WorldWideScience

Sample records for archaeon pyrbaculum aerophilum

  1. Amylomaltase of Pyrobaculum aerophilum IM2 produces thermoreversible starch gels

    NARCIS (Netherlands)

    Kaper, T.; Talik, B.; Ettema, T.J.; Bos, H.; Maarel, M.J.E.C. van der; Dijkhuizen, L.

    2005-01-01

    Amylomaltases are 4-α-glucanotransferases (EC 2.4.1.25) of glycoside hydrolase family 77 that transfer α-1,4-linked glucans to another acceptor, which can be the 4-OH group of an α-1,4-linked glucan or glucose. The amylomaltase-encoding gene (PAE1209) from the hyperthermophilic archaeon Pyrobaculum

  2. 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

  3. 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.

    2017-06-01

    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.

  4. Purification, crystallization and preliminary crytallographic analysis of phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    Akerboom, A.P.; Turnbull, A.P.; Hargreaves, D.; Fischer, M.; Geus, de D.; Sedelnikova, S.E.; Berrisford, J.M.; Baker, P.J.; Verhees, C.H.; Oost, van der J.; Rice, D.W.

    2003-01-01

    The glycolytic enzyme phosphoglucose isomerase catalyses the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate. The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus, which shows no sequence similarity to any known bacterial or eukaryotic

  5. 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.

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

    Science.gov (United States)

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

    1997-05-15

    Archaea (archaebacteria) constitute a domain of life that is distinct from Bacteria (eubacteria) and Eucarya (eukaryotes). Although archaeal cells share many morphological features with eubacteria, their transcriptional apparatus is more akin to eukaryotic RNA polymerases I, II and III than it is to eubacterial transcription systems. Thus, in addition to possessing a 10 subunit RNA polymerase and a homologue of the TATA-binding protein (TBP), Archaea possess a polypeptide termed TFB that is homologous to eukaryotic TFIIB. Here, we investigate the factor requirements for transcription of several promoters of the archaeon Sulfolobus shibatae and its associated virus SSV. Through in vitro transcription and immunodepletion, we demonstrate that S. shibatae TBP, TFB and RNA polymerase are not complexed tightly with one another and that each is required for efficient transcription of all promoters tested. Furthermore, full transcription is restored by supplementing respective depleted extracts with recombinant TBP or TFB, indicating that TBP-associated factors or TFB-associated factors are not required. Indeed, gel-filtration suggests that Sulfolobus TBP and TFB are not associated stably with other proteins. Finally, all promoters analysed are transcribed accurately and efficiently in an in vitro system comprising recombinant TBP and TFB, together with essentially homogeneous preparation of RNA polymerase. Transcription in Archaea is therefore fundamentally homologous to that in eukaryotes, although factor requirements appear to be much less complex.

  7. Metabolic flux analysis of the halophilic archaeon Haladaptatus paucihalophilus.

    Science.gov (United States)

    Liu, Guangxiu; Zhang, Manxiao; Mo, Tianlu; He, Lian; Zhang, Wei; Yu, Yi; Zhang, Qi; Ding, Wei

    2015-11-27

    This work reports the (13)C-assisted metabolic flux analysis of Haladaptatus paucihalophilus, a halophilic archaeon possessing an intriguing osmoadaption mechanism. We showed that the carbon flow is through the oxidative tricarboxylic acid (TCA) cycle whereas the reductive TCA cycle is not operative in H. paucihalophilus. In addition, both threonine and the citramalate pathways contribute to isoleucine biosynthesis, whereas lysine is synthesized through the diaminopimelate pathway and not through the α-aminoadipate pathway. Unexpected, the labeling patterns of glycine from the cells grown on [1-(13)C]pyruvate and [2-(13)C]pyruvate suggest that, unlike all the organisms investigated so far, in which glycine is produced exclusively from the serine hydroxymethyltransferase (SHMT) pathway, glycine biosynthesis in H. paucihalophilus involves different pathways including SHMT, threonine aldolase (TA) and the reverse reaction of glycine cleavage system (GCS), demonstrating for the first time that other pathways instead of SHMT can also make a significant contribution to the cellular glycine pool. Transcriptional analysis confirmed that both TA and GCS genes were transcribed in H. paucihalophilus, and the transcriptional level is independent of salt concentrations in the culture media. This study expands our understanding of amino acid biosynthesis and provides valuable insights into the metabolism of halophilic archaea. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. 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.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    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. (paper)

  10. 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.

  11. Metabolic flux analysis of the halophilic archaeon Haladaptatus paucihalophilus

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Guangxiu; Zhang, Manxiao [Key Laboratory of Desert and Desertification, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, 730000 (China); Key Laboratory of Extreme Environmental Microbial Resources and Engineering, Gansu Province, Lanzhou, 730000 (China); Mo, Tianlu [Department of Chemistry, Fudan University, Shanghai, 200433 (China); He, Lian [Key Laboratory of Combinatory Biosynthesis and Drug Discovery (Ministry of Education), School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430071 (China); Zhang, Wei [Key Laboratory of Desert and Desertification, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, 730000 (China); Key Laboratory of Extreme Environmental Microbial Resources and Engineering, Gansu Province, Lanzhou, 730000 (China); Yu, Yi, E-mail: yu_yi@whu.edu.cn [Key Laboratory of Combinatory Biosynthesis and Drug Discovery (Ministry of Education), School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430071 (China); Zhang, Qi, E-mail: qizhang@sioc.ac.cn [Department of Chemistry, Fudan University, Shanghai, 200433 (China); Ding, Wei, E-mail: dingw@lzu.edu.cn [Key Laboratory of Desert and Desertification, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou, 730000 (China); Key Laboratory of Extreme Environmental Microbial Resources and Engineering, Gansu Province, Lanzhou, 730000 (China); Department of Chemistry, Fudan University, Shanghai, 200433 (China)

    2015-11-27

    This work reports the {sup 13}C-assisted metabolic flux analysis of Haladaptatus paucihalophilus, a halophilic archaeon possessing an intriguing osmoadaption mechanism. We showed that the carbon flow is through the oxidative tricarboxylic acid (TCA) cycle whereas the reductive TCA cycle is not operative in H. paucihalophilus. In addition, both threonine and the citramalate pathways contribute to isoleucine biosynthesis, whereas lysine is synthesized through the diaminopimelate pathway and not through the α-aminoadipate pathway. Unexpected, the labeling patterns of glycine from the cells grown on [1-{sup 13}C]pyruvate and [2-{sup 13}C]pyruvate suggest that, unlike all the organisms investigated so far, in which glycine is produced exclusively from the serine hydroxymethyltransferase (SHMT) pathway, glycine biosynthesis in H. paucihalophilus involves different pathways including SHMT, threonine aldolase (TA) and the reverse reaction of glycine cleavage system (GCS), demonstrating for the first time that other pathways instead of SHMT can also make a significant contribution to the cellular glycine pool. Transcriptional analysis confirmed that both TA and GCS genes were transcribed in H. paucihalophilus, and the transcriptional level is independent of salt concentrations in the culture media. This study expands our understanding of amino acid biosynthesis and provides valuable insights into the metabolism of halophilic archaea. - Highlights: • Serine hydroxymethyltransferase, threonine aldolase, and glycine cleavage system all contribute to the glycine pool of H. paucihalophilus. • Threonine and the citramalate pathways contribute equally to the isoleucine biosynthesis in H. paucihalophilus. • Lysine in H. paucihalophilus is synthesized through the diaminopimelate pathway and not through the α-aminoadipate pathway. • Glycine biosynthesis is likely unrelated to the cell osmoadaption mechanism.

  12. Metabolic flux analysis of the halophilic archaeon Haladaptatus paucihalophilus

    International Nuclear Information System (INIS)

    Liu, Guangxiu; Zhang, Manxiao; Mo, Tianlu; He, Lian; Zhang, Wei; Yu, Yi; Zhang, Qi; Ding, Wei

    2015-01-01

    This work reports the "1"3C-assisted metabolic flux analysis of Haladaptatus paucihalophilus, a halophilic archaeon possessing an intriguing osmoadaption mechanism. We showed that the carbon flow is through the oxidative tricarboxylic acid (TCA) cycle whereas the reductive TCA cycle is not operative in H. paucihalophilus. In addition, both threonine and the citramalate pathways contribute to isoleucine biosynthesis, whereas lysine is synthesized through the diaminopimelate pathway and not through the α-aminoadipate pathway. Unexpected, the labeling patterns of glycine from the cells grown on [1-"1"3C]pyruvate and [2-"1"3C]pyruvate suggest that, unlike all the organisms investigated so far, in which glycine is produced exclusively from the serine hydroxymethyltransferase (SHMT) pathway, glycine biosynthesis in H. paucihalophilus involves different pathways including SHMT, threonine aldolase (TA) and the reverse reaction of glycine cleavage system (GCS), demonstrating for the first time that other pathways instead of SHMT can also make a significant contribution to the cellular glycine pool. Transcriptional analysis confirmed that both TA and GCS genes were transcribed in H. paucihalophilus, and the transcriptional level is independent of salt concentrations in the culture media. This study expands our understanding of amino acid biosynthesis and provides valuable insights into the metabolism of halophilic archaea. - Highlights: • Serine hydroxymethyltransferase, threonine aldolase, and glycine cleavage system all contribute to the glycine pool of H. paucihalophilus. • Threonine and the citramalate pathways contribute equally to the isoleucine biosynthesis in H. paucihalophilus. • Lysine in H. paucihalophilus is synthesized through the diaminopimelate pathway and not through the α-aminoadipate pathway. • Glycine biosynthesis is likely unrelated to the cell osmoadaption mechanism.

  13. The nif Gene Operon of the Methanogenic Archaeon Methanococcus maripaludis

    Science.gov (United States)

    Kessler, Peter S.; Blank, Carrine; Leigh, John A.

    1998-01-01

    Nitrogen fixation occurs in two domains, Archaea and Bacteria. We have characterized a nif (nitrogen fixation) gene cluster in the methanogenic archaeon Methanococcus maripaludis. Sequence analysis revealed eight genes, six with sequence similarity to known nif genes and two with sequence similarity to glnB. The gene order, nifH, ORF105 (similar to glnB), ORF121 (similar to glnB), nifD, nifK, nifE, nifN, and nifX, was the same as that found in part in other diazotrophic methanogens and except for the presence of the glnB-like genes, also resembled the order found in many members of the Bacteria. Using transposon insertion mutagenesis, we determined that an 8-kb region required for nitrogen fixation corresponded to the nif gene cluster. Northern analysis revealed the presence of either a single 7.6-kb nif mRNA transcript or 10 smaller mRNA species containing portions of the large transcript. Polar effects of transposon insertions demonstrated that all of these mRNAs arose from a single promoter region, where transcription initiated 80 bp 5′ to nifH. Distinctive features of the nif gene cluster include the presence of the six primary nif genes in a single operon, the placement of the two glnB-like genes within the cluster, the apparent physical separation of the cluster from any other nif genes that might be in the genome, the fragmentation pattern of the mRNA, and the regulation of expression by a repression mechanism described previously. Our study and others with methanogenic archaea reporting multiple mRNAs arising from gene clusters with only a single putative promoter sequence suggest that mRNA processing following transcription may be a common occurrence in methanogens. PMID:9515920

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

    Science.gov (United States)

    Auernik, Kathryne S.; Kelly, Robert M.

    2010-01-01

    Hydrogen served as a competitive inorganic energy source, impacting the CuFeS2 bioleaching efficiency of the extremely thermoacidophilic archaeon Metallosphaera sedula. Open reading frames encoding key terminal oxidase and electron transport chain components were triggered by CuFeS2. Evidence of heterotrophic metabolism was noted after extended periods of bioleaching, presumably related to cell lysis. PMID:20190092

  15. Genome Sequence of a Hyperthermophilic Archaeon, Thermococcus nautili 30-1, That Produces Viral Vesicles.

    Science.gov (United States)

    Oberto, Jacques; Gaudin, Marie; Cossu, Matteo; Gorlas, Aurore; Slesarev, Alexeï; Marguet, Evelyne; Forterre, Patrick

    2014-03-27

    Thermococcus nautili 30-1 (formerly Thermococcus nautilus), an anaerobic hyperthermophilic marine archaeon, was isolated in 1999 from a deep-sea hydrothermal vent during the Amistad campaign. Here, we present the complete sequence of T. nautili, which is able to produce membrane vesicles containing plasmid DNA. This property makes T. nautili a model organism to study horizontal gene transfer.

  16. Protein complexes in the archaeon Methanothermobacter thermautotrophicus analyzed by blue native/SDS-PAGE and mass spectrometry.

    NARCIS (Netherlands)

    Farhoud, M.H.; Wessels, H.C.T.; Steenbakkers, P.J.M.; Mattijssen, S.; Wevers, R.A.; Engelen, B.G.M. van; Jetten, M.S.M.; Smeitink, J.A.M.; Heuvel, L.P.W.J. van den; Keltjens, J.T.M.

    2005-01-01

    Methanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism. The biochemistry of methane formation has been extensively studied and is catalyzed by individual enzymes and proteins that are organized in protein complexes.

  17. 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.

  18. Purification, crystallization and preliminary crystallographic analysis of GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Wu Hao,; Sun, L.; Brouns, S.J.J.; Fu, S.; Akerboom, A.P.; Li, X.; Oost, van der J.

    2007-01-01

    A predicted GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus, termed SsGBP, has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized using the hanging-drop vapour-diffusion technique in the presence of 0.05 M cadmium sulfate and 0.8

  19. Adaptation, Ecology, and Evolution of the Halophilic Stromatolite Archaeon Halococcus hamelinensis Inferred through Genome Analyses

    Directory of Open Access Journals (Sweden)

    Reema K. Gudhka

    2015-01-01

    Full Text Available Halococcus hamelinensis was the first archaeon isolated from stromatolites. These geomicrobial ecosystems are thought to be some of the earliest known on Earth, yet, despite their evolutionary significance, the role of Archaea in these systems is still not well understood. Detailed here is the genome sequencing and analysis of an archaeon isolated from stromatolites. The genome of H. hamelinensis consisted of 3,133,046 base pairs with an average G+C content of 60.08% and contained 3,150 predicted coding sequences or ORFs, 2,196 (68.67% of which were protein-coding genes with functional assignments and 954 (29.83% of which were of unknown function. Codon usage of the H. hamelinensis genome was consistent with a highly acidic proteome, a major adaptive mechanism towards high salinity. Amino acid transport and metabolism, inorganic ion transport and metabolism, energy production and conversion, ribosomal structure, and unknown function COG genes were overrepresented. The genome of H. hamelinensis also revealed characteristics reflecting its survival in its extreme environment, including putative genes/pathways involved in osmoprotection, oxidative stress response, and UV damage repair. Finally, genome analyses indicated the presence of putative transposases as well as positive matches of genes of H. hamelinensis against various genomes of Bacteria, Archaea, and viruses, suggesting the potential for horizontal gene transfer.

  20. Modeling of the structure of ribosomal protein L1 from the archaeon Haloarcula marismortui

    Science.gov (United States)

    Nevskaya, N. A.; Kljashtorny, V. G.; Vakhrusheva, A. V.; Garber, M. B.; Nikonov, S. V.

    2017-07-01

    The halophilic archaeon Haloarcula marismortui proliferates in the Dead Sea at extremely high salt concentrations (higher than 3 M). This is the only archaeon, for which the crystal structure of the ribosomal 50S subunit was determined. However, the structure of the functionally important side protuberance containing the abnormally negatively charged protein L1 (HmaL1) was not visualized. Attempts to crystallize HmaL1 in the isolated state or as its complex with RNA using normal salt concentrations (≤500 mM) failed. A theoretical model of HmaL1 was built based on the structural data for homologs of the protein L1 from other organisms, and this model was refined by molecular dynamics methods. Analysis of this model showed that the protein HmaL1 can undergo aggregation due to the presence of a cluster of positive charges unique for proteins L1. This cluster is located at the RNA-protein interface, which interferes with the crystallization of HmaL1 and the binding of the latter to RNA.

  1. 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

  2. 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.

  3. Metabolism Dealing with Thermal Degradation of NAD+ in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

    Science.gov (United States)

    Hachisuka, Shin-Ichi; Sato, Takaaki; Atomi, Haruyuki

    2017-10-01

    NAD + is an important cofactor for enzymatic oxidation reactions in all living organisms, including (hyper)thermophiles. However, NAD + is susceptible to thermal degradation at high temperatures. It can thus be expected that (hyper)thermophiles harbor mechanisms that maintain in vivo NAD + concentrations and possibly remove and/or reuse undesirable degradation products of NAD + Here we confirmed that at 85°C, thermal degradation of NAD + results mostly in the generation of nicotinamide and ADP-ribose, the latter known to display toxicity by spontaneously linking to proteins. The hyperthermophilic archaeon Thermococcus kodakarensis possesses a putative ADP-ribose pyrophosphatase (ADPR-PPase) encoded by the TK2284 gene. ADPR-PPase hydrolyzes ADP-ribose to ribose 5-phosphate (R5P) and AMP. The purified recombinant TK2284 protein exhibited activity toward ADP-ribose as well as ADP-glucose. Kinetic analyses revealed a much higher catalytic efficiency toward ADP-ribose, suggesting that ADP-ribose was the physiological substrate. To gain insight into the physiological function of TK2284, a TK2284 gene disruption strain was constructed and examined. Incubation of NAD + in the cell extract of the mutant strain at 85°C resulted in higher ADP-ribose accumulation and lower AMP production compared with those in experiments with the host strain cell extract. The mutant strain also exhibited lower cell yield and specific growth rates in a synthetic amino acid medium compared with those of the host strain. The results obtained here suggest that the ADPR-PPase in T. kodakarensis is responsible for the cleavage of ADP-ribose to R5P and AMP, providing a means to utilize the otherwise dead-end product of NAD + breakdown. IMPORTANCE Hyperthermophilic microorganisms living under high temperature conditions should have mechanisms that deal with the degradation of thermolabile molecules. NAD + is an important cofactor for enzymatic oxidation reactions and is susceptible to thermal

  4. Thermococcus prieurii sp. nov., a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent.

    Science.gov (United States)

    Gorlas, Aurore; Alain, Karine; Bienvenu, Nadège; Geslin, Claire

    2013-08-01

    A novel hyperthermophilic, anaerobic archaeon, strain Bio-pl-0405IT2(T), was isolated from a hydrothermal chimney sample collected from the East Pacific Rise at 2700 m depth in the 'Sarah Spring' area (7° 25' 24" S 107° 47' 66" W). Cells were irregular, motile cocci (0.8-1.5 µm in diameter) and divided by constriction. Growth was observed at temperatures between 60 °C and 95 °C with an optimum at 80 °C. The pH range for growth was between pH 4.0 and pH 8.0 with an optimum around pH 7.0. Strain Bio-pl-0405IT2(T) grew at salt concentrations of 1-5 % (w/v) NaCl with an optimum at 2 %. The novel isolate grew by fermentation or sulphur respiration on a variety of organic compounds. It was a chemoorganoheterotrophic archaeon growing preferentially with yeast extract, peptone and tryptone as carbon and energy sources and sulphur and organic compounds as electron acceptors; it also grew on maltose and starch. Sulphur or l-cystine were required for growth and were reduced to hydrogen sulfide. The strain was resistant to rifampicin, chloramphenicol, vancomycin and kanamycin (all at 100 µg ml(-1)) but was sensitive to tetracycline. The G+C content of its genomic DNA was 53.6 mol%. Phylogenetic analysis of the almost complete 16S rRNA gene sequence (1450 bp) of strain Bio-pl-0405IT2(T) showed that the novel isolate belonged to the genus Thermococcus. DNA-DNA hybridization values with the two closest relatives Thermococcus hydrothermalis AL662(T) and Thermococcus celer JCM 8558(T) were below the threshold value of 70 %. On the basis of the physiological and genotypic distinctness, we propose a novel species, Thermococcus prieurii sp. nov. The type strain is Bio-pl-0405IT2(T) ( = CSUR P577(T)= JCM 16307(T)).

  5. Hyperthermophilic Archaeon Thermococcus kodakarensis Utilizes a Four-Step Pathway for NAD+ Salvage through Nicotinamide Deamination.

    Science.gov (United States)

    Hachisuka, Shin-Ichi; Sato, Takaaki; Atomi, Haruyuki

    2018-06-01

    Many organisms possess pathways that regenerate NAD + from its degradation products, and two pathways are known to salvage NAD + from nicotinamide (Nm). One is a four-step pathway that proceeds through deamination of Nm to nicotinic acid (Na) by Nm deamidase and phosphoribosylation to nicotinic acid mononucleotide (NaMN), followed by adenylylation and amidation. Another is a two-step pathway that does not involve deamination and directly proceeds with the phosphoribosylation of Nm to nicotinamide mononucleotide (NMN), followed by adenylylation. Judging from genome sequence data, the hyperthermophilic archaeon Thermococcus kodakarensis is supposed to utilize the four-step pathway, but the fact that the adenylyltransferase encoded by TK0067 recognizes both NMN and NaMN also raises the possibility of a two-step salvage mechanism. Here, we examined the substrate specificity of the recombinant TK1676 protein, annotated as nicotinic acid phosphoribosyltransferase. The TK1676 protein displayed significant activity toward Na and phosphoribosyl pyrophosphate (PRPP) and only trace activity with Nm and PRPP. We further performed genetic analyses on TK0218 (quinolinic acid phosphoribosyltransferase) and TK1650 (Nm deamidase), involved in de novo biosynthesis and four-step salvage of NAD + , respectively. The ΔTK0218 mutant cells displayed growth defects in a minimal synthetic medium, but growth was fully restored with the addition of Na or Nm. The ΔTK0218 ΔTK1650 mutant cells did not display growth in the minimal medium, and growth was restored with the addition of Na but not Nm. The enzymatic and genetic analyses strongly suggest that NAD + salvage in T. kodakarensis requires deamination of Nm and proceeds through the four-step pathway. IMPORTANCE Hyperthermophiles must constantly deal with increased degradation rates of their biomolecules due to their high growth temperatures. Here, we identified the pathway that regenerates NAD + from nicotinamide (Nm) in the

  6. Nicotinamidase from the thermophilic archaeon Acidilobus saccharovorans: structural and functional characteristics.

    Science.gov (United States)

    Stekhanova, T N; Bezsudnova, E Y; Mardanov, A V; Osipov, E M; Ravin, N V; Skryabin, K G; Popov, V O

    2014-01-01

    Nicotinamidase is involved in the maintenance of NAD+ homeostasis and in the NAD+ salvage pathway of most prokaryotes, and it is considered as a possible drug target. The gene (ASAC_0847) encoding a hypothetical nicotinamidase has been found in the genome of the thermophilic archaeon Acidilobus saccharovorans. The product of this gene, NA_As0847, has been expressed in Escherichia coli, isolated, and characterized as a Fe(2+)-containing nicotinamidase (k(cat)/K(m) = 427 mM(-1)·sec(-1))/pyrazinamidase (k(cat)/K(m) = 331 mM(-1)·sec(-1)). NA_As0847 is a homodimer with molecular mass 46.4 kDa. The enzyme has high thermostability (T(1/2) (60°C) = 180 min, T(1/2) (80°C) = 35 min) and thermophilicity (T(opt) = 90°C, E(a) = 30.2 ± 1.0 kJ/mol) and broad pH interval of activity, with the optimum at pH 7.5. Special features of NA_As0847 are the presence of Fe2+ instead of Zn2+ in the active site of the enzyme and inhibition of the enzyme activity by Zn2+ at micromolar concentrations. Analysis of the amino acid sequence revealed a new motif of the metal-binding site (DXHXXXDXXEXXXWXXH) for homological archaeal nicotinamidases.

  7. Two DNA polymerase sliding clamps from the thermophilic archaeon Sulfolobus solfataricus.

    Science.gov (United States)

    De Felice, M; Sensen, C W; Charlebois, R L; Rossi, M; Pisani, F M

    1999-08-06

    Herein, we report the identification and characterization of two DNA polymerase processivity factors from the thermoacidophilic archaeon Sulfolobus solfataricus. They, referred to as 039p (244 amino acid residues, 27 kDa) and 048p (249 amino acid residues, 27 kDa), present significant primary structure similarity to eukaryotic proliferating cell nuclear antigen (PCNA). We demonstrate that both 039p and 048p form oligomers in solution and are able to substantially activate the synthetic activity of the single-subunit family B DNA polymerase from S. solfataricus (Sso DNA pol B1) on poly(dA)-oligo(dT) as a primer-template. This stimulatory effect is the result of enhanced DNA polymerase processivity, as indicated by the analysis of the elongation products on polyacrylamide gels. Activation of Sso DNA pol B1 synthetic activity was also observed on linear primed single-stranded M13 mp18 DNA as a template. By immunoblot analysis using specific rabbit antisera, 039p and 048p were both detected in the logarithmic and stationary phases of S. solfataricus growth curve. This is the first report of the identification and biochemical characterization of two distinct DNA polymerase processivity factors from the same organism. The significance of these findings for the understanding of the DNA replication process in Archaea is discussed. Copyright 1999 Academic Press.

  8. Random mutagenesis of the hyperthermophilic archaeon Pyrococcus furiosus using in vitro mariner transposition and natural transformation.

    Science.gov (United States)

    Guschinskaya, Natalia; Brunel, Romain; Tourte, Maxime; Lipscomb, Gina L; Adams, Michael W W; Oger, Philippe; Charpentier, Xavier

    2016-11-08

    Transposition mutagenesis is a powerful tool to identify the function of genes, reveal essential genes and generally to unravel the genetic basis of living organisms. However, transposon-mediated mutagenesis has only been successfully applied to a limited number of archaeal species and has never been reported in Thermococcales. Here, we report random insertion mutagenesis in the hyperthermophilic archaeon Pyrococcus furiosus. The strategy takes advantage of the natural transformability of derivatives of the P. furiosus COM1 strain and of in vitro Mariner-based transposition. A transposon bearing a genetic marker is randomly transposed in vitro in genomic DNA that is then used for natural transformation of P. furiosus. A small-scale transposition reaction routinely generates several hundred and up to two thousands transformants. Southern analysis and sequencing showed that the obtained mutants contain a single and random genomic insertion. Polyploidy has been reported in Thermococcales and P. furiosus is suspected of being polyploid. Yet, about half of the mutants obtained on the first selection are homozygous for the transposon insertion. Two rounds of isolation on selective medium were sufficient to obtain gene conversion in initially heterozygous mutants. This transposition mutagenesis strategy will greatly facilitate functional exploration of the Thermococcales genomes.

  9. 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.

  10. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

  12. 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.

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    Alvaro Orell

    2013-01-01

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

  15. 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.

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

    Science.gov (United States)

    Moon, Yoon-Jung; Kwon, Joseph; Yun, Sung-Ho; Lim, Hye Li; Kim, Jonghyun; Kim, Soo Jung; Kang, Sung Gyun; Lee, Jung-Hyun; Kim, Seung Il; Chung, Young-Ho

    2015-01-01

    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. PMID:25915030

  17. 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.

  18. 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

    -xylosidase stabilities, approximately 55% and 83% of the initial beta-xylanase and beta-xylosidase activities, respectively, remained after 24 h incubation at 20% NaCl. The enzymes were also shown to be slightly thermophilic: P-xylanase activity exhibiting two optima at 55degrees and 70degreesC, while beta......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...... for beta-xylanase activity between 5% and 15% NaCl, while beta-xylosidase activity was highest at 5% NaCl. Almost half of the maximum activities remained at 27%-30% NaCl for both enzyme activities. When dialyzed culture supernatant and culture broth were employed for determination of beta-xylanase and beta...

  19. 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

    By generating a specialized cDNA library from the archaeon Sulfolobus solfataricus, we have identified 57 novel small non-coding RNA (ncRNA) candidates and confirmed their expression by Northern blot analysis. The majority was found to belong to one of two classes, either antisense or antisense...... elements by inhibiting expression of the transposase mRNA. Surprisingly, the class of antisense RNAs also contained RNAs complementary to tRNAs or sRNAs (small-nucleolar-like RNAs). For the antisense-box ncRNAs, the majority could be assigned to the class of C/D sRNAs, which specify 2'-O-methylation sites...... on rRNAs or tRNAs. Five C/D sRNAs of this group are predicted to target methylation at six sites in 13 different tRNAs, thus pointing to the widespread role of these sRNA species in tRNA modification in Archaea. Another group of antisense-box RNAs, lacking typical C/D sRNA motifs, was predicted...

  20. 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.

  1. 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. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  2. Halorubrum depositum sp. nov., a Novel Halophilic Archaeon Isolated from a Salt Deposit.

    Science.gov (United States)

    Chen, Shaoxing; Sun, Siqi; Xu, Yao; Lv, Jinting; Chen, Linan; Liu, Liu

    2018-06-01

    A non-motile, pleomorphic rod-shaped or oval, red-pigmented (nearly scarlet), extremely halophilic archaeon, strain Y78 T , was isolated from a salt deposit of Yunnan salt mine, China. Analysis of the 16S rRNA gene sequence showed that it was phylogenetically related to species of the genus Halorubrum, with a close relationship to Halorubrum rutilum YJ-18-S1 T (98.6%), Halorubrum yunnanense Q85 T (98.3%), and Halorubrum lipolyticum 9-3 T (98.1%). The temperature, NaCl, and pH ranges for growth were 25-50 °C, 12-30% (w/v), and 6.5-9.0, respectively. Mg 2+ was required for growth. The polar lipids of strain Y78 T were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, and a sulfated diglycosyl diether. The DNA G+C content was 66.6 mol%. DNA-DNA hybridization values between strain Y78 T and two closely related species of the genus Halorubrum were far below 70%. Based on the data presented in this study, strain Y78 T represents a novel species for which the name Halorubrum depositum sp. nov. is proposed; the type strain is Y78 T (= CGMCC 1.15456 T  = JCM 31272 T ).

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Purification and characterisation of a salt-stable protease from the halophilic archaeon Halogranum rubrum.

    Science.gov (United States)

    Gao, Ruichang; Shi, Tong; Liu, Xiangdong; Zhao, Mengqin; Cui, Henglin; Yuan, Li

    2017-03-01

    Because proteases play an important role in the fermentation of fish sauce, the purification and characterisation of an extracellular protease from the halophilic archaeon Halogranum rubrum was investigated. The molecular mass of the protease was estimated to be approximately 47 kDa based on sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) and native-PAGE analysis. The optimum conditions for catalytic activity were pH 8.0 and 50°C. The protease showed alkaline stability (pH 7.0-10.0). The protease also exhibited novel catalytic ability over a broad range of salinity (NaCl 0-3 mol L -1 ). Calcium ion enhanced the proteolytic activity of the enzyme. The K m and V max values of the purified protease for casein were calculated to be 4.89 mg mL -1 and 1111.11 U mL -1 , respectively. The protease was strongly inhibited by ethylenediamine tetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Meanwhile, the protease was stable in the presence of Triton X-100, isopropanol, ethanol or dithio-bis-nitrobenzoic (DTNB), but was inhibited by sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO) or methanol. MALDI -TOF/TOF MS analysis revealed that the protease shared some functional traits with protease produced by Halogranum salarium. Furthermore, it exhibited high hydrolytic activity on silver carp myosin protein. The protease is an alkaline and salt-tolerant enzyme that hydrolyses silver carp myosin with high efficiency. These excellent characteristics make this protease an attractive candidate for industrial use in low-salt fish sauce fermentation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  8. A Heme-based Redox Sensor in the Methanogenic Archaeon Methanosarcina acetivorans*

    Science.gov (United States)

    Molitor, Bastian; Stassen, Marc; Modi, Anuja; El-Mashtoly, Samir F.; Laurich, Christoph; Lubitz, Wolfgang; Dawson, John H.; Rother, Michael; Frankenberg-Dinkel, Nicole

    2013-01-01

    Based on a bioinformatics study, the protein MA4561 from the methanogenic archaeon Methanosarcina acetivorans was originally predicted to be a multidomain phytochrome-like photosensory kinase possibly binding open-chain tetrapyrroles. Although we were able to show that recombinantly produced and purified protein does not bind any known phytochrome chromophores, UV-visible spectroscopy revealed the presence of a heme tetrapyrrole cofactor. In contrast to many other known cytoplasmic heme-containing proteins, the heme was covalently attached via one vinyl side chain to cysteine 656 in the second GAF domain. This GAF domain by itself is sufficient for covalent attachment. Resonance Raman and magnetic circular dichroism data support a model of a six-coordinate heme species with additional features of a five-coordination structure. The heme cofactor is redox-active and able to coordinate various ligands like imidazole, dimethyl sulfide, and carbon monoxide depending on the redox state. Interestingly, the redox state of the heme cofactor has a substantial influence on autophosphorylation activity. Although reduced protein does not autophosphorylate, oxidized protein gives a strong autophosphorylation signal independent from bound external ligands. Based on its genomic localization, MA4561 is most likely a sensor kinase of a two-component system effecting regulation of the Mts system, a set of three homologous corrinoid/methyltransferase fusion protein isoforms involved in methyl sulfide metabolism. Consistent with this prediction, an M. acetivorans mutant devoid of MA4561 constitutively synthesized MtsF. On the basis of our results, we postulate a heme-based redox/dimethyl sulfide sensory function of MA4561 and propose to designate it MsmS (methyl sulfide methyltransferase-associated sensor). PMID:23661702

  9. Palaeococcus pacificus sp. nov., an archaeon from deep-sea hydrothermal sediment.

    Science.gov (United States)

    Zeng, Xiang; Zhang, Xiaobo; Jiang, Lijing; Alain, Karine; Jebbar, Mohamed; Shao, Zongze

    2013-06-01

    A hyperthermophilic, anaerobic, piezophilic archaeon (strain DY20341(T)) was isolated from a sediment sample collected from an East Pacific Ocean hydrothermal field (1° 37' S 102° 45' W) at a depth of 2737 m. The cells were irregular cocci, 0.8-1.5 µm in diameter. Growth was observed between 50 and 90 °C (optimum 80 °C), pH 5.0 and 8.0 (optimum pH 7.0), 1% and 7% (w/v) sea salts (Sigma, optimum 3%), 1% and 4% (w/v) NaCl (optimum 3%) and 0.1 and 80 MPa (optimum 30 MPa). The minimum doubling time was 66 min at 30 MPa and 80 °C. The isolate was an obligate chemoorganoheterotroph, capable of utilizing complex organic compounds and organic acids including yeast extract, peptone, tryptone, casein, starch, Casamino acids, citrate, lactate, acetate, fumarate, propanoate and pyruvate for growth. It was strictly anaerobic and facultatively dependent on elemental sulfur or sulfate as electron acceptors, but did not reduce sulfite, thiosulfate, Fe(III) or nitrate. The presence of elemental sulfur enhanced growth. The G+C content of the genomic DNA was 43.6 ± 1 mol%. 16S rRNA gene sequence analysis revealed that the closest relative of the isolated organism was Palaeococcus ferrophilus DMJ(T) (95.7% 16S rRNA gene similarity). On the basis of its physiological properties and phylogenetic analyses, the isolate is considered to represent a novel species, for which the name Palaeococcus pacificus sp. nov. is proposed. The type strain is strain DY20341(T) (=JCM 17873(T)=DSM 24777(T)).

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

    International Nuclear Information System (INIS)

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

    2010-01-01

    Crystal structures of ModA from M. acetivorans in the apo and ligand-bound conformations confirm domain rotation upon ligand binding. 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 Å resolution and in the molybdate-bound conformation at 2.25 and 2.45 Å resolution. The overall domain structure of MaModA is similar to other ModA proteins in that it has a bilobal structure in which two mixed α/β 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

  11. 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.

  12. Generation of comprehensive transposon insertion mutant library for the model archaeon, Haloferax volcanii, and its use for gene discovery.

    Science.gov (United States)

    Kiljunen, Saija; Pajunen, Maria I; Dilks, Kieran; Storf, Stefanie; Pohlschroder, Mechthild; Savilahti, Harri

    2014-12-09

    Archaea share fundamental properties with bacteria and eukaryotes. Yet, they also possess unique attributes, which largely remain poorly characterized. Haloferax volcanii is an aerobic, moderately halophilic archaeon that can be grown in defined media. It serves as an excellent archaeal model organism to study the molecular mechanisms of biological processes and cellular responses to changes in the environment. Studies on haloarchaea have been impeded by the lack of efficient genetic screens that would facilitate the identification of protein functions and respective metabolic pathways. Here, we devised an insertion mutagenesis strategy that combined Mu in vitro DNA transposition and homologous-recombination-based gene targeting in H. volcanii. We generated an insertion mutant library, in which the clones contained a single genomic insertion. From the library, we isolated pigmentation-defective and auxotrophic mutants, and the respective insertions pinpointed a number of genes previously known to be involved in carotenoid and amino acid biosynthesis pathways, thus validating the performance of the methodologies used. We also identified mutants that had a transposon insertion in a gene encoding a protein of unknown or putative function, demonstrating that novel roles for non-annotated genes could be assigned. We have generated, for the first time, a random genomic insertion mutant library for a halophilic archaeon and used it for efficient gene discovery. The library will facilitate the identification of non-essential genes behind any specific biochemical pathway. It represents a significant step towards achieving a more complete understanding of the unique characteristics of halophilic archaea.

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

    International Nuclear Information System (INIS)

    Byrne-Steele, Miranda L.; Hughes, Ronny C.; Ng, Joseph D.

    2009-01-01

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

  14. The 60 kDa heat shock proteins in the hyperthermophilic archaeon Sulfolobus shibatae.

    Science.gov (United States)

    Kagawa, H K; Osipiuk, J; Maltsev, N; Overbeek, R; Quaite-Randall, E; Joachimiak, A; Trent, J D

    1995-11-10

    One of the most abundant proteins in the hyperthermophilic archaeon Sulfolobus shibatae is the 59 kDa heat shock protein (TF55) that is believed to form a homo-oligomeric double ring complex structurally similar to the bacterial chaperonins. We discovered a second protein subunit in the S. shibatae ring complex (referred to as alpha) that is stoichiometric with TF55 (renamed beta). The gene and flanking regions of alpha were cloned and sequenced and its inferred amino acid sequence has 54.4% identity and 74.4% similarity to beta. Transcription start sites for both alpha and beta were mapped and three potential transcription regulatory regions were identified. Northern analyses of cultures shifted from normal growth temperatures (70 to 75 degrees C) to heat shock temperatures (85 to 90 degrees C) indicated that the levels of alpha and beta mRNAs increased during heat shock, but at all temperatures their relative proportions remained constant. Monitoring protein synthesis by autoradiography of total proteins from cultures pulse labeled with L(-)[35S]methionine at normal and heat shock temperatures indicated significant increases in alpha and beta synthesis during heat shock. Under extreme heat shock conditions (> or = 90 degrees C) alpha and beta appeared to be the only two proteins synthesized. The purified alpha and beta subunits combined to form high molecular mass complexes with similar mobilities on native polyacrylamide gels to the complexes isolated directly from cells. Equal proportions of the two subunits gave the greatest yield of the complex, which we refer to as a "rosettasome". It is argued that the rosettasome consists of two homo-oligomeric rings; one of alpha and the other of beta. Polyclonal antibodies against alpha and beta from S. shibatae cross-reacted with proteins of similar molecular mass in 10 out of the 17 archaeal species tested, suggesting that the two rosettasome proteins are highly conserved among the archaea. The archaeal sequences were

  15. Draft genome sequence of the extremely halophilic archaeon Haladaptatus cibarius type strain D43(T) isolated from fermented seafood.

    Science.gov (United States)

    Lee, Hae-Won; Kim, Dae-Won; Lee, Mi-Hwa; Kim, Byung-Yong; Cho, Yong-Joon; Yim, Kyung June; Song, Hye Seon; Rhee, Jin-Kyu; Seo, Myung-Ji; Choi, Hak-Jong; Choi, Jong-Soon; Lee, Dong-Gi; Yoon, Changmann; Nam, Young-Do; Roh, Seong Woon

    2015-01-01

    An extremely halophilic archaeon, Haladaptatus cibarius D43(T), was isolated from traditional Korean salt-rich fermented seafood. Strain D43(T) shows the highest 16S rRNA gene sequence similarity (98.7 %) with Haladaptatus litoreus RO1-28(T), is Gram-negative staining, motile, and extremely halophilic. Despite potential industrial applications of extremely halophilic archaea, their genome characteristics remain obscure. Here, we describe the whole genome sequence and annotated features of strain D43(T). The 3,926,724 bp genome includes 4,092 protein-coding and 57 RNA genes (including 6 rRNA and 49 tRNA genes) with an average G + C content of 57.76 %.

  16. Non-essential MCM-related proteins mediate a response to DNA damage in the archaeon Methanococcus maripaludis.

    Science.gov (United States)

    Walters, Alison D; Chong, James P J

    2017-05-01

    The single minichromosome maintenance (MCM) protein found in most archaea has been widely studied as a simplified model for the MCM complex that forms the catalytic core of the eukaryotic replicative helicase. Organisms of the order Methanococcales are unusual in possessing multiple MCM homologues. The Methanococcus maripaludis S2 genome encodes four MCM homologues, McmA-McmD. DNA helicase assays reveal that the unwinding activity of the three MCM-like proteins is highly variable despite sequence similarities and suggests additional motifs that influence MCM function are yet to be identified. While the gene encoding McmA could not be deleted, strains harbouring individual deletions of genes encoding each of the other MCMs display phenotypes consistent with these proteins modulating DNA damage responses. M. maripaludis S2 is the first archaeon in which MCM proteins have been shown to influence the DNA damage response.

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    The upp gene, encoding uracil phosphoribosyltransferase (UPRTase) from the thermoacidophilic archaeon Sulfolobus solfataricus, was cloned and expressed in Escherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH 5.5 when...... assayed at 60 °C. Enzyme activity was strongly stimulated by GTP and inhibited by CTP. GTP caused an approximately 20-fold increase in the turnover number kcat and raised the Km values for 5-phosphoribosyl-1-diphosphate (PRPP) and uracil by two- and >10-fold, respectively. The inhibition by CTP...... was complex as it depended on the presence of the reaction product UMP. Neither CTP nor UMP were strong inhibitors of the enzyme, but when present in combination their inhibition was extremely powerful. Ligand binding analyses showed that GTP and PRPP bind cooperatively to the enzyme and that the inhibitors...

  18. 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

  19. Functional Role of MrpA in the MrpABCDEFG Na+/H+ Antiporter Complex from the Archaeon Methanosarcina acetivorans

    OpenAIRE

    Jasso-Ch?vez, Ricardo; Diaz-Perez, C?sar; Rodr?guez-Zavala, Jos? S.; Ferry, James G.

    2016-01-01

    The multisubunit cation/proton antiporter 3 family, also called Mrp, is widely distributed in all three phylogenetic domains (Eukarya, Bacteria, and Archaea). Investigations have focused on Mrp complexes from the domain Bacteria to the exclusion of Archaea, with a consensus emerging that all seven subunits are required for Na+/H+ antiport activity. The MrpA subunit from the MrpABCDEFG Na+/H+ antiporter complex of the archaeon Methanosarcina acetivorans was produced in antiporter-deficient Esc...

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

    Directory of Open Access Journals (Sweden)

    Roberta V. Branco

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Jiannan Ding

    2011-06-01

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

  2. Multistage bioassociation of uranium onto an extremely halophilic archaeon revealed by a unique combination of spectroscopic and microscopic techniques.

    Science.gov (United States)

    Bader, Miriam; Müller, Katharina; Foerstendorf, Harald; Drobot, Björn; Schmidt, Matthias; Musat, Niculina; Swanson, Juliet S; Reed, Donald T; Stumpf, Thorsten; Cherkouk, Andrea

    2017-04-05

    The interactions of two extremely halophilic archaea with uranium were investigated at high ionic strength as a function of time, pH and uranium concentration. Halobacterium noricense DSM-15987 and Halobacterium sp. putatively noricense, isolated from the Waste Isolation Pilot Plant repository, were used for these investigations. The kinetics of U(VI) bioassociation with both strains showed an atypical multistage behavior, meaning that after an initial phase of U(VI) sorption, an unexpected interim period of U(VI) release was observed, followed by a slow reassociation of uranium with the cells. By applying in situ attenuated total reflection Fourier-transform infrared spectroscopy, the involvement of phosphoryl and carboxylate groups in U(VI) complexation during the first biosorption phase was shown. Differences in cell morphology and uranium localization become visible at different stages of the bioassociation process, as shown with scanning electron microscopy in combination with energy dispersive X-ray spectroscopy. Our results demonstrate for the first time that association of uranium with the extremely halophilic archaeon is a multistage process, beginning with sorption and followed by another process, probably biomineralization. Copyright © 2016. Published by Elsevier B.V.

  3. Redox stress proteins are involved in adaptation response of the hyperthermoacidophilic archaeon Sulfolobus solfataricus to nickel challenge

    Directory of Open Access Journals (Sweden)

    Scaloni Andrea

    2007-08-01

    Full Text Available Abstract Background Exposure to nickel (Ni and its chemical derivatives has been associated with severe health effects in human. On the contrary, poor knowledge has been acquired on target physiological processes or molecular mechanisms of this metal in model organisms, including Bacteria and Archaea. In this study, we describe an analysis focused at identifying proteins involved in the recovery of the archaeon Sulfolobus solfataricus strain MT4 from Ni-induced stress. Results To this purpose, Sulfolobus solfataricus was grown in the presence of the highest nickel sulphate concentration still allowing cells to survive; crude extracts from treated and untreated cells were compared at the proteome level by using a bi-dimensional chromatography approach. We identified several proteins specifically repressed or induced as result of Ni treatment. Observed up-regulated proteins were largely endowed with the ability to trigger recovery from oxidative and osmotic stress in other biological systems. It is noteworthy that most of the proteins induced following Ni treatment perform similar functions and a few have eukaryal homologue counterparts. Conclusion These findings suggest a series of preferential gene expression pathways activated in adaptation response to metal challenge.

  4. Immunogenic properties of the human gut-associated archaeon Methanomassiliicoccus luminyensis and its susceptibility to antimicrobial peptides.

    Directory of Open Access Journals (Sweden)

    Corinna Bang

    Full Text Available The methanogenic archaeon Methanomassiliicoccus luminyensis strain B10T was isolated from human feces just a few years ago. Due to its remarkable metabolic properties, particularly the degradation of trimethylamines, this strain was supposed to be used as "Archaebiotic" during metabolic disorders of the human intestine. However, there is still no data published regarding adaptations to the natural habitat of M. luminyensis as it has been shown for the other two reported mucosa-associated methanoarchaea. This study aimed at unraveling susceptibility of M. luminyensis to antimicrobial peptides as well as its immunogenicity. By using the established microtiter plate assay adapted to the anaerobic growth requirements of methanogenic archaea, we demonstrated that M. luminyensis is highly sensitive against LL32, a derivative of human cathelicidin (MIC = 2 μM. However, the strain was highly resistant against the porcine lysin NK-2 (MIC = 10 μM and the synthetic antilipopolysaccharide peptide (Lpep (MIC>10 μM and overall differed from the two other methanoarchaea, Methanobrevibacter smithii and Methanosphaera stadtmanae in respect to AMP sensitivity. Moreover, only weak immunogenic potential of M. luminyensis was demonstrated using peripheral blood mononuclear cells (PBMCs and monocyte-derived dendritic cells (moDCs by determining release of pro-inflammatory cytokines. Overall, our findings clearly demonstrate that the archaeal gut inhabitant M. luminyensis is susceptible to the release of human-derived antimicrobial peptides and exhibits low immunogenicity towards human immune cells in vitro-revealing characteristics of a typical commensal gut microbe.

