DOCSLIB.ORG

Sulfolobus Acidocaldarius & Sulfolobus Solfataricus

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Sulfolobus Acidocaldarius & Sulfolobus Solfataricus Sulfolobus acidocaldarius & Sulfolobus solfataricus: Exploitation of thermoacidophilic Archaea for biotechnological applications Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften - Dr. rer. nat. - vorgelegt von JULIA CHRISTIN VERHEYEN geboren am 09.08.1982 in Dresden Biofilm Centre, Molekulare Enzymtechnologie und Biochemie Fachbereich Chemie der Universität Duisburg-Essen 2015 Die vorliegende Arbeit wurde im Zeitraum von Oktober 2009 bis April 2015 im Arbeitskreis von Prof. Dr. Bettina Siebers in der Fakultät Chemie, Biofilm Centre, Molekulare Enzymtechnologie und Biochemie der Universität Duisburg-Essen durchgeführt. Tag der Disputation: 24.06.2015 Gutachter: Prof. Dr. Bettina Siebers Prof. Dr. Peter Bayer Vorsitzender: Prof. Dr. Thomas Schrader Meinen Kindern – die schönste Bereicherung meines Lebens TABLE OF CONTENTS i TABLE OF CONTENTS TABLE OF CONTENTS .................................................................................................... I! LIST OF FIGURES ......................................................................................................... IV! LIST OF TABLES ........................................................................................................... VI! CHAPTER 1 INTRODUCTION ......................................................................................... 2! 1.1 (HYPER)THERMOPHILES, THEIR ENZYMES AND THE BIOTECHNOLOGICAL RELEVANCE .................................................................................................................................... 2! 1.2 MINING FOR NOVEL ENZYMES - METAGENOMICS .................................................................. 5! 1.3 ARCHAEA AS ALTERNATIVE EXPRESSION HOSTS FOR METAGENOMICS ............................ 8! 1.4 GLYCOLYSIS IN SULFOLOBUS SOLFATARICUS – THE ROLE OF GDH .................................. 12! 1.5 AIM OF THIS WORK ...................................................................................................................... 16! CHAPTER 2 MATERIALS & METHODS ........................................................................ 19! 2.1 CHEMICALS, VECTORS AND COMMERCIAL KITS .................................................................... 19! 2.2 INSTRUMENTS ............................................................................................................................. 20! 2.3 MICROBIOLOGICAL METHODS .................................................................................................. 21! 2.3.1 Strains of Escherichia coli & growth conditions ...................................................................... 21! 2.3.2 Strains of Sulfolobus acidocaldarius & growth conditions .................................................... 21! 2.4. MOLECULAR BIOLOGICAL METHODS ..................................................................................... 23! 2.4.1 Preparation of genomic DNA ................................................................................................... 23! 2.4.2 Isolation of plasmid DNA from E. coli .................................................................................... 23! 2.4.3 Quantification of DNA ............................................................................................................. 23! 2.4.4 Agarose gel electrophoresis ..................................................................................................... 23! 2.4.5 Purification of DNA fragments ................................................................................................ 24! 2.4.6 Amplification of DNA by Polymerase Chain Reaction (PCR) ................................................. 24! 2.4.7 Restriction of DNA ................................................................................................................... 27! 2.4.8 Ligation .................................................................................................................................... 27! 2.4.9 Preparation of chemically competent E. coli cells .................................................................. 28! 2.4.10 Transformation of competent E. coli cells ............................................................................ 28! 2.4.11 Identification of correctly ligated recombinant plasmid DNA ............................................. 28! 2.4.12 DNA sequencing ..................................................................................................................... 29! 2.4.13 Preparation of competent S. acidocaldarius cells ................................................................. 29! 2.4.14 Transformation of competent S. acidocaldarius cells via electroporation ........................... 29! 2.4.15 Identification of S. acidocaldarius clones harboring recombinant expression plasmid ...... 29! 2.4.16 RNA isolation from S. acidocaldarius cells and DNaseI digestion ...................................... 30! 2.4.17 cDNA synthesis and PCR ....................................................................................................... 30! 2.5 BIOCHEMICAL METHODS ........................................................................................................... 31! 2.5.1 Heterologous expression of recombinant proteins in E. coli Rosetta (DE3) and preparation of cell-free extracts .............................................................................................. 31! TABLE OF CONTENTS ii 2.5.2 Purification of recombinant target proteins from E. coli cells ................................................ 31! 