wiL75233_fm_i-xiv.indd Page i 11/28/07 10:28:45 AM epg /Volumes/ve401/MHIY034/mhwiL1%0/wiL1fm Prescott’s Principles of MICROBIOLOGY Joanne M Willey Hofstra University Linda M Sherwood Montana State University Christopher J Woolverton Kent State University wiL75233_fm_i-xiv.indd Page ii 11/28/07 10:28:46 AM epg /Volumes/ve401/MHIY034/mhwiL1%0/wiL1fm PRESCOTT’S PRINCIPLES OF MICROBIOLOGY Published by McGraw-Hill, a business unit of The McGraw-Hill Companies, Inc., 1221 Avenue of the Americas, New York, NY 10020 Copyright © 2009 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 acid-free paper DOW/DOW ISBN 978–0–07–337523–6 MHID 0–07–337523–3 Publisher: Michelle Watnick Senior Sponsoring Editor: James F Connely Senior Developmental Editor: Lisa A Bruflodt Senior Marketing Manager: Tami Petsche Project Coordinator: Mary Jane Lampe Lead Production Supervisor: Sandy Ludovissy Lead Media Project Manager: Stacy A Patch Designer: John Joran Lead Photo Research Coordinator: Carrie Burger Photo Research: Mary Reeg Compositor: Aptara Typeface: 10/12 Times Roman Printer: R R Donnelley Willard, OH (USE) Cover Image (Front and Back): ©Dennis Kunkel Microscopy, Inc 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’s principles of microbiology / Joanne M Willey, Linda M Sherwood, Christopher J Woolverton.— 1st ed p cm “In using the seventh edition of PHK’s microbiology as the foundation for the development of principles, we identified two overarching goals.” Includes index ISBN 978–0–07–337523–6 — ISBN 0–07–337523–3 (hard copy : alk paper) Microbiology I Sherwood, Linda II Woolverton, Christopher J III Prescott, Lansing M Microbiology IV Title V Title: Microbiology QR41.2.W545 2009 616.9’041—dc22 2007040352 www.mhhe.com wiL75233_fm_i-xiv.indd Page iii 11/28/07 10:28:46 AM epg /Volumes/ve401/MHIY034/mhwiL1%0/wiL1fm Brief Table of Contents Part One INTRODUCTION TO MICROBIOLOGY Part Six The History and Scope of Microbiology Microscopes and the Study of Microbial Structure 13 Procaryotic Cell Structure and Function 33 Eucaryotic Cell Structure and Function 65 Viruses and Other Acellular Agents 87 25 26 27 Biogeochemical Cycling and the Study of Microbial Ecology 593 Microorganisms in Natural Environments 608 Microbial Interactions 641 Part Seven Part Two MICROBIAL NUTRITION, GROWTH, AND CONTROL Microbial Nutrition 109 Microbial Growth 126 Control of Microorganisms 28 29 153 10 11 MICROBIAL METABOLISM Introduction to Metabolism 169 Catabolism: Energy Release and Conservation Anabolism: The Use of Energy in Biosynthesis 30 31 32 33 12 13 14 15 16 17 18 19 20 21 22 23 24 MICROBIAL DISEASES AND THEIR CONTROL 188 219 MICROBIAL MOLECULAR BIOLOGY AND GENETICS Genes: Structure, Replication, and Expression 240 Regulation of Gene Expression 277 Mechanisms of Genetic Variation 300 Microbial Genomics 332 Biotechnology and Industrial Microbiology 351 Part Five 661 Pathogenicity of Microorganisms 726 Antimicrobial Chemotherapy 746 Clinical Microbiology and Immunology 768 The Epidemiology of Infectious Disease 787 Part Nine Part Four HOST DEFENSES Nonspecific (Innate) Host Resistance Specific (Adaptive) Immunity 689 Part Eight Part Three ECOLOGY AND SYMBIOSIS THE DIVERSITY OF THE MICROBIAL WORLD Microbial Evolution, Taxonomy, and Diversity 381 The Archaea 405 The Deinococci and Gram-Negative Nonproteobacteria 420 The Proteobacteria 439 The Low G + C Gram-Positive Bacteria 474 The High G + C Gram-Positive Bacteria 499 Eucaryotic Microbes 517 Viral Diversity 554 34 35 APPLIED MICROBIOLOGY Microbiology of Food 809 Applied Environmental Microbiology Appendix I A Review of the Chemistry of Biological Molecules A-1 Appendix II Common Metabolic Pathways Glossary Credits Index 831 A-13 G-1 C-1 I-1 iii wiL75233_fm_i-xiv.indd Page iv 11/28/07 10:28:47 AM epg /Volumes/ve401/MHIY034/mhwiL1%0/wiL1fm Table of Contents About the Authors Preface x Part One INTRODUCTION TO MICROBIOLOGY The History and Scope of Microbiology 1.1 Members of the Microbial World 1.2 Scope and Relevance of Microbiology 1.3 Discovery of Microorganisms 1.4 Conflict over Spontaneous Generation 1.5 Golden Age of Microbiology Disease 1.1: A Molecular Approach to Koch’s Postulates 10 1.6 Development of Industrial Microbiology and Microbial Ecology 11 Microscopes and the Study of Microbial Structure 13 2.1 Lenses and the Bending of Light 14 2.2 Light Microscopes 14 2.3 Preparation and Staining of Specimens 21 2.4 Electron Microscopy 24 2.5 Newer Techniques in Microscopy 29 iv ix Procaryotic Cell Structure and Function 33 3.1 Overview of Procaryotic Cell Structure 34 Microbial Diversity & Ecology 3.1: Monstrous Microbes 36 3.2 Procaryotic Cell Membranes 38 3.3 Procaryotic Cytoplasm 42 3.4 Bacterial Cell Walls 46 3.5 Archaeal Cell Walls 53 3.6 Components External to the Cell Wall 53 3.7 Bacterial Motility and Chemotaxis 57 3.8 Bacterial Endospores 60 Eucaryotic Cell Structure and Function 65 4.1 Overview of Eucaryotic Cell Structure 66 4.2 Eucaryotic Membranes 67 4.3 Eucaryotic Cytoplasm 67 4.4 Organelles of the Biosynthetic-Secretory and Endocytic Pathways 70 4.5 Organelles Involved in Genetic Control of the Cell 73 4.6 Organelles Involved in Energy Conservation 75 4.7 Structures External to the Plasma Membrane 76 4.8 Comparison of Procaryotic and Eucaryotic Cells 79 4.9 Overview of Protist Structure and Function 79 4.10 Overview of Fungal Structure and Function 82 Viruses and Other Acellular Agents 87 5.1 Introduction to Viruses 88 5.2 Structure of Viruses 88 Microbial Diversity & Ecology 5.1: Host-Independent Growth of an Archaeal Virus 89 5.3 Viral Multiplication 95 5.4 Types of Viral Infections 99 5.5 Cultivation and Enumeration of Viruses 103 5.6 Viroids and Virusoids 105 5.7 Prions 106 Part Two MICROBIAL NUTRITION, GROWTH, AND CONTROL Microbial Nutrition 109 6.1 Elements of Life 110 6.2 Requirements for Carbon, Hydrogen, Oxygen, and Electrons 110 6.3 Nutritional Types of Microorganisms 111 6.4 Requirements for Nitrogen, Phosphorus, and Sulfur 113 6.5 Growth Factors 113 6.6 Uptake of Nutrients 114 6.7 Culture Media 118 Techniques & Applications 6.1: Enrichment Cultures 121 6.8 Isolation of Pure Cultures 121 Microbial Growth 126 7.1 Bacterial Cell Cycle 127 7.2 Growth Curve 130 7.3 Measurement of Microbial Growth 134 7.4 Continuous Culture of Microorganisms 136 7.5 Influences of Environmental Factors on Growth 138 Microbial Diversity & Ecology 7.1: Life Above 100°C 144 7.6 Microbial Growth in Natural Environments 148 Control of Microorganisms 153 8.1 Definitions of Frequently Used Terms 154 8.2 The Pattern of Microbial Death 154 8.3 Conditions Influencing the Effectiveness of Antimicrobial Agents 156 8.4 The Use of Physical Methods in Control 157 wiL75233_fm_i-xiv.indd Page v 11/28/07 10:29:45 AM epg /Volumes/ve401/MHIY034/mhwiL1%0/wiL1fm Table of Contents 12.3 12.4 12.5 12.6 12.7 12.8 8.5 The Use of Chemical Agents in Control 160 Techniques & Applications 8.1: Standard Microbiological Practices 162 8.6 Evaluation of Antimicrobial Agent Effectiveness 165 8.7 Biological Control of Microorganisms 167 Part Three 10 11 Catabolism: Energy Release and Conservation 188 10.1 Chemoorganotrophic Fueling Processes 189 10.2 Aerobic Respiration 190 10.3 Breakdown of Glucose to Pyruvate 192 10.4 Tricarboxylic Acid Cycle 195 10.5 Electron Transport and Oxidative Phosphorylation 197 10.6 Anaerobic Respiration 200 10.7 Fermentation 202 10.8 Catabolism of Carbohydrates and Intracellular Reserve Polymers 206 10.9 Lipid Catabolism 207 10.10 Protein and Amino Acid Catabolism 207 10.11 Chemolithotrophy 208 Microbial Diversity & Ecology 10.1: Acid Mine