  5. Effects of a Squalene Epoxidase Inhibitor, Terbinafine, on Ether Lipid Biosyntheses in a Thermoacidophilic Archaeon, Thermoplasma acidophilum

    Science.gov (United States)

    Kon, Takahide; Nemoto, Naoki; Oshima, Tairo; Yamagishi, Akihiko

    2002-01-01

    The archaeal plasma membrane consists mainly of diether lipids and tetraether lipids instead of the usual ester lipids found in other organisms. Although a molecule of tetraether lipid is thought to be synthesized from two molecules of diether lipids, there is no direct information about the biosynthetic pathway(s) or intermediates of tetraether lipid biosynthesis. In this study, we examined the effects of the fungal squalene epoxidase inhibitor terbinafine on the growth and ether lipid biosyntheses in the thermoacidophilic archaeon Thermoplasma acidophilum. Terbinafine was found to inhibit the growth of T. acidophilum in a concentration-dependent manner. When growing T. acidophilum cells were pulse-labeled with [2-14C]mevalonic acid in the presence of terbinafine, incorporation of radioactivity into the tetraether lipid fraction was strongly suppressed, while accumulation of radioactivity was noted at the position corresponding to diether lipids, depending on the concentration of terbinafine. After the cells were washed with fresh medium and incubated further without the radiolabeled substrate and the inhibitor, the accumulated radioactivity in the diether lipid fraction decreased quickly while that in the tetraether lipids increased simultaneously, without significant changes in the total radioactivity of ether lipids. These results strongly suggest that terbinafine inhibits the biosynthesis of tetraether lipids from a diether-type precursor lipid(s). The terbinafine treatment will be a tool for dissecting tetraether lipid biosynthesis in T. acidophilum. PMID:11844769

  6. Overexpression, purification and crystallization of tyrosyl-tRNA synthetase from the hyperthermophilic archaeon Aeropyrum pernix K1

    International Nuclear Information System (INIS)

    Iwaki, Jun; Suzuki, Ryuichiro; Fujimoto, Zui; Momma, Mitsuru; Kuno, Atsushi; Hasegawa, Tsunemi

    2005-01-01

    Tyrosyl-tRNA synthetase from the hyperthermophilic archaeon A. pernix K1 was cloned, purified and crystallized. The crystals belonged to the tetragonal space group P4 3 2 1 2, with unit-cell parameters a = b = 66.1, c = 196.2 Å, and diffracted to beyond 2.15 Å resolution at 100 K. Hyperthermophilic archaeal tyrosyl-tRNA synthetase from Aeropyrum pernix K1 was cloned and overexpressed in Escherichia coli. The expressed protein was purified by Cibacron Blue affinity chromatography following heat treatment at 363 K. Crystals suitable for X-ray diffraction studies were obtained under optimized crystallization conditions in the presence of 1.5 M ammonium sulfate using the hanging-drop vapour-diffusion method. The crystals belonged to the tetragonal space group P4 3 2 1 2, with unit-cell parameters a = b = 66.1, c = 196.2 Å, and diffracted to beyond 2.15 Å resolution at 100 K

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

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Bin, E-mail: ren@csb.ki.se [Center for Structural Biochemistry, Karolinska Institute, NOVUM, S-141 57 Huddinge (Sweden); Kuhn, Joëlle; Meslet-Cladiere, Laurence; Myllykallio, Hannu [Université Paris-Sud, Institut de Génétique et Microbiologie, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8621, F-91405 Orsay CEDEX (France); Ladenstein, Rudolf [Center for Structural Biochemistry, Karolinska Institute, NOVUM, S-141 57 Huddinge (Sweden)

    2007-05-01

    A RecB-like nuclease from the archaeon Pyrococcus abyssi was expressed, purified and crystallized. The crystals belong to the orthorhombic space group C222{sub 1} with a = 81.5, b = 159.8, c = 100.8 Å, and a native data set was collected to 2.65 Å resolution. Nucleases are required to process and repair DNA damage in living cells. One of the best studied nucleases is the RecB protein, which functions in Escherichia coli as a component of the RecBCD enzyme complex that amends double-strand breaks in DNA. Although archaea do not contain the RecBCD complex, a RecB-like nuclease from Pyrococcus abyssi has been cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant. The crystals belong to the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 81.5, b = 159.8, c = 100.8 Å. Self-rotation function and native Patterson map calculations revealed that there is a dimer in the asymmetric unit with its local twofold axis running parallel to the crystallographic twofold screw axis. The crystals diffracted to about 2 Å and a complete native data set was collected to 2.65 Å resolution.

  8. Unusual Starch Degradation Pathway via Cyclodextrins in the Hyperthermophilic Sulfate-Reducing Archaeon Archaeoglobus fulgidus Strain 7324▿

    Science.gov (United States)

    Labes, Antje; Schönheit, Peter

    2007-01-01

    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, α-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 β-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. PMID:17921308

  9. Palaeococcus helgesonii sp. nov., a facultatively anaerobic, hyperthermophilic archaeon from a geothermal well on Vulcano Island, Italy.

    Science.gov (United States)

    Amend, Jan P; Meyer-Dombard, D'Arcy R; Sheth, Seema N; Zolotova, Natalya; Amend, Andrea C

    2003-06-01

    A novel, hyperthermophilic archaeon was isolated from a shallow geothermal well that taps marine waters on the Island of Vulcano in the southern Tyrrhenian Sea, Italy. The cells were irregular cocci, 0.6-1.5 microm in diameter, with multiple polar flagella. Growth was observed at temperatures from 45 to 85 degrees C (optimum at approximately 80 degrees C), pH 5-8 (optimum at 6.5), and 0.5-6.0% NaCl (optimum at approximately 2.8%). The minimum doubling time was 50 min. The isolate was obligately chemoheterotrophic, utilizing complex organic compounds including yeast or beef extract, peptone, tryptone, or casein for best growth. The presence of elemental sulfur enhanced growth. The isolate grew anaerobically as well as microaerobically. The G+C content of the genomic DNA was 42.5 mol%. The 16S rRNA sequence indicated that the new isolate was a member of the Thermococcales within the euryarchaeota, representing the second species in the genus Palaeococcus. Its physiology and phylogeny differed in several key characteristics from those of Palaeococcus ferrophilus, justifying the establishment of a new species; the name Palaeococcus helgesonii sp. nov. is proposed, type strain PI1 (DSM 15127).

  10. Multistage bioassociation of uranium onto an extremely halophilic archaeon revealed by a unique combination of spectroscopic and microscopic techniques

    Energy Technology Data Exchange (ETDEWEB)

    Bader, Miriam; Müller, Katharina; Foerstendorf, Harald; Drobot, Björn [Helmholtz-Zentrum Dresden – Rossendorf, Institute of Resource Ecology, Bautzner Landstraße 400, 01328 Dresden (Germany); Schmidt, Matthias; Musat, Niculina [Helmholtz Centre for Environmental Research–UFZ, Department of Isotope Biogeochemistry, Permoserstraße 15, 04318 Leipzig (Germany); Swanson, Juliet S.; Reed, Donald T. [Los Alamos National Laboratory, Repository Science and Operations, 1400 University Drive, Carlsbad, NM, 88220 (United States); Stumpf, Thorsten [Helmholtz-Zentrum Dresden – Rossendorf, Institute of Resource Ecology, Bautzner Landstraße 400, 01328 Dresden (Germany); Cherkouk, Andrea, E-mail: a.cherkouk@hzdr.de [Helmholtz-Zentrum Dresden – Rossendorf, Institute of Resource Ecology, Bautzner Landstraße 400, 01328 Dresden (Germany)

    2017-04-05

    Highlights: • First prolonged kinetics study of uranium to halophilic archaea was performed. • An atypical time-dependent bioassociation behavior of uranium was observed. • Unique combination of spectroscopic and microscopic methods was used. • In situ ATR FT-IR showed association of U(VI) to phosphoryl and carboxylate groups. • Time-dependent changes of U(VI) localization could be monitored by SEM/EDX. - Abstract: The interactions of two extremely halophilic archaea with uranium were investigated at high ionic strength as a function of time, pH and uranium concentration. Halobacterium noricense DSM-15987 and Halobacterium sp. putatively noricense, isolated from the Waste Isolation Pilot Plant repository, were used for these investigations. The kinetics of U(VI) bioassociation with both strains showed an atypical multistage behavior, meaning that after an initial phase of U(VI) sorption, an unexpected interim period of U(VI) release was observed, followed by a slow reassociation of uranium with the cells. By applying in situ attenuated total reflection Fourier-transform infrared spectroscopy, the involvement of phosphoryl and carboxylate groups in U(VI) complexation during the first biosorption phase was shown. Differences in cell morphology and uranium localization become visible at different stages of the bioassociation process, as shown with scanning electron microscopy in combination with energy dispersive X-ray spectroscopy. Our results demonstrate for the first time that association of uranium with the extremely halophilic archaeon is a multistage process, beginning with sorption and followed by another process, probably biomineralization.

  11. Cloning and Molecular Characterization of an Alpha-Glucosidase (MalH) from the Halophilic Archaeon Haloquadratum walsbyi.

    Science.gov (United States)

    Cuebas-Irizarry, Mara F; Irizarry-Caro, Ricardo A; López-Morales, Carol; Badillo-Rivera, Keyla M; Rodríguez-Minguela, Carlos M; Montalvo-Rodríguez, Rafael

    2017-11-21

    We report the heterologous expression and molecular characterization of the first extremely halophilic alpha-glucosidase (EC 3.2.1.20) from the archaeon Haloquadratum walsbyi . A 2349 bp region ( Hqrw_2071 ) from the Hqr. walsbyi C23 annotated genome was PCR-amplified and the resulting amplicon ligated into plasmid pET28b(+), expressed in E. coli Rosetta cells, and the resulting protein purified by Ni-NTA affinity chromatography. The recombinant protein showed an estimated molecular mass of 87 kDa, consistent with the expected value of the annotated protein, and an optimal activity for the hydrolysis of α-PNPG was detected at 40 °C, and at pH 6.0. Enzyme activity values were the highest in the presence of 3 M NaCl or 3-4 M KCl. However, specific activity values were two-fold higher in the presence of 3-4 M KCl when compared to NaCl suggesting a cytoplasmic localization. Phylogenetic analyses, with respect to other alpha-glucosidases from members of the class Halobacteria, showed that the Hqr. walsbyi MalH was most similar (up to 41%) to alpha-glucosidases and alpha-xylosidases of Halorubrum . Moreover, computational analyses for the detection of functional domains, active and catalytic sites, as well as 3D structural predictions revealed a close relationship with an E. coli YicI-like alpha-xylosidase of the GH31 family. However, the purified enzyme did not show alpha-xylosidase activity. This narrower substrate range indicates a discrepancy with annotations from different databases and the possibility of specific substrate adaptations of halophilic glucosidases due to high salinity. To our knowledge, this is the first report on the characterization of an alpha-glucosidase from the halophilic Archaea, which could serve as a new model to gain insights into carbon metabolism in this understudied microbial group.

  12. Multistage bioassociation of uranium onto an extremely halophilic archaeon revealed by a unique combination of spectroscopic and microscopic techniques

    International Nuclear Information System (INIS)

    Bader, Miriam; Müller, Katharina; Foerstendorf, Harald; Drobot, Björn; Schmidt, Matthias; Musat, Niculina; Swanson, Juliet S.; Reed, Donald T.; Stumpf, Thorsten; Cherkouk, Andrea

    2017-01-01

    Highlights: • First prolonged kinetics study of uranium to halophilic archaea was performed. • An atypical time-dependent bioassociation behavior of uranium was observed. • Unique combination of spectroscopic and microscopic methods was used. • In situ ATR FT-IR showed association of U(VI) to phosphoryl and carboxylate groups. • Time-dependent changes of U(VI) localization could be monitored by SEM/EDX. - Abstract: The interactions of two extremely halophilic archaea with uranium were investigated at high ionic strength as a function of time, pH and uranium concentration. Halobacterium noricense DSM-15987 and Halobacterium sp. putatively noricense, isolated from the Waste Isolation Pilot Plant repository, were used for these investigations. The kinetics of U(VI) bioassociation with both strains showed an atypical multistage behavior, meaning that after an initial phase of U(VI) sorption, an unexpected interim period of U(VI) release was observed, followed by a slow reassociation of uranium with the cells. By applying in situ attenuated total reflection Fourier-transform infrared spectroscopy, the involvement of phosphoryl and carboxylate groups in U(VI) complexation during the first biosorption phase was shown. Differences in cell morphology and uranium localization become visible at different stages of the bioassociation process, as shown with scanning electron microscopy in combination with energy dispersive X-ray spectroscopy. Our results demonstrate for the first time that association of uranium with the extremely halophilic archaeon is a multistage process, beginning with sorption and followed by another process, probably biomineralization.

  13. Cloning and Molecular Characterization of an Alpha-Glucosidase (MalH from the Halophilic Archaeon Haloquadratum walsbyi

    Directory of Open Access Journals (Sweden)

    Mara F. Cuebas-Irizarry

    2017-11-01

    Full Text Available We report the heterologous expression and molecular characterization of the first extremely halophilic alpha-glucosidase (EC 3.2.1.20 from the archaeon Haloquadratum walsbyi. A 2349 bp region (Hqrw_2071 from the Hqr. walsbyi C23 annotated genome was PCR-amplified and the resulting amplicon ligated into plasmid pET28b(+, expressed in E. coli Rosetta cells, and the resulting protein purified by Ni-NTA affinity chromatography. The recombinant protein showed an estimated molecular mass of 87 kDa, consistent with the expected value of the annotated protein, and an optimal activity for the hydrolysis of α-PNPG was detected at 40 °C, and at pH 6.0. Enzyme activity values were the highest in the presence of 3 M NaCl or 3–4 M KCl. However, specific activity values were two-fold higher in the presence of 3–4 M KCl when compared to NaCl suggesting a cytoplasmic localization. Phylogenetic analyses, with respect to other alpha-glucosidases from members of the class Halobacteria, showed that the Hqr. walsbyi MalH was most similar (up to 41% to alpha-glucosidases and alpha-xylosidases of Halorubrum. Moreover, computational analyses for the detection of functional domains, active and catalytic sites, as well as 3D structural predictions revealed a close relationship with an E. coli YicI-like alpha-xylosidase of the GH31 family. However, the purified enzyme did not show alpha-xylosidase activity. This narrower substrate range indicates a discrepancy with annotations from different databases and the possibility of specific substrate adaptations of halophilic glucosidases due to high salinity. To our knowledge, this is the first report on the characterization of an alpha-glucosidase from the halophilic Archaea, which could serve as a new model to gain insights into carbon metabolism in this understudied microbial group.

  14. Natronolimnobius aegyptiacus sp. nov., an extremely halophilic alkalithermophilic archaeon isolated from the athalassohaline Wadi An Natrun, Egypt.

    Science.gov (United States)

    Zhao, Baisuo; Hu, Qingping; Guo, Xiaomeng; Liao, Ziya; Sarmiento, Felipe; Mesbah, Noha M; Yan, Yanchun; Li, Jun; Wiegel, Juergen

    2018-02-01

    An obligately aerobic extremely halophilic alkalithermophilic archaeon, strain JW/NM-HA 15 T , was isolated from the sediments of Wadi An Natrun in Egypt. Phylogenetic analysis based on 16S rRNA and rpoB' gene sequences indicated that it belongs to the family Natrialbaceae of the order Natrialbales. The closest relatives were Natronolimnobius baerhuensis IHC-005 T and Natronolimnobius innermongolicus N-1311 T (95.3 and 94.5 % 16S rRNA gene sequence similarity, respectively). Genome relatedness between strain JW/NM-HA 15 T and its neighbours was evaluated using average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity with the values of 75.7-85.0, 18.1-20.0, and 70.2-71.0%, respectively. Cells were obligately aerobic, rod-shaped, non-motile, Gram-stain-negative and chemo-organotrophic. The strain grew in the presence of 2.57 M to saturating Na + (optimum 3.25-4.60 M Na + ), at pH 55 °C 7.5-10.5 (optimum pH 55 °C 9.0-9.5), and at 30-56 °C (optimum 52 °C). The major polar lipids consisted of phosphatidylglycerol, methylated phosphatidylglycerolphosphate and two phospholipids. The complete genome size of strain JW/NM-HA 15 T is approximately 3.93 Mb, with a DNA G+C content of 64.1 mol%. On the basis of phylogenetic features, genomic relatedness, phenotypic and chemotaxonomic data, strain JW/NM-HA 15 T was thus considered to represent a novel species within the genus Natronolimnobius, for which the name Natronolimnobius aegyptiacus sp. nov. is proposed. The type strain is JW/NM-HA 15 T (=ATCC BAA-2088 T =DSM 23470 T ).

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

    International Nuclear Information System (INIS)

    Fukunaga, Ryuya; Ishitani, Ryuichiro; Nureki, Osamu; Yokoyama, Shigeyuki

    2004-01-01

    The leucyl-tRNA synthetase (LeuRS) from P. horikoshii has been overexpressed in Escherichia coli and purified, and cocrystallizations with each of the tRNA Leu isoacceptors have been attempted. Cocrystals were obtained by the hanging-drop vapour-diffusion method, but only when the tRNA Leu isoacceptor with the anticodon CAA was used. All five tRNA Leu isoacceptors from the archaeon Pyrococcus horikoshii have been transcribed in vitro and purified. The leucyl-tRNA synthetase (LeuRS) from P. horikoshii was overexpressed in Escherichia coli and purified, and cocrystallizations with each of the tRNA Leu isoacceptors were attempted. Cocrystals were obtained by the hanging-drop vapour-diffusion method, but only when the tRNA Leu isoacceptor with the anticodon CAA was used. Electrophoretic analyses revealed that the crystals contain both LeuRS and tRNA Leu , suggesting that they are LeuRS–tRNA Leu complex crystals. A data set diffracting to 3.3 Å resolution was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 118.18, b = 120.55, c = 231.13 Å. The asymmetric unit is expected to contain two complexes of LeuRS–tRNA Leu , with a corresponding crystal volume per protein weight of 2.9 Å 3 Da −1 and a solvent content of 57.3%

  16. Haloprofundus marisrubri gen. nov., sp. nov., an extremely halophilic archaeon isolated from a brine-seawater interface.

    Science.gov (United States)

    Zhang, Guishan; Gu, Jingang; Zhang, Ruifu; Rashid, Mamoon; Haroon, Mohamed Fauzi; Xun, Weibing; Ruan, Zhiyong; Dong, Xiuzhu; Stingl, Ulrich

    2017-01-01

    We isolated a Gram-stain-negative, pink-pigmented, motile, pleomorphic, extremely halophilic archaeon from the brine-seawater interface of Discovery Deep in the Saudi Arabian Red Sea. This strain, designated SB9T, was capable of growth within a wide range of temperatures and salinity, but required MgCl2. Cells lysed in distilled water, but at 7.0 % (w/v) NaCl cell lysis was prevented. The major polar lipids from strain SB9T were phosphatidylglycerol, phosphatidylglycerolphosphate methyl ester, sulfated mannosyl glucosyl diether, mannosyl glucosyl diether, an unidentified glycolipid and two unidentified phospholipids. The major respiratory quinones of strain SB9T were menaquinones MK8 (66 %) and MK8 (VIII-H2) (34 %). Analysis of the 16S rRNA gene sequence revealed that strain SB9T was closely related to species in the genera Halogranum and Haloplanus; in particular, it shared highest sequence similarity with the type strain of Halogranum rubrum (93.4 %), making it its closest known relative. The unfinished draft genome of strain SB9Twas 3 931 127 bp in size with a total G+C content of 62.53 mol% and contained 3917 ORFs, 50 tRNAs and eight rRNAs. Based on comparisons with currently available genomes, the highest average nucleotide identity value was 83 % to Halogranum salarium B-1T (GenBank accession no. GCA_000283335.1). These data indicate that this new isolate cannot be classified into any recognized genera of the family Haloferacaceae, and therefore strain SB9T is considered to be a representative of a novel species of a new genus within this family, for which the name Haloprofundus marisrubri gen. nov., sp. nov. is proposed. The type strain of Haloprofundus marisrubri is SB9T (=JCM 19565T=CGMCC 1.14959T).

  17. Thermococcus sulfurophilus sp. nov., a New Hyperthermophilic, Sulfur-Reducing Archaeon Isolated from Deep-Sea Hydrothermal Vent

    Science.gov (United States)

    Pikuta, Elena V.; Hoover, Richard B.; Whitman, William B.; Marsic, Damien; Garriott, Owen; Six, N. Frank (Technical Monitor)

    2002-01-01

    A new hyperthermophilic, anaerobic, sulfur-reducing, organo-heterotrophic archaeon, strain OGL-20P, was isolated from "black smoker" chimney material at the Rainbow hydrothermal vent site in the Atlantic Ocean (36.2 N; 33.9 W). The cells of strain OGL-20P have irregular coccoid shape and are motile with a single flagellum. Growth occurs within pH range of 5.5-8.2 (optimal at pH 7.0-7.2), salinity range of 1-5% NaCl (optimal concentration 3% NaCl wt/vol), and temperature range of +55 C to +94 C (optimal growth at +83 C to +85 C). Strain OGL-20P is resistant to freezing (at -20 C). New isolate is strictly anaerobic with sulfur-type of respiration. A limited number of compounds are utilized as electron donors, including peptone, becto-tryptone, casamino-acids, and yeast extract but does not grow with separate amino acids. Sulfur and Iron can be used as electron acceptors; but not sulfate, sulfite, thiosulfate or nitrate. Strain OGL-20P is resistant to chloramphenicol, kanamycin, and gentamycin. Growth of str. OGL20P is inhibited by tetracyclin but not by Na2MoO4. The G+C content of DNA is 57.2 mol%. The 16S ribosomal RNA sequence analysis allows one to classify strain OGL-20P as a representative of a now species of Thermococcus genus. The name Thermococcus sulfurophilus op. nov., was suggested for the new isolate, type strain OGL-20P (sup T) (= ATCC BAA_394 (sup T) = DSM...(supT)).

  18. 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 was isolated from black smoker chimney material from the Rainbow hydrothermal vent site on the Mid-Atlantic Ridge (36.2 N, 33.9 W). The cells of strain OGL-20P(sup T) have an irregular coccoid shape and are motile with a single flagellum. Growth was observed within the pH range 5.0-8.5 (optimum pH 7.0), NaCl concentration range 1-5 % (w/v) (optimum 3%), and temperature range 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, iron (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, chloramphenicol, kanamycin, and gentamycin, but sensitive to tetracycline and rifampicin. The G+C content of 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, and propose the name Thermococcus thioreducens sp. nov. The type strain is OGL-20P(sup T) (= ATCC BAA-394(sup T) = JCM 12859(sup T) = DSM 14981(sup T)).

  19. 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

  20. 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

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

    DEFF Research Database (Denmark)

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

    1999-01-01

    The hsp70 (dnaK) locus of the moderate thermophilic archaeon Methanosarcina thermophila TM-1 was cloned, sequenced, and tested in vitro to measure gene induction by heat and ammonia, i.e., stressors pertinent to the biotechnological ecosystem of this methanogen that plays a key role in anaerobic...... thermoautotrophicum Delta H, from another genus, in which trkA is not part of the locus. The proteins encoded in the TM-1 genes are very similar to the S-6 homologs, but considerably less similar to the Delta H proteins. The TM-1 Hsp70(DnaK) protein has the 23-amino acid deletion-by comparison with homologs from Gram...

  2. 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.

  3. Archaeoglobus infectus sp. nov., a novel thermophilic, chemolithoheterotrophic archaeon isolated from a deep-sea rock collected at Suiyo Seamount, Izu-Bonin Arc, western Pacific Ocean.

    Science.gov (United States)

    Mori, Koji; Maruyama, Akihiko; Urabe, Tetsuro; Suzuki, Ken-Ichiro; Hanada, Satoshi

    2008-04-01

    A novel thermophilic, strictly anaerobic archaeon, designated strain Arc51T, was isolated from a rock sample collected from a deep-sea hydrothermal field in Suiyo Seamount, Izu-Bonin Arc, western Pacific Ocean. Cells of the isolate were irregular cocci with single flagella and exhibited blue-green fluorescence at 436 nm. The optimum temperature, pH and NaCl concentration for growth were 70 degrees C, pH 6.5 and 3 % (w/v), respectively. Strain Arc51T could grow on thiosulfate or sulfite as an electron acceptor in the presence of hydrogen. This strain required acetate as a carbon source for its growth, suggesting that the reductive acetyl CoA pathway for CO2 fixation was incomplete. In addition, coenzyme M (2-mercaptoethanesulfonic acid), which is a known methyl carrier in methanogenesis, was also a requirement for growth of the strain. Analysis of the 16S rRNA gene sequence revealed that the isolate was similar to members of the genus Archaeoglobus, with sequence similarities of 93.6-97.2 %; the closest relative was Archaeoglobus veneficus. Phylogenetic analyses of the dsrAB and apsA genes, encoding the alpha and beta subunits of dissimilatory sulfite reductase and the alpha subunit of adenosine-5'-phosphosulfate reductase, respectively, produced results similar to those inferred from comparisons based on the 16S rRNA gene sequence. On the basis of phenotypic and phylogenetic data, strain Arc51T represents a novel species of the genus Archaeoglobus, for which the name Archaeoglobus infectus sp. nov. is proposed. The type strain is Arc51T (=NBRC 100649T=DSM 18877T).

  4. 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.

  5. Crystal structure of the NADP+ and tartrate-bound complex of L-serine 3-dehydrogenase from the hyperthermophilic archaeon Pyrobaculum calidifontis.

    Science.gov (United States)

    Yoneda, Kazunari; Sakuraba, Haruhiko; Araki, Tomohiro; Ohshima, Toshihisa

    2018-05-01

    A gene encoding L-serine dehydrogenase (L-SerDH) that exhibits extremely low sequence identity to the Agrobacterium tumefaciens L-SerDH was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The predicted amino acid sequence showed 36% identity with that of Pseudomonas aeruginosa L-SerDH, suggesting that P. calidifontis L-SerDH is a novel type of L-SerDH, like Ps. aeruginosa L-SerDH. The overexpressed enzyme appears to be the most thermostable L-SerDH described to date, and no loss of activity was observed by incubation for 30 min at temperatures up to 100 °C. The enzyme showed substantial reactivity towards D-serine, in addition to L-serine. Two different crystal structures of P. calidifontis L-SerDH were determined using the Se-MAD and MR method: the structure in complex with NADP + /sulfate ion at 1.18 Å and the structure in complex with NADP + /L-tartrate (substrate analog) at 1.57 Å. The fold of the catalytic domain showed similarity with that of Ps. aeruginosa L-SerDH. However, the active site structure significantly differed between the two enzymes. Based on the structure of the tartrate, L- and D-serine and 3-hydroxypropionate molecules were modeled into the active site and the substrate binding modes were estimated. A structural comparison suggests that the wide cavity at the substrate binding site is likely responsible for the high reactivity of the enzyme toward both L- and D-serine enantiomers. This is the first description of the structure of the novel type of L-SerDH with bound NADP + and substrate analog, and it provides new insight into the substrate binding mechanism of L-SerDH. The results obtained here may be very informative for the creation of L- or D-serine-specific SerDH by protein engineering.

  6. The RecJ2 protein in the thermophilic archaeon Thermoplasma acidophilum is a 3'-5' exonuclease that associates with a DNA replication complex.

    Science.gov (United States)

    Ogino, Hiromi; Ishino, Sonoko; Kohda, Daisuke; Ishino, Yoshizumi

    2017-05-12

    RecJ/cell division cycle 45 (Cdc45) proteins are widely conserved in the three domains of life, i.e. in bacteria, Eukarya, and Archaea. Bacterial RecJ is a 5'-3' exonuclease and functions in DNA repair pathways by using its 5'-3' exonuclease activity. Eukaryotic Cdc45 has no identified enzymatic activity but participates in the CMG complex, so named because it is composed of Cdc45, minichromosome maintenance protein complex (MCM) proteins 2-7, and GINS complex proteins (Sld5, Psf11-3). Eukaryotic Cdc45 and bacterial/archaeal RecJ share similar amino acid sequences and are considered functional counterparts. In Archaea, a RecJ homolog in Thermococcus kodakarensis was shown to associate with GINS and accelerate its nuclease activity and was, therefore, designated GAN ( G INS- a ssociated n uclease); however, to date, no archaeal RecJ·MCM·GINS complex has been isolated. The thermophilic archaeon Thermoplasma acidophilum has two RecJ-like proteins, designated TaRecJ1 and TaRecJ2. TaRecJ1 exhibited DNA-specific 5'-3' exonuclease activity, whereas TaRecJ2 had 3'-5' exonuclease activity and preferred RNA over DNA. TaRecJ2, but not TaRecJ1, formed a stable complex with TaGINS in a 2:1 molar ratio. Furthermore, the TaRecJ2·TaGINS complex stimulated activity of TaMCM ( T. acidophilum MCM) helicase in vitro , and the TaRecJ2·TaMCM·TaGINS complex was also observed in vivo However, TaRecJ2 did not interact with TaMCM directly and was not required for the helicase activation in vitro These findings suggest that the function of archaeal RecJ in DNA replication evolved divergently from Cdc45 despite conservation of the CMG-like complex formation between Archaea and Eukarya. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Saccharolobus caldissimus gen. nov., sp. nov., a facultatively anaerobic iron-reducing hyperthermophilic archaeon isolated from an acidic terrestrial hot spring, and reclassification of Sulfolobus solfataricus as Saccharolobus solfataricus comb. nov. and Sulfolobus shibatae as Saccharolobus shibatae comb. nov.

    Science.gov (United States)

    Sakai, Hiroyuki D; Kurosawa, Norio

    2018-04-01

    A novel hyperthermophilic archaeon of strain HS-3 T , belonging to the family Sulfolobaceae, was isolated from an acidic terrestrial hot spring in Hakone Ohwaku-dani, Japan. Based on 16S rRNA gene sequence analysis, the closest phylogenetic relatives of strain HS-3 T were, first, Sulfolobus solfataricus (96.4 %) and, second, Sulfolobus shibatae (96.2 %), indicating that the strain belongs to the genus Sulfolobus. However, the sequence similarity to the type species of the genus Sulfolobus (Sulfolobus acidocaldarius) was remarkably low (91.8 %). In order to determine whether strain HS-3 T belongs to the genus Sulfolobus, its morphological, biochemical and physiological characteristics were examined in parallel with those of S. solfataricus and S. shibatae. Although there were some differences in chemolithotrophic growth between strain HS-3 T , S. solfataricus and S. shibatae, their temperature, pH and facultatively anaerobic characteristics of growth, and their utilization of various sugars were almost identical. In contrast, the utilization of various sugars by S. acidocaldarius was quite different from that of HS-3 T , S. solfataricus and S. shibatae. Phylogenetic evidence based on the 16S and the 23S rRNA gene sequences also clearly distinguished the monophyletic clade composed of strain HS-3 T , S. solfataricus, and S. shibatae from S. acidocaldarius. Based on these results, we propose a new genus and species, Saccharolobus caldissimus gen. nov., sp. nov., for strain HS-3 T , as well as two reclassifications, Saccharolobus solfataricus comb. nov. and Saccharolobus shibatae comb. nov. The type strain of Saccharolobus caldissimus is HS-3 T (=JCM 32116 T and InaCC Ar80 T ). The type species of the genus is Saccharolobus solfataricus.

  8. Crystal structure of the regulatory subunit of archaeal initiation factor 2B (aIF2B) from hyperthermophilic archaeon Pyrococcus horikoshii OT3: a proposed structure of the regulatory subcomplex of eukaryotic IF2B

    International Nuclear Information System (INIS)

    Kakuta, Yoshimitsu; Tahara, Maino; Maetani, Shigehiro; Yao, Min; Tanaka, Isao; Kimura, Makoto

    2004-01-01

    Eukaryotic translation initiation factor 2B (eIF2B) is the guanine-nucleotide exchange factor for eukaryotic initiation factor 2 (eIF2). eIF2B is a heteropentameric protein composed of α-ε subunits. The α, β, and δ subunits form a regulatory subcomplex, while the γ and ε form a catalytic subcomplex. Archaea possess homologues of α, β, and δ subunits of eIF2B. Here, we report the three-dimensional structure of an archaeal regulatory subunit (aIF2Bα) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 determined by X-ray crystallography at 2.2 A resolution. aIF2Bα consists of two subdomains, an N-domain (residues 1-95) and a C-domain (residues 96-276), connected by a long α-helix (α5: 78-106). The N-domain contains a five helix bundle structure, while the C-domain folds into the α/β structure, thus showing similarity to D-ribose-5-phosphate isomerase structure. The presence of two molecules in the crystallographic asymmetric unit and the gel filtration analysis suggest a dimeric structure of aIF2Bα in solution, interacting with each other by C-domains. Furthermore, the crystallographic 3-fold symmetry generates a homohexameric structure of aIF2Bα; the interaction is primarily mediated by the long α-helix at the N-domains. This structure suggests an architecture of the three subunits, α, β, and δ, in the regulatory subcomplex within eIF2B

  9. Respiration of arsenate and selenate by hyperthermophilic archaea.

    Science.gov (United States)

    Huber, R; Sacher, M; Vollmann, A; Huber, H; Rose, D

    2000-10-01

    A novel, strictly anaerobic, hyperthermophilic, facultative organotrophic archaeon was isolated from a hot spring at Pisciarelli Solfatara, Naples, Italy. The rod-shaped cells grew chemolithoautotrophically with carbon dioxide as carbon source, hydrogen as electron donor and arsenate, thiosulfate or elemental sulfur as electron acceptor. H2S was formed from sulfur or thiosulfate, arsenite from arsenate. Organotrophically, the new isolate grew optimally in the presence of an inorganic electron acceptor like sulfur, selenate or arsenate. Cultures, grown on arsenate and thiosulfate or arsenate and L-cysteine, precipitated realgar (As2S2). During growth on selenate, elemental selenium was produced. The G+C content of the DNA was 58.3 mol%. Due to 16S rRNA gene sequence analysis combined with physiological and morphological criteria, the new isolate belongs to the Thermoproteales order. It represents a new species within the genus Pyrobaculum, the type species of which we name Pyrobaculum arsenaticum (type strain PZ6*, DSM 13514, ATCC 700994). Comparative studies with different Pyrobaculum-species showed, that Pyrobaculum aerophilum was also able to grow organotrophically under anaerobic culture conditions in the presence of arsenate, selenate and selenite. During growth on selenite, elemental selenium was formed as final product. In contrast to P. arsenaticum, P. aerophilum could use selenate or arsenate for lithoautotrophic growth with carbon dioxide and hydrogen.

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

    OpenAIRE

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

    1997-01-01

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

  11. 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.

  12. Components of calcium homeostasis in Archaeon Methanobacterium thermoautotrophicum

    International Nuclear Information System (INIS)

    Varecka, L.; Smigan, P.; Vancek, M.; Greksak, M.

    1998-01-01

    The cells of Archaea are interesting from several points of view. Among others there are: (a) the evolutionary relationship to procaryotes and eucaryotes and (b) the involvement of Na + and H + gradient in archaeal bio-energetics. The observations are presented which are devoted to the description of components of Ca 2+ homeostasis, an apparatus is vital for both procaryotic and eukaryotic organisms, in obligate anaerobe Methanobacterium thermoautotrophicum. This is, after the demonstration of the ATP-dependent Ca 2+ transport in Halobacterium halobium membrane vesicles, the first complex description of processes of Ca 2+ homeostasis in Archaea. The Ca 2+ influx and efflux was measured using radionuclide 4 5 Ca 2+ . The experiment were performed under strictly anaerobic conditions. The measurement of the membrane potential by means of 3 H-tetraphenyl phosphonium chloride showed that the presence of Na + depolarized the membrane from -110 to -60 mV. The growth of M. thermoautotrophicum and methanogenesis was suppressed but nor arrested by the presence EGTA suggesting that the Ca 2+ homeostasis may be involved in controlling these cellular functions. The results indicate the presence of three components involved in establishing the Ca 2+ homeostasis in cell of M. thermoautotrophicum. The first is the Ca 2+ -carrier mediating the CA 2+ influx driven by the proton motive force or the membrane potential. The Ca 2+ efflux is mediated by two transport systems, Na + /Ca 2+ and H + /Ca 2+ anti-porters. The evidence for the presence of the Ca 2+ -transporting ATPase was not obtained so far. (authors)

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

    NARCIS (Netherlands)

    Zaparty, M.; Esser, D.; Gertig, S.; Haferkamp, P.; Kouril, T.; Sierocinski, P.; Pham, T.K.; Manica, A.; Reimann, J.; Schreiber, K.; Teichmann, D.; Wolferen, van M.E.; Jan, von M.; Wieloch, P.; Albers, S.V.; Driessen, A.J.M.; Klenk, H.P.; Schleper, C.; Schomburg, D.; Oost, van der J.; Wright, P.C.; Siebers, B.

    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

  14. "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

    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

  15. 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.

  16. ORF Alignment: NC_003364 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available initiation factor IIB (TFIIB) [Pyrobaculum aerophilum ... str. IM2] ... Length = 52 ... Query: 2 ... TRKLIFELEEYACPACGAVNDVI...VDYERGQVICKSCGVVLKDGIADLGPEWR 53 ... TRKLIFELEEYACPACGAVNDVI...VDYERGQVICKSCGVVLKDGIADLGPEWR Sbjct: 1 ... TRKLIFELEEYACPACGAVNDVIVDYERGQVICKSCGVVLKDGIADLGPEWR 52

  17. ORF Alignment: NC_003364 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available ... aerophilum str. IM2] ... Length = 291 ... Query: 25 ... AYLGFEPLWPIHIGWLIWAYKLAELKEAGFDVIVLVATWHAWIND...KGSIEELRAHGERVR 84 ... AYLGFEPLWPIHIGWLIWAYKLAELKEAGFDVIVLVATWHAWINDKGS...IEELRAHGERVR Sbjct: 1 ... AYLGFEPLWPIHIGWLIWAYKLAELKEAGFDVIVLVATWHAWINDKGSIEELRAHGERVR 60 ... Query: 145 MYPLMQVA

  18. Complete Genome Sequence of the Halophilic Methylotrophic Methanogen Archaeon Methanohalophilus portucalensis Strain FDF-1T

    KAUST Repository

    L’Haridon, Stéphane

    2018-01-17

    We report here the complete genome sequence (2.08 Mb) of Methanohalophilus portucalensis strain FDF-1T, a halophilic methylotrophic methanogen isolated from the sediment of a saltern in Figeria da Foz, Portugal. The average nucleotide identity and DNA-DNA hybridization analyses show that Methanohalophilus mahii, M. halophilus, and M. portucalensis are three different species within the Methanosarcinaceae family.

  19. Divergent Roles of RPA Homologs of the Model Archaeon Halobacterium salinarum in Survival of DNA Damage.

    Science.gov (United States)

    Evans, Jessica J; Gygli, Patrick E; McCaskill, Julienne; DeVeaux, Linda C

    2018-04-20

    The haloarchaea are unusual in possessing genes for multiple homologs to the ubiquitous single-stranded DNA binding protein (SSB or replication protein A, RPA) found in all three domains of life. Halobacterium salinarum contains five homologs: two are eukaryotic in organization, two are prokaryotic and are encoded on the minichromosomes, and one is uniquely euryarchaeal. Radiation-resistant mutants previously isolated show upregulation of one of the eukaryotic-type RPA genes. Here, we have created deletions in the five RPA operons. These deletion mutants were exposed to DNA-damaging conditions: ionizing radiation, UV radiation, and mitomycin C. Deletion of the euryarchaeal homolog, although not lethal as in Haloferax volcanii , causes severe sensitivity to all of these agents. Deletion of the other RPA/SSB homologs imparts a variable sensitivity to these DNA-damaging agents, suggesting that the different RPA homologs have specialized roles depending on the type of genomic insult encountered.

  20. 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.

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

    Science.gov (United States)

    Sublimi Saponetti, Matilde; Bobba, Fabrizio; Salerno, Grazia; Scarfato, Alessandro; Corcelli, Angela; Cucolo, Annamaria

    2011-04-29

    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.

  2. Morphological and Structural Aspects of the Extremely Halophilic Archaeon Haloquadratum walsbyi

    OpenAIRE

    Sublimi Saponetti, Matilde; Bobba, Fabrizio; Salerno, Grazia; Scarfato, Alessandro; Corcelli, Angela; Cucolo, Annamaria

    2011-01-01

    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 asymmet...

  3. Natronorubrum sediminis sp. nov., an archaeon isolated from a saline lake.

    Science.gov (United States)

    Gutiérrez, M C; Castillo, A M; Corral, P; Minegishi, H; Ventosa, A

    2010-08-01

    Two novel haloalkaliphilic archaea, strains CG-6T and CG-4, were isolated from sediment of the hypersaline Lake Chagannor in Inner Mongolia, China. Cells of the two strains were pleomorphic, non-motile and strictly aerobic. They required at least 2.5 M NaCl for growth, with optimum growth at 3.4 M NaCl. They grew at pH 8.0-11.0, with optimum growth at pH 9.0. Hypotonic treatment with less than 1.5 M NaCl caused cell lysis. The two strains had similar polar lipid compositions, possessing C20C20 and C20C25 derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. No glycolipids were detected. Comparison of 16S rRNA gene sequences and morphological features placed them in the genus Natronorubrum. 16S rRNA gene sequence similarities to strains of recognized species of the genus Natronorubrum were 96.2-93.8%. Detailed phenotypic characterization and DNA-DNA hybridization studies revealed that the two strains belong to a novel species in the genus Natronorubrum, for which the name Natronorubrum sediminis sp. nov. is proposed; the type strain is CG-6T (=CECT 7487T =CGMCC 1.8981T =JCM 15982T).

  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. Replication termination and chromosome dimer resolution in the archaeon Sulfolobus solfataricus.

    Science.gov (United States)

    Duggin, Iain G; Dubarry, Nelly; Bell, Stephen D

    2011-01-05

    Archaea of the genus Sulfolobus have a single-circular chromosome with three replication origins. All three origins fire in every cell in every cell cycle. Thus, three pairs of replication forks converge and terminate in each replication cycle. Here, we report 2D gel analyses of the replication fork fusion zones located between origins. These indicate that replication termination involves stochastic fork collision. In bacteria, replication termination is linked to chromosome dimer resolution, a process that requires the XerC and D recombinases, FtsK and the chromosomal dif site. Sulfolobus encodes a single-Xer homologue and its deletion gave rise to cells with aberrant DNA contents and increased volumes. Identification of the chromosomal dif site that binds Xer in vivo, and biochemical characterization of Xer/dif recombination revealed that, in contrast to bacteria, dif is located outside the fork fusion zones. Therefore, it appears that replication termination and dimer resolution are temporally and spatially distinct processes in Sulfolobus.

  6. Haloplanus salinarum sp. nov., an extremely halophilic archaeon isolated from a solar saltern.

    Science.gov (United States)

    Hwang, Han-Bit; Kim, Ye-Eun; Koh, Hyeon-Woo; Song, Hye Seon; Roh, Seong Woon; Kim, So-Jeong; Nam, Seung Won; Park, Soo-Je

    2017-11-01

    An extremely halophilic archaeal strain SP28 T was isolated from the Gomso solar saltern, Republic of Korea. Cells of the new strain SP28 T were pleomorphic and Gram stain negative, and produced red-pigmented colonies. These grew in medium with 2.5-4.5 M NaCl (optimum 3.1 M) and 0.05-0.5 M MgCl2 (optimum 0.1 M), at 25-50 °C (optimum 37 °C) and at a pH of 6.5-8.5 (optimum pH 8.0). Mg 2+ was required for growth. A concentration of at least 2 M NaCl was required to prevent cell lysis. Polar lipids included phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and one glycolipid chromatographically identical to sulfated mannosyl glucosyl diether. 16S rRNA and rpoB' gene sequence analyses showed that strain SP28 T is closely related to Haloplanus ruber R35 T (97.3 and 94.1 %, 16S rRNA and rpoB' gene sequence similarity, respectively), Haloplanus litoreus GX21 T (97.0 and 92.1 %), Haloplanus salinus YGH66 T (96.0 and 91.9 %), Haloplanus vescus RO5-8 T (95.9 and 90.9 %), Haloplanus aerogenes TBN37 T (95.6 and 90.3 %) and Haloplanus natans RE-101 T (95.3 and 89.8 %). The DNA G+C content of the novel strain SP28 T was 66.2 mol%, which is slightly higher than that of Hpn.litoreus GX21 T (65.8 mol%) and Hpn.ruber R35 T (66.0 mol%). DNA-DNA hybridization values betweenHpn.ruber R35 T and strain SP28 T and between Hpn.litoreus GX21 T and strain SP28 T were about 24.8 and 20.7 %, respectively. We conclude that strain SP28 T represents a novel species of the genus Haloplanus and propose the name Haloplanus salinarum sp. nov. The type strain is SP28 T (=JCM 31424 T =KCCM 43210 T ).

  7. 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.

    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

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

    Czech Academy of Sciences Publication Activity Database

    Čejchan, Petr

    2004-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Amber L Hartman

    2010-03-01

    Full Text Available 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.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.The completed genome sequence, presented here, provides an invaluable tool for further in vivo and in vitro studies of Hfx. volcanii.

  10. Candidatus Nitrosocaldus cavascurensis, an Ammonia Oxidizing, Extremely Thermophilic Archaeon with a Highly Mobile Genome

    Directory of Open Access Journals (Sweden)

    Sophie S. Abby

    2018-01-01

    Full Text Available Ammonia oxidizing archaea (AOA of the phylum Thaumarchaeota are widespread in moderate environments but their occurrence and activity has also been demonstrated in hot springs. Here we present the first enrichment of a thermophilic representative with a sequenced genome, which facilitates the search for adaptive strategies and for traits that shape the evolution of Thaumarchaeota. Candidatus Nitrosocaldus cavascurensis has been enriched from a hot spring in Ischia, Italy. It grows optimally at 68°C under chemolithoautotrophic conditions on ammonia or urea converting ammonia stoichiometrically into nitrite with a generation time of approximately 23 h. Phylogenetic analyses based on ribosomal proteins place the organism as a sister group to all known mesophilic AOA. The 1.58 Mb genome of Ca. N. cavascurensis harbors an amoAXCB gene cluster encoding ammonia monooxygenase and genes for a 3-hydroxypropionate/4-hydroxybutyrate pathway for autotrophic carbon fixation, but also genes that indicate potential alternative energy metabolisms. Although a bona fide gene for nitrite reductase is missing, the organism is sensitive to NO-scavenging, underlining the potential importance of this compound for AOA metabolism. Ca. N. cavascurensis is distinct from all other AOA in its gene repertoire for replication, cell division and repair. Its genome has an impressive array of mobile genetic elements and other recently acquired gene sets, including conjugative systems, a provirus, transposons and cell appendages. Some of these elements indicate recent exchange with the environment, whereas others seem to have been domesticated and might convey crucial metabolic traits.

  11. 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 (<50 μM) compared to growth at 1 mM. Although the ammonia oxidation pathway has not been fully elucidated, we have shown that nitric oxide (NO) appears to be a key intermediate: exponentially growing HL72 produces significant NO and the removal of NO using a scavenger reversibly inhibits growth. In addition to AMO, the HL72 genome also contains sequences for a urease encoded by subunits ureABC and an active 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.

  12. Candidatus Nitrosocaldus cavascurensis, an Ammonia Oxidizing, Extremely Thermophilic Archaeon with a Highly Mobile Genome

    OpenAIRE

    Abby, Sophie S.; Melcher, Michael; Kerou, Melina; Krupovic, Mart; Stieglmeier, Michaela; Rossel, Claudia; Pfeifer, Kevin; Schleper, Christa

    2018-01-01

    Ammonia oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread in moderate environments but their occurrence and activity has also been demonstrated in hot springs. Here we present the first enrichment of a thermophilic representative with a sequenced genome, which facilitates the search for adaptive strategies and for traits that shape the evolution of Thaumarchaeota. Candidatus Nitrosocaldus cavascurensis has been enriched from a hot spring in Ischia, Italy. It grows optimally ...

  13. Candidatus Nitrosocaldus cavascurensis, an Ammonia Oxidizing, Extremely Thermophilic Archaeon with a Highly Mobile Genome.

    Science.gov (United States)

    Abby, Sophie S; Melcher, Michael; Kerou, Melina; Krupovic, Mart; Stieglmeier, Michaela; Rossel, Claudia; Pfeifer, Kevin; Schleper, Christa

    2018-01-01

    Ammonia oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread in moderate environments but their occurrence and activity has also been demonstrated in hot springs. Here we present the first enrichment of a thermophilic representative with a sequenced genome, which facilitates the search for adaptive strategies and for traits that shape the evolution of Thaumarchaeota. Candidatus Nitrosocaldus cavascurensis has been enriched from a hot spring in Ischia, Italy. It grows optimally at 68°C under chemolithoautotrophic conditions on ammonia or urea converting ammonia stoichiometrically into nitrite with a generation time of approximately 23 h. Phylogenetic analyses based on ribosomal proteins place the organism as a sister group to all known mesophilic AOA. The 1.58 Mb genome of Ca. N. cavascurensis harbors an amo AXCB gene cluster encoding ammonia monooxygenase and genes for a 3-hydroxypropionate/4-hydroxybutyrate pathway for autotrophic carbon fixation, but also genes that indicate potential alternative energy metabolisms. Although a bona fide gene for nitrite reductase is missing, the organism is sensitive to NO-scavenging, underlining the potential importance of this compound for AOA metabolism. Ca. N. cavascurensis is distinct from all other AOA in its gene repertoire for replication, cell division and repair. Its genome has an impressive array of mobile genetic elements and other recently acquired gene sets, including conjugative systems, a provirus, transposons and cell appendages. Some of these elements indicate recent exchange with the environment, whereas others seem to have been domesticated and might convey crucial metabolic traits.