2.5.2.1 Ion exchange chromatography (IC) ...................................................................................... 33! 2.5.2.2 Hydrophobic interaction chromatography (HIC) ................................................................ 33! 2.5.2.3 Gel filtration (GF) ................................................................................................................. 33! 2.5.2.4 Ni-TED affinity chromatography .......................................................................................... 34! 2.5.3 Expression and purification of the thermophilic auxiliary enzyme GAPN ............................. 34! 2.5.4 Heterologous expression in S. acidocaldarius, preparation of cell-free extracts and purification of recombinant target proteins ........................................................................... 34! 2.5.5 Protein quantification .............................................................................................................. 35! 2.5.6 Protein precipitation ................................................................................................................ 35! 2.5.7 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ........................................................... 35! 2.5.8 Immuno-blotting ..................................................................................................................... 36! 2.6 ENZYME ASSAYS ........................................................................................................................... 36! 2.6.1 Phosphofructokinase (PFK) ..................................................................................................... 36! 2.6.2 Esterase .................................................................................................................................... 37! 2.6.3 Glucose dehydrogenase (GDH) ............................................................................................... 37! 2.6.4 β-galactosidase ......................................................................................................................... 38! 2.7 KINETIC PARAMETERS ................................................................................................................ 38! 2.8 SOFTWARE, INTERNET DATABASES AND TOOLS ................................................................... 39! CHAPTER 3 RESULTS .................................................................................................. 41! 3.1 ESTABLISHMENT OF AN ARCHAEAL EXPRESSION HOST FOR BIOTECHNOLOGICAL APPLICATIONS ............................................................................................................................. 41! 3.1.1 Promoter selectivity – Comparison of bacterial and archaeal promoters for heterologous expression in Sulfolobus acidocaldarius ................................................................................ 41! 3.1.2 Functional Metagenomics – Establishment of Sulfolobus acidocaldarius as screening host ........................................................................................................................................... 44! 3.1.2.1 Examination of esterase activity in Sulfolobus acidocaldarius ............................................ 44! 3.1.2.2 Recombinant expression of Saci_1105 and Saci_1116 in Escherichia coli and protein characterization ....................................................................................................................... 46! 3.1.2.3 Heterologous expression of selected metagenomic clones in Sulfolobus acidocaldarius MW001Δ1105Δ1116 ................................................................................................................. 48! 3.1.2.4 Transcription analysis of selected metagenomic clones in Sulfolobus acidocaldarius MW001Δ1105Δ1116 .................................................................................................................. 51! 3.2
Load more

Recommended publications
  • CRISPR Loci Reveal Networks of Gene Exchange in Archaea Avital Brodt, Mor N Lurie-Weinberger and Uri Gophna*
    Brodt et al. Biology Direct 2011, 6:65 http://www.biology-direct.com/content/6/1/65 RESEARCH Open Access CRISPR loci reveal networks of gene exchange in archaea Avital Brodt, Mor N Lurie-Weinberger and Uri Gophna* Abstract Background: CRISPR (Clustered, Regularly, Interspaced, Short, Palindromic Repeats) loci provide prokaryotes with an adaptive immunity against viruses and other mobile genetic elements. CRISPR arrays can be transcribed and processed into small crRNA molecules, which are then used by the cell to target the foreign nucleic acid. Since spacers are accumulated by active CRISPR/Cas systems, the sequences of these spacers provide a record of the past “infection history” of the organism. Results: Here we analyzed all currently known spacers present in archaeal genomes and identified their source by DNA similarity. While nearly 50% of archaeal spacers matched mobile genetic elements, such as plasmids or viruses, several others matched chromosomal genes of other organisms, primarily other archaea. Thus, networks of gene exchange between archaeal species were revealed by the spacer analysis, including many cases of inter-genus and inter-species gene transfer events. Spacers that recognize viral sequences tend to be located further away from the leader sequence, implying that there exists a selective pressure for their retention. Conclusions: CRISPR spacers provide direct evidence for extensive gene exchange in archaea, especially within genera, and support the current dogma where the primary role of the CRISPR/Cas system is anti-viral and anti- plasmid defense. Open peer review: This article was reviewed by: Profs. W. Ford Doolittle, John van der Oost, Christa Schleper (nominated by board member Prof.