Drainage 210 10.12 Phototrophy 211 Anabolism: The Use of Energy in Biosynthesis 219 11.1 Principles Governing Biosynthesis 220 11.2 Precursor Metabolites 221 11.3 CO2 Fixation 222 11.4 Synthesis of Sugars and Polysaccharides 224 11.5 Synthesis of Amino Acids 228 11.6 Synthesis of Purines, Pyrimidines, and Nucleotides 233 11.7 Lipid Synthesis 236 Part Four 12 MICROBIAL MOLECULAR BIOLOGY AND GENETICS Genes: Structure, Replication, and Expression 240 12.1 Flow of Genetic Information 241 12.2 Nucleic Acid Structure 242 DNA Replication 245 Gene Structure 253 Transcription 255 The Genetic Code 262 Translation 263 Protein Maturation and Secretion 270 13 Regulation of Gene Expression 277 13.1 Levels of Regulation of Gene Expression 278 13.2 Regulation of Transcription Initiation 279 13.3 Regulation of Transcription Elongation 287 13.4 Regulation at the Level of Translation 289 13.5 Global Regulatory Systems 290 13.6 Regulation of Gene Expression in Eucarya and Archaea 296 14 Mechanisms of Genetic Variation 300 14.1 Mutations and Their Chemical Basis 301 14.2 Detection and Isolation of Mutants 306 14.3 DNA Repair 308 14.4 Creating Genetic Variability 311 14.5 Transposable Elements 312 14.6 Bacterial Plasmids 316 14.7 Bacterial Conjugation 317 14.8 Bacterial Transformation 322 14.9 Transduction 324 14.10 Mapping the Genome 326 15 Microbial Genomics 332 15.1 Introduction 333 15.2 Determining DNA Sequences 333 15.3 Whole-Genome Shotgun Sequencing 335 15.4 Bioinformatics 337 15.5 Functional Genomics 337 15.6 Proteomics 344 15.7 Comparative Genomics 346 15.8 Environmental Genomics 348 16 Biotechnology and Industrial Microbiology 351 16.1 Key Developments in Recombinant DNA Technology 352 16.2 Polymerase Chain Reaction 357 16.3 Gel Electrophoresis 357 16.4 Cloning Vectors and Creating Recombinant DNA 359 16.5 Construction of Genomic Libraries 363 16.6 Introducing Recombinant DNA into Host Cells 365 16.7 Expressing Foreign Genes in Host Cells 365 16.8 Microorganisms Used in Industrial Microbiology 366 Techniques & Applications 16.1: Visualizing Proteins with Green Fluorescence 367 16.9 Microorganism Growth in Controlled Environments 370 16.10 Major Products of Industrial Microbiology 371 16.11 Recombinant DNA Technology in Agriculture 374 MICROBIAL METABOLISM Introduction to Metabolism 169 9.1 Energy and Work 170 9.2 Laws of Thermodynamics 170 9.3 Free Energy and Reactions 170 9.4 ATP 171 9.5 Oxidation-Reduction Reactions 173 9.6 Electron Transport Chains 174 9.7 Enzymes 176 9.8 Ribozymes 180 9.9 Regulation of Metabolism 181 9.10 Posttranslational Regulation of Enzyme Activity 182 v wiL75233_fm_i-xiv.indd Page vi 11/28/07 10:30:11 AM epg vi /Volumes/ve401/MHIY034/mhwiL1%0/wiL1fm Table of Contents 16.12 Microbes as Products 376 Techniques & Applications 16.2: Streptavidin-Biotin Binding and Biotechnology 378 Part Five 17 22 The High G + C Gram-Positive Bacteria 499 22.1 General Properties of the Actinomycetes 500 22.2 Suborder Actinomycineae 503 22.3 Suborder Micrococcineae 503 22.4 Suborder Corynebacterineae 505 22.5 Suborder Micromonosporineae 511 22.6 Suborder Propionibacterineae 511 22.7 Suborder Streptomycineae 512 22.8 Suborder Streptosporangineae 514 22.9 Suborder Frankineae 514 22.10 Order Bifidobacteriales 514 23 Eucaryotic Microbes 517 23.1 Introduction 519 23.2 Protist Classification 519 Disease 23.1: A Brief History of Malaria 534 23.3 Characteristics of the Fungal Divisions 540 24 Viral Diversity 554 24.1 Principles of Virus Taxonomy 555 24.2 Viruses with Double-Stranded DNA Genomes (Group I) 555 24.3 Viruses with Single-Stranded DNA Genomes (Group II) 571 24.4 Viruses with Double-Stranded RNA Genomes (Group III) 573 24.5 Viruses with Plus-Strand RNA Genomes (Group IV) 574 Microbial Diversity & Ecology 24.1: SARS: Evolution of a Virus 579 24.6 Viruses with Minus-Strand RNA Genomes (Group V) 580 24.7 Viruses with Single-Stranded RNA Genomes (Group VI-Retroviruses) 584 24.8 Viruses with Gapped DNA Genomes (Group VII) 589 THE DIVERSITY OF THE MICROBIAL WORLD Microbial Evolution, Taxonomy, and Diversity 381 17.1 Microbial Evolution 382 17.2 Introduction to Microbial Classification and Taxonomy 389 17.3 Taxonomic Ranks 390 17.4 Techniques for Determining Microbial Taxonomy and Phylogeny 392 17.5 Phylogenetic Trees 398 17.6 The Major Divisions of Life 399 17.7 Bergey’s Manual of Systematic Bacteriology 400 Microbial Diversity & Ecology 17.1: “Official” Nomenclature Lists— A Letter from Bergey’s 401 18 The Archaea 405 18.1 Introduction to the Archaea 405 18.2 Phylum Crenarchaeota 411 18.3 Phylum Euryarchaeota 413 Microbial Diversity & Ecology 18.1: Methanotrophic Archaea 414 19 The Deinococci and Gram-Negative Nonproteobacteria 420 19.1 Aquificae and Thermotogae 421 19.2 Deinococcus-Thermus 421 19.3 Photosynthetic Bacteria 422 19.4 Phylum Planctomycetes 429 19.5 Phylum Chlamydiae 429 19.6 Phylum Spirochaetes 432 19.7 Phylum Bacteroidetes 436 Part Six 20 21 The Proteobacteria 439 20.1 Class Alphaproteobacteria 440 20.2 Class Betaproteobacteria 448 20.3 Class Gammaproteobacteria 453 Microbial Diversity & Ecology 20.1: Bacterial Bioluminescence 459 20.4 Class Deltaproteobacteria 467 20.5 Class Epsilonproteobacteria 471 The Low G + C Gram-Positive Bacteria 474 21.1 Class Mollicutes (The Mycoplasmas) 475 21.2 Peptidoglycan and Endospore Structure 477 Microbial Tidbits 21.1: Spores in Space 479 21.3 Class Clostridia 479 21.4 Class Bacilli 483 ECOLOGY AND SYMBIOSIS 25 Biogeochemical Cycling and the Study of Microbial Ecology 593 Microbial Diversity & Ecology 25.1: Microbial Ecology versus Environmental Microbiology 594 25.1 Biogeochemical Cycling 594 25.2 Microbial Ecology and Its Methods: An Overview 601 26 Microorganisms in Natural Environments 608 26.1 Marine and Freshwater Microbiology 609 26.2 Microorganisms in Terrestrial Environments 621 Microbial Diversity & Ecology 26.1: Mycorrhizae and the Evolution of Vascular Plants 628 wiL75233_fm_i-xiv.indd Page vii 11/28/07 10:31:08 AM epg /Volumes/ve401/MHIY034/mhwiL1%0/wiL1fm Table of Contents 27 Part Seven 28 29 31 HOST DEFENSES Nonspecific (Innate) Host Resistance 661 28.1 Overview of Host Resistance 662 28.2 Cells, Tissues, and Organs of the Immune System 663 28.3 Phagocytosis 670 28.4 Inflammation 673 28.5 Physical Barriers in Nonspecific (Innate) Resistance 675 28.6 Chemical Mediators in Nonspecific (Innate) Resistance 679 32 Clinical Microbiology and Immunology 768 32.1 Overview of the Clinical Microbiology Laboratory 769 32.2 Identification of Microorganisms from Specimens 769 Techniques & Applications 32.1: Standard Microbiological Practices 770 32.3 Clinical Immunology 779 33 The Epidemiology of Infectious Disease 787 33.1 Epidemiological Terminology 788 Historical Highlights 33.1: John Snow—The First Epidemiologist 788 33.2 Measuring Frequency 789 33.3 Recognition of an Infectious Disease in a Population 789 Historical Highlights 33.2: “Typhoid Mary” 791 33.4 Recognition of an Epidemic 791 33.5 The Infectious Disease Cycle: Story of a Disease 793 33.6 Virulence and the Mode of Transmission 797 33.7 Emerging and Reemerging Infectious Diseases and Pathogens 798 33.8 Control of Epidemics 801 Historical Highlights 33.3: The First Immunizations 802 33.9 Bioterrorism Preparedness 804 33.10 Nosocomial Infections 806 Specific (Adaptive) Immunity 689 29.1 Overview of Specific (Adaptive) Immunity 690 29.2 Antigens 691 29.3 Types of Specific (Adaptive) Immunity 694 29.4 Recognition of Foreignness 695 29.5 T-Cell Biology 697 29.6 B-Cell Biology 701 29.7 Antibodies 704 29.8 Action of Antibodies 712 Techniques & Applications 29.1: Monoclonal Antibody Technology 713 29.9 Summary: The Role of Antibodies and Lymphocytes in Immune Defense 715 29.10 Acquired Immune Tolerance 716 29.11 Immune Disorders 716 Part Eight 30 31.5 Factors Influencing Antimicrobial Drug Effectiveness 757 31.6 Drug Resistance 758 Disease 31.1: Antibiotic Misuse and Drug Resistance 758 31.7 Antifungal Drugs 762 31.8 Antiviral Drugs 763 31.9 Antiprotozoan Drugs 765 Microbial Interactions 641 27.1 Microbial Interactions 642 Microbial Diversity & Ecology 27.1: Wolbachia pipientis: The World’s Most Infectious Microbe? 