  14. The common ancestor of archaea and eukarya was not an archaeon.

    Science.gov (United States)

    Forterre, Patrick

    2013-01-01

    It is often assumed that eukarya originated from archaea. This view has been recently supported by phylogenetic analyses in which eukarya are nested within archaea. Here, I argue that these analyses are not reliable, and I critically discuss archaeal ancestor scenarios, as well as fusion scenarios for the origin of eukaryotes. Based on recognized evolutionary trends toward reduction in archaea and toward complexity in eukarya, I suggest that their last common ancestor was more complex than modern archaea but simpler than modern eukaryotes (the bug in-between scenario). I propose that the ancestors of archaea (and bacteria) escaped protoeukaryotic predators by invading high temperature biotopes, triggering their reductive evolution toward the "prokaryotic" phenotype (the thermoreduction hypothesis). Intriguingly, whereas archaea and eukarya share many basic features at the molecular level, the archaeal mobilome resembles more the bacterial than the eukaryotic one. I suggest that selection of different parts of the ancestral virosphere at the onset of the three domains played a critical role in shaping their respective biology. Eukarya probably evolved toward complexity with the help of retroviruses and large DNA viruses, whereas similar selection pressure (thermoreduction) could explain why the archaeal and bacterial mobilomes somehow resemble each other.

  15. The Common Ancestor of Archaea and Eukarya Was Not an Archaeon

    Directory of Open Access Journals (Sweden)

    Patrick Forterre

    2013-01-01

    Full Text Available It is often assumed that eukarya originated from archaea. This view has been recently supported by phylogenetic analyses in which eukarya are nested within archaea. Here, I argue that these analyses are not reliable, and I critically discuss archaeal ancestor scenarios, as well as fusion scenarios for the origin of eukaryotes. Based on recognized evolutionary trends toward reduction in archaea and toward complexity in eukarya, I suggest that their last common ancestor was more complex than modern archaea but simpler than modern eukaryotes (the bug in-between scenario. I propose that the ancestors of archaea (and bacteria escaped protoeukaryotic predators by invading high temperature biotopes, triggering their reductive evolution toward the “prokaryotic” phenotype (the thermoreduction hypothesis. Intriguingly, whereas archaea and eukarya share many basic features at the molecular level, the archaeal mobilome resembles more the bacterial than the eukaryotic one. I suggest that selection of different parts of the ancestral virosphere at the onset of the three domains played a critical role in shaping their respective biology. Eukarya probably evolved toward complexity with the help of retroviruses and large DNA viruses, whereas similar selection pressure (thermoreduction could explain why the archaeal and bacterial mobilomes somehow resemble each other.

  16. Interactions between nitrogen fixation and osmoregulation in the methanogenic archaeon Methanosarcina barkeri 227

    Energy Technology Data Exchange (ETDEWEB)

    Brabban, A.D.; Orcutt, E.N.; Zinder, S.H. [Cornell Univ., Ithaca, NY (United States). Section of Microbiology

    1999-03-01

    The nitrogenase enzyme complex of Methanosarcina barkeri 227 was found to be more sensitive to NaCl than previously studied molybdenum nitrogenases are, with total inhibition of activity occurring at 190 mM NaCl, compared with >600 mM NaCl for Azotobacter vinelandii and Clostridium pasteurianum nitrogenases. Na{sup +} and K{sup +} had equivalent effects, whereas Mg{sup 2+} was more inhibitory than either monovalent cation, even on a per-charge basis. The anion Cl{sup {minus}} was more inhibitory than acetate was. Because M. barkeri 227 is a facultative halophile, the authors examined the effects of external salt on growth and diazotrophy and found that inhibition of growth was not greater with N{sub 2} than with NH{sub 4}{sup +}. Cells grown with N{sub 2} and cells grown with NH{sub 4}{sup +} produced equal concentrations of {alpha}-glutamate at low salt concentrations and equal concentrations of N{sup {var_epsilon}}-acetyl-{beta}-lysine at NaCl concentrations greater than 500 mM. Despite the high energetic cost of fixing nitrogen for these osmolytes, the authors obtained no evidence that there is a shift towards nonnitrogenous osmolytes during diazotrophic growth. In vitro nitrogenase enzyme assays showed that at a low concentration potassium glutamate enhanced activity but at higher concentrations this compound inhibited activity; 50% inhibition occurred at a potassium glutamate concentration of approximately 400 mM.

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

    DEFF Research Database (Denmark)

    Studdert, C A; Herrera Seitz, M K; Plasencia, I

    2001-01-01

    A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5.5-12), is rat......A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5.......5-12), is rather thermophilic (optimal activity at 60 degrees C in 1-2 M NaCl) and is dependent on high salt concentrations for activity and stability (1-2 M NaCl or KCl). Polyclonal antibodies were raised against the purified protease. In Western blots, they presented no cross-reactivity with culture medium from...... other halobacteria nor with commercial proteases except subtilisin. The amino acid sequences of three tryptic peptides obtained from Natronococcus occultus protease did not show significant similarity to other known proteolytic enzymes. This fact, in addition to its high molecular mass suggests...

  18. 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

  19. 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

    -dependent methyltransferase 144, GTP cyclohydrolase 398, DNA topoisomerase VI subunit A 209, DNA topoisomerase VI subunit B 192, Type A Flavoprotein 911, NAD(P)H:rubredoxin oxidoreductase (Fatty acid metabolism) 120, NAD(P)H:rubredoxin oxidoreductase 120, cofactor-independent phosphoglycerate mutase 909, bis(5'-adenosyl...... subunit 2 255, glycerol kinase 257, phosphomannomutase-related protein 321, ribose-5-phosphate isomerase A 107, phosphate transport regulator 193, isopentenyl pyrophosphate isomerase (mevanolate Pathway) 500, amino acid kinase 203, NADH:polysulfide oxidoreductase 203, 5'-methylthioadenosine phosphorylase......, cysteine desulfurase 521, hydrogenase maturation protein HypF 235, iron-molybdenum cofactor-binding protein 192, ATPase 260, 4Fe-4S cluster-binding protein 254, phosphopyruvate hydratase 650, fructose-1,6-bisphosphatase 140, aspartate carbamoyltransferase catalytic subunit 158, Bipolar DNA helicase 448...

  20. Complete Genome Sequence of the Halophilic Methylotrophic Methanogen Archaeon Methanohalophilus portucalensis Strain FDF-1T

    KAUST Repository

    L’ Haridon, Sté phane; Corre, Erwan; Guan, Yue; Vinu, Manikandan; La Cono, Violetta; Yakimov, Michail; Stingl, Ulrich; Toffin, Laurent; Jebbar, Mohamed

    2018-01-01

    We report here the complete genome sequence (2.08 Mb) of Methanohalophilus portucalensis strain FDF-1T, a halophilic methylotrophic methanogen isolated from the sediment of a saltern in Figeria da Foz, Portugal. The average nucleotide identity and DNA-DNA hybridization analyses show that Methanohalophilus mahii, M. halophilus, and M. portucalensis are three different species within the Methanosarcinaceae family.

  1. 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.

  2. 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

    The molecular structures, biosynthetic pathways and physiological functions of membrane lipids produced by organisms in the domain Archaea are poorly characterized as compared with that of counterparts in Bacteria and Eukaryota. Here we report on the use of high-resolution shotgun lipidomics......-resolution Fourier transform mass spectrometry using an ion trap-orbitrap mass spectrometer. This analysis identified five clusters of molecular ions that matched ether lipids in the database with sub-ppm mass accuracy. To structurally characterize and validate the identities of the potential lipid species, we...... performed structural analysis using multistage activation on the ion trap-orbitrap instrument as well as tandem mass analysis using a quadrupole time-of-flight machine. Our analysis identified four ether lipid species previously reported in Archaea, and one ether lipid species that had not been described...

  3. A novel carbohydrate-binding surface layer protein from the hyperthermophilic archaeon Pyrococcus horikoshii.

    Science.gov (United States)

    Goda, Shuichiro; Koga, Tomoyuki; Yamashita, Kenichiro; Kuriura, Ryo; Ueda, Toshifumi

    2018-04-08

    In Archaea and Bacteria, surface layer (S-layer) proteins form the cell envelope and are involved in cell protection. In the present study, a putative S-layer protein was purified from the crude extract of Pyrococcus horikoshii using affinity chromatography. The S-layer gene was cloned and expressed in Escherichia coli. Isothermal titration calorimetry analyses showed that the S-layer protein bound N-acetylglucosamine and induced agglutination of the gram-positive bacterium Micrococcus lysodeikticus. The protein comprised a 21-mer structure, with a molecular mass of 1,340 kDa, as determined using small-angle X-ray scattering. This protein showed high thermal stability, with a midpoint of thermal denaturation of 79 °C in dynamic light scattering experiments. This is the first description of the carbohydrate-binding archaeal S-layer protein and its characteristics.

  4. ORF Alignment: NC_003364 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available domains ... [Pyrobaculum aerophilum str. IM2] ... Length = 128 ... Query: 2 ... KCGEIASRPVITVTPDTPLEKAVELMADHD...VGILVLVDKENPKRVVGVLSERDIVRSLAG 61 ... KCGEIASRPVITVTPDTPLEKAVELMADHD...VGILVLVDKENPKRVVGVLSERDIVRSLAG Sbjct: 1 ... KCGEIASRPVITVTPDTPLEKAVELMADHDVGILVLVDKENPKRVVGVLSERDIVRSLAG 60 ... Query: 122 KRIGGLRK 129 ... KRIGGLRK Sbjct: 121 KRIGGLRK 128

  5. Crystallization and preliminary X-ray characterization of a ferritin from the hyperthermophilic archaeon and anaerobe Pyrococcus furiosus

    International Nuclear Information System (INIS)

    Matias, Pedro M.; Tatur, Jana; Carrondo, Maria Arménia; Hagen, Wilfred R.

    2005-01-01

    Ferritin from P. furiosus crystallizes in space group C222 1 , with unit-cell parameters a = 258.1, b = 340.1, c = 266.5 Å and 36 monomers in the asymmetric unit, corresponding to one and a half 24-mers. Crystals of the title protein have been produced and preliminary structural analysis has been carried out. The crystals belong to the orthorhombic space group C222 1 , with unit-cell parameters a = 258.1, b = 340.1, c = 266.5 Å. The protein forms a 24-mer of 20 kDa subunits, which assemble with 432 non-crystallographic symmetry. A total of 36 monomers are found in the asymmetric unit, corresponding to one and a half 24-mers

  6. The chaperonin of the archaeon Sulfolobus solfataricus is an RNA-binding protein that participates in ribosomal RNA processing.

    OpenAIRE

    Ruggero, D; Ciammaruconi, A; Londei, P

    1998-01-01

    The 60 kDa molecular chaperones (chaperonins) are high molecular weight protein complexes having a characteristic double-ring toroidal shape; they are thought to aid the folding of denatured or newly synthesized polypeptides. These proteins exist as two functionally similar, but distantly related families, one comprising the bacterial and organellar chaperonins and another (the so-called CCT-TRiC family) including the chaperonins of the archaea and the eukaryotes. Although some evidence exist...

  7. 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.

  8. 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.

  9. 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.

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

    KAUST Repository

    Thennarasu, Sugumar; Poli Reddy, Dinesh Reddy; Antony, Aju; Yada, Madhava; Alqarawi, Sami; Neelamegam, Sivakumar

    2013-01-01

    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.

  11. 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.

  12. 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

    Despite major progresses in genetic studies of hyperthermophilic archaea, recombinant protein production in these organisms always suffers from low yields and a robust expression system is still in great demand. Here we report a versatile vector that confers high levels of protein expression...... to remove the peptide tags from expressed recombinant proteins. While pEXA employed an araS promoter for protein expression, pSeSD utilized P(araS-SD), an araS derivative promoter carrying an engineered ribosome-binding site (RBS; a Shine-Dalgarno [SD] sequence). We found that P(araS-SD) directed high...... 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...

  13. Insights into high-temperature nitrogen cycling from studies of the thermophilic ammonia-oxidizing archaeon Nitrosocaldus yellowstonii. (Invited)

    Science.gov (United States)

    de la Torre, J. R.

    2010-12-01

    Our understanding of the nitrogen cycle has advanced significantly in recent years with the discovery of new metabolic processes and the recognition that key processes such as aerobic ammonia oxidation are more broadly distributed among extant organisms and habitat ranges. Nitrification, the oxidation of ammonia to nitrite and nitrate, is a key component of the nitrogen cycle and, until recently, was thought to be mediated exclusively by the ammonia-oxidizing bacteria (AOB). The discovery that mesophilic marine archaea, some of the most abundant microorganisms on the planet, are capable of oxidizing ammonia to nitrite fundamentally changed our perception of the global nitrogen cycle. Ammonia-oxidizing archaea (AOA) are now thought to be significant drivers of nitrification in many marine and terrestrial environments. Most studies, however, have focused on the contribution of AOA to nitrogen cycling in mesophilic environments. Our recent discovery of a thermophilic AOA, Nitrosocaldus yellowstonii, has expanded the role and habitat range of AOA to include high temperature environments. Numerous studies have shown that AOA are widely distributed in geothermal habitats with a wide range of temperature and pH. The availability of multiple AOA genome sequences, combined with metagenomic studies from mesophilic and thermophilic environments gives us a better understanding of the physiology, ecology and evolution of these organisms. Recent studies have proposed that the AOA represent the most deeply branching lineage within the Archaea, the Thaumarchaeota. Furthermore, genomic comparisons between AOA and AOB reveal significant differences in the proposed pathways for ammonia oxidation. These genetic differences likely explain fundamental physiological differences such as the resistance of N. yellowstonii and other AOA to the classical nitrification inhibitors allylthiourea and acetylene. Physiological studies suggest that the marine AOA are adapted to oligotrophic environments. Our studies, however, point to a greater metabolic versatility in N. yellowstonii, including the ability to utilize alternative sources of energy. Understanding the biology of N. yellowstonii, the most deeply branching cultivated AOA to date, gives us a better understanding of the ecological and evolutionary significance of these organisms and sheds new light on nitrogen cycling at high temperature.

  14. Cultivation and characterization of Candidatus Nitrosocosmicus exaquare, an ammonia-oxidizing archaeon from a municipal wastewater treatment system.

    Science.gov (United States)

    Sauder, Laura A; Albertsen, Mads; Engel, Katja; Schwarz, Jasmin; Nielsen, Per H; Wagner, Michael; Neufeld, Josh D

    2017-05-01

    Thaumarchaeota have been detected in several industrial and municipal wastewater treatment plants (WWTPs), despite the fact that ammonia-oxidizing archaea (AOA) are thought to be adapted to low ammonia environments. However, the activity, physiology and metabolism of WWTP-associated AOA remain poorly understood. We report the cultivation and complete genome sequence of Candidatus Nitrosocosmicus exaquare, a novel AOA representative from a municipal WWTP in Guelph, Ontario (Canada). In enrichment culture, Ca. N. exaquare oxidizes ammonia to nitrite stoichiometrically, is mesophilic, and tolerates at least 15 mm of ammonium chloride or sodium nitrite. Microautoradiography (MAR) for enrichment cultures demonstrates that Ca. N. exaquare assimilates bicarbonate in association with ammonia oxidation. However, despite using inorganic carbon, the ammonia-oxidizing activity of Ca. N. exaquare is greatly stimulated in enrichment culture by the addition of organic compounds, especially malate and succinate. Ca. N. exaquare cells are coccoid with a diameter of ~1-2 μm. Phylogenetically, Ca. N. exaquare belongs to the Nitrososphaera sister cluster within the Group I.1b Thaumarchaeota, a lineage which includes most other reported AOA sequences from municipal and industrial WWTPs. The 2.99 Mbp genome of Ca. N. exaquare encodes pathways for ammonia oxidation, bicarbonate fixation, and urea transport and breakdown. In addition, this genome encodes several key genes for dealing with oxidative stress, including peroxidase and catalase. Incubations of WWTP biofilm demonstrate partial inhibition of ammonia-oxidizing activity by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), suggesting that Ca. N. exaquare-like AOA may contribute to nitrification in situ. However, CARD-FISH-MAR showed no incorporation of bicarbonate by detected Thaumarchaeaota, suggesting that detected AOA may incorporate non-bicarbonate carbon sources or rely on an alternative and yet unknown metabolism.

  15. 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....... volcanii also encodes an NAD+-dependent DNA ligase family member, LigN, the first such enzyme to be identified in the archaea, and present phylogenetic analysis indicating that the gene encoding this protein has been acquired by lateral gene transfer (LGT) from eubacteria. As with LigA, we show that Lig...

  16. Haladaptatus pallidirubidus sp. nov., a halophilic archaeon isolated from saline soil samples in Yunnan and Xinjiang, China.

    Science.gov (United States)

    Liu, Bing-Bing; Zhao, Wan-Yu; Chu, Xiao; Hozzein, Wael N; Prabhu, Deene Manik; Wadaan, Mohammed A M; Tang, Shu-Kun; Zhang, Li-Li; Li, Wen-Jun

    2014-11-01

    Two extremely halophilic archaea, designated YIM 90917 and YIM 93656(T), were isolated from saline soils in Yunnan province and Lup nur region in Xinjiang province, western China, respectively. Colonies of the two strains were observed to be pink-pigmented. The cells were found to be Gram-stain negative, coccoid and non-motile. The organisms were found to be aerobic and could grow in an NaCl range of 6-35 % (optimum 18 %), temperatures ranging from 25 to 50 °C (optimum 37-42 °C), pH range from 6.0-8.5 (optimum pH 7.0-7.5) and Mg(2+) range from 0 to 1.5 M (optimum 0.5-1.0 M); Mg(2+) was not necessary for growth. Cells were not observed to lyse in distilled water. Strains YIM 90917 and YIM 93656(T) showed the highest 16S rRNA gene sequence similarities to Haladaptatus cibarius JCM 15962(T) (97.6 and 97.9 %, respectively). In addition, the DNA-DNA hybridizations of strains YIM 90917 and YIM 93656(T) with type strains H. cibarius JCM 15962(T), Haladaptatus litoreus JCM 15771(T) and Haladaptatus paucihalophilus JCM 13897(T) were 37.2 and 38.2 %, 36.6 and 39.0 % and 27.9 and 27.7 %, respectively. The DNA G+C contents of strains YIM 90917 and YIM 93656(T) were determined to be 56.0 and 57.4 mol%. The major polar lipids of the two strains were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, sulfated mannosyl glucosyl diether and other four unidentified glycolipids. On the basis of physiological, chemotaxonomic data and phylogenetic analysis, the strains YIM 90917 and YIM 93656(T) can be classified as a novel species of the genus Haladaptatus, for which the name Haladaptatus pallidirubidus sp. nov. is proposed. The type strain is YIM 93656(T) (=JCM 17504(T) = CCTCC AB2010454(T)).

  17. Draft Genome Sequence of Halostagnicola sp. A56, an Extremely Halophilic Archaeon Isolated from the Andaman Islands

    Science.gov (United States)

    Kanekar, Sagar P.; Saxena, Neha; Pore, Soham D.; Arora, Preeti; Kanekar, P. P.

    2015-01-01

    The first draft genome of Halostagnicola sp. A56, isolated from the Andaman Islands is reported here. The A56 genome comprises 3,178,490 bp in 26 contigs with a G+C content of 60.8%. The genome annotation revealed that A56 could have potential applications for the production of polyhydroxyalkanoate or bioplastics. PMID:26564049

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

    Directory of Open Access Journals (Sweden)

    Bourne Philip E

    2011-02-01

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

  19. Thioredoxin-linked redox control of metabolism in Methanocaldococcus jannaschii, an evolutionarily deeply-rooted hyperthermophilic methanogenic archaeon

    Science.gov (United States)

    Thioredoxin (Trx), a small redox protein, controls multiple processes in eukaryotes and bacteria by changing the thiol redox status of selected proteins. We have investigated this aspect in methanarchaea. These ancient methanogens produce methane almost exclusively from H2 plus CO2 carried approxima...

  20. An Integrative Genomic Island Affects the Adaptations of Piezophilic Hyperthermophilic Archaeon Pyrococcus yayanosii to High Temperature and High Hydrostatic Pressure

    Directory of Open Access Journals (Sweden)

    Zhen Li

    2016-11-01

    Full Text Available Deep-sea hydrothermal vent environments are characterized by high hydrostatic pressure and sharp temperature and chemical gradients. Horizontal gene transfer is thought to play an important role in the microbial adaptation to such an extreme environment. In this study, a 21.4-kb DNA fragment was identified as a genomic island, designated PYG1, in the genomic sequence of the piezophilic hyperthermophile Pyrococcus yayanosii. According to the sequence alignment and functional annotation, the genes in PYG1 could tentatively be divided into five modules, with functions related to mobility, DNA repair, metabolic processes and the toxin-antitoxin system. Integrase can mediate the site-specific integration and excision of PYG1 in the chromosome of P. yayanosii A1. Gene replacement of PYG1 with a SimR cassette was successful. The growth of the mutant strain ∆PYG1 was compared with its parent strain P. yayanosii A2 under various stress conditions, including different pH, salinity, temperature and hydrostatic pressure. The ∆PYG1 mutant strain showed reduced growth when grown at 100 °C, while the biomass of ∆PYG1 increased significantly when cultured at 80 MPa. Differential expression of the genes in module Ⅲ of PYG1 was observed under different temperature and pressure conditions. This study demonstrates the first example of an archaeal integrative genomic island that could affect the adaptation of the hyperthermophilic piezophile P. yayanosii to high temperature and high hydrostatic pressure.

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

    KAUST Repository

    Antunes, Andre; Alam, Intikhab; Bajic, Vladimir B.; Stingl, Ulrich

    2011-01-01

    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.

  2. Dicty_cDB: Contig-U11181-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available id:none) Pyrobaculum aerophilum str. IM2,... 94 2e-17 AM944373_1( AM944373 |pid:n...one) Propionibacterium freudenreichii s... 93 3e-17 CP001114_2205( CP001114 |pid:none) Deinococcus deserti V

  3. 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

  4. Aromatic residues located close to the active center are essential for the catalytic reaction of flap endonuclease-1 from hyperthermophilic archaeon Pyrococcus horikoshii.

    Science.gov (United States)

    Matsui, Eriko; Abe, Junko; Yokoyama, Hideshi; Matsui, Ikuo

    2004-04-16

    Flap endonuclease-1 (FEN-1) possessing 5'-flap endonuclease and 5'-->3' exonuclease activity plays important roles in DNA replication and repair. In this study, the kinetic parameters of mutants at highly conserved aromatic residues, Tyr33, Phe35, Phe79, and Phe278-Phe279, in the vicinity of the catalytic centers of FEN-1 were examined. The substitution of these aromatic residues with alanine led to a large reduction in kcat values, although these mutants retained Km values similar to that of the wild-type enzyme. Notably, the kcat of Y33A and F79A decreased 333-fold and 71-fold, respectively, compared with that of the wild-type enzyme. The aromatic residues Tyr33 and Phe79, and the aromatic cluster Phe278-Phe279 mainly contributed to the recognition of the substrates without the 3' projection of the upstream strand (the nick, 5'-recess-end, single-flap, and pseudo-Y substrates) for the both exo- and endo-activities, but played minor roles in recognizing the substrates with the 3' projection (the double flap substrate and the nick substrate with the 3' projection). The replacement of Tyr33, Phe79, and Phe278-Phe279, with non-charged aromatic residues, but not with aliphatic hydrophobic residues, recovered the kcat values almost fully for the substrates without the 3' projection of the upstream strand, suggesting that the aromatic groups of Tyr33, Phe79, and Phe278-Phe279 might be involved in the catalytic reaction, probably via multiple stacking interactions with nucleotide bases. The stacking interactions of Tyr33 and Phe79 might play important roles in fixing the template strand and the downstream strand, respectively, in close proximity to the active center to achieve the productive transient state leading to the hydrolysis.

  5. 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...... phosphoribosyltransferase (PRTase), the protein product turned out to be a PRTase highly specific for adenine and we suggest that the reading frame should be renamed apT. The other reading frame SSO2424 (gpT-2) proved to be a true 6-oxopurine PRTase active with hypoxanthine, xanthine and guanine as substrates, and we.......5, while maximal activity with xanthine was observed at pH 7.5. We discuss likely reasons why SSO2341 in S. solfataricus and similar open reading frames in other Crenarchaeota could not be identified as genes encoding APRTase....

  6. 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. Keywords: Halophilic archaea, Henome sequence, Puerto Rico

  7. 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...... results suggest that the enzyme may serve roles in chromosomal segregation and control of the level of supercoiling in the cell....

  8. 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.

  9. 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.

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

    Science.gov (United States)

    Lebedeva, Elena V; Hatzenpichler, Roland; Pelletier, Eric; Schuster, Nathalie; Hauzmayer, Sandra; Bulaev, Aleksandr; Grigor'eva, Nadezhda V; Galushko, Alexander; Schmid, Markus; Palatinszky, Marton; Le Paslier, Denis; Daims, Holger; Wagner, Michael

    2013-01-01

    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. 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, B.; Sierocinski, P.; Steuer, R.; van der Oost, 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

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

    OpenAIRE

    Müller, Jochen A.; DasSarma, Shiladitya

    2005-01-01

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

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

    NARCIS (Netherlands)

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

    2000-01-01

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

  14. 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

    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

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

    Science.gov (United States)

    Sánchez-Nieves, Rubén; Facciotti, Marc T; Saavedra-Collado, Sofía; Dávila-Santiago, Lizbeth; Rodríguez-Carrero, Roy; Montalvo-Rodríguez, Rafael

    2016-03-01

    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.

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

    Science.gov (United States)

    Sánchez-Nieves, Rubén; Facciotti, Marc; Saavedra-Collado, Sofía; Dávila-Santiago, Lizbeth; Rodríguez-Carrero, Roy; Montalvo-Rodríguez, Rafael

    2016-03-01

    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.

  17. Evidence that biosynthesis of the second and third sugars of the archaellin Tetrasaccharide in the archaeon Methanococcus maripaludis occurs by the same pathway used by Pseudomonas aeruginosa to make a di-N-acetylated sugar.

    Science.gov (United States)

    Siu, Sarah; Robotham, Anna; Logan, Susan M; Kelly, John F; Uchida, Kaoru; Aizawa, Shin-Ichi; Jarrell, Ken F

    2015-05-01

    Methanococcus maripaludis has two surface appendages, archaella and type IV pili, which are composed of glycoprotein subunits. Archaellins are modified with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose. The pilin glycan has an additional hexose attached to GalNAc. In this study, genes located in two adjacent, divergently transcribed operons (mmp0350-mmp0354 and mmp0359-mmp0355) were targeted for study based on annotations suggesting their involvement in biosynthesis of N-glycan sugars. Mutants carrying deletions in mmp0350, mmp0351, mmp0352, or mmp0353 were nonarchaellated and synthesized archaellins modified with a 1-sugar glycan, as estimated from Western blots. Mass spectroscopy analysis of pili purified from the Δmmp0352 strain confirmed a glycan with only GalNAc, suggesting mmp0350 to mmp0353 were all involved in biosynthesis of the second sugar (GlcNAc3NAcA). The Δmmp0357 mutant was archaellated and had archaellins with a 2-sugar glycan, as confirmed by mass spectroscopy of purified archaella, indicating a role for MMP0357 in biosynthesis of the third sugar (ManNAc3NAmA6Thr). M. maripaludis mmp0350, mmp0351, mmp0352, mmp0353, and mmp0357 are proposed to be functionally equivalent to Pseudomonas aeruginosa wbpABEDI, involved in converting UDP-N-acetylglucosamine to UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, an O5-specific antigen sugar. Cross-domain complementation of the final step of the P. aeruginosa pathway with mmp0357 supports this hypothesis. This work identifies a series of genes in adjacent operons that are shown to encode the enzymes that complete the entire pathway for generation of the second and third sugars of the N-linked tetrasaccharide that modifies archaellins of Methanococcus maripaludis. This posttranslational modification of archaellins is important, as it is necessary for archaellum assembly. Pilins are modified with a different N-glycan consisting of the archaellin tetrasaccharide but with an additional hexose attached to the linking sugar. Mass spectrometry analysis of the pili of one mutant strain provided insight into how this different glycan might ultimately be assembled. This study includes a rare example of an archaeal gene functionally replacing a bacterial gene in a complex sugar biosynthesis pathway. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. JGEN-D-15-00296R2_MOESM3_ESM.xls

    Indian Academy of Sciences (India)

    SSS

    5, Crenarchaea, Hyperthermus butylicus DSM 5456, 0.139, 51.15, 54.2, 0.224, 47.32, 50.21, -0.085, 3.83, 3.99. 6, Crenarchaea, Metallosphaera sedula DSM 5348, 0.109, 51.62, 52.99, 0.137, 51.08, 50.47, -0.028, 0.54, 2.52. 7, Crenarchaea, Pyrobaculum aerophilum str. IM2, 0.145, 52.89, 54.34, 0.152, 52.53, 53.87, -0.007 ...

  19. CC1, a novel crenarchaeal DNA binding protein.

    Science.gov (United States)

    Luo, Xiao; Schwarz-Linek, Uli; Botting, Catherine H; Hensel, Reinhard; Siebers, Bettina; White, Malcolm F

    2007-01-01

    The genomes of the related crenarchaea Pyrobaculum aerophilum and Thermoproteus tenax lack any obvious gene encoding a single-stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of T. tenax. An assay for proteins capable of binding to a single-stranded DNA oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind single-stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the P. aerophilum and Aeropyrum pernix genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in T. tenax. This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein.

  20. Environmental genomics of "Haloquadratum walsbyi" in a saltern crystallizer indicates a large pool of accessory genes in an otherwise coherent species

    NARCIS (Netherlands)

    Legault, Boris A.; Lopez-Lopez, Arantxa; Alba-Casado, Jose Carlos; Doolittle, W. Ford; Bolhuis, Henk; Rodriguez-Valera, Francisco; Papke, R. Thane

    2006-01-01

    Background: Mature saturated brine (crystallizers) communities are largely dominated (> 80% of cells) by the square halophilic archaeon "Haloquadratum walsbyi". The recent cultivation of the strain HBSQ001 and thesequencing of its genome allows comparison with the metagenome of this taxonomically

  1. 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...

  2. Extreme genomes

    OpenAIRE

    DeLong, Edward F

    2000-01-01

    The complete genome sequence of Thermoplasma acidophilum, an acid- and heat-loving archaeon, has recently been reported. Comparative genomic analysis of this 'extremophile' is providing new insights into the metabolic machinery, ecology and evolution of thermophilic archaea.

  3. Extraction, isolation and NMR data of the tetraether lipid calditoglycerocaldarchaeol (GDNT) from Sulfolobus metallicus harvested from a bioleaching reactor

    CSIR Research Space (South Africa)

    Bode, ML

    2008-08-01

    Full Text Available The successful extraction and isolation of the hydrolysed tetraether lipid calditoglycerocaldarchaeol (GDNT) from Sulfolobus etallicus, a key thermophilic bioleaching archaeon, is described. The archaeal biomasswas recovered directly from a...

  4. Engineering and Characterization of a Superfolder Green Fluorescent Protein

    International Nuclear Information System (INIS)

    Pedelacq, J.; Cabantous, S.; Tran, T.; Terwilliger, T.; Waldo, G.

    2006-01-01

    Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP

  5. Geranylfarnesyl diphosphate synthase from Methanosarcina mazei: Different role, different evolution

    International Nuclear Information System (INIS)

    Ogawa, Takuya; Yoshimura, Tohru; Hemmi, Hisashi

    2010-01-01

    The gene of (all-E) geranylfarnesyl diphosphate synthase that is responsible for the biosynthesis of methanophenazine, an electron carrier utilized for methanogenesis, was cloned from a methanogenic archaeon Methanosarcina mazei Goe1. The properties of the recombinant enzyme and the results of phylogenetic analysis suggest that the enzyme is closely related to (all-E) prenyl diphosphate synthases that are responsible for the biosynthesis of respiratory quinones, rather than to the enzymes involved in the biosynthesis of archaeal membrane lipids, including (all-E) geranylfarnesyl diphosphate synthase from a thermophilic archaeon.

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

    International Nuclear Information System (INIS)

    Cho, Ching-Chang; Luo, Ching-Wei; Hsu, Chun-Hua

    2008-01-01

    PAT, an acetyltransferase from the archaeon S. solfataricus that specifically acetylates the chromatin protein Alba, was expressed, purified and crystallized. PAT is an acetyltransferase from the archaeon Sulfolobus solfataricus that specifically acetylates the chromatin protein Alba. The enzyme was expressed, purified and subsequently crystallized using the sitting-drop vapour-diffusion technique. Native diffraction data were collected to 1.70 Å resolution on the BL13C1 beamline of NSRRC from a flash-frozen crystal at 100 K. The crystals belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 44.30, b = 46.59, c = 68.39 Å

  7. 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

  8. Main: FEB4 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available akaraensis and two novel enzymes involved in its carbon fixation pathway. Here, we will investigate the structures...archaeon Thermococcus kodakaraensis (Tk) and two novel enzymes involved in its carbon fixation pathway - We have determined the struc...tures and functions of the two novel enzymes - Design of

  9. Quantitative Proteomic Analysis of Sulfolobus solfataricus Membrane Proteins

    NARCIS (Netherlands)

    Pham, T.K.; Sierocinski, P.; Oost, van der J.; Wright, P.C.

    2010-01-01

    A quantitative proteomic analysis of the membrane of the archaeon Sulfolobus solfataricus P2 using iTRAQ was successfully demonstrated in this technical note. The estimated number of membrane proteins of this organism is 883 (predicted based on Gravy score), corresponding to 30 % of the total

  10. How oxygen gave rise to eukaryotic sex

    NARCIS (Netherlands)

    Hörandl, Elvira; Speijer, Dave

    2018-01-01

    9years ago. The large amount of ROS coming from a bacterial endosymbiont gave rise to DNA damage and vast increases in host genome mutation rates. Eukaryogenesis and chromosome evolution represent adaptations to oxidative stress. The host, an archaeon, most probably already had repair mechanisms

  11. Tertiary structure in 7.9 M guanidinium chloride: the role of Glu-53 and Asp-287 in Pyrococcus furiosus endo-beta-1,3-glucanase

    NARCIS (Netherlands)

    Chiaraluce, R.; Florio, R.; Angelaccio, S.; Gianese, G.; Lieshout, van J.F.T.; Oost, van der J.; Consalvi, V.

    2007-01-01

    The thermodynamic stability of family 16 endo-ß-1,3-glucanase (EC 3.2.1.39) from the hyperthermophilic archaeon Pyrococcus furiosus is decreased upon single (D287A, E53A) and double (E53A/D287A) mutation of Asp287 and Glu53. In accordance with the homology model prediction, both carboxylic acids are

  12. Crystal structure of a family 16 endoglucanase from the hyperthermophile Pyrococcus furiosus--structural basis of substrate recognition

    NARCIS (Netherlands)

    Ilari, A.; Fiorillo, A.; Angelaccio, S.; Florio, R.; Chiaraluce, R.; Oost, van der J.; Consalvi, V.

    2009-01-01

    Bacterial and archaeal endo-beta-1,3-glucanases that belong to glycoside hydrolase family 16 share a beta-jelly-roll fold, but differ significantly in sequence and in substrate specificity. The crystal structure of the laminarinase (EC 3.2.1.39) from the hyperthermophilic archaeon Pyrococcus

  13. Calcium-induced tertiary structure modifications of endo-B-1,3-glucanase form Pyrococcus furiosus in 7.9 M guanidinium chloride

    NARCIS (Netherlands)

    Chiaraluce, R.; Gianese, G.; Angelaccio, S.; Florio, R.; Lieshout, van J.F.T.; Oost, van der J.; Consalvi, V.

    2005-01-01

    The family 16 endo-b-1,3 glucanase from the extremophilic archaeon Pyrococcus furiosus is a laminarinase, which in 7.9 M GdmCl (guanidinium chloride) maintains a significant amount of tertiary structure without any change of secondary structure. The addition of calcium to the enzyme in 7.9 M GdmCl

  14. RNAi: prokaryotes get in on the act

    NARCIS (Netherlands)

    Oost, van der J.; Brouns, S.J.J.

    2009-01-01

    The small CRISPR-derived RNAs of bacteria and archaea provide adaptive immunity by targeting the DNA of invading viruses and plasmids. Hale et al. (2009) now report on a new variant CRISPR/Cas complex in the archaeon Pyrococcus furiosus that uses guide RNAs to specifically target and cleave RNA not

  15. 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 ...

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

    KAUST Repository

    Guan, Yue; Ngugi, David; Blom, J.; Ali, Shahjahan; Ferry, J. G.; Stingl, Ulrich

    2014-01-01

    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%.

  17. 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...

  18. 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%.

  19. Characterization of different crystal forms of the alpha-glucosidase MalA from Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Ernst, Heidi Asschenfeldt; Willemoës, Martin; Lo Leggio, Leila

    2005-01-01

    MalA is an alpha-glucosidase from the hyperthermophilic archaeon Sulfolobus solfataricus. It belongs to glycoside hydrolase family 31, which includes several medically interesting alpha-glucosidases. MalA and its selenomethionine derivative have been overproduced in Escherichia coli...

  20. Production of cellulose nanofibrils from bleached eucalyptus fibers by hyperthermostable endoglucanase treatment and subsequent microfluidization

    Science.gov (United States)

    Wangxia Wang; Michael D. Mozuch; Ronald C. Sabo; Phil Kersten; J.Y. Zhu; Yongcan Jin

    2015-01-01

    A GH5 hyperthermostable endoglucanase from the archaeon Pyrococcus honkoshii (ph-GH5) and a commercial endoglucanase FR were used to treat bleached eucalyptus pulp (BEP) fibers to produce cellulose nanofibrils (CNFs) through subsequent microfluidization Enzymatic treatments facilitated CNF production due to the reduced degree of polymerization (DP)...

  1. Physical and Mechanical Properties of Cellulose Nanofibril Films from Bleached Eucalyptus Pulp by Endoglucanase Treatment and Microfluidization

    Science.gov (United States)

    Wangxia Wang; Ronald C. Sabo; Michael D. Mozuch; Phil Kersten; J. Y. Zhu; Yongcan Jin

    2015-01-01

    A GH5 hyperthermostable endoglucanase (Ph-GH5) from the archaeon Pyrococcus horikoshii and a commercial endoglucanase (FR) were used to treat bleached eucalyptus pulp (BEP) fibers to produce cellulose nanofibrils (CNF) and subsequently to CNF films. TEM imaging indicated that Ph-GH5 produced longer and more entangled CNF than FR with the same number...

  2. Purification, crystallization and preliminary X-ray diffraction analysis of an archaeal ABC-ATPase

    NARCIS (Netherlands)

    Verdon, Grégory; Albers, Sonja-V.; Dijkstra, Bauke W.; Driessen, Arnold J.M.; Thunnissen, Andy-Mark W.H.

    2002-01-01

    In the archaeon Sulfolobus solfataricus glucose uptake is mediated by an ABC transport system. The ABC-ATPase of this transporter (GlcV) has been overproduced in Escherichia coli and purified. Crystals of GlcV suitable for data collection were obtained in the absence of nucleotide by microseeding

  3. Methanomethylovorans thermophila sp. nov., a thermophilic, methylotrophic methanogen form an anaerobic reactor fed with methanol

    NARCIS (Netherlands)

    Jiang, B.; Parshina, S.N.; Doesburg, van W.C.J.; Lomans, B.P.; Stams, A.J.M.

    2005-01-01

    A novel thermophilic, obligately methylotrophic, methanogenic archaeon, strain L2FAWT, was isolated from a thermophilic laboratory-scale upflow anaerobic sludge blanket reactor fed with methanol as the carbon and energy source. Cells of strain L2FAWT were non-motile, irregular cocci, 0·7¿1·5 µm in

  4. Production and characterization of a thermostable alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily

    NARCIS (Netherlands)

    Machielsen, M.P.; Uria, A.R.; Kengen, S.W.M.; Oost, van der J.

    2006-01-01

    The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The gene, referred to as adhD, was functionally expressed in Escherichia coli and subsequently purified to homogeneity. The

  5. 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...

  6. Crystallization and preliminary X-ray analysis of membrane-bound pyrophosphatases.

    Science.gov (United States)

    Kellosalo, Juho; Kajander, Tommi; Honkanen, Riina; Goldman, Adrian

    2013-02-01

    Membrane-bound pyrophosphatases (M-PPases) are enzymes that enhance the survival of plants, protozoans and prokaryotes in energy constraining stress conditions. These proteins use pyrophosphate, a waste product of cellular metabolism, as an energy source for sodium or proton pumping. To study the structure and function of these enzymes we have crystallized two membrane-bound pyrophosphatases recombinantly produced in Saccharomyces cerevisae: the sodium pumping enzyme of Thermotoga maritima (TmPPase) and the proton pumping enzyme of Pyrobaculum aerophilum (PaPPase). Extensive crystal optimization has allowed us to grow crystals of TmPPase that diffract to a resolution of 2.6 Å. The decisive step in this optimization was in-column detergent exchange during the two-step purification procedure. Dodecyl maltoside was used for high temperature solubilization of TmPPase and then exchanged to a series of different detergents. After extensive screening, the new detergent, octyl glucose neopentyl glycol, was found to be the optimal for TmPPase but not PaPPase.

  7. [Prokaryote diversity in water environment of land-ocean ecotone of Zhuhai City].

    Science.gov (United States)

    Huang, Xiao-Lan; Chen, Jian-Yao; Zhou, Shi-Ning; Xie, Li-Chun; Fu, Cong-Sheng

    2010-02-01

    By constructing 16S rDNA clone library with PCR-RFLP, the prokaryote diversity in the seawater and groundwater of land-ocean ecotone of Zhuhai City was investigated, and the similarity and cluster analyses were implemented with the database of the sequences in Genbank. In the seawater, Proteobacteria was dominant, followed by Archaeon, Gemmatimonadetes, Candidate division OP3 and OP8, and Planctomycetes, etc.; while in the groundwater, Archaeon was dominant, followed by Proteobacteria, Sphingobacteria, Candidate division OP3, Actinobacterium, and Pseudomonas. The dominant taxa in the groundwater had high similarity to the unculturable groups of marine microorganisms. Large amount of bacteria capable of degrading organic matter and purifying water body existed in the groundwater, suggesting that after long-term evolution, the land-ocean ecotone of Zhuhai City had the characteristics of both land and ocean.

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

    International Nuclear Information System (INIS)

    Cao, Yi; Lou, Zhiyong; Sun, Yuna; Xue, Fei; Feng, Changzeng; Gong, Xiaocui; Yang, Dongmei; Bartlam, Mark; Meng, Zhaohui; Zhang, Keqin

    2009-01-01

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

  9. On the Archaeal Origins of Eukaryotes and the Challenges of Inferring Phenotype from Genotype.

    Science.gov (United States)

    Dey, Gautam; Thattai, Mukund; Baum, Buzz

    2016-07-01

    If eukaryotes arose through a merger between archaea and bacteria, what did the first true eukaryotic cell look like? A major step toward an answer came with the discovery of Lokiarchaeum, an archaeon whose genome encodes small GTPases related to those used by eukaryotes to regulate membrane traffic. Although 'Loki' cells have yet to be seen, their existence has prompted the suggestion that the archaeal ancestor of eukaryotes engulfed the future mitochondrion by phagocytosis. We propose instead that the archaeal ancestor was a relatively simple cell, and that eukaryotic cellular organization arose as the result of a gradual transfer of bacterial genes and membranes driven by an ever-closer symbiotic partnership between a bacterium and an archaeon. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Comparison of two extreme halophilic Halobacterium noricense strains on DNA and protein level

    Energy Technology Data Exchange (ETDEWEB)

    Bader, Miriam; Cherkouk, Andrea [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). HZDR Young Investigator Group; Flemming, Katrin [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Biogeochemistry; Swanson, J.S. [Los Alamos National Laboratory, Carlsbad, NM (United States)

    2017-06-01

    Two strains of the halophilic archaeon Halobacterium noricense isolated from rock salt of different locations were used for interaction studies with uranium. It was found that both strains showed similar, atypical bioassociation kinetics accompanied by cell agglomeration as a stress response. The 16S rRNA gene sequences of both strains had a high similarity (> 99 %). However, differences in the whole protein pattern were apparent.

  11. Identification of a mismatch-specific endonuclease in hyperthermophilic Archaea

    OpenAIRE

    Ishino, Sonoko; Nishi, Yuki; Oda, Soichiro; Uemori, Takashi; Sagara, Takehiro; Takatsu, Nariaki; Yamagami, Takeshi; Shirai, Tsuyoshi; Ishino, Yoshizumi

    2016-01-01

    The common mismatch repair system processed by MutS and MutL and their homologs was identified in Bacteria and Eukarya. However, no evidence of a functional MutS/L homolog has been reported for archaeal organisms, and it is not known whether the mismatch repair system is conserved in Archaea. Here, we describe an endonuclease that cleaves double-stranded DNA containing a mismatched base pair, from the hyperthermophilic archaeon Pyrococcus furiosus. The corresponding gene revealed that the act...

  12. Role of Mn2+ and Compatible Solutes in the Radiation Resistance of Thermophilic Bacteria and Archaea

    OpenAIRE

    Webb, Kimberly M.; DiRuggiero, Jocelyne

    2012-01-01

    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-m...

  13. Cloning, expression, and crystallization of Cpn60 proteins from Thermococcus litoralis.

    Science.gov (United States)

    Osipiuk, J; Sriram, M; Mai, X; Adams, M W; Joachimiak, A

    2000-01-01

    Two genes of the extreme thermophilic archaeon Thermococcus litoralis homologous to those that code for Cpn60 chaperonins were cloned and expressed in Escherichia coli. Each of the Cpn60 subunits as well as the entire Cpn60 complex crystallize in a variety of morphological forms. The best crystals diffract to 3.6 A resolution at room temperature and belong to the space group 1422 with unit cell parameters a = b = 193.5 A, c = 204.2 A.

  14. A simple laser-based device for simultaneous microbial culture and absorbance measurement

    Science.gov (United States)

    Abrevaya, X. C.; Cortón, E.; Areso, O.; Mauas, P. J. D.

    2013-07-01

    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, a halophilic archaeon.

  15. Comparison of two extreme halophilic Halobacterium noricense strains on DNA and protein level

    International Nuclear Information System (INIS)

    Bader, Miriam; Cherkouk, Andrea

    2017-01-01

    Two strains of the halophilic archaeon Halobacterium noricense isolated from rock salt of different locations were used for interaction studies with uranium. It was found that both strains showed similar, atypical bioassociation kinetics accompanied by cell agglomeration as a stress response. The 16S rRNA gene sequences of both strains had a high similarity (> 99 %). However, differences in the whole protein pattern were apparent.

  16. Enzymology and Evolution of the Pyruvate Pathway to 2-Oxobutyrate in Methanocaldococcus jannaschii▿

    OpenAIRE

    Drevland, Randy M.; Waheed, Abdul; Graham, David E.

    2007-01-01

    The archaeon Methanocaldococcus jannaschii uses three different 2-oxoacid elongation pathways, which extend the chain length of precursors in leucine, isoleucine, and coenzyme B biosyntheses. In each of these pathways an aconitase-type hydrolyase catalyzes an hydroxyacid isomerization reaction. The genome sequence of M. jannaschii encodes two homologs of each large and small subunit that forms the hydrolyase, but the genes are not cotranscribed. The genes are more similar to each other than t...

  17. Cloning, Sequencing, and Expression of the Gene Encoding Cyclic 2,3-Diphosphoglycerate Synthetase, the Key Enzyme of Cyclic 2,3-Diphosphoglycerate Metabolism in Methanothermus fervidus

    OpenAIRE

    Matussek, Karl; Moritz, Patrick; Brunner, Nina; Eckerskorn, Christoph; Hensel, Reinhard

    1998-01-01

    Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and...

