Prescott harley kleins microbiology 7th ed j willey, l sherwood, c woolverton (mcgraw hill, 2008) 1

MD DALIM 933354 10/15/07 CYAN MAG YELO BLK wil92913_FM_00i_xx.qxd 11/6/06 11:53 AM Page i Prescott, Harley, and Klein’s Microbiology Seventh Edition Joanne M Willey Hofstra University Linda M Sherwood Montana State University Christopher J Woolverton Kent State University wil92913_FM_00i_xx.qxd 11/6/06 11:53 AM Page ii PRESCOTT, HARLEY, AND KLEIN’S MICROBIOLOGY, SEVENTH EDITION Published by McGraw-Hill, a business unit of The McGraw-Hill Companies, Inc., 1221 Avenue of the Americas, New York, NY 10020 Copyright © 2008 by The McGraw-Hill Companies, Inc All rights reserved No part of this publication may be reproduced or distributed in any form or by any means, or stored in a database or retrieval system, without the prior written consent of The McGraw-Hill Companies, Inc., including, but not limited to, in any network or other electronic storage or transmission, or broadcast for distance learning Some ancillaries, including electronic and print components, may not be available to customers outside the United States This book is printed on recycled, acid-free paper containing 10% postconsumer waste DOW/DOW ISBN 978–0–07–299291–5 MHID 0–07–299291–3 Publisher: Colin Wheatley/Janice Roerig-Blong Senior Developmental Editor: Lisa A Bruflodt Senior Marketing Manager: Tami Petsche Senior Project Manager: Jayne Klein Lead Production Supervisor: Sandy Ludovissy Senior Media Project Manager: Jodi K Banowetz Senior Media Producer: Eric A Weber Designer: John Joran (USE) Cover Image: Dennis Kunkel Microscopy, Inc Lead Photo Research Coordinator: Carrie K Burger Photo Research: Mary Reeg Supplement Producer: Mary Jane Lampe Compositor: Carlisle Publishing Services Typeface: 10/12 Times Roman Printer: R R Donnelley Willard, OH The credits section for this book begins on page C-1 and is considered an extension of the copyright page Library of Congress Cataloging-in-Publication Data Willey, Joanne M Prescott, Harley, and Klein’s microbiology / Joanne M Willey, Linda M Sherwood, Christopher J Woolverton — 7th ed p cm Includes index ISBN 978–0–07–299291–5 — ISBN 0–07–299291–3 (hard copy : alk paper) Microbiology I Sherwood, Linda M II Woolverton, Christopher J III Prescott, Lansing M Microbiology IV Title QR41.2.P74 2008 616.9’041—dc22 www.mhhe.com 2006027152 CIP wil92913_FM_00i_xx.qxd 11/6/06 11:53 AM Page iii This text is dedicated to our mentors—John Waterbury, Richard Losick, Thomas Bott, Hank Heath, Pete Magee, Lou Rigley, Irv Snyder, and R Balfour Sartor And to our students —Joanne M Willey —Linda M Sherwood —Christopher J Woolverton wil92913_FM_00i_xx.qxd 11/6/06 11:53 AM Page iv Brief Contents Part I The History and Scope of Microbiology The Study of Microbial Structure: Microscopy and Specimen Preparation Procaryotic Cell Structure and Function Eucaryotic Cell Structure and Function Part II Introduction to Microbiology 17 39 79 Microbial Nutrition, Growth, and Control Microbial Metabolism Metabolism: Energy, Enzymes, and Regulation Metabolism: Energy Release and Conservation 10 Metabolism: The Use of Energy in Biosynthesis Part IV 167 191 225 Microbial Molecular Biology and Genetics DNA Technology and Genomics 14 Recombinant DNA Technology 15 Microbial Genomics Part VI 27 Biogeochemical Cycling and Introductory Microbial Ecology 28 Microorganism in Marine and Freshwater Environments 29 Microorganisms in Terrestrial Environments 30 Microbial Interactions Part IX 357 383 Part X 33 34 35 36 37 38 39 16 The Viruses: Introduction and General Characteristics 407 17 The Viruses: Viruses of Bacteria and Archaea 427 18 The Viruses: Eucaryotic Viruses and Other Acellular Infectious Agents 447 667 687 717 743 773 Microbial Diseases and Their Control Pathogenicity of Microorganisms Antimicrobial Chemotherapy Clinical Microbiology and Immunology The Epidemiology of Infectious Disease Human Diseases Caused by Viruses and Prions Human Diseases Caused by Bacteria Human Diseases Caused by Fungi and Protists Part XI The Viruses 643 Nonspecific (Innate) Resistance and the Immune Response 31 Nonspecific (Innate) Host Resistance 32 Specific (Adaptive) Immunity 11 Microbial Genetics: Gene Structure, Replication, and Expression 247 12 Microbial Genetics: Regulation of Gene Expression 291 13 Microbial Genetics: Mechanisms of Genetic Variation 317 Part V 519 539 571 589 605 629 Part VIII Ecology and Symbiosis Microbial Nutrition 101 Microbial Growth 119 Control of Microorganisms by Physical and Chemical Agents 149 Part III 21 Bacteria: The Deinococci and Nonproteobacteria Gram Negatives 22 Bacteria: The Proteobacteria 23 Bacteria: The Low G ϩ C Gram Positives 24 Bacteria: The High G ϩ C Gram Positives 25 The Protists 26 The Fungi (Eumycota) 815 835 859 885 913 947 997 Food and Industrial Microbiology 40 Microbiology of Food 41 Applied and Industrial Microbiology Appendix I A Review of the Chemistry of Biological Molecules 1023 1049 A-1 Part VII The Diversity of the Microbial World 19 Microbial Evolution, Taxonomy, and Diversity 20 The Archaea iv 471 503 Appendix II Common Metabolic Pathways A-13 wil92913_FM_00i_xx.qxd 11/6/06 11:53 AM Page v Contents About the Authors xi Preface xii Part I Members of the Microbial World The Discovery of Microorganisms The Conflict over Spontaneous Generation The Golden Age of Microbiology ■ Techniques & Applications 1.1: The Scientific Method ■ Disease 1.2: Koch’s Molecular Postulates 10 11 The Development of Industrial Microbiology and Microbial Ecology The Scope and Relevance of Microbiology The Future of Microbiology 12 13 14 The Study of Microbial Structure: Microscopy and Specimen Preparation 17 2.1 2.2 2.3 2.4 2.5 17 18 25 28 31 39 3.1 3.2 39 42 3.4 3.5 3.6 3.7 3.8 3.9 3.10 3.11 Lenses and the Bending of Light The Light Microscope Preparation and Staining of Specimens Electron Microscopy Newer Techniques in Microscopy Procaryotic Cell Structure and Function 3.3 4.8 4.9 4.10 4.11 Introduction to Microbiology The History and Scope of Microbiology 1.6 1.7 An Overview of Procaryotic Cell Structure Procaryotic Cell Membranes ■ Microbial Diversity & Ecology 3.1: Monstrous Microbes 43 The Cytoplasmic Matrix 48 ■ Microbial Diversity & Ecology 3.2: Living Magnets 51 The Nucleoid Plasmids The Bacterial Cell Wall Archaeal Cell Walls Protein Secretion in Procaryotes Components External to the Cell Wall Chemotaxis The Bacterial Endospore 52 53 55 62 63 65 71 73 Part II 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 6.6 79 An Overview of Eucaryotic Cell Structure The Plasma Membrane and Membrane Structure The Cytoplasmic Matrix, Microfilaments, Intermediate Filaments, and Microtubules 79 81 7.2 7.3 83 ■ Disease 4.1: Getting Around 84 7.4 7.5 Organelles of the Biosynthetic-Secretory and Endocytic Pathways Eucaryotic Ribosomes Mitochondria 84 88 88 7.6 91 91 94 95 96 101 101 102 102 104 105 105 110 ■ Historical Highlights 5.1: The Discovery of Agar as a Solidifying Agent and the Isolation of Pure Cultures 112 Isolation of Pure Cultures 113 ■ Techniques & Applications 5.2: The Enrichment and Isolation of Pure Cultures 116 The Procaryotic Cell Cycle The Growth Curve Measurement of Microbial Growth The Continuous Culture of Microorganisms The Influence of Environmental Factors on Growth 119 119 123 128 131 132 ■ Microbial Diversity & Ecology 6.1: Life Above 100°C 138 Microbial Growth in Natural Environments 142 Definitions of Frequently Used Terms ■ Techniques & Applications 7.1: Safety in the Microbiology Laboratory Eucaryotic Cell Structure and Function 4.5 4.6 The Common Nutrient Requirements Requirements for Carbon, Hydrogen, Oxygen, and Electrons Nutritional Types of Microorganisms Requirements for Nitrogen, Phosphorus, and Sulfur Growth Factors Uptake of Nutrients by the Cell Culture Media Control of Microorganisms by Physical and Chemical Agents 7.1 90 Microbial Nutrition, Growth, and Control Microbial Growth 6.1 6.2 6.3 6.4 6.5 The Nucleus and Cell Division External Cell Coverings Cilia and Flagella Comparison of Procaryotic and Eucaryotic Cells Microbial Nutrition 4.1 4.2 4.3 4.4 Chloroplasts ■ Microbial Diversity & Ecology 4.2: The Origin of the Eucaryotic Cell 1.1 1.2 1.3 1.4 1.5 4.7 149 149 150 The Pattern of Microbial Death Conditions Influencing the Effectiveness of Antimicrobial Agents The Use of Physical Methods in Control The Use of Chemical Agents in Control 151 152 153 158 ■ Techniques & Applications 7.2: Universal Precautions for Microbiology Laboratories 160 Evaluation of Antimicrobial Agent Effectiveness 164 v wil92913_FM_00i_xx.qxd 11/6/06 11:53 AM Page vi vi Contents Part III Microbial Metabolism Metabolism: Energy, Enzymes, and Regulation 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 8.10 Metabolism: Energy Release and Conservation 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9 9.10 9.11 9.12 10 An Overview of Metabolism Energy and Work The Laws of Thermodynamics Free Energy and Reactions The Role of ATP in Metabolism Oxidation-Reduction Reactions, Electron Carriers, and Electron Transport Systems Enzymes The Nature and Significance of Metabolic Regulation Metabolic Channeling Control of Enzyme Activity Chemoorganotrophic Fueling Processes Aerobic Respiration The Breakdown of Glucose to Pyruvate The Tricarboxylic Acid Cycle Electron Transport and Oxidative Phosphorylation Anaerobic Respiration Fermentations 10.7 167 169 169 170 171 12 12.1 12.2 12.3 12.4 12.5 12.6 180 180 181 13 191 193 194 198 13.1 13.2 13.3 13.4 13.5 13.6 13.7 13.8 13.9 13.10 13.11 200 205 207 210 210 211 212 212 214 Part V ■ Microbial Diversity & Ecology 9.2: Acid Mine Drainage 215 14 14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 225 226 227 228 230 235 241 242 Part IV Microbial Molecular Biology and Genetics Microbial Genetics: Gene Structure, Replication, and Expression 11.1 11.2 11.3 11.4 11.5 11.6 14.10 247 ■ Historical Highlights 11.1: The Elucidation of DNA Structure 248 DNA as Genetic Material The Flow of Genetic Information Nucleic Acid Structure DNA Replication Gene Structure Transcription 249 251 252 253 264 268 15 Regulation of Transcription Elongation Regulation at the Level of Translation Global Regulatory Systems Regulation of Gene Expression in Eucarya and Archaea Mutations and Their Chemical Basis Detection and Isolation of Mutants DNA Repair Creating Genetic Variability Transposable Elements Bacterial Plasmids Bacterial Conjugation DNA Transformation Transduction Mapping the Genome Recombination and Genome Mapping in Viruses Historical Perspectives Synthetic DNA The Polymerase Chain Reaction Gel Electrophoresis Cloning Vectors and Creating Recombinant DNA Construction of Genomic Libraries Inserting Recombinant DNA into Host Cells Expressing Foreign Genes in Host Cells 292 293 294 302 305 307 313 317 317 324 326 329 332 334 337 342 345 349 350 357 357 361 362 366 366 370 371 371 ■ Techniques & Applications 14.1: Visualizing Proteins with Green Fluorescence 374 Applications of Genetic Engineering 375 ■ Techniques & Applications 14.2: Plant Tumors and Nature’s Genetic Engineer 378 Social Impact of Recombinant DNA Technology 380 Microbial Genomics 15.1 15.2 15.3 15.4 15.5 15.6 15.7 15.8 15.9 291 DNA Technology and Genomics Recombinant DNA Technology 14.9 11 Levels of Regulation of Gene Expression Regulation of Transcription Initiation Microbial Genetics: Mechanisms of Genetic Variation Catabolism of Carbohydrates and Intracellular Reserve Polymers Lipid Catabolism Protein and Amino Acid Catabolism Chemolithotrophy Phototrophy Principles Governing Biosynthesis The Precursor Metabolites The Fixation of CO2 by Autotrophs Synthesis of Sugars and Polysaccharides Synthesis of Amino Acids Synthesis of Purines, Pyrimidines, and Nucleotides Lipid Synthesis 275 276 ■ Historical Highlights 12.1: The Discovery of Gene Regulation 172 174 191 268 The Genetic Code Translation Microbial Genetics: Regulation of Gene Expression ■ Historical Highlights 9.1: Microbiology and World War I Metabolism: The Use of Energy in Biosynthesis 10.1 10.2 10.3 10.4 10.5 10.6 11.7 11.8 167 ■ Microbial Tidbits 11.2: Catalytic RNA (Ribozymes) Introduction Determining DNA Sequences Whole-Genome Shotgun Sequencing Bioinformatics Functional Genomics Comparative Genomics Proteomics Insights from Microbial Genomes Environmental Genomics 383 383 384 384 388 388 391 393 395 402 wil92913_FM_00i_xx.qxd 11/6/06 11:53 AM Page vii Contents Part VI The Viruses 16 The Viruses: Introduction and General Characteristics 16.1 16.2 16.3 16.4 16.5 16.6 16.7 17 17.3 17.4 17.5 17.6 408 409 409 417 417 419 423 ■ Microbial Tidbits 16.2: The Origin of Viruses 423 429 436 437 438 444 18.3 18.4 18.5 18.6 18.7 18.8 18.9 18.10 21.4 21.5 21.6 21.7 22 447 447 448 ■ Microbial Diversity & Ecology 18.1: SARS: Evolution of a Virus ■ Techniques & Applications 18.2: Constructing a Virus 458 Cytocidal Infections and Cell Damage Persistent, Latent, and Slow Virus Infections Viruses and Cancer Plant Viruses Viruses of Fungi and Protists Insect Viruses Viroids and Virusoids Prions 459 461 461 463 466 466 467 468 22.4 22.5 Microbial Evolution, Taxonomy, and Diversity 19.1 19.2 19.3 19.4 19.5 19.6 19.7 19.8 Microbial Evolution Introduction to Microbial Classification and Taxonomy Taxonomic Ranks Techniques for Determining Microbial Taxonomy and Phylogeny Assessing Microbial Phylogeny The Major Divisions of Life Bergey’s Manual of Systematic Bacteriology ■ Microbial Diversity & Ecology 20.1: Archaeal Phylogeny: More Than Just the Crenarchaeota and Euryarchaeota? ■ Microbial Diversity & Ecology 20.2: Methanotrophic Archaea 511 Aquificae and Thermotogae Deinococcus-Thermus Photosynthetic Bacteria 513 519 519 520 520 ■ Microbial Diversity & Ecology 21.1: The Mechanism of Gliding Motility 527 Phylum Planctomycetes Phylum Chlamydiae Phylum Spirochaetes Phylum Bacteroidetes 530 531 532 534 Class Alphaproteobacteria Class Betaproteobacteria Class Gammaproteobacteria 539 540 546 551 ■ Microbial Diversity & Ecology 22.1: Bacterial Bioluminescence 559 Class Deltaproteobacteria Class Epsilonproteobacteria 562 567 451 23 24 471 471 477 480 481 488 489 493 ■ Microbial Diversity & Ecology 19.1: “Official” Nomenclature Lists—A Letter from Bergey’s 494 A Survey of Procaryotic Phylogeny and Diversity 494 Bacteria: The Low G ϩ C Gram Positives 25 571 23.1 23.2 23.3 General Introduction Class Mollicutes (The Mycoplasmas) Peptidoglycan and Endospore Structure ■ Microbial Tidbits 23.1: Spores in Space 576 23.4 23.5 Class Clostridia Class Bacilli 576 578 Bacteria: The High G ϩ C Gram Positives 24.1 24.2 24.3 24.4 24.5 24.6 24.7 24.8 24.9 24.10 Part VII The Diversity of the Microbial World 19 503 507 508 Bacteria: The Proteobacteria 22.1 22.2 22.3 503 Introduction to the Archaea Phylum Crenarchaeota Phylum Euryarchaeota Bacteria: The Deinococci and Nonproteobacteria Gram Negatives 21.1 21.2 21.3 428 428 Single-Stranded DNA Phages RNA Phages Temperate Bacteriophages and Lysogeny Bacteriophage Genomes Taxonomy of Eucaryotic Viruses Reproduction of Vertebrate Viruses 21 427 ■ Microbial Diversity & Ecology 17.1: Host-Independent Growth of an Archaeal Virus The Viruses: Eucaryotic Viruses and Other Acellular Infectious Agents 18.1 18.2 407 General Properties of Viruses The Structure of Viruses Virus Reproduction The Cultivation of Viruses Virus Purification and Assays Principles of Virus Taxonomy Classification of Bacterial and Archaeal Viruses Virulent Double-Stranded DNA Phages The Archaea 20.1 20.2 20.3 407 ■ Historical Highlights 16.1: Disease and the Early Colonization of America The Viruses: Viruses of Bacteria and Archaea 17.1 17.2 18 Early Development of Virology 20 vii General Properties of the Actinomycetes Suborder Actinomycineae Suborder Micrococcineae Suborder Corynebacterineae Suborder Micromonosporineae Suborder Propionibacterineae Suborder Streptomycineae Suborder Streptosporangineae Suborder Frankineae Order Bifidobacteriales The Protists 25.1 25.2 25.3 25.4 25.5 25.6 Distribution Nutrition Morphology Encystment and Excystment Reproduction Protist Classification 571 571 572 589 589 593 593 595 597 598 598 601 601 602 605 606 606 607 608 608 609 wil92913_FM_00i_xx.qxd 11/6/06 11:54 AM Page viii viii Contents ■ Disease 25.1: Harmful Algal Blooms (HABs) ■ Techniques & Applications 25.2: Practical Importance of Diatoms 26 The Fungi (Eumycota) 26.1 26.2 26.3 26.4 26.5 26.6 Distribution Importance Structure Nutrition and Metabolism Reproduction Characteristics of the Fungal Divisions 621 624 30.2 30.3 629 630 630 631 632 632 635 Biogeochemical Cycling and Introductory Microbial Ecology 27.1 Foundations in Microbial Diversity and Ecology ■ Microbial Diversity & Ecology 27.1: Microbial Ecology Versus Environmental Microbiology 27.2 27.3 27.4 Biogeochemical Cycling The Physical Environment Microbial Ecology and Its Methods: An Overview ■ Techniques & Applications 27.2: Thermophilic Microorganisms and Modern Biotechnology 28 Microorganisms in Marine and Freshwater Environments 28.1 28.2 28.3 28.4 29 31 29.1 29.2 31.1 31.2 31.3 31.4 31.5 29.3 29.4 29.5 29.6 31.6 644 32 32.1 32.2 32.3 32.4 659 660 667 32.5 32.6 32.7 32.8 667 668 Microbial Adaptations to Marine and Freshwater Environments Microorganisms in Marine Environments Microorganisms in Freshwater Environments 671 673 682 32.9 687 32.10 32.11 687 689 ■ Microbial Tidbits 29.1: An Unintended Global-Scale Nitrogen Experiment 691 Microorganisms in the Soil Environment Microorganisms and the Formation of Different Soils Microorganism Associations with Vascular Plants 33 693 697 708 33.5 33.6 710 34 Microbial Interactions 30.1 717 Microbial Interactions 717 ■ Microbial Diversity & Ecology 30.1: Wolbachia pipientis: The World’s Most Infectious Microbe? 720 34.2 34.3 34.4 743 743 744 752 756 758 762 773 774 774 776 778 ■ Techniques & Applications 32.1: Donor Selection for Tissue or Organ Transplants 779 T Cell Biology B Cell Biology Antibodies Action of Antibodies 781 786 789 799 ■ Techniques & Applications 32.2: Monoclonal Antibody Technology 800 Summary: The Role of Antibodies and Lymphocytes in Immune Defense Acquired Immune Tolerance Immune Disorders 802 802 803 Microbial Diseases and Their Control Host-Parasite Relationships Pathogenesis of Viral Diseases Overview of Bacterial Pathogenesis Toxigenicity Host Defense Against Microbial Invasion Microbial Mechanisms for Escaping Host Defenses Antimicrobial Chemotherapy 34.1 30 Overview of Specific (Adaptive) Immunity Antigens Types of Specific (Adaptive) Immunity Recognition of Foreignness ■ Techniques & Applications 33.1: Detection and Removal of Endotoxins 709 711 713 Overview of Host Resistance Cells, Tissues, and Organs of the Immune System Phagocytosis Inflammation Physical Barriers in Nonspecific (Innate) Resistance Chemical Mediators in Nonspecific (Innate) Resistance Pathogenicity of Microorganisms 33.1 33.2 33.3 33.4 696 Soil Microorganisms and the Atmosphere The Subsurface Biosphere Soil Microorganisms and Human Health Part X 692 ■ Microbial Diversity & Ecology 29.2: Mycorrhizae and the Evolution of Vascular Plants 739 Specific (Adaptive) Immunity ■ Disease 28.1: New Agents in Medicine— The Sea as the New Frontier ■ Microbial Diversity & Ecology 29.3: Soils, Termites, Intestinal Microbes, and Atmospheric Methane ■ Techniques & Applications 29.4: Keeping Inside Air Fresh with Soil Microorganisms 29.7 29.8 643 Soils as an Environment for Microorganisms Soils, Plants, and Nutrients ■ Techniques & Applications 30.3: Probiotics for Humans and Animals Nonspecific (Innate) Host Resistance Marine and Freshwater Environments Microorganisms in Terrestrial Environments 734 735 and the Immune Response 643 644 653 725 Human-Microbe Interactions Normal Microbiota of the Human Body Part IX Nonspecific (Innate) Resistance Part VIII Ecology and Symbiosis 27 ■ Microbial Diversity & Ecology 30.2: Coevolution of Animals and Their Gut Microbial Communities The Development of Chemotherapy 815 815 818 820 824 830 830 832 835 835 ■ Techniques & Applications 34.1: The Use of Antibiotics in Microbiological Research 837 General Characteristics of Antimicrobial Drugs Determining the Level of Antimicrobial Activity Antibacterial Drugs 837 840 841 wil92913_FM_00i_xx.qxd 11/6/06 11:54 AM Page ix Contents 34.5 34.6 34.7 34.8 34.9 35 35.2 35.3 35.4 35.5 36.2 36.3 36.4 36.5 36.6 36.7 859 ■ Techniques & Applications 35.1: Standard Microbial Practices 861 Identification of Microorganisms from Specimens 864 ■ Microbial Tidbits 35.2: Biosensors: The Future Is Now 871 Clinical Immunology 875 ■ Techniques & Applications 35.3: History and Importance of Serotyping 876 Susceptibility Testing Computers in Clinical Microbiology 882 882 885 Epidemiological Terminology 886 ■ Historical Highlights 36.1: John Snow—The First Epidemiologist 886 39 ■ Disease 39.1: A Brief History of Malaria 1002 1008 1012 1016 ■ Disease 39.2: The Emergence of Candidiasis 1018 Part XI Food and Industrial Microbiology Microbiology of Food 40.1 40.2 40.3 40.4 ■ Historical Highlights 36.3:The First Indications of Person-to-Person Spread of an Infectious Disease 896 40.5 40.6 907 908 Human Diseases Caused by Viruses and Prions 897 914 ■ Disease 37.1: Reye’s and Guillain-Barré Syndromes 918 37.2 Arthropod-Borne Diseases 922 41.2 41.3 1033 Detection of Food-Borne Pathogens Microbiology of Fermented Foods 1035 1036 Water Purification and Sanitary Analysis Wastewater Treatment Microorganisms Used in Industrial Microbiology ■ Techniques & Applications 41.2: The Potential of Thermophilic Archaea in Biotechnology 41.4 ■ Historical Highlights 37.3: A Brief History of Polio 941 Zoonotic Diseases Prion Diseases 941 944 41.5 41.6 947 960 ■ Historical Highlights 40.2: Typhoid Fever and Canned Meat ■ Techniques & Applications 41.1: Waterborne Diseases, Water Supplies, and Slow Sand Filtration 923 ■ Historical Highlights 38.1: The Hazards of Microbiological Research 1032 1049 925 939 948 960 1030 Food-Borne Diseases Applied and Industrial Microbiology 41.1 Direct Contact Diseases Food-Borne and Waterborne Diseases Airborne Diseases Arthropod-Borne Diseases ■ Historical Highlights 40.1: An Army Travels on Its Stomach 1046 ■ Disease 37.2: Viral Hemorrhagic Fevers— A Microbial History Lesson Human Diseases Caused by Bacteria 1024 1026 1028 1039 913 Airborne Diseases 1023 Microorganisms as Foods and Food Amendments 40.7 41 Microorganism Growth in Foods Microbial Growth and Food Spoilage Controlling Food Spoilage ■ Techniques & Applications 40.3: Chocolate: The Sweet Side of Fermentation ■ Techniques & Applications 40.4: Starter Cultures, Bacteriophage Infections, and Plasmids 897 900 37.1 987 987 991 Direct Contact Diseases Food-Borne and Waterborne Diseases Opportunistic Diseases 889 891 Global Travel and Health Considerations Nosocomial Infections 983 Sepsis and Septic Shock Zoonotic Diseases Dental Infections 39.4 39.5 39.6 Recognition of an Epidemic The Infectious Disease Cycle: Story of a Disease 905 979 ■ Techniques & Applications 38.5: Clostridial Toxins as Therapeutic Agents—Benefits of Nature’s Most Toxic Proteins 997 999 1001 889 ■ Historical Highlights 36.5: 1346—The First Recorded Biological Warfare Attack Food-Borne and Waterborne Diseases 997 888 902 969 972 974 Pathogenic Fungi and Protists Airborne Diseases Arthropod-Borne Diseases ■ Historical Highlights 36.2:“Typhoid Mary” 905 964 ■ Disease 38.2: Biofilms ■ Disease 38.3: Antibiotic-Resistant Staphylococci ■ Disease 38.4: A Brief History of Syphilis Human Diseases Caused by Fungi and Protists 887 ■ Historical Highlights 36.4:The First Immunizations Direct Contact Diseases 39.1 39.2 39.3 Measuring Frequency: The Epidemiologist’s Tools Recognition of an Infectious Disease in a Population Bioterrorism Preparedness 38.1 38.2 38.5 38.6 38.7 40 36.9 37.5 37.6 38.4 859 Specimens 36.8 37.3 37.4 38 854 855 856 Virulence and the Mode of Transmission Emerging and Reemerging Infectious Diseases and Pathogens Control of Epidemics 36.10 36.11 37 850 Antifungal Drugs Antiviral Drugs Antiprotozoan Drugs The Epidemiology of Infectious Disease 36.1 38.3 849 849 ■ Disease 34.2: Antibiotic Misuse and Drug Resistance Clinical Microbiology and Immunology 35.1 36 Factors Influencing Antimicrobial Drug Effectiveness Drug Resistance ix 41.7 Microorganism Growth in Controlled Environments Major Products of Industrial Microbiology Biodegradation and Bioremediation by Natural Communities 1037 1050 1051 1054 1060 1061 1064 1070 1075 ■ Microbial Diversity & Ecology 41.3: Methanogens— A New Role for a Unique Microbial Group 1078 Bioaugmentation 1080 ■ Microbial Diversity & Ecology 41.4: A Fungus with a Voracious Appetite 1081 wil92913_ch03.qxd 9/21/06 10:34 AM Page 65 Components External to the Cell Wall Gram-negative bacteria use the type II and type V pathways to transport proteins across the outer membrane after the protein has first been translocated across the plasma membrane by the Secdependant pathway The type I and type III pathways not interact with proteins that are first translocated by the Sec system, so they are said to be Sec-independent The type IV pathway sometimes is linked to the Sec-dependent pathway but usually functions on its own The type II protein secretion pathway is present in a number of plant and animal pathogens, including Erwinia carotovora, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Vibrio cholerae It is responsible for the secretion of proteins such as the degradative enzymes pullulanases, cellulases, pectinases, proteases, and lipases, as well as other proteins like cholera toxin and pili proteins Type II systems are quite complex and can contain as many as 12 to 14 proteins, most of which appear to be integral membrane proteins (figure 3.33) Even though some components of type II systems span the plasma membrane, they apparently translocate proteins only across the outer membrane In most cases, the Sec-dependent pathway first translocates the protein across the plasma membrane and then the type II system completes the secretion process In gram-negative and gram-positive bacteria, another plasma membrane translocation system called the Tat pathway can move proteins across the plasma membrane In gram-negatives, these proteins are then delivered to the type II system The Tat pathway is distinct from the Sec system in that it translocates already folded proteins The type V protein secretion pathways are the most recently discovered protein secretion systems They, too, rely on the Secdependent pathway to move proteins across the plasma membrane However, once in the periplasmic space, many of these proteins are able to form a channel in the outer membrane through which they transport themselves; these proteins are referred to as autotransporters Other proteins are secreted by the type V pathway with the aid of a separate helper protein The ABC protein secretion pathway, which derives its name from ATP binding cassette, is ubiquitous in procaryotes—that is, it is present in gram-positive and gram-negative bacteria as well as Archaea It is sometimes called the type I protein secretion pathway (figure 3.33) In pathogenic gram-negative bacteria, it is involved in the secretion of toxins (␣-hemolysin), as well as proteases, lipases, and specific peptides Secreted proteins usually contain C-terminal secretion signals that help direct the newly synthesized protein to the type I machinery, which spans the plasma membrane, the periplasmic space, and the outer membrane These systems translocate proteins in one step across both membranes, bypassing the Sec-dependent pathway Gram-positive bacteria use a modified version of the type I system to translocate proteins across the plasma membrane Analysis of the Bacillus subtilis genome has identified 77 ABC transporters This may reflect the fact that ABC transporters transport a wide variety of solutes in addition to proteins, including sugars and amino acids, as well as exporting drugs from the cell interior Several gram-negative pathogens have the type III protein secretion pathway, another secretion system that bypasses the Sec-dependent pathway Most type III systems inject virulence 65 factors directly into the plant and animal host cells these pathogens attack These virulence factors include toxins, phagocytosis inhibitors, stimulators of cytoskeleton reorganization in the host cell, and promoters of host cell suicide (apoptosis) However, in some cases the virulence factor is simply secreted into the extracellular milieu Type III systems also transport other proteins, including (1) some of the proteins from which the system is built, (2) proteins that regulate the secretion process, and (3) proteins that aid in the insertion of secreted proteins into target cells Type III systems are structurally complex and often are shaped like a syringe (figure 3.33) The slender, needlelike portion extends from the cell surface; a cylindrical base is connected to both the outer membrane and the plasma membrane and looks somewhat like the flagellar basal body (see p 67) It is thought that proteins may move through a translocation channel Important examples of bacteria with type III systems are Salmonella, Yersinia, Shigella, E coli, Bordetella, Pseudomonas aeruginosa, and Erwinia The participation of type III systems in bacterial virulence is further discussed in chapter 33 Type IV protein secretion pathways are unique in that they are used to secrete proteins as well as to transfer DNA from a donor bacterium to a recipient during bacterial conjugation Type IV systems are composed of many different proteins, and like the type III systems, these proteins form a syringelike structure Type IV systems and conjugation are described in more detail in chapter 13 Give the major characteristics and functions of the protein secretion pathways described in this section Which secretion pathway is most widespread? What is a signal peptide? Why you think a protein’s signal peptide is not removed until after the protein is translocated across the plasma membrane? 3.9 COMPONENTS EXTERNAL TO THE CELL WALL Procaryotes have a variety of structures outside the cell wall that can function in protection, attachment to objects, and cell movement Several of these are discussed Capsules, Slime Layers, and S-Layers Some procaryotes have a layer of material lying outside the cell wall This layer has different names depending on its characteristics When the layer is well organized and not easily washed off, it is called a capsule (figure 3.34a) It is called a slime layer when it is a zone of diffuse, unorganized material that is removed easily When the layer consists of a network of polysaccharides extending from the surface of the cell, it is referred to as the glycocalyx (figure 3.34b), a term that can encompass both capsules and slime layers because they usually are composed of polysaccharides However, some slime layers and capsules are constructed of other materials For example, Bacillus anthracis has a wil92913_ch03.qxd 8/8/06 3:02 PM Page 66 66 Chapter Procaryotic Cell Structure and Function Intestinal tissue Bacteria Glycocalyx (a) K pneumoniae Figure 3.35 Bacterial Glycocalyx Bacteria connected to each other and to the intestinal wall, by their glycocalyxes, the extensive networks of fibers extending from the cells (ϫ17,500) (b) Bacteroides Figure 3.34 Bacterial Capsules (a) Klebsiella pneumoniae with its capsule stained for observation in the light microscope (ϫ1,500) (b) Bacteroides glycocalyx (gly), TEM (ϫ71,250) proteinaceous capsule composed of poly-D-glutamic acid Capsules are clearly visible in the light microscope when negative stains or special capsule stains are employed (figure 3.34a); they also can be studied with the electron microscope (figure 3.34b) Although capsules are not required for growth and reproduction in laboratory cultures, they confer several advantages when procaryotes grow in their normal habitats They help pathogenic bacteria resist phagocytosis by host phagocytes Streptococcus pneumoniae provides a dramatic example When it lacks a capsule, it is destroyed easily and does not cause disease, whereas the capsulated variant quickly kills mice Capsules contain a great deal of water and can protect against desiccation They exclude viruses and most hydrophobic toxic materials such as detergents The glycocalyx also aids in attachment to solid surfaces, including tissue surfaces in plant and animal hosts (figure 3.35) Gliding bacteria often produce slime, which in some cases, has been shown to facilitate motility Microbial Diversity & Ecology 21.1: The mechanism of gliding motility; Phagocytosis (section 31.3); Overview of bacterial pathogenesis (section 33.3) Many procaryotes have a regularly structured layer called an Slayer on their surface In bacteria, the S-layer is external to the cell wall In archaea, the S-layer may be the only wall structure outside the plasma membrane The S-layer has a pattern something like floor tiles and is composed of protein or glycoprotein (figure 3.36) In gram-negative bacteria the S-layer adheres directly to the outer membrane; it is associated with the peptidoglycan surface in grampositive bacteria It may protect the cell against ion and pH fluctuations, osmotic stress, enzymes, or the predacious bacterium Bdellovibrio The S-layer also helps maintain the shape and envelope rigidity of some cells It can promote cell adhesion to surfaces Finally, the S-layer seems to protect some bacterial pathogens against host defenses, thus contributing to their virulence Class Deltaproteobacteria: Order Bdellovibrionales (section 22.4) Pili and Fimbriae Many procaryotes have short, fine, hairlike appendages that are thinner than flagella These are usually called fimbriae (s., fimbria) Although many people use the terms fimbriae and pili interchangeably, we shall distinguish between fimbriae and sex pili A cell may be covered with up to 1,000 fimbriae, but they are only visible in an electron microscope due to their small size (figure 3.37) They are slender tubes composed of helically arranged protein subunits and are about to 10 nm in diameter and up to several micrometers long At least some types of fimbriae attach bacteria to solid surfaces such as rocks in streams and host tissues Fimbriae are responsible for more than attachment Type IV fimbriae are present at one or both poles of bacterial cells They can aid in attachment to objects, and also are required for the twitching motility that occurs in some bacteria such as P aeruginosa, Neisseria gonorrhoeae, and some strains of E coli Movement is by short, intermittent jerky motions of up to several micrometers in length and normally is seen on very moist surfaces There is evidence that the fimbriae actively retract to move these bacteria Type wil92913_ch03.qxd 7/20/06 12:02 PM Page 67 Components External to the Cell Wall 67 plasmids and are required for conjugation Some bacterial viruses attach specifically to receptors on sex pili at the start of their reproductive cycle Bacterial conjugation (section 13.7) Flagella and Motility Figure 3.36 The S-Layer An electron micrograph of the S-layer of the bacterium Deinococcus radiodurans after shadowing Fimbriae Flagella Figure 3.37 Flagella and Fimbriae The long flagella and the numerous shorter fimbriae are very evident in this electron micrograph of the bacterium Proteus vulgaris (ϫ39,000) IV fimbriae are also involved in gliding motility by myxobacteria These bacteria are also of interest because they have complex life cycles that include the formation of a fruiting body Class Most motile procaryotes move by use of flagella (s., flagellum), threadlike locomotor appendages extending outward from the plasma membrane and cell wall Bacterial flagella are the best studied and they are the focus of this discussion Bacterial flagella are slender, rigid structures, about 20 nm across and up to 15 or 20 ␮m long Flagella are so thin they cannot be observed directly with a bright-field microscope, but must be stained with special techniques designed to increase their thickness The detailed structure of a flagellum can only be seen in the electron microscope (figure 3.37) Bacterial species often differ distinctively in their patterns of flagella distribution and these patterns are useful in identifying bacteria Monotrichous bacteria (trichous means hair) have one flagellum; if it is located at an end, it is said to be a polar flagellum (figure 3.38a) Amphitrichous bacteria (amphi means on both sides) have a single flagellum at each pole In contrast, lophotrichous bacteria (lopho means tuft) have a cluster of flagella at one or both ends (figure 3.38b) Flagella are spread fairly evenly over the whole surface of peritrichous (peri means around) bacteria (figure 3.38c) Flagellar Ultrastructure Transmission electron microscope studies have shown that the bacterial flagellum is composed of three parts (1) The longest and most obvious portion is the flagellar filament, which extends from the cell surface to the tip (2) A basal body is embedded in the cell; and (3) a short, curved segment, the flagellar hook, links the filament to its basal body and acts as a flexible coupling The filament is a hollow, rigid cylinder constructed of subunits of the protein flagellin, which ranges in molecular weight from 30,000 to 60,000 daltons, depending on the bacterial species The filament ends with a capping protein Some bacteria have sheaths surrounding their flagella For example, Bdellovibrio has a membranous structure surrounding the filament Vibrio cholerae has a lipopolysaccharide sheath The hook and basal body are quite different from the filament (figure 3.39) Slightly wider than the filament, the hook is made of different protein subunits The basal body is the most complex part of a flagellum In E coli and most gram-negative bacteria, the basal body has four rings connected to a central rod (figure 3.39a,d) The outer L and P rings associate with the lipopolysaccharide and peptidoglycan layers, respectively The inner M ring contacts the plasma membrane Gram-positive bacteria have only two basal body rings—an inner ring connected to the plasma membrane and an outer one probably attached to the peptidoglycan (figure 3.39b) Deltaproteobacteria: Order Myxococcales (section 22.4) Many bacteria have about 1-10 sex pili (s., pilus) per cell These are hairlike structures that differ from fimbriae in the following ways Pili often are larger than fimbriae (around to 10 nm in diameter) They are genetically determined by conjugative Flagellar Synthesis The synthesis of bacterial flagella is a complex process involving at least 20 to 30 genes Besides the gene for flagellin, 10 or more genes code for hook and basal body proteins; other genes wil92913_ch03.qxd 7/20/06 12:02 PM Page 68 68 Chapter Procaryotic Cell Structure and Function are concerned with the control of flagellar construction or function How the cell regulates or determines the exact location of flagella is not known When flagella are removed, the regeneration of the flagellar filament can then be studied Transport of many flagellar components is carried out by an apparatus in the basal body that is a specialized type III protein secretion system It is thought that flagellin subunits are transported through the filament’s hollow internal core When they reach the tip, the subunits spontaneously aggregate under the direction of a special filament cap so that the filament grows at its tip rather than at the base (figure 3.40) Filament synthesis is an excellent example of selfassembly Many structures form spontaneously through the association of their component parts without the aid of any special enzymes or other factors The information required for filament construction is present in the structure of the flagellin subunit itself mm (a) Pseudomonas—monotrichous polar flagellation mm (b) Spirillum—lophotrichous flagellation (c) P vulgaris—peritrichous flagellation Figure 3.38 Flagellar Distribution Examples of various patterns of flagellation as seen in the light microscope (a) Monotrichous polar (Pseudomonas) (b) Lophotrichous (Spirillum) (c) Peritrichous (Proteus vulgaris, ϫ600) The Mechanism of Flagellar Movement Procaryotic flagella operate differently from eucaryotic flagella The filament is in the shape of a rigid helix, and the cell moves when this helix rotates Considerable evidence shows that flagella act just like propellers on a boat Bacterial mutants with straight flagella or abnormally long hook regions cannot swim When bacteria are tethered to a glass slide using antibodies to filament or hook proteins, the cell body rotates rapidly about the stationary flagellum If polystyrene-latex beads are attached to flagella, the beads spin about the flagellar axis due to flagellar rotation The flagellar motor can rotate very rapidly The E coli motor rotates 270 revolutions per second; Vibrio alginolyticus averages 1,100 rps Cilia and flagella (section 4.10) The direction of flagellar rotation determines the nature of bacterial movement Monotrichous, polar flagella rotate counterclockwise (when viewed from outside the cell) during normal forward movement, whereas the cell itself rotates slowly clockwise The rotating helical flagellar filament thrusts the cell forward in a run with the flagellum trailing behind (figure 3.41) Monotrichous bacteria stop and tumble randomly by reversing the direction of flagellar rotation Peritrichously flagellated bacteria operate in a somewhat similar way To move forward, the flagella rotate counterclockwise As they so, they bend at their hooks to form a rotating bundle that propels the cell forward Clockwise rotation of the flagella disrupts the bundle and the cell tumbles Because bacteria swim through rotation of their rigid flagella, there must be some sort of motor at the base A rod extends from the hook and ends in the M ring, which can rotate freely in the plasma membrane (figure 3.42) It is thought that the S ring is attached to the cell wall in gram-positive cells and does not rotate The P and L rings of gram-negative bacteria would act as bearings for the rotating rod There is some evidence that the basal body is a passive structure and rotates within a membrane-embedded protein complex much like the rotor of an electrical motor turns in the center of a ring of electromagnets (the stator) The exact mechanism that drives basal body rotation is not entirely clear Figure 3.42 provides a more detailed depiction of wil92913_ch03.qxd 7/20/06 12:02 PM Page 69 Components External to the Cell Wall 69 Filament Hook L ring Outer membrane P ring Rod S ring Peptidoglycan layer Periplasmic space Plasma membrane M ring (a) 22 nm (b) 30 nm (c) Arrows indicate hooks and basal bodies (d) Figure 3.39 The Ultrastructure of Bacterial Flagella Flagellar basal bodies and hooks in (a) gram-negative and (b) gram-positive bacteria (c) Negatively stained flagella from Escherichia coli (ϫ66,000) (d) An enlarged view of the basal body of an E coli flagellum (ϫ485,000) All four rings (L, P, S, and M) can be clearly seen The uppermost arrow is at the junction of the hook and filament the basal body in gram-negative bacteria The rotor portion of the motor seems to be made primarily of a rod, the M ring, and a C ring joined to it on the cytoplasmic side of the basal body These two rings are made of several proteins; FliG is particularly important in generating flagellar rotation The two most important proteins in the stator part of the motor are MotA and MotB These form a proton channel through the plasma membrane, and MotB also anchors the Mot complex to cell wall peptidoglycan There is some evidence that MotA and FliG directly interact during flagellar rotation This rotation is driven by proton or sodium gradients in procaryotes, not directly by ATP as is the case with eucaryotic flagella The electron transport chain and oxidative phosphorylation (section 9.5) The flagellum is a very effective swimming device From the bacterium’s point of view, swimming is quite a task because the surrounding water seems as thick and viscous as molasses The cell wil92913_ch03.qxd 8/8/06 3:02 PM Page 70 70 Chapter Procaryotic Cell Structure and Function LPS Flagellin Filament cap protein Outer membrane Peptidoglycan Plasma membrane mRNA Ribosome Figure 3.40 Growth of Flagellar Filaments Flagellin subunits travel through the flagellar core and attach to the growing tip Their attachment is directed by the filament cap protein Forward run (a) (a) Tumble (b) (b) Forward run (c) (c) Tumble (d) Figure 3.41 Flagellar Motility The relationship of flagellar rotation to bacterial movement Parts (a) and (b) describe the motion of monotrichous, polar bacteria Parts (c) and (d) illustrate the movements of peritrichous organisms must bore through the water with its corkscrew-shaped flagella, and if flagellar activity ceases, it stops almost instantly Despite such environmental resistance to movement, bacteria can swim from 20 to almost 90 ␮m/second This is equivalent to traveling from to over 100 cell lengths per second In contrast, an exceptionally fast 6-ft human might be able to run around body lengths per second Bacteria can move by mechanisms other than flagellar rotation Spirochetes are helical bacteria that travel through viscous substances such as mucus or mud by flexing and spinning movements caused by a special axial filament composed of periplasmic flagella The swimming motility of the helical bacterium Spiroplasma is accomplished by the formation of kinks in the cell body that travel the length of the bacterium A very different type of motility, gliding motility, is employed by many bacteria: cyanobacteria, myxobacteria and cytophagas, and some mycoplasmas Although there are no visible external structures associated with gliding motility, it enables movement along solid surfaces at rates up to ␮m/second Microbial Diversity & Ecology 21.1: The mechanism of gliding motility; Phylum Spirochaetes (section 21.6); Photosynthetic bacteria (section 21.3); Class Deltaproteobacteria: Order Myxococcales (section 22.4); Class Mollicutes (the Mycoplasmas) (section 23.2) Briefly describe capsules, slime layers, glycocalyxes, and S-layers.What are their functions? Distinguish between fimbriae and sex pili,and give the function of each Be able to discuss the following:flagella distribution patterns,flagella structure and synthesis,and the way in which flagella operate to move a bacterium What is self-assembly? Why does it make sense that the flagellar filament is assembled in this way? wil92913_ch03.qxd 9/6/06 2:14 PM Page 71 Chemotaxis 71 Filament Hook L ring P ring Rod + H Outer membrane Figure 3.42 Mechanism of Flagellar Movement Peptidoglycan layer This diagram of a gram-negative flagellum shows some of the more important components and the flow of protons that drives rotation Five of the many flagellar proteins are labeled (MotA, MotB, FliG, FliM, FliN) Periplasmic space S ring M ring Plasma membrane MotB MotA FliG C ring FliM, N 3.10 CHEMOTAXIS Bacteria not always move aimlessly but are attracted by such nutrients as sugars and amino acids, and are repelled by many harmful substances and bacterial waste products Bacteria also can respond to other environmental cues such as temperature (thermotaxis), light (phototaxis), oxygen (aerotaxis), osmotic pressure (osmotaxis), and gravity; (Microbial Diversity & Ecology 3.2.) Movement toward chemical attractants and away from repellents is known as chemotaxis Such behavior is of obvious advantage to bacteria Chemotaxis may be demonstrated by observing bacteria in the chemical gradient produced when a thin capillary tube is filled with an attractant and lowered into a bacterial suspension As the attractant diffuses from the end of the capillary, bacteria collect and swim up the tube The number of bacteria within the capillary after a short length of time reflects the strength of attraction and rate of chemotaxis Positive and negative chemotaxis also can be studied with petri dish cultures (figure 3.43) If bacteria are placed in the center of a dish of semisolid agar containing an attractant, the bacteria will exhaust the local supply and then swim outward following the attractant gradient they have created The result is an expanding ring of bacteria When a disk of repellent is placed in a petri dish of semisolid agar and bacteria, the bacteria will swim away from the repellent, creating a clear zone around the disk (figure 3.44) Bacteria can respond to very low levels of attractants (about 10Ϫ8 M for some sugars), the magnitude of their response increasing with attractant concentration Usually they sense repellents only at higher concentrations If an attractant and a repellent are present together, the bacterium will compare both signals and respond to the chemical with the most effective concentration Attractants and repellents are detected by chemoreceptors, special proteins that bind chemicals and transmit signals to the other components of the chemosensing system About 20 attractant chemoreceptors and 10 chemoreceptors for repellents have been discovered thus far These chemoreceptor proteins may be located in the periplasmic space or the plasma membrane Some receptors participate in the initial stages of sugar transport into the cell The chemotactic behavior of bacteria has been studied using the tracking microscope, a microscope with a moving stage that automatically keeps an individual bacterium in view In the absence of a chemical gradient, E coli and other bacteria move randomly For a few seconds, the bacterium will travel in a straight or slightly curved line called a run When a bacterium is running, its flagella are organized into a coordinated, corkscrew-shaped bundle (figure 3.41c) Then the flagella “fly apart” and the bacterium will stop and tumble The tumble results in the random reorientation of the bacterium so that it often is facing in a different direction Therefore when it begins the next run, it usually goes in a different direction (figure 3.45a) In contrast, when the bacterium is exposed to an attractant, it tumbles less frequently (or has longer runs) when traveling towards the attractant Although the tumbles can still orient the bacterium away from the attractant, over time, the bacterium gets closer and closer to the attractant (figure 3.45b) The opposite response occurs with a repellent Tumbling frequency decreases (the run time lengthens) when the bacterium moves away from the repellent wil92913_ch03.qxd 7/20/06 12:02 PM Page 72 72 Chapter Procaryotic Cell Structure and Function Figure 3.43 Positive Bacterial Chemotaxis Chemotaxis can be demonstrated on an agar plate that contains various nutrients Positive chemotaxis by E coli on the left The outer ring is composed of bacteria consuming serine The second ring was formed by E coli consuming aspartate, a less powerful attractant The upper right colony is composed of motile, but nonchemotactic mutants The bottom right colony is formed by nonmotile bacteria Colony of motile but nonchemotactic bacteria Colony of chemotactic motile bacteria Colony of nonmotile bacteria Tumble Run (a) (b) Figure 3.44 Negative Bacterial Chemotaxis Negative chemotaxis by E coli in response to the repellent acetate The bright disks are plugs of concentrated agar containing acetate that have been placed in dilute agar inoculated with E coli Acetate concentration increases from zero at the top right to M at top left Note the increasing size of bacteria-free zones with increasing acetate The bacteria have migrated for 30 minutes Figure 3.45 Directed Movement in Bacteria (a) Random movement of a bacterium in the absence of a concentration gradient Tumbling frequency is fairly constant (b) Movement in an attractant gradient Tumbling frequency is reduced when the bacterium is moving up the gradient Therefore, runs in the direction of increasing attractant are longer wil92913_ch03.qxd 9/6/06 2:14 PM Page 73 The Bacterial Endospore Clearly, the bacterium must have some mechanism for sensing that it is getting closer to the attractant (or is moving away from the repellent) The behavior of the bacterium is shaped by temporal changes in chemical concentration The bacterium moves toward the attractant because it senses that the concentration of the attractant is increasing Likewise, it moves away from a repellent because it senses that the concentration of the repellent is decreasing The bacterium’s chemoreceptors play a critical role in this process The molecular events that enable bacterial cells to sense a chemical gradient and respond appropriately are presented in chapter 73 The cortex, which may occupy as much as half the spore volume, rests beneath the spore coat It is made of a peptidoglycan that is less cross-linked than that in vegetative cells The spore cell wall (or core wall) is inside the cortex and surrounds the protoplast or spore core The core has normal cell structures such as ribosomes and a nucleoid, but is metabolically inactive It is still not known precisely why the endospore is so resistant to heat and other lethal agents As much as 15% of the spore’s dry weight consists of dipicolinic acid complexed with calcium ions (figure 3.48), which is located in the core It has long been thought that dipicolinic acid was directly involved in Define chemotaxis, run, and tumble Explain in a general way how bacteria move toward substances like nutrients and away from toxic materials Central 3.11 THE BACTERIAL ENDOSPORE A number of gram-positive bacteria can form a special resistant, dormant structure called an endospore Endospores develop within vegetative bacterial cells of several genera: Bacillus and Clostridium (rods), Sporosarcina (cocci), and others These structures are extraordinarily resistant to environmental stresses such as heat, ultraviolet radiation, gamma radiation, chemical disinfectants, and desiccation In fact, some endospores have remained viable for around 100,000 years Because of their resistance and the fact that several species of endospore-forming bacteria are dangerous pathogens, endospores are of great practical importance in food, industrial, and medical microbiology This is because it is essential to be able to sterilize solutions and solid objects Endospores often survive boiling for an hour or more; therefore autoclaves must be used to sterilize many materials Endospores are also of considerable theoretical interest Because bacteria manufacture these intricate structures in a very organized fashion over a period of a few hours, spore formation is well suited for research on the construction of complex biological structures In the environment, endospores aid in survival when moisture or nutrients are scarce The use of physical methods in control: Heat (section 7.4) Endospores can be examined with both light and electron microscopes Because endospores are impermeable to most stains, they often are seen as colorless areas in bacteria treated with methylene blue and other simple stains; special endospore stains are used to make them clearly visible Endospore position in the mother cell (sporangium) frequently differs among species, making it of considerable value in identification Endospores may be centrally located, close to one end (subterminal), or definitely terminal (figure 3.46) Sometimes an endospore is so large that it swells the sporangium Preparation and staining of specimens (section 2.3) Electron micrographs show that endospore structure is complex (figure 3.47) The spore often is surrounded by a thin, delicate covering called the exosporium A spore coat lies beneath the exosporium, is composed of several protein layers, and may be fairly thick It is impermeable to many toxic molecules and is responsible for the spore’s resistance to chemicals The coat also is thought to contain enzymes that are involved in germination Subterminal Swollen sporangium Terminal Figure 3.46 Examples of Endospore Location and Size Ribosomes Nucleoid Core wall Cortex Spore coat Exosporium Figure 3.47 Endospore Structure Bacillus anthracis endospore (ϫ151,000) HOOC N Figure 3.48 Dipicolinic Acid COOH wil92913_ch03.qxd 7/20/06 12:02 PM Page 74 74 Chapter Procaryotic Cell Structure and Function N N 0.25 hrs Cell division Wall Free spore Spore coat Cortex Core SC OFM 10.5 hrs N Exosporium C VII Lysis of sporangium, spore liberation Spore coat VI Completion of coat synthesis, increase in refractility and heat resistance Exosporium V Coat synthesis Cortex C I Axial filament formation S M hrs Plasma membrane DNA II Septum formation and forespore development III Engulfment of forespore IV Cortex formation OFM N SC hrs IFM IFM OFM N 5.5 hrs N 6.5 hrs Figure 3.49 Endospore Formation: Life Cycle of Bacillus megaterium The stages are indicated by Roman numerals The circled numbers in the photographs refer to the hours from the end of the logarithmic phase of growth: 0.25 h—a typical vegetative cell; h–stage II cell, septation; 5.5 h–stage III cell, engulfment; 6.5 h–stage IV cell, cortex formation; h–stage V cell, coat formation; 10.5 h–stage VI cell, mature spore in sporangium Abbreviations used: C, cortex; IFM and OFM, inner and outer forespore membranes; M, mesosome; N, nucleoid; S, septum; SC, spore coats Bars ϭ 0.5 ␮m wil92913_ch03.qxd 7/20/06 12:02 PM Page 75 The Bacterial Endospore heat resistance, but heat-resistant mutants lacking dipicolinic acid have been isolated Calcium does aid in resistance to wet heat, oxidizing agents, and sometimes dry heat It may be that calcium-dipicolinate stabilizes the spore’s nucleic acids In addition, specialized small, acid-soluble DNA-binding proteins (SASPs), are found in the endospore They saturate spore DNA and protect it from heat, radiation, dessication, and chemicals Dehydration of the protoplast appears to be very important in heat resistance The cortex may osmotically remove water from the protoplast, thereby protecting it from both heat and radiation damage The spore coat also seems to protect against enzymes and chemicals such as hydrogen peroxide Finally, spores contain some DNA repair enzymes DNA is repaired once the spore germinates and the cell becomes active again In summary, endospore heat resistance probably is due to several factors: calcium-dipicolinate and acid-soluble protein stabilization of DNA, protoplast dehydration, the spore coat, DNA repair, the greater stability of cell proteins in bacteria adapted to growth at high temperatures, and others Endospore formation, also called sporogenesis or sporulation, normally commences when growth ceases due to lack of nutrients It is a complex process and may be divided into seven stages (figure 3.49) An axial filament of nuclear material forms (stage I), followed by an inward folding of the cell membrane to enclose part of the DNA and produce the forespore septum (stage II) The membrane continues to grow and engulfs the immature endospore in a second membrane (stage III) Next, cortex is laid down in the space between the two membranes, and both calcium and dipicolinic acid are accumulated (stage IV) Protein coats then are formed around the cortex (stage V), and maturation of the endospore occurs (stage VI) Finally, lytic enzymes destroy the sporangium releasing the spore (stage VII) Sporulation requires about 10 hours in Bacillus megaterium Global regulatory systems: Sporulation in Bacillus subtilus (section 12.5) The transformation of dormant spores into active vegetative cells seems almost as complex a process as sporogenesis It occurs in three stages: (1) activation, (2) germination, and (3) outgrowth (figure 3.50) Often a spore will not germinate successfully, even in a nutrient-rich medium, unless it has been activated Activation 75 0.5 mm Figure 3.50 Endospore Germination Clostridium pectinovorum emerging from the spore during germination is a process that prepares spores for germination and usually results from treatments like heating It is followed by germination, the breaking of the spore’s dormant state This process is characterized by spore swelling, rupture or absorption of the spore coat, loss of resistance to heat and other stresses, loss of refractility, release of spore components, and increase in metabolic activity Many normal metabolites or nutrients (e.g., amino acids and sugars) can trigger germination after activation Germination is followed by the third stage, outgrowth The spore protoplast makes new components, emerges from the remains of the spore coat, and develops again into an active bacterium Describe the structure of the bacterial endospore using a labeled diagram Briefly describe endospore formation and germination.What is the importance of the endospore? What might account for its heat resistance? How might one go about showing that a bacterium forms true endospores? Why you think dehydration of the protoplast is an important factor in the ability of endospores to resist environmental stress? Summary 3.1 An Overview of Procaryotic Cell Structure a Procaryotes may be spherical (cocci), rod-shaped (bacilli), spiral, or filamentous; they may form buds and stalks; or they may even have no characteristic shape at all (pleomorphic) (figure 3.1 and 3.2) b Procaryotic cells can remain together after division to form pairs, chains, and clusters of various sizes and shapes c Procaryotes are much simpler structurally than eucaryotes, but they have unique structures Table 3.1 summarizes the major functions of procaryotic cell structures 3.2 Procaryotic Cell Membranes a The plasma membrane fulfills many roles, including acting as a semipermeable barrier, carrying out respiration and photosynthesis, and detecting and responding to chemicals in the environment b The fluid mosaic model proposes that cell membranes are lipid bilayers in which integral proteins are buried (figure 3.5) Peripheral proteins are loosely associated with the membrane c Bacterial membranes are composed of phospholipids constructed of fatty acids connected to glycerol by ester linkages (figure 3.6) Bacterial membranes usually lack sterols, but often contain hopanoids (figure 3.7) wil92913_ch03.qxd 7/20/06 12:02 PM Page 76 76 Chapter Procaryotic Cell Structure and Function d The plasma membrane of some bacteria invaginates to form simple membrane systems containing photosynthetic and respiratory assemblies Other bacteria, like the cyanobacteria, have internal membranes (figure 3.8) e Archaeal membranes are composed of glycerol diether and diglycerol tetraether lipids (figure 3.9) Membranes composed of glycerol diether are lipid bilayers Membranes composed of diglycerol tetraethers are lipid monolayers (figure 3.11) The overall structure of a monolayer membrane is similar to that of the bilayer membrane in that the membrane has a hydrophobic core and its surfaces are hydrophilic 3.3 The Cytoplasmic Matrix a The cytoplasm of procaryotes contains proteins that are similar in structure and function to the cytoskeletal proteins observed in eucaryotes b The cytoplasmic matrix of procaryotes contains inclusion bodies Most are used for storage (glycogen inclusions, PHB inclusions, cyanophycin granules, carboxysomes, and polyphosphate granules), but others are used for other purposes (magnetosomes and gas vacuoles) c The cytoplasm of procaryotes is packed with 70S ribosomes (figure 3.15) 3.4 The Nucleoid a Procaryotic genetic material is located in an area called the nucleoid and is not usually enclosed by a membrane (figure 3.16) b In most procaryotes, the nucleoid contains a single chromosome The chromsosome consists of a double-stranded, covalently closed, circular DNA molecule 3.5 Plasmids a Plasmids are extrachromosomal DNA molecules They are found in many procaryotes b Although plasmids are not required for survival in most conditions, they can encode traits that confer selective advantage in some environments c Episomes are plasmids that are able to exist freely in the cytoplasm or can be integrated into the chromosome d Conjugative plasmids encode genes that promote their transfer from one cell to another e Resistance factors are plasmids that have genes conferring resistance to antibiotics f Col plasmids contain genes for the synthesis of colicins, proteins that kill E coli Other plasmids encode virulence factors or metabolic capabilities 3.6 The Bacterial Cell Wall a The vast majority of procaryotes have a cell wall outside the plasma membrane to give them shape and protect them from osmotic stress b Bacterial walls are chemically complex and usually contain peptidoglycan (figures 3.17–3.21) c Bacteria often are classified as either gram-positive or gram-negative based on differences in cell wall structure and their response to Gram staining d Gram-positive walls have thick, homogeneous layers of peptidoglycan and teichoic acids (figure 3.23) Gram-negative bacteria have a thin peptidoglycan layer surrounded by a complex outer membrane containing lipopolysaccharides (LPSs) and other components (figure 3.25) e The mechanism of the Gram stain is thought to depend on the peptidoglycan, which binds crystal violet tightly, preventing the loss of crystal violet during the ethanol wash 3.7 Archaeal Cell Walls a Archaeal cell walls not contain peptidoglycan (figure 3.30) b Archaea exhibit great diversity in their cell wall make-up Some archaeal cell walls are composed of heteropolysaccharides, some are composed of glycoprotein, and some are composed of protein 3.8 Protein Secretion in Procaryotes a The Sec-dependent protein secretion pathway (figure 3.32) has been observed in all domains of life It transports proteins across or into the cytoplasmic membrane b Gram-negative bacteria have additional protein secretion systems that allow them to move proteins from the cytoplasm, across both the cytoplasmic and outer membranes, to the outside of the cell (figure 3.33) Some of these systems work with the Sec-dependent pathway to accomplish this (Type II, Type V, and usually Type IV) Some pathways function alone to move proteins across both membranes (Types I and III) c ABC transporters (Type I protein secretion system) are used by all procaryotes for protein translocation 3.9 Components External to the Cell Wall a Capsules, slime layers, and glycocalyxes are layers of material lying outside the cell wall They can protect procaryotes from certain environmental conditions, allow procaryotes to attach to surfaces, and protect pathogenic bacteria from host defenses (figures 3.34 and 3.35) b S-layers are observed in some bacteria and many archaea They are composed of proteins or glycoprotein and have a characteristic geometric shape In many archaea the S-layer serves as the cell wall (figure 3.36) c Pili and fimbriae are hairlike appendages Fimbriae function primarily in attachment to surfaces, but some types of bacterial fimbriae are involved in a twitching motility Sex pili participate in the transfer of DNA from one bacterium to another (figure 3.37) d Many procaryotes are motile, usually by means of threadlike, locomotory organelles called flagella (figure 3.38) e Bacterial species differ in the number and distribution of their flagella f In bacteria, the flagellar filament is a rigid helix that rotates like a propeller to push the bacterium through water (figure 3.41) 3.10 Chemotaxis Motile procaryotes can respond to gradients of attractants and repellents, a phenomenon known as chemotaxis b A bacterium accomplishes movement toward an attractant by increasing the length of time it spends moving toward the attractant, shortening the time it spends tumbling Conversely, a bacterium increases its run time when it moves away from a repellent a 3.11 The Bacterial Endospore a Some bacteria survive adverse environmental conditions by forming endospores, dormant structures resistant to heat, desiccation, and many chemicals (figure 3.47) b Both endospore formation and germination are complex processes that begin in response to certain environmental signals and involve numerous stages (figures 3.49 and 3.50) wil92913_ch03.qxd 7/20/06 12:02 PM Page 77 Summary 77 Key Terms ABC protein secretion pathway 65 activation 75 amphipathic 45 amphitrichous 67 axial filament 70 bacillus 40 bacteriocin 53 basal body 67 capsule 65 carboxysomes 49 cell envelope 55 chemoreceptors 71 chemotaxis 71 coccus 39 Col plasmid 53 conjugative plasmid 53 core polysaccharide 58 cortex 73 curing 53 cyanophycin granules 49 cytoplasmic matrix 48 deoxyribonucleic acid (DNA) 52 diplococcus 39 endospore 73 episome 53 exoenzyme 58 exosporium 73 F factor 53 fimbriae 66 flagellar filament 67 flagellar hook 67 flagellin 67 flagellum 67 fluid mosaic model 44 gas vacuole 50 gas vesicles 50 germination 75 gliding motility 70 glycocalyx 65 glycogen 49 hopanoids 46 hydrophilic 45 hydrophobic 45 inclusion body 48 integral proteins 45 lipid A 58 lipopolysaccharides (LPSs) 58 lophotrichous 67 lysis 61 lysozyme 61 magnetosomes 50 metabolic plasmid 54 metachromatic granules 50 monotrichous 67 murein 55 mycelium 40 nucleoid 52 O antigen 60 O side chain 60 outer membrane 55 outgrowth 75 penicillin 61 peptide interbridge 56 peptidoglycan 55 peripheral proteins 45 periplasm 55 periplasmic space 55 peritrichous 67 plasma membrane 42 plasmid 53 plasmolysis 61 pleomorphic 41 polar flagellum 67 poly-␤-hydroxybutyrate (PHB) 49 polyphosphate granules 50 porin proteins 60 protoplast 48 pseudomurein 62 resistance factor (R factor, R plasmid) 53 ribosome 50 rod 40 run 71 Sec-dependent pathway 63 self-assembly 68 sex pili 67 signal peptide 63 S-layer 66 slime layer 65 spheroplast 61 spirilla 40 spirochete 40 sporangium 73 spore cell wall 73 spore coat 73 spore core 73 sporogenesis 75 sporulation 75 Svedberg unit 50 teichoic acid 57 tumble 71 type I protein secretion pathway 65 type II protein secretion pathway 65 type III protein secretion pathway 65 type IV protein secretion pathway 65 type V protein secretion pathway 65 vibrio 40 virulence plasmid 54 volutin granules 50 Critical Thinking Questions Propose a model for the assembly of a flagellum in a gram-positive cell envelope How would that model need to be modified for the assembly of a flagellum in a gram-negative cell envelope? If you could not use a microscope, how would you determine whether a cell is procaryotic or eucaryotic? Assume the organism can be cultured easily in the laboratory The peptidoglycan of bacteria has been compared with the chain mail worn beneath a medieval knight’s suit of armor It provides both protection and flexibility Can you describe other structures in biology that have an analogous function? How are they replaced or modified to accommodate the growth of the inhabitant? Learn More Cannon, G C.; Bradburne, C E.; Aldrich, H C.; Baker, S H.; Heinhorst, S.; and Shively, J M 2001 Microcompartments in prokaryotes: Carboxysomes and related polyhedra Appl Env Microbiol 67(12):5351–61 Drews, G 1992 Intracytoplasmic membranes in bacterial cells: Organization, function and biosynthesis In Prokaryotic structure and function, S Mohan, C Dow, and J A Coles, editors, 249–74 New York: Cambridge University Press Frankel, R B., and Bazylinski, D A 2004 Magnetosome mysteries ASM News 70(4):176–83 Ghuysen, J.-M., and Hekenbeck, R., editors 1994 Bacterial cell wall New York: Elsevier Gital, Z 2005 The new bacterial cell biology: Moving parts and subcellular architecture Cell 120:577–86 wil92913_ch03.qxd 7/20/06 12:02 PM Page 78 78 Chapter Procaryotic Cell Structure and Function Harshey, R M 2003 Bacterial motility on a surface: Many ways to a common goal Annu Rev Microbiol 57:249–73 Nikaido, H 2003 Molecular basis of bacterial outer membrane permeability revisited Microbiol Mol Biol Rev 67(4):593–656 Henderson, I R.; Navarro-Garcia, F.; Desvaux, M.; Fernandez, R C.; and Ala’Aldeen, D 2004 Type V protein secretion pathway: The autotransporter story Microbiol Mol Biol Rev 68(4):692–744 Parkinson, J S 2004 Signal amplification in bacterial chemotaxis through receptor teamwork ASM News 70(12):575–82 Hoppert, M., and Mayer, F 1999 Prokaryotes American Scientist 87:518–25 Robinow, C., and Kellenberger, E 1994 The bacterial nucleoid revisited Microbiol Rev 58(2):211–32 Kerfeld, C A.; Sawaya, M R.; Tanaka, A.; Nguyen, C V.; Phillips, M.; Beeby, M.; and Yeates, T O 2005 Protein structures forming the shell of primitive bacterial organelles Science 309:936–38 Sára, M., and Sleytr, U B 2000 S-layer proteins J Bacteriol 182(4):859–68 Kostakioti, M.; Newman, C L.; Thanassi, D G.; and Stathopoulos, C 2005 Mechanisms of protein export across the bacterial outer membrane J Bacteriol 187(13):4306–14 Schulz, H N., and Jorgensen, B B 2001 Big bacteria Annu Rev Microbiol 55:105–37 Macnab, R M 2003 How bacteria assemble flagella Annu Rev Microbiol 57:77–100 Scherrer, R 1984 Gram’s staining reaction, Gram types and cell walls of bacteria Trends Biochem Sci 9:242–45 Trun, N J., and Marko, J F 1998 Architecture of a bacterial chromosome ASM News 64(5):276–83 Walsby, A E 1994 Gas vesicles Microbiol Rev 58(1):94–144 Mattick, J S 2002 Type IV pili and twitching motility Annu Rev Microbiol 56:289–314 Walsby, A E 2005 Archaea with square cells Trends Microbiol 13(5):193–95 Nicholson, W L.; Munakata, N.; Horneck, G.; Melosh, H J.; and Setlow, P 2000 Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments Microbiol Mol Biol Rev 64(3):548–72 Wätermann, M., and Steinbüchel, A 2005 Neutral lipid bodies in prokaryotes: Recent insights into structure, formation, and relationship to eukaryotic lipid depots J Bacteriol 187(11):3607–19 Please visit the Prescott website at www.mhhe.com/prescott7 for additional references wil92913_ch04.qxd 7/27/06 12:35 PM Page 79 Eucaryotic Cell Structure and Function Often we emphasize procaryotes and viruses, but eucaryotic microorganisms also have major impacts on human welfare For example, the protozoan parasite Trypanosoma brucei gambiense is a cause of African sleeping sickness The organism invades the nervous system and the victim frequently dies after suffering several years from symptoms such as weakness, headache, apathy, emaciation, sleepiness, and coma PREVIEW • Eucaryotic cells differ most obviously from procaryotic cells in having a variety of complex membranous organelles in the cytoplasmic matrix and the majority of their genetic material within membrane-delimited nuclei Each organelle has a distinctive structure directly related to specific functions • A cytoskeleton composed of microtubules, microfilaments, and intermediate filaments helps give eucaryotic cells shape; the cytoskeleton is also involved in cell movements, intracellular transport, and reproduction • When eucaryotes reproduce, genetic material is distributed between cells by the highly organized, complex processes called mitosis and meiosis • Despite great differences between eucaryotes and procaryotes with respect to such things as morphology, they are similar on the biochemical level I n chapter considerable attention is devoted to procaryotic cell structure and function because procaryotes are immensely important in microbiology and have occupied a large portion of microbiologists’ attention in the past Nevertheless, protists and fungi also are microorganisms and have been extensively studied These eucaryotes often are extraordinarily complex, interesting in their own right, and prominent members of ecosystems (figure 4.1) In addition, many protists and fungi are important model organisms, as well as being exceptionally useful in industrial microbiology A number of protists and fungi are also major human pathogens; one only need think of candidiasis, malaria, or African sleeping sickness to appreciate the significance of eucaryotes in medical microbiology So although this text emphasizes procaryotes, eucaryotic microorganisms also demand attention and are briefly discussed in this chapter Chapter focuses on eucaryotic cell structure and its relationship to cell function Because many valuable studies on eucaryotic cell ultrastructure have used organisms other than microorganisms, some work on nonmicrobial cells is presented At the end of the chapter, procaryotic and eucaryotic cells are compared in some depth 4.1 AN OVERVIEW OF EUCARYOTIC CELL STRUCTURE The most obvious difference between eucaryotic and procaryotic cells is in their use of membranes Eucaryotic cells have membrane-delimited nuclei, and membranes also play a prominent part in the structure of many other organelles (figures 4.2 and 4.3) Organelles are intracellular structures that perform specific functions in cells analogous to the functions of organs in the body The name organelle (little organ) was coined because biologists saw a parallel between the relationship of organelles to a cell and that of organs to the whole body It is not satisfactory to define organelles as membrane-bound structures because this would exclude such components as ribosomes and bacterial flagella A comparison of figures 4.2 and 4.3 with figures 3.4 and 3.13a shows how structurally complex the eucaryotic cell is This complexity is due chiefly to the use of internal membranes for several purposes The partitioning of the eucaryotic cell interior by membranes makes possible the placement of different biochemical and physiological functions in separate compartments so that they can more easily take place simultaneously under independent control and proper coordination Large membrane surfaces The key to every biological problem must finally be sought in the cell —E B Wilson ... Fermentations 10 .7 16 7 16 9 16 9 17 0 17 1 12 12 .1 12.2 12 .3 12 .4 12 .5 12 .6 18 0 18 0 18 1 13 19 1 19 3 19 4 19 8 13 .1 13.2 13 .3 13 .4 13 .5 13 .6 13 .7 13 .8 13 .9 13 .10 13 .11 200 205 207 210 210 211 212 212 214 Part... Genetics: Gene Structure, Replication, and Expression 11 .1 11. 2 11 .3 11 .4 11 .5 11 .6 14 .10 247 ■ Historical Highlights 11 .1: The Elucidation of DNA Structure 248 DNA as Genetic Material The Flow... copyright page Library of Congress Cataloging-in-Publication Data Willey, Joanne M Prescott, Harley, and Klein’s microbiology / Joanne M Willey, Linda M Sherwood, Christopher J Woolverton — 7th