    [Show full text]
  • Genome-Resolved Meta-Analysis of the Microbiome in Oil Reservoirs Worldwide
    microorganisms Article Genome-Resolved Meta-Analysis of the Microbiome in Oil Reservoirs Worldwide Kelly J. Hidalgo 1,2,* , Isabel N. Sierra-Garcia 3 , German Zafra 4 and Valéria M. de Oliveira 1 1 Microbial Resources Division, Research Center for Chemistry, Biology and Agriculture (CPQBA), University of Campinas–UNICAMP, Av. Alexandre Cazellato 999, 13148-218 Paulínia, Brazil; [email protected] 2 Graduate Program in Genetics and Molecular Biology, Institute of Biology, University of Campinas (UNICAMP), Rua Monteiro Lobato 255, Cidade Universitária, 13083-862 Campinas, Brazil 3 Biology Department & CESAM, University of Aveiro, Aveiro, Portugal, Campus de Santiago, Avenida João Jacinto de Magalhães, 3810-193 Aveiro, Portugal; [email protected] 4 Grupo de Investigación en Bioquímica y Microbiología (GIBIM), Escuela de Microbiología, Universidad Industrial de Santander, Cra 27 calle 9, 680002 Bucaramanga, Colombia; [email protected] * Correspondence: [email protected]; Tel.: +55-19981721510 Abstract: Microorganisms inhabiting subsurface petroleum reservoirs are key players in biochemical transformations. The interactions of microbial communities in these environments are highly complex and still poorly understood. This work aimed to assess publicly available metagenomes from oil reservoirs and implement a robust pipeline of genome-resolved metagenomics to decipher metabolic and taxonomic profiles of petroleum reservoirs worldwide. Analysis of 301.2 Gb of metagenomic information derived from heavily flooded petroleum reservoirs in China and Alaska to non-flooded petroleum reservoirs in Brazil enabled us to reconstruct 148 metagenome-assembled genomes (MAGs) of high and medium quality. At the phylum level, 74% of MAGs belonged to bacteria and 26% to archaea. The profiles of these MAGs were related to the physicochemical parameters and recovery management applied.
    [Show full text]
  • Marsarchaeota Are an Aerobic Archaeal Lineage Abundant in Geothermal Iron Oxide Microbial Mats
    Marsarchaeota are an aerobic archaeal lineage abundant in geothermal iron oxide microbial mats Authors: Zackary J. Jay, Jacob P. Beam, Mansur Dlakic, Douglas B. Rusch, Mark A. Kozubal, and William P. Inskeep This is a postprint of an article that originally appeared in Nature Microbiology on May 14, 2018. The final version can be found at https://dx.doi.org/10.1038/s41564-018-0163-1. Jay, Zackary J. , Jacob P. Beam, Mensur Dlakic, Douglas B. Rusch, Mark A. Kozubal, and William P. Inskeep. "Marsarchaeota are an aerobic archaeal lineage abundant in geothermal iron oxide microbial mats." Nature Microbiology 3, no. 6 (May 2018): 732-740. DOI: 10.1038/ s41564-018-0163-1. Made available through Montana State University’s ScholarWorks scholarworks.montana.edu Marsarchaeota are an aerobic archaeal lineage abundant in geothermal iron oxide microbial mats Zackary J. Jay1,4,7, Jacob P. Beam1,5,7, Mensur Dlakić2, Douglas B. Rusch3, Mark A. Kozubal1,6 and William P. Inskeep 1* The discovery of archaeal lineages is critical to our understanding of the universal tree of life and evolutionary history of the Earth. Geochemically diverse thermal environments in Yellowstone National Park provide unprecedented opportunities for studying archaea in habitats that may represent analogues of early Earth. Here, we report the discovery and character- ization of a phylum-level archaeal lineage proposed and herein referred to as the ‘Marsarchaeota’, after the red planet. The Marsarchaeota contains at least two major subgroups prevalent in acidic, microaerobic geothermal Fe(III) oxide microbial mats across a temperature range from ~50–80 °C. Metagenomics, single-cell sequencing, enrichment culturing and in situ transcrip- tional analyses reveal their biogeochemical role as facultative aerobic chemoorganotrophs that may also mediate the reduction of Fe(III).