  18. ABC Transporter for Corrinoids in Halobacterium sp. Strain NRC-1†

    OpenAIRE

    Woodson, Jesse D.; Reynolds, April A.; Escalante-Semerena, Jorge C.

    2005-01-01

    We report evidence for the existence of a putative ABC transporter for corrinoid utilization in the extremely halophilic archaeon Halobacterium sp. strain NRC-1. Results from genetic and nutritional analyses of Halobacterium showed that mutants with lesions in open reading frames (ORFs) Vng1370G, Vng1371Gm, and Vng1369G required a 105-fold higher concentration of cobalamin for growth than the wild-type or parent strain. The data support the conclusion that these ORFs encode orthologs of the b...

  19. Comparative analysis of uranium bioassociation with halophilic bacteria and archaea

    Science.gov (United States)

    Bader, Miriam; Müller, Katharina; Foerstendorf, Harald; Schmidt, Matthias; Simmons, Karen; Swanson, Juliet S.; Reed, Donald T.; Stumpf, Thorsten

    2018-01-01

    Rock salt represents a potential host rock formation for the final disposal of radioactive waste. The interactions between indigenous microorganisms and radionuclides, e.g. uranium, need to be investigated to better predict the influence of microorganisms on the safety assessment of the repository. Hence, the association process of uranium with two microorganisms isolated from rock salt was comparatively studied. Brachybacterium sp. G1, which was isolated from the German salt dome Gorleben, and Halobacterium noricense DSM15987T, were selected as examples of a moderately halophilic bacterium and an extremely halophilic archaeon, respectively. The microorganisms exhibited completely different association behaviors with uranium. While a pure biosorption process took place with Brachybacterium sp. G1 cells, a multistage association process occurred with the archaeon. In addition to batch experiments, in situ attenuated total reflection Fourier-transform infrared spectroscopy was applied to characterize the U(VI) interaction process. Biosorption was identified as the dominating process for Brachybacterium sp. G1 with this method. Carboxylic functionalities are the dominant interacting groups for the bacterium, whereas phosphoryl groups are also involved in U(VI) association by the archaeon H. noricense. PMID:29329319

  20. Intermolecular ion pairs maintain the toroidal structure of Pyrococcus furiosus PCNA

    OpenAIRE

    Matsumiya, Shigeki; Ishino, Sonoko; Ishino, Yoshizumi; Morikawa, Kosuke

    2003-01-01

    Two mutant proliferating cell nuclear antigens from the hyperthermophilic archaeon Pyrococcus furiosus, PfuPCNA(D143A) and PfuPCNA(D143A/D147A), were prepared by site-specific mutagenesis. The results from gel filtration showed that mutations at D143 and D147 drastically affect the stability of the trimeric structure of PfuPCNA. The PfuPCNA(D143A) still retained the activity to stimulate the DNA polymerase reaction, but PfuPCNA(D143A/D147A) lost the activity. Crystal structures of the mutant ...

  1. First insights in the Eu(III) speciation in Halobacterium noricense DSM-15987 suspensions

    Energy Technology Data Exchange (ETDEWEB)

    Bader, Miriam; Moll, Henry; Cherkouk, Andrea [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Div. Biogeochemistry

    2016-07-01

    The association of Eu(III) on the halophilic archaeon Halobacterium (Hbt.) noricense DSM-15987 was investigated between pC{sub H+} 4 to 8 in 3 M NaCl. In contrast to uranium [1], a lower affinity of Eu(III) towards Hbt. noricense was observed. Three different Eu(III)- species could be isolated in this system, whereas one is the Eu(III) aquo ion and one represents a Hbt. noricense associated species. The third species might belong to a dissolved species.

  2. 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....

  3. 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...... technique, demonstrated that a general tightening of the C-terminal domain occurred on rRNA binding, while thiostrepton produced a footprint centred on tyrosine 62 at the junction of the N and C-terminal domains of protein L11 complexed to rRNA. The intensity of this protein footprint was strongly reduced...

  4. Processivity and Subcellular Localization of Glycogen Synthase Depend on a Non-catalytic High Affinity Glycogen-binding Site*

    OpenAIRE

    Díaz, Adelaida; Martínez-Pons, Carlos; Fita, Ignacio; Ferrer, Juan C.; Guinovart, Joan J.

    2011-01-01

    Glycogen synthase, a central enzyme in glucose metabolism, catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of a growing glycogen molecule. A non-catalytic glycogen-binding site, identified by x-ray crystallography on the surface of the glycogen synthase from the archaeon Pyrococcus abyssi, has been found to be functionally conserved in the eukaryotic enzymes. The disruption of this binding site in both the archaeal and the human muscle glycogen synth...

  5. 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.

  6. Cryo-EM Structure of the Archaeal 50S Ribosomal Subunit in Complex with Initiation Factor 6 and Implications for Ribosome Evolution

    Science.gov (United States)

    Greber, Basil J.; Boehringer, Daniel; Godinic-Mikulcic, Vlatka; Crnkovic, Ana; Ibba, Michael; Weygand-Durasevic, Ivana; Ban, Nenad

    2013-01-01

    Translation of mRNA into proteins by the ribosome is universally conserved in all cellular life. The composition and complexity of the translation machinery differ markedly between the three domains of life. Organisms from the domain Archaea show an intermediate level of complexity, sharing several additional components of the translation machinery with eukaryotes that are absent in bacteria. One of these translation factors is initiation factor 6 (IF6), which associates with the large ribosomal subunit. We have reconstructed the 50S ribosomal subunit from the archaeon Methanothermobacter thermautotrophicus in complex with archaeal IF6 at 6.6 Å resolution using cryo-electron microscopy (EM). The structure provides detailed architectural insights into the 50S ribosomal subunit from a methanogenic archaeon through identification of the rRNA expansion segments and ribosomal proteins that are shared between this archaeal ribosome and eukaryotic ribosomes but are mostly absent in bacteria and in some archaeal lineages. Furthermore, the structure reveals that, in spite of highly divergent evolutionary trajectories of the ribosomal particle and the acquisition of novel functions of IF6 in eukaryotes, the molecular binding of IF6 on the ribosome is conserved between eukaryotes and archaea. The structure also provides a snapshot of the reductive evolution of the archaeal ribosome and offers new insights into the evolution of the translation system in archaea. PMID:22306461

  7. Global transcriptome analysis of Halolamina sp. to decipher the salt tolerance in extremely halophilic archaea.

    Science.gov (United States)

    Kurt-Kızıldoğan, Aslıhan; Abanoz, Büşra; Okay, Sezer

    2017-02-15

    Extremely halophilic archaea survive in the hypersaline environments such as salt lakes or salt mines. Therefore, these microorganisms are good sources to investigate the molecular mechanisms underlying the tolerance to high salt concentrations. In this study, a global transcriptome analysis was conducted in an extremely halophilic archaeon, Halolamina sp. YKT1, isolated from a salt mine in Turkey. A comparative RNA-seq analysis was performed using YKT1 isolate grown either at 2.7M NaCl or 5.5M NaCl concentrations. A total of 2149 genes were predicted to be up-regulated and 1638 genes were down-regulated in the presence of 5.5M NaCl. The salt tolerance of Halolamina sp. YKT1 involves the up-regulation of genes related with membrane transporters, CRISPR-Cas systems, osmoprotectant solutes, oxidative stress proteins, and iron metabolism. On the other hand, the genes encoding the proteins involved in DNA replication, transcription, translation, mismatch and nucleotide excision repair were down-regulated. The RNA-seq data were verified for seven up-regulated genes as well as six down-regulated genes via qRT-PCR analysis. This comprehensive transcriptome analysis showed that the halophilic archaeon canalizes its energy towards keeping the intracellular osmotic balance minimizing the production of nucleic acids and peptides. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. 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.

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

    International Nuclear Information System (INIS)

    Morgunova, Ekaterina; Gray, Fiona C.; MacNeill, Stuart A.; Ladenstein, Rudolf

    2009-01-01

    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 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

  10. Cloning, purification, crystallization and preliminary crystallographic analysis of a penicillin-binding protein homologue from Pyrococcus abyssi

    International Nuclear Information System (INIS)

    Delfosse, Vanessa; Hugonnet, Jean-Emmanuel; Sougakoff, Wladimir; Mayer, Claudine

    2005-01-01

    The crystallization of a hypothetical penicillin-binding protein from the archaeon P. abyssi in space group C2 by hanging-drop vapour diffusion is reported. The genome of the hyperthermophilic archaeon Pyrococcus abyssi contains a gene (pab0087) encoding a penicillin-binding protein (PBP) homologue. This sequence consists of 447 residues and shows significant sequence similarity to low-molecular-weight PBPs and class C β-lactamases. The Pab0087 protein was overexpressed, purified and crystallized. Diffraction data from two different crystal forms were collected to 2.7 and 2.0 Å resolution. Both crystals belong to space group C2, with unit-cell parameters a = 160.59, b = 135.74, c = 113.02 Å, β = 117.36° and a = 166.97, b = 131.25, c = 189.39 Å, β = 113.81°, respectively. The asymmetric unit contains four and eight molecules, respectively, with fourfold non-crystallographic symmetry

  11. 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 pri......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...... of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene...

  12. Insights into archaeal evolution and symbiosis from the genomes of a Nanoarchaeon and its crenarchaeal host from Yellowstone National Park

    Energy Technology Data Exchange (ETDEWEB)

    Podar, Mircea [ORNL; Graham, David E [ORNL; Reysenbach, Anna-Louise [Portland State University; Koonin, Eugene [National Center for Biotechnology Information; Wolf, Yuri [National Center for Biotechnology Information; Makarova, Kira S. [National Center for Biotechnology Information

    2013-01-01

    A hyperthemophilic member of the Nanoarchaeota from Obsidian Pool, a thermal feature in Yellowstone National Park was characterized using single cell isolation and sequencing, together with its putative host, a Sulfolobales archaeon. This first representative of a non-marine Nanoarchaeota (Nst1) resembles Nanoarchaeum equitans by lacking most biosynthetic capabilities, the two forming a deep-branching archaeal lineage. However, the Nst1 genome is over 20% larger, encodes a complete gluconeogenesis pathway and a full complement of archaeal flagellum proteins. Comparison of the two genomes suggests that the marine and terrestrial Nanoarchaeota lineages share a common ancestor that was already a symbiont of another archaeon. With a larger genome, a smaller repertoire of split protein encoding genes and no split non-contiguous tRNAs, Nst1 appears to have experienced less severe genome reduction than N. equitans. The inferred host of Nst1 is potentially autotrophic, with a streamlined genome and simplified central and energetic metabolism as compared to other Sulfolobales. The two distinct Nanoarchaeota-host genomic data sets offer insights into the evolution of archaeal symbiosis and parasitism and will further enable studies of the cellular and molecular mechanisms of these relationships.

  13. Metagenomic Analysis of Hot Springs in Central India Reveals Hydrocarbon Degrading Thermophiles and Pathways Essential for Survival in Extreme Environments

    Science.gov (United States)

    Saxena, Rituja; Dhakan, Darshan B.; Mittal, Parul; Waiker, Prashant; Chowdhury, Anirban; Ghatak, Arundhuti; Sharma, Vineet K.

    2017-01-01

    Extreme ecosystems such as hot springs are of great interest as a source of novel extremophilic species, enzymes, metabolic functions for survival and biotechnological products. India harbors hundreds of hot springs, the majority of which are not yet explored and require comprehensive studies to unravel their unknown and untapped phylogenetic and functional diversity. The aim of this study was to perform a large-scale metagenomic analysis of three major hot springs located in central India namely, Badi Anhoni, Chhoti Anhoni, and Tattapani at two geographically distinct regions (Anhoni and Tattapani), to uncover the resident microbial community and their metabolic traits. Samples were collected from seven distinct sites of the three hot spring locations with temperature ranging from 43.5 to 98°C. The 16S rRNA gene amplicon sequencing of V3 hypervariable region and shotgun metagenome sequencing uncovered a unique taxonomic and metabolic diversity of the resident thermophilic microbial community in these hot springs. Genes associated with hydrocarbon degradation pathways, such as benzoate, xylene, toluene, and benzene were observed to be abundant in the Anhoni hot springs (43.5–55°C), dominated by Pseudomonas stutzeri and Acidovorax sp., suggesting the presence of chemoorganotrophic thermophilic community with the ability to utilize complex hydrocarbons as a source of energy. A high abundance of genes belonging to methane metabolism pathway was observed at Chhoti Anhoni hot spring, where methane is reported to constitute >80% of all the emitted gases, which was marked by the high abundance of Methylococcus capsulatus. The Tattapani hot spring, with a high-temperature range (61.5–98°C), displayed a lower microbial diversity and was primarily dominated by a nitrate-reducing archaeal species Pyrobaculum aerophilum. A higher abundance of cell metabolism pathways essential for the microbial survival in extreme conditions was observed at Tattapani. Taken together

  14. Crystallization and preliminary X-ray analysis of PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins

    International Nuclear Information System (INIS)

    Shirokane, Michio; Sawano, Yoriko; Miyazono, Ken-ichi; Nagata, Koji; Tanokura, Masaru

    2007-01-01

    PH1010, a DUF54-family protein from the hyperthermophilic archaeon P. horikoshii OT3, was crystallized and X-ray diffraction data were collected to 1.90 Å resolution. PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins, was expressed, purified and crystallized. Crystallization was performed by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.90 Å resolution using a synchrotron-radiation source. The space group of the crystal was determined to be P2 1 2 1 2 1 , with unit-cell parameters a = 46.9, b = 49.5, c = 132.7 Å. The crystal contained two PH1010 molecules in the asymmetric unit (V M = 2.4 Å 3 Da −1 ) and had a solvent content of 48%

  15. Crystallization and preliminary X-ray analysis of PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins

    Energy Technology Data Exchange (ETDEWEB)

    Shirokane, Michio; Sawano, Yoriko; Miyazono, Ken-ichi; Nagata, Koji; Tanokura, Masaru, E-mail: amtanok@mail.ecc.u-tokyo.ac.jp [Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

    2007-06-01

    PH1010, a DUF54-family protein from the hyperthermophilic archaeon P. horikoshii OT3, was crystallized and X-ray diffraction data were collected to 1.90 Å resolution. PH1010 from Pyrococcus horikoshii OT3, a member of the archaeal DUF54 family of proteins, was expressed, purified and crystallized. Crystallization was performed by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystal diffracted X-rays to 1.90 Å resolution using a synchrotron-radiation source. The space group of the crystal was determined to be P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.9, b = 49.5, c = 132.7 Å. The crystal contained two PH1010 molecules in the asymmetric unit (V{sub M} = 2.4 Å{sup 3} Da{sup −1}) and had a solvent content of 48%.

  16. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    Science.gov (United States)

    Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B.

    2011-07-01

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Å resolution.

  17. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    Energy Technology Data Exchange (ETDEWEB)

    Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B., E-mail: garber@vega.protres.ru [Institute of Protein Research RAS (Russian Federation)

    2011-07-15

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Angstrom-Sign resolution.

  18. Expression, purification, crystallization and preliminary X-ray analysis of a nucleoside kinase from the hyperthermophile Methanocaldococcus jannaschii

    International Nuclear Information System (INIS)

    Arnfors, Linda; Hansen, Thomas; Meining, Winfried; Schönheit, Peter; Ladenstein, Rudolf

    2005-01-01

    Nucleoside kinase from the hyperthermophilic archaeon M. jannaschii is a member of the PFK-B family which belongs to the ribokinase superfamily. Here, its expression, purification, crystallization and preliminary X-ray analysis are described. Methanocaldococcus jannaschii nucleoside kinase (MjNK) is an ATP-dependent non-allosteric phosphotransferase that shows high catalytic activity for guanosine, inosine and cytidine. MjNK is a member of the phosphofructokinase B family, but participates in the biosynthesis of nucleoside monophosphates rather than in glycolysis. MjNK was crystallized as the apoenzyme as well as in complex with an ATP analogue and Mg 2+ . The latter crystal form was also soaked with fructose-6-phosphate. Synchrotron-radiation data were collected to 1.70 Å for the apoenzyme crystals and 1.93 Å for the complex crystals. All crystals exhibit orthorhombic symmetry; however, the apoenzyme crystals contain one monomer per asymmetric unit whereas the complex crystals contain a dimer

  19. Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase.

    Science.gov (United States)

    Liew, Li Phing; Lim, Zun Yi; Cohen, Matan; Kong, Ziqing; Marjavaara, Lisette; Chabes, Andrei; Bell, Stephen D

    2016-11-01

    In many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. 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.

  1. 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.

  2. 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.

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

    DEFF Research Database (Denmark)

    Becker, F.; Schnorr, K.; Wilting, R.

    2004-01-01

    To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less beta-lactamase ('bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The 'bla gene was cloned into a bacteriophage MU...... minitransposon enabling translational fusions between 'bla and target genes. Fusion of TnSig in the correct reading frame to a protein carrying transmembrane domains or signal peptides resulted in ampicillin resistance of the corresponding clone. Prokaryotic gene libraries from the alkaliphilic bacterium...... Bacillus halodurans C125 and the hyperthermophilic archaeon Sulfolobus solfataricus P2 were tagged with TnSig. The genomic sequences, which are publicly available (EMBL BA000004 and EMBL AE006641), were used for rapid open reading frame (ORF) identification and prediction of protein localisation...

  4. Gas Vesicle Nanoparticles for Antigen Display

    Directory of Open Access Journals (Sweden)

    Shiladitya DasSarma

    2015-09-01

    Full Text Available Microorganisms like the halophilic archaeon Halobacterium sp. NRC-1 produce gas-filled buoyant organelles, which are easily purified as protein nanoparticles (called gas vesicles or GVNPs. GVNPs are non-toxic, exceptionally stable, bioengineerable, and self-adjuvanting. A large gene cluster encoding more than a dozen proteins has been implicated in their biogenesis. One protein, GvpC, found on the exterior surface of the nanoparticles, can accommodate insertions near the C-terminal region and results in GVNPs displaying the inserted sequences on the surface of the nanoparticles. Here, we review the current state of knowledge on GVNP structure and biogenesis as well as available studies on immunogenicity of pathogenic viral, bacterial, and eukaryotic proteins and peptides displayed on the nanoparticles. Recent improvements in genetic tools for bioengineering of GVNPs are discussed, along with future opportunities and challenges for development of vaccines and other applications.

  5. Identification and experimental characterization of an extremophilic brine pool alcohol dehydrogenase from single amplified genomes

    KAUST Repository

    Grö tzinger, Stefan W.; Karan, Ram; Strillinger, Eva; Bader, Stefan; Frank, Annika; Al Rowaihi, Israa; Akal, Anastassja; Wackerow, Wiebke; Archer, John A.C.; Rueping, Magnus; Weuster-Botz, Dirk; Groll, Michael; Eppinger, Jö rg; Arold, Stefan T.

    2017-01-01

    Because only 0.01% of prokaryotic genospecies can be cultured and in situ observations are often impracticable, culture-independent methods are required to understand microbial life and harness potential applications of microbes. Here, we report a methodology for the production of proteins with desired functions based on single amplified genomes (SAGs) from unculturable species. We use this method to resurrect an alcohol dehydrogenase (ADH/D1) from an uncharacterized halo-thermophilic archaeon collected from a brine pool at the bottom of the Red Sea. Our crystal structure of 5,6-dihydroxy NADPH-bound ADH/D1 combined with biochemical analyses reveal the molecular features of its halo-thermophily, its unique habitat adaptations, and its possible reaction mechanism for atypical oxygen activation. Our strategy offers a general guide for using SAGs as a source for scientific and industrial investigations of ‘microbial dark matter’.

  6. 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.)

  7. 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...

  8. 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...

  9. Dicty_cDB: Contig-U05908-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available uilegia formo... 44 4.4 1 ( DR944473 ) EST1136012 Aquilegia cDNA library Aquilegia formo... 44 4.4 1 >( AU261433 ) Dic...i strain CBS... 46 4e-04 AM114193_389( AM114193 |pid:none) Uncultured methanogenic archaeon... 46 5e-04 CP00... SP6 en... 44 4.4 1 ( DT754699 ) EST1188548 Aquilegia cDNA library Aquilegia formo... 44 4.4 1 ( DT748890 ) ...EST1182739 Aquilegia cDNA library Aquilegia formo... 44 4.4 1 ( DT743646 ) EST1177495 Aquilegia cDNA library... Aquilegia formo... 44 4.4 1 ( DT742533 ) EST1176382 Aquilegia cDNA library Aquil

  10. The 1.8-Å crystal structure of the N-terminal domain of an archaeal MCM as a right-handed filament.

    Science.gov (United States)

    Fu, Yang; Slaymaker, Ian M; Wang, Junfeng; Wang, Ganggang; Chen, Xiaojiang S

    2014-04-03

    Mini-chromosome maintenance (MCM) proteins are the replicative helicase necessary for DNA replication in both eukarya and archaea. Most of archaea only have one MCM gene. Here, we report a 1.8-Å crystal structure of the N-terminal MCM from the archaeon Thermoplasma acidophilum (tapMCM). In the structure, the MCM N-terminus forms a right-handed filament that contains six subunits in each turn, with a diameter of 25Å of the central channel opening. The inner surface is highly positively charged, indicating DNA binding. This filament structure with six subunits per turn may also suggests a potential role for an open-ring structure for hexameric MCM and dynamic conformational changes in initiation and elongation stages of DNA replication. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. 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...... of different types of mutation and possible rearrangements that can occur in the genome, the pyrEF locus was examined for mutations that were isolated after selection with 5-fluoroorotic acid. About two-thirds of the 130 mutations resulted from insertions of mobile elements, including insertion sequence (IS...... deletions, insertions, and a duplication, were observed, and about one-fifth of the mutations occurred elsewhere in the genome, possibly in an orotate transporter gene. One mutant exhibited a 5-kb genomic rearrangement at the pyrEF locus involving a two-step IS element-dependent reaction, and its boundaries...

  12. In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag

    DEFF Research Database (Denmark)

    Visone, Valeria; Han, Wenyuan; Perugino, Giuseppe

    2017-01-01

    Protein imaging, allowing a wide variety of biological studies both in vitro and in vivo, is of great importance in modern biology. Protein and peptide tags fused to proteins of interest provide the opportunity to elucidate protein location and functions, detect protein-protein interactions, and ......, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms....... to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase endowed with positive supercoiling activity...

  13. Cryo-EM structure of the archaeal 50S ribosomal subunit in complex with initiation factor 6 and implications for ribosome evolution

    DEFF Research Database (Denmark)

    Greber, Basil J; Boehringer, Daniel; Godinic-Mikulcic, Vlatka

    2012-01-01

    additional components of the translation machinery with eukaryotes that are absent in bacteria. One of these translation factors is initiation factor 6 (IF6), which associates with the large ribosomal subunit. We have reconstructed the 50S ribosomal subunit from the archaeon Methanothermobacter...... between this archaeal ribosome and eukaryotic ribosomes but are mostly absent in bacteria and in some archaeal lineages. Furthermore, the structure reveals that, in spite of highly divergent evolutionary trajectories of the ribosomal particle and the acquisition of novel functions of IF6 in eukaryotes......, the molecular binding of IF6 on the ribosome is conserved between eukaryotes and archaea. The structure also provides a snapshot of the reductive evolution of the archaeal ribosome and offers new insights into the evolution of the translation system in archaea....

  14. Identification and experimental characterization of an extremophilic brine pool alcohol dehydrogenase from single amplified genomes

    KAUST Repository

    Grötzinger, Stefan W.

    2017-11-30

    Because only 0.01% of prokaryotic genospecies can be cultured and in situ observations are often impracticable, culture-independent methods are required to understand microbial life and harness potential applications of microbes. Here, we report a methodology for the production of proteins with desired functions based on single amplified genomes (SAGs) from unculturable species. We use this method to resurrect an alcohol dehydrogenase (ADH/D1) from an uncharacterized halo-thermophilic archaeon collected from a brine pool at the bottom of the Red Sea. Our crystal structure of 5,6-dihydroxy NADPH-bound ADH/D1 combined with biochemical analyses reveal the molecular features of its halo-thermophily, its unique habitat adaptations, and its possible reaction mechanism for atypical oxygen activation. Our strategy offers a general guide for using SAGs as a source for scientific and industrial investigations of ‘microbial dark matter’.

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

    KAUST Repository

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

    2016-01-01

    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.

  16. The history of aerobic ammonia oxidizers: from the first discoveries to today.

    Science.gov (United States)

    Monteiro, Maria; Séneca, Joana; Magalhães, Catarina

    2014-07-01

    Nitrification, the oxidation of ammonia to nitrite and nitrate, has long been considered a central biological process in the global nitrogen cycle, with its first description dated 133 years ago. Until 2005, bacteria were considered the only organisms capable of nitrification. However, the recent discovery of a chemoautotrophic ammonia-oxidizing archaeon, Nitrosopumilus maritimus, changed our concept of the range of organisms involved in nitrification, highlighting the importance of ammonia-oxidizing archaea (AOA) as potential players in global biogeochemical nitrogen transformations. The uniqueness of these archaea justified the creation of a novel archaeal phylum, Thaumarchaeota. These recent discoveries increased the global scientific interest within the microbial ecology society and have triggered an analysis of the importance of bacterial vs archaeal ammonia oxidation in a wide range of natural ecosystems. In this mini review we provide a chronological perspective of the current knowledge on the ammonia oxidation pathway of nitrification, based on the main physiological, ecological and genomic discoveries.

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

    Science.gov (United States)

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

    2016-05-01

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

  18. Annotating functional RNAs in genomes using Infernal.

    Science.gov (United States)

    Nawrocki, Eric P

    2014-01-01

    Many different types of functional non-coding RNAs participate in a wide range of important cellular functions but the large majority of these RNAs are not routinely annotated in published genomes. Several programs have been developed for identifying RNAs, including specific tools tailored to a particular RNA family as well as more general ones designed to work for any family. Many of these tools utilize covariance models (CMs), statistical models of the conserved sequence, and structure of an RNA family. In this chapter, as an illustrative example, the Infernal software package and CMs from the Rfam database are used to identify RNAs in the genome of the archaeon Methanobrevibacter ruminantium, uncovering some additional RNAs not present in the genome's initial annotation. Analysis of the results and comparison with family-specific methods demonstrate some important strengths and weaknesses of this general approach.

  19. Model Construction and Analysis of Respiration in Halobacterium salinarum.

    Science.gov (United States)

    Talaue, Cherryl O; del Rosario, Ricardo C H; Pfeiffer, Friedhelm; Mendoza, Eduardo R; Oesterhelt, Dieter

    2016-01-01

    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.

  20. Insights into archaeal evolution and symbiosis from the genomes of a nanoarchaeon and its inferred crenarchaeal host from Obsidian Pool, Yellowstone National Park.

    Science.gov (United States)

    Podar, Mircea; Makarova, Kira S; Graham, David E; Wolf, Yuri I; Koonin, Eugene V; Reysenbach, Anna-Louise

    2013-04-22

    A single cultured marine organism, Nanoarchaeum equitans, represents the Nanoarchaeota branch of symbiotic Archaea, with a highly reduced genome and unusual features such as multiple split genes. The first terrestrial hyperthermophilic member of the Nanoarchaeota was collected from Obsidian Pool, a thermal feature in Yellowstone National Park, separated by single cell isolation, and sequenced together with its putative host, a Sulfolobales archaeon. Both the new Nanoarchaeota (Nst1) and N. equitans lack most biosynthetic capabilities, and phylogenetic analysis of ribosomal RNA and protein sequences indicates that the two form a deep-branching archaeal lineage. However, the Nst1 genome is more than 20% larger, and encodes a complete gluconeogenesis pathway as well as the full complement of archaeal flagellum proteins. With a larger genome, a smaller repertoire of split protein encoding genes and no split non-contiguous tRNAs, Nst1 appears to have experienced less severe genome reduction than N. equitans. These findings imply that, rather than representing ancestral characters, the extremely compact genomes and multiple split genes of Nanoarchaeota are derived characters associated with their symbiotic or parasitic lifestyle. The inferred host of Nst1 is potentially autotrophic, with a streamlined genome and simplified central and energetic metabolism as compared to other Sulfolobales. Comparison of the N. equitans and Nst1 genomes suggests that the marine and terrestrial lineages of Nanoarchaeota share a common ancestor that was already a symbiont of another archaeon. The two distinct Nanoarchaeota-host genomic data sets offer novel insights into the evolution of archaeal symbiosis and parasitism, enabling further studies of the cellular and molecular mechanisms of these relationships. This article was reviewed by Patrick Forterre, Bettina Siebers (nominated by Michael Galperin) and Purification Lopez-Garcia.

  1. 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)

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

    2013-01-01

    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 CH4 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. PMID:23183975

  2. Viruses in the Oceanic Basement

    Directory of Open Access Journals (Sweden)

    Olivia D. Nigro

    2017-03-01

    Full Text Available 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.

  3. 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.

  4. 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.

  5. In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag.

    Science.gov (United States)

    Visone, Valeria; Han, Wenyuan; Perugino, Giuseppe; Del Monaco, Giovanni; She, Qunxin; Rossi, Mosè; Valenti, Anna; Ciaramella, Maria

    2017-01-01

    Protein imaging, allowing a wide variety of biological studies both in vitro and in vivo, is of great importance in modern biology. Protein and peptide tags fused to proteins of interest provide the opportunity to elucidate protein location and functions, detect protein-protein interactions, and measure protein activity and kinetics in living cells. Whereas several tags are suitable for protein imaging in mesophilic organisms, the application of this approach to microorganisms living at high temperature has lagged behind. Archaea provide an excellent and unique model for understanding basic cell biology mechanisms. Here, we present the development of a toolkit for protein imaging in the hyperthermophilic archaeon Sulfolobus islandicus. The system relies on a thermostable protein tag (H5) constructed by engineering the alkylguanine-DNA-alkyl-transferase protein of Sulfolobus solfataricus, which can be covalently labeled using a wide range of small molecules. As a suitable host, we constructed, by CRISPR-based genome-editing technology, a S. islandicus mutant strain deleted for the alkylguanine-DNA-alkyl-transferase gene (Δogt). Introduction of a plasmid-borne H5 gene in this strain led to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase endowed with positive supercoiling activity, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms.

  6. Deletion of acetyl-CoA synthetases I and II increases production of 3-hydroxypropionate by the metabolically-engineered hyperthermophile Pyrococcus furiosus.

    Science.gov (United States)

    Thorgersen, Michael P; Lipscomb, Gina L; Schut, Gerrit J; Kelly, Robert M; Adams, Michael W W

    2014-03-01

    The heterotrophic, hyperthermophilic archaeon Pyrococcus furiosus is a new addition to the growing list of genetically-tractable microorganisms suitable for metabolic engineering to produce liquid fuels and industrial chemicals. P. furiosus was recently engineered to generate 3-hydroxypropionate (3-HP) from CO₂ and acetyl-CoA by the heterologous-expression of three enzymes from the CO₂ fixation cycle of the thermoacidophilic archaeon Metallosphaera sedula using a thermally-triggered induction system. The acetyl-CoA for this pathway is generated from glucose catabolism that in wild-type P. furiosus is converted to acetate with concurrent ATP production by the heterotetrameric (α₂β₂) acetyl-CoA synthetase (ACS). Hence ACS in the engineered 3-HP production strain (MW56) competes with the heterologous pathway for acetyl-CoA. Herein we show that strains of MW56 lacking the α-subunit of either of the two ACSs previously characterized from P. furiosus (ACSI and ACSII) exhibit a three-fold increase in specific 3-HP production. The ΔACSIα strain displayed only a minor defect in growth on either maltose or peptides, while no growth defect on these substrates was observed with the ΔACSIIα strain. Deletion of individual and multiple ACS subunits was also shown to decrease CoA release activity for several different CoA ester substrates in addition to acetyl-CoA, information that will be extremely useful for future metabolic engineering endeavors in P. furiosus. Copyright © 2014 International Metabolic Engineering Society. All rights reserved.

  7. Meteorite-associated growth physiology of the iron oxidising extremophile Metallosphaera sedula

    Science.gov (United States)

    Zebec, Ziga; Rittmann, Simon; Schleper, Christa; Milojevic, Tetyana

    2015-04-01

    Extremophiles cherry pick the habitats at the edge of living limits, shaping the life under inhospitable conditions. Such microbes are characterised by functional capabilities required for survival in harsh and extreme environments. These living entities serve as models for a life on early Earth with its severe and ancient habitats, providing an understanding of the extent of biology on Earth, and enabling a discovery of its wider presence in the universe. The Fe-oxidising archaeon Metallosphaera sedula inhabits extreme environments, flourishing in hot acid and exhibiting unusual heavy-metal resistance. This chemolithotrophic archaea thrives at 73°C and pH 2, utilizing energy derived from metal oxidation. Iron and sulphur compounds are preferentially required for its growth. Owing its physiological versatility, M. sedula is able to grow efficiently chemoauto- and chemomixotrophically using pyrite, chalcopyrite or sulfur compounds as an energy source and can also grow heterotrophically using yeast extract representing an excellent model organism for basic research into bioleaching processes. Stimulated by its exceptional physiological properties, we have set out to assess the survival potential of M. sedula by investigating the viability of this archaeon living on and interacting with minerals of non-terrestrial origin. Initial results demonstrate that the iron oxidising thermoacidophile M. sedula utilizes metals trapped within stony meteorites as the most preferential energy source, resulting in i) one order of magnitude higher growth rate comparatively to the other energy substrates of terrestrial origin (sulfide ores, hydrogen, iron sulfate) ii) a drastic shift in a lower temperature limit for this microbe. A comprehensive complex of genetic, biochemical and geochemical techniques will be applied to analyze microbial-meteorite liaison.

  8. 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.

  9. Viruses in the Oceanic Basement.

    Science.gov (United States)

    Nigro, Olivia D; Jungbluth, Sean P; Lin, Huei-Ting; Hsieh, Chih-Chiang; Miranda, Jaclyn A; Schvarcz, Christopher R; Rappé, Michael S; Steward, Grieg F

    2017-03-07

    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 × 10 5 to 2 × 10 5  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. IMPORTANCE The hydrothermally active ocean basement is voluminous and likely provided conditions critical to the origins of life, but the microbiology of this vast habitat is not

  10. Structures of three members of Pfam PF02663 (FmdE) implicated in microbial methanogenesis reveal a conserved α+β core domain and an auxiliary C-terminal treble-clef zinc finger

    International Nuclear Information System (INIS)

    Axelrod, Herbert L.; Das, Debanu; Abdubek, Polat; Astakhova, Tamara; Bakolitsa, Constantina; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Farr, Carol L.; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Lam, Winnie W.; Marciano, David; McMullan, Daniel; Miller, Mitchell D.; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; Bedem, Henry van den; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    The first structures from the FmdE Pfam family (PF02663) reveal that some members of this family form tightly intertwined dimers consisting of two domains (N-terminal α+β core and C-terminal zinc-finger domains), whereas others contain only the core domain. The presence of the zinc-finger domain suggests that some members of this family may perform functions associated with transcriptional regulation, protein–protein interaction, RNA binding or metal-ion sensing. Examination of the genomic context for members of the FmdE Pfam family (PF02663), such as the protein encoded by the fmdE gene from the methanogenic archaeon Methanobacterium thermoautotrophicum, indicates that 13 of them are co-transcribed with genes encoding subunits of molybdenum formylmethanofuran dehydrogenase (EC 1.2.99.5), an enzyme that is involved in microbial methane production. Here, the first crystal structures from PF02663 are described, representing two bacterial and one archaeal species: B8FYU2-DESHY from the anaerobic dehalogenating bacterium Desulfitobacterium hafniense DCB-2, Q2LQ23-SYNAS from the syntrophic bacterium Syntrophus aciditrophicus SB and Q9HJ63-THEAC from the thermoacidophilic archaeon Thermoplasma acidophilum. Two of these proteins, Q9HJ63-THEAC and Q2LQ23-SYNAS, contain two domains: an N-terminal thioredoxin-like α+β core domain (NTD) consisting of a five-stranded, mixed β-sheet flanked by several α-helices and a C-terminal zinc-finger domain (CTD). B8FYU2-DESHY, on the other hand, is composed solely of the NTD. The CTD of Q9HJ63-THEAC and Q2LQ23-SYNAS is best characterized as a treble-clef zinc finger. Two significant structural differences between Q9HJ63-THEAC and Q2LQ23-SYNAS involve their metal binding. First, zinc is bound to the putative active site on the NTD of Q9HJ63-THEAC, but is absent from the NTD of Q2LQ23-SYNAS. Second, whereas the structure of the CTD of Q2LQ23-SYNAS shows four Cys side chains within coordination distance of the Zn atom, the structure

  11. Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus.

    Science.gov (United States)

    Lian, Hong; Zeldes, Benjamin M; Lipscomb, Gina L; Hawkins, Aaron B; Han, Yejun; Loder, Andrew J; Nishiyama, Declan; Adams, Michael W W; Kelly, Robert M

    2016-12-01

    Acetyl-Coenzyme A carboxylase (ACC), malonyl-CoA reductase (MCR), and malonic semialdehyde reductase (MRS) convert HCO 3 - and acetyl-CoA into 3-hydroxypropionate (3HP) in the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle resident in the extremely thermoacidophilic archaeon Metallosphaera sedula. These three enzymes, when introduced into the hyperthermophilic archaeon Pyrococcus furiosus, enable production of 3HP from maltose and CO 2 . Sub-optimal function of ACC was hypothesized to be limiting for production of 3HP, so accessory enzymes carbonic anhydrase (CA) and biotin protein ligase (BPL) from M. sedula were produced recombinantly in Escherichia coli to assess their function. P. furiosus lacks a native, functional CA, while the M. sedula CA (Msed_0390) has a specific activity comparable to other microbial versions of this enzyme. M. sedula BPL (Msed_2010) was shown to biotinylate the β-subunit (biotin carboxyl carrier protein) of the ACC in vitro. Since the native BPLs in E. coli and P. furiosus may not adequately biotinylate the M. sedula ACC, the carboxylase was produced in P. furiosus by co-expression with the M. sedula BPL. The baseline production strain, containing only the ACC, MCR, and MSR, grown in a CO 2 -sparged bioreactor reached titers of approximately 40 mg/L 3HP. Strains in which either the CA or BPL accessory enzyme from M. sedula was added to the pathway resulted in improved titers, 120 or 370 mg/L, respectively. The addition of both M. sedula CA and BPL, however, yielded intermediate titers of 3HP (240 mg/L), indicating that the effects of CA and BPL on the engineered 3HP pathway were not additive, possible reasons for which are discussed. While further efforts to improve 3HP production by regulating gene dosage, improving carbon flux and optimizing bioreactor operation are needed, these results illustrate the ancillary benefits of accessory enzymes for incorporating CO 2 into 3HP production in metabolically engineered P

  12. Solution Structure of Pfu RPP21, a Component of the Archaeal RNase P Holoenzyme, and Interactions with its RPP29 Protein Partner

    Science.gov (United States)

    Amero, Carlos D; Boomershine, William P; Xu, Yiren; Foster, Mark

    2009-01-01

    RNase P is the ubiquitous ribonucleoprotein metalloenzyme responsible for cleaving the 5′-leader sequence of precursor tRNAs during their maturation. While the RNA subunit is catalytically active on its own at high monovalent and divalent ion concentration, four proteins subunits are associated with archaeal RNase P activity in vivo: RPP21, RPP29, RPP30 and POP5. These proteins have been shown to function in pairs: RPP21-RPP29 and POP5-RPP30. We have determined the solution structure of RPP21 from the hyperthermophilic archaeon Pyrococcus furiosus (Pfu) using conventional and paramagnetic NMR techniques. Pfu RPP21 in solution consists of an unstructured N-terminus, two alpha helices, a zinc binding motif, and an unstructured C-terminus. Moreover, we have used chemical shift perturbations to characterize the interaction of RPP21 with Pfu RPP29. The data show that the primary contact with RPP29 is localized to the two helices of RPP21. This information represents a fundamental step towards understanding structure-function relationships of the archaeal RNase P holoenzyme. PMID:18922021

  13. Construction, Expression, and Characterization of Recombinant Pfu DNA Polymerase in Escherichia coli.

    Science.gov (United States)

    Zheng, Wenjun; Wang, Qingsong; Bi, Qun

    2016-04-01

    Pfu DNA polymerase (Pfu) is a DNA polymerase isolated from the hyperthermophilic archaeon Pyrococcus furiosus. With its excellent thermostability and high fidelity, Pfu is well known as one of the enzymes widely used in the polymerase chain reaction. In this study, the recombinant plasmid pLysS His6-tagged Pfu-pET28a was constructed. His-tagged Pfu was expressed in Escherichia coli BL21 (DE3) competent cells and then successfully purified with the ÄKTAprime plus compact one-step purification system by Ni(2+) chelating affinity chromatography after optimization of the purification conditions. The authenticity of the purified Pfu was further confirmed by peptide mass fingerprinting. A bio-assay indicated that its activity in the polymerase chain reaction was equivalent to that of commercial Pfu and its isoelectric point was found to be between 6.85 and 7.35. These results will be useful for further studies on Pfu and its wide application in the future.

  14. Biological methane production under putative Enceladus-like conditions.

    Science.gov (United States)

    Taubner, Ruth-Sophie; Pappenreiter, Patricia; Zwicker, Jennifer; Smrzka, Daniel; Pruckner, Christian; Kolar, Philipp; Bernacchi, Sébastien; Seifert, Arne H; Krajete, Alexander; Bach, Wolfgang; Peckmann, Jörn; Paulik, Christian; Firneis, Maria G; Schleper, Christa; Rittmann, Simon K-M R

    2018-02-27

    The detection of silica-rich dust particles, as an indication for ongoing hydrothermal activity, and the presence of water and organic molecules in the plume of Enceladus, have made Saturn's icy moon a hot spot in the search for potential extraterrestrial life. Methanogenic archaea are among the organisms that could potentially thrive under the predicted conditions on Enceladus, considering that both molecular hydrogen (H 2 ) and methane (CH 4 ) have been detected in the plume. Here we show that a methanogenic archaeon, Methanothermococcus okinawensis, can produce CH 4 under physicochemical conditions extrapolated for Enceladus. Up to 72% carbon dioxide to CH 4 conversion is reached at 50 bar in the presence of potential inhibitors. Furthermore, kinetic and thermodynamic computations of low-temperature serpentinization indicate that there may be sufficient H 2 gas production to serve as a substrate for CH 4 production on Enceladus. We conclude that some of the CH 4 detected in the plume of Enceladus might, in principle, be produced by methanogens.

  15. Every OGT Is Illuminated … by Fluorescent and Synchrotron Lights

    Directory of Open Access Journals (Sweden)

    Riccardo Miggiano

    2017-12-01

    Full Text Available O6-DNA-alkyl-guanine-DNA-alkyl-transferases (OGTs are evolutionarily conserved, unique proteins that repair alkylation lesions in DNA in a single step reaction. Alkylating agents are environmental pollutants as well as by-products of cellular reactions, but are also very effective chemotherapeutic drugs. OGTs are major players in counteracting the effects of such agents, thus their action in turn affects genome integrity, survival of organisms under challenging conditions and response to chemotherapy. Numerous studies on OGTs from eukaryotes, bacteria and archaea have been reported, highlighting amazing features that make OGTs unique proteins in their reaction mechanism as well as post-reaction fate. This review reports recent functional and structural data on two prokaryotic OGTs, from the pathogenic bacterium Mycobacterium tuberculosis and the hyperthermophilic archaeon Sulfolobus solfataricus, respectively. These studies provided insight in the role of OGTs in the biology of these microorganisms, but also important hints useful to understand the general properties of this class of proteins.

  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. Microbial diversity from chlorophyll maximum, oxygen minimum and bottom zones in the southwestern Atlantic Ocean

    Science.gov (United States)

    Medina-Silva, Renata; de Oliveira, Rafael R.; Pivel, Maria A. G.; Borges, Luiz G. A.; Simão, Taiz L. L.; Pereira, Leandro M.; Trindade, Fernanda J.; Augustin, Adolpho H.; Valdez, Fernanda P.; Eizirik, Eduardo; Utz, Laura R. P.; Groposo, Claudia; Miller, Dennis J.; Viana, Adriano R.; Ketzer, João M. M.; Giongo, Adriana

    2018-02-01

    Conspicuous physicochemical vertical stratification in the deep sea is one of the main forces driving microbial diversity in the oceans. Oxygen and sunlight availability are key factors promoting microbial diversity throughout the water column. Ocean currents also play a major role in the physicochemical stratification, carrying oxygen down to deeper zones as well as moving deeper water masses up towards shallower depths. Water samples within a 50-km radius in a pockmark location of the southwestern Atlantic Ocean were collected and the prokaryotic communities from different water depths - chlorophyll maximum, oxygen minimum and deep-sea bottom (down to 1355 m) - were described. At phylum level, Proteobacteria were the most frequent in all water depths, Cyanobacteria were statistically more frequent in chlorophyll maximum zone, while Thaumarchaeota were significantly more abundant in both oxygen minimum and bottom waters. The most frequent microorganism in the chlorophyll maximum and oxygen minimum zones was a Pelagibacteraceae operational taxonomic unit (OTU). At the bottom, the most abundant genus was the archaeon Nitrosopumilus. Beta diversity analysis of the 16S rRNA gene sequencing data uncovered in this study shows high spatial heterogeneity among water zones communities. Our data brings important contribution for the characterisation of oceanic microbial diversity, as it consists of the first description of prokaryotic communities occurring in different oceanic water zones in the southwestern Atlantic Ocean.

  18. VapC toxins drive cellular dormancy under uranium stress for the extreme thermoacidophile Metallosphaera prunae.

    Science.gov (United States)

    Mukherjee, Arpan; Wheaton, Garrett H; Counts, James A; Ijeomah, Brenda; Desai, Jigar; Kelly, Robert M

    2017-07-01

    When abruptly exposed to toxic levels of hexavalent uranium, the extremely thermoacidophilic archaeon Metallosphaera prunae, originally isolated from an abandoned uranium mine, ceased to grow, and concomitantly exhibited heightened levels of cytosolic ribonuclease activity that corresponded to substantial degradation of cellular RNA. The M. prunae transcriptome during 'uranium-shock' implicated VapC toxins as possible causative agents of the observed RNA degradation. Identifiable VapC toxins and PIN-domain proteins encoded in the M. prunae genome were produced and characterized, three of which (VapC4, VapC7, VapC8) substantially degraded M. prunae rRNA in vitro. RNA cleavage specificity for these VapCs mapped to motifs within M. prunae rRNA. Furthermore, based on frequency of cleavage sequences, putative target mRNAs for these VapCs were identified; these were closely associated with translation, transcription, and replication. It is interesting to note that Metallosphaera sedula, a member of the same genus and which has a nearly identical genome sequence but not isolated from a uranium-rich biotope, showed no evidence of dormancy when exposed to this metal. M. prunae utilizes VapC toxins for post-transcriptional regulation under uranium stress to enter a cellular dormant state, thereby providing an adaptive response to what would otherwise be a deleterious environmental perturbation. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  19. 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.

  20. 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.

  1. An archaebacterial homologue of the essential eubacterial cell division protein FtsZ.

    Science.gov (United States)

    Baumann, P; Jackson, S P

    1996-06-25

    Life falls into three fundamental domains--Archaea, Bacteria, and Eucarya (formerly archaebacteria, eubacteria, and eukaryotes,. respectively). Though Archaea lack nuclei and share many morphological features with Bacteria, molecular analyses, principally of the transcription and translation machineries, have suggested that Archaea are more related to Eucarya than to Bacteria. Currently, little is known about the archaeal cell division apparatus. In Bacteria, a crucial component of the cell division machinery is FtsZ, a GTPase that localizes to a ring at the site of septation. Interestingly, FtsZ is distantly related in sequence to eukaryotic tubulins, which also interact with GTP and are components of the eukaryotic cell cytoskeleton. By screening for the ability to bind radiolabeled nucleotides, we have identified a protein of the hyperthermophilic archaeon Pyrococcus woesei that interacts tightly and specifically with GTP. Furthermore, through screening an expression library of P. woesei genomic DNA, we have cloned the gene encoding this protein. Sequence comparisons reveal that the P. woesei GTP-binding protein is strikingly related in sequence to eubacterial FtsZ and is marginally more similar to eukaryotic tubulins than are bacterial FtsZ proteins. Phylogenetic analyses reinforce the notion that there is an evolutionary linkage between FtsZ and tubulins. These findings suggest that the archaeal cell division apparatus may be fundamentally similar to that of Bacteria and lead us to consider the evolutionary relationships between Archaea, Bacteria, and Eucarya.

