DOCSLIB.ORG

Production and Folding of Proteins in the Periplasm of Escherichia Coli

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Production and Folding of Proteins in the Periplasm of Escherichia Coli Rageia Elfageih Production and folding of proteins in the periplasm of Escherichia coli Production and folding of proteins in the periplasm of in and folding of proteins Production Rageia Elfageih Escherichia coli Escherichia ISBN 978-91-7911-464-0 Department of Biochemistry and Biophysics Doctoral Thesis in Biochemistry at Stockholm University, Sweden 2021 Production and folding of proteins in the periplasm of Escherichia coli Rageia Elfageih Academic dissertation for the Degree of Doctor of Philosophy in Biochemistry at Stockholm University to be publicly defended on Friday 14 May 2021 at 10.00 online via Zoom, public link is available at the department website. Abstract The Gram-negative bacterium E. coli is the most widely used host for the production of recombinant proteins. Disulfide bond containing recombinant proteins are usually produced in the periplasm of E. coli since in this compartment of the cell - in contrast to the cytoplasm - disulfide bond formation is promoted. To reach the periplasm recombinant proteins have to be translocated across the cytoplasmic membrane by the protein translocation machinery. To obtain sufficient yields of active recombinant protein in the periplasm is always challenging. The Ph.D. studies have aimed at developing strategies to enhance recombinant protein production yields in the periplasm, to better understand what happens when a protein is produced in the periplasm, and to shed light on the protein folding process in the periplasm. It has been shown that evolving translation initiation regions (TIRs) can enhance periplasmic protein production yields of a variety of proteins. Furthermore, it has been shown that the protein translocation machinery can adapt for enhanced periplasmic recombinant protein production. Force profile analysis was used to study co-translational folding of the periplasmic disulfide-bond containing protein alkaline phosphatase (PhoA) in the periplasm. It was shown that folding-induced forces can be transmitted via the nascent chain from the periplasm to the peptidyl transferase center in the ribosome and that PhoA appears to fold co- translationally via disulfide-stabilized folding intermediates. Finally, the S. pneumoniae neuraminidases NanA, NanB, and NanC were produced in E. coli and subsequently isolated. The activity of these neuraminidases was monitored at different pH as well as their oligomeric state was studied. Keywords: Escherichia coli, periplasm, recombinant protein production, disulfide bond containing proteins, translation initiation region, protein translocation machinery, co-translational folding, neuraminidases. Stockholm 2021 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-191529 ISBN 978-91-7911-464-0 ISBN 978-91-7911-465-7 Department of Biochemistry and Biophysics Stockholm University, 106 91 Stockholm PRODUCTION AND FOLDING OF PROTEINS IN THE PERIPLASM OF ESCHERICHIA COLI Rageia Elfageih Production and folding of proteins in the periplasm of Escherichia coli Rageia Elfageih ©Rageia Elfageih, Stockholm University 2021 ISBN print 978-91-7911-464-0 ISBN PDF 978-91-7911-465-7 The cover image is created using BioRender.com. Images and image modifications in comprehensive summary by Rageia Elfageih Printed in Sweden by Universitetsservice US-AB, Stockholm 2021 “Anything you can imagine you can create.” Oprah Winfrey Dedication To my parents and family For their endless love, support, and encouragements List of papers: I. Elfageih R, Karyolaimos A, Kemp G, de Gier J-W, von Heijne G, Kudva R (2020). Cotranslational folding of alkaline phospha- tase in the periplasm of Escherichia coli. Protein Science 29(10):2028–37. II. Karyolaimos A*, Dolata KM*, Antelo-Varela M*, Mestre Borras A, Elfageih R, Sievers S, Becher D, Riedel K and de Gier J-W (2020). Escherichia coli can adapt its protein translocation ma- chinery for enhanced periplasmic recombinant protein produc- tion. Frontiers in Bioengineering and Biotechnology 7: 465. *Shared first author III. MirZadeh K*, Shilling PJ*, Elfageih R, Cumming AJ, Cui HL, Rennig M, Nørholm MHH, Daley DO (2020). Increased produc- tion of periplasmic proteins in Escherichia coli by directed evo- lution of the translation initiation region. Microbial Cell Factories 19(1):85. *Shared first author IV. Elfageih R, de Gier J-W, Daniels R (2021). CharacteriZation of the Streptococcus pneumoniae neuraminidases NanA, NanB, and NanC. Manuscript in preparation Contents Introduction ................................................................................................... 1 1. Gram-negative bacteria .......................................................................... 3 1.1. The cytoplasm ....................................................................................... 3 1.2. The inner membrane .............................................................................. 4 1.3. The periplasm ......................................................................................... 4 1.4. The peptidoglycan layer ......................................................................... 4 1.5. The outer membrane .............................................................................. 5 2. Gram-positive bacteria ........................................................................... 5 3. Biogenesis of cell envelope proteins ....................................................... 5 3.1. The ribosome and the translation initiation region in mRNA ............. 6 3.1.1. Initiation of translation ..................................................... 7 3.1.2. Elongation of translation ................................................... 8 3.1.3. Termination of translation and ribosomal recycling ............. 9 4. Biogenesis of bacterial cell envelope proteins .................................... 10 4.1. Protein targeting .......................................................................... 11 4.2. Protein translocation ..................................................................... 12 4.2.1. Regulation of secA expression ......................................... 13 4.3. Formation of disulfide bonds in the periplasm ................................ 13 5. Neuraminidases ..................................................................................... 16 5.1. Streptococcus pneumoniae and influenza virus neuraminidases ....... 16 5.1.2. Catalytic site characteristics, substrate specificity and product formation .................................................................. 17 6. Recombinant protein production in E. coli ........................................ 19 6.1. Production strains and expression vectors ...................................... 20 6.1.1. The T7 RNA polymerase/promoter .................................. 21 6.1.2. The tac promoter ............................................................ 22 6.1.3. The rhaBAD promoter .................................................... 23 6.1.4. The araBAD promoter .................................................... 24 7. Summaries of chapters I-IV ............................................................. 25 8. Future perspectives ........................................................................... 28 Sammanfattning på svenska ....................................................................... 30 32 ..................................................... ﺺﺨﻠﻤﻟا( ﺔﻐﻠﻟﺎﺑ ﺔﯿﺑﺮﻌﻟا ) Summary in Arabic Acknowledgments ....................................................................................... 33 References .................................................................................................... 35 Introduction Bacteria are single-cell based organisms and they can have different shapes (they can be e.g., spherical or rod-shaped) (1). On average, bacteria are 1 – 2 µm in diameter/length, and their mass (dry weight) can range from 1 – 10 pg (1). The bacterial chromosome is localized in the central region of the cell and bacteria lack a membrane-based nucleus; the region within the bacterial cell containing its genetic information is often referred to as the nucleoid (2). In addition, bacteria can also contain plasmids, which are independently repli- cating pieces of DNA that contain additional genetic information (3). There are Gram-negative and Gram-positive bacteria (4)(Figure 1). Gram- negative bacteria have two membranes, i.e., the inner or cytoplasmic mem- brane and the outer membrane. Between the two membranes is the periplasm, which contains a thin layer of peptidoglycan (5). Figure 1. Schematic representation of the cell envelope of a Gram-negative bacterium and the one of a Gram-positive bacterium. a) The basic setup (from the outside to the inside) of the Gram-negative bacterial cell envelope consists of the outer membrane, the periplasm and the inner/cytoplasmic membrane. In the periplasm, there is a thin layer of peptidoglycan that is anchored to the outer membrane via the lipoprotein Lpp (for the sake of simplicity not specified in the cartoon). The outer membrane contains lipopolysaccharides (LPS) in the outer leaflet and lipids in the inner leaflet. The inner/cytoplasmic membrane consists of a lipid bilayer. Both membrane systems contain (integral) membrane proteins, including lipoproteins. b) In Gram- positive bacteria, the basic setup of the cell envelope (from the outside to the inside) consists of a thick layer of peptidoglycan that contains an anionic polymer called teichoic acid and sur- face proteins are attached to it, and the cytoplasmic membrane. The cytoplasmic membrane 1 consists
Load more

Recommended publications
  • Some English Terms Used in Microbiology 1
    Some English terms used in Microbiology 1 Shapes & Structures General terms Antibiotics and related Bacillus (pl. bacilli) Acid fast (acid fastness) ácido-alcohol resistente Acetylases Capsule Bacterial (adj.) Ampicillin Cell wall pared celular Bacterium (pl., bacteria): Beta-lactamase Coccus (cocci; and hence Staphylococcus, Bench: poyata Beta-lactamic Staphylococci) Biofilm Cephalosporin Core oligosaccharide núcleo oligosacarídico Burner (Bunsen Burner): mechero (Bunsen) Chloramphenicol Cortex Colony: colonia Colistin Fimbria (pl. fimbriae) Coverslip: (vidrio) cubreobjetos D-Cycloserine Flagellum (pl. flagella) Dye colorante DNA-gyrase Glycocalix Eukaryote or eucaryote Erythromycin Lipid A Incubator: estufa de incubación Ethambuthol Lipopolysaccharide Inoculating loop asa de siembra Fluoroquinolone Murein mureína Inoculum (inocula): Gentamicin (formerly gentamycin) Omp: outer membrane major protein proteína To flame: flamear Isoniazide de membrane externa Flask (Erlenmeyer flask): matraz Methicillin Outer membrane membrana externa Volumetric flask: matraz aforado Methylases PAMP (pathogen associated molecular pattern): Microorganism Nalidixic acid patrón molecular asociado a patógeno Motility movilidad Penicillin Peptidoglycan peptidoglucano Mycoplasm Penicillin binding protein (PBP) Periplasm periplasma Negative staining tinción negativa Phosphonomycin Periplasmic space espacio periplásmico Petri dish: placa de Petri Phosphorylases Permeability barrier barrera de permeabilidad Prokaryote or procaryote Polymyxin Pilus (pl. pili) Rack:
    [Show full text]
  • The Regulation of Arsenic Metabolism in Rhizobium Sp. NT-26
    The regulation of arsenic metabolism in Rhizobium sp. NT-26 Paula Corsini Madeira University College London Thesis submitted for the degree of Doctor of Philosophy 2016 I, Paula Corsini Madeira, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Date: Signed: II Abstract Arsenic (As) is a toxic metalloid and a major contaminant in terrestrial and aquatic environments. The two soluble forms, arsenite (AsIII) and arsenate (AsV) are toxic to most organisms. A range of phylogenetically distant bacteria are able to oxidize AsIII to the less toxic form, arsenate AsV using the periplasmic arsenite oxidase (AioBA). The two-component signal transduction system AioS/AioR and the AsIII-binding periplasmic protein AioX are required for AsIII oxidation and are involved in the transcriptional regulation of the aioBA operon. Most AsIII oxidisers can also reduce AsV to AsIII via the As (Ars) resistance system. The focus of this work was to understand the regulation of genes involved in AsIII oxidation and As resistance together with those involved in phosphate metabolism in the facultative chemolithoautotrophic AsIII oxidiser NT-26 grown under different conditions. Gene expression was studied by quantitative PCR in cells grown heterotrophically with and without AsIII or AsV in late-log and stationary phases. qPCR was optimised and suitable reference genes were chosen. The expression of genes involved in phosphate transport, sensing As and the genes aioX, aioS, aioR (AsIII-sensing and regulation) and aioB, aioA (AsIII oxidation) and cytC (cytochrome c) were also analysed in NT-26 grown heterotrophically in the presence or absence of AsIII or AsV at different growth stages (i.e., late-log and stationary phases).
    [Show full text]
  • Cell Structure and Function in the Bacteria and Archaea
    4 Chapter Preview and Key Concepts 4.1 1.1 DiversityThe Beginnings among theof Microbiology Bacteria and Archaea 1.1. •The BacteriaThe are discovery classified of microorganismsinto several Cell Structure wasmajor dependent phyla. on observations made with 2. theThe microscope Archaea are currently classified into two 2. •major phyla.The emergence of experimental 4.2 Cellscience Shapes provided and Arrangements a means to test long held and Function beliefs and resolve controversies 3. Many bacterial cells have a rod, spherical, or 3. MicroInquiryspiral shape and1: Experimentation are organized into and a specific Scientificellular c arrangement. Inquiry in the Bacteria 4.31.2 AnMicroorganisms Overview to Bacterialand Disease and Transmission Archaeal 4.Cell • StructureEarly epidemiology studies suggested how diseases could be spread and 4. Bacterial and archaeal cells are organized at be controlled the cellular and molecular levels. 5. • Resistance to a disease can come and Archaea 4.4 External Cell Structures from exposure to and recovery from a mild 5.form Pili allowof (or cells a very to attach similar) to surfacesdisease or other cells. 1.3 The Classical Golden Age of Microbiology 6. Flagella provide motility. Our planet has always been in the “Age of Bacteria,” ever since the first 6. (1854-1914) 7. A glycocalyx protects against desiccation, fossils—bacteria of course—were entombed in rocks more than 3 billion 7. • The germ theory was based on the attaches cells to surfaces, and helps observations that different microorganisms years ago. On any possible, reasonable criterion, bacteria are—and always pathogens evade the immune system. have been—the dominant forms of life on Earth.
    [Show full text]
  • Gram Staining Staining & Acid Fast & Acid Fast Staining
    GRAM STAINING && AACCIIDD FFAASSTT SSTTAAIINNIINNGG Dr. R. Haritha Lecturer in Biotechnology Visakha Government Degree College for Women Visakhapatnam Staining Stains Principle Types Staining methods Smear Preparation Smear- Distribution of Bacterial cells on a slide Objective-To kill the microorganism & fix bacteria Method- Air Dry, Heat Fixation GRAM STAINING Gram staining is most widely Hans Christian Joachim Gram used differential staining in Microbiology. It classifies bacteria into two major groups: Gram positive Gram negative Appears violet Appears red after Gram’s stain after Gram’s stain REAGENTS 1. CRYSTAL VIOLET Primary stain Violet colored, stains all micro-organism 2. GRAM IODINE Mordant Forms Crystal violet iodine complexes 3. DECOLORIZER Acetone + Methanol Removes Crystal violet iodine complex from thin peptidoglycan layers 4. GRAM SAFRANINE Counter stain Red colored Step 1: Crystal Violet Step 2: Gram’’s Iodine Step 3: Decolorization (Aceton-Alcohol) Step 4: Safranin Red 7 PRINCIPLE : Composition of the cell wall Gram-positive bacteria Cell wall has a thick peptidoglycan layer The Crystal Violet stain gets trapped into this layer and the bacteria turned purple. Retains the color of the primary stain (crystal violet) after decolorization with alcohol. Gram-negative bacteria Cell wall has a thin peptidoglycan layer that does not retain crystal violet stain. Cell wall has a thick lipid layer which dissolves easily upon decoulorization with Aceton-Alcohol. Therefore, cells will be counterstained with safranin and turned red. Gram’s +ve Bacteria Gram’s -ve Bacteria 9 RESULT Bacteria that manage to keep the original purple are called Gram positive. Bacteria that lose the original purple dye and can therefore take up the second red dye are called Gram negative APPLICATIONS 1.Rapid preliminary diagnosis of diseases such as Bacterial meningitis.
    [Show full text]
  • Metabolic Flexibility of Enigmatic SAR324 Revealed Through
    bs_bs_banner Environmental Microbiology (2014) 16(1), 304–317 doi:10.1111/1462-2920.12165 Metabolic flexibility of enigmatic SAR324 revealed through metagenomics and metatranscriptomics Cody S. Sheik,1 Sunit Jain1 and Gregory J. Dick1,2,3* deep ocean (ArÍstegui et al., 2009) is thought to constrain 1Department of Earth and Environmental Sciences and heterotrophic microbial activity. However, recent identifi- 2Center for Computational Medicine and Bioinformatics cation of carbon fixation genes in the dark ocean (> 200 m and 3Department of Ecology and Evolutionary Biology, below surface) (Swan et al., 2011) underscores auto- University of Michigan, Ann Arbor, MI 48109, USA. trophy as a metabolic strategy for many dark ocean- associated micro-organisms (Karl et al., 1984). Because autotrophy is energetically taxing (Hügler and Sievert, Summary 2011), organisms must couple carbon fixation to an Chemolithotrophy is a pervasive metabolic lifestyle electron-donating reaction such as the oxidation of for microorganisms in the dark ocean. The SAR324 ammonia, nitrite, sulfur, iron, methane, hydrogen or group of Deltaproteobacteria is ubiquitous in the formate. Yet such electron donors required to power ocean and has been implicated in sulfur oxidation and carbon fixation in the deep ocean are scarce (Reinthaler carbon fixation, but also contains genomic signatures and Van, 2010), suggesting that mixotrophy (Sorokin, of C1 utilization and heterotrophy. Here, we recon- 2003; Moran et al., 2004; Dick et al., 2008) may be nec- structed the metagenome and metatranscriptome of a essary for micro-organisms to persist until preferred population of SAR324 from a hydrothermal plume and energy sources are encountered. surrounding waters in the deep Gulf of California to Within the deep ocean, hydrothermal vents are crucial gain insight into the genetic capability and transcrip- sources of reduced compounds such as sulfur, ammonia, tional dynamics of this enigmatic group.
    [Show full text]
  • Perchlorate Bioremediation: Controlling Media Loss in Ex-Situ
    UNLV Theses, Dissertations, Professional Papers, and Capstones 5-1-2016 Perchlorate Bioremediation: Controlling Media Loss in Ex-Situ Fluidized Bed Reactors and In-Situ Biological Reduction by Slow-Release Electron Donor Sichu Shrestha University of Nevada, Las Vegas, [email protected] Follow this and additional works at: https://digitalscholarship.unlv.edu/thesesdissertations Part of the Civil Engineering Commons, and the Environmental Engineering Commons Repository Citation Shrestha, Sichu, "Perchlorate Bioremediation: Controlling Media Loss in Ex-Situ Fluidized Bed Reactors and In-Situ Biological Reduction by Slow-Release Electron Donor" (2016). UNLV Theses, Dissertations, Professional Papers, and Capstones. 2744. https://digitalscholarship.unlv.edu/thesesdissertations/2744 This Dissertation is brought to you for free and open access by Digital Scholarship@UNLV. It has been accepted for inclusion in UNLV Theses, Dissertations, Professional Papers, and Capstones by an authorized administrator of Digital Scholarship@UNLV. For more information, please contact [email protected]. PERCHLORATE BIOREMEDIATION: CONTROLLING MEDIA LOSS IN EX-SITU FLUIDIZED BED REACTORS AND IN-SITU BIOLOGICAL REDUCTION BY SLOW- RELEASE ELECTRON DONOR By Sichu Shrestha Bachelor of Engineering in Civil Engineering, Institute of Engineering, Tribhuvan University, Nepal 2004 Master of Science in Civil and Environmental Engineering Carnegie Mellon University 2011 A dissertation submitted in partial fulfillment of the requirements for the Doctor of Philosophy-
    [Show full text]
  • Metapangenomics of the Oral Microbiome Provides Insights Into Habitat Adaptation and Cultivar Diversity Daniel R
    Utter et al. Genome Biology (2020) 21:293 https://doi.org/10.1186/s13059-020-02200-2 RESEARCH Open Access Metapangenomics of the oral microbiome provides insights into habitat adaptation and cultivar diversity Daniel R. Utter1* , Gary G. Borisy2, A. Murat Eren3,4, Colleen M. Cavanaugh1* and Jessica L. Mark Welch3* * Correspondence: dutter@g. harvard.edu; [email protected]. Abstract edu; [email protected] 1Department of Organismic and Background: The increasing availability of microbial genomes and environmental Evolutionary Biology, Harvard shotgun metagenomes provides unprecedented access to the genomic differences University, Cambridge, MA 02138, within related bacteria. The human oral microbiome with its diverse habitats and USA 3The Josephine Bay Paul Center for abundant, relatively well-characterized microbial inhabitants presents an opportunity Comparative Molecular Biology and to investigate bacterial population structures at an ecosystem scale. Evolution, Marine Biological Laboratory, Woods Hole, MA 02543, Results: Here, we employ a metapangenomic approach that combines public USA genomes with Human Microbiome Project (HMP) metagenomes to study the Full list of author information is diversity of microbial residents of three oral habitats: tongue dorsum, buccal mucosa, available at the end of the article and supragingival plaque. For two exemplar taxa, Haemophilus parainfluenzae and the genus Rothia, metapangenomes reveal distinct genomic groups based on shared genome content. H. parainfluenzae genomes separate into three distinct subgroups with differential abundance between oral habitats. Functional enrichment analyses identify an operon encoding oxaloacetate decarboxylase as diagnostic for the tongue-abundant subgroup. For the genus Rothia, grouping by shared genome content recapitulates species-level taxonomy and habitat preferences. However, while most R.
    [Show full text]
  • Fate of Arsenate Following Arsenite Oxidation in Agrobacterium Tumefaciens GW4
    bs_bs_banner Environmental Microbiology (2014) doi:10.1111/1462-2920.12465 Fate of arsenate following arsenite oxidation in Agrobacterium tumefaciens GW4 Qian Wang,1 Dong Qin,1 Shengzhe Zhang,1 Introduction Lu Wang,1 Jingxin Li,1 Christopher Rensing,2 Arsenic (As) is the most common toxic element in the Timothy R. McDermott3** and Gejiao Wang1* environment, ranking first on the U.S. Superfund List 1State Key Laboratory of Agricultural Microbiology, of Hazardous Substances and is responsible for mass College of Life Science and Technology, Huazhong poisoning throughout Asia (Chakraborti et al., 2009; Agricultural University, Wuhan 430070, China. Rodríguez-Lado et al., 2013). In the environment, trans- 2Department of Plant and Environmental Sciences, port, bioavailability and accumulation of As in biological University of Copenhagen, Frederiksberg, Denmark. endpoints is dependent on chemical speciation, with 3Department of Land Resources and Environmental arsenite (AsIII) and arsenate (AsV) being the primary Sciences, Montana State University, Bozeman, MT arsenicals found in the environment. Microbial redox 59717, USA. transformations are recognized as being important con- tributors to equilibrium levels of AsIII and AsV (Cullen and Summary Reimer, 1989; Inskeep et al., 2001; Oremland and Stolz, 2005; Stolz et al., 2006). The fate of arsenate (AsV) generated by microbial Microbial AsIII oxidation and AsV reduction both serve arsenite (AsIII) oxidation is poorly understood. as detoxification and/or energy-generating functions, Agrobacterium tumefaciens wild-type strain (GW4) depending on the organism (Stolz et al., 2006; Páez- was studied to determine how the cell copes with AsV Espino et al., 2009). Reasonable models exist for under- generated in batch culture.
    [Show full text]
  • Extreme Environments and High-Level Bacterial Tellurite Resistance
    microorganisms Review Extreme Environments and High-Level Bacterial Tellurite Resistance Chris Maltman 1,* and Vladimir Yurkov 2 1 Department of Biology, Slippery Rock University, Slippery Rock, PA 16001, USA 2 Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; [email protected] * Correspondence: [email protected]; Tel.: +724-738-4963 Received: 28 October 2019; Accepted: 20 November 2019; Published: 22 November 2019 Abstract: Bacteria have long been known to possess resistance to the highly toxic oxyanion tellurite, most commonly though reduction to elemental tellurium. However, the majority of research has focused on the impact of this compound on microbes, namely E. coli, which have a very low level of resistance. Very little has been done regarding bacteria on the other end of the spectrum, with three to four orders of magnitude greater resistance than E. coli. With more focus on ecologically-friendly methods of pollutant removal, the use of bacteria for tellurite remediation, and possibly recovery, further highlights the importance of better understanding the effect on microbes, and approaches for resistance/reduction. The goal of this review is to compile current research on bacterial tellurite resistance, with a focus on high-level resistance by bacteria inhabiting extreme environments. Keywords: tellurite; tellurite resistance; extreme environments; metalloids; bioremediation; biometallurgy 1. Introduction Microorganisms possess a wide range of extraordinary abilities, from the production of bioactive molecules [1] to resistance to and transformation of highly toxic compounds [2–5]. Of great interest are bacteria which can convert the deleterious oxyanion tellurite to elemental tellurium (Te) through reduction. Currently, research into bacterial interactions with tellurite has been lagging behind investigation of the oxyanions of other metals such as nickel (Ni), molybdenum (Mo), tungsten (W), iron (Fe), and cobalt (Co).
    [Show full text]
  • Population Structure and Dynamics of Polyphosphate Accumulating Organisms in a Communal Wastewater Treatment Plant
    Population structure and dynamics of polyphosphate accumulating organisms in a communal wastewater treatment plant DISSERTATION zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Fakultät Mathematik und Naturwissenschaften der Technischen Universität Dresden von Dipl. Biol. Susanne Günther geboren am 26.05.1983 in Leipzig Leipzig, 2011 Dissertation der Technischen Universität Dresden Datum der mündlichen Prüfungen: 12.12.2011 Gutachter: Prof. Dr. Isolde Röske Prof. Dr. Thomas Bley II Table of contents Table of contents Abbreviations VI List of Figures VIII List of Tables XI Introduction 1 1. Eutrophication and the phosphorous cycle 1 2. Wastewater treatment and phosphate elimination 3 2.1 General wastewater treatment 3 2.2 Phosphate removal 4 3. Polyphosphates and organisms responsible for the phosphate elimination 4 3.1 Ubiquitous distribution of polyphosphates and their cellular localization 5 3.2 Molecular composition of polyphosphate granules 5 3.3 Enzymes involved in polyphosphate synthesis and degradation 5 3.4 Polyphosphate functions 6 3.5 Probable evolutionary functions of polyphosphates 7 4. Microorganisms participating in the EBPR Process 7 5. Detection of PAOs 8 5.1 Polyphosphate granule visualization and detection of possible PAOs 8 5.2 Combinations of the afore mentioned techniques and their application 9 6. Phosphate recycling from wastewater 10 Aim of this study 11 Material and Methods 12 1. Microorganisms and culture conditions 12 1.1 Pure cultures 12 1.2 Mixed cultures 14 1.2.1 Batch experiments 14 1.2.2 Bench top bioreactor experiments 14 1.2.3 Direct sampling from WWTP Eilenburg 14 2. Cell fixation 15 2.1 Test of fixation efficiency 16 2.2 Polyphosphate staining experiments and WWTP samples 16 3.
    [Show full text]
  • The Role of Outer Membrane Proteins of Porphyromonas Gingivalis in Host-Pathogen Interactions Kathryn Lucy Naylor
    The Role of Outer Membrane Proteins of Porphyromonas gingivalis in Host-Pathogen Interactions By: Kathryn Lucy Naylor A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy The University of Sheffield Faculty of Medicine, Dentistry and Health School of Clinical Dentistry 23 / 08 / 2016 Summary The keystone periodontal pathogen, P. gingivalis, is a Gram-negative oral anaerobe that is strongly implicated as the prime etiological agent in the initiation and progression of periodontal disease. P. gingivalis contains a plethora of virulence factors, including fimbriae, proteases, lipopolysaccharides and outer membrane proteins that contribute to the pathogenesis of the disease. The bacterium also displays the ability to interact with host cells through the adherence and invasion both in vitro and in vivo. In this thesis new light has been shed on the role of the heterotrimeric outer membrane protein A (OmpA). Through the creation of ompA mutants and recombinant complementation plasmids, alongside the use of standard antibiotic protection assays in an oral epithelial cell line, this research has demonstrated the importance of OmpA, specifically the OmpA2 subunit, in the invasion of the host and in the ability of the bacteria to form biofilms. Structural analysis of the protein identified extracellular loops, which when synthetic versions were applied to host cells, demonstrated successful interruption of wild-type P. gingivalis adherence and invasion of the host, indicating a direct interaction of OmpA2 with oral epithelial cells. In particular, this research demonstrates that OmpA2-loop 4 plays an important role in the interaction with the host through significantly increased binding to host cells when applied to fluorescent latex beads.
    [Show full text]
  • Molecular Analyses of Changes Induced in the Microbial Populations of Murine Colon After As(III) Exposure Rishu Dheer
    Duquesne University Duquesne Scholarship Collection Electronic Theses and Dissertations Summer 2011 Molecular Analyses of Changes Induced in the Microbial Populations of Murine Colon After As(III) Exposure Rishu Dheer Follow this and additional works at: https://dsc.duq.edu/etd Recommended Citation Dheer, R. (2011). Molecular Analyses of Changes Induced in the Microbial Populations of Murine Colon After As(III) Exposure (Doctoral dissertation, Duquesne University). Retrieved from https://dsc.duq.edu/etd/484 This Immediate Access is brought to you for free and open access by Duquesne Scholarship Collection. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Duquesne Scholarship Collection. For more information, please contact [email protected]. MOLECULAR ANALYSES OF CHANGES INDUCED IN THE MICROBIAL POPULATIONS OF MURINE COLON AFTER As (III) EXPOSURE A Dissertation Submitted to Bayer School of Natural and Environmental Sciences Duquesne University In partial fulfillment of the requirements for the degree of Doctor of Philosophy By Rishu Dheer August, 2011 Copyright by Rishu Dheer 2011 MOLECULAR ANALYSES OF CHANGES INDUCED IN THE MICROBIAL POPULATIONS OF MURINE COLON AFTER As (III) EXPOSURE By Rishu Dheer Approved June 14, 2011 ________________________________ ________________________________ Dr. John F. Stolz Dr. Aaron Barchowsky Director, Center of Environmental Associate Professor of Biology Research and Education (Committee Member) Professor of Biology (Committee Chair) ________________________________
    [Show full text]