David R. Wessner is a Professor of Biology at Davidson College. David teaches introductory biology and courses on microbiology, genetics, and HIV/AIDS. His research focuses on viral pathogenesis. He is a member of the American Society for Microbiology Committee for K-12 Education. He also is a coauthor of Vision and Change in Undergraduate Education: A Call to Action. Prior to joining the faculty at Davidson, David conducted research at the Navy Medical Center. He earned his Ph.D. in Microbiology and Molecular Genetics from Harvard University and his B.A. in Biology from Franklinand Marshall College.

Christine Dupont is a Lecturer in the Department of Biology at the University of Waterloo in southern Ontario, Canada, Christine teaches undergraduate courses in genetics, biotechnology, virology, and bacterial pathogenesis. She earned her Ph.D. from Massey University, Faculty of Veterinary Science, New Zealand; B.Ed. from the University of Windsor, Ontario; and M.Sc. and B.Sc. in Microbiology from the University of Guelph, Ontario. Prior to her Ph.D. studies, Christine taught high school science for several years, developing a passion for teaching and working with students.

Trevor C. Charles is a Professor in the Department of Biology, University of Waterloo, Trevor teaches undergraduate courses in microbiology and synthetic biology, and runs a research program that focuses on plant microbe interactions and functional metagenomics. Prior to joining the faculty at Waterloo, he held a faculty position at McGill University and did postdoctoral research at the University of Washington. He earned his Ph.D. from McMaster University, and his B.Sc. in Microbiology from the University of British Columbia.

Josh D. Neufeld, as a Professor in the Department of Biology at the University of Waterloo, explores microbiology with undergraduate students in the classroom and both undergraduate and graduate students in his microbial ecology research program. Josh earned his B.Sc. and M.Sc. in Microbiology from McGill University's Macdonald Campus prior to completing a Ph.D. at the University of British Columbia and postdoctoral research at the University of Warwick.

PART I THE MICROBES

1 The Microbial World 2

1.1 The Microbes 4

Mini-Paper: A Focus on the Research

THE THREE DOMAINS OF LIFE 10

Toolbox 1.1 POLYMERASE CHAIN REACTION AMPLIFICATION OF rRNA GENES 12

1.2 Microbial Genetics 15

Perspective 1.1 CREATING LIFE IN THE LABORATORY: THE MILLER-UREY EXPERIMENT 18

1.3 Microbial Physiology and Ecology 23

1.4 Microbes and Disease 26

2 Bacteria 34

2.1 Morphology of Bacterial Cells 36

2.2 The Cytoplasm 38

2.3 The Bacterial Cytoskeleton 41

2.4 The Cell Envelope 43

Perspective 2.1 MARVELOUS MAGNETOSOMES! 44

Toolbox 2.1 THE GRAM STAIN 53

Perspective 2.2 THE PROTECTIVE SHELLS OF ENDOSPORES 54

Mini-Paper: A Focus on the Research NEW MICROSCOPY METHODS REVEAL A PERIPLASM IN GRAM POSITIVE BACTERIAL CELLS 58

2.5 The Bacterial Cell Surface 59

2.6 Diversity of Bacteria 66

3 Eukaryal Microorganisms 72

3.1 The Morphology of Typical Eukaryal Cells 74

Mini-Paper: A Focus on the Research LIPID RAFTS: ORGANIZED CLUSTERING OF LIPIDS WITHIN A MEMBRANE 80

Toolbox 3.1 USING MICROSCOPY TO EXAMINE CELL STRUCTURE 82

Perspective 3.1 HIJACKING THE CYTOSKELETON 86

3.2 Diversity of Eukaryal Microorganisms 86

3.3 Replication of Eukaryal Microorganisms 91

3.4 The Origin of Eukaryal Cells 94

Perspective 3.2 SECONDARY ENDOSYMBIOSIS: THE ORIGINS OF AN ORGANELLE WITH FOUR MEMBRANES 97

3.5 Interactions Between Eukaryal Microorganisms and Animals, Plants, and the Environment 98

4 Archaea 106

4.1 Evolution of Archaea 108

4.2 Archaeal Cell Structure 110

Toolbox 4.1 VACCINE DELIVERY STRATEGIES 115

4.3 Diversity of Archaea 118

Perspective 4.1 EXTREMOPHILES AND BIOTECHNOLOGY 120

Mini-Paper: A Focus on the Research THE ROLE OF ARCHAEA IN OUR DIGESTIVE SYSTEM 124