  2. The Adaptive Immune System of Haloferax volcanii

    Directory of Open Access Journals (Sweden)

    Lisa-Katharina Maier

    2015-02-01

    Full Text Available To fight off invading genetic elements, prokaryotes have developed an elaborate defence system that is both adaptable and heritable—the CRISPR-Cas system (CRISPR is short for: clustered regularly interspaced short palindromic repeats and Cas: CRISPR associated. Comprised of proteins and multiple small RNAs, this prokaryotic defence system is present in 90% of archaeal and 40% of bacterial species, and enables foreign intruders to be eliminated in a sequence-specific manner. There are three major types (I–III and at least 14 subtypes of this system, with only some of the subtypes having been analysed in detail, and many aspects of the defence reaction remaining to be elucidated. Few archaeal examples have so far been analysed. Here we summarize the characteristics of the CRISPR-Cas system of Haloferax volcanii, an extremely halophilic archaeon originally isolated from the Dead Sea. It carries a single CRISPR-Cas system of type I-B, with a Cascade like complex composed of Cas proteins Cas5, Cas6b and Cas7. Cas6b is essential for CRISPR RNA (crRNA maturation but is otherwise not required for the defence reaction. A systematic search revealed that six protospacer adjacent motif (PAM sequences are recognised by the Haloferax defence system. For successful invader recognition, a non-contiguous seed sequence of 10 base-pairs between the crRNA and the invader is required.

  3. 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.

  4. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. CRISPR-Cas type I-A Cascade complex couples viral infection surveillance to host transcriptional regulation in the dependence of Csa3b.

    Science.gov (United States)

    He, Fei; Vestergaard, Gisle; Peng, Wenfang; She, Qunxin; Peng, Xu

    2017-02-28

    CRISPR-Cas (clustered regularly interspaced short palindromic repeats and the associated genes) constitute adaptive immune systems in bacteria and archaea and they provide sequence specific immunity against foreign nucleic acids. CRISPR-Cas systems are activated by viral infection. However, little is known about how CRISPR-Cas systems are activated in response to viral infection or how their expression is controlled in the absence of viral infection. Here, we demonstrate that both the transcriptional regulator Csa3b, and the type I-A interference complex Cascade, are required to transcriptionally repress the interference gene cassette in the archaeon Sulfolobus. Csa3b binds to two palindromic repeat sites in the promoter region of the cassette and facilitates binding of the Cascade to the promoter region. Upon viral infection, loading of Cascade complexes onto crRNA-matching protospacers leads to relief of the transcriptional repression. Our data demonstrate a mechanism coupling CRISPR-Cas surveillance of protospacers to transcriptional regulation of the interference gene cassette thereby allowing a fast response to viral infection. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Exploring Fingerprints of the Extreme Thermoacidophile Metallosphaera sedula Grown on Synthetic Martian Regolith Materials as the Sole Energy Sources

    Directory of Open Access Journals (Sweden)

    Denise Kölbl

    2017-10-01

    Full Text Available The biology of metal transforming microorganisms is of a fundamental and applied importance for our understanding of past and present biogeochemical processes on Earth and in the Universe. The extreme thermoacidophile Metallosphaera sedula is a metal mobilizing archaeon, which thrives in hot acid environments (optimal growth at 74°C and pH 2.0 and utilizes energy from the oxidation of reduced metal inorganic sources. These characteristics of M. sedula make it an ideal organism to further our knowledge of the biogeochemical processes of possible life on extraterrestrial planetary bodies. Exploring the viability and metal extraction capacity of M. sedula living on and interacting with synthetic extraterrestrial minerals, we show that M. sedula utilizes metals trapped in the Martian regolith simulants (JSC Mars 1A; P-MRS; S-MRS; MRS07/52 as the sole energy sources. The obtained set of microbiological and mineralogical data suggests that M. sedula actively colonizes synthetic Martian regolith materials and releases free soluble metals. The surface of bioprocessed Martian regolith simulants is analyzed for specific mineralogical fingerprints left upon M. sedula growth. The obtained results provide insights of biomining of extraterrestrial material as well as of the detection of biosignatures implementing in life search missions.

  7. Structure of D-tagatose 3-epimerase-like protein from Methanocaldococcus jannaschii.

    Science.gov (United States)

    Uechi, Keiko; Takata, Goro; Yoneda, Kazunari; Ohshima, Toshihisa; Sakuraba, Haruhiko

    2014-07-01

    The crystal structure of a D-tagatose 3-epimerase-like protein (MJ1311p) encoded by a hypothetical open reading frame, MJ1311, in the genome of the hyperthermophilic archaeon Methanocaldococcus jannaschii was determined at a resolution of 2.64 Å. The asymmetric unit contained two homologous subunits, and the dimer was generated by twofold symmetry. The overall fold of the subunit proved to be similar to those of the D-tagatose 3-epimerase from Pseudomonas cichorii and the D-psicose 3-epimerases from Agrobacterium tumefaciens and Clostridium cellulolyticum. However, the situation at the subunit-subunit interface differed substantially from that in D-tagatose 3-epimerase family enzymes. In MJ1311p, Glu125, Leu126 and Trp127 from one subunit were found to be located over the metal-ion-binding site of the other subunit and appeared to contribute to the active site, narrowing the substrate-binding cleft. Moreover, the nine residues comprising a trinuclear zinc centre in endonuclease IV were found to be strictly conserved in MJ1311p, although a distinct groove involved in DNA binding was not present. These findings indicate that the active-site architecture of MJ1311p is quite unique and is substantially different from those of D-tagatose 3-epimerase family enzymes and endonuclease IV.

  8. Spatial organization of the gastrointestinal microbiota in urban Canada geese

    Science.gov (United States)

    Drovetski, Sergei V.; O'Mahoney, Michael; Ransome, Emma J.; Matterson, Kenan O.; Lim, Haw Chuan; Chesser, Terry; Graves, Gary R.

    2018-01-01

    Recent reviews identified the reliance on fecal or cloacal samples as a significant limitation hindering our understanding of the avian gastrointestinal (gut) microbiota and its function. We investigated the microbiota of the esophagus, duodenum, cecum, and colon of a wild urban population of Canada goose (Branta canadensis). From a population sample of 30 individuals, we sequenced the V4 region of the 16S SSU rRNA on an Illumina MiSeq and obtained 8,628,751 sequences with a median of 76,529 per sample. These sequences were assigned to 420 bacterial OTUs and a single archaeon. Firmicutes, Proteobacteria, and Bacteroidetes accounted for 90% of all sequences. Microbiotas from the four gut regions differed significantly in their richness, composition, and variability among individuals. Microbial communities of the esophagus were the most distinctive whereas those of the colon were the least distinctive, reflecting the physical downstream mixing of regional microbiotas. The downstream mixing of regional microbiotas was also responsible for the majority of observed co-occurrence patterns among microbial families. Our results indicate that fecal and cloacal samples inadequately represent the complex patterns of richness, composition, and variability of the gut microbiota and obscure patterns of co-occurrence of microbial lineages.

  9. A system-level model for the microbial regulatory genome.

    Science.gov (United States)

    Brooks, Aaron N; Reiss, David J; Allard, Antoine; Wu, Wei-Ju; Salvanha, Diego M; Plaisier, Christopher L; Chandrasekaran, Sriram; Pan, Min; Kaur, Amardeep; Baliga, Nitin S

    2014-07-15

    Microbes can tailor transcriptional responses to diverse environmental challenges despite having streamlined genomes and a limited number of regulators. Here, we present data-driven models that capture the dynamic interplay of the environment and genome-encoded regulatory programs of two types of prokaryotes: Escherichia coli (a bacterium) and Halobacterium salinarum (an archaeon). The models reveal how the genome-wide distributions of cis-acting gene regulatory elements and the conditional influences of transcription factors at each of those elements encode programs for eliciting a wide array of environment-specific responses. We demonstrate how these programs partition transcriptional regulation of genes within regulons and operons to re-organize gene-gene functional associations in each environment. The models capture fitness-relevant co-regulation by different transcriptional control mechanisms acting across the entire genome, to define a generalized, system-level organizing principle for prokaryotic gene regulatory networks that goes well beyond existing paradigms of gene regulation. An online resource (http://egrin2.systemsbiology.net) has been developed to facilitate multiscale exploration of conditional gene regulation in the two prokaryotes. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

  10. Identification and characterization of an archaeal ketopantoate reductase and its involvement in regulation of coenzyme A biosynthesis.

    Science.gov (United States)

    Tomita, Hiroya; Imanaka, Tadayuki; Atomi, Haruyuki

    2013-10-01

    Coenzyme A (CoA) biosynthesis in bacteria and eukaryotes is regulated primarily by feedback inhibition towards pantothenate kinase (PanK). As most archaea utilize a modified route for CoA biosynthesis and do not harbour PanK, the mechanisms governing regulation of CoA biosynthesis are unknown. Here we performed genetic and biochemical studies on the ketopantoate reductase (KPR) from the hyperthermophilic archaeon Thermococcus kodakarensis. KPR catalyses the second step in CoA biosynthesis, the reduction of 2-oxopantoate to pantoate. Gene disruption of TK1968, whose product was 20-29% identical to previously characterized KPRs from bacteria/eukaryotes, resulted in a strain with growth defects that were complemented by addition of pantoate. The TK1968 protein (Tk-KPR) displayed reductase activity specific for 2-oxopantoate and preferred NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH-dependent enzymes. Tk-KPR activity decreased dramatically in the presence of CoA and KPR activity in cell-free extracts was also inhibited by CoA. Kinetic studies indicated that CoA inhibits KPR by competing with NADH. Inhibition of ketopantoate hydroxymethyltransferase, the first enzyme of the pathway, by CoA was not observed. Our results suggest that CoA biosynthesis in T. kodakarensis is regulated by feedback inhibition of KPR, providing a feasible regulation mechanism of CoA biosynthesis in archaea. © 2013 John Wiley & Sons Ltd.

  11. The impact of genomics on research in diversity and evolution of archaea.

    Science.gov (United States)

    Mardanov, A V; Ravin, N V

    2012-08-01

    Since the definition of archaea as a separate domain of life along with bacteria and eukaryotes, they have become one of the most interesting objects of modern microbiology, molecular biology, and biochemistry. Sequencing and analysis of archaeal genomes were especially important for studies on archaea because of a limited availability of genetic tools for the majority of these microorganisms and problems associated with their cultivation. Fifteen years since the publication of the first genome of an archaeon, more than one hundred complete genome sequences of representatives of different phylogenetic groups have been determined. Analysis of these genomes has expanded our knowledge of biology of archaea, their diversity and evolution, and allowed identification and characterization of new deep phylogenetic lineages of archaea. The development of genome technologies has allowed sequencing the genomes of uncultivated archaea directly from enrichment cultures, metagenomic samples, and even from single cells. Insights have been gained into the evolution of key biochemical processes in archaea, such as cell division and DNA replication, the role of horizontal gene transfer in the evolution of archaea, and new relationships between archaea and eukaryotes have been revealed.

  12. A functional endonuclease Q exists in the bacterial domain: identification and characterization of endonuclease Q from Bacillus pumilus.

    Science.gov (United States)

    Shiraishi, Miyako; Ishino, Sonoko; Cann, Isaac; Ishino, Yoshizumi

    2017-05-01

    DNA base deamination occurs spontaneously under physiological conditions and is promoted by high temperature. Therefore, hyperthermophiles are expected to have efficient repair systems of the deaminated bases in their genomes. Endonuclease Q (EndoQ) was originally identified from the hyperthermophlic archaeon, Pyrococcus furiosus, as a hypoxanthine-specific endonuclease recently. Further biochemical analyses revealed that EndoQ also recognizes uracil, xanthine, and the AP site in DNA, and is probably involved in a specific repair process for damaged bases. Initial phylogenetic analysis showed that an EndoQ homolog is found only in the Thermococcales and some of the methanogens in Archaea, and is not present in most members of the domains Bacteria and Eukarya. A better understanding of the distribution of the EndoQ-mediated repair system is, therefore, of evolutionary interest. We showed here that an EndoQ-like polypeptide from Bacillus pumilus, belonging to the bacterial domain, is functional and has similar properties with the archaeal EndoQs.

  13. A Single-Culture Bioprocess of Methanothermobacter thermautotrophicus to Upgrade Digester Biogas by CO2-to-CH4 Conversion with H2

    Science.gov (United States)

    Martin, Matthew R.; Fornero, Jeffrey J.; Angenent, Largus T.

    2013-01-01

    We optimized and tested a postbioprocessing step with a single-culture archaeon to upgrade biogas (i.e., increase methane content) from anaerobic digesters via conversion of CO2 into CH4 by feeding H2 gas. We optimized a culture of the thermophilic methanogen Methanothermobacter thermautotrophicus using: (1) a synthetic H2/CO2 mixture; (2) the same mixture with pressurization; (3) a synthetic biogas with different CH4 contents and H2; and (4) an industrial, untreated biogas and H2. A 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. PMID:24194675

  14. Role of Mn2+ and compatible solutes in the radiation resistance of thermophilic bacteria and archaea.

    Science.gov (United States)

    Webb, Kimberly M; DiRuggiero, Jocelyne

    2012-01-01

    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 Mn(2+)-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.

  15. Engineering substrate promiscuity in halophilic alcohol dehydrogenase (HvADH2 by in silico design.

    Directory of Open Access Journals (Sweden)

    Jennifer Cassidy

    Full Text Available An alcohol dehydrogenase from the halophilic archaeon Haloferax volcanii (HvADH2 has been engineered by rational design to broaden its substrate scope towards the conversion of a range of aromatic substrates, including flurbiprofenol, that is an intermediate of the non-steroidal anti-inflammatory drug, flurbiprofen. Wild-type HvADH2 showed minimal activity with flurbiprofenol (11.1 mU/mg. A homology model of HvADH2 was built and docking experiments with this substrate revealed that the biphenyl rings of flurbiprofenol formed strong interactions with residues F85 and F108, preventing its optimal binding in the active site. Mutations at position 85 however did not increase activity. Site directed mutagenesis at position F108 allowed the identification of three variants showing a significant (up to 2.3-fold enhancement of activity towards flurbiprofenol, when compared to wild-type HvADH2. Interestingly, F108G variant did not show the classic inhibition in the presence of (R-enantiomer when tested with rac-1-phenylethanol, underling its potential in racemic resolution of secondary alcohols.

  16. A systematic review of studies on the faecal microbiota in anorexia nervosa: future research may need to include microbiota from the small intestine.

    Science.gov (United States)

    Schwensen, Hanna Ferløv; Kan, Carol; Treasure, Janet; Høiby, Niels; Sjögren, Magnus

    2018-03-14

    Anorexia nervosa (AN) is a poorly understood and often chronic condition. Deviations in the gut microbiota have been reported to influence the gut-brain axis in other disorders. Therefore, if present in AN, it may impact on symptoms and illness progression. A review of the gut microbiota studies in AN is presented. A literature search on PubMed yielded 27 articles; 14 were selected and based on relevance, 9 articles were included. The findings were interpreted in the larger context of preclinical research and clinical observations. 8 out of 9 included studies analysed microbiota from faeces samples, while the last analysed a protein in plasma produced by the gut. Two studies were longitudinal and included an intervention (i.e., weight restoration), five were cross-sectional, one was a case report, and the last was a case series consisting of three cases. Deviations in abundance, diversity, and microbial composition of the faecal microbiota in AN were found. There are currently only a few studies on the gut microbiota in AN, all done on faeces samples, and not all describe the microbiota at the species level extensively. The Archaeon Methanobrevibacter smithii was increased in participants with a BMI study and specifically in AN patients in three studies. Methanobrevibacter smithii may, if detected, be a benchmark biomarker for future studies. We propose that microbiota samples could also be collected from the small intestine, where a major exchange of nutrients takes place and where the microbiota may have a biological impact on AN.

  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. Monitoring bacterial community of human gut microbiota reveals an increase in Lactobacillus in obese patients and Methanogens in anorexic patients.

    Directory of Open Access Journals (Sweden)

    Fabrice Armougom

    Full Text Available BACKGROUND: Studies of the bacterial communities of the gut microbiota have revealed a shift in the ratio of Firmicutes and Bacteroidetes in obese patients. Determining the variations of microbial communities in feces may be beneficial for the identification of specific profiles in patients with abnormal weights. The roles of the archaeon Methanobrevibacter smithii and Lactobacillus species have not been described in these studies. METHODS AND FINDINGS: We developed an efficient and robust real-time PCR tool that includes a plasmid-based internal control and allows for quantification of the bacterial divisions Bacteroidetes, Firmicutes, and Lactobacillus as well as the methanogen M. smithii. We applied this technique to the feces of 20 obese subjects, 9 patients with anorexia nervosa, and 20 normal-weight healthy controls. Our results confirmed a reduction in the Bacteroidetes community in obese patients (p<0.01. We found a significantly higher Lactobacillus species concentration in obese patients than in lean controls (p=0.0197 or anorexic patients (p=0.0332. The M. smithii concentration was much higher in anorexic patients than in the lean population (p=0.0171. CONCLUSIONS: Lactobacillus species are widely used as growth promoters in the farm industry and are now linked to obesity in humans. The study of the bacterial flora in anorexic patients revealed an increase in M. smithii. This increase might represent an adaptive use of nutrients in this population.

  19. Conserved TRAM Domain Functions as an Archaeal Cold Shock Protein via RNA Chaperone Activity

    Directory of Open Access Journals (Sweden)

    Bo Zhang

    2017-08-01

    Full Text Available Cold shock proteins (Csps enable organisms to acclimate to and survive in cold environments and the bacterial CspA family exerts the cold protection via its RNA chaperone activity. However, most Archaea do not contain orthologs to the bacterial csp. TRAM, a conserved domain among RNA modification proteins ubiquitously distributed in organisms, occurs as an individual protein in most archaeal phyla and has a structural similarity to Csp proteins, yet its biological functions remain unknown. Through physiological and biochemical studies on four TRAM proteins from a cold adaptive archaeon Methanolobus psychrophilus R15, this work demonstrated that TRAM is an archaeal Csp and exhibits RNA chaperone activity. Three TRAM encoding genes (Mpsy_0643, Mpsy_3043, and Mpsy_3066 exhibited remarkable cold-shock induced transcription and were preferentially translated at lower temperature (18°C, while the fourth (Mpsy_2002 was constitutively expressed. They were all able to complement the cspABGE mutant of Escherichia coli BX04 that does not grow in cold temperatures and showed transcriptional antitermination. TRAM3066 (gene product of Mpsy_3066 and TRAM2002 (gene product of Mpsy_2002 displayed sequence-non-specific RNA but not DNA binding activity, and TRAM3066 assisted RNases in degradation of structured RNA, thus validating the RNA chaperone activity of TRAMs. Given the chaperone activity, TRAM is predicted to function beyond a Csp.

  20. First characterization of extremely halophilic 2-deoxy-D-ribose-5-phosphate aldolase.

    Science.gov (United States)

    Ohshida, Tatsuya; Hayashi, Junji; Satomura, Takenori; Kawakami, Ryushi; Ohshima, Toshihisa; Sakuraba, Haruhiko

    2016-10-01

    2-Deoxy-d-ribose-5-phosphate aldolase (DERA) catalyzes the aldol reaction between two aldehydes and is thought to be a potential biocatalyst for the production of a variety of stereo-specific materials. A gene encoding DERA from the extreme halophilic archaeon, Haloarcula japonica, was overexpressed in Escherichia coli. The gene product was successfully purified, using procedures based on the protein's halophilicity, and characterized. The expressed enzyme was stable in a buffer containing 2 M NaCl and exhibited high thermostability, retaining more than 90% of its activity after heating at 70 °C for 10 min. The enzyme was also tolerant to high concentrations of organic solvents, such as acetonitrile and dimethylsulfoxide. Moreover, H. japonica DERA was highly resistant to a high concentration of acetaldehyde and retained about 35% of its initial activity after 5-h' exposure to 300 mM acetaldehyde at 25 °C, the conditions under which E. coli DERA is completely inactivated. The enzyme exhibited much higher activity at 25 °C than the previously characterized hyperthermophilic DERAs (Sakuraba et al., 2007). Our results suggest that the extremely halophilic DERA has high potential to serve as a biocatalyst in organic syntheses. This is the first description of the biochemical characterization of a halophilic DERA. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. 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.

  2. Evaluation of three automated genome annotations for Halorhabdus utahensis.

    Directory of Open Access Journals (Sweden)

    Peter Bakke

    2009-07-01

    Full Text Available Genome annotations are accumulating rapidly and depend heavily on automated annotation systems. Many genome centers offer annotation systems but no one has compared their output in a systematic way to determine accuracy and inherent errors. Errors in the annotations are routinely deposited in databases such as NCBI and used to validate subsequent annotation errors. We submitted the genome sequence of halophilic archaeon Halorhabdus utahensis to be analyzed by three genome annotation services. We have examined the output from each service in a variety of ways in order to compare the methodology and effectiveness of the annotations, as well as to explore the genes, pathways, and physiology of the previously unannotated genome. The annotation services differ considerably in gene calls, features, and ease of use. We had to manually identify the origin of replication and the species-specific consensus ribosome-binding site. Additionally, we conducted laboratory experiments to test H. utahensis growth and enzyme activity. Current annotation practices need to improve in order to more accurately reflect a genome's biological potential. We make specific recommendations that could improve the quality of microbial annotation projects.

  3. 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.

  4. Survival and death of the haloarchaeon Natronorubrum strain HG-1 in a simulated martian environment

    Science.gov (United States)

    Peeters, Z.; Vos, D.; ten Kate, I. L.; Selch, F.; van Sluis, C. A.; Sorokin, D. Yu.; Muijzer, G.; Stan-Lotter, H.; van Loosdrecht, M. C. M.; Ehrenfreund, P.

    2010-11-01

    Halophilic archaea are of interest to astrobiology due to their survival capabilities in desiccated and high salt environments. The detection of remnants of salty pools on Mars stimulated investigations into the response of haloarchaea to martian conditions. Natronorubrum sp. strain HG-1 is an extremely halophilic archaeon with unusual metabolic pathways, growing on acetate and stimulated by tetrathionate. We exposed Natronorubrum strain HG-1 to ultraviolet (UV) radiation, similar to levels currently prevalent on Mars. In addition, the effects of low temperature (4, -20, and -80 °C), desiccation, and exposure to a Mars soil analogue from the Atacama desert on the viability of Natronorubrum strain HG-1 cultures were investigated. The results show that Natronorubrum strain HG-1 cannot survive for more than several hours when exposed to UV radiation equivalent to that at the martian equator. Even when protected from UV radiation, viability is impaired by a combination of desiccation and low temperature. Desiccating Natronorubrum strain HG-1 cells when mixed with a Mars soil analogue impaired growth of the culture to below the detection limit. Overall, we conclude that Natronorubrum strain HG-1 cannot survive the environment currently present on Mars. Since other halophilic microorganisms were reported to survive simulated martian conditions, our results imply that survival capabilities are not necessarily shared between phylogenetically related species.

  5. 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.

  6. CRISPR-Cas type I-A Cascade complex couples viral infection surveillance to host transcriptional regulation in the dependence of Csa3b

    Science.gov (United States)

    He, Fei; Vestergaard, Gisle; Peng, Wenfang; She, Qunxin

    2017-01-01

    Abstract CRISPR-Cas (clustered regularly interspaced short palindromic repeats and the associated genes) constitute adaptive immune systems in bacteria and archaea and they provide sequence specific immunity against foreign nucleic acids. CRISPR-Cas systems are activated by viral infection. However, little is known about how CRISPR-Cas systems are activated in response to viral infection or how their expression is controlled in the absence of viral infection. Here, we demonstrate that both the transcriptional regulator Csa3b, and the type I-A interference complex Cascade, are required to transcriptionally repress the interference gene cassette in the archaeon Sulfolobus. Csa3b binds to two palindromic repeat sites in the promoter region of the cassette and facilitates binding of the Cascade to the promoter region. Upon viral infection, loading of Cascade complexes onto crRNA-matching protospacers leads to relief of the transcriptional repression. Our data demonstrate a mechanism coupling CRISPR-Cas surveillance of protospacers to transcriptional regulation of the interference gene cassette thereby allowing a fast response to viral infection. PMID:27980065

  7. 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.

  8. Cloning, Sequencing, and Expression of the Gene Encoding Cyclic 2,3-Diphosphoglycerate Synthetase, the Key Enzyme of Cyclic 2,3-Diphosphoglycerate Metabolism in Methanothermus fervidus

    Science.gov (United States)

    Matussek, Karl; Moritz, Patrick; Brunner, Nina; Eckerskorn, Christoph; Hensel, Reinhard

    1998-01-01

    Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and expressed it in Escherichia coli. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in M. fervidus, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the Vmax of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from E. coli revealed a twofold-higher Vmax of the enzyme from M. fervidus in the synthesizing direction. PMID:9811660

  9. Genome sequence of Thermofilum pendens reveals an exceptional loss of biosynthetic pathways without genome reduction

    Energy Technology Data Exchange (ETDEWEB)

    Kyrpides, Nikos; Anderson, Iain; Rodriguez, Jason; Susanti, Dwi; Porat, Iris; Reich, Claudia; Ulrich, Luke E.; Elkins, James G.; Mavromatis, Kostas; Lykidis, Athanasios; Kim, Edwin; Thompson, Linda S.; Nolan, Matt; Land, Miriam; Copeland, Alex; Lapidus, Alla; Lucas, Susan; Detter, Chris; Zhulin, Igor B.; Olsen, Gary J.; Whitman, William; Mukhopadhyay, Biswarup; Bristow, James; Kyrpides, Nikos

    2008-01-01

    We report the complete genome of Thermofilum pendens, a deep-branching, hyperthermophilic member of the order Thermoproteales within the archaeal kingdom Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It is an extracellular commensal, requiring an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. In fact T. pendens has fewer biosynthetic enzymes than obligate intracellular parasites, although it does not display other features common among obligate parasites and thus does not appear to be in the process of becoming a parasite. It appears that T. pendens has adapted to life in an environment rich in nutrients. T. pendens was known to utilize peptides as an energy source, but the genome reveals substantial ability to grow on carbohydrates. T. pendens is the first crenarchaeote and only the second archaeon found to have a transporter of the phosphotransferase system. In addition to fermentation, T. pendens may gain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogenlyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time this enzyme has been found outside of Methanosarcinales, and a presenilin-related protein. Predicted highly expressed proteins do not include housekeeping genes, and instead include ABC transporters for carbohydrates and peptides, and CRISPR-associated proteins.

  10. 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.

  11. Patterns of gene flow define species of thermophilic Archaea.

    Directory of Open Access Journals (Sweden)

    Hinsby Cadillo-Quiroz

    2012-02-01

    Full Text Available Despite a growing appreciation of their vast diversity in nature, mechanisms of speciation are poorly understood in Bacteria and Archaea. Here we use high-throughput genome sequencing to identify ongoing speciation in the thermoacidophilic Archaeon Sulfolobus islandicus. Patterns of homologous gene flow among genomes of 12 strains from a single hot spring in Kamchatka, Russia, demonstrate higher levels of gene flow within than between two persistent, coexisting groups, demonstrating that these microorganisms fit the biological species concept. Furthermore, rates of gene flow between two species are decreasing over time in a manner consistent with incipient speciation. Unlike other microorganisms investigated, we do not observe a relationship between genetic divergence and frequency of recombination along a chromosome, or other physical mechanisms that would reduce gene flow between lineages. Each species has its own genetic island encoding unique physiological functions and a unique growth phenotype that may be indicative of ecological specialization. Genetic differentiation between these coexisting groups occurs in large genomic "continents," indicating the topology of genomic divergence during speciation is not uniform and is not associated with a single locus under strong diversifying selection. These data support a model where species do not require physical barriers to gene flow but are maintained by ecological differentiation.

  12. Differential stability of TATA box binding proteins from archaea with different optimal growth temperatures

    Science.gov (United States)

    Kopitz, Annette; Soppa, Jörg; Krejtschi, Carsten; Hauser, Karin

    2009-09-01

    The TATA box binding protein (TBP) is involved in promoter recognition, the first step of transcription initiation. TBP is universally conserved and essential in archaea and eukaryotes. In archaea, TBPs have to be stable and to function in species that cover an extremely wide range of optimal growth temperatures (OGTs), from below 0 °C to more than 100 °C. Thus, the archaeal TBP family is ideally suited to study the evolutionary adaptation of proteins to an extremely wide range of temperatures. We characterized the thermostability of one mesophilic and one thermophilic TBP by infrared spectroscopy. Transition temperatures ( Tms) of thermal unfolding have been determined using TBPs from Methanosarcina mazei (OGT 37 °C) and from Methanothermobacter thermautotrophicus (OGT 65 °C). Furthermore, the influence of protein and salt concentration on thermostability has been characterized. Together with previous studies, our results reveal that the Tms of archaeal TBPs are closely correlated with the OGTs of the respective species. Noteworthy, this is also true for the TBP from M. mazei representing the first characterized TBP from a mesophilic archaeon. In contrast, the only characterized eukaryotic TBP of the mesophilic plant Arabidopsis thaliana has a Tm more than 40 °C above the OGT.

  13. Patterns of gene flow define species of thermophilic Archaea.

    Science.gov (United States)

    Cadillo-Quiroz, Hinsby; Didelot, Xavier; Held, Nicole L; Herrera, Alfa; Darling, Aaron; Reno, Michael L; Krause, David J; Whitaker, Rachel J

    2012-02-01

    Despite a growing appreciation of their vast diversity in nature, mechanisms of speciation are poorly understood in Bacteria and Archaea. Here we use high-throughput genome sequencing to identify ongoing speciation in the thermoacidophilic Archaeon Sulfolobus islandicus. Patterns of homologous gene flow among genomes of 12 strains from a single hot spring in Kamchatka, Russia, demonstrate higher levels of gene flow within than between two persistent, coexisting groups, demonstrating that these microorganisms fit the biological species concept. Furthermore, rates of gene flow between two species are decreasing over time in a manner consistent with incipient speciation. Unlike other microorganisms investigated, we do not observe a relationship between genetic divergence and frequency of recombination along a chromosome, or other physical mechanisms that would reduce gene flow between lineages. Each species has its own genetic island encoding unique physiological functions and a unique growth phenotype that may be indicative of ecological specialization. Genetic differentiation between these coexisting groups occurs in large genomic "continents," indicating the topology of genomic divergence during speciation is not uniform and is not associated with a single locus under strong diversifying selection. These data support a model where species do not require physical barriers to gene flow but are maintained by ecological differentiation.

  14. Stability of the 'L12 stalk' in ribosomes from mesophilic and (hyper)thermophilic Archaea and Bacteria.

    Science.gov (United States)

    Shcherbakov, D; Dontsova, M; Tribus, M; Garber, M; Piendl, W

    2006-01-01

    The ribosomal stalk complex, consisting of one molecule of L10 and four or six molecules of L12, is attached to 23S rRNA via protein L10. This complex forms the so-called 'L12 stalk' on the 50S ribosomal subunit. Ribosomal protein L11 binds to the same region of 23S rRNA and is located at the base of the 'L12 stalk'. The 'L12 stalk' plays a key role in the interaction of the ribosome with translation factors. In this study stalk complexes from mesophilic and (hyper)thermophilic species of the archaeal genus Methanococcus and from the Archaeon Sulfolobus solfataricus, as well as from the Bacteria Escherichia coli, Geobacillus stearothermophilus and Thermus thermophilus, were overproduced in E.coli and purified under non-denaturing conditions. Using filter-binding assays the affinities of the archaeal and bacterial complexes to their specific 23S rRNA target site were analyzed at different pH, ionic strength and temperature. Affinities of both archaeal and bacterial complexes for 23S rRNA vary by more than two orders of magnitude, correlating very well with the growth temperatures of the organisms. A cooperative effect of binding to 23S rRNA of protein L11 and the L10/L12(4) complex from mesophilic and thermophilic Archaea was shown to be temperature-dependent.

  15. 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.

  16. High salinity facilitates dolomite precipitation mediated by Haloferax volcanii DS52

    Science.gov (United States)

    Qiu, Xuan; Wang, Hongmei; Yao, Yanchen; Duan, Yong

    2017-08-01

    Although most modern dolomites occur in hypersaline environments, the effects of elevated salinity on the microbial mediation of dolomite precipitation have not been fully evaluated. Here we report results of dolomite precipitation in association with a batch culture of Haloferax volcanii DS52, a halophilic archaeon, under various salinities (from 120‰ to 360‰) and the impact of salinity on microbe-mediated dolomite formation. The mineral phases, morphology and atomic arrangement of the precipitates were analyzed by XRD, SEM and TEM, respectively. The amount of amino acids on the archaeal cell surface was quantified by HPLC/MS. The XRD analysis indicated that disordered dolomite formed successfully with the facilitation of cells harvested from cultures with relatively high salinities (200‰ and 280‰) but was not observed in association with cells harvested from cultures with lower salinity (120‰) or the lysates of cells harvested from extremely high salinity (360‰). The TEM analysis demonstrated that the crystals from cultures with a salinity of 200‰ closely matched that of dolomite. Importantly, we found that more carboxyl groups were presented on the cell surface under high salinity conditions to resist the high osmotic pressure, which may result in the subsequent promotion of dolomite formation. Our finding suggests a link between variations in the hydro-chemical conditions and the formation of dolomite via microbial metabolic activity and enhances our understanding about the mechanism of microbially mediated dolomite formation under high salinity conditions.

  17. Specificity and Function of Archaeal DNA Replication Initiator Proteins

    Directory of Open Access Journals (Sweden)

    Rachel Y. Samson

    2013-02-01

    Full Text Available Chromosomes with multiple DNA replication origins are a hallmark of Eukaryotes and some Archaea. All eukaryal nuclear replication origins are defined by the origin recognition complex (ORC that recruits the replicative helicase MCM(2-7 via Cdc6 and Cdt1. We find that the three origins in the single chromosome of the archaeon Sulfolobus islandicus are specified by distinct initiation factors. While two origins are dependent on archaeal homologs of eukaryal Orc1 and Cdc6, the third origin is instead reliant on an archaeal Cdt1 homolog. We exploit the nonessential nature of the orc1-1 gene to investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels the protein’s structure rather than that of the DNA template.

  18. Halobacterium salinarum NRC-1 PeptideAtlas: toward strategies for targeted proteomics and improved proteome coverage.

    Science.gov (United States)

    Van, Phu T; Schmid, Amy K; King, Nichole L; Kaur, Amardeep; Pan, Min; Whitehead, Kenia; Koide, Tie; Facciotti, Marc T; Goo, Young Ah; Deutsch, Eric W; Reiss, David J; Mallick, Parag; Baliga, Nitin S

    2008-09-01

    The relatively small numbers of proteins and fewer possible post-translational modifications in microbes provide a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a PeptideAtlas (PA) covering 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636 000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has highlighted plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore, we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics.

  19. Halobacterium salinarum NRC-1 PeptideAtlas: strategies for targeted proteomics

    Science.gov (United States)

    Van, Phu T.; Schmid, Amy K.; King, Nichole L.; Kaur, Amardeep; Pan, Min; Whitehead, Kenia; Koide, Tie; Facciotti, Marc T.; Goo, Young-Ah; Deutsch, Eric W.; Reiss, David J.; Mallick, Parag; Baliga, Nitin S.

    2009-01-01

    The relatively small numbers of proteins and fewer possible posttranslational modifications in microbes provides a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a Peptide Atlas (PA) for 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636,000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has helped highlight plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics. PMID:18652504

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    Sarit Edelheit

    2013-06-01

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

  2. 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.

  3. Crystal Structures of Two Isozymes of Citrate Synthase from Sulfolobus tokodaii Strain 7

    Directory of Open Access Journals (Sweden)

    Midori Murakami

    2016-01-01

    Full Text Available Thermoacidophilic archaeon Sulfolobus tokodaii strain 7 has two citrate synthase genes (ST1805-CS and ST0587-CS in the genome with 45% sequence identity. Because they exhibit similar optimal temperatures of catalytic activity and thermal inactivation profiles, we performed structural comparisons between these isozymes to elucidate adaptation mechanisms to high temperatures in thermophilic CSs. The crystal structures of ST1805-CS and ST0587-CS were determined at 2.0 Å and 2.7 Å resolutions, respectively. Structural comparison reveals that both of them are dimeric enzymes composed of two identical subunits, and these dimeric structures are quite similar to those of citrate synthases from archaea and eubacteria. ST0587-CS has, however, 55 ion pairs within whole dimer structure, while having only 36 in ST1805-CS. Although the number and distributions of ion pairs are distinct from each other, intersubunit ion pairs between two domains of each isozyme are identical especially in interterminal region. Because the location and number of ion pairs are in a trend with other CSs from thermophilic microorganisms, the factors responsible for thermal adaptation of ST-CS isozymes are characterized by ion pairs in interterminal region.

  4. Construction of an Immobilized Thermophilic Esterase on Epoxy Support for Poly(ε-caprolactone) Synthesis.

    Science.gov (United States)

    Ren, Hui; Xing, Zhen; Yang, Jiebing; Jiang, Wei; Zhang, Gang; Tang, Jun; Li, Quanshun

    2016-06-18

    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 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.

  5. Protein-DNA binding dynamics predict transcriptional response to nutrients in archaea.

    Science.gov (United States)

    Todor, Horia; Sharma, Kriti; Pittman, Adrianne M C; Schmid, Amy K

    2013-10-01

    Organisms across all three domains of life use gene regulatory networks (GRNs) to integrate varied stimuli into coherent transcriptional responses to environmental pressures. However, inferring GRN topology and regulatory causality remains a central challenge in systems biology. Previous work characterized TrmB as a global metabolic transcription factor in archaeal extremophiles. However, it remains unclear how TrmB dynamically regulates its ∼100 metabolic enzyme-coding gene targets. Using a dynamic perturbation approach, we elucidate the topology of the TrmB metabolic GRN in the model archaeon Halobacterium salinarum. Clustering of dynamic gene expression patterns reveals that TrmB functions alone to regulate central metabolic enzyme-coding genes but cooperates with various regulators to control peripheral metabolic pathways. Using a dynamical model, we predict gene expression patterns for some TrmB-dependent promoters and infer secondary regulators for others. Our data suggest feed-forward gene regulatory topology for cobalamin biosynthesis. In contrast, purine biosynthesis appears to require TrmB-independent regulators. We conclude that TrmB is an important component for mediating metabolic modularity, integrating nutrient status and regulating gene expression dynamics alone and in concert with secondary regulators.

  6. Diphthamide biosynthesis requires an organic radical generated by an iron-sulphur enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yang; Zhu, Xuling; Torelli, Andrew T; Lee, Michael; Dzikovski, Boris; Koralewski, Rachel M; Wang, Eileen; Freed, Jack; Krebs, Carsten; Ealick, Steve E; Lin, Hening [Cornell; (Penn)

    2010-08-30

    Archaeal and eukaryotic translation elongation factor 2 contain a unique post-translationally modified histidine residue called diphthamide, which is the target of diphtheria toxin. The biosynthesis of diphthamide was proposed to involve three steps, with the first being the formation of a C-C bond between the histidine residue and the 3-amino-3-carboxypropyl group of S-adenosyl-l-methionine (SAM). However, further details of the biosynthesis remain unknown. Here we present structural and biochemical evidence showing that the first step of diphthamide biosynthesis in the archaeon Pyrococcus horikoshii uses a novel iron-sulphur-cluster enzyme, Dph2. Dph2 is a homodimer and each of its monomers can bind a [4Fe-4S] cluster. Biochemical data suggest that unlike the enzymes in the radical SAM superfamily, Dph2 does not form the canonical 5'-deoxyadenosyl radical. Instead, it breaks the Cγ,Met-S bond of SAM and generates a 3-amino-3-carboxypropyl radical. Our results suggest that P. horikoshii Dph2 represents a previously unknown, SAM-dependent, [4Fe-4S]-containing enzyme that catalyses unprecedented chemistry.

  7. Microbial redox processes in deep subsurface environments and the potential application of (perchlorate in oil reservoirs

    Directory of Open Access Journals (Sweden)

    Martin G Liebensteiner

    2014-09-01

    Full Text Available The ability of microorganisms to thrive under oxygen-free conditions in subsurface environments relies on the enzymatic reduction of oxidized elements, such as sulfate, ferric iron or CO2, coupled to the oxidation of inorganic or organic compounds. A broad phylogenetic and functional diversity of microorganisms from subsurface environments has been described using isolation-based and advanced molecular ecological techniques. The physiological groups reviewed here comprise iron-, manganese- and nitrate-reducing microorganisms. In the context of recent findings also the potential of chlorate and perchlorate [jointly termed (perchlorate] reduction in oil reservoirs will be discussed. Special attention is given to elevated temperatures that are predominant in the deep subsurface. Microbial reduction of (perchlorate is a thermodynamically favorable redox process, also at high temperature. However, knowledge about (perchlorate reduction at elevated temperatures is still scarce and restricted to members of the Firmicutes and the archaeon Archaeoglobus fulgidus. By analyzing the diversity and phylogenetic distribution of functional genes in (metagenome databases and combining this knowledge with extrapolations to earlier-made physiological observations we speculate on the potential of (perchlorate reduction in the subsurface and more precisely oil fields. In addition, the application of (perchlorate for bioremediation, souring control and microbial enhanced oil recovery are addressed.

  8. Dynamic fluorescence studies of beta-glycosidase mutants from Sulfolobus solfataricus: effects of single mutations on protein thermostability.

    Science.gov (United States)

    Bismuto, Ettore; Febbraio, Ferdinando; Limongelli, Simona; Briante, Raffaella; Nucci, Roberto

    2003-04-01

    Multiple sequence alignment on 73 proteins belonging to glycosyl hydrolase family 1 reveals the occurrence of a segment (83-124) in the enzyme sequences from hyperthermophilic archaea bacteria, which is absent in all the mesophilic members of the family. The alignment of the known three-dimensional structures of hyperthermophilic glycosidases with the known ones from mesophilic organisms shows a similar spatial organizations of beta-glycosidases except for this sequence segment whose structure is located on the external surface of each of four identical subunits, where it overlaps two alpha-helices. Site-directed mutagenesis substituting N97 or S101 with a cysteine residue in the sequence of beta-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus caused some changes in the structural and dynamic properties as observed by circular dichroism in far- and near-UV light, as well as by frequency domain fluorometry, with a simultaneous loss of thermostability. The results led us to hypothesize an important role of the sequence segment present only in hyperthermophilic beta-glycosidases, in the thermal adaptation of archaea beta-glycosidases. The thermostabilization mechanism could occur as a consequence of numerous favorable ionic interactions of the 83-124 sequence with the other part of protein matrix that becomes more rigid and less accessible to the insult of thermal-activated solvent molecules. Copyright 2003 Wiley-Liss, Inc.

  9. 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.

  10. Hydrogen or formate: Alternative key players in methanogenic degradation.

    Science.gov (United States)

    Schink, Bernhard; Montag, Dominik; Keller, Anja; Müller, Nicolai

    2017-06-01

    Hydrogen and formate are important electron carriers in methanogenic degradation in anoxic environments such as sediments, sewage sludge digestors and biogas reactors. Especially in the terminal steps of methanogenesis, they determine the energy budgets of secondary (syntrophically) fermenting bacteria and their methanogenic partners. The literature provides considerable data on hydrogen pool sizes in such habitats, but little data exist for formate concentrations due to technical difficulties in formate determination at low concentration. Recent evidence from biochemical and molecular biological studies indicates that several secondary fermenters can use both hydrogen and formate for electron release, and may do so even simultaneously. Numerous strictly anaerobic bacteria contain enzymes which equilibrate hydrogen and formate pools to energetically equal values, and recent measurements in sewage digestors and biogas reactors indicate that - beyond occasional fluctuations - the pool sizes of hydrogen and formate are indeed energetically nearly equivalent. Nonetheless, a thermophilic archaeon from a submarine hydrothermal vent, Thermococcus onnurineus, can obtain ATP from the conversion of formate to hydrogen plus bicarbonate at 80°C, indicating that at least in this extreme environment the pools of formate and hydrogen are likely to be sufficiently different to support such an unusual type of energy conservation. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  11. Identification and characterisation of a novel acylpeptide hydrolase from Sulfolobus solfataricus: structural and functional insights.

    Directory of Open Access Journals (Sweden)

    Marta Gogliettino

    Full Text Available A novel acylpeptide hydrolase, named APEH-3(Ss, was isolated from the hypertermophilic archaeon Sulfolobus solfataricus. APEH is a member of the prolyl oligopeptidase family which catalyzes the removal of acetylated amino acid residues from the N terminus of oligopeptides. The purified enzyme shows a homotrimeric structure, unique among the associate partners of the APEH cluster and, in contrast to the archaeal APEHs which show both exo/endo peptidase activities, it appears to be a "true" aminopeptidase as exemplified by its mammalian counterparts, with which it shares a similar substrate specificity. Furthermore, a comparative study on the regulation of apeh gene expression, revealed a significant but divergent alteration in the expression pattern of apeh-3(Ss and apeh(Ss (the gene encoding the previously identified APEH(Ss from S. solfataricus, which is induced in response to various stressful growth conditions. Hence, both APEH enzymes can be defined as stress-regulated proteins which play a complementary role in enabling the survival of S. solfataricus cells under different conditions. These results provide new structural and functional insights into S. solfataricus APEH, offering a possible explanation for the multiplicity of this enzyme in Archaea.

  12. Deep sequencing of Salmonella RNA associated with heterologous Hfq proteins in vivo reveals small RNAs as a major target class and identifies RNA processing phenotypes.

    Science.gov (United States)

    Sittka, Alexandra; Sharma, Cynthia M; Rolle, Katarzyna; Vogel, Jörg

    2009-01-01

    The bacterial Sm-like protein, Hfq, is a key factor for the stability and function of small non-coding RNAs (sRNAs) in Escherichia coli. Homologues of this protein have been predicted in many distantly related organisms yet their functional conservation as sRNA-binding proteins has not entirely been clear. To address this, we expressed in Salmonella the Hfq proteins of two eubacteria (Neisseria meningitides, Aquifex aeolicus) and an archaeon (Methanocaldococcus jannaschii), and analyzed the associated RNA by deep sequencing. This in vivo approach identified endogenous Salmonella sRNAs as a major target of the foreign Hfq proteins. New Salmonella sRNA species were also identified, and some of these accumulated specifically in the presence of a foreign Hfq protein. In addition, we observed specific RNA processing defects, e.g., suppression of precursor processing of SraH sRNA by Methanocaldococcus Hfq, or aberrant accumulation of extracytoplasmic target mRNAs of the Salmonella GcvB, MicA or RybB sRNAs. Taken together, our study provides evidence of a conserved inherent sRNA-binding property of Hfq, which may facilitate the lateral transmission of regulatory sRNAs among distantly related species. It also suggests that the expression of heterologous RNA-binding proteins combined with deep sequencing analysis of RNA ligands can be used as a molecular tool to dissect individual steps of RNA metabolism in vivo.

  13. MTH187 from Methanobacterium thermoautotrophicum has three HEAT-like Repeats

    Energy Technology Data Exchange (ETDEWEB)

    Julien, Olivier; Gignac, Isabelle [Universite Laval, Departement de biochimie et de microbiologie and CREFSIP (Canada); Hutton, Anna [University of Alberta, Institute for Biomolecular Design (Canada); Yee, Adelinda; Arrowsmith, Cheryl H. [University of Toronto, Division of Molecular and Structural Biology, Department of Medical Biophysics, Ontario Cancer Institute (Canada); Gagne, Stephane M. [Universite Laval, Departement de biochimie et de microbiologie and CREFSIP (Canada)], E-mail: sgagne@rsvs.ulaval.ca

    2006-06-15

    With the completion of genome sequencing projects, there are a large number of proteins for which we have little or no functional information. Since protein function is closely related to three-dimensional conformation, structural proteomics is one avenue where the role of proteins with unknown function can be investigated. In the present structural project, the structure of MTH187 has been determined by solution-state NMR spectroscopy. This protein of 12.4 kDa is one of the 424 non-membrane proteins that were cloned and purified for the structural proteomic project of Methanobacterium thermoautotrophicum [Christendat, D., Yee, A., Dharamsi, A., Kluger, Y., Gerstein, M., Arrowsmith, C.H. and Edwards, A.M. (2000) Prog. Biophys. Mol. Biol., 73, 339-345]. Methanobacterium thermoautotrophicum is a thermophilic archaeon that grows optimally at 65 deg. C. A particular characteristic of this microorganism is its ability to generate methane from carbon dioxide and hydrogen [Smith, D.R., Doucette-Stamm, L.A., Deloughery, C., Lee, H., Dubois, J., Aldredge, T., Bashirzadeh, R., Blakely, D., Cook, R., Gilbert, K., Harrison, D., Hoang, L., Keagle, P., Lumm, W., Pothier, B., Qiu, D., Spadafora, R., Vicaire, R., Wang, Y., Wierzbowski, J., Gibson, R., Jiwani, N., Caruso, A., Bush, D., Reeve, J. N. et al. (1997) J. Bacteriol., 179, 7135-7155].

  14. Bacteriophage T5 encodes a homolog of the eukaryotic transcription coactivator PC4 implicated in recombination-dependent DNA replication.

    Science.gov (United States)

    Steigemann, Birthe; Schulz, Annina; Werten, Sebastiaan

    2013-11-15

    The RNA polymerase II cofactor PC4 globally regulates transcription of protein-encoding genes through interactions with unwinding DNA, the basal transcription machinery and transcription activators. Here, we report the surprising identification of PC4 homologs in all sequenced representatives of the T5 family of bacteriophages, as well as in an archaeon and seven phyla of eubacteria. We have solved the crystal structure of the full-length T5 protein at 1.9Å, revealing a striking resemblance to the characteristic single-stranded DNA (ssDNA)-binding core domain of PC4. Intriguing novel structural features include a potential regulatory region at the N-terminus and a C-terminal extension of the homodimerisation interface. The genome organisation of T5-related bacteriophages points at involvement of the PC4 homolog in recombination-dependent DNA replication, strongly suggesting that the protein corresponds to the hitherto elusive replicative ssDNA-binding protein of the T5 family. Our findings imply that PC4-like factors intervene in multiple unwinding-related processes by acting as versatile modifiers of nucleic acid conformation and raise the possibility that the eukaryotic transcription coactivator derives from ancestral DNA replication, recombination and repair factors. © 2013.

  15. Exploring Fingerprints of the Extreme Thermoacidophile Metallosphaera sedula Grown on Synthetic Martian Regolith Materials as the Sole Energy Sources.

    Science.gov (United States)

    Kölbl, Denise; Pignitter, Marc; Somoza, Veronika; Schimak, Mario P; Strbak, Oliver; Blazevic, Amir; Milojevic, Tetyana

    2017-01-01

    The biology of metal transforming microorganisms is of a fundamental and applied importance for our understanding of past and present biogeochemical processes on Earth and in the Universe. The extreme thermoacidophile Metallosphaera sedula is a metal mobilizing archaeon, which thrives in hot acid environments (optimal growth at 74°C and pH 2.0) and utilizes energy from the oxidation of reduced metal inorganic sources. These characteristics of M. sedula make it an ideal organism to further our knowledge of the biogeochemical processes of possible life on extraterrestrial planetary bodies. Exploring the viability and metal extraction capacity of M. sedula living on and interacting with synthetic extraterrestrial minerals, we show that M. sedula utilizes metals trapped in the Martian regolith simulants (JSC Mars 1A; P-MRS; S-MRS; MRS07/52) as the sole energy sources. The obtained set of microbiological and mineralogical data suggests that M. sedula actively colonizes synthetic Martian regolith materials and releases free soluble metals. The surface of bioprocessed Martian regolith simulants is analyzed for specific mineralogical fingerprints left upon M. sedula growth. The obtained results provide insights of biomining of extraterrestrial material as well as of the detection of biosignatures implementing in life search missions.

  16. Cloning, sequencing, and expression of the gene encoding cyclic 2, 3-diphosphoglycerate synthetase, the key enzyme of cyclic 2, 3-diphosphoglycerate metabolism in Methanothermus fervidus.

    Science.gov (United States)

    Matussek, K; Moritz, P; Brunner, N; Eckerskorn, C; Hensel, R

    1998-11-01

    Cyclic 2,3-diphosphoglycerate synthetase (cDPGS) catalyzes the synthesis of cyclic 2,3-diphosphoglycerate (cDPG) by formation of an intramolecular phosphoanhydride bond in 2,3-diphosphoglycerate. cDPG is known to be accumulated to high intracellular concentrations (>300 mM) as a putative thermoadapter in some hyperthermophilic methanogens. For the first time, we have purified active cDPGS from a methanogen, the hyperthermophilic archaeon Methanothermus fervidus, sequenced the coding gene, and expressed it in Escherichia coli. cDPGS purification resulted in enzyme preparations containing two isoforms differing in their electrophoretic mobility under denaturing conditions. Since both polypeptides showed the same N-terminal amino acid sequence and Southern analyses indicate the presence of only one gene coding for cDPGS in M. fervidus, the two polypeptides originate from the same gene but differ by a not yet identified modification. The native cDPGS represents a dimer with an apparent molecular mass of 112 kDa and catalyzes the reversible formation of the intramolecular phosphoanhydride bond at the expense of ATP. The enzyme shows a clear preference for the synthetic reaction: the substrate affinity and the Vmax of the synthetic reaction are a factor of 8 to 10 higher than the corresponding values for the reverse reaction. Comparison with the kinetic properties of the electrophoretically homogeneous, apparently unmodified recombinant enzyme from E. coli revealed a twofold-higher Vmax of the enzyme from M. fervidus in the synthesizing direction.

  17. 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.

  18. 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

    2016-01-01

    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. PMID:27226589

  19. Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols

    Science.gov (United States)

    Wu, Xi; Zhang, Chong; Orita, Izumi; Imanaka, Tadayuki

    2013-01-01

    A novel thermostable alcohol dehydrogenase (ADH) showing activity toward aromatic secondary alcohols was identified from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkADH). The gene, tk0845, which encodes an aldo-keto reductase, was heterologously expressed in Escherichia coli. The enzyme was found to be a monomer with a molecular mass of 31 kDa. It was highly thermostable with an optimal temperature of 90°C and a half-life of 4.5 h at 95°C. The apparent Km values for the cofactors NAD(P)+ and NADPH were similar within a range of 66 to 127 μM. TkADH preferred secondary alcohols and accepted various ketones and aldehydes as substrates. Interestingly, the enzyme could oxidize 1-phenylethanol and its derivatives having substituents at the meta and para positions with high enantioselectivity, yielding the corresponding (R)-alcohols with optical purities of greater than 99.8% enantiomeric excess (ee). TkADH could also reduce 2,2,2-trifluoroacetophenone to (R)-2,2,2-trifluoro-1-phenylethanol with high enantioselectivity (>99.6% ee). Furthermore, the enzyme showed high resistance to organic solvents and was particularly highly active in the presence of H2O–20% 2-propanol and H2O–50% n-hexane or n-octane. This ADH is expected to be a useful tool for the production of aromatic chiral alcohols. PMID:23354700

  20. The relationship of protein conservation and sequence length

    Directory of Open Access Journals (Sweden)

    Panchenko Anna R

    2002-11-01

    Full Text Available Abstract Background In general, the length of a protein sequence is determined by its function and the wide variance in the lengths of an organism's proteins reflects the diversity of specific functional roles for these proteins. However, additional evolutionary forces that affect the length of a protein may be revealed by studying the length distributions of proteins evolving under weaker functional constraints. Results We performed sequence comparisons to distinguish highly conserved and poorly conserved proteins from the bacterium Escherichia coli, the archaeon Archaeoglobus fulgidus, and the eukaryotes Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens. For all organisms studied, the conserved and nonconserved proteins have strikingly different length distributions. The conserved proteins are, on average, longer than the poorly conserved ones, and the length distributions for the poorly conserved proteins have a relatively narrow peak, in contrast to the conserved proteins whose lengths spread over a wider range of values. For the two prokaryotes studied, the poorly conserved proteins approximate the minimal length distribution expected for a diverse range of structural folds. Conclusions There is a relationship between protein conservation and sequence length. For all the organisms studied, there seems to be a significant evolutionary trend favoring shorter proteins in the absence of other, more specific functional constraints.

  1. Development of a 16S rRNA-targeted fluorescence in situ hybridization probe for quantification of the ammonia-oxidizer Nitrosotalea devanaterra and its relatives.

    Science.gov (United States)

    Restrepo-Ortiz, C X; Merbt, S N; Barrero-Canossa, J; Fuchs, B M; Casamayor, E O

    2018-04-28

    The Thaumarchaeota SAGMCG-1 group and, in particular, members of the genus Nitrosotalea have high occurrence in acidic soils, the rhizosphere, groundwater and oligotrophic lakes, and play a potential role in nitrogen cycling. In this study, the specific oligonucleotide fluorescence in situ hybridization probe SAG357 was designed for this Thaumarchaeota group based on the available 16S rRNA gene sequences in databases, and included the ammonia-oxidizing species Nitrosotalea devanaterra. Cell permeabilization for catalyzed reporter deposition fluorescence in situ detection and the hybridization conditions were optimized on enrichment cultures of the target species N. devanaterra, as well as the non-target ammonia-oxidizing archaeon Nitrosopumilus maritimus. Probe specificity was improved with a competitor oligonucleotide, and fluorescence intensity and cell visualization were enhanced by the design and application of two adjacent helpers. Probe performance was tested in soil samples along a pH gradient, and counting results matched the expected in situ distributions. Probe SAG357 and the CARD-FISH protocol developed in the present study will help to improve the current understanding of the ecology and physiology of N. devanaterra and its relatives in natural environments. Copyright © 2018 Elsevier GmbH. All rights reserved.

  2. Investigation of sliding DNA clamp dynamics by single-molecule fluorescence, mass spectrometry and structure-based modeling

    Science.gov (United States)

    Gadkari, Varun V; Harvey, Sophie R; Raper, Austin T; Chu, Wen-Ting; Wang, Jin; Wysocki, Vicki H; Suo, Zucai

    2018-01-01

    Abstract Proliferating cell nuclear antigen (PCNA) is a trimeric ring-shaped clamp protein that encircles DNA and interacts with many proteins involved in DNA replication and repair. Despite extensive structural work to characterize the monomeric, dimeric, and trimeric forms of PCNA alone and in complex with interacting proteins, no structure of PCNA in a ring-open conformation has been published. Here, we use a multidisciplinary approach, including single-molecule Förster resonance energy transfer (smFRET), native ion mobility-mass spectrometry (IM-MS), and structure-based computational modeling, to explore the conformational dynamics of a model PCNA from Sulfolobus solfataricus (Sso), an archaeon. We found that Sso PCNA samples ring-open and ring-closed conformations even in the absence of its clamp loader complex, replication factor C, and transition to the ring-open conformation is modulated by the ionic strength of the solution. The IM-MS results corroborate the smFRET findings suggesting that PCNA dynamics are maintained in the gas phase and further establishing IM-MS as a reliable strategy to investigate macromolecular motions. Our molecular dynamic simulations agree with the experimental data and reveal that ring-open PCNA often adopts an out-of-plane left-hand geometry. Collectively, these results implore future studies to define the roles of PCNA dynamics in DNA loading and other PCNA-mediated interactions. PMID:29529283

  3. 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.

  4. Anaerobic Ammonium-Oxidizing Bacteria: Unique Microorganisms with Exceptional Properties

    Science.gov (United States)

    Jetten, Mike S. M.

    2012-01-01

    Summary: Anaerobic ammonium-oxidizing (anammox) bacteria defy many microbiological concepts and share numerous properties with both eukaryotes and archaea. Among their most intriguing characteristics are their compartmentalized cell plan and archaeon-like cell wall. Here we review our current knowledge about anammox cell biology. The anammox cell is divided into three separate compartments by bilayer membranes. The anammox cell consists of (from outside to inside) the cell wall, paryphoplasm, riboplasm, and anammoxosome. Not much is known about the composition or function of both the anammox cell wall and the paryphoplasm compartment. The cell wall is proposed to be proteinaceous and to lack both peptidoglycan and an outer membrane typical of Gram-negative bacteria. The function of the paryphoplasm is unknown, but it contains the cell division ring. The riboplasm resembles the standard cytoplasmic compartment of other bacteria; it contains ribosomes and the nucleoid. The anammoxosome occupies most of the cell volume and is a so-called “prokaryotic organelle” analogous to the eukaryotic mitochondrion. This is the site where the anammox reaction takes place, coupled over the curved anammoxosome membrane, possibly giving rise to a proton motive force and subsequent ATP synthesis. With these unique properties, anammox bacteria are food for thought concerning the early evolution of the domains Bacteria, Archaea, and Eukarya. PMID:22933561

  5. An SH2 domain-based tyrosine kinase assay using biotin ligase modified with a terbium(III) complex.

    Science.gov (United States)

    Sueda, Shinji; Shinboku, Yuki; Kusaba, Takeshi

    2013-01-01

    Src homology 2 (SH2) domains are modules of approximately 100 amino acids and are known to bind phosphotyrosine-containing sequences with high affinity and specificity. In the present work, we developed an SH2 domain-based assay for Src tyrosine kinase using a unique 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). Here, an SH2 domain from lymphocyte-specific tyrosine kinase was genetically fused to a truncated BCCP, and the resulting fusion protein was labeled through biotinylation with BPL carrying multiple copies of a luminescent Tb(3+) complex. The labeled SH2 fusion proteins were employed to detect a phosphorylated peptide immobilized on the surface of the microtiter plate, where the phosphorylated peptide was produced by phosphorylation to the substrate peptide by Src tyrosine kinase. Our assay allows for a reliable determination of the activity of Src kinase lower than 10 pg/μL by a simple procedure.

  6. 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.

  7. Evolution of context dependent regulation by expansion of feast/famine regulatory proteins.

    Science.gov (United States)

    Plaisier, Christopher L; Lo, Fang-Yin; Ashworth, Justin; Brooks, Aaron N; Beer, Karlyn D; Kaur, Amardeep; Pan, Min; Reiss, David J; Facciotti, Marc T; Baliga, Nitin S

    2014-11-14

    Expansion of transcription factors is believed to have played a crucial role in evolution of all organisms by enabling them to deal with dynamic environments and colonize new environments. We investigated how the expansion of the Feast/Famine Regulatory Protein (FFRP) or Lrp-like proteins into an eight-member family in Halobacterium salinarum NRC-1 has aided in niche-adaptation of this archaeon to a complex and dynamically changing hypersaline environment. We mapped genome-wide binding locations for all eight FFRPs, investigated their preference for binding different effector molecules, and identified the contexts in which they act by analyzing transcriptional responses across 35 growth conditions that mimic different environmental and nutritional conditions this organism is likely to encounter in the wild. Integrative analysis of these data constructed an FFRP regulatory network with conditionally active states that reveal how interrelated variations in DNA-binding domains, effector-molecule preferences, and binding sites in target gene promoters have tuned the functions of each FFRP to the environments in which they act. We demonstrate how conditional regulation of similar genes by two FFRPs, AsnC (an activator) and VNG1237C (a repressor), have striking environment-specific fitness consequences for oxidative stress management and growth, respectively. This study provides a systems perspective into the evolutionary process by which gene duplication within a transcription factor family contributes to environment-specific adaptation of an organism.

  8. 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.

  9. 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...... 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...... 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...

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

    Science.gov (United States)

    Gruber-Vodicka, Harald Ronald; Dirks, Ulrich; Leisch, Nikolaus; 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. The symbionts of Paracatenula galateia are provisionally classified as “Candidatus Riegeria galateiae” based on 16S ribosomal RNA sequencing confirmed by fluorescence in situ hybridization together with functional gene and sulfur metabolite evidence. 16S rRNA gene phylogenetic analysis shows that all 16 Paracatenula species examined harbor host species-specific intracellular Candidatus Riegeria bacteria that form a monophyletic group within the order Rhodospirillales. Comparing host and symbiont phylogenies reveals strict cocladogenesis and points to vertical transmission of the symbionts. Between 33% and 50% of the body volume of the various worm species is composed of bacterial symbionts, by far the highest proportion among all known endosymbiotic associations between bacteria and metazoans. This symbiosis, which likely originated more than 500 Mya during the early evolution of flatworms, is the oldest known animal–chemoautotrophic bacteria association. The distant phylogenetic position of the symbionts compared with other mutualistic or parasitic Alphaproteobacteria promises to illuminate the common genetic predispositions that have allowed several members of this class to successfully colonize eukaryote cells. PMID:21709249

  11. 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.

  12. Anion binding in biological systems

    International Nuclear Information System (INIS)

    Feiters, Martin C; Meyer-Klaucke, Wolfram; Kostenko, Alexander V; Soldatov, Alexander V; Leblanc, Catherine; Michel, Gurvan; Potin, Philippe; Kuepper, Frithjof C; Hollenstein, Kaspar; Locher, Kaspar P; Bevers, Loes E; Hagedoorn, Peter-Leon; Hagen, Wilfred R

    2009-01-01

    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 L 3 (2p 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.

  13. 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.

  14. 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.

  15. Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position.

    Science.gov (United States)

    Honda, Yuki; Zang, Qian; Shimizu, Yasuhiro; Dadashipour, Mohammad; Zhang, Zilian; Kawarabayasi, Yutaka

    2017-02-01

    The ST0452 protein is a bifunctional protein exhibiting sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) and amino-sugar-1-phosphate acetyltransferase activities and was isolated from the thermophilic archaeon Sulfolobus tokodaii Based on the previous observation that five single mutations increased ST0452 sugar-1-P NTase activity, nine double-mutant ST0452 proteins were generated with the intent of obtaining enzymes exhibiting a further increase in catalysis, but all showed less than 15% of the wild-type N-acetyl-d-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity. The Y97A mutant exhibited the highest activity of the single-mutant proteins, and thus site saturation mutagenesis of the 97th position (Tyr) was conducted. Six mutants showed both increased GlcNAc-1-P UTase and glucose-1-phosphate uridyltransferase activities, eight mutants showed only enhanced GlcNAc-1-P UTase activity, and six exhibited higher GlcNAc-1-P UTase activity than that of the Y97A mutant. Kinetic analyses of three typical mutants indicated that the increase in sugar-1-P NTase activity was mainly due to an increase in the apparent k cat value. We hypothesized that changing the 97th position (Tyr) to a smaller amino acid with similar electronic properties would increase activity, and thus the Tyr at the corresponding 103rd position of the Escherichia coli GlmU (EcGlmU) enzyme was replaced with the same residues. The Y103N mutant EcGlmU showed increased GlcNAc-1-P UTase activity, revealing that the Tyr at the 97th position of the ST0452 protein (103rd position in EcGlmU) plays an important role in catalysis. The present results provide useful information regarding how to improve the activity of natural enzymes and how to generate powerful enzymes for the industrial production of sugar nucleotides. It is typically difficult to increase enzymatic activity by introducing substitutions into a natural enzyme. However, it was previously found that the ST0452 protein

  16. Regulation of methane genes and genome expression

    Energy Technology Data Exchange (ETDEWEB)

    John N. Reeve

    2009-09-09

    , designated TFE, that had sequences in common with the eukaryotic general transcription factor TFIIE, stimulated archaeal transcription initiation and that the archaeal TATA-box binding protein (TBP) remained attached to the promoter region whereas the transcription factor TFB dissociated from the template DNA following initiation. DNA sequences that directed the localized assembly of archaeal histones into archaeal nucleosomes were identified, and we established that transcription by an archaeal RNA polymerase was slowed but not blocked by archaeal nucleosomes. We developed a new protocol to purify archaeal RNA polymerases and with this enzyme and additional improvements to the in vitro transcription system, we established the template requirements for archaeal transcription termination, investigated the activities of proteins predicted to be methane gene regulators, and established how TrpY, a novel archaeal regulator of expression of the tryptophan biosynthetic operon functions in M. thermautotrophicus. This also resulted in the discovery that almost all M. thermautotrophicus mutants isolated as spontaneously resistant to 5-methyl tryptophan (5MTR) had mutations in trpY and were therefore 5MTR through de-repressed trp operon expression. This established a very simple, practical procedure to determine and quantify the DNA sequence changes that result from exposure of this Archaeon to any experimental mutagenesis protocol. Following the discovery that the Thermococcus kodakaraensis was amenable to genetic manipulation, we established this technology at OSU and subsequently added plasmid expression, a reporter system and additional genetic selections to the T. kodakaraensis genetic toolbox. We established that transcription and translation are coupled in this Archaeon, and by combining in vitro transcription and in vivo genetics, we documented that both TFB1 and TFB2 support transcription initiation in T. kodakaraensis. We quantified the roles of ribosome binding sequences

  17. Nanobody®-based chromatin immunoprecipitation/micro-array analysis for genome-wide identification of transcription factor DNA binding sites

    Science.gov (United States)

    Nguyen-Duc, Trong; Peeters, Eveline; Muyldermans, Serge; Charlier, Daniel; Hassanzadeh-Ghassabeh, Gholamreza

    2013-01-01

    Nanobodies® are single-domain antibody fragments derived from camelid heavy-chain antibodies. Because of their small size, straightforward production in Escherichia coli, easy tailoring, high affinity, specificity, stability and solubility, nanobodies® have been exploited in various biotechnological applications. A major challenge in the post-genomics and post-proteomics era is the identification of regulatory networks involving nucleic acid–protein and protein–protein interactions. Here, we apply a nanobody® in chromatin immunoprecipitation followed by DNA microarray hybridization (ChIP-chip) for genome-wide identification of DNA–protein interactions. The Lrp-like regulator Ss-LrpB, arguably one of the best-studied specific transcription factors of the hyperthermophilic archaeon Sulfolobus solfataricus, was chosen for this proof-of-principle nanobody®-assisted ChIP. Three distinct Ss-LrpB-specific nanobodies®, each interacting with a different epitope, were generated for ChIP. Genome-wide ChIP-chip with one of these nanobodies® identified the well-established Ss-LrpB binding sites and revealed several unknown target sequences. Furthermore, these ChIP-chip profiles revealed auxiliary operator sites in the open reading frame of Ss-lrpB. Our work introduces nanobodies® as a novel class of affinity reagents for ChIP. Taking into account the unique characteristics of nanobodies®, in particular, their short generation time, nanobody®-based ChIP is expected to further streamline ChIP-chip and ChIP-Seq experiments, especially in organisms with no (or limited) possibility of genetic manipulation. PMID:23275538

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

    Science.gov (United States)

    Kirpekar, F; Douthwaite, S; Roepstorff, P

    2000-02-01

    We present a method to screen RNA for posttranscriptional modifications based on Matrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS). After the RNA is digested to completion with a nucleotide-specific RNase, the fragments are analyzed by mass spectrometry. A comparison of the observed mass data with the data predicted from the gene sequence identifies fragments harboring modified nucleotides. Fragments larger than dinucleotides were valuable for the identification of posttranscriptional modifications. A more refined mapping of RNA modifications can be obtained by using two RNases in parallel combined with further fragmentation by Post Source Decay (PSD). This approach allows fast and sensitive screening of a purified RNA for posttranscriptional modification, and has been applied on 5S rRNA from two thermophilic microorganisms, the bacterium Bacillus stearothermophilus 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 that is clearly conserved with respect to both sequence and position in B. stearothermophilus and H. halobium and to some degree also in H. marismortui. However, no analogous modification was identified in the latter three organisms. We further find that the 5' end of H. halobium 5S rRNA is dephosphorylated, in contrast to the other 5S rRNA species investigated. The method additionally gives an immediate indication of whether the expected RNA sequence is in agreement with the observed fragment masses. Discrepancies with two of the published 5S rRNA sequences were identified and are reported here.

  19. Effects of cattle husbandry on abundance and activity of methanogenic archaea in upland soils.

    Science.gov (United States)

    Radl, Viviane; Gattinger, Andreas; Chronáková, Alica; Nemcová, Anna; Cuhel, Jiri; Simek, Miloslav; Munch, Jean Charles; Schloter, Michael; Elhottová, Dana

    2007-09-01

    In the present study, we tested the hypothesis that animal treading associated with a high input of organic matter would favour methanogenesis in soils used as overwintering pasture. Hence, methane emissions and methanogen populations were examined at sections with different degree of cattle impact in a Farm in South Bohemia, Czech Republic. In spring, methane emission positively corresponded to the gradient of animal impact. Applying phospholipid etherlipid analysis, the highest archaeal biomass was found in section severe impact (SI), followed by moderate impact (MI) and no impact. The same trend was observed for the methanogens as showed by real-time quantitative PCR analyses of methyl coenzyme M reductase (mcrA) genes. The detection of monounsaturated isoprenoid side chain hydrocarbons (i20:1) indicated the presence of acetoclastic methanogens in the cattle-impacted sites. This result was corroborated by the phylogenetic analysis of mcrA gene sequences obtained from section SI, which showed that 33% of the analysed clones belonged to the genus Methanosarcina. The majority of the sequenced clones (41%) showed close affiliations with uncultured rumen archaeons. This leads to the assumption that a substantial part of the methanogenic community in plot SI derived from the grazing cattle itself. Compared to the spring sampling, in autumn, a significant reduction in archaeal biomass and number of copies of mcrA genes was observed mainly for section MI. It can be concluded that after 5 months without cattle impact, the severely impact section maintained its methane production potential, whereas the methane production potential under moderate impact returned to background values.

  20. High sensitive RNA detection by one-step RT-PCR using the genetically engineered variant of DNA polymerase with reverse transcriptase activity from hyperthermophilies.

    Science.gov (United States)

    Okano, Hiroyuki; Baba, Misato; Kawato, Katsuhiro; Hidese, Ryota; Yanagihara, Itaru; Kojima, Kenji; Takita, Teisuke; Fujiwara, Shinsuke; Yasukawa, Kiyoshi

    2018-03-01

    One-step RT-PCR has not been widely used even though some thermostable DNA polymerases with reverse transcriptase (RT) activity were developed from bacterial and archaeal polymerases, which is owing to low cDNA synthesis activity from RNA. In the present study, we developed highly-sensitive one-step RT-PCR using the single variant of family A DNA polymerase with RT activity, K4pol L329A (L329A), from the hyperthermophilic bacterium Thermotoga petrophila K4 or the 16-tuple variant of family B DNA polymerase with RT activity, RTX, from the hyperthermophilic archaeon Thermococcus kodakarensis. Optimization of reaction condition revealed that the activities for cDNA synthesis and PCR of K4pol L329A and RTX were highly affected by the concentrations of MgCl 2 and Mn(OCOCH 3 ) 2 as well as those of K4pol L329A or RTX. Under the optimized condition, 300 copies/μl of target RNA in 10 μl reaction volumes were successfully detected by the one-step RT-PCR with K4pol L329A or RTX, which was almost equally sensitive enough compared with the current RT-PCR condition using retroviral RT and thermostable DNA polymerase. Considering that K4pol L329A and RTX are stable even at 90-100°C, our results suggest that the one-step RT-PCR with K4pol L329A or RTX is more advantageous than the current one. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  1. First description of giant Archaea (Thaumarchaeota) associated with putative bacterial ectosymbionts in a sulfidic marine habitat.

    Science.gov (United States)

    Muller, Félix; Brissac, Terry; Le Bris, Nadine; Felbeck, Horst; Gros, Olivier

    2010-08-01

    Archaea may be involved in global energy cycles, and are known for their ability to interact with eukaryotic species (sponges, corals and ascidians) or as archaeal-bacterial consortia. The recently proposed phylum Thaumarchaeota may represent the deepest branching lineage in the archaeal phylogeny emerging before the divergence between Euryarchaeota and Crenarchaeota. Here we report the first characterization of two marine thaumarchaeal species from shallow waters that consist of multiple giant cells. One species is coated with sulfur-oxidizing γ-Proteobacteria. These new uncultured thaumarchaeal species are able to live in the sulfide-rich environments of a tropical mangrove swamp, either on living tissues such as roots or on various kinds of materials such as stones, sunken woods, etc. These archaea and archaea/bacteria associations have been studied using light microscopy, transmission electron microscopy and scanning electron microscopy. Species identification of archaeons and the putative bacterial symbiont have been assessed by 16S small subunit ribosomal RNA analysis. The sulfur-oxidizing ability of the bacteria has been assessed by genetic investigation on alpha-subunit of the adenosine-5'-phosphosulfate reductase/oxidase's (AprA). Species identifications have been confirmed by fluorescence in situ hybridization using specific probes designed in this study. In this article, we describe two new giant archaeal species that form the biggest archaeal filaments ever observed. One of these species is covered by a specific biofilm of sulfur-oxidizing γ-Proteobacteria. This study highlights an unexpected morphological and genetic diversity of the phylum Thaumarchaeota. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

  2. Two Strategies for Microbial Production of an Industrial Enzyme-Alpha-Amylase

    Science.gov (United States)

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

    2003-01-01

    Extremophiles are microorganisms that thrive in, from an anthropocentric view, extreme environments including hot springs, soda lakes and arctic water. This ability of survival at extreme conditions has rendered extremophiles to be of interest in astrobiology, evolutionary biology as well as in industrial applications. Of particular interest to the biotechnology industry are the biological catalysts of the extremophiles, the extremozymes, whose unique stabilities at extreme conditions make them potential sources of novel enzymes in industrial applications. There are two major approaches to microbial enzyme production. This entails enzyme isolation directly from the natural host or creating a recombinant expression system whereby the targeted enzyme can be overexpressed in a mesophilic host. We are employing both methods in the effort to produce alpha-amylases from a hyperthermophilic archaeon (Thermococcus) isolated from a hydrothermal vent in the Atlantic Ocean, as well as from alkaliphilic bacteria (Bacillus) isolated from a soda lake in Tanzania. Alpha-amylases catalyze the hydrolysis of internal alpha-1,4-glycosidic linkages in starch to produce smaller sugars. Thermostable alpha-amylases are used in the liquefaction of starch for production of fructose and glucose syrups, whereas alpha-amylases stable at high pH have potential as detergent additives. The alpha-amylase encoding gene from Thermococcus was PCR amplified using carefully designed primers and analyzed using bioinformatics tools such as BLAST and Multiple Sequence Alignment for cloning and expression in E.coli. Four strains of Bacillus were grown in alkaline starch-enriched medium of which the culture supernatant was used as enzyme source. Amylolytic activity was detected using the starch-iodine method.

  3. Factors to Enable Crystallization of Environmentally Stable Bioscorodite from Dilute As(III-Contaminated Waters

    Directory of Open Access Journals (Sweden)

    Masahito Tanaka

    2018-01-01

    Full Text Available Applicability of the bioscorodite method (use of the thermo-acidophilic Fe(II-oxidizing archaeon Acidianus brierleyi for arsenic (As oxidation and immobilization at 70 °C was tested for synthetic copper refinery wastewaters of a wide range of dilute initial As(III concentrations ([As(III]ini = 3.3–20 mM with varying initial [Fe(II]/[As(III] molar ratios ([Fe(II]ini/[As(III]ini = 0.8–6.0. Crystallization of scorodite (FeAsO4·2H2O tends to become increasingly challenging at more dilute As(III solutions. Optimization of conditions such as initial pH, seed feeding and initial [Fe(II]/[As(III] molar ratio was found critical in improving final As removal and product stability: Whilst setting the initial pH at 1.2 resulted in an immediate single-stage precipitation of crystalline bioscorodite, the initial pH 1.5 led to a two-stage As precipitation (generation of brown amorphous precursors followed by whitish crystalline bioscorodite particles with a greater final As removal. The formation process of bioscorodite precipitates differed significantly depending on the type of seed crystals fed (bio- versus chemical- scorodite seeds. Feeding the former was found effective not only in accelerating the reaction, but also in forming more recalcitrant bioscorodite products (0.59 mg/L; Toxicity Characteristic Leaching Procedure (TCLP test. Under such favorable conditions, 94–99% of As was successfully removed as crystalline bioscorodite at all dilute As(III concentrations tested by setting [Fe(II]ini/[As(III]ini at 1.4–2.0. Providing an excess Fe(II (closer to [Fe(II]ini/[As(III]ini = 2.0 was found beneficial to improve the final As removal (up to 98–99% especially from more dilute As(III solutions.

  4. Analysis of dissimilatory sulfite reductase and 16S rRNA gene fragments from deep-sea hydrothermal sites of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific.

    Science.gov (United States)

    Nakagawa, Tatsunori; Ishibashi, Jun-Ichiro; Maruyama, Akihiko; Yamanaka, Toshiro; Morimoto, Yusuke; Kimura, Hiroyuki; Urabe, Tetsuro; Fukui, Manabu

    2004-01-01

    This study describes the occurrence of unique dissimilatory sulfite reductase (DSR) genes at a depth of 1,380 m from the deep-sea hydrothermal vent field at the Suiyo Seamount, Izu-Bonin Arc, Western Pacific, Japan. The DSR genes were obtained from microbes that grew in a catheter-type in situ growth chamber deployed for 3 days on a vent and from the effluent water of drilled holes at 5 degrees C and natural vent fluids at 7 degrees C. DSR clones SUIYOdsr-A and SUIYOdsr-B were not closely related to cultivated species or environmental clones. Moreover, samples of microbial communities were examined by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA gene. The sequence analysis of 16S rRNA gene fragments obtained from the vent catheter after a 3-day incubation revealed the occurrence of bacterial DGGE bands affiliated with the Aquificae and gamma- and epsilon-Proteobacteria as well as the occurrence of archaeal phylotypes affiliated with the Thermococcales and of a unique archaeon sequence that clustered with "Nanoarchaeota." The DGGE bands obtained from drilled holes and natural vent fluids from 7 to 300 degrees C were affiliated with the delta-Proteobacteria, genus Thiomicrospira, and Pelodictyon. The dominant DGGE bands retrieved from the effluent water of casing pipes at 3 and 4 degrees C were closely related to phylotypes obtained from the Arctic Ocean. Our results suggest the presence of microorganisms corresponding to a unique DSR lineage not detected previously from other geothermal environments.

  5. An Uncharacterized Member of the Ribokinase Family in Thermococcus kodakarensis Exhibits myo-Inositol Kinase Activity*

    Science.gov (United States)

    Sato, Takaaki; Fujihashi, Masahiro; Miyamoto, Yukika; Kuwata, Keiko; Kusaka, Eriko; Fujita, Haruo; Miki, Kunio; Atomi, Haruyuki

    2013-01-01

    Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate. PMID:23737529

  6. Genome-wide mRNA processing in methanogenic archaea reveals post-transcriptional regulation of ribosomal protein synthesis.

    Science.gov (United States)

    Qi, Lei; Yue, Lei; Feng, Deqin; Qi, Fengxia; Li, Jie; Dong, Xiuzhu

    2017-07-07

    Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an 'all-or-none' pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Identification of an archaeal presenilin-like intramembrane protease.

    Science.gov (United States)

    Torres-Arancivia, Celia; Ross, Carolyn M; Chavez, Jose; Assur, Zahra; Dolios, Georgia; Mancia, Filippo; Ubarretxena-Belandia, Iban

    2010-09-29

    The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the γ-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of β-amyloid peptides (Aβ) implicated in Alzheimer's disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs). The presence of presenilin-like enzymes outside eukaryots has not been demonstrated. Here we report the existence of presenilin-like GXGD-type diaspartyl intramembrane proteases in archaea. We have employed in vitro activity assays to show that MCMJR1, a polytopic membrane protein from the archaeon Methanoculleus marisnigri JR1, is an intramembrane protease bearing the signature YD and GXGD catalytic motifs of presenilin-like enzymes. Mass spectrometry analysis showed MCMJR1 could cleave model intramembrane protease substrates at several sites within their transmembrane region. Remarkably, MCMJR1 could also cleave substrates derived from the β-amyloid precursor protein (APP) without the need of protein co-factors, as required by presenilin. Two distinct cleavage sites within the transmembrane domain of APP could be identified, one of which coincided with Aβ40, the predominant site processed by γ-secretase. Finally, an established presenilin and SPP transition-state analog inhibitor could inhibit MCMJR1. Our findings suggest that a primitive GXGD-type diaspartyl intramembrane protease from archaea can recapitulate key biochemical properties of eukaryotic presenilins and SPPs. MCMJR1 promises to be a more tractable, simpler system for in depth structural and mechanistic studies of GXGD-type diaspartyl intramembrane proteases.

  8. Identification of a mismatch-specific endonuclease in hyperthermophilic Archaea.

    Science.gov (United States)

    Ishino, Sonoko; Nishi, Yuki; Oda, Soichiro; Uemori, Takashi; Sagara, Takehiro; Takatsu, Nariaki; Yamagami, Takeshi; Shirai, Tsuyoshi; Ishino, Yoshizumi

    2016-04-20

    The common mismatch repair system processed by MutS and MutL and their homologs was identified in Bacteria and Eukarya. However, no evidence of a functional MutS/L homolog has been reported for archaeal organisms, and it is not known whether the mismatch repair system is conserved in Archaea. Here, we describe an endonuclease that cleaves double-stranded DNA containing a mismatched base pair, from the hyperthermophilic archaeon Pyrococcus furiosus The corresponding gene revealed that the activity originates from PF0012, and we named this enzyme Endonuclease MS (EndoMS) as the mismatch-specific Endonuclease. The sequence similarity suggested that EndoMS is the ortholog of NucS isolated from Pyrococcus abyssi, published previously. Biochemical characterizations of the EndoMS homolog from Thermococcus kodakarensis clearly showed that EndoMS specifically cleaves both strands of double-stranded DNA into 5'-protruding forms, with the mismatched base pair in the central position. EndoMS cleaves G/T, G/G, T/T, T/C and A/G mismatches, with a more preference for G/T, G/G and T/T, but has very little or no effect on C/C, A/C and A/A mismatches. The discovery of this endonuclease suggests the existence of a novel mismatch repair process, initiated by the double-strand break generated by the EndoMS endonuclease, in Archaea and some Bacteria. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Identification of an archaeal presenilin-like intramembrane protease.

    Directory of Open Access Journals (Sweden)

    Celia Torres-Arancivia

    Full Text Available BACKGROUND: The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the γ-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of β-amyloid peptides (Aβ implicated in Alzheimer's disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs. The presence of presenilin-like enzymes outside eukaryots has not been demonstrated. Here we report the existence of presenilin-like GXGD-type diaspartyl intramembrane proteases in archaea. METHODOLOGY AND PRINCIPAL FINDINGS: We have employed in vitro activity assays to show that MCMJR1, a polytopic membrane protein from the archaeon Methanoculleus marisnigri JR1, is an intramembrane protease bearing the signature YD and GXGD catalytic motifs of presenilin-like enzymes. Mass spectrometry analysis showed MCMJR1 could cleave model intramembrane protease substrates at several sites within their transmembrane region. Remarkably, MCMJR1 could also cleave substrates derived from the β-amyloid precursor protein (APP without the need of protein co-factors, as required by presenilin. Two distinct cleavage sites within the transmembrane domain of APP could be identified, one of which coincided with Aβ40, the predominant site processed by γ-secretase. Finally, an established presenilin and SPP transition-state analog inhibitor could inhibit MCMJR1. CONCLUSIONS AND SIGNIFICANCE: Our findings suggest that a primitive GXGD-type diaspartyl intramembrane protease from archaea can recapitulate key biochemical properties of eukaryotic presenilins and SPPs. MCMJR1 promises to be a more tractable, simpler system for in depth structural and mechanistic studies of GXGD-type diaspartyl intramembrane proteases.

  10. Genomic Comparison of Two Family-Level Groups of the Uncultivated NAG1 Archaeal Lineage from Chemically and Geographically Disparate Hot Springs

    Directory of Open Access Journals (Sweden)

    Eric D. Becraft

    2017-10-01

    Full Text Available Recent progress based on single-cell genomics and metagenomic investigations of archaea in a variety of extreme environments has led to significant advances in our understanding of the diversity, evolution, and metabolic potential of archaea, yet the vast majority of archaeal diversity remains undersampled. In this work, we coordinated single-cell genomics with metagenomics in order to construct a near-complete genome from a deeply branching uncultivated archaeal lineage sampled from Great Boiling Spring (GBS in the U.S. Great Basin, Nevada. This taxon is distantly related (distinct families to an archaeal genome, designated “Novel Archaeal Group 1” (NAG1, which was extracted from a metagenome recovered from an acidic iron spring in Yellowstone National Park (YNP. We compared the metabolic predictions of the NAG1 lineage to better understand how these archaea could inhabit such chemically distinct environments. Similar to the NAG1 population previously studied in YNP, the NAG1 population from GBS is predicted to utilize proteins as a primary carbon source, ferment simple carbon sources, and use oxygen as a terminal electron acceptor under oxic conditions. However, GBS NAG1 populations contained distinct genes involved in central carbon metabolism and electron transfer, including nitrite reductase, which could confer the ability to reduce nitrite under anaerobic conditions. Despite inhabiting chemically distinct environments with large variations in pH, GBS NAG1 populations shared many core genomic and metabolic features with the archaeon identified from YNP, yet were able to carve out a distinct niche at GBS.

  11. Environmental genomics of "Haloquadratum walsbyi" in a saltern crystallizer indicates a large pool of accessory genes in an otherwise coherent species

    Directory of Open Access Journals (Sweden)

    Bolhuis Henk

    2006-07-01

    Full Text Available Abstract Background Mature saturated brine (crystallizers communities are largely dominated (>80% of cells by the square halophilic archaeon "Haloquadratum walsbyi". The recent cultivation of the strain HBSQ001 and thesequencing of its genome allows comparison with the metagenome of this taxonomically simplified environment. Similar studies carried out in other extreme environments have revealed very little diversity in gene content among the cell lineages present. Results The metagenome of the microbial community of a crystallizer pond has been analyzed by end sequencing a 2000 clone fosmid library and comparing the sequences obtained with the genome sequence of "Haloquadratum walsbyi". The genome of the sequenced strain was retrieved nearly complete within this environmental DNA library. However, many ORF's that could be ascribed to the "Haloquadratum" metapopulation by common genome characteristics or scaffolding to the strain genome were not present in the specific sequenced isolate. Particularly, three regions of the sequenced genome were associated with multiple rearrangements and the presence of different genes from the metapopulation. Many transposition and phage related genes were found within this pool which, together with the associated atypical GC content in these areas, supports lateral gene transfer mediated by these elements as the most probable genetic cause of this variability. Additionally, these sequences were highly enriched in putative regulatory and signal transduction functions. Conclusion These results point to a large pan-genome (total gene repertoire of the genus/species even in this highly specialized extremophile and at a single geographic location. The extensive gene repertoire is what might be expected of a population that exploits a diverse nutrient pool, resulting from the degradation of biomass produced at lower salinities.

  12. Deletion of the Sm1 encoding motif in the lsm gene results in distinct changes in the transcriptome and enhanced swarming activity of Haloferax cells.

    Science.gov (United States)

    Maier, Lisa-Katharina; Benz, Juliane; Fischer, Susan; Alstetter, Martina; Jaschinski, Katharina; Hilker, Rolf; Becker, Anke; Allers, Thorsten; Soppa, Jörg; Marchfelder, Anita

    2015-10-01

    Members of the Sm protein family are important for the cellular RNA metabolism in all three domains of life. The family includes archaeal and eukaryotic Lsm proteins, eukaryotic Sm proteins and archaeal and bacterial Hfq proteins. While several studies concerning the bacterial and eukaryotic family members have been published, little is known about the archaeal Lsm proteins. Although structures for several archaeal Lsm proteins have been solved already more than ten years ago, we still do not know much about their biological function, however one can confidently propose that the archaeal Lsm proteins will also be involved in RNA metabolism. Therefore, we investigated this protein in the halophilic archaeon Haloferax volcanii. The Haloferax genome encodes a single Lsm protein, the lsm gene overlaps and is co-transcribed with the gene for the ribosomal L37.eR protein. Here, we show that the reading frame of the lsm gene contains a promoter which regulates expression of the overlapping rpl37R gene. This rpl37R specific promoter ensures high expression of the rpl37R gene in exponential growth phase. To investigate the biological function of the Lsm protein we generated a lsm deletion mutant that had the coding sequence for the Sm1 motif removed but still contained the internal promoter for the downstream rpl37R gene. The transcriptome of this deletion mutant was compared to the wild type transcriptome, revealing that several genes are down-regulated and many genes are up-regulated in the deletion strain. Northern blot analyses confirmed down-regulation of two genes. In addition, the deletion strain showed a gain of function in swarming, in congruence with the up-regulation of transcripts encoding proteins required for motility. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Halophilic mechanism of the enzymatic function of a moderately halophilic dihydrofolate reductase from Haloarcula japonica strain TR-1.

    Science.gov (United States)

    Miyashita, Yurina; Ohmae, Eiji; Ikura, Teikichi; Nakasone, Kaoru; Katayanagi, Katsuo

    2017-05-01

    Dihydrofolate (DHF) reductase coded by a plasmid of the extremely halophilic archaeon Haloarcula japonica strain TR-1 (HjDHFR P1) shows moderate halophilicity on enzymatic activity at pH 6.0, although there is no significant effect of NaCl on its secondary structure. To elucidate the salt-activation and -inactivation mechanisms of this enzyme, we investigated the effects of pH and salt concentration, deuterium isotope effect, steady-state kinetics, and rapid-phase ligand-binding kinetics. Enzyme activity was increased eightfold by the addition of 500 mM NaCl at pH 6.0, fourfold by 250 mM at pH 8.0, and became independent of salt concentration at pH 10.0. Full isotope effects observed at pH 10.0 under 0-1000 mM NaCl indicated that the rate of hydride transfer, which was the rate-determining step at the basic pH region, was independent of salt concentration. Conversely, rapid-phase ligand-binding experiments showed that the amplitude of the DHF-binding reaction increased and the tetrahydrofolate (THF)-releasing rate decreased with increasing NaCl concentration. These results suggested that the salt-activation mechanism of HjDHFR P1 is via the population change of the anion-unbound and anion-bound conformers, which are binding-incompetent and -competent conformations for DHF, respectively, while that of salt inactivation is via deceleration of the THF-releasing rate, which is the rate-determining step at the neutral pH region.

  14. Investigating the Effects of Simulated Space conditions on Novel Extremely Halophilic Archaea: Halovarius Luteus gen. nov., sp. nov.

    Science.gov (United States)

    Feshangsaz, Niloofar; Van Loon, ing.. Jack J. W. A.; Nazmi, Kamran; Semsarha, Farid

    2016-07-01

    Studying halophiles from different environments of Earth provide new insights into our search for life in the universe. Haloarchaea show some unique characteristics and physiological adaptations like acidic proteins against harsh environments such as natural brine with salt concentration approaching saturation (5 M) and regions with low active water. These properties make haloarchaea interesting candidate for astrobiological studies. Halovarius luteus gen. nov., sp. nov. a novel extremely halophilic archaeon from Urmia salt lake, in Iran has been chosen to explore its resistance against a series of extreme conditions. The aim of this study is to assess the resistance of strain DA50T under the effects of simulated space conditions like simulated microgravity, hypergravity, and desiccation. In this paper we will discuss the results of these studies where we specifically focus on changes in carotenoid pigments production and whole cell proteome. This is the first report of very novel Iranian archaea in response to extreme space conditions. The pigments were extracted by acetone and methanol. Pigments were analyzed by scanning the absorbance spectrum in the UV-VIS spectrophotometer. And they were separated by TLC. Whole protein from cell lysate supernatant was extracted after lysis with Bacterial Protein Extraction Reagent and fractionated by RP-HPLC using C18 column. Proteome analyzed by electrophoresis (SDS-PAGE), and MALDI-TOF. Carotenoid pigments are formed under different extreme conditions such as dry environment and gravitational changes. Also the protein composition exhibits alterations after exposure to the same conditions. Our conclusion is that pigments and proteins formation depend on the growth circumstances. Halophiles use this as an adaptation to survive under different environmental conditions.

  15. Identification of a haloalkaliphilic and thermostable cellulase with improved ionic liquid tolerance

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Tao; Datta, Supratim; Eichler, Jerry; Ivanova, Natalia; Axen, Seth D.; Kerfeld, Cheryl A.; Chen, Feng; Kyrpides, Nikos; Hugenholtz, Philip; Cheng, Jan-Fang; Sale, Kenneth L.; Simmons, Blake; Rubin, Eddy

    2011-02-17

    Some ionic liquids (ILs) have been shown to be very effective solvents for biomass pretreatment. It is known that some ILs can have a strong inhibitory effect on fungal cellulases, making the digestion of cellulose inefficient in the presence of ILs. The identification of IL-tolerant enzymes that could be produced as a cellulase cocktail would reduce the costs and water use requirements of the IL pretreatment process. Due to their adaptation to high salinity environments, halophilic enzymes are hypothesized to be good candidates for screening and identifying IL-resistant cellulases. Using a genome-based approach, we have identified and characterized a halophilic cellulase (Hu-CBH1) from the halophilic archaeon, Halorhabdus utahensis. Hu-CBH1 is present in a gene cluster containing multiple putative cellulolytic enzymes. Sequence and theoretical structure analysis indicate that Hu-CBH1 is highly enriched with negatively charged acidic amino acids on the surface, which may form a solvation shell that may stabilize the enzyme, through interaction with salt ions and/or water molecules. Hu-CBH1 is a heat tolerant haloalkaliphilic cellulase and is active in salt concentrations up to 5 M NaCl. In high salt buffer, Hu-CBH1 can tolerate alkali (pH 11.5) conditions and, more importantly, is tolerant to high levels (20percent w/w) of ILs, including 1-allyl-3-methylimidazolium chloride ([Amim]Cl). Interestingly, the tolerances to heat, alkali and ILs are found to be salt-dependent, suggesting that the enzyme is stabilized by the presence of salt. Our results indicate that halophilic enzymes are good candidates for the screening of IL-tolerant cellulolytic enzymes.

  16. Bacterial and archaeal resistance to ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Confalonieri, F; Sommer, S, E-mail: fabrice.confalonieri@u-psud.fr, E-mail: suzanne.sommer@u-psud.fr [University Paris-Sud, CNRS UMR8621, Institut de Genetique et Microbiologie, Batiments 400-409, Universite Paris-Sud, 91405 Orsay (France)

    2011-01-01

    Organisms living in extreme environments must cope with large fluctuations of temperature, high levels of radiation and/or desiccation, conditions that can induce DNA damage ranging from base modifications to DNA double-strand breaks. The bacterium Deinococcus radiodurans is known for its resistance to extremely high doses of ionizing radiation and for its ability to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Recently, extreme ionizing radiation resistance was also generated by directed evolution of an apparently radiation-sensitive bacterial species, Escherichia coli. Radioresistant organisms are not only found among the Eubacteria but also among the Archaea that represent the third kingdom of life. They present a set of particular features that differentiate them from the Eubacteria and eukaryotes. Moreover, Archaea are often isolated from extreme environments where they live under severe conditions of temperature, pressure, pH, salts or toxic compounds that are lethal for the large majority of living organisms. Thus, Archaea offer the opportunity to understand how cells are able to cope with such harsh conditions. Among them, the halophilic archaeon Halobacterium sp and several Pyrococcus or Thermococcus species, such as Thermococcus gammatolerans, were also shown to display high level of radiation resistance. The dispersion, in the phylogenetic tree, of radioresistant prokaryotes suggests that they have independently acquired radioresistance. Different strategies were selected during evolution including several mechanisms of radiation byproduct detoxification and subtle cellular metabolism modifications to help cells recover from radiation-induced injuries, protection of proteins against oxidation, an efficient DNA repair tool box, an original pathway of DNA double-strand break repair, a condensed nucleoid that may prevent the dispersion of the DNA fragments and specific radiation-induced proteins involved in

  17. Resistance of the Extreme Halophile Halobacterium sp. NRC-1 to Multiple Stresses

    International Nuclear Information System (INIS)

    Gygli, Patrick E.; Prajapati, Surendra; DeVeaux, Linda C.; DasSarma, Shiladitya; DasSarma, Priya; Mestari, Mohammed Amine; Wells, Douglas P.

    2009-01-01

    The model Archaeon Halobacterium sp. NRC-1 is an extreme halophile known for its resistance to multiple stressors, including electron-beam and ultraviolet radiation. It is a well-developed system with a completely sequenced genome and extensive post-genomic tools for the study of a variety of biological processes. To further understand the mechanisms of Halobacterium's, radiation resistance, we previously reported the selection for multiple independent highly resistant mutants using repeated exposure to high doses of 18-20 MeV electrons using a medical S-band Linac. Molecular analysis of the transcriptional profile of several of these mutants revealed a single common change: upregulation of the rfa3 operon. These genes encode proteins homologous to the subunits of eukaryotic Replication Protein A (RPA), a DNA binding protein with major roles in DNA replication, recombination, and repair. This operon has also been implicated in a somewhat lesser role in resistance of wild type Halobacterium to ultraviolet radiation, suggesting common mechanisms for resistance. To further understand the mechanism of radiation resistance in the mutant strains, we measured the survival after exposure to both electron-beam and ultraviolet radiation, UV-A, B, and C All mutant strains showed increased resistance to electrons when compared with the parent. However, the mutant strains do not display increased UV resistance, and in one case is more sensitive than the parent strain. Thus, the protective role of increased RPA expression within a cell may be specific to the DNA damage caused by the different physical effects induced by high energy electron-beam radiation.

  18. Roles of thermophilic thiosulfate-reducing bacteria and methanogenic archaea in the biocorrosion of oil pipelines

    Directory of Open Access Journals (Sweden)

    Renxing eLiang

    2014-03-01

    Full Text Available Thermophilic sulfide-producing microorganisms from an oil pipeline network were enumerated with different sulfur oxyanions as electron acceptors at 55 oC. Most-probable number (MPN analysis showed that thiosulfate-reducing bacteria were the most numerous sulfidogenic microorganisms in pipeline inspection gauge (PIG scrapings. Thiosulfate-reducing and methanogenic enrichments were obtained from the MPN cultures that were able to use yeast extract as the electron donor. Molecular analysis revealed that both enrichments harbored the same dominant bacterium, which belonged to the genus Anaerobaculum. The dominant archaeon in the methanogenic enrichment was affiliated with the genus Methanothermobacter. With yeast extract as the electron donor, the general corrosion rate by the thiosulfate-reducing enrichment (8.43 ± 1.40 milli-inch per year, abbreviated as mpy was about 5.5 times greater than the abiotic control (1.49 ± 0.15 mpy, while the comparable measures for the methanogenic culture were 2.03 ± 0.49 mpy and 0.62 ± 0.07 mpy, respectively. Total iron analysis in the cultures largely accounted for the mass loss of iron measured in the weight loss determinations. Profilometry analysis of polished steel coupons incubated in the presence of the thiosulfate-reducing enrichment revealed 59 pits over an area of 71.16 mm2, while only 6 pits were evident in the corresponding methanogenic incubations. The results show the importance of thiosulfate-utilizing, sulfide-producing fermentative bacteria such as Anaerobaculum sp. in the corrosion of carbon steel, but also suggest that Anaerobaculum sp. are of far less concern when growing syntrophically with methanogens.

  19. Purification and characterization of an extracellular halophilic and organic solvent-tolerant amylopullulanase from a haloarchaeon, Halorubrum sp. strain Ha25.

    Directory of Open Access Journals (Sweden)

    Mostafa Fazeli

    2013-01-01

    Full Text Available Introduction: Halophiles, especially haloarchaea are one of the most important groups of extremophiles. Halophilic hydrolases have been studied worldwide and have been considered for biotechnology and industrial technologies. This study is the first report in amylopullulanase production in halophilic microorganisms.Materials and methods: A halophilic archaeon, Halorubrum sp. strain Ha25, produced extracellular halophilic organic solvent-tolerant amylopullulanase. The enzyme was purified using ethanol precipitation and anion exchange chromatography method. Molecular mass of purified enzyme was determined by SDS–PAGE method. After purification, the enzyme was characterized. To study the effects of organic solvents in the stability of the enzyme, the enzyme solution was incubated in the presence of various organic compounds and then, residual enzyme activity was measured. Mode of action of the enzyme was determined by thin-layer chromatography.Results: Molecular weight of the purified enzyme was estimated to be 140 kDa by SDS–PAGE method. Optimum temperature for amylolitic and pullulytic activities was 50 °C. Optimum pH for amylolitic activity was 7.0 and for pullulytic activity was 7.5. This enzyme was active over a wide range of concentrations (0-4.5 M of NaCl. The effect of organic solvents on the amylolitic and pullulytic activities showed that this enzyme was more stable in the presence of non-polar organic solvents than polar solvents. The enzyme solely hydrolyzed pullulan and soluble starch to glucose.Discussion and conclusion: Halorubrum sp. strain Ha25 produces thermophilic and extremely halophilic amylopullulanase. The catalytic function under multi extreme condition of high temperature, high salinity, and low water activity might possess biotechnological and commercial values such as treatment waste solutions with starch residues, high salt content and solvents.

  20. Metagenomic analysis and functional characterization of the biogas microbiome using high throughput shotgun sequencing and a novel binning strategy.

    Science.gov (United States)

    Campanaro, Stefano; Treu, Laura; Kougias, Panagiotis G; De Francisci, Davide; Valle, Giorgio; Angelidaki, Irini

    2016-01-01

    Biogas production is an economically attractive technology that has gained momentum worldwide over the past years. Biogas is produced by a biologically mediated process, widely known as "anaerobic digestion." This process is performed by a specialized and complex microbial community, in which different members have distinct roles in the establishment of a collective organization. Deciphering the complex microbial community engaged in this process is interesting both for unraveling the network of bacterial interactions and for applicability potential to the derived knowledge. In this study, we dissect the bioma involved in anaerobic digestion by means of high throughput Illumina sequencing (~51 gigabases of sequence data), disclosing nearly one million genes and extracting 106 microbial genomes by a novel strategy combining two binning processes. Microbial phylogeny and putative taxonomy performed using >400 proteins revealed that the biogas community is a trove of new species. A new approach based on functional properties as per network representation was developed to assign roles to the microbial species. The organization of the anaerobic digestion microbiome is resembled by a funnel concept, in which the microbial consortium presents a progressive functional specialization while reaching the final step of the process (i.e., methanogenesis). Key microbial genomes encoding enzymes involved in specific metabolic pathways, such as carbohydrates utilization, fatty acids degradation, amino acids fermentation, and syntrophic acetate oxidation, were identified. Additionally, the analysis identified a new uncultured archaeon that was putatively related to Methanomassiliicoccales but surprisingly having a methylotrophic methanogenic pathway. This study is a pioneer research on the phylogenetic and functional characterization of the microbial community populating biogas reactors. By applying for the first time high-throughput sequencing and a novel binning strategy, the

  1. Ammonia oxidation driven by archaea rather than bacteria in the hot spring at Tengchong geothermal field, China.

    Science.gov (United States)

    Chen, Shun; Peng, Xiaotong; Xu, Hengchao; Li, Jiwei; Ta, Kaiwen

    2015-04-01

    The occurrence of microbial mediated ammonia oxidation and these organisms are present in large numbers in natural environments indicated a potential biogeochemical role for them in the global nitrogen cycle. However, very little is understood about their role and contribution to nitrification in the high temperature extreme environments. Here we explore the ammonia oxidation rates and abundance of potential ammonia-oxidizing archaea (AOA) in upper and bottom sediments from Gongxiaoshe hot spring, Tengchong, Yunnan, China. The 15N-incorporating AOA cells and cell aggregated were detected with Fluorescence in situ hybridization (FISH) and Nano secondary ion mass spectrometry (Nano-SIMS). Ammonia oxidation rates measured using 15N-NO3- pool dilution in upper and bottom sediments (without NH4+ stimulated) were 4.8 and 5.3 nmol N g-1h-1, respectively. Close relatives of the autotrophic, ammonia-oxidizing archaeon 'Candidatus Nitrosocaldus yellowstonii' represented the most abundant OTU in both of the two spring sediments by 16S rRNA gene analysis. Furthermore, it should be noted that no ammonia-oxidizing bacterial clones detected in this study. Quantitative PCR (qPCR) indicated that AOA and 16S rRNA genes were present at 2.75-9.80×105 and 0.128-1.96×108 gene copies g-1 sediment. Based on the reaction rates and AOA abundance, we estimated the cell-specific nitrification rates were 0.41 to 0.79 fmol N archaeal cell-1 h-1, which are comparable to those observed in estuary environment. We suggest that AOA have the responsibility in nitrification in this hot spring, and these archaea rather than bacteria may be considered as a driver in nitrogen cycling in terrestrial hot ecosystems. Key words: ammonia-oxidizing archaea (AOA); nitrification; ammonia-oxidizing rate; hot spring;

  2. Preliminary X-ray analysis of twinned crystals of the Q88Y25-Lacpl esterase from Lactobacillus plantarum WCFS1

    International Nuclear Information System (INIS)

    Álvarez, Yanaisis; Esteban-Torres, María; Acebrón, Iván; Rivas, Blanca de las; Muñoz, Rosario; Martínez-Ripoll, Martín; Mancheño, José M.

    2011-01-01

    The Q88Y25-Lacpl esterase from L. plantarum WCFS1 has been recombinantly expressed, purified and crystallized. A native diffraction data set has been collected to 2.24 Å resolution. Q88Y25-Lacpl is an esterase produced by the lactic acid bacterium Lactobacillus plantarum WCFS1 that shows amino-acid sequence similarity to carboxylesterases from the hormone-sensitive lipase family, in particular the AFEST esterase from the archaeon Archaeoglobus fulgidus and the hyperthermophilic esterase EstEI isolated from a metagenomic library. N-terminally His 6 -tagged Q88Y25-Lacpl has been overexpressed in Escherichia coli BL21 (DE3) cells, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. Mass spectrometry was used to determine the purity and homogeneity of the enzyme. Crystals of His 6 -tagged Q88Y25-Lacpl were prepared in a solution containing 2.8 M sodium acetate trihydrate pH 7.0. X-ray diffraction data were collected to 2.24 Å resolution on beamline ID29 at the ESRF. The apparent crystal point group was 422; however, initial global analysis of the intensity statistics (data processed with high symmetry in space group I422) and subsequent tests on data processed with low symmetry (space group I4) showed that the crystals were almost perfectly merohedrally twinned. Most probably, the true space group is I4, with unit-cell parameters a = 169.05, b = 169.05, c = 183.62 Å

  3. Phylogenetic diversity and spatial distribution of the microbial community associated with the Caribbean deep-water sponge Polymastia cf. corticata by 16S rRNA, aprA, and amoA gene analysis.

    Science.gov (United States)

    Meyer, Birte; Kuever, Jan

    2008-08-01

    Denaturing gradient gel electrophoresis (DGGE)-based analyses of 16S rRNA, aprA, and amoA genes demonstrated that a phylogenetically diverse and complex microbial community was associated with the Caribbean deep-water sponge Polymastia cf. corticata Ridley and Dendy, 1887. From the 38 archaeal and bacterial 16S rRNA phylotypes identified, 53% branched into the sponge-specific, monophyletic sequence clusters determined by previous studies (considering predominantly shallow-water sponge species), whereas 26% appeared to be P. cf. corticata specifically associated microorganisms ("specialists"); 21% of the phylotypes were confirmed to represent seawater- and sediment-derived proteobacterial species ("contaminants") acquired by filtration processes from the host environment. Consistently, the aprA and amoA gene-based analyses indicated the presence of environmentally derived sulfur- and ammonia-oxidizers besides putative sponge-specific sulfur-oxidizing Gammaproteobacteria and Alphaproteobacteria and a sulfate-reducing archaeon. A sponge-specific, endosymbiotic sulfur cycle as described for marine oligochaetes is proposed to be also present in P. cf. corticata. Overall, the results of this work support the recent studies that demonstrated the sponge species specificity of the associated microbial community while the biogeography of the host collection site has only a minor influence on the composition. In P. cf. corticata, the specificity of the sponge-microbe associations is even extended to the spatial distribution of the microorganisms within the sponge body; distinct bacterial populations were associated with the different tissue sections, papillae, outer and inner cortex, and choanosome. The local distribution of a phylotype within P. cf. corticata correlated with its (1) phylogenetic affiliation, (2) classification as sponge-specific or nonspecifically associated microorganism, and (3) potential ecological role in the host sponge.

  4. Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding

    Directory of Open Access Journals (Sweden)

    Brewster Aaron S

    2010-08-01

    Full Text Available Abstract Background The mini-chromosome maintenance protein (MCM complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7, the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM, six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket. Results In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp. We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity. Conclusions These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.

  5. A Metagenomics-Based Metabolic Model of Nitrate-Dependent Anaerobic Oxidation of Methane by Methanoperedens-Like Archaea

    Science.gov (United States)

    Arshad, Arslan; Speth, Daan R.; de Graaf, Rob M.; Op den Camp, Huub J. M.; Jetten, Mike S. M.; Welte, Cornelia U.

    2015-01-01

    Methane oxidation is an important process to mitigate the emission of the greenhouse gas methane and further exacerbating of climate forcing. Both aerobic and anaerobic microorganisms have been reported to catalyze methane oxidation with only a few possible electron acceptors. Recently, new microorganisms were identified that could couple the oxidation of methane to nitrate or nitrite reduction. Here we investigated such an enrichment culture at the (meta) genomic level to establish a metabolic model of nitrate-driven anaerobic oxidation of methane (nitrate-AOM). Nitrate-AOM is catalyzed by an archaeon closely related to (reverse) methanogens that belongs to the ANME-2d clade, tentatively named Methanoperedens nitroreducens. Methane may be activated by methyl-CoM reductase and subsequently undergo full oxidation to carbon dioxide via reverse methanogenesis. All enzymes of this pathway were present and expressed in the investigated culture. The genome of the archaeal enrichment culture encoded a variety of enzymes involved in an electron transport chain similar to those found in Methanosarcina species with additional features not previously found in methane-converting archaea. Nitrate reduction to nitrite seems to be located in the pseudoperiplasm and may be catalyzed by an unusual Nar-like protein complex. A small part of the resulting nitrite is reduced to ammonium which may be catalyzed by a Nrf-type nitrite reductase. One of the key questions is how electrons from cytoplasmically located reverse methanogenesis reach the nitrate reductase in the pseudoperiplasm. Electron transport in M. nitroreducens probably involves cofactor F420 in the cytoplasm, quinones in the cytoplasmic membrane and cytochrome c in the pseudoperiplasm. The membrane-bound electron transport chain includes F420H2 dehydrogenase and an unusual Rieske/cytochrome b complex. Based on genome and transcriptome studies a tentative model of how central energy metabolism of nitrate-AOM could work is

  6. Efficient Fludarabine-Activating PNP From Archaea as a Guidance for Redesign the Active Site of E. Coli PNP.

    Science.gov (United States)

    Cacciapuoti, Giovanna; Bagarolo, Maria Libera; Martino, Elisa; Scafuri, Bernardina; Marabotti, Anna; Porcelli, Marina

    2016-05-01

    The combination of the gene of purine nucleoside phosphorylase (PNP) from Escherichia coli and fludarabine represents one of the most promising systems in the gene therapy of solid tumors. The use of fludarabine in gene therapy is limited by the lack of an enzyme that is able to efficiently activate this prodrug which, consequently, has to be administered in high doses that cause serious side effects. In an attempt to identify enzymes with a better catalytic efficiency than E. coli PNP towards fludarabine to be used as a guidance on how to improve the activity of the bacterial enzyme, we have selected 5'-deoxy-5'-methylthioadenosine phosphorylase (SsMTAP) and 5'-deoxy-5'-methylthioadenosine phosphorylase II (SsMTAPII), two PNPs isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. Substrate specificity and catalytic efficiency of SsMTAP and SsMTAPII for fludarabine were analyzed by kinetic studies and compared with E. coli PNP. SsMTAP and SsMTAPII share with E. coli PNP a comparable low affinity for the arabinonucleoside but are better catalysts of fludarabine cleavage with k(cat)/K(m) values that are 12.8-fold and 6-fold higher, respectively, than those reported for the bacterial enzyme. A computational analysis of the interactions of fludarabine in the active sites of E. coli PNP, SsMTAP, and SsMTAPII allowed to identify the crucial residues involved in the binding with this substrate, and provided structural information to improve the catalytic efficiency of E. coli PNP by enzyme redesign. © 2015 Wiley Periodicals, Inc.

  7. Avoiding dangerous missense: thermophiles display especially low mutation rates.

    Directory of Open Access Journals (Sweden)

    John W Drake

    2009-06-01

    Full Text Available Rates of spontaneous mutation have been estimated under optimal growth conditions for a variety of DNA-based microbes, including viruses, bacteria, and eukaryotes. When expressed as genomic mutation rates, most of the values were in the vicinity of 0.003-0.004 with a range of less than two-fold. Because the genome sizes varied by roughly 10(4-fold, the mutation rates per average base pair varied inversely by a similar factor. Even though the commonality of the observed genomic rates remains unexplained, it implies that mutation rates in unstressed microbes reach values that can be finely tuned by evolution. An insight originating in the 1920s and maturing in the 1960s proposed that the genomic mutation rate would reflect a balance between the deleterious effect of the average mutation and the cost of further reducing the mutation rate. If this view is correct, then increasing the deleterious impact of the average mutation should be countered by reducing the genomic mutation rate. It is a common observation that many neutral or nearly neutral mutations become strongly deleterious at higher temperatures, in which case they are called temperature-sensitive mutations. Recently, the kinds and rates of spontaneous mutations were described for two microbial thermophiles, a bacterium and an archaeon. Using an updated method to extrapolate from mutation-reporter genes to whole genomes reveals that the rate of base substitutions is substantially lower in these two thermophiles than in mesophiles. This result provides the first experimental support for the concept of an evolved balance between the total genomic impact of mutations and the cost of further reducing the basal mutation rate.

  8. Roles of thermophilic thiosulfate-reducing bacteria and methanogenic archaea in the biocorrosion of oil pipelines.

    Science.gov (United States)

    Liang, Renxing; Grizzle, Robert S; Duncan, Kathleen E; McInerney, Michael J; Suflita, Joseph M

    2014-01-01

    Thermophilic sulfide-producing microorganisms from an oil pipeline network were enumerated with different sulfur oxyanions as electron acceptors at 55°C. Most-probable number (MPN) analysis showed that thiosulfate-reducing bacteria were the most numerous sulfidogenic microorganisms in pipeline inspection gauge (PIG) scrapings. Thiosulfate-reducing and methanogenic enrichments were obtained from the MPN cultures that were able to use yeast extract as the electron donor. Molecular analysis revealed that both enrichments harbored the same dominant bacterium, which belonged to the genus Anaerobaculum. The dominant archaeon in the methanogenic enrichment was affiliated with the genus Methanothermobacter. With yeast extract as the electron donor, the general corrosion rate by the thiosulfate-reducing enrichment (8.43 ± 1.40 milli-inch per year, abbreviated as mpy) was about 5.5 times greater than the abiotic control (1.49 ± 0.15 mpy), while the comparable measures for the methanogenic culture were 2.03 ± 0.49 mpy and 0.62 ± 0.07 mpy, respectively. Total iron analysis in the cultures largely accounted for the mass loss of iron measured in the weight loss determinations. Profilometry analysis of polished steel coupons incubated in the presence of the thiosulfate-reducing enrichment revealed 59 pits over an area of 71.16 mm(2), while only 6 pits were evident in the corresponding methanogenic incubations. The results show the importance of thiosulfate-utilizing, sulfide-producing fermentative bacteria such as Anaerobaculum sp. in the corrosion of carbon steel, but also suggest that Anaerobaculum sp. are of far less concern when growing syntrophically with methanogens.

  9. Microbiological monitoring in geothermal plants and a cold storage

    Science.gov (United States)

    Alawi, Mashal; Lerm, Stephanie; Vieth, Andrea; Vetter, Alexandra; Miethling-Graff, Rona; Seibt, Andrea; Wolfgramm, Markus; Würdemann, Hilke

    2010-05-01

    Enhanced process understanding of engineered geothermal systems is mandatory to optimize plant reliability and economy. In the scope of the research project 'AquiScreen' we investigated geothermally used groundwater systems under microbial, geochemical, mineralogical and petrological aspects. Geothermal systems located in the North German Basin and the Molasse Basin were analyzed by sampling of fluids and solid phases. The investigated sites were characterized by different temperatures, salinities and potential microbial substrates. The microbial population was analyzed by the use of genetic fingerprinting techniques based on PCR-amplified 16S rRNA genes. Sequencing of dominant bands of fingerprints from different sites and the subsequent comparison on public databases enables a correlation to metabolic classes and provides information about the biochemical processes. In all investigated geothermal plants covering a temperature range from 45° to 120° C microorganisms were found. Phylogenetic gene analyses indicate a broad diversity of microorganisms adapted to the specific conditions in the engineered system. Beside characterized bacteria like Thermus scotoductus, Siderooxidans lithoautotrophicus and the archaeon Methanothermobacter thermoautotrophicus a high number of so far uncultivated microorganisms was detected. As it is known that -in addition to abiotic factors- microbes like sulfate-reducing bacteria (SRB) are involved in the processes of corrosion and scaling in plant components we identified SRB by specific analyses of dissimilatoric sulfite reductase genes. The SRB detected are closely related to thermotolerant and thermophilic species of Desulfotomaculum, Thermodesulfovibrio and Thermodesulfobacterium, respectively. Overall, the detection of microbes known to be involved in biocorrosion and examined precipitation products like iron sulfides are indicating that microorganisms play an important role for the understanding of processes in engineered

  10. Microbiological Monitoring in Geothermal Plants

    Science.gov (United States)

    Alawi, M.; Lerm, S.; Linder, R.; Vetter, A.; Vieth-Hillebrand, A.; Miethling-Graff, R.; Seibt, A.; Wolfgramm, M.; Wuerdemann, H.

    2010-12-01

    In the scope of the research projects “AquiScreen” and “MiProTherm” we investigated geothermally used groundwater systems under microbial, geochemical, mineralogical and petrological aspects. On one side an enhanced process understanding of engineered geothermal systems is mandatory to optimize plant reliability and economy, on the other side this study provides insights into the microbiology of terrestrial thermal systems. Geothermal systems located in the North German Basin and the Molasse Basin were analyzed by sampling of fluids and solid phases. The investigated sites were characterized by different temperatures, salinities and potential microbial substrates. The microbial population was monitored by the use of genetic fingerprinting techniques and PCR-cloning based on PCR-amplified 16S rRNA and dissimilatory sulfite reductase (DSR) genes. DNA-sequences of fingerprints and cloned PCR-products were compared to public databases and correlated with metabolic classes to provide information about the biogeochemical processes. In all investigated geothermal plants, covering a temperature range from 5° to 120°C, microorganisms were found. Phylogenetic gene analyses indicate a broad diversity of microorganisms adapted to the specific conditions in the engineered system. Beside characterized bacteria like Thermus scotoductus, Siderooxidans lithoautotrophicus and the archaeon Methanothermobacter thermoautotrophicus a high number of so far uncultivated microorganisms was detected. As it is known that - in addition to abiotic factors - microbes like sulfate-reducing bacteria (SRB) are involved in the processes of corrosion and scaling in plant components, we identified SRB by specific analyses of DSR genes. The SRB detected are closely related to thermotolerant and thermophilic species of Desulfotomaculum, Thermodesulfovibrio, Desulfohalobium and Thermodesulfobacterium, respectively. Overall, the detection of microbes known to be involved in biocorrosion and the

  11. Two functionally distinct NADP+-dependent ferredoxin oxidoreductases maintain the primary redox balance of Pyrococcus furiosus.

    Science.gov (United States)

    Nguyen, Diep M N; Schut, Gerrit J; Zadvornyy, Oleg A; Tokmina-Lukaszewska, Monika; Poudel, Saroj; Lipscomb, Gina L; Adams, Leslie A; Dinsmore, Jessica T; Nixon, William J; Boyd, Eric S; Bothner, Brian; Peters, John W; Adams, Michael W W

    2017-09-01

    Electron bifurcation has recently gained acceptance as the third mechanism of energy conservation in which energy is conserved through the coupling of exergonic and endergonic reactions. A structure-based mechanism of bifurcation has been elucidated recently for the flavin-based enzyme NADH-dependent ferredoxin NADP + oxidoreductase I (NfnI) from the hyperthermophillic archaeon Pyrococcus furiosus. NfnI is thought to be involved in maintaining the cellular redox balance, producing NADPH for biosynthesis by recycling the two other primary redox carriers, NADH and ferredoxin. The P. furiosus genome encodes an NfnI paralog termed NfnII, and the two are differentially expressed, depending on the growth conditions. In this study, we show that deletion of the genes encoding either NfnI or NfnII affects the cellular concentrations of NAD(P)H and particularly NADPH. This results in a moderate to severe growth phenotype in deletion mutants, demonstrating a key role for each enzyme in maintaining redox homeostasis. Despite their similarity in primary sequence and cofactor content, crystallographic, kinetic, and mass spectrometry analyses reveal that there are fundamental structural differences between the two enzymes, and NfnII does not catalyze the NfnI bifurcating reaction. Instead, it exhibits non-bifurcating ferredoxin NADP oxidoreductase-type activity. NfnII is therefore proposed to be a bifunctional enzyme and also to catalyze a bifurcating reaction, although its third substrate, in addition to ferredoxin and NADP(H), is as yet unknown. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Reconstructing viral genomes from the environment using fosmid clones: the case of haloviruses.

    Directory of Open Access Journals (Sweden)

    Inmaculada Garcia-Heredia

    Full Text Available BACKGROUND: Metaviriomes, the viral genomes present in an environment, have been studied by direct sequencing of the viral DNA or by cloning in small insert libraries. The short reads generated by both approaches make it very difficult to assemble and annotate such flexible genomic entities. Many environmental viruses belong to unknown groups or prey on uncultured and little known cellular lineages, and hence might not be present in databases. METHODOLOGY AND PRINCIPAL FINDINGS: Here we have used a different approach, the cloning of viral DNA into fosmids before sequencing, to obtain natural contigs that are close to the size of a viral genome. We have studied a relatively low diversity extreme environment: saturated NaCl brines, which simplifies the analysis and interpretation of the data. Forty-two different viral genomes were retrieved, and some of these were almost complete, and could be tentatively identified as head-tail phages (Caudovirales. CONCLUSIONS AND SIGNIFICANCE: We found a cluster of phage genomes that most likely infect Haloquadratum walsbyi, the square archaeon and major component of the community in these hypersaline habitats. The identity of the prey could be confirmed by the presence of CRISPR spacer sequences shared by the virus and one of the available strain genomes. Other viral clusters detected appeared to prey on the Nanohaloarchaea and on the bacterium Salinibacter ruber, covering most of the diversity of microbes found in this type of environment. This approach appears then as a viable alternative to describe metaviriomes in a much more detailed and reliable way than by the more common approaches based on direct sequencing. An example of transfer of a CRISPR cluster including repeats and spacers was accidentally found supporting the dynamic nature and frequent transfer of this peculiar prokaryotic mechanism of cell protection.

  13. Nucleotide fluctuation of radiation-resistant Halobacterium sp. NRC-1 single-stranded DNA-binding protein (RPA) genes

    Science.gov (United States)

    Holden, Todd; Tremberger, G., Jr.; Cheung, E.; Subramaniam, R.; Gadura, N.; Schneider, P.; Sullivan, R.; Flamholz, A.; Lieberman, D.; Cheung, T. D.

    2009-08-01

    The Single-Stranded DNA-Binding Protein (RPA) Genes in gamma ray radiation-resistant halophilic archaeon Halobacterium sp. NRC-1 were analyzed in terms of their nucleotide fluctuations. In an ATCG sequence, each base was assigned a number equal to its atomic number. The resulting numerical sequence was the basis of the statistical analysis in this study. Fractal analysis using the Higuchi method gave fractal dimensions of 2.04 and 2.06 for the gene sequences VNG2160 and VNG2162, respectively. The 16S rRNA sequence has a fractal dimension of 1.99. The di-nucleotide Shannon entropy values were found to be negatively correlated with the observed fractal dimensions (R2~ 0.992, N=3). Inclusion of Deinococcus radiodurans Rad-A in the regression analysis decreases the R2 slightly to 0.98 (N=4). A third VNG2163 RPA gene of unknown function but with upregulation activity under irradiation was found to have a fractal dimension of 2.05 and a Shannon entropy of 3.77 bits. The above results are similar to those found in bacterial Deinococcus radiodurans and suggest that their high radiation resistance property would have favored selection of CG di-nucleotide pairs. The two transcription factors TbpD (VNG7114) and TfbA (VNG 2184) were also studied. Using VNG7114, VNG2184, and VNG2163; the regression analysis of fractal dimension versus Shannon entropy shows that R2 ~ 0.997 for N =3. The VNG2163 unknown function may be related to the pathways with transcriptions closely regulated to sequences VNG7114 and VNG2184.

  14. Cloning, overexpression, purification, and characterization of a polyextremophilic β-galactosidase from the Antarctic haloarchaeon Halorubrum lacusprofundi

    Directory of Open Access Journals (Sweden)

    Karan Ram

    2013-01-01

    Full Text Available Abstract Background Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, a perennially cold and hypersaline lake in Antarctica. Its genome sequencing project was recently completed, providing access to many genes predicted to encode polyextremophilic enzymes active in both extremely high salinity and cold temperatures. Results Analysis of the genome sequence of H. lacusprofundi showed a gene cluster for carbohydrate utilization containing a glycoside hydrolase family 42 β-galactosidase gene, named bga. In order to study the biochemical properties of the β-galactosidase enzyme, the bga gene was PCR amplified, cloned, and expressed in the genetically tractable haloarchaeon Halobacterium sp. NRC-1 under the control of a cold shock protein (cspD2 gene promoter. The recombinant β-galactosidase protein was produced at 20-fold higher levels compared to H. lacusprofundi, purified using gel filtration and hydrophobic interaction chromatography, and identified by SDS-PAGE, LC-MS/MS, and ONPG hydrolysis activity. The purified enzyme was found to be active over a wide temperature range (−5 to 60°C with an optimum of 50°C, and 10% of its maximum activity at 4°C. The enzyme also exhibited extremely halophilic character, with maximal activity in either 4 M NaCl or KCl. The polyextremophilic β-galactosidase was also stable and active in 10–20% alcohol-aqueous solutions, containing methanol, ethanol, n-butanol, or isoamyl alcohol. Conclusion The H. lacusprofundi β-galactosidase is a polyextremophilic enzyme active in high salt concentrations and low and high temperature. The enzyme is also active in aqueous-organic mixed solvents, with potential applications in synthetic chemistry. H. lacuprofundi proteins represent a significant biotechnology resource and for developing insights into enzyme catalysis under water limiting conditions. This study provides a system for better understanding how H. lacusprofundi is

  15. A Purple Cupredoxin from Nitrosopumilus maritimus Containing a Mononuclear Type 1 Copper Center with an Open Binding Site

    Energy Technology Data Exchange (ETDEWEB)

    Hosseinzadeh, Parisa; Tian, Shiliang; Marshall, Nicholas M.; Hemp, James; Mullen, Timothy; Nilges, Mark J.; Gao, Yi-Gui; Robinson, Howard; Stahl, David A.; Gennis, Robert B.; Lu, Yi

    2016-05-25

    Mononuclear cupredoxin proteins usually contain a coordinately saturated type 1 copper (T1Cu) center and function exclusively as electron carriers. Here we report a cupredoxin isolated from the nitrifying archaeon Nitrosopumilus maritimus SCM1, called Nmar1307, that contains a T1Cu center with an open binding site containing water. It displays a deep purple color due to strong absorptions around 413 nm (1880 M–1 cm–1) and 558 nm (2290 M–1 cm–1) in the UV–vis electronic spectrum. EPR studies suggest the protein contains two Cu(II) species of nearly equal population, one nearly axial, with hyperfine constant A∥ = 98 × 10–4 cm–1, and another more rhombic, with a smaller A∥ value of 69 × 10–4 cm–1. The X-ray crystal structure at 1.6 Å resolution confirms that it contains a Cu atom coordinated by two His and one Cys in a trigonal plane, with an axial H2O at 2.25 Å. Both UV–vis absorption and EPR spectroscopic studies suggest that the Nmar1307 can oxidize NO to nitrite, an activity that is attributable to the high reduction potential (354 mV vs SHE) of the copper site. These results suggest that mononuclear cupredoxins can have a wide range of structural features, including an open binding site containing water, making this class of proteins even more versatile.

  16. Diversity and stratification of archaea in a hypersaline microbial mat.

    Science.gov (United States)

    Robertson, Charles E; Spear, John R; Harris, J Kirk; Pace, Norman R

    2009-04-01

    The Guerrero Negro (GN) hypersaline microbial mats have become one focus for biogeochemical studies of stratified ecosystems. The GN mats are found beneath several of a series of ponds of increasing salinity that make up a solar saltern fed from Pacific Ocean water pumped from the Laguna Ojo de Liebre near GN, Baja California Sur, Mexico. Molecular surveys of the laminated photosynthetic microbial mat below the fourth pond in the series identified an enormous diversity of bacteria in the mat, but archaea have received little attention. To determine the bulk contribution of archaeal phylotypes to the pond 4 study site, we determined the phylogenetic distribution of archaeal rRNA gene sequences in PCR libraries based on nominally universal primers. The ratios of bacterial/archaeal/eukaryotic rRNA genes, 90%/9%/1%, suggest that the archaeal contribution to the metabolic activities of the mat may be significant. To explore the distribution of archaea in the mat, sequences derived using archaeon-specific PCR primers were surveyed in 10 strata of the 6-cm-thick mat. The diversity of archaea overall was substantial albeit less than the diversity observed previously for bacteria. Archaeal diversity, mainly euryarchaeotes, was highest in the uppermost 2 to 3 mm of the mat and decreased rapidly with depth, where crenarchaeotes dominated. Only 3% of the sequences were specifically related to known organisms including methanogens. While some mat archaeal clades corresponded with known chemical gradients, others did not, which is likely explained by heretofore-unrecognized gradients. Some clades did not segregate by depth in the mat, indicating broad metabolic repertoires, undersampling, or both.

  17. Characterization of the archaeal ribonuclease P proteins from Pyrococcus horikoshii OT3.

    Science.gov (United States)

    Terada, Atsushi; Honda, Takashi; Fukuhara, Hideo; Hada, Kazumasa; Kimura, Makoto

    2006-08-01

    Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5'-leader sequence of precursor tRNA (pre-tRNA). Our earlier study revealed that RNase P RNA (pRNA) and five proteins (PhoPop5, PhoRpp38, PhoRpp21, PhoRpp29, and PhoRpp30) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 reconstituted RNase P activity that exhibits enzymatic properties like those of the authentic enzyme. In present study, we investigated involvement of the individual proteins in RNase P activity. Two particles (R-3Ps), in which pRNA was mixed with three proteins, PhoPop5, PhoRpp30, and PhoRpp38 or PhoPop5, PhoRpp30, and PhoRpp21 showed a detectable RNase P activity, and five reconstituted particles (R-4Ps) composed of pRNA and four proteins exhibited RNase P activity, albeit at reduced level compared to that of the reconstituted particle (R-5P) composed of pRNA and five proteins. Time-course analysis of the RNase P activities of R-4Ps indicated that the R-4Ps lacking PhoPop5, PhoRpp21, or PhoRpp30 had virtually reduced activity, while omission of PhoRpp29 or PhoRpp38 had a slight effect on the activity. The results indicate that the proteins contribute to RNase P activity in order of PhoPop5 > PhoRpp30 > PhoRpp21 > PhoRpp29 > PhoRpp38. It was further found that R-4Ps showed a characteristic Mg2+ ion dependency approximately identical to that of R-5P. However, R-4Ps had optimum temperature of around at 55 degrees C which is lower than 70 degrees C for R-5P. Together, it is suggested that the P. horikoshii RNase P proteins are predominantly involved in optimization of the pRNA conformation, though they are individually dispensable for RNase P activity in vitro.

  18. A comprehensive census of microbial diversity in hot springs of Tengchong, Yunnan Province China using 16S rRNA gene pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Weiguo Hou

    Full Text Available The Rehai and Ruidian geothermal fields, located in Tengchong County, Yunnan Province, China, host a variety of geochemically distinct hot springs. In this study, we report a comprehensive, cultivation-independent census of microbial communities in 37 samples collected from these geothermal fields, encompassing sites ranging in temperature from 55.1 to 93.6°C, in pH from 2.5 to 9.4, and in mineralogy from silicates in Rehai to carbonates in Ruidian. Richness was low in all samples, with 21-123 species-level OTUs detected. The bacterial phylum Aquificae or archaeal phylum Crenarchaeota were dominant in Rehai samples, yet the dominant taxa within those phyla depended on temperature, pH, and geochemistry. Rehai springs with low pH (2.5-2.6, high temperature (85.1-89.1°C, and high sulfur contents favored the crenarchaeal order Sulfolobales, whereas those with low pH (2.6-4.8 and cooler temperature (55.1-64.5°C favored the Aquificae genus Hydrogenobaculum. Rehai springs with neutral-alkaline pH (7.2-9.4 and high temperature (>80°C with high concentrations of silica and salt ions (Na, K, and Cl favored the Aquificae genus Hydrogenobacter and crenarchaeal orders Desulfurococcales and Thermoproteales. Desulfurococcales and Thermoproteales became predominant in springs with pH much higher than the optimum and even the maximum pH known for these orders. Ruidian water samples harbored a single Aquificae genus Hydrogenobacter, whereas microbial communities in Ruidian sediment samples were more diverse at the phylum level and distinctly different from those in Rehai and Ruidian water samples, with a higher abundance of uncultivated lineages, close relatives of the ammonia-oxidizing archaeon "Candidatus Nitrosocaldus yellowstonii", and candidate division O1aA90 and OP1. These differences between Ruidian sediments and Rehai samples were likely caused by temperature, pH, and sediment mineralogy. The results of this study significantly expand the current

  19. A comprehensive census of microbial diversity in hot springs of Tengchong, Yunnan Province China using 16S rRNA gene pyrosequencing.

    Science.gov (United States)

    Hou, Weiguo; Wang, Shang; Dong, Hailiang; Jiang, Hongchen; Briggs, Brandon R; Peacock, Joseph P; Huang, Qiuyuan; Huang, Liuqin; Wu, Geng; Zhi, Xiaoyang; Li, Wenjun; Dodsworth, Jeremy A; Hedlund, Brian P; Zhang, Chuanlun; Hartnett, Hilairy E; Dijkstra, Paul; Hungate, Bruce A

    2013-01-01

    The Rehai and Ruidian geothermal fields, located in Tengchong County, Yunnan Province, China, host a variety of geochemically distinct hot springs. In this study, we report a comprehensive, cultivation-independent census of microbial communities in 37 samples collected from these geothermal fields, encompassing sites ranging in temperature from 55.1 to 93.6°C, in pH from 2.5 to 9.4, and in mineralogy from silicates in Rehai to carbonates in Ruidian. Richness was low in all samples, with 21-123 species-level OTUs detected. The bacterial phylum Aquificae or archaeal phylum Crenarchaeota were dominant in Rehai samples, yet the dominant taxa within those phyla depended on temperature, pH, and geochemistry. Rehai springs with low pH (2.5-2.6), high temperature (85.1-89.1°C), and high sulfur contents favored the crenarchaeal order Sulfolobales, whereas those with low pH (2.6-4.8) and cooler temperature (55.1-64.5°C) favored the Aquificae genus Hydrogenobaculum. Rehai springs with neutral-alkaline pH (7.2-9.4) and high temperature (>80°C) with high concentrations of silica and salt ions (Na, K, and Cl) favored the Aquificae genus Hydrogenobacter and crenarchaeal orders Desulfurococcales and Thermoproteales. Desulfurococcales and Thermoproteales became predominant in springs with pH much higher than the optimum and even the maximum pH known for these orders. Ruidian water samples harbored a single Aquificae genus Hydrogenobacter, whereas microbial communities in Ruidian sediment samples were more diverse at the phylum level and distinctly different from those in Rehai and Ruidian water samples, with a higher abundance of uncultivated lineages, close relatives of the ammonia-oxidizing archaeon "Candidatus Nitrosocaldus yellowstonii", and candidate division O1aA90 and OP1. These differences between Ruidian sediments and Rehai samples were likely caused by temperature, pH, and sediment mineralogy. The results of this study significantly expand the current understanding of

  20. Physiology and genetics of sulfur-oxidizing bacteria.

    Science.gov (United States)

    Friedrich, C G

    1998-01-01

    Reduced inorganic sulfur compounds are oxidized by members of the domains Archaea and Bacteria. These compounds are used as electron donors for anaerobic phototrophic and aerobic chemotrophic growth, and are mostly oxidized to sulfate. Different enzymes mediate the conversion of various reduced sulfur compounds. Their physiological function in sulfur oxidation is considered (i) mostly from the biochemical characterization of the enzymatic reaction, (ii) rarely from the regulation of their formation, and (iii) only in a few cases from the mutational gene inactivation and characterization of the resulting mutant phenotype. In this review the sulfur-metabolizing reactions of selected phototrophic and of chemotrophic prokaryotes are discussed. These comprise an archaeon, a cyanobacterium, green sulfur bacteria, and selected phototrophic and chemotrophic proteobacteria. The genetic systems are summarized which are presently available for these organisms, and which can be used to study the molecular basis of their dissimilatory sulfur metabolism. Two groups of thiobacteria can be distinguished: those able to grow with tetrathionate and other reduced sulfur compounds, and those unable to do so. This distinction can be made irrespective of their phototrophic or chemotrophic metabolism, neutrophilic or acidophilic nature, and may indicate a mechanism different from that of thiosulfate oxidation. However, the core enzyme for tetrathionate oxidation has not been identified so far. Several phototrophic bacteria utilize hydrogen sulfide, which is considered to be oxidized by flavocytochrome c owing to its in vitro activity. However, the function of flavocytochrome c in vivo may be different, because it is missing in other hydrogen sulfide-oxidizing bacteria, but is present in most thiosulfate-oxidizing bacteria. A possible function of flavocytochrome c is discussed based on biophysical studies, and the identification of a flavocytochrome in the operon encoding enzymes involved

  1. Genetic and Biochemical Identification of a Novel Single-Stranded DNA-Binding Complex in Haloferax volcanii.

    Science.gov (United States)

    Stroud, Amy; Liddell, Susan; Allers, Thorsten

    2012-01-01

    Single-stranded DNA (ssDNA)-binding proteins play an essential role in DNA replication and repair. They use oligonucleotide/oligosaccharide-binding (OB)-folds, a five-stranded β-sheet coiled into a closed barrel, to bind to ssDNA thereby protecting and stabilizing the DNA. In eukaryotes the ssDNA-binding protein (SSB) is known as replication protein A (RPA) and consists of three distinct subunits that function as a heterotrimer. The bacterial homolog is termed SSB and functions as a homotetramer. In the archaeon Haloferax volcanii there are three genes encoding homologs of RPA. Two of the rpa genes (rpa1 and rpa3) exist in operons with a novel gene specific to Euryarchaeota; this gene encodes a protein that we have termed RPA-associated protein (rpap). The rpap genes encode proteins belonging to COG3390 group and feature OB-folds, suggesting that they might cooperate with RPA in binding to ssDNA. Our genetic analysis showed that rpa1 and rpa3 deletion mutants have differing phenotypes; only Δrpa3 strains are hypersensitive to DNA damaging agents. Deletion of the rpa3-associated gene rpap3 led to similar levels of DNA damage sensitivity, as did deletion of the rpa3 operon, suggesting that RPA3 and RPAP3 function in the same pathway. Protein pull-downs involving recombinant hexahistidine-tagged RPAs showed that RPA3 co-purifies with RPAP3, and RPA1 co-purifies with RPAP1. This indicates that the RPAs interact only with their respective associated proteins; this was corroborated by the inability to construct rpa1 rpap3 and rpa3 rpap1 double mutants. This is the first report investigating the individual function of the archaeal COG3390 RPA-associated proteins (RPAPs). We have shown genetically and biochemically that the RPAPs interact with their respective RPAs, and have uncovered a novel single-stranded DNA-binding complex that is unique to Euryarchaeota.

  2. Ammonia oxidation, denitrification and dissimilatory nitrate reduction to ammonium in two US Great Basin hot springs with abundant ammonia-oxidizing archaea.

    Science.gov (United States)

    Dodsworth, Jeremy A; Hungate, Bruce A; Hedlund, Brian P

    2011-08-01

    Many thermophiles catalyse free energy-yielding redox reactions involving nitrogenous compounds; however, little is known about these processes in natural thermal environments. Rates of ammonia oxidation, denitrification and dissimilatory nitrate reduction to ammonium (DNRA) were measured in source water and sediments of two ≈ 80°C springs in the US Great Basin. Ammonia oxidation and denitrification occurred mainly in sediments. Ammonia oxidation rates measured using (15)N-NO(3)(-) pool dilution ranged from 5.5 ± 0.8 to 8.6 ± 0.9 nmol N g(-1) h(-1) and were unaffected or only mildly stimulated by amendment with NH(4) Cl. Denitrification rates measured using acetylene block ranged from 15.8 ± 0.7 to 51 ± 12 nmol N g(-1) h(-1) and were stimulated by amendment with NO(3)(-) and complex organic compounds. The DNRA rate in one spring sediment measured using an (15)N-NO(3)(-) tracer was 315 ± 48 nmol N g(-1) h(-1). Both springs harboured distinct planktonic and sediment microbial communities. Close relatives of the autotrophic, ammonia-oxidizing archaeon 'Candidatus Nitrosocaldus yellowstonii' represented the most abundant OTU in both spring sediments by 16S rRNA gene pyrotag analysis. Quantitative PCR (qPCR) indicated that 'Ca. N. yellowstonii'amoA and 16S rRNA genes were present at 3.5-3.9 × 10(8) and 6.4-9.0 × 10(8) copies g(-1) sediment. Potential denitrifiers included members of the Aquificales and Thermales. Thermus spp. comprised <1% of 16S rRNA gene pyrotags in both sediments and qPCR for T. thermophilus narG revealed sediment populations of 1.3-1.7 × 10(6) copies g(-1) sediment. These data indicate a highly active nitrogen cycle (N-cycle) in these springs and suggest that ammonia oxidation may be a major source of energy fuelling primary production. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

  3. Experimental assessment of the importance of amino acid positions identified by an entropy-based correlation analysis of multiple-sequence alignments.

    Science.gov (United States)

    Dietrich, Susanne; Borst, Nadine; Schlee, Sandra; Schneider, Daniel; Janda, Jan-Oliver; Sterner, Reinhard; Merkl, Rainer

    2012-07-17

    The analysis of a multiple-sequence alignment (MSA) with correlation methods identifies pairs of residue positions whose occupation with amino acids changes in a concerted manner. It is plausible to assume that positions that are part of many such correlation pairs are important for protein function or stability. We have used the algorithm H2r to identify positions k in the MSAs of the enzymes anthranilate phosphoribosyl transferase (AnPRT) and indole-3-glycerol phosphate synthase (IGPS) that show a high conn(k) value, i.e., a large number of significant correlations in which k is involved. The importance of the identified residues was experimentally validated by performing mutagenesis studies with sAnPRT and sIGPS from the archaeon Sulfolobus solfataricus. For sAnPRT, five H2r mutant proteins were generated by replacing nonconserved residues with alanine or the prevalent residue of the MSA. As a control, five residues with conn(k) values of zero were chosen randomly and replaced with alanine. The catalytic activities and conformational stabilities of the H2r and control mutant proteins were analyzed by steady-state enzyme kinetics and thermal unfolding studies. Compared to wild-type sAnPRT, the catalytic efficiencies (k(cat)/K(M)) were largely unaltered. In contrast, the apparent thermal unfolding temperature (T(M)(app)) was lowered in most proteins. Remarkably, the strongest observed destabilization (ΔT(M)(app) = 14 °C) was caused by the V284A exchange, which pertains to the position with the highest correlation signal [conn(k) = 11]. For sIGPS, six H2r mutant and four control proteins with alanine exchanges were generated and characterized. The k(cat)/K(M) values of four H2r mutant proteins were reduced between 13- and 120-fold, and their T(M)(app) values were decreased by up to 5 °C. For the sIGPS control proteins, the observed activity and stability decreases were much less severe. Our findings demonstrate that positions with high conn(k) values have an

  4. Genetic and biochemical identification of a novel single-stranded DNA binding complex in Haloferax volcanii

    Directory of Open Access Journals (Sweden)

    Amy eStroud

    2012-06-01

    Full Text Available Single-stranded DNA binding proteins play an essential role in DNA replication and repair. They use oligosaccharide-binding folds, a five-stranded ß-sheet coiled into a closed barrel, to bind to single-stranded DNA thereby protecting and stabilizing the DNA. In eukaryotes the single-stranded DNA binding protein is known as replication protein A (RPA and consists of three distinct subunits that function as a heterotrimer. The bacterial homolog is termed single-stranded DNA-binding protein (SSB and functions as a homotetramer. In the archaeon Haloferax volcanii there are three genes encoding homologs of RPA. Two of the rpa genes (rpa1 and rpa3 exist in operons with a novel gene specific to Euryarchaeota, this gene encodes a protein that we have termed rpa-associated protein (RPAP. The rpap genes encode proteins belonging to COG3390 group and feature oligosaccharide-binding folds, suggesting that they might cooperate with RPA in binding to single-stranded DNA. Our genetic analysis showed that rpa1 and rpa3 deletion mutants have differing phenotypes; only ∆rpa3 strains are hypersensitive to DNA damaging agents. Deletion of the rpa3-associated gene rpap3 led to similar levels of DNA damage sensitivity, as did deletion of the rpa3 operon, suggesting that RPA3 and RPAP3 function in the same pathway. Protein pull-downs involving recombinant hexahistidine-tagged RPAs showed that RPA3 co-purifies with RPAP3, and RPA1 co-purifies with RPAP1. This indicates that the RPAs interact only with their respective associated proteins; this was corroborated by the inability to construct rpa1 rpap3 and rpa3 rpap1 double mutants. This is the first report investigating the individual function of the archaeal COG3390 RPA-associated proteins. We have shown genetically and biochemically that the RPAPs interact with their respective RPAs, and have uncovered a novel single-stranded DNA binding complex that is unique to Euryarchaeota.

  5. Seryl-tRNA Synthetases in Translation and Beyond

    Directory of Open Access Journals (Sweden)

    Marko Močibob

    2016-06-01

    Full Text Available For a long time seryl-tRNA synthetases (SerRSs stood as an archetypal, canonical aminoacyl-tRNA synthetases (aaRS, exhibiting only basic tRNA aminoacylation activity and with no moonlighting functions beyond protein biosynthesis. The picture has changed substantially in recent years after the discovery that SerRSs play an important role in antibiotic production and resistance and act as a regulatory factor in vascular development, as well as after the discovery of mitochondrial morphogenesis factor homologous to SerRS in insects. In this review we summarize the recent research results from our laboratory, which advance the understanding of seryl-tRNA synthetases and further paint the dynamic picture of unexpected SerRS activities. SerRS from archaeon Methanothermobacter thermautotrophicus was shown to interact with the large ribosomal subunit and it was postulated to contribute to a more efficient translation by the"tRNA channeling" hypothesis. Discovery of the atypical SerRS in a small number of methanogenic archaea led to the discovery of a new family of enzymes in numerous bacteria - amino acid:[carrier protein] ligases (aa:CP ligases. These SerRS homologues resigned tRNA aminoacylation activity, and instead adopted carrier proteins as the acceptors of activated amino acids. The crystal structure of the aa:CP ligase complex with the carrier protein revealed that the interactions between two macromolecules are incomparable to tRNA binding by the aaRS and consequently represent a true evolutionary invention. Kinetic investigations of SerRSs and the accuracy of amino acid selection revealed that SerRSs possess pre-transfer proofreading activity, challenging the widely accepted presumption that hydrolytic proofreading activity must reside in an additional, separate editing domain, not present in SerRSs. Finally, the plant tRNA serylation system is discussed, which is particularly interesting due to the fact that protein biosynthesis takes place

  6. Evolutionary consequences of polyploidy in prokaryotes and the origin of mitosis and meiosis.

    Science.gov (United States)

    Markov, Alexander V; Kaznacheev, Ilya S

    2016-06-08

    The origin of eukaryote-specific traits such as mitosis and sexual reproduction remains disputable. There is growing evidence that both mitosis and eukaryotic sex (i.e., the alternation of syngamy and meiosis) may have already existed in the basal eukaryotes. The mating system of the halophilic archaeon Haloferax volcanii probably represents an intermediate stage between typical prokaryotic and eukaryotic sex. H. volcanii is highly polyploid, as well as many other Archaea. Here, we use computer simulation to explore genetic and evolutionary outcomes of polyploidy in amitotic prokaryotes and its possible role in the origin of mitosis, meiosis and eukaryotic sex. Modeling suggests that polyploidy can confer strong short-term evolutionary advantage to amitotic prokaryotes. However, it also promotes the accumulation of recessive deleterious mutations and the risk of extinction in the long term, especially in highly mutagenic environment. There are several possible strategies that amitotic polyploids can use in order to reduce the genetic costs of polyploidy while retaining its benefits. Interestingly, most of these strategies resemble different components or aspects of eukaryotic sex. They include asexual ploidy cycles, equalization of genome copies by gene conversion, high-frequency lateral gene transfer between relatives, chromosome exchange coupled with homologous recombination, and the evolution of more accurate chromosome distribution during cell division (mitosis). Acquisition of mitosis by an amitotic polyploid results in chromosome diversification and specialization. Ultimately, it transforms a polyploid cell into a functionally monoploid one with multiple unique, highly redundant chromosomes. Specialization of chromosomes makes the previously evolved modes of promiscuous chromosome shuffling deleterious. This can result in selective pressure to develop accurate mechanisms of homolog pairing, and, ultimately, meiosis. Emergence of mitosis and the first

  7. Structures of SRP54 and SRP19, the two proteins that organize the ribonucleic core of the signal recognition particle from Pyrococcus furiosus.

    Directory of Open Access Journals (Sweden)

    Pascal F Egea

    Full Text Available In all organisms the Signal Recognition Particle (SRP, binds to signal sequences of proteins destined for secretion or membrane insertion as they emerge from translating ribosomes. In Archaea and Eucarya, the conserved ribonucleoproteic core is composed of two proteins, the accessory protein SRP19, the essential GTPase SRP54, and an evolutionarily conserved and essential SRP RNA. Through the GTP-dependent interaction between the SRP and its cognate receptor SR, ribosomes harboring nascent polypeptidic chains destined for secretion are dynamically transferred to the protein translocation apparatus at the membrane. We present here high-resolution X-ray structures of SRP54 and SRP19, the two RNA binding components forming the core of the signal recognition particle from the hyper-thermophilic archaeon Pyrococcus furiosus (Pfu. The 2.5 A resolution structure of free Pfu-SRP54 is the first showing the complete domain organization of a GDP bound full-length SRP54 subunit. In its ras-like GTPase domain, GDP is found tightly associated with the protein. The flexible linker that separates the GTPase core from the hydrophobic signal sequence binding M domain, adopts a purely alpha-helical structure and acts as an articulated arm allowing the M domain to explore multiple regions as it scans for signal peptides as they emerge from the ribosomal tunnel. This linker is structurally coupled to the GTPase catalytic site and likely to propagate conformational changes occurring in the M domain through the SRP RNA upon signal sequence binding. Two different 1.8 A resolution crystal structures of free Pfu-SRP19 reveal a compact, rigid and well-folded protein even in absence of its obligate SRP RNA partner. Comparison with other SRP19*SRP RNA structures suggests the rearrangement of a disordered loop upon binding with the RNA through a reciprocal induced-fit mechanism and supports the idea that SRP19 acts as a molecular scaffold and a chaperone, assisting the SRP

  8. Enhanced selection of micro-aerobic pentachlorophenol degrading granular sludge

    International Nuclear Information System (INIS)

    Lv, Yuancai; Chen, Yuancai; Song, Wenzhe; Hu, Yongyou

    2014-01-01

    Graphical abstract: In this work, an aerobic column reactor was placed before the USB to maintain micro-oxygen condition in the reactor and the micro-aerobic pentachlorophenol (PCP) degrading granular sludge under oxygen-limited conditions (0.1–0.2 mg L −1 ) was successfully obtained. PCP degradation by the micro-aerobic system was studied and the variance of microbial community was also discussed by using PCR-DGGE analysis. - Highlights: • Micro-aerobic granular sludge was cultivated in column-type combined reactors. • PCP biodegradation, VFA accumulation and biogas production were studied. • The function of Methanogenic archaeon in the system was investigated. • Fluctuation and diversity of microbial community were discussed by DGGE analysis. • The dominated microorganisms were identified by 16S rDNA sequences. - Abstract: Column-type combined reactors were designed to cultivate micro-aerobic pentachlorophenol (PCP) degrading granular sludge under oxygen-limited conditions (0.1–0.2 mg L −1 ) over 39-day experimental period. Micro-aerobic granular had both anaerobic activity (SMA: 2.34 mMCH 4 /h g VSS) and aerobic activity (SOUR: 2.21 mMO 2 /h g VSS). Metabolite analysis results revealed that PCP was sequentially dechlorinated to TCP, DCP, and eventually to MCP. Methanogens were not directly involved in the dechlorination of PCP, but might played a vital role in stabilizing the overall structure of the granule sludge. For Eubacteria, the Shannon Index (2.09 in inoculated granular sludge) increased both in micro-aerobic granular sludge (2.61) and PCP-degradation granular sludge (2.55). However, for Archaea, it decreased from 2.53 to 1.85 and 1.84, respectively. Although the Shannon Index demonstrated slight difference between micro-aerobic granular sludge and PCP-degradation granular sludge, the Principal Component Analysis (PCA) indicated obvious variance of the microbial composition, revealing significant effect of micro-aerobic condition and

  9. MAGGIE Component 1: Identification and Purification of Native and Recombinant Multiprotein Complexes and Modified Proteins from Pyrococcus furiosus

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Michael W. [University of Georgia; W. W. Adams, Michael

    2014-01-07

    Virtualy all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes (PCs), the composition of which is largely unknown. Structural genomics efforts have demonstrated that less than 25% of the genes in a given prokaryotic genome will yield stable, soluble proteins when expressed using a one-ORF-at-a-time approach. We proposed that much of the remaining 75% of the genes encode proteins that are part of multiprotein complexes or are modified post-translationally, for example, with metals. The problem is that PCs and metalloproteins (MPs) cannot be accurately predicted on a genome-wide scale. The only solution to this dilemma is to experimentally determine PCs and MPs in biomass of a model organism and to develop analytical tools that can then be applied to the biomass of any other organism. In other words, organisms themselves must be analyzed to identify their PCs and MPs: “native proteomes” must be determined. This information can then be utilized to design multiple ORF expression systems to produce recombinant forms of PCs and MPs. Moreover, the information and utility of this approach can be enhanced by using a hyperthermophile, one that grows optimally at 100°C, as a model organism. By analyzing the native proteome at close to 100 °C below the optimum growth temperature, we will trap reversible and dynamic complexes, thereby enabling their identification, purification, and subsequent characterization. The model organism for the current study is Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100°C. It is grown up to 600-liter scale and kg quantities of biomass are available. In this project we identified native PCs and MPs using P. furiosus biomass (with MS/MS analyses to identify proteins by component 4). In addition, we provided samples of abundant native PCs and MPs for structural characterization (using SAXS by component 5). We also designed and evaluated generic bioinformatics and

  10. Physiological and molecular studies of the resistance to ionizing radiations of hyper-thermophilic archaea isolated from deep ocean hydrothermal sources

    International Nuclear Information System (INIS)

    Jolivet, E.

    2002-10-01

    In this study, we have first tested in vivo the effect of gamma irradiation on Pyrococcus abyssi, a hyper-thermophilic archaeon, isolated from a deep-sea hydrothermal vent. We have shown that this strain was as radioresistant as P. furiosus but less than Deinococcus radiodurans. The rates of double stranded breaks provoked into DNA following irradiation were monitored by the pulsed-field gel electrophoresis technique (P.F.G.E.) with P. abyssi, P. furiosus, D. radiodurans and Escherichia coli. Results clearly showed that all these rates were similar suggesting that no specific DNA protection system exits in Pyrococcus species. The growth of P. abyssi was efficiently recovered within two hours following the exposure to 2.5 kGy of gamma irradiation. As revealed by P.F.G.E., genomic DNA of P. abyssi totally fragmented after irradiation was efficiently restored within two hours presumably by inter chromosomal homologous recombination. The DNA replication in P. abyssi cells following irradiation at 2.5 kGy was blocked for 90 minutes that corresponds to the decay for repairing damaged DNA. Moreover, following irradiation P. abyssi actively expulse damaged DNA material before DNA replication resumes, preventing the amplification of genetic mutations. We have also showed that at least a subset cf P. abyssi DNA repair and replication proteins, such as RadA, RPA-41 and RFC-S. were constitutively expressed in chromatin bound forms in stationary phase cells. Our results were in agreement with the view that P. abyssi contains a very efficient DNA repair system, which is continuously ready to counteract the DNA damaged caused by the high temperature and/or ionizing radiation. For the first time, three novel hyper-thermophilic archaea species from deep-sea hydrothermal vents more radioresistant than P. abyssi were isolated and characterized, after 'y-irradiation exposures of some enrichment cultures. Thermococcus marinus, Thermococcus radiophilus and Thermococcus gammafolerans

  11. Enhanced selection of micro-aerobic pentachlorophenol degrading granular sludge

    Energy Technology Data Exchange (ETDEWEB)

    Lv, Yuancai, E-mail: donkey1204@hotmail.com [State Key Laboratory of Pulp and Paper Engineering, College of Light Industry and Food Science, South China University of Technology, Guangzhou 510640 (China); Chen, Yuancai, E-mail: chenyc@scut.edu.cn [State Key Laboratory of Pulp and Paper Engineering, College of Light Industry and Food Science, South China University of Technology, Guangzhou 510640 (China); Ministry of Education Key Laboratory of Pollution Control and Ecological Remediation for Industrial Agglomeration Area, College of Environment and Energy, South China University of Technology, Guangzhou 510006 (China); Song, Wenzhe, E-mail: songwenzhe007@126.com [Ministry of Education Key Laboratory of Pollution Control and Ecological Remediation for Industrial Agglomeration Area, College of Environment and Energy, South China University of Technology, Guangzhou 510006 (China); Hu, Yongyou, E-mail: ppyyhu@scut.edu.cn [State Key Laboratory of Pulp and Paper Engineering, College of Light Industry and Food Science, South China University of Technology, Guangzhou 510640 (China); Ministry of Education Key Laboratory of Pollution Control and Ecological Remediation for Industrial Agglomeration Area, College of Environment and Energy, South China University of Technology, Guangzhou 510006 (China)

    2014-09-15

    Graphical abstract: In this work, an aerobic column reactor was placed before the USB to maintain micro-oxygen condition in the reactor and the micro-aerobic pentachlorophenol (PCP) degrading granular sludge under oxygen-limited conditions (0.1–0.2 mg L{sup −1}) was successfully obtained. PCP degradation by the micro-aerobic system was studied and the variance of microbial community was also discussed by using PCR-DGGE analysis. - Highlights: • Micro-aerobic granular sludge was cultivated in column-type combined reactors. • PCP biodegradation, VFA accumulation and biogas production were studied. • The function of Methanogenic archaeon in the system was investigated. • Fluctuation and diversity of microbial community were discussed by DGGE analysis. • The dominated microorganisms were identified by 16S rDNA sequences. - Abstract: Column-type combined reactors were designed to cultivate micro-aerobic pentachlorophenol (PCP) degrading granular sludge under oxygen-limited conditions (0.1–0.2 mg L{sup −1}) over 39-day experimental period. Micro-aerobic granular had both anaerobic activity (SMA: 2.34 mMCH{sub 4}/h g VSS) and aerobic activity (SOUR: 2.21 mMO{sub 2}/h g VSS). Metabolite analysis results revealed that PCP was sequentially dechlorinated to TCP, DCP, and eventually to MCP. Methanogens were not directly involved in the dechlorination of PCP, but might played a vital role in stabilizing the overall structure of the granule sludge. For Eubacteria, the Shannon Index (2.09 in inoculated granular sludge) increased both in micro-aerobic granular sludge (2.61) and PCP-degradation granular sludge (2.55). However, for Archaea, it decreased from 2.53 to 1.85 and 1.84, respectively. Although the Shannon Index demonstrated slight difference between micro-aerobic granular sludge and PCP-degradation granular sludge, the Principal Component Analysis (PCA) indicated obvious variance of the microbial composition, revealing significant effect of micro

  12. Evolution of Molybdenum Nitrogenase during the Transition from Anaerobic to Aerobic Metabolism

    Science.gov (United States)

    Boyd, Eric S.; Costas, Amaya M. Garcia; Hamilton, Trinity L.; Mus, Florence

    2015-01-01

    ABSTRACT Molybdenum nitrogenase (Nif), which catalyzes the reduction of dinitrogen to ammonium, has modulated the availability of fixed nitrogen in the biosphere since early in Earth's history. Phylogenetic evidence indicates that oxygen (O2)-sensitive Nif emerged in an anaerobic archaeon and later diversified into an aerobic bacterium. Aerobic bacteria that fix N2 have adapted a number of strategies to protect Nif from inactivation by O2, including spatial and temporal segregation of Nif from O2 and respiratory consumption of O2. Here we report the complement of Nif-encoding genes in 189 diazotrophic genomes. We show that the evolution of Nif during the transition from anaerobic to aerobic metabolism was accompanied by both gene recruitment and loss, resulting in a substantial increase in the number of nif genes. While the observed increase in the number of nif genes and their phylogenetic distribution are strongly correlated with adaptation to utilize O2 in metabolism, the increase is not correlated with any of the known O2 protection mechanisms. Rather, gene recruitment appears to have been in response to selective pressure to optimize Nif synthesis to meet fixed N demands associated with aerobic productivity and to more efficiently regulate Nif under oxic conditions that favor protein turnover. Consistent with this hypothesis, the transition of Nif from anoxic to oxic environments is associated with a shift from posttranslational regulation in anaerobes to transcriptional regulation in obligate aerobes and facultative anaerobes. Given that fixed nitrogen typically limits ecosystem productivity, our observations further underscore the dynamic interplay between the evolution of Earth's oxygen, nitrogen, and carbon biogeochemical cycles. IMPORTANCE Molybdenum nitrogenase (Nif), which catalyzes the reduction of dinitrogen to ammonium, has modulated the availability of fixed nitrogen in the biosphere since early in Earth's history. Nif emerged in an anaerobe and

  13. Growth-Phase-Specific Modulation of Cell Morphology and Gene Expression by an Archaeal Histone Protein.

    Science.gov (United States)

    Dulmage, Keely A; Todor, Horia; Schmid, Amy K

    2015-09-08

    histone also use these proteins to package DNA. In contrast, here we demonstrate that the sole histone encoded in the genome of the salt-adapted archaeon Halobacterium salinarum is both unessential and unlikely to be involved in DNA compaction despite conservation of residues important for eukaryotic histones. Rather, H. salinarum histone is required for global regulation of gene expression and cell shape. These data are consistent with the hypothesis that H. salinarum histone, strongly conserved across all other known salt-adapted archaea, serves a novel role in gene regulation and cell shape maintenance. Given that archaea possess the ancestral form of eukaryotic histone, this study has important implications for understanding the evolution of histone function. Copyright © 2015 Dulmage et al.

  14. Roles of the conserved aspartate and arginine in the catalytic mechanism of an archaeal beta-class carbonic anhydrase.

    Science.gov (United States)

    Smith, Kerry S; Ingram-Smith, Cheryl; Ferry, James G

    2002-08-01

    The roles of an aspartate and an arginine, which are completely conserved in the active sites of beta-class carbonic anhydrases, were investigated by steady-state kinetic analyses of replacement variants of the beta-class enzyme (Cab) from the archaeon Methanobacterium thermoautotrophicum. Previous kinetic analyses of wild-type Cab indicated a two-step zinc-hydroxide mechanism of catalysis in which the k(cat)/K(m) value depends only on the rate constants for the CO(2) hydration step, whereas k(cat) also depends on rate constants from the proton transfer step (K. S. Smith, N. J. Cosper, C. Stalhandske, R. A. Scott, and J. G. Ferry, J. Bacteriol. 182:6605-6613, 2000). The recently solved crystal structure of Cab shows the presence of a buffer molecule within hydrogen bonding distance of Asp-34, implying a role for this residue in the proton transport step (P. Strop, K. S. Smith, T. M. Iverson, J. G. Ferry, and D. C. Rees, J. Biol. Chem. 276:10299-10305, 2001). The k(cat)/K(m) values of Asp-34 variants were decreased relative to those of the wild type, although not to an extent which supports an essential role for this residue in the CO(2) hydration step. Parallel decreases in k(cat) and k(cat)/K(m) values for the variants precluded any conclusions regarding a role for Asp-34 in the proton transfer step; however, the k(cat) of the D34A variant was chemically rescued by replacement of 2-(N-morpholino)propanesulfonic acid buffer with imidazole at pH 7.2, supporting a role for the conserved aspartate in the proton transfer step. The crystal structure of Cab also shows Arg-36 with two hydrogen bonds to Asp-34. Arg-36 variants had both k(cat) and k(cat)/K(m) values that were decreased at least 250-fold relative to those of the wild type, establishing an essential function for this residue. Imidazole was unable to rescue the k(cat) of the R36A variant; however, partial rescue of the kinetic parameter was obtained with guanidine-HCl indicating that the guanido group of this

  15. Extracellular enzymes facilitate electron uptake in biocorrosion and bioelectrosynthesis.

    Science.gov (United States)

    Deutzmann, Jörg S; Sahin, Merve; Spormann, Alfred M

    2015-04-21

    Direct, mediator-free transfer of electrons between a microbial cell and a solid phase in its surrounding environment has been suggested to be a widespread and ecologically significant process. The high rates of microbial electron uptake observed during microbially influenced corrosion of iron [Fe(0)] and during microbial electrosynthesis have been considered support for a direct electron uptake in these microbial processes. However, the underlying molecular mechanisms of direct electron uptake are unknown. We investigated the electron uptake characteristics of the Fe(0)-corroding and electromethanogenic archaeon Methanococcus maripaludis and discovered that free, surface-associated redox enzymes, such as hydrogenases and presumably formate dehydrogenases, are sufficient to mediate an apparent direct electron uptake. In genetic and biochemical experiments, we showed that these enzymes, which are released from cells during routine culturing, catalyze the formation of H2 or formate when sorbed to an appropriate redox-active surface. These low-molecular-weight products are rapidly consumed by M. maripaludis cells when present, thereby preventing their accumulation to any appreciable or even detectable level. Rates of H2 and formate formation by cell-free spent culture medium were sufficient to explain the observed rates of methane formation from Fe(0) and cathode-derived electrons by wild-type M. maripaludis as well as by a mutant strain carrying deletions in all catabolic hydrogenases. Our data collectively show that cell-derived free enzymes can mimic direct extracellular electron transfer during Fe(0) corrosion and microbial electrosynthesis and may represent an ecologically important but so far overlooked mechanism in biological electron transfer. The intriguing trait of some microbial organisms to engage in direct electron transfer is thought to be widespread in nature. Consequently, direct uptake of electrons into microbial cells from solid surfaces is assumed

  16. Characterization of an archaeal two-component system that regulates methanogenesis in Methanosaeta harundinacea.

    Directory of Open Access Journals (Sweden)

    Jie Li

    Full Text Available Two-component signal transduction systems (TCSs are a major mechanism used by bacteria in response to environmental changes. Although many sequenced archaeal genomes encode TCSs, they remain poorly understood. Previously, we reported that a methanogenic archaeon, Methanosaeta harundinacea, encodes FilI, which synthesizes carboxyl-acyl homoserine lactones, to regulate transitions of cellular morphology and carbon metabolic fluxes. Here, we report that filI, the cotranscribed filR2, and the adjacent filR1 constitute an archaeal TCS. FilI possesses a cytoplasmic kinase domain (histidine kinase A and histidine kinase-like ATPase and its cognate response regulator. FilR1 carries a receiver (REC domain coupled with an ArsR-related domain with potential DNA-binding ability, while FilR2 carries only a REC domain. In a phosphorelay assay, FilI was autophosphorylated and specifically transferred the phosphoryl group to FilR1 and FilR2, confirming that the three formed a cognate TCS. Through chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR using an anti-FilR1 antibody, FilR1 was shown to form in vivo associations with its own promoter and the promoter of the filI-filR2 operon, demonstrating a regulatory pattern common among TCSs. ChIP-qPCR also detected FilR1 associations with key genes involved in acetoclastic methanogenesis, acs4 and acs1. Electrophoretic mobility shift assays confirmed the in vitro tight binding of FilR1 to its own promoter and those of filI-filR2, acs4, and mtrABC. This also proves the DNA-binding ability of the ArsR-related domain, which is found primarily in Archaea. The archaeal promoters of acs4, filI, acs1, and mtrABC also initiated FilR1-modulated expression in an Escherichia coli lux reporter system, suggesting that FilR1 can up-regulate both archaeal and bacterial transcription. In conclusion, this work identifies an archaeal FilI/FilRs TCS that regulates the methanogenesis of M. harundinacea.

  17. Microbial metatranscriptomics in a permanent marine oxygen minimum zone.

    Science.gov (United States)

    Stewart, Frank J; Ulloa, Osvaldo; DeLong, Edward F

    2012-01-01

    Simultaneous characterization of taxonomic composition, metabolic gene content and gene expression in marine oxygen minimum zones (OMZs) has potential to broaden perspectives on the microbial and biogeochemical dynamics in these environments. Here, we present a metatranscriptomic survey of microbial community metabolism in the Eastern Tropical South Pacific OMZ off northern Chile. Community RNA was sampled in late austral autumn from four depths (50, 85, 110, 200 m) extending across the oxycline and into the upper OMZ. Shotgun pyrosequencing of cDNA yielded 180,000 to 550,000 transcript sequences per depth. Based on functional gene representation, transcriptome samples clustered apart from corresponding metagenome samples from the same depth, highlighting the discrepancies between metabolic potential and actual transcription. BLAST-based characterizations of non-ribosomal RNA sequences revealed a dominance of genes involved with both oxidative (nitrification) and reductive (anammox, denitrification) components of the marine nitrogen cycle. Using annotations of protein-coding genes as proxies for taxonomic affiliation, we observed depth-specific changes in gene expression by key functional taxonomic groups. Notably, transcripts most closely matching the genome of the ammonia-oxidizing archaeon Nitrosopumilus maritimus dominated the transcriptome in the upper three depths, representing one in five protein-coding transcripts at 85 m. In contrast, transcripts matching the anammox bacterium Kuenenia stuttgartiensis dominated at the core of the OMZ (200 m; 1 in 12 protein-coding transcripts). The distribution of N. maritimus-like transcripts paralleled that of transcripts matching ammonia monooxygenase genes, which, despite being represented by both bacterial and archaeal sequences in the community DNA, were dominated (> 99%) by archaeal sequences in the RNA, suggesting a substantial role for archaeal nitrification in the upper OMZ. These data, as well as those

  18. A nuclear magnetic resonance based approach to accurate functional annotation of putative enzymes in the methanogen Methanosarcina acetivorans

    Directory of Open Access Journals (Sweden)

    Nikolau Basil J

    2011-06-01

    Full Text Available Abstract Background Correct annotation of function is essential if one is to take full advantage of the vast amounts of genomic sequence data. The accuracy of sequence-based functional annotations is often variable, particularly if the sequence homology to a known function is low. Indeed recent work has shown that even proteins with very high sequence identity can have different folds and functions, and therefore caution is needed in assigning functions by sequence homology in the absence of experimental validation. Experimental methods are therefore needed to efficiently evaluate annotations in a way that complements current high throughput technologies. Here, we describe the use of nuclear magnetic resonance (NMR-based ligand screening as a tool for testing functional assignments of putative enzymes that may be of variable reliability. Results The target genes for this study are putative enzymes from the methanogenic archaeon Methanosarcina acetivorans (MA that have been selected after manual genome re-annotation and demonstrate detectable in vivo expression at the level of the transcriptome. The experimental approach begins with heterologous E. coli expression and purification of individual MA gene products. An NMR-based ligand screen of the purified protein then identifies possible substrates or products from a library of candidate compounds chosen from the putative pathway and other related pathways. These data are used to determine if the current sequence-based annotation is likely to be correct. For a number of case studies, additional experiments (such as in vivo genetic complementation were performed to determine function so that the reliability of the NMR screen could be independently assessed. Conclusions In all examples studied, the NMR screen was indicative of whether the functional annotation was correct. Thus, the case studies described demonstrate that NMR-based ligand screening is an effective and rapid tool for confirming or

  19. Evolution of molybdenum nitrogenase during the transition from anaerobic to aerobic metabolism.

    Science.gov (United States)

    Boyd, Eric S; Costas, Amaya M Garcia; Hamilton, Trinity L; Mus, Florence; Peters, John W

    2015-05-01

    Molybdenum nitrogenase (Nif), which catalyzes the reduction of dinitrogen to ammonium, has modulated the availability of fixed nitrogen in the biosphere since early in Earth's history. Phylogenetic evidence indicates that oxygen (O2)-sensitive Nif emerged in an anaerobic archaeon and later diversified into an aerobic bacterium. Aerobic bacteria that fix N2 have adapted a number of strategies to protect Nif from inactivation by O2, including spatial and temporal segregation of Nif from O2 and respiratory consumption of O2. Here we report the complement of Nif-encoding genes in 189 diazotrophic genomes. We show that the evolution of Nif during the transition from anaerobic to aerobic metabolism was accompanied by both gene recruitment and loss, resulting in a substantial increase in the number of nif genes. While the observed increase in the number of nif genes and their phylogenetic distribution are strongly correlated with adaptation to utilize O2 in metabolism, the increase is not correlated with any of the known O2 protection mechanisms. Rather, gene recruitment appears to have been in response to selective pressure to optimize Nif synthesis to meet fixed N demands associated with aerobic productivity and to more efficiently regulate Nif under oxic conditions that favor protein turnover. Consistent with this hypothesis, the transition of Nif from anoxic to oxic environments is associated with a shift from posttranslational regulation in anaerobes to transcriptional regulation in obligate aerobes and facultative anaerobes. Given that fixed nitrogen typically limits ecosystem productivity, our observations further underscore the dynamic interplay between the evolution of Earth's oxygen, nitrogen, and carbon biogeochemical cycles. Molybdenum nitrogenase (Nif), which catalyzes the reduction of dinitrogen to ammonium, has modulated the availability of fixed nitrogen in the biosphere since early in Earth's history. Nif emerged in an anaerobe and later diversified into

  20. Permuting the PGF Signature Motif Blocks both Archaeosortase-Dependent C-Terminal Cleavage and Prenyl Lipid Attachment for the Haloferax volcanii S-Layer Glycoprotein.

    Science.gov (United States)

    Abdul Halim, Mohd Farid; Karch, Kelly R; Zhou, Yitian; Haft, Daniel H; Garcia, Benjamin A; Pohlschroder, Mechthild

    2015-12-28

    For years, the S-layer glycoprotein (SLG), the sole component of many archaeal cell walls, was thought to be anchored to the cell surface by a C-terminal transmembrane segment. Recently, however, we demonstrated that the Haloferax volcanii SLG C terminus is removed by an archaeosortase (ArtA), a novel peptidase. SLG, which was previously shown to be lipid modified, contains a C-terminal tripartite structure, including a highly conserved proline-glycine-phenylalanine (PGF) motif. Here, we demonstrate that ArtA does not process an SLG variant where the PGF motif is replaced with a PFG motif (slg(G796F,F797G)). Furthermore, using radiolabeling, we show that SLG lipid modification requires the PGF motif and is ArtA dependent, lending confirmation to the use of a novel C-terminal lipid-mediated protein-anchoring mechanism by prokaryotes. Similar to the case for the ΔartA strain, the growth, cellular morphology, and cell wall of the slg(G796F,F797G) strain, in which modifications of additional H. volcanii ArtA substrates should not be altered, are adversely affected, demonstrating the importance of these posttranslational SLG modifications. Our data suggest that ArtA is either directly or indirectly involved in a novel proteolysis-coupled, covalent lipid-mediated anchoring mechanism. Given that archaeosortase homologs are encoded by a broad range of prokaryotes, it is likely that this anchoring mechanism is widely conserved. Prokaryotic proteins bound to cell surfaces through intercalation, covalent attachment, or protein-protein interactions play critical roles in essential cellular processes. Unfortunately, the molecular mechanisms that anchor proteins to archaeal cell surfaces remain poorly characterized. Here, using the archaeon H. volcanii as a model system, we report the first in vivo studies of a novel protein-anchoring pathway involving lipid modification of a peptidase-processed C terminus. Our findings not only yield important insights into poorly understood

  1. Non-extremophilic 'extremophiles' - Archaeal dominance in the subsurface and their implication for life

    Science.gov (United States)

    Reitschuler, Christoph; Lins, Philipp; Illmer, Paul

    2014-05-01

    Archaea - besides bacteria and eukaryota constituting the third big domain of life - were so far regarded as typical inhabitants of extreme environments, as indicated by the name (Archaeon, Greek: 'original', 'primal'). Previous research and cultivation successes were basically carried out in habitats characterized by extreme temperature, pH and salinity regimes. Such extreme conditions, as expected at the beginning of the Earth's evolution, are occasionally also prevalent on extraterrestrial planets and moons and make the Archaeal domain a key group to be studied concerning life's evolution and the most likely pioneer organisms to colonize environments that are regarded as hostile. However, in recent years it became obvious that Archaea, in particular non-extremophilic species, can be found almost ubiquitously in marine, freshwater, terrestrial and also subsurface habitats and occasionally outnumber other microbial domains and hold key positions in globally relevant energy and nutrient cycles. Besides extreme environments - the big question remains how to define a parameter as extreme - subsurface and cave environments present a window to the past, where adaptions to early life's conditions can be studied and how microbiomes may be structured in a habitat that represents a refugium on extraterrestrial celestial bodies, were surface conditions might be at first sight too extreme for life. The lower part of the alpine Hundsalm cave in Tyrol (Austria) offered a unique opportunity to study an almost pristine cave habitat, which is separated from the touristic part of the ice cave. The main focus of our research was laid on the microbial communities that were supposed to be in connection with secondary carbonate precipitations ('moonmilk'). For the ascertainment of these so far poorly evaluated structures a multiple approach assessment was chosen to generate a virtually complete picture of these subsurface microbiomes. Thereby, a combination of different cultivation

  2. MICROBIAL FERMENTATION OF ABUNDANT BIOPOLYMERS: CELLULOSE AND CHITIN

    Energy Technology Data Exchange (ETDEWEB)

    Leschine, Susan

    2009-10-31

    that the chitinase and cellulase systems of this bacterium are distinct in terms of the proteins involved and the regulation of their production. 4. Characterization of the chitinase system of C. uda. A 70,000-Mr endochitinase, designated ChiA, was purified from C. uda culture supernatant fluids and characterized. 5. Analysis of chiA, which codes for the major enzymatic component of the chitinase system of C. uda. The gene encoding the endochitinase ChiA in C. uda was cloned, its complete nucleotide sequence was determined and its implications were investigated. 6. Formation of biofilms by C. uda on cellulose and chitin. Microscopic observations indicated that, under conditions of nitrogen limitation, C. uda cells grew as a biofilm attached tightly to the surface of cellulose or chitin. 7. Development of tools for a genetic approach to studies of cellulose fermentation by cellulolytic clostridia. We have explored the potential of various techniques, and obtained evidence indicating that Tn916 mutagenesis may be particularly effective in this regard. As part of this research, we identified the presence of a plasmid in one strain, which was cloned, sequenced, and analyzed for its utility in the development of vectors for genetic studies. 8. Effects of humic substances on cellulose degradation by anaerobic cellulolytic microbes. We determined that humic substances play an important role in the anaerobic cellulose decomposition and in the physiology of cellulose-fermenting soil bacteria. 9. Nitrogenases of cellulolytic clostridia. We described a nitrogenase gene from a cellulolytic clostridium and presented evidence, based on sequence analyses and conserved gene order, for lateral gene transfer between this bacterium and a methanogenic archaeon. 10. Characterization of Clostridium hungatei, a new N2-fixing cellulolytic species isolated from a methanogenic consortium from soil. 11. Understanding the molecular architecture of the multicomplex cellulase-xylanase system of

  3. Transmission of the PabI family of restriction DNA glycosylase genes: mobility and long-term inheritance.

    Science.gov (United States)

    Kojima, Kenji K; Kobayashi, Ichizo

    2015-10-19

    R.PabI is an exceptional restriction enzyme that functions as a DNA glycosylase. The enzyme excises an unmethylated base from its recognition sequence to generate apurinic/apyrimidinic (AP) sites, and also displays AP lyase activity, cleaving the DNA backbone at the AP site to generate the 3'-phospho alpha, beta-unsaturated aldehyde end in addition to the 5'-phosphate end. The resulting ends are difficult to religate with DNA ligase. The enzyme was originally isolated in Pyrococcus, a hyperthermophilic archaeon, and additional homologs subsequently identified in the epsilon class of the Gram-negative bacterial phylum Proteobacteria, such as Helicobacter pylori. Systematic analysis of R.PabI homologs and their neighboring genes in sequenced genomes revealed co-occurrence of R.PabI with M.PabI homolog methyltransferase genes. R.PabI and M.PabI homolog genes are occasionally found at corresponding (orthologous) loci in different species, such as Helicobacter pylori, Helicobacter acinonychis and Helicobacter cetorum, indicating long-term maintenance of the gene pair. One R.PabI and M.PabI homolog gene pair is observed immediately after the GMP synthase gene in both Campylobacter and Helicobacter, representing orthologs beyond genera. The mobility of the PabI family of restriction-modification (RM) system between genomes is evident upon comparison of genomes of sibling strains/species. Analysis of R.PabI and M.PabI homologs in H. pylori revealed an insertion of integrative and conjugative elements (ICE), and replacement with a gene of unknown function that may specify a membrane-associated toxin (hrgC). In view of the similarity of HrgC with toxins in type I toxin-antitoxin systems, we addressed the biological significance of this substitution. Our data indicate that replacement with hrgC occurred in the common ancestor of hspAmerind and hspEAsia. Subsequently, H. pylori with and without hrgC were intermixed at this locus, leading to complex distribution of hrgC in East

  4. Lessons from Suiyo Seamount studies, for understanding extreme (ancient?) microbial ecosystems in the deep-sea hydrothermal fields

    Science.gov (United States)

    Maruyama, A.; Higashi, Y.; Sunamura, M.; Urabe, T.

    2004-12-01

    Deep-sea hydrothermal ecosystems are driven with various geo-thermally modified, mainly reduced, compounds delivered from extremely hot subsurface environments. To date, several unique microbes including thermophilic archaeons have been isolated from/around vent chimneys. However, there is little information about microbes in over-vent and sub-vent fields. Here, we report several new findings on microbial diversity and ecology of the Suiyo Seamount that locates on the Izu-Bonin Arc in the northwest Pacific Ocean, as a result of the Japanese Archaean Park project, with special concern to the sub-vent biosphere. At first, we succeeded to reveal a very unique microbial ecosystem in hydrothermal plume reserved within the outer rim of the seamount crater, that is, it consisted of almost all metabolically active microbes belonged to only two Bacteria phylotypes, probably of sulfur oxidizers. In the center of the caldera seafloor (ca. 1,388-m deep) consisted mainly of whitish sands and pumices, we found many small chimneys (ca. 5-10 cm) and bivalve colonies distributed looking like gray to black patches. These geo/ecological features of the seafloor were supposed to be from a complex mixing of hydrothermal venting and strong water current near the seafloor. Through quantitative FISH analysis for various environmental samples, one of the two representative groups in the plume was assessed to be from some of the bivalve colonies. Using the Benthic Multi-coring System (BMS), total 10 points were drilled and 6 boreholes were maintained with stainless or titanium casing pipes. In the following submersible surveys, newly developed catheter- and column-type in situ growth chambers were deployed in and on the boreholes, respectively, for collecting indigenous sub-vent microbes. Finally, we succeeded to detect several new phylotypes of microbes in these chamber samples, e.g., within epsilon-Proteobacteria, a photosynthetic group of alpha-Proteobacteria, and hyperthermophile

  5. Autotrophic Ecosystems on the Early Earth

    Science.gov (United States)

    Schulte, M.

    2003-01-01

    resulting from aqueous alteration of olivine resides in veins that are see to cross cut the primary mineral grains. There are several generations of alteration products, comprised mostly of serpentines that are magnesium rich, with magnetite, brucite and carbonates observed as accessory minerals. The formation of carbonates can be taken to indicate the presence of CO2 in the altering fluids. We collected samples from a spring in the Coast Range Ophiolite in order to determine whether the geochemical environment serves as a habitat for chemotrophic microorganisms. DNA was extracted from the sediment samples and the 16s rRNA gene was PCR amplified using universal Archaeal primers. Denaturing gradient gel electrophoresis (DGGE) was used to determine the community of Archaea thriving in these samples. Our results indicate that there were 8 different genera of Archaea from a single sample. A sequence was obtained from one of these eight. The sequence is of an organism similar to Halorubrum tibetense, and alkalophilic Archaeon. This result suggests that these environments are likely hosts for communities of organisms that are adapted for the unique chemistry provided by the alkaline spring.

  6. Joint project. Retention of radionuclides relevant for final disposal in natural clay rock and saline systems. Subproject 2. Geochemical behavior and transport of radionuclides in saline systems in the presence of repository-relevant organics. Final report

    International Nuclear Information System (INIS)

    Schmeide, Katja; Fritsch, Katharina; Lippold, Holger

    2016-01-01

    The objective of this project was to study the influence of increased salinities on interaction processes in the system radionuclide - organics - clay - aquifer. For this purpose, complexation, redox, sorption, and diffusion studies were performed under variation of the ionic strength (up to 4 mol kg -1 ) and the background electrolyte. The U(VI) complexation by propionate was studied in dependence on ionic strength (up to 4 mol kg -1 NaClO 4 ) by TRLFS, ATR FT-IR spectroscopy, and DFT calculations. An influence of ionic strength on stability constants was detected, depending on the charge of the respective complexes. The conditional stability constants, determined for 1:1, 1:2, and 1:3 complexes at specific ionic strengths, were extrapolated to zero ionic strength. The interaction of the bacteria Sporomusa sp. MT-2.99 and Paenibacillus sp. MT-2.2 cells, isolated from Opalinus Clay, with Pu was studied. The experiments can be divided into such without an electron donor where biosorption is favored and such with addition of Na-pyruvate as an electron donor stimulating also bioreduction processes. Moreover, experiments were performed to study the interactions of the halophilic archaeon Halobacterium noricense DSM-15987 with U(VI), Eu(III), and Cm(III) in 3 M NaCl solutions. Research for improving process understanding with respect to the mobility of multivalent metals in systems containing humic matter was focused on the reversibility of elementary processes and on their interaction. Kinetic stabilization processes in the dynamics of humate complexation equilibria were quantified in isotope exchange studies. The influence of high salinity on the mobilizing potential of humic-like clay organics was systematically investigated and was described by modeling. The sorption of Tc(VII)/Tc(IV) onto the iron(II)-containing minerals magnetite and siderite was studied by means of batch sorption experiments, ATR FT-IR and X-ray absorption spectroscopy. The strong Tc retention at

  7. Joint project. Retention of radionuclides relevant for final disposal in natural clay rock and saline systems. Subproject 2. Geochemical behavior and transport of radionuclides in saline systems in the presence of repository-relevant organics. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Schmeide, Katja; Fritsch, Katharina; Lippold, Holger [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Inst. of Ressource Ecology; and others

    2016-08-01

    The objective of this project was to study the influence of increased salinities on interaction processes in the system radionuclide - organics - clay - aquifer. For this purpose, complexation, redox, sorption, and diffusion studies were performed under variation of the ionic strength (up to 4 mol kg{sup -1}) and the background electrolyte. The U(VI) complexation by propionate was studied in dependence on ionic strength (up to 4 mol kg{sup -1} NaClO{sub 4}) by TRLFS, ATR FT-IR spectroscopy, and DFT calculations. An influence of ionic strength on stability constants was detected, depending on the charge of the respective complexes. The conditional stability constants, determined for 1:1, 1:2, and 1:3 complexes at specific ionic strengths, were extrapolated to zero ionic strength. The interaction of the bacteria Sporomusa sp. MT-2.99 and Paenibacillus sp. MT-2.2 cells, isolated from Opalinus Clay, with Pu was studied. The experiments can be divided into such without an electron donor where biosorption is favored and such with addition of Na-pyruvate as an electron donor stimulating also bioreduction processes. Moreover, experiments were performed to study the interactions of the halophilic archaeon Halobacterium noricense DSM-15987 with U(VI), Eu(III), and Cm(III) in 3 M NaCl solutions. Research for improving process understanding with respect to the mobility of multivalent metals in systems containing humic matter was focused on the reversibility of elementary processes and on their interaction. Kinetic stabilization processes in the dynamics of humate complexation equilibria were quantified in isotope exchange studies. The influence of high salinity on the mobilizing potential of humic-like clay organics was systematically investigated and was described by modeling. The sorption of Tc(VII)/Tc(IV) onto the iron(II)-containing minerals magnetite and siderite was studied by means of batch sorption experiments, ATR FT-IR and X-ray absorption spectroscopy. The strong Tc