645 27.2 Human-Microbe Interactions 653 27.3 Normal Microbiota of the Human Body 654 MICROBIAL DISEASES AND THEIR CONTROL Pathogenicity of Microorganisms 726 30.1 Host-Parasite Relationships 727 30.2 Pathogenesis of Viral Diseases 728 30.3 Overview of Bacterial Pathogenesis 730 30.4 Toxigenicity 736 Techniques & Applications 30.1: Detection and Removal of Endotoxins 741 30.5 Polymicrobial Disease 742 Antimicrobial Chemotherapy 746 31.1 The Development of Chemotherapy 747 31.2 General Characteristics of Antimicrobial Drugs 748 31.3 Determining the Level of Antimicrobial Activity 748 31.4 Antibacterial Drugs 752 Part Nine 34 APPLIED MICROBIOLOGY Microbiology of Food 809 34.1 Microorganism Growth in Foods 810 34.2 Microbial Growth and Food Spoilage 811 34.3 Controlling Food Spoilage 814 34.4 Food-Borne Diseases 816 34.5 Detection of Food-Borne Pathogens 820 34.6 Microbiology of Fermented Foods 821 Techniques & Applications 34.1: Chocolate: The Sweet Side of Fermentation 822 34.7 Microorganisms as Foods and Food Amendments 829 vii wiL75233_fm_i-xiv.indd Page viii 11/28/07 10:32:47 AM epg viii /Volumes/ve401/MHIY034/mhwiL1%0/wiL1fm Table of Contents 35 Applied Environmental Microbiology 831 35.1 Water Purification and Sanitary Analysis 832 Techniques & Applications 35.1: Waterborne Diseases, Water Supplies, and Slow Sand Filtration 833 35.2 Wastewater Treatment 836 35.3 Biodegradation and Bioremediation by Natural Communities 842 35.4 Bioaugmentation 845 Microbial Diversity & Ecology 35.2: A Fungus with a Voracious Appetite 846 Appendix I A Review of the Chemistry of Biological Molecules A-1 Appendix II Common Metabolic Pathways Glossary G-1 Credits C-1 Index I-1 A-13 wiL75233_ch03_033-064.indd Page 49 10/22/07 1:57:11 PM e /Volumes/ju103/HCAC039/sxn_m5_SM_indd%0/H5_TX_SM_L111-120_Inv12 3.4 Lipoteichoic acid Bacterial Cell Walls Teichoic acid 49 O O P O- Peptidoglycan O CH2 H C O R CH2 O O P O- O Periplasmic space CH2 H C O R Plasma membrane CH2 O O P O- O Figure 3.24 Teichoic Acid Structure Figure 3.23 The Gram-Positive Envelope glycerol or ribitol joined by phosphate groups (figure 3.23 and figure 3.24) Amino acids such as D-alanine or sugars such as glucose are attached to the glycerol and ribitol groups The teichoic acids are covalently connected to the peptidoglycan itself or to plasma membrane lipids; in the latter case, they are called lipoteichoic acids Teichoic acids appear to extend to the surface of the peptidoglycan Because they are negatively charged, they help give the gram-positive cell wall its negative charge The functions of techoic acids are still unclear, but they may be important in maintaining the structure of the wall Teichoic acids are not present in gram-negative bacteria The periplasmic space of gram-positive bacteria lies between the plasma membrane and the cell wall, and is smaller than that of gram-negative bacteria The periplasm has relatively few proteins; this is probably because the peptidoglycan sac is porous and any proteins secreted by the cell usually pass through it Enzymes secreted by gram-positive bacteria are called exoenzymes They often serve to degrade polymeric nutrients that would otherwise be too large for transport across the plasma membrane Those proteins that remain in the periplasmic space are usually attached to the plasma membrane Staphylococci and most other gram-positive bacteria have a layer of proteins on the surface of the peptidoglycan These proteins are involved in interactions of the cell with its environment Some are noncovalently attached by binding to the peptidoglycan, teichoic acids, or other receptors For example, the S-layer proteins (p 54) bind noncovalently to polymers scattered throughout the cell wall Enzymes involved in peptidoglycan synthesis and turnover also seem to interact noncovalently with the cell wall Other surface proteins are covalently attached to the peptidoglycan Many covalently attached proteins, such as the M protein of pathogenic streptococci, The segment of a teichoic acid made of phosphate, glycerol, and a side chain, R R may represent D-alanine, glucose, or other molecules have roles in virulence, such as aiding in adhesion to host tissues and interfering with host defenses In staphylococci, these surface proteins are covalently joined to the pentaglycine interbridge of the peptidoglycan (figure 3.20b) An enzyme called sortase catalyzes the attachment of these surface proteins to the peptidoglycan Sortases are attached to the plasma membrane of the cell Gram-Negative Cell Walls Even a brief inspection of figure 3.17 shows that gram-negative cell walls are much more complex than gram-positive walls The thin peptidoglycan layer next to the plasma membrane and bounded on either side by the periplasmic space usually constitutes only to 10% of the wall weight In E coli, it is about nm thick and contains only one or two sheets of peptidoglycan The periplasmic space of gram-negative bacteria is also strikingly different from that of gram-positive bacteria It ranges in width from nm to as great as 71 nm Some recent studies indicate that it may constitute about 20 to 40% of the total cell volume, and it is usually 30 to 70 nm wide When cell walls are disrupted carefully or removed without disturbing the underlying plasma membrane, periplasmic enzymes and other proteins are released and may be easily studied Some periplasmic proteins participate in nutrient acquisition—for example, hydrolytic enzymes and transport proteins Some periplasmic proteins are involved in energy conservation For example, the denitrifying bacteria, which convert nitrate to nitrogen gas, and bacteria that use inorganic molecules as energy sources (chemolithotrophs) have electron transport proteins in their periplasm Other periplasmic proteins are involved in peptidoglycan synthesis and the modification of toxic compounds that could harm the wiL75233_ch03_033-064.indd Page 50 10/22/07 1:57:12 PM e 50 Chapter /Volumes/ju103/HCAC039/sxn_m5_SM_indd%0/H5_TX_SM_L111-120_Inv12 Procaryotic Cell Structure and Function >> Chemolithotrophy (section 10.11); Biogeochemical cycling: The nitrogen cycle (section 25.1) cell extending outward from the core It has several peculiar sugars and varies in composition between bacterial strains LPS has many important functions Because the core polysaccharide usually contains charged sugars and phosphate (figure 3.26), LPS contributes to the negative charge on the bacterial surface LPS helps stabilize outer membrane structure because lipid A is a major constituent of the exterior leaflet of the outer membrane LPS may contribute to bacterial attachment to surfaces and biofilm formation A major function of LPS is that it helps create a permeability barrier The geometry of LPS (figure 3.26b) and interactions between neighboring LPS molecules are thought to restrict the entry of bile salts, antibiotics, and other toxic substances that might kill or injure the bacterium LPS also plays a role in protecting pathogenic gram-negative bacteria from host