5 Viruses 130

5.1 A Basic Overview of Viruses 132

5.2 Origins of Viruses 140

Perspective 5.1 RIBOZYMES: EVIDENCE FOR AN RNA-BASED WORLD 141

5.3 Cultivation, Purification, and Quantification of Viruses 143

Toolbox 5.1 CELL CULTURE TECHNIQUES 144

Perspective 5.2 MEASUREMENT OF HIV VIRAL LOAD 147

5.4 Diversity of Viruses 150

Toolbox 5.2 REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION (RT-PCR) 154

5.5 Virus-Like Particles 155

5.6 Virology Today 158

Mini-Paper: A Focus on the Research NEW FINDINGS IN THE PACKAGING OF DNA BY THE MODEL BACTERIOPHAGE T4 160

6 Cultivating Microorganisms 164

6.1 Nutritional Requirements of Microorganisms 166

6.2 Factors Affecting Microbial Growth 168

Toolbox 6.1 PHENOTYPE MICROARRAYS FOR EXAMINING MICROBIAL GROWTH 169

6.3 Growing Microorganisms in the Laboratory 173

Perspective 6.1 THE DISCOVERY OF HELICOBACTER PYLORI 178

Toolbox 6.2 FISHING FOR UNCULTIVATED MICROORGANISMS 180

6.4 Measuring Microbial Population Growth 181

Mini-Paper: A Focus on the Research BRINGING TO LIFE THE PREVIOUSLY UNCULTURABLE USING THE SOIL SUBSTRATE MEMBRANE SYSTEM (SSMS) 182

Perspective 6.2 MYCOBACTERIUM LEPRAE, AN EXTRAORDINARILY SLOW-GROWING PATHOGEN 189

Perspective 6.3 THE HUMAN INTESTINE-A CONTINUOUS CULTURE 191

6.5 Eliminating Microbes and Preventing Their Growth 192

PART II MICROBIAL GENETICS

7 DNA Replication and Gene Expression 202

7.1 The Role of DNA 204

7.2 DNA Replication 210

7.3 Transcription 217

Toolbox 7.1 USING A GEL SHIFT ASSAY TO IDENTIFY DNA-BINDING PROTEINS 219

7.4 Translation 222

7.5 The Effects of Mutations 229

Mini-Paper: A Focus on the Research TELOMERES WITH PROMOTER ACTIVITY 232

Perspective 7.1 USING MUTATIONS TO CONTROL VIRAL INFECTIONS 234

8 Viral Replication Strategies 238

8.1 Recognition of Host Cells 240

Perspective 8.1 DNA MICROARRAYS AND THE SARS VIRUS 242

Toolbox 8.1 THE WESTERN BLOT 244

8.2 Viral Entry and Uncoating 246

8.3 Viral Replication 249

Perspective 8.2 PHAGE THERAPY: BIOCONTROL FOR INFECTIONS 256

8.4 Viral Assembly and Egress 258

Mini-Paper: A Focus on the Research THE DISCOVERY OF REVERSE TRANSCRIPTASE 260

9 Bacterial Genetic Analysis and Manipulation 264

9.1 Bacteria as Subjects of Genetic Research 266

9.2 Mutations, Mutants, and Strains 269

Toolbox 9.1 ISOLATING NUTRITIONAL MUTANTS 271

9.3 Restriction Enzymes, Vectors, and Cloning 276

9.4 Recombination and DNA Transfer 283

Perspective 9.1 PLASMIDS THAT PRODUCE PATHOGENS 287

Mini-Paper: A Focus on the Research THE DISCOVERY OF TRANSDUCTION 294

10 Microbial Genomics 300

10.1 Genome Sequencing 302

Perspective 10.1 RATE OF DNA SEQUENCING 306

Toolbox 10.1 GENOME DATABASES 310

10.2 Genomic Analysis of Gene Expression 312

10.3 Comparative Genomics 318

Perspective 10.2 THE MINIMAL GENOME 318

Mini-Paper: A Focus on the Research GENOME SEQUENCE OF A KILLER BUG 320

10.4 Metagenomics and Related Analyses 323

11 Regulation of Gene Expression 328

11.1 Differential Gene Expression 330

11.2 The Operon 332

11.3 Global Gene Regulation 337

Mini-Paper: A Focus on the Research TUNING PROMOTERS FOR USE IN SYNTHETIC BIOLOGY 338