    [Show full text]
  • Sulfolobus Acidocaldarius Claus AAGAARD, JACOB Z
    Proc. Natl. Acad. Sci. USA Vol. 92, pp. 12285-12289, December 1995 Biochemistry Intercellular mobility and homing of an archaeal rDNA intron confers a selective advantage over intron- cells of Sulfolobus acidocaldarius CLAus AAGAARD, JACOB Z. DALGAARD*, AND ROGER A. GARRETr Institute of Molecular Biology, Copenhagen University, S0lvgade 83 H, 1307 Copenhagen K, Denmark Communicated by Carl R. Woese, University of Illinois at Urbana-Champaign, Urbana, IL, August 28, 1995 ABSTRACT Some intron-containing rRNA genes of ar- experiments suggest, but do not establish, that the intron is chaea encode homing-type endonucleases, which facilitate mobile between yeast cells. intron insertion at homologous sites in intron- alleles. These To test whether the archaeal introns constitute mobile archaeal rRNA genes, in contrast to their eukaryotic coun- elements, we electroporated the intron-containing 23S rRNA terparts, are present in single copies per cell, which precludes gene from the archaeal hyperthermophile Desulfurococcus intron homing within one cell. However, given the highly mobilis on nonreplicating bacterial vectors into an intron- conserved nature of the sequences flanking the intron, hom- culture of Sulfolobus acidocaldarius. In the presence of I-Dmo ing may occur in intron- rRNA genes of other archaeal cells. I, the endonuclease encoded by the D. mobilis intron (20), the To test whether this occurs, the intron-containing 23S rRNA intron was shown to home in the chromosomal DNA of intron- gene of the archaeal hyperthermophile Desulfurococcus mobi- cells of S. acidocaldarius. Moreover, using a double drug- lis, carried on nonreplicating bacterial vectors, was electro- resistant mutant (21), it was demonstrated that the intron can porated into an intron- culture of Sulfolobus acidocaldarius.
    [Show full text]
  • Archaeology of Eukaryotic DNA Replication
    Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Archaeology of Eukaryotic DNA Replication Kira S. Makarova and Eugene V. Koonin National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894 Correspondence: [email protected] Recent advances in the characterization of the archaeal DNA replication system together with comparative genomic analysis have led to the identification of several previously un- characterized archaeal proteins involved in replication and currently reveal a nearly com- plete correspondence between the components of the archaeal and eukaryotic replication machineries. It can be inferred that the archaeal ancestor of eukaryotes and even the last common ancestor of all extant archaea possessed replication machineries that were compa- rable in complexity to the eukaryotic replication system. The eukaryotic replication system encompasses multiple paralogs of ancestral components such that heteromeric complexes in eukaryotes replace archaeal homomeric complexes, apparently along with subfunctionali- zation of the eukaryotic complex subunits. In the archaea, parallel, lineage-specific dupli- cations of many genes encoding replication machinery components are detectable as well; most of these archaeal paralogs remain to be functionally characterized. The archaeal rep- lication system shows remarkable plasticity whereby even some essential components such as DNA polymerase and single-stranded DNA-binding protein are displaced by unrelated proteins with analogous activities in some lineages. ouble-stranded DNA is the molecule that Okazaki fragments (Kornberg and Baker 2005; Dcarries genetic information in all cellular Barry and Bell 2006; Hamdan and Richardson life-forms; thus, replication of this genetic ma- 2009; Hamdan and van Oijen 2010).
    [Show full text]
  • The Isolation of Viruses Infecting Archaea
    Portland State University PDXScholar Biology Faculty Publications and Presentations Biology 2010 The Isolation of Viruses Infecting Archaea Kenneth M. Stedman Portland State University Kate Porter Mike L. Dyall-Smith Follow this and additional works at: https://pdxscholar.library.pdx.edu/bio_fac Part of the Bacteria Commons, Biology Commons, and the Viruses Commons Let us know how access to this document benefits ou.y Citation Details Stedman, Kenneth M., Kate Porter, and Mike L. Dyall-Smith. "The isolation of viruses infecting Archaea." Manual of aquatic viral ecology. American Society for Limnology and Oceanography (ASLO) (2010): 57-64. This Article is brought to you for free and open access. It has been accepted for inclusion in Biology Faculty Publications and Presentations by an authorized administrator of PDXScholar. Please contact us if we can make this document more accessible: [email protected]. MANUAL of MAVE Chapter 6, 2010, 57–64 AQUATIC VIRAL ECOLOGY © 2010, by the American Society of Limnology and Oceanography, Inc. The isolation of viruses infecting Archaea Kenneth M. Stedman1, Kate Porter2, and Mike L. Dyall-Smith3 1Department of Biology, Center for Life in Extreme Environments, Portland State University, P.O. Box 751, Portland, OR 97207, USA 2Biota Holdings Limited, 10/585 Blackburn Road, Notting Hill Victoria 3168, Australia 3Max Planck Institute of Biochemistry, Department of Membrane Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany Abstract A mere 50 viruses of Archaea have been reported to date; these have been investigated mostly by adapting methods used to isolate bacteriophages to the unique growth conditions of their archaeal hosts. The most numer- ous are viruses of thermophilic Archaea.
    [Show full text]
  • The Role of Polyphosphate in Motility, Adhesion, and Biofilm Formation in Sulfolobales. Microorganisms 2021, 9
    microorganisms Article The Role of Polyphosphate in Motility, Adhesion, and Biofilm Formation in Sulfolobales Alejandra Recalde 1,2 , Marleen van Wolferen 2 , Shamphavi Sivabalasarma 2 , Sonja-Verena Albers 2, Claudio A. Navarro 1 and Carlos A. Jerez 1,* 1 Laboratory of Molecular Microbiology and Biotechnology, Department of Biology, Faculty of Sciences, University of Chile, Santiago 8320000, Chile; [email protected] (A.R.); [email protected] (C.A.N.) 2 Laboratory of Molecular Biology of Archaea, Institute of Biology II-Microbiology, University of Freiburg, 79085 Freiburg, Germany; [email protected] (M.v.W.); [email protected] (S.S.); [email protected] (S.-V.A.) * Correspondence: [email protected] Abstract: Polyphosphates (polyP) are polymers of orthophosphate residues linked by high-energy phosphoanhydride bonds that are important in all domains of life and function in many different processes, including biofilm development. To study the effect of polyP in archaeal biofilm formation, our previously described Sa. solfataricus polyP (−) strain and a new polyP (−) S. acidocaldarius strain generated in this report were used. These two strains lack the polymer due to the overexpression of their respective exopolyphosphatase gene (ppx). Both strains showed a reduction in biofilm formation, decreased motility on semi-solid plates and a diminished adherence to glass surfaces as seen by DAPI (40,6-diamidino-2-phenylindole) staining using fluorescence microscopy. Even though arlB (encoding the archaellum subunit) was highly upregulated in S. acidocardarius polyP (−), no archaellated cells were observed. These results suggest that polyP might be involved in the regulation of the expression of archaellum components and their assembly, possibly by affecting energy availability, phosphorylation or other phenomena.