defenses The O side chain of LPS is also called the O antigen because it elicits an immune response by an infected host This response involves the production of antibodies that bind the strain-specific form of LPS that elicited the response However, many gram-negative bacteria can rapidly change the antigenic nature of their O side chains, thus thwarting host defenses Importantly, the lipid A portion of LPS is toxic; as a result, LPS can act as an endotoxin and cause some of the symptoms that arise in gram-negative bacterial infections If LPS or lipid A enters the bloodstream, a form of septic shock develops, for which there is no direct treatment >> Antibodies (section 29.7); The outer membrane lies outside the thin peptidoglycan layer and is linked to the cell in two ways (figure 3.25) The first is by Braun’s lipoprotein, the most abundant protein in the outer membrane This small lipoprotein is covalently joined to the underlying peptidoglycan and is embedded in the outer membrane by its hydrophobic end The second linking mechanism involves the many adhesion sites joining the outer membrane and the plasma membrane The two membranes appear to be in direct contact at these sites In E coli, 20 to 100 nm areas of contact between the two membranes can be seen Adhesion sites may be regions of direct contact or possibly true membrane fusions Possibly the most unusual constituents of the outer membrane are its lipopolysaccharides (LPSs) These large, complex molecules contain both lipid and carbohydrate, and consist of three parts: (1) lipid A, (2) the core polysaccharide, and (3) the O side chain The LPS from Salmonella has been studied most, and its general structure is described here (figure 3.26) The lipid A region contains two glucosamine sugar derivatives, each with three fatty acids and phosphate or pyrophosphate attached The fatty acids of lipid A are embedded in the outer membrane, while the remainder of the LPS molecule projects from the surface The core polysaccharide is joined to lipid A In Salmonella, it is constructed of 10 sugars, many of them unusual in structure The O side chain or O antigen is a polysaccharide chain O-specific side chains of Lipopolysaccharide (LPS) Overview of bacterial pathogenesis (section 30.3) Porin Braun’s lipoprotein Outer membrane Periplasmic space and peptidoglycan Plasma membrane Phospholipid Peptidoglycan Integral protein Figure 3.25 The Gram-Negative Envelope wiL75233_ch03_033-064.indd Page 51 10/22/07 1:57:13 PM e /Volumes/ju103/HCAC039/sxn_m5_SM_indd%0/H5_TX_SM_L111-120_Inv12 3.4 Man Bacterial Cell Walls 51 Abe Rha Gal Man n O side chain Abe Rha Gal Glc NAG Gal Glc Gal Core polysaccharide Hep Hep P ethanolamine P KDO KDO P GlcN KDO GlcN P ethanolamine P Fatty acid (a) Lipid A (b) Figure 3.26 Lipopolysaccharide Structure (a) The lipopolysaccharide from Salmonella This slightly simplified diagram illustrates one form of the LPS Abbreviations: Abe, abequose; Gal, galactose; Glc, glucose; GlcN, glucosamine; Hep, heptulose; KDO, 2-keto-3-deoxyoctonate; Man, mannose; NAG, N-acetylglucosamine; P, phosphate; Rha, L-rhamnose Lipid A is buried in the outer membrane (b) Molecular model of an Escherichia coli lipopolysaccharide The lipid A and core polysaccharide are straight; the O side chain is bent at an angle in this model Despite the role of LPS in creating a permeability barrier, the outer membrane is more permeable than the plasma membrane and permits the passage of small molecules such as glucose and other monosaccharides This is due to the presence of porin proteins Most porin proteins cluster together to form a trimer in the outer membrane (figure 3.25 and figure 3.27) Each porin protein spans the outer membrane and is more or less tube-shaped; its narrow channel allows passage of molecules smaller than about 600 to 700 daltons However, larger molecules such as vitamin B12 also cross the outer membrane Such large molecules not pass through porins; instead, specific carriers transport them across the outer membrane >> Uptake of nutrients (section 6.6) Mechanism of Gram Staining The difference between gram-positive and gram-negative bacteria is thought to be due to the physical nature of their cell walls If the cell wall is removed from gram-positive bacteria, they stain gram negative Furthermore, genetically wall-less bacteria such as the mycoplasmas also stain gram negative During the procedure, bacteria are first stained with crystal violet and next treated with iodine to promote dye retention When bacteria are treated with ethanol in the decolorization step, the alcohol is thought to shrink the pores of the thick peptidoglycan found in gram-positive bacteria, causing the peptidoglycan to act as a permeability barrier that prevents loss of crystal violet Thus the dye-iodine complex is retained during the decolorization step and the bacteria remain purple In contrast, gram-negative peptidoglycan is very thin, not as highly cross-linked, and has larger pores Alcohol treatment also may extract enough lipid from the outer membrane to increase the cell wall’s porosity further For these reasons, alcohol more readily removes the crystal violet-iodine complex from gram-negative bacteria Thus gram-negative bacteria are easily stained red or pink by the counterstain safranin Cell Walls and Osmotic Protection Microbes have several mechanisms for responding to changes in osmotic pressure This pressure arises when the concentration of solutes inside the cell differs from that outside, and the adaptive responses work to equalize the solute concentrations However, in certain situations, the osmotic pressure can exceed the cell’s ability to adapt In these cases, additional protection is provided by the cell wall When cells are in hypotonic solutions—ones in which the solute concentration is less than that in the cytoplasm—water moves into the cell, causing it to swell Without the cell wall, the pressure on the plasma membrane would become so great that the membrane would be disrupted and the cell would burst—a process wiL75233_ch03_033-064.indd Page 52 10/22/07 1:57:14 PM e 52 Chapter /Volumes/ju103/HCAC039/sxn_m5_SM_indd%0/H5_TX_SM_L111-120_Inv12 Procaryotic Cell Structure and Function called lysis Conversely, in hypertonic solutions, water flows out and the cytoplasm shrivels up—a process called plasmolysis The protective nature of the cell wall is most clearly demonstrated when bacterial cells are treated with lysozyme or penicillin The enzyme lysozyme attacks peptidoglycan by hydrolyzing the bond that connects N-acetylmuramic acid with N-acetylglucosamine (figure 3.18) Penicillin works by a different mechanism It inhibits the enzyme transpeptidase, which is responsible for making the cross-links between peptidoglycan chains If bacteria are treated with either of these substances while in a hypotonic solution, they lyse However, if they are in an isotonic solution, they can survive and grow normally If they are gram positive, treatment with lysozyme or penicillin results in the complete loss of the cell wall, and the cell becomes a protoplast When gramnegative bacteria are exposed to lysozyme or penicillin, the peptidoglycan layer is lost, but the outer membrane remains These cells are called spheroplasts Because they lack a complete cell wall, both protoplasts and spheroplasts are osmotically sensitive If they are transferred to a hypotonic solution, they lyse due to uncontrolled water influx (figure 3.28) >> Antibacterial (a) Porin trimer drugs (section 31.4) Although most bacteria require an intact cell wall for survival, some have