Perspective 11.1 THE USE OF LACTOSE ANALOGS IN GENE EXPRESSION STUDIES 342

11.4 Post-initiation Control of Gene Expression 345

Toolbox 11.1 USING RNA MOLECULES TO DECREASE GENE EXPRESSION 347

11.5 Quorum Sensing 348

11.6 Two-Component Regulatory Systems 351

11.7 Chemotaxis 354

12 Microbial Biotechnology 360

12.1 Microbes for Biotechnology 362

Perspective 12.1 BIOPROSPECTING: WHO OWNS THE MICROBES? 364

12.2 Molecular Genetic Modification 366

Toolbox 12.1 SITE-DIRECTED MUTAGENESIS 368

Toolbox 12.2 FUSION PROTEIN PURIFICATION 374

Perspective 12.2 THE INTERNATIONAL GENETICALLY ENGINEERED MACHINE (IGEM) COMPETITION, STANDARD BIOLOGICAL PARTS, AND SYNTHETIC BIOLOGY 378

12.3 Red Biotechnology 379

12.4 White Biotechnology 381

Perspective 12.3 BIOFUELS: BIODIESEL AND ALGAE 384

12.5 Green Biotechnology 390

Toolbox 12.3 PLANT TRANSFORMATION USING BACTERIA 392

PART III MICROBIAL PHYSIOLOGY AND ECOLOGY

13 Metabolism 400

13.1 Energy, Enzymes, and ATP 402

Perspective 13.1 WHO NEEDS VITAMINS? 405

13.2 Central Processes in ATP Synthesis 406

13.3 Carbon Utilization in Microorganisms 412

13.4 Respiration and the Electron Transport System 421

Perspective 13.2 ELECTRICIGENIC BACTERIA AND MICROBIAL FUEL CELLS 425

13.5 Metabolism of Non-glucose Carbon Sources 429

Toolbox 13.1 METABOLISM AND RAPID BACTERIAL IDENTIFICATION SYSTEMS 431

13.6 Phototrophy and Photosynthesis 433

13.7 Nitrogen and Sulfur Metabolism 442

Mini-Paper: A Focus on the Research GENOME SEQUENCE OF A DEEP SEA SYMBIONT 443

13.8 Biosynthesis of Cellular Components 448

14 Biogeochemical Cycles 456

14.1 Nutrient Cycling 459

Toolbox 14.1 USING MICROARRAYS TO EXAMINE MICROBIAL COMMUNITIES: The Geochip 461

14.2 Cycling Driven by Carbon Metabolism 462

Perspective 14.1 CO2 AS A GREENHOUSE GAS AND ITS INFLUENCE ON CLIMATE CHANGE 464

14.3 Cycling Driven by Nitrogen Metabolism 471

Mini-Paper: A Focus on the Research THE FIRST ISOLATION AND CULTIVATION OF A MARINE ARCHAEON 476

14.4 Other Cycles and their Connections 476

Perspective 14.2 LIFE IN A WORLD WITHOUT MICROBES 478

Perspective 14.3 THE MICROBIOLOGY OF ENVIRONMENTALLY TOXIC ACID MINE DRAINAGE 479

Toolbox 14.2 BIOGEOCHEMISTRY IN A BOTTLE: THE WINOGRADSKY COLUMN 481

15 Microbial Ecosystems 486

15.1 Microbes in the Environment 488

15.2 Microbial Community Analysis 493

Toolbox 15.1 FLOW CYTOMETRY 498

Mini-Paper: A Focus on the Research INSIGHTS INTO THE PHYLOGENY AND CODING POTENTIAL OF MICROBIAL DARK MATTER 500

Perspective 15.1 NAMING THE UNCULTURED AND UNCHARACTERIZED 502

15.3 Aquatic Ecosystems 502

Perspective 15.2 DEAD ZONES 503

15.4 Terrestrial Ecosystems 509

15.5 Deep Subsurface and Geothermal Ecosystems 515

Mini-Paper: A Focus on the Research MAKING A SYNTHETIC GENOME 376

16 The Microbiology of Food and Water 524

16.1 Food Spoilage 526

16.2 Food Preservation 530

16.3 Food Fermentation 535

16.4 Foodborne and Waterborne Illness 542

16.5 Microbiological Aspects of Water Quality 545

Perspective 16.1 IMPLICATIONS OF SLUDGE BULKING 550

Toolbox 16.1 MEASURING BIOCHEMICAL OXYGEN DEMAND (BