DOCSLIB.ORG

Domesticating Predatory Bacteria for Biotechnological Tools

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Domesticating Predatory Bacteria for Biotechnological Tools UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE CIENCIAS QUÍMICAS Departamento de Bioquímica y Biología Molecular TESIS DOCTORAL Domesticating predatory bacteria for biotechnological tools Bacterias depredadoras para ser aplicadas en procesos biotecnológicos MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Cristina Herencias Rodríguez Directora Mª Auxiliadora Prieto Jiménez Madrid Ed. electrónica 2019 © Cristina Herencias Rodríguez, 2019 UNIVERSIDAD COMPLUTENSE DE MADRID Facultad de Ciencias Químicas Departamento de Bioquímica y Biología Molecular DOMESTICATING PREDATORY BACTERIA FOR BIOTECHNOLOGICAL TOOLS BACTERIAS DEPREDADORAS PARA SER APLICADAS EN PROCESOS BIOTECNOLÓGICOS Tesis doctoral Cristina Herencias Rodríguez Directora Mª Auxiliadora Prieto Jiménez Madrid, 2019 1 UNIVERSIDAD COMPLUTENSE DE MADRID Facultad de Ciencias Químicas Departamento de Bioquímica y Biología Molecular DOMESTICATING PREDATORY BACTERIA FOR BIOTECHNOLOGICAL TOOLS BACTERIAS DEPREDADORAS PARA SER APLICADAS EN PROCESOS BIOTECNOLÓGICOS Tesis doctoral Cristina Herencias Rodríguez Directora María Auxiliadora Prieto Jiménez Consejo Superior de Investigaciones Científicas – CSIC Centro de Investigaciones Biológicas - CIB Madrid, 2019 3 And those who were seen dancing were thought to be insane by those who couldn’t hear the music. Aquellos que eran vistos bailando, eran considerados locos por quienes no podían escuchar la música. F. Nietzsche 5 ACKNOWLEDGEMENTS First and foremost, I would like to express my wide and sincere gratitude to my research supervisor, Dr. Mª Auxiliadora Prieto Jiménez, for giving me the opportunity to research and providing invaluable guidance throughout this thesis. Her dynamism, vision, and motivation have deeply inspired me during these years. I would like to acknowledge Dr. Aljoscha Wahl for accepting me into his research group in TUDelft and leading me to work on the fascinating field of metabolism. I would also like to thank Prof. José Luis García, Dr. Eduardo Díaz, Dr. Pedro García, and Prof. Ernesto García for their constructive advice during these years. Thanks to Virginia Martínez and Juan Nogales for introducing me to the interesting fields of predatory bacteria and metabolism. I would also like to thank to Rosa del Campo for her enthusiasm with which she understands the microbiology and for her fruitful advices. I am indebted to the institutions that have financially supported this work: Ministerio de Economía y Competitividad and EMBO which supported over FPI fellowship the accomplishment of this Ph.D. Thesis at CIB and the short stay at Delft University of Technology. I appreciate the work of CIB facilities such as microscopy, photography and technical support team, as well as ICTP facilities (SEM). I would like to give special thanks to Natalia, Vir, Sergio, Gonn, Fran and Roberto, without whom it would not be possible. To Elena and Edu for their inconditional support whatever happens. Wilson, thanks for listening to my music and making me dance. Lastly, I would like to thank my family for all the support during my whole life. Special thanks to Jose, who design the beautiful cover of this book. To my mum and my grandfather who always encourage me to get my dreams. 7 INDEX I. ABBREVIATIONS .................................................................................................................. 13 II. SUMMARY ........................................................................................................................... 15 III. RESUMEN ............................................................................................................................. 19 IV. GENERAL INTRODUCTION ................................................................................................... 23 1. PREDATION: A GENERAL OVERVIEW ............................................................................... 23 2. BACTERIAL PREDATION ................................................................................................... 24 3. BDELLOVIBRIO AND LIKE ORGANISMS ............................................................................ 27 4. BDELLOVIBRIO BACTERIOVORUS HD100 ......................................................................... 28 4.1 Prey range and predatory efficiency ....................................................................... 30 4.2 Epibiotic predation by B. bacteriovorus .................................................................. 31 4.3 Resistance to B. bacteriovorus ................................................................................ 32 5. HOST-INDEPENDENT B. BACTERIOVORUS ....................................................................... 32 6. BDELLOVIBRIO AS A TOOL FOR BIOTECHNOLOGICAL APPLICATIONS ............................. 34 6.1 B. bacteriovorus as a source of biocatalysts............................................................ 34 6.2 Bdellovibrio strains as a living antibiotics ................................................................ 34 6.3 Bdellovibrio strains as a biocontrol agents .............................................................. 35 6.4 Genetic tools for B. bacteriovorus ........................................................................... 36 V. MOTIVATIONS AND AIMS .................................................................................................... 39 VI. COMMON MATERIALS AND METHODS ............................................................................... 41 1. GROWTH CONDITIONS OF B. BACTERIOVORUS HD100 .................................................. 41 2. B. BACTERIOVORUS HD100 AND PREY VIABILITY CALCULATION .................................... 41 3. BIOMASS CALCULATION .................................................................................................. 42 4. PHASE CONTRAST AND FLUORESCENCE MICROSCOPIC TECHNIQUES ........................... 42 5. SCANNING ELECTRON AND TRANSMISSION ELECTRON MICROSCOPIC TECHNIQUES ... 43 6. STATISTICAL ANALYSES .................................................................................................... 43 RESULTS ....................................................................................................................................... 45 VII. COMPREHENSIVE ANALYSIS OF THE PREDATORY BACTERIA BDELLOVIBRIO BACTERIOVORUS THROUGH A GENOME SCALE METABOLIC MODEL ........................................ 47 1. INTRODUCTION ............................................................................................................... 49 2. MATERIALS AND METHODS ............................................................................................ 51 2.1 General concepts in the metabolic reconstruction ................................................. 51 2.2 Genome-scale metabolic network reconstruction: iCH457 .................................... 54 2.3 Biomass Function .................................................................................................... 54 9 2.4 Reaction essentiality analysis .................................................................................. 56 2.5 Generation of condition-specific models: iCHAP and iCHGP .................................. 56 2.6 Software .................................................................................................................. 57 3. RESULTS ........................................................................................................................... 59 3.1 Characteristics of B. bacteriovorus metabolic reconstruction ................................ 59 3.2 Model-driven assessment of auxotrophies and biomass building block transport systems highlight the predatory lifestyle of B. bacteriovorus ................................. 61 3.3 iCH457 exhibits high accuracy by predicting physiological states of B. bacteriovorus under different nutrients scenarios ........................................................................ 62 3.4 Reaction essentiality towards understanding the predator´s lifestyle ................... 64 3.5 Analysis of the predator´s lifestyle using condition-specific models: Attack Phase (iCHAP) and Growth Phase (iCHGP) models ............................................................ 66 VIII. DEVELOPMENT OF AN ACTIVE AND FUNCTIONAL AXENIC CULTURE OF B. BACTERIOVORUS HD100 ............................................................................................................. 71 1. INTRODUCTION ............................................................................................................... 73 2. MATERIALS AND METHODS ............................................................................................ 75 2.1 Strain media and axenic growth conditions ............................................................ 75 2.2 Quantification of the genome number of B. bacteriovorus .................................... 75 2.3 Amplification of DNA and hit locus sequencing ...................................................... 76 2.4 Analysis of extracellular metabolites by GC-TOF-MS .............................................. 77 2.5 Intracellular ATP measurements of B. bacteriovorus cells growing in rich medium………………………………………………………………………………………………………………79 2.6 Consumption, production rate and growth rate calculations in BdQ10 cells ......... 79 3. RESULTS ........................................................................................................................... 81 3.1 Feasibility of developing axenic cultures of B. bacteriovorus HD100: Quantification
Load more

Recommended publications
  • Genomic Signatures of Predatory Bacteria
    The ISME Journal (2013) 7, 756–769 & 2013 International Society for Microbial Ecology All rights reserved 1751-7362/13 www.nature.com/ismej ORIGINAL ARTICLE By their genes ye shall know them: genomic signatures of predatory bacteria Zohar Pasternak1, Shmuel Pietrokovski2, Or Rotem1, Uri Gophna3, Mor N Lurie-Weinberger3 and Edouard Jurkevitch1 1Department of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Rehovot, Israel; 2Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel and 3Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel Predatory bacteria are taxonomically disparate, exhibit diverse predatory strategies and are widely distributed in varied environments. To date, their predatory phenotypes cannot be discerned in genome sequence data thereby limiting our understanding of bacterial predation, and of its impact in nature. Here, we define the ‘predatome,’ that is, sets of protein families that reflect the phenotypes of predatory bacteria. The proteomes of all sequenced 11 predatory bacteria, including two de novo sequenced genomes, and 19 non-predatory bacteria from across the phylogenetic and ecological landscapes were compared. Protein families discriminating between the two groups were identified and quantified, demonstrating that differences in the proteomes of predatory and non-predatory bacteria are large and significant. This analysis allows predictions to be made, as we show by confirming from genome data an over-looked bacterial predator. The predatome exhibits deficiencies in riboflavin and amino acids biosynthesis, suggesting that predators obtain them from their prey. In contrast, these genomes are highly enriched in adhesins, proteases and particular metabolic proteins, used for binding to, processing and consuming prey, respectively.
    [Show full text]
  • Characterization of Nudix Hydrolases: a Utilitarian
    CHARACTERIZATION OF NUDIX HYDROLASES: A UTILITARIAN SUPERFAMILY OF ENZYMES By Andres Hernandez de la Peña A dissertation submitted to the Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland August, 2015 Abstract The present work details the structural and enzymatic characterization of Nudix hydrolases from three different organisms – Bdellovibrio bacteriovorus, Mycobacterium tuberculosis, and Tetrahymena thermophila. Each of these Nudix enzymes presents unique questions about their physiological function within their organism which are answered with a combination of structural biology, genetic manipulation, enzyme kinetics, and a wide range of protein assays. We demonstrate that RenU, from M. tuberculosis, is part of Redox Homeostasis Control System (RHOCS), which senses and regulates NADH concentrations. This control systems involves two other proteins, the serine/threonine protein kinase G (pknG) and the L13 ribosomal subunits, without which the bacterium fails to evade lysosomal delivery and falls prey to the oxidative arsenal of the macrophage host. Bd-NDPSase, a Nudix enzyme encoded by the B. bacteriovorus gene BD3179, localizes to the periplasmic space of the bacterium and hydrolyses at least four nucleoside diphosphate sugars in vitro. Through atomic-resolution models from X-ray diffraction, we identified a motif that differentiates this hydrolase from the similar, but more substrate specific, ADP- ribose hydrolase from E. coli. Lastly, we show that Nud1p from T. thermophila is a member of the Ezl1p complex, the histone methyltransferase Polycomb Group homologue of this protozoan. With the use of in vitro enzymatic assays we show, in addition, that Nu1dp hydrolyses CoA preferentially over acetyl-CoA and other nucleoside derivatives.
    [Show full text]
  • There Ae Currently Three Fully Sequenced Deltaproteobacteria
    עבודת גמר )תזה( לתואר Thesis for the degree דוקטור לפילוסופיה Doctor of Philosophy מוגשת למועצה המדעית של Submitted to the Scientific Council of the מכון ויצמן למדע Weizmann Institute of Science רחובות, ישראל Rehovot, Israel מאת By אופיר אבידן Ofir Avidan התגובה הראשונית של Bdellovibrio לטרף Early response of Bdellovibrio to its prey מנחים: :Advisors פרופסור שמואל פיטרוקובסקי )מכון Professor Shmuel Pietrokovski וויצמן( (WIS) פרופסור אדוארד יורקביץ Professor Edouard Jurkevitch )האוניברסיטה העברית בירושלים( The Hebrew University of) Jerusalem) חודש ושנה עבריים Month and Year שבט, תשע"ו January, 2016 Table of contents 1. List of abbreviations .................................................................................... 2 2. Abstract................................................................................................................ 3 4 ...................................................................................................................... תקציר .3 4. Introduction ...................................................................................................... 5 5. Research aims ................................................................................................ 12 6. Materials and methods ............................................................................ 13 7. Results ............................................................................................................... 18 7.1 Genetic manipulations in Bdellovibrio ................................................................
    [Show full text]
  • New 16S Rrna Primers to Uncover Bdellovibrio and Like Organisms Diversity and Abundance Jade Ezzedine, Cécile Chardon, Stéphan Jacquet
    New 16S rRNA primers to uncover Bdellovibrio and like organisms diversity and abundance Jade Ezzedine, Cécile Chardon, Stéphan Jacquet To cite this version: Jade Ezzedine, Cécile Chardon, Stéphan Jacquet. New 16S rRNA primers to uncover Bdellovibrio and like organisms diversity and abundance. Journal of Microbiological Methods, Elsevier, 2020, 10.1016/j.mimet.2020.105996. hal-02935301 HAL Id: hal-02935301 https://hal.inrae.fr/hal-02935301 Submitted on 10 Sep 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Journal of Microbiological Methods 175 (2020) 105996 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth New 16S rRNA primers to uncover Bdellovibrio and like organisms diversity T and abundance ⁎ Jade A. Ezzedine, Cécile Chardon, Stéphan Jacquet Université Savoie Mont-Blanc, INRAE, UMR CARRTEL, Thonon-les-Bains, France ARTICLE INFO ABSTRACT Keywords: Appropriate use and specific primers are important in assessing the diversity and abundance of microbial groups Bdellovibrio and like organisms of interest. Bdellovibrio and like organisms (BALOs), that refer to obligate Gram-negative bacterial predators of Primer design other Gram-negative bacteria, evolved in terms of taxonomy and classification over the past two decades.
    [Show full text]
  • Association of Gene Ontology Categories with Decay Rate for Hepg2 Experiments These Tables Show Details for All Gene Ontology Categories
    Supplementary Table 1: Association of Gene Ontology Categories with Decay Rate for HepG2 Experiments These tables show details for all Gene Ontology categories. Inferences for manual classification scheme shown at the bottom. Those categories used in Figure 1A are highlighted in bold. Standard Deviations are shown in parentheses. P-values less than 1E-20 are indicated with a "0". Rate r (hour^-1) Half-life < 2hr. Decay % GO Number Category Name Probe Sets Group Non-Group Distribution p-value In-Group Non-Group Representation p-value GO:0006350 transcription 1523 0.221 (0.009) 0.127 (0.002) FASTER 0 13.1 (0.4) 4.5 (0.1) OVER 0 GO:0006351 transcription, DNA-dependent 1498 0.220 (0.009) 0.127 (0.002) FASTER 0 13.0 (0.4) 4.5 (0.1) OVER 0 GO:0006355 regulation of transcription, DNA-dependent 1163 0.230 (0.011) 0.128 (0.002) FASTER 5.00E-21 14.2 (0.5) 4.6 (0.1) OVER 0 GO:0006366 transcription from Pol II promoter 845 0.225 (0.012) 0.130 (0.002) FASTER 1.88E-14 13.0 (0.5) 4.8 (0.1) OVER 0 GO:0006139 nucleobase, nucleoside, nucleotide and nucleic acid metabolism3004 0.173 (0.006) 0.127 (0.002) FASTER 1.28E-12 8.4 (0.2) 4.5 (0.1) OVER 0 GO:0006357 regulation of transcription from Pol II promoter 487 0.231 (0.016) 0.132 (0.002) FASTER 6.05E-10 13.5 (0.6) 4.9 (0.1) OVER 0 GO:0008283 cell proliferation 625 0.189 (0.014) 0.132 (0.002) FASTER 1.95E-05 10.1 (0.6) 5.0 (0.1) OVER 1.50E-20 GO:0006513 monoubiquitination 36 0.305 (0.049) 0.134 (0.002) FASTER 2.69E-04 25.4 (4.4) 5.1 (0.1) OVER 2.04E-06 GO:0007050 cell cycle arrest 57 0.311 (0.054) 0.133 (0.002)
    [Show full text]
  • Growth and Gene Expression Profile Analyses of Endometrial Cancer Cells Expressing Exogenous PTEN
    [CANCER RESEARCH 61, 3741–3749, May 1, 2001] Growth and Gene Expression Profile Analyses of Endometrial Cancer Cells Expressing Exogenous PTEN Mieko Matsushima-Nishiu, Motoko Unoki, Kenji Ono, Tatsuhiko Tsunoda, Takeo Minaguchi, Hiroyuki Kuramoto, Masato Nishida, Toyomi Satoh, Toshihiro Tanaka, and Yusuke Nakamura1 Laboratories of Molecular Medicine [M. M-N., M. U., K. O., T. M., T. Ta., Y. N.] and Genome Database [T. Ts.], Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan; Department of Obstetrics and Gynecology, School of Medicine, Kitasato University, Sagamihara 228-8555, Japan [H. K.]; Department of Obstetrics and Gynecology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba 305-8576, Japan [M. N.]; and Department of Obstetrics and Gynecology, Ibaraki Seinan Central Hospital, Tsukuba 306-0433, Japan [T. S.] ABSTRACT Akt/protein kinase B, cell survival, and cell proliferation (8). Over- expression of PTEN can decrease cell proliferation and tumorigenicity The PTEN tumor suppressor gene encodes a multifunctional phospha- (9, 10), an observation attributed to the ability of PTEN to induce cell tase that plays an important role in inhibiting the phosphatidylinositol-3- cycle arrest and apoptosis (11, 12). kinase pathway and downstream functions that include activation of Akt/protein kinase B, cell survival, and cell proliferation. Enforced ex- Thus, lack of PTEN expression may affect a complex set of pression of PTEN in various cancer cell lines decreases cell proliferation transcriptional targets. However, no systematic assessment of PTEN- through arrest of the cell cycle, accompanied in some cases by induction regulated targets in cancer cells has been reported to date.
    [Show full text]
  • Impact of Sideways and Bottom-Up Control Factors on Bacterial Community Succession Over a Tidal Cycle
    Impact of sideways and bottom-up control factors on bacterial community succession over a tidal cycle Ashvini Chauhan1, Jennifer Cherrier, and Henry N. Williams Environmental Sciences Institute, Florida A&M University, Frederick S. Humphries Science Research Building, Suite 305-D, Tallahassee, FL 32307 Edited by David M. Karl, University of Hawaii, Honolulu, HI, and approved January 22, 2009 (received for review October 21, 2008) In aquatic systems, bacterial community succession is a function of tandem, top-down factors shape bacterial community structure top-down and bottom-up factors, but little information exists on through a variety of processes, including protistan grazing (10) and ‘‘sideways’’ controls, such as bacterial predation by Bdellovibrio- viral lysis (11). Therefore, in all likelihood at any given time, like organisms (BLOs), which likely impacts nutrient cycling within combinations of these factors drive bacterial community succession the microbial loop and eventual export to higher trophic groups. in aquatic ecosystems (12–14). Here we report transient response of estuarine microbiota and BLO Recently, Mou et al. (2) proposed that in marine systems, spp. to tidal-associated dissolved organic matter supply in a river- transient changes in the dissolved organic carbon (DOC) pool are dominated estuary, Apalachicola Bay, Florida. Both dissolved or- less critical in structuring bacterial communities than those that ganic carbon and dissolved organic nitrogen concentrations oscil- result from viral lysis, protistan grazing, or even physicochemical lated over the course of the tidal cycle with relatively higher conditions. Both grazing and viral lyses are selective, such that concentrations observed at low tide. Concurrent with the shift in factors including nonsusceptibility, morphology, size, and motility dissolved organic matter (DOM) supply at low tide, a synchronous offer protection to certain bacterial groups (12).
    [Show full text]
  • Generated by SRI International Pathway Tools Version 25.0, Authors S
    Authors: Pallavi Subhraveti Ron Caspi Quang Ong Peter D Karp An online version of this diagram is available at BioCyc.org. Biosynthetic pathways are positioned in the left of the cytoplasm, degradative pathways on the right, and reactions not assigned to any pathway are in the far right of the cytoplasm. Transporters and membrane proteins are shown on the membrane. Ingrid Keseler Periplasmic (where appropriate) and extracellular reactions and proteins may also be shown. Pathways are colored according to their cellular function. Gcf_000725805Cyc: Streptomyces xanthophaeus Cellular Overview Connections between pathways are omitted for legibility.
    [Show full text]
  • Downloaded from the National Center for Biotechnology Information (NCBI)
    microorganisms Article Phylogenomic Insights into Distribution and Adaptation of Bdellovibrionota in Marine Waters Qing-Mei Li 1,2, Ying-Li Zhou 1,2, Zhan-Fei Wei 1,2 and Yong Wang 1,* 1 Institute of Deep Sea Science and Engineering, Chinese Academy of Sciences, Sanya 572000, China; [email protected] (Q.-M.L.); [email protected] (Y.-L.Z.); [email protected] (Z.-F.W.) 2 University of Chinese Academy of Sciences, Beijing 100049, China * Correspondence: [email protected] Abstract: Bdellovibrionota is composed of obligate predators that can consume some Gram-negative bacteria inhabiting various environments. However, whether genomic traits influence their distribu- tion and marine adaptation remains to be answered. In this study, we performed phylogenomics and comparative genomics studies using 132 Bdellovibrionota genomes along with five metagenome- assembled genomes (MAGs) from deep sea zones. Four phylogenetic groups, Oligoflexia, Bdello- group1, Bdello-group2 and Bacteriovoracia, were revealed by constructing a phylogenetic tree, of which 53.84% of Bdello-group2 and 48.94% of Bacteriovoracia were derived from the ocean. Bacteri- ovoracia was more prevalent in deep sea zones, whereas Bdello-group2 was largely distributed in the epipelagic zone. Metabolic reconstruction indicated that genes involved in chemotaxis, flagellar (mobility), type II secretion system, ATP-binding cassette (ABC) transporters and penicillin-binding protein were necessary for the predatory lifestyle of Bdellovibrionota. Genes involved in glycerol metabolism, hydrogen peroxide (H2O2) degradation, cell wall recycling and peptide utilization were ubiquitously present in Bdellovibrionota genomes. Comparative genomics between marine and Citation: Li, Q.-M.; Zhou, Y.-L.; Wei, non-marine Bdellovibrionota demonstrated that betaine as an osmoprotectant is probably widely Z.-F.; Wang, Y.
    [Show full text]
  • (10) Patent No.: US 8119385 B2
    US008119385B2 (12) United States Patent (10) Patent No.: US 8,119,385 B2 Mathur et al. (45) Date of Patent: Feb. 21, 2012 (54) NUCLEICACIDS AND PROTEINS AND (52) U.S. Cl. ........................................ 435/212:530/350 METHODS FOR MAKING AND USING THEMI (58) Field of Classification Search ........................ None (75) Inventors: Eric J. Mathur, San Diego, CA (US); See application file for complete search history. Cathy Chang, San Diego, CA (US) (56) References Cited (73) Assignee: BP Corporation North America Inc., Houston, TX (US) OTHER PUBLICATIONS c Mount, Bioinformatics, Cold Spring Harbor Press, Cold Spring Har (*) Notice: Subject to any disclaimer, the term of this bor New York, 2001, pp. 382-393.* patent is extended or adjusted under 35 Spencer et al., “Whole-Genome Sequence Variation among Multiple U.S.C. 154(b) by 689 days. Isolates of Pseudomonas aeruginosa” J. Bacteriol. (2003) 185: 1316 1325. (21) Appl. No.: 11/817,403 Database Sequence GenBank Accession No. BZ569932 Dec. 17. 1-1. 2002. (22) PCT Fled: Mar. 3, 2006 Omiecinski et al., “Epoxide Hydrolase-Polymorphism and role in (86). PCT No.: PCT/US2OO6/OOT642 toxicology” Toxicol. Lett. (2000) 1.12: 365-370. S371 (c)(1), * cited by examiner (2), (4) Date: May 7, 2008 Primary Examiner — James Martinell (87) PCT Pub. No.: WO2006/096527 (74) Attorney, Agent, or Firm — Kalim S. Fuzail PCT Pub. Date: Sep. 14, 2006 (57) ABSTRACT (65) Prior Publication Data The invention provides polypeptides, including enzymes, structural proteins and binding proteins, polynucleotides US 201O/OO11456A1 Jan. 14, 2010 encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides.
    [Show full text]
  • The Microbiota-Produced N-Formyl Peptide Fmlf Promotes Obesity-Induced Glucose
    Page 1 of 230 Diabetes Title: The microbiota-produced N-formyl peptide fMLF promotes obesity-induced glucose intolerance Joshua Wollam1, Matthew Riopel1, Yong-Jiang Xu1,2, Andrew M. F. Johnson1, Jachelle M. Ofrecio1, Wei Ying1, Dalila El Ouarrat1, Luisa S. Chan3, Andrew W. Han3, Nadir A. Mahmood3, Caitlin N. Ryan3, Yun Sok Lee1, Jeramie D. Watrous1,2, Mahendra D. Chordia4, Dongfeng Pan4, Mohit Jain1,2, Jerrold M. Olefsky1 * Affiliations: 1 Division of Endocrinology & Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California, USA. 2 Department of Pharmacology, University of California, San Diego, La Jolla, California, USA. 3 Second Genome, Inc., South San Francisco, California, USA. 4 Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, VA, USA. * Correspondence to: 858-534-2230, [email protected] Word Count: 4749 Figures: 6 Supplemental Figures: 11 Supplemental Tables: 5 1 Diabetes Publish Ahead of Print, published online April 22, 2019 Diabetes Page 2 of 230 ABSTRACT The composition of the gastrointestinal (GI) microbiota and associated metabolites changes dramatically with diet and the development of obesity. Although many correlations have been described, specific mechanistic links between these changes and glucose homeostasis remain to be defined. Here we show that blood and intestinal levels of the microbiota-produced N-formyl peptide, formyl-methionyl-leucyl-phenylalanine (fMLF), are elevated in high fat diet (HFD)- induced obese mice. Genetic or pharmacological inhibition of the N-formyl peptide receptor Fpr1 leads to increased insulin levels and improved glucose tolerance, dependent upon glucagon- like peptide-1 (GLP-1). Obese Fpr1-knockout (Fpr1-KO) mice also display an altered microbiome, exemplifying the dynamic relationship between host metabolism and microbiota.
    [Show full text]
  • Cultivation of Bdellovibrios
    Special Instructions Cultivation of Bdellovibrios Bdellovibrios are unique microorganisms that prey upon a wide variety of susceptible Gram-negative bacteria. Their predatory life style is characterized by two distinct phases, a free-living attack phase and an intraperiplasmic growth phase. Although members of the order Bdellovibrionales are phenotypically quite similar, they do not form a coherent phylogenetic group. Currently, they are classified into five different genera, Bdellovibrio, Bacteriovorax, Peredibacter, Halobacteriovorax and Pseudobacteriovorax. Bdellovibrios can be found in a wide variety of habitats, ranging from soil to sewage, provided these environments are densely populated with bacteria. Some strains represent prey-independent mutants that have lost their requirement for prey cells and hence are facultatively predacious (e.g., Bacteriovorax stolpii DSM 12778) or adapted to an obligate saprophytic life-style (e.g., Bdellovibrio bacteriovorus DSM 12732). Prey-dependent bdellovibrios are usually sensitive to lyophilization and consequently delivered as actively growing cultures from the DSMZ. Fresh samples of attack phase bdellovibrios are shipped either on double-layered agar plates or in liquid broth culture. Presumably, due to the high endogenous respiration rates of bdellovibrios, their viability rapidly decreases after complete lysis of prey cells. Therefore, it is important to transfer the obtained cultures immediately upon receipt into freshly prepared media containing suspensions of susceptible prey cells. An axenic culture of prey cells is shipped along with prey-dependent strains of bdellovibrios. A detailed description of the cultivation of Bdellovibrio bacteriovorus DSM 50701T follows below to exemplify the recommended handling of prey-dependent strains. You will receive from the DSMZ a double layer agar plate of DSMZ medium 257 containing predator and prey cells in the top layer and a tube of slant agar (DSMZ medium 54) with an axenic culture of the prey bacterium Pseudomonas sp.
    [Show full text]