none at all For example, the mycoplasmas lack a cell wall and are osmotically sensitive, yet often can grow in dilute media or terrestrial environments because their plasma membranes are more resistant to osmotic pressure than those of bacteria having walls The precise reason for this is not clear, although the presence of sterols in the membranes of many species may provide added strength Without a rigid cell wall, mycoplasmas tend to be pleomorphic or variable in shape (see figure 21.3) List the functions of the cell wall Describe in detail the composition and structure of peptidoglycan Why does peptidoglycan contain the unusual D-isomers of alanine and glutamic acid rather than the L-isomers observed in proteins? Compare and contrast the cell walls of gram-positive bacteria and gram-negative bacteria Include labeled drawings in your discussion When protoplasts and spheroplasts are made, the shape of the cell becomes spherical regardless of the original cell shape Why does this occur? (b) OmpF side view Figure 3.27 Porin Proteins Two views of the OmpF porin of E coli (a) Porin structure observed when looking down at the outer surface of the outer membrane (i.e., top view) The three porin proteins forming the protein each form a channel Each OmpF porin can be divided into three loops: the green loop forms the channel, the blue loop interacts with other porin proteins to help form the trimer, and the orange loop narrows the channel The arrow indicates the area of a porin molecule viewed from the side in panel (b) Side view of a porin monomer showing the β-barrel structure characteristic of porin proteins Penicillin inhibition of wall synthesis Incubation in isotonic medium Transfer to hypotonic medium Swelling due to H2O influx Lysis Protoplast H2O Figure 3.28 Protoplast Formation and Lysis Protoplast formation induced by incubation with penicillin in an isotonic medium Transfer to hypotonic medium will result in lysis wiL75233_ch03_033-064.indd Page 53 10/22/07 1:57:15 PM e /Volumes/ju103/HCAC039/sxn_m5_SM_indd%0/H5_TX_SM_L111-120_Inv12 3.6 Design an experiment that illustrates the cell wall’s role in protecting against lysis With a few exceptions, the cell walls of gram-positive bacteria lack porins Why is this the case? 3.5 H O CH2OH O H H OH β(1→3) H Glu Phylum Euryarchaeota (section 18.3) Many archaea that stain gram negative have either a layer of glycoprotein or protein outside their plasma membrane (figure 3.29b) The layer may be as thick as 20 to 40 nm Sometimes there are two layers—an electron-dense layer and a sheath surrounding it Some methanogens (Methanolobus), salt-loving archaea (Halobacterium), and extreme thermophiles (Sulfolobus, CW 0.1 μm CM 53 NHAc O OH β(1→3) H CO H H H NHAc ARCHAEAL CELL WALLS Before they were distinguished as a unique domain of life, the Archaea were characterized as being either gram positive or gram negative However, their staining reaction does not correlate as reliably with a particular cell wall structure Archaeal wall structure and chemistry differ from those of the Bacteria Archaeal cell walls lack peptidoglycan and exhibit considerable variety in terms of their chemical makeup Some of the major features of archaeal cell walls are described in this section Many archaea have a wall with a single, thick homogeneous layer resembling that in gram-positive bacteria (figure 3.29a) These archaea often stain gram positive Their wall chemistry varies from species to species but usually consists of complex heteropolysaccharides For example, Methanobacterium and some other methane-generating archaea (methanogens) have walls containing pseudomurein, a peptidoglycan-like polymer that has L-amino acids instead of D-amino acids in its cross-links, N-acetyltalosaminuronic acid instead of N-acetylmuramic acid, and β(1→3) glycosidic bonds instead of β(1→4) glycosidic bonds (figure 3.30) Other archaea, such as Methanosarcina and the salt-loving Halococcus, contain complex polysaccharides similar to the chondroitin sulfate of animal connective tissue >> Components External to the Cell Wall H O H O β(1→3) (NH2) Ala Lys Glu (Glu) Lys (Ala) Ala (NH2) Glu H H CO H β(1→3) OH O O Hβ (1→3) OH H O NHAc N-acetyltalosaminuronic acid H H H NHAc H H β(1→3) O O CH2OH N-acetylglucosamine Figure 3.30 The Structure of Pseudomurein The amino acids and amino groups in parentheses are not always present Ac represents the acetyl group Thermoproteus, and Pyrodictium) have glycoproteins in their walls In contrast, other methanogens (Methanococcus, Methanomicrobium, and Methanogenium) and the extreme thermophile Desulfurococcus have protein walls >> Phylum Crenarchaeota (section 18.2); Phylum Euryarchaeota (section 18.3) How the cells walls of Archaea differ from those of Bacteria? What is pseudomurein? How is it similar to peptidoglycan? How is it different? Archaea with cell walls consisting of a thick, homogeneous layer of complex polysaccharides often retain the crystal violet dye when stained using the Gramstaining procedure Why you think this is so? CPL 3.6 (a) SL 0.1 μm CM CPL (b) Figure 3.29 Cell Envelopes of Archaea Schematic representations and electron micrographs of (a) Methanobacterium formicicum and (b) Thermoproteus tenax CW, cell wall; SL, surface layer; CM, cell membrane or plasma membrane; CPL, cytoplasm 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 in this section Capsules and Slime 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 wiL75233_ch03_033-064.indd Page 54 10/22/07 1:57:16 PM e 54 Chapter Procaryotic Cell Structure and Function called a capsule (figure 3.31a) 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 3.31b), 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 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 3.31a); they also can be studied with the electron microscope (figure 3.31b) 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 On the other hand, 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 3.32) Gliding bacteria often produce slime, which in some cases has been shown to facilitate motility (section 3.7) >> Overview of bacterial pathogenesis (a) K pneumoniae (b) Bacteroides Figure 3.31 /Volumes/ju103/HCAC039/sxn_m5_SM_indd%0/H5_TX_SM_L111-120_Inv12 (section 30.3) Bacterial Capsules (a) Klebsiella pneumoniae with its capsule stained for observation in the light microscope (×1,500) (b) Bacteroides glycocalyx (gly), TEM (×71,250) Intestinal tissue Bacteria Glycocalyx Figure 3.32 Bacterial Glycocalyx Bacteria connected to each other and to the intestinal wall by their glycocalyxes, the extensive networks of fibers extending from the cells (×17,500) S-Layers Many procaryotes have a regularly structured layer called an S-layer on their surface In bacteria, the S-layer is external to the cell wall In some archaea, the S-layer is the only structure outside the plasma membrane where it serves as the cell wall The S-layer has a pattern something like floor tiles and is composed of protein or glycoprotein (figure 3.33) In gram-negative bacteria, the S-layer adheres directly to the outer membrane; it is associated with the peptidoglycan surface in gram-positive bacteria Currently S-layers are of considerable interest not only for their biological roles but also in the growing field of nanotechnology Their biological roles include protecting the cell against ion and pH fluctuations, osmotic stress, enzymes, or predacious bacteria The S-layer also helps maintain the shape and envelope rigidity of some cells, and 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 The potential use of S-layers in nanotechnology is due to the ability of S-layer proteins to self-assemble That is, the S-layer proteins contain the information required to associate and form the S-layer without the aid of any special enzymes or other factors Thus S-layer proteins could be used as building blocks for the creation of technologies such as drug-delivery systems and novel detection systems for toxic chemicals or bioterrorism agents wiL75233_ch03_033-064.indd Page 55 10/22/07 1:57:17 PM e /Volumes/ju103/HCAC039/sxn_m5_SM_indd%0/H5_TX_SM_L111-120_Inv12 3.6 Components External to the Cell Wall 55 Fimbriae Flagella Figure 3.34 Flagella and Fimbriae The long flagella and the numerous shorter fimbriae are evident in this electron micrograph of the bacterium Proteus vulgaris (×39,000) Figure 3.33 The S-Layer An electron micrograph of the S-layer of the bacterium Deinococcus radiodurans after shadowing Pili and Fimbriae Many procaryotes have short, fine, hairlike appendages that are thinner than flagella These are usually called fimbriae (s., fimbria) or pili (s., pilus) Although many people use the terms fimbriae and pili interchangeably, we 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 3.34) They are slender tubes composed of helically arranged protein subunits and are about to 10 nm in diameter and up to several micrometers long Some types of fimbriae attach bacteria to solid surfaces such as rocks in streams and host tissues, and some are involved in motility (section 3.7) Many bacteria have about one to 10 sex pili (s., sex pilus) per cell These hairlike structures 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 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 14.7) Flagella 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 are the focus of this discussion Bacterial flagella are slender, rigid structures, about 20 nm across and up to 20 μm 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 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 3.35a) 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 3.35b) Flagella are spread evenly over the whole surface of peritrichous (peri means around) bacteria (figure 3.35c) 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) The 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, Vibrio cholerae has a lipopolysaccharide sheath The hook and basal body are quite different from the filament (figure 3.36) 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 transmission electron micrographs of the basal bodies of E coli and most other gram-negative bacteria, the basal body appears to have four rings, L ring, P ring, S ring, and M ring, connected to a central rod (figure 3.36a) It is now known that the S ring and M ring are different portions of the same protein, and they are now referred to as the MS ring On the cytoplasmic side of the MS ring is the C ring, which was discovered wiL75233_ch03_033-064.indd Page 56 11/10/07 4:20:58 PM epg 56 Chapter /Volumes/ve401/MHIY034/mhwiL1%0/wiL1ch03 Procaryotic Cell Structure and Function mm (a) Pseudomonas—monotrichous polar flagellation (c) P vulgaris—peritrichous flagellation Figure 3.35 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, ×600) mm (b) Spirillum—lophotrichous flagellation Filament Hook L ring Outer membrane P ring Periplasmic space Rod 30 nm Peptidoglycan layer 30 nm Plasma membrane MS ring (a) 22 nm Figure 3.36 The Ultrastructure of Bacterial Flagella (b) 30 nm Flagellar basal bodies and hooks in (a) gram-negative and (b) gram-positive bacteria The inset photo shows an enlarged view of the basal body of an E coli flagellum All three rings (L, P, and MS) can be clearly seen The uppermost arrow is at the junction of the hook and filament later Gram-positive bacteria have only two rings—an inner ring connected to the plasma membrane and an outer one probably attached to the peptidoglycan (figure 3.36b) 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 are concerned with the control of flagellar construction or function How the cell regulates or determines the exact location of flagella is not known Because many components of the flagellum lie outside the cell wall, they must be transported across the plasma membrane and cell wall Transport of many flagellar components is carried out by an apparatus in the basal body 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 3.37) Thus filament synthesis, like S-layer formation, is an example of self-assembly wiL75233_ch03_033-064.indd Page 57 10/22/07 1:57:20 PM e /Volumes/ju103/HCAC039/sxn_m5_SM_indd%0/H5_TX_SM_L111-120_Inv12 3.7 Bacterial Motility and Chemotaxis 57 LPS Flagellin Filament cap protein Outer membrane Peptidoglycan Plasma membrane mRNA Ribosome Figure 3.37 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 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 Discuss flagella distribution patterns and structure and synthesis of flagella What is self-assembly? Why does it make sense that the flagellar filament is assembled in this way? 3.7 BACTERIAL MOTILITY AND CHEMOTAXIS As we note in section 3.6, several structures outside the cell wall contribute to the motility of procaryotes Four major methods of movement have been observed in Bacteria: the swimming movement conferred by flagella; the corkscrew movement of spirochetes; the twitching motility associated with fimbriae; and gliding motility Bacteria have not evolved motility to move aimlessly Rather, motility is used to move toward nutrients such as sugars and amino acids and away from many harmful substances and bacterial waste products Bacteria also can respond to environmental cues such as temperature (thermotaxis), light (phototaxis), oxygen (aerotaxis), osmotic pressure (osmotaxis), and gravity Movement toward chemical attractants and away from repellents is known as chemotaxis Flagellar Movement Procaryotic flagella operate differently from eucaryotic flagella Eucaryotic flagella flex and bend, resulting in a whiplash that moves the cell The filament of a procaryotic flagellum is in the shape of a rigid helix, and the cell moves when this helix rotates like a propeller on a boat The flagellar motor can rotate very rapidly The E coli motor rotates 270 revolutions per second (rps); Vibrio alginolyticus averages 1,100 rps >> Structures external to the plasma membrane: Cilia and flagella (section 4.7) 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 with the flagellum trailing behind (figure 3.38) 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 The motor that drives flagellar rotation is located at the base of the flagellum, where it is associated with the basal body Torque generated by the motor is transmitted by the basal body to the hook and filament The motor is composed of two components: the rotor and the stator It is thought to function like an electrical motor, where the rotor turns in the center of a ring of electromagnets, the stator In gram-negative bacteria, the rotor is composed of the MS ring and the C ring (figure 3.39) The flagellar protein FliG is a particularly important component of the rotor as it is thought to interact with the stator The stator is composed of the proteins MotA and MotB Both form a channel through the plasma membrane, and MotB also anchors MotA to cell wall peptidoglycan As with all motors, the flagellar motor must have a power source that allows it to generate torque and cause flagellar rotation The power used by most flagellar motors is a difference in charge wiL75233_ch03_033-064.indd Page 58 10/22/07 1:57:21 PM e 58 Chapter /Volumes/ju103/HCAC039/sxn_m5_SM_indd%0/H5_TX_SM_L111-120_Inv12 