    [Show full text]
  • The Stability of Lytic Sulfolobus Viruses
    The Stability of Lytic Sulfolobus Viruses A thesis submitted to the Graduate School of the University of Cincinnati In partial fulfillment of The requirements for the degree of Master of Sciences in the Department of Biological Sciences of the College of Arts and Sciences 2017 Khaled S. Gazi B.S. Umm Al-Qura University, 2011 Committee Chair: Dennis W. Grogan, Ph.D. i Abstract Among the three domains of cellular life, archaea are the least understood, and functional information about archaeal viruses is very limited. For example, it is not known whether many of the viruses that infect hyperthermophilic archaea retain infectivity for long periods of time under the extreme conditions of geothermal environments. To investigate the capability of viruses to Infect under the extreme conditions of geothermal environments. A number of plaque- forming viruses related to Sulfolobus islandicus rod-shaped viruses (SIRVs), isolated from Yellowstone National Park in a previous study, were evaluated for stability under different stress conditions including high temperature, drying, and extremes of pH. Screening of 34 isolates revealed a 95-fold range of survival with respect to boiling for two hours and 94-fold range with respect to drying for 24 hours. Comparison of 10 viral strains chosen to represent the extremes of this range showed little correlation of stability with respect to different stresses. For example, three viral strains survived boiling but not drying. On the other hand, five strains that survived the drying stress did not survive the boiling temperature, whereas one strain survived both treatments and the last strain showed low survival of both.
    [Show full text]
  • Insights Into Archaeal Evolution and Symbiosis from the Genomes of a Nanoarchaeon and Its Inferred Crenarchaeal Host from Obsidian Pool, Yellowstone National Park
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Microbiology Publications and Other Works Microbiology 4-22-2013 Insights into archaeal evolution and symbiosis from the genomes of a nanoarchaeon and its inferred crenarchaeal host from Obsidian Pool, Yellowstone National Park Mircea Podar University of Tennessee - Knoxville, [email protected] Kira S. Makarova National Institutes of Health David E. Graham University of Tennessee - Knoxville, [email protected] Yuri I. Wolf National Institutes of Health Eugene V. Koonin National Institutes of Health See next page for additional authors Follow this and additional works at: https://trace.tennessee.edu/utk_micrpubs Part of the Microbiology Commons Recommended Citation Biology Direct 2013, 8:9 doi:10.1186/1745-6150-8-9 This Article is brought to you for free and open access by the Microbiology at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Microbiology Publications and Other Works by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. Authors Mircea Podar, Kira S. Makarova, David E. Graham, Yuri I. Wolf, Eugene V. Koonin, and Anna-Louise Reysenbach This article is available at TRACE: Tennessee Research and Creative Exchange: https://trace.tennessee.edu/ utk_micrpubs/44 Podar et al. Biology Direct 2013, 8:9 http://www.biology-direct.com/content/8/1/9 RESEARCH Open Access Insights into archaeal evolution and symbiosis from the genomes of a nanoarchaeon and its inferred crenarchaeal host from Obsidian Pool, Yellowstone National Park Mircea Podar1,2*, Kira S Makarova3, David E Graham1,2, Yuri I Wolf3, Eugene V Koonin3 and Anna-Louise Reysenbach4 Abstract Background: 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.
    [Show full text]
  • Orthologs of the Small RPB8 Subunit of the Eukaryotic RNA Polymerases
    Biology Direct BioMed Central Discovery notes Open Access Orthologs of the small RPB8 subunit of the eukaryotic RNA polymerases are conserved in hyperthermophilic Crenarchaeota and "Korarchaeota" Eugene V Koonin*1, Kira S Makarova1 and James G Elkins2 Address: 1National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA and 2Microbial Ecology and Physiology Group, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA Email: Eugene V Koonin* - [email protected]; Kira S Makarova - [email protected]; James G Elkins - [email protected] * Corresponding author Published: 14 December 2007 Received: 13 December 2007 Accepted: 14 December 2007 Biology Direct 2007, 2:38 doi:10.1186/1745-6150-2-38 This article is available from: http://www.biology-direct.com/content/2/1/38 © 2007 Koonin et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract : Although most of the key components of the transcription apparatus, and in particular, RNA polymerase (RNAP) subunits, are conserved between archaea and eukaryotes, no archaeal homologs of the small RPB8 subunit of eukaryotic RNAP have been detected. We report that orthologs of RPB8 are encoded in all sequenced genomes of hyperthermophilic Crenarchaeota and a recently sequenced "korarchaeal" genome, but not in Euryarchaeota or the mesophilic crenarchaeon Cenarchaeum symbiosum. These findings suggest that all 12 core subunits of eukaryotic RNAPs were already present in the last common ancestor of the extant archaea.
