take advantage of the causal influence of sunlight (via temperature on O(2 diffusivity in a closed hypersaline environment to streamline their physiology and operate oxically during nighttime and anoxically during daytime.

  4. A small basic protein from the brz-brb operon is involved in regulation of bop transcription in Halobacterium salinarum

    Directory of Open Access Journals (Sweden)

    Dyall-Smith Mike

    2011-09-01

    Full Text Available Abstract Background The halophilic archaeon Halobacterium salinarum expresses bacteriorhodopsin, a retinal-protein that allows photosynthetic growth. Transcription of the bop (bacterioopsin gene is controlled by two transcription factors, Bat and Brz that induce bop when cells are grown anaerobically and under light. Results A new gene was identified that is transcribed together with the brz gene that encodes a small basic protein designated as Brb (bacteriorhodopsin-regulating basic protein. The translation activity of the start codon of the brb gene was confirmed by BgaH reporter assays. In vivo site-directed mutagenesis of the brb gene showed that the Brb protein cooperates with Brz in the regulation of bop expression. Using a GFP reporter assay, it was demonstrated that Brb cooperates with both Brz and Bat proteins to activate bop transcription under phototrophic growth conditions. Conclusions The activation of the bop promoter was shown to be dependent not only on two major factors, Bat and Brz, but is also tuned by the small basic protein, Brb.

  5. Transcription start site associated RNAs (TSSaRNAs are ubiquitous in all domains of life.

    Directory of Open Access Journals (Sweden)

    Livia S Zaramela

    Full Text Available A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq and a primary-transcript enriched (dRNA-seq strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (∼7% of the annotated genome. Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.

  6. Formation of the conserved pseudouridine at position 55 in archaeal tRNA.

    Science.gov (United States)

    Roovers, Martine; Hale, Caryn; Tricot, Catherine; Terns, Michael P; Terns, Rebecca M; Grosjean, Henri; Droogmans, Louis

    2006-01-01

    Pseudouridine (Psi) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Psi55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are (pfu)Cbf5, a protein known to play a role in RNA-guided modification of rRNA, and (pfu)PsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. (pfu)PsuX is hereafter renamed (pfu)Pus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, (pfu)Pus10 efficiently complements an Escherichia coli strain deficient in the bacterial Psi55 synthase TruB. These results indicate that it is probable that (pfu)Cbf5 or (pfu)Pus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein.

  7. Recycling of Waste Streams of the Biotechnological Poly(hydroxyalkanoate Production by Haloferax mediterranei on Whey

    Directory of Open Access Journals (Sweden)

    Martin Koller

    2015-01-01

    Full Text Available For manufacturing “bioplastics” such as poly(hydroxyalkanoates (PHA, the combination of utilization of inexpensive carbon sources with the application of robust microbial production strains is considered a decisive step to make this process more cost-efficient and sustainable. PHA production based on surplus whey from dairy industry was accomplished by the extremely halophile archaeon Haloferax mediterranei. After fermentative production of PHA-rich biomass and the subsequent cell harvest and downstream processing for PHA recovery, environmentally hazardous, highly saline residues, namely spent fermentation broth and cell debris, remain as residues. These waste streams were used for recycling experiments to assess their recyclability in subsequent production processes. It was demonstrated that spent fermentation broth can be used to replace a considerable part of fresh saline fermentation medium in subsequent production processes. In addition, 29% of the expensive yeast extract, needed as nitrogen and phosphate source for efficient cultivation of the microorganism, can be replaced by cell debris from prior cultivations. The presented study provides strategies to combine the reduction of costs for biomediated PHA production with minimizing ecological risks by recycling precarious waste streams. Overall, the presented work shall contribute to the quick economic success of these promising biomaterials.

  8. Harnessing Type I and Type III CRISPR-Cas systems for genome editing.

    Science.gov (United States)

    Li, Yingjun; Pan, Saifu; Zhang, Yan; Ren, Min; Feng, Mingxia; Peng, Nan; Chen, Lanming; Liang, Yun Xiang; She, Qunxin

    2016-02-29

    CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are widespread in archaea and bacteria, and research on their molecular mechanisms has led to the development of genome-editing techniques based on a few Type II systems. However, there has not been any 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-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 the protospacer adjacent motif (PAM) of an uncharacterized PAM-dependent CRISPR-Cas system can be predicted by bioinformatic analysis.

  9. The viral eukaryogenesis hypothesis: a key role for viruses in the emergence of eukaryotes from a prokaryotic world environment.

    Science.gov (United States)

    Bell, Philip John Livingstone

    2009-10-01

    Understanding how the gulf between prokaryotic and eukaryotic cellular design arose is a major challenge. The viral eukaryogenesis (VE) hypothesis addresses the challenge of eukaryotic origins by suggesting the first eukaryotic cell was a multimember consortium consisting of a viral ancestor of the nucleus, an archaeal ancestor of the eukaryotic cytoplasm, and a bacterial ancestor of the mitochondria. Using only prokaryotes and their viruses, and invoking selective pressures observed in modern organisms, the VE hypothesis can explain the origins of the eukaryotic cell, sex, and meiosis. In the VE hypothesis, a cell wall-less archaeon and an alpha-proteobacterium established a syntrophic relationship, and then a complex DNA virus permanently lysogenized the archaeal syntroph to produce a consortium of three organisms that evolved into the eukaryotic cell. The mechanisms by which the virus replicated, controlled its copy number, and segregated to daughter cells led to the evolution of the asexual mitotic replication cycle and the sexual meiotic replication cycle. The VE hypothesis conceptually unifies prokaryotic and eukaryotic sex into variants of a single process.

  10. Probing the two-domain structure of homodimeric prokaryotic and eukaryotic catalase-peroxidases.

    Science.gov (United States)

    Banerjee, Srijib; Zamocky, Marcel; Furtmüller, Paul G; Obinger, Christian

    2010-11-01

    Catalase-peroxidases (KatGs) are ancestral bifunctional heme peroxidases found in archaeons, bacteria and lower eukaryotes. In contrast to homologous cytochrome c peroxidase (CcP) and ascorbate peroxidase (APx) homodimeric KatGs have a two-domain monomeric structure with a catalytic N-terminal heme domain and a C-terminal domain of high sequence and structural similarity but without obvious function. Nevertheless, without its C-terminal counterpart the N-terminal domain exhibits neither catalase nor peroxidase activity. Except some hybrid-type proteins all other members of the peroxidase-catalase superfamily lack this C-terminal domain. In order to probe the role of the two-domain monomeric structure for conformational and thermal stability urea and temperature-dependent unfolding experiments were performed by using UV-Vis-, electronic circular dichroism- and fluorescence spectroscopy, as well as differential scanning calorimetry. Recombinant prokaryotic (cyanobacterial KatG from Synechocystis sp. PCC6803) and eukaryotic (fungal KatG from Magnaporthe grisea) were investigated. The obtained data demonstrate that the conformational and thermal stability of bifunctional KatGs is significantly lower compared to homologous monofunctional peroxidases. The N- and C-terminal domains do not unfold independently. Differences between the cyanobacterial and the fungal enzyme are relatively small. Data will be discussed with respect to known structure and function of KatG, CcP and APx.

  11. New role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.

    Science.gov (United States)

    Unno, Hideaki; Yamashita, Satoshi; Ikeda, Yosuke; Sekiguchi, Shin-Ya; Yoshida, Norie; Yoshimura, Tohru; Kusunoki, Masami; Nakayama, Toru; Nishino, Tokuzo; Hemmi, Hisashi

    2009-04-03

    Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.

  12. Isolation of an extremely halophilic arhaeon Natrialba sp. C21 able to degrade aromatic compounds and to produce stable biosurfactant at high salinity.

    Science.gov (United States)

    Khemili-Talbi, Souad; Kebbouche-Gana, Salima; Akmoussi-Toumi, Siham; Angar, Yassmina; Gana, Mohamed Lamine

    2015-11-01

    Natrialba sp. strain C21 was isolated from oil contaminated saline water in Ain Salah (Algeria) and has exhibited a good potential for degrading phenol (3% v/v), naphthalene (3% v/v), and pyrene (3% v/v) at high salinity with high growth, enzymatic activity and biosurfactant production. Successful metabolism of aromatic hydrocarbon compounds of the strain Natrialba sp. C21 appears to require the ortho-cleavage pathway. Indeed, assays of the key enzymes involved in the ring cleavage of catechol 1, 2-dioxygenase indicated that degradation of the phenol, naphthalene and pyrene by strain Natrialba sp. C21 was via the ortho-cleavage pathway. Cells grown on aromatic hydrocarbons displayed greater ortho-activities mainly towards catechol, while the meta-activity was very low. Besides, biosurfactants derived from the strain C21 were capable of effectively emulsifying both aromatic and aliphatic hydrocarbons and seem to be particularly promising since they have particular adaptations like the increased stability at high temperature and salinity conditions. This study clearly demonstrates for the first time that strain belonging to the genera Natrialba is able to grow at 25% (w/v) NaCl, utilizing phenol, naphthalene, and pyrene as the sole carbon sources. The results suggest that the isolated halophilic archaeon could be a good candidate for the remediation process in extreme environments polluted by aromatic hydrocarbons. Moreover, the produced biosurfactant offers a multitude of interesting potential applications in various fields of biotechnology.

  13. Metal-ion dependent catalytic properties of Sulfolobus solfataricus class II α-mannosidase

    DEFF Research Database (Denmark)

    Nielsen, Jonas Willum; Poulsen, Nina Rødtness; Johnsson, Anna Margit Susanne;

    2012-01-01

    The active site for the family GH38 class II α-mannosidase is constituted in part by a divalent metal ion, mostly Zn(2+), as revealed in the crystal structures of enzymes from both animal and bacterial sources. The metal ion coordinates to the bound substrate and side chains of conserved amino acid...... residues. Recently, evidence has accumulated that class II α-mannosidase is active in complex with a range of divalent metal ions. In the present work, with employment of the class II α-mannosidase, ManA, from the hyperthermophilic archaeon Sulfolobus solfataricus, we explored the influence of the divalent...... metal ion on the associated steady-state kinetic parameters, K(M) and k(cat), for various substrates. With p-nitrophenyl-α-d-mannoside as a substrate, the enzyme showed activity in the presence of Co(2+), Cd(2+), Mn(2+), and Zn(2+), whereas Ni(2+) and Cu(2+) were inhibitory and nonactivating. Co(2...

  14. A Single-Culture Bioprocess of Methanothermobacter thermautotrophicus to Upgrade Digester Biogas by CO2-to-CH4 Conversion with H2

    Directory of Open Access Journals (Sweden)

    Matthew R. Martin

    2013-01-01

    Full Text Available 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 laboratory culture with a robust growth (dry weight of 6.4–7.4 g/L; OD600 of 13.6–15.4, a volumetric methane production rate of 21 L/L culture-day, and a H2 conversion efficiency of 89% was moved to an industrial anaerobic digester facility, where it was restarted and fed untreated biogas with a methane content of ~70% at a rate such that CO2 was in excess of the stoichiometric requirements in relation to H2. Over an 8-day operating period, the dry weight of the culture initially decreased slightly before stabilizing at an elevated level of ~8 g/L to achieve a volumetric methane production rate of 21 L/L culture-day and a H2 conversion efficiency of 62%. While some microbial contamination of the culture was observed via microscopy, it did not affect the methane production rate of the culture.

  15. 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.

  16. RNomics and Modomics in the halophilic archaea Haloferax volcanii: identification of RNA modification genes

    Directory of Open Access Journals (Sweden)

    Decatur Wayne A

    2008-10-01

    Full Text Available Abstract Background Naturally occurring RNAs contain numerous enzymatically altered nucleosides. Differences in RNA populations (RNomics and pattern of RNA modifications (Modomics depends on the organism analyzed and are two of the criteria that distinguish the three kingdoms of life. If the genomic sequences of the RNA molecules can be derived from whole genome sequence information, the modification profile cannot and requires or direct sequencing of the RNAs or predictive methods base on the presence or absence of the modifications genes. Results By employing a comparative genomics approach, we predicted almost all of the genes coding for the t+rRNA modification enzymes in the mesophilic moderate halophile Haloferax volcanii. These encode both guide RNAs and enzymes. Some are orthologous to previously identified genes in Archaea, Bacteria or in Saccharomyces cerevisiae, but several are original predictions. Conclusion The number of modifications in t+rRNAs in the halophilic archaeon is surprisingly low when compared with other Archaea or Bacteria, particularly the hyperthermophilic organisms. This may result from the specific lifestyle of halophiles that require high intracellular salt concentration for survival. This salt content could allow RNA to maintain its functional structural integrity with fewer modifications. We predict that the few modifications present must be particularly important for decoding, accuracy of translation or are modifications that cannot be functionally replaced by the electrostatic interactions provided by the surrounding salt-ions. This analysis also guides future experimental validation work aiming to complete the understanding of the function of RNA modifications in Archaeal translation.

  17. Extracellular DNA metabolism in Haloferax volcanii

    Directory of Open Access Journals (Sweden)

    Scott eChimileski

    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.

  18. 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.

  19. Study of microbial community structures in UASB sludge treating municipal wastewater by denaturing gradient gel electrophoresis of 16S rDNA

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ying; Sunny Aiyuk; XU Hui; CHEN Guanxiong; Willy Verstraete

    2005-01-01

    The structures of microbial communities in lab-scale upflow anaerobic sludge blanket (UASB) reactors for treating municipal wastewater with different ratios of COD soluble/COD total were studied using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes.The microbial structure of the inoculum sludge obtained from a full-scale UASB reactor of treating potato processing wastewater was compared with the structures of sludges collected from three lab-scale UASB reactors after eight months feeding with raw municipal wastewater, with CEPS (chemically enhanced primary sedimentation) pretreated municipal wastewater, and with a synthetic municipal sewage, respectively. Computer-aided numerical analysis of the DGGE fingerprints showed that the bacterial community underwent major changes. The sludges for treating raw and CEPS pretreated wastewater had very similar bacterial and archaeal communities (82%and 96% similarity) but were different from that for treating the synthetic sewage. Hence, despite similar % COD in the particulate form in the synthetic and the real wastewater, the two wastewaters were selected for different microbial communities. Prominent DGGE bands of Bacteria and Archaea were purified and sequenced. The 16S rRNA gene sequences of the dominant archaeal bands found in the inoculum, and UASB sludge fed with raw sewage, CEPS pretreated wastewater, and synthetic sewage were closely associated with Methanosaeta concilii. In the UASB sludge fed with synthetic sewage, another dominant band associated with an uncultured archaeon 39-2 was found together with M. concilii.

  20. Sequence, Structure, and Binding Analysis of Cyclodextrinase (TK1770 from T. kodakarensis (KOD1 Using an In Silico Approach

    Directory of Open Access Journals (Sweden)

    Ramzan Ali

    2015-01-01

    Full Text Available Thermostable cyclodextrinase (Tk1770 CDase from hyperthermophilic archaeon Thermococcus kodakarensis (KOD1 hydrolyzes cyclodextrins into linear dextrins. The sequence of Tk1770 CDase retrieved from UniProt was aligned with sequences of sixteen CD hydrolyzing enzymes and a phylogenetic tree was constructed using Bayesian inference. The homology model of Tk1770 CDase was constructed and optimized with Modeller v9.14 program. The model was validated with ProSA server and PROCHECK analysis. Four conserved regions and the catalytic triad consisting of Asp411, Glu437, and Asp502 of GH13 family were identified in catalytic site. Also an additional fifth conserved region downstream to the fourth region was also identified. The structure of Tk1770 CDase consists of an additional N′-domain and a helix-loop-helix motif that is conserved in all archaeal CD hydrolyzing enzymes. The N′-domain contains an extended loop region that forms a part of catalytic domain and plays an important role in stability and substrate binding. The docking of substrate into catalytic site revealed the interactions with different conserved residues involved in substrate binding and formation of enzyme-substrate complex.