Procaryotic Cell Structure and Function Filament Forward run (a) Hook Tumble L ring P ring (b) Outer membrane Forward run Peptidoglycan layer Rod + H (c) Periplasmic space MS ring Plasma membrane MotB MotA FliG C ring FliM, N Tumble Figure 3.39 Mechanism of Flagellar Movement This diagram of a (d) 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) Figure 3.38 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 Indeed, the speed of flagellar rotation is proportional to the magnitude of the PMF >> Electron transport and oxidative phosphorylation (section 10.5); Uptake of nutrients (section 6.6) and pH across the plasma membrane This difference is called the proton motive force (PMF) PMF is largely created by the metabolic activities of organisms as described in chapter 10 One important metabolic process carried out by cells is the transfer of electrons from an electron donor to a terminal electron acceptor via a chain of electron carriers called the electron transport chain (ETC) In procaryotes, the ETC is located in the plasma membrane As electrons are transported down the ETC, protons are transported from the cytoplasm to the outside of the cell Because there are more protons outside the cell than inside, the outside has more positively charged ions (the protons) and has a lower pH PMF is a type of potential energy that can be used to work: mechanical work, as in the case of flagellar rotation; transport work, the movement of materials into or out of the cell; or chemical work such as the synthesis of ATP, the cell’s energy currency So how can PMF be used to power the flagellar motor? The channels created by the MotA and MotB proteins allow protons to move across the plasma membrane from the outside to the inside Thus they move down the charge and pH gradient This movement releases energy that is used to rotate the flagellum In essence, the entry of a proton into the channel is like the entry of a person into a revolving door The “power” of the proton generates torque, rather like a person pushing the revolving door The flagellum is a very effective swimming device From the bacterium’s point of view, swimming is quite a difficult task because the surrounding water seems as viscous as molasses The cell 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 μm/second This is equivalent to traveling from to over 100 cell lengths per second In contrast, an exceptionally fast human might be able to run around to body lengths per second Spirochete Motility Although spirochetes have flagella, they work in a different manner In many spirochetes, multiple flagella arise from each end of the cell and associate to form an axial fibril, which winds around the cell (figure 3.40) The flagella not extend outside the cell wall but rather remain in the periplasmic space and are covered by an outer sheath The way in which axial fibrils propel the cell has not been fully established They are thought to rotate like the external flagella of other bacteria, causing the corkscrew-shaped outer sheath to rotate and move the cell through the surrounding liquid, even very viscous liquids Flagellar rotation may also flex wiL75233_ch03_033-064.indd Page 59 10/22/07 1:57:22 PM e /Volumes/ju103/HCAC039/sxn_m5_SM_indd%0/H5_TX_SM_L111-120_Inv12 3.7 AF PC OS AF axial fibril PC protoplasmic cylinder OS outer sheath IP insertion pore IP Figure 3.40 Spirochete Flagella Generally, numerous flagella arise from each end of the spirochete These intertwine to form an axial fibril The axial fibril winds around the cell, usually overlapping in the middle or bend the cell and account for the creeping or crawling movement observed when spirochetes are in contact with a solid surface >> Phylum Spirochaetes (section 19.6) Twitching and Gliding Motility Twitching and gliding motility occur when cells are on a solid surface Both types of motility can involve fimbriae, the production of slime, or both Thus they are considered together Several types of fimbriae have been identified on procaryotic cells Type IV fimbriae are present at one or both poles of some bacteria and are involved in twitching motility and in the gliding motility of some bacteria Twitching motility is characterized by short, intermittent, jerky motions of up to several micrometers in length and is normally seen on very moist surfaces It occurs only when cells are in contact with each other; isolated cells rarely move by this mechanism Considerable evidence exists that the fimbriae alternately extend and retract to move bacteria during twitching motility Gliding motility is smooth and varies greatly in rate (from to over 600 μm per minute) and in the nature of the motion Although first observed over 100 years ago, the mechanism by which many bacteria glide remains a mystery Some glide along in a direction parallel to the longitudinal axis of their cells Others travel with a screwlike motion or even move in a direction perpendicular to the long axis of the cells Still others rotate around their longitudinal axis while gliding Such diversity in gliding movement correlates with the observation that more than one mechanism for gliding motility exists Some types involve type IV fimbriae, some involve slime, and some involve mechanisms that have not yet been elucidated Gliding motility is best understood in the bacterium Myxococcus xanthus This rod-shaped microbe has a complex life cycle that includes the aggregation of cells to form a complex fruiting body in response to nutrient starvation M xanthus exhibits two types of motility The first is called social (S) motility because it occurs when large groups of cells move together in a coordinated fashion The second is called adventurous (A) motility, and it is observed when single cells move independently Both S and A motility can occur during aggregation and fruiting body formation S motility is mediated by the extension and retraction of type IV fimbriae at the front pole of the cell To reverse its direction, the bacterium disassembles fimbriae at one pole and moves them to the opposite pole The mechanism of A motility is not as well understood One hypothesis is that the cells contain pores through which slime is secreted Bacterial Motility and Chemotaxis 59 and that this propels the cell forward A more recent hypothesis is that adhesion complexes are located along the length of the cell and that these attach the cell to the surface The adhesion complexes are thought to span all the layers of the cell envelope, such that some portions are external and in contact with the surface and other portions are in the cytoplasm The adhesion complexes remain stationary relative to the surface on which the cell is gliding but move along a “track” within the cell >> Class Deltaproteobacteria: Order Myxococcales (section 20.4) Chemotaxis The movement of cells toward chemical attractants or away from chemical repellents is called chemotaxis Chemotaxis is readily observed in petri dish cultures If bacteria are placed in the center of a dish of semisolid agar containing an attractant, the bacteria will exhaust the local supply of the nutrient and swim outward following the attractant gradient they have created The result is an expanding ring of bacteria (figure 3.41a) 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 3.41b) Attractants and repellents are detected by chemoreceptors, proteins that bind chemicals and transmit signals to other components of the chemosensing system The chemosensing systems are very sensitive and allow the cell to respond to very low levels of attractants (about 10−8 M for some sugars) In gram-negative bacteria, the chemoreceptor proteins are located in the periplasmic space or in the plasma membrane Some receptors also 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, bacteria move randomly, switching back and forth between a phase called a run and a phase called a tumble For a bacterium with peritrichous flagella, a run occurs when its flagella are organized into a coordinated, corkscrewshaped bundle (figure 3.38c) During a run, the bacterium travels in a straight or slightly curved line After a few seconds, the flagella “fly apart” and the bacterium will stop and tumble The tumble randomly reorients 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 3.42a) In contrast, when the bacterium is exposed to an attractant, it tumbles less frequently (or has longer runs) when traveling toward 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 3.42b) The opposite response occurs with a repellent Tumbling frequency decreases (the run time lengthens) when the bacterium moves away from the repellent Clearly, the bacterium must have some mechanism for sensing that it is getting closer to the attractant (or 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 wiL75233_ch03_033-064.indd Page 60 10/22/07 1:57:22 PM e 60 Chapter /Volumes/ju103/HCAC039/sxn_m5_SM_indd%0/H5_TX_SM_L111-120_Inv12 Procaryotic Cell Structure and Function Colony of motile but nonchemotactic bacteria Colony of chemotactic motile bacteria Colony of nonmotile bacteria (a) Positive chemotaxis (b) Negative chemotaxis Figure 3.41 Positive and Negative Bacterial Chemotaxis (a) Positive chemotaxis can be demonstrated on an agar plate that contains various nutrients; positive chemotaxis by E coli is shown 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 (b) 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 Tumble Run Describe the way flagella operate to move a bacterium Define chemotaxis, run, and tumble Explain in a general way how bacteria move toward substances such as nutrients and away from toxic materials Why you think chemotaxis is sometimes called a “biased random walk”? (a) 3.8 (b) Figure 3.42 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 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 BACTERIAL ENDOSPORES Several genera of gram-positive bacteria, including Bacillus and Clostridium (rods), and Sporosarcina (cocci), can form a resistant, dormant structure called an endospore Endospores develop within vegetative bacterial cells and 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 In the environment, endospores aid in survival when moisture or nutrients are scarce Endospores are also of considerable theoretical interest Because bacteria manufacture these intricate structures in a very organized wiL75233_ch03_033-064.indd Page 61 10/22/07 1:57:23 PM e /Volumes/ju103/HCAC039/sxn_m5_SM_indd%0/H5_TX_SM_L111-120_Inv12 3.8 fashion over a period of a few hours, spore formation is well suited for research on the construction of complex biological structures This has made the endospore-forming Bacillus subtilis an important model organism >> The use of physical methods in control: Heat (section 8.4) 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 value in identification Endospores may be centrally located, close to one end (subterminal), or terminal (figure 3.43) Sometimes an endospore is so large that it swells the sporangium > Global regulatory systems: Sporulation in Bacillus subtilis (section 13.5) Core wall Cortex Spore coat Exosporium HOOC Figure 3.44 Endospore Structure spore (×151,000) 61 Bacillus anthracis endo- Figure 3.45 N Dipicolinic Acid COOH wiL75233_ch03_033-064.indd Page 62 10/22/07 1:57:24 PM e 62 Chapter /Volumes/ju103/HCAC039/sxn_m5_SM_indd%0/H5_TX_SM_L111-120_Inv12 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 Spore coat VII Lysis of sporangium, spore liberation VI Completion of coat synthesis, increase in refractility and heat resistance Exosporium V Coat synthesis Cortex C OFM I Axial filament formation S M hrs Plasma membrane DNA II Septum formation and forespore development III Engulfment of forespore IV Cortex formation N SC hrs IFM OFM N IFM 5.5 hrs N 6.5 hrs Figure 3.46 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 = 0.5 μm wiL75233_ch03_033-064.indd Page 63 10/22/07 1:57:25 PM e /Volumes/ju103/HCAC039/sxn_m5_SM_indd%0/H5_TX_SM_L111-120_Inv12 Summary The transformation of dormant spores into active vegetative cells seems almost as complex a process as sporulation It occurs in three stages: (1) activation, (2) germination, and (3) outgrowth (figure 3.47) Activation is a process that prepares spores for ger- 0.5 mm Figure 3.47 Endospore Germination rum emerging from the spore during germination Clostridium pectinovo- 63 mination and usually results from treatments such as heating This 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 the bilayer membrane in that the membrane has a hydrophobic core and its surfaces are hydrophilic 3.1 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 have no characteristic shape (pleomorphic) (figures 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 (figure 3.4) 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 Peripheral proteins are loosely associated with the membrane (figure 3.5) c Bacterial membranes are bilayers 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) d Some bacteria have simple internal membrane systems containing photosynthetic and respiratory machinery (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 3.3 Procaryotic Cytoplasm a The cytoplasm of procaryotes contains proteins that are similar in structure and function to the cytoskeletal proteins observed in eucaryotes (figure 3.12) b The cytoplasm of procaryotes contains inclusion bodies Most are used for storage (glycogen inclusions, PHB inclusions, cyanophycin granules, carboxysomes, and polyphosphate granules) (figure 3.13), but some are used for other purposes (magnetosomes and gas vacuoles) (figure 3.14) c The cytoplasm of procaryotes is packed with 70S ribosomes (figure 3.15) d Procaryotic genetic material is located in an area within the cytoplasm called the nucleoid The nucleoid is not usually enclosed by a membrane (figure 3.16) e In most procaryotes, the nucleoid contains a single chromosome The chromsosome usually consists of a double-stranded, covalently closed, circular DNA molecule f Plasmids are extrachromosomal DNA molecules found in many procaryotes Some are episomes—plasmids that are able to exist freely in the cytoplasm or can be integrated into the chromosome g Although plasmids are not required for survival in most conditions, they can encode traits that confer selective advantage in some environments h Many types of plasmids have been identified Conjugative plasmids encode genes that promote their transfer from one cell to another Resistance factors have genes conferring resistance to antibiotics Col plasmids contain genes for the synthesis of ... Space 479 21. 3 Class Clostridia 479 21. 4 Class Bacilli 483 ECOLOGY AND SYMBIOSIS 25 Biogeochemical Cycling and the Study of Microbial Ecology 593 Microbial Diversity & Ecology 25 .1: Microbial Ecology... Procaryotic Cell Structure 34 Microbial Diversity & Ecology 3 .1: Monstrous Microbes 36 3.2 Procaryotic Cell Membranes 38 3.3 Procaryotic Cytoplasm 42 3.4 Bacterial Cell Walls 46 3.5 Archaeal Cell... Sulfur 11 3 6.5 Growth Factors 11 3 6.6 Uptake of Nutrients 11 4 6.7 Culture Media 11 8 Techniques & Applications 6 .1: Enrichment Cultures 12 1 6.8 Isolation of Pure Cultures 12 1 Microbial Growth 12 6