    [Show full text]
  • Resolution of Carbon Metabolism and Sulfur-Oxidation Pathways of Metallosphaera Cuprina Ar-4 Via Comparative Proteomics
    JOURNAL OF PROTEOMICS 109 (2014) 276– 289 Available online at www.sciencedirect.com ScienceDirect www.elsevier.com/locate/jprot Resolution of carbon metabolism and sulfur-oxidation pathways of Metallosphaera cuprina Ar-4 via comparative proteomics Cheng-Ying Jianga, Li-Jun Liua, Xu Guoa, Xiao-Yan Youa, Shuang-Jiang Liua,c,⁎, Ansgar Poetschb,⁎⁎ aState Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, PR China bPlant Biochemistry, Ruhr University Bochum, Bochum, Germany cEnvrionmental Microbiology and Biotechnology Research Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing, PR China ARTICLE INFO ABSTRACT Article history: Metallosphaera cuprina is able to grow either heterotrophically on organics or autotrophically Received 16 March 2014 on CO2 with reduced sulfur compounds as electron donor. These traits endowed the species Accepted 6 July 2014 desirable for application in biomining. In order to obtain a global overview of physiological Available online 14 July 2014 adaptations on the proteome level, proteomes of cytoplasmic and membrane fractions from cells grown autotrophically on CO2 plus sulfur or heterotrophically on yeast extract Keywords: were compared. 169 proteins were found to change their abundance depending on growth Quantitative proteomics condition. The proteins with increased abundance under autotrophic growth displayed Bioleaching candidate enzymes/proteins of M. cuprina for fixing CO2 through the previously identified Autotrophy 3-hydroxypropionate/4-hydroxybutyrate cycle and for oxidizing elemental sulfur as energy Heterotrophy source. The main enzymes/proteins involved in semi- and non-phosphorylating Entner– Industrial microbiology Doudoroff (ED) pathway and TCA cycle were less abundant under autotrophic growth. Also Extremophile some transporter proteins and proteins of amino acid metabolism changed their abundances, suggesting pivotal roles for growth under the respective conditions.
    [Show full text]
  • Sulfolobus As a Model Organism for the Study of Diverse
    SULFOLOBUS AS A MODEL ORGANISM FOR THE STUDY OF DIVERSE BIOLOGICAL INTERESTS; FORAYS INTO THERMAL VIROLOGY AND OXIDATIVE STRESS by Blake Alan Wiedenheft A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Microbiology MONTANA STATE UNIVERSITY Bozeman, Montana November 2006 © COPYRIGHT by Blake Alan Wiedenheft 2006 All Rights Reserved ii APPROVAL of a dissertation submitted by Blake Alan Wiedenheft This dissertation has been read by each member of the dissertation committee and has been found to be satisfactory regarding content, English usage, format, citations, bibliographic style, and consistency, and is ready for submission to the Division of Graduate Education. Dr. Mark Young and Dr. Trevor Douglas Approved for the Department of Microbiology Dr.Tim Ford Approved for the Division of Graduate Education Dr. Carl A. Fox iii STATEMENT OF PERMISSION TO USE In presenting this dissertation in partial fulfillment of the requirements for a doctoral degree at Montana State University – Bozeman, I agree that the Library shall make it available to borrowers under rules of the Library. I further agree that copying of this dissertation is allowable only for scholarly purposes, consistent with “fair use” as prescribed in the U.S. Copyright Law. Requests for extensive copying or reproduction of this dissertation should be referred to ProQuest Information and Learning, 300 North Zeeb Road, Ann Arbor, Michigan 48106, to whom I have granted “the exclusive right to reproduce and distribute my dissertation in and from microfilm along with the non-exclusive right to reproduce and distribute my abstract in any format in whole or in part.” Blake Alan Wiedenheft November, 2006 iv DEDICATION This work was funded in part through grants from the National Aeronautics and Space Administration Program (NAG5-8807) in support of Montana State University’s Center for Life in Extreme Environments (MCB-0132156), and the National Institutes of Health (R01 EB00432 and DK57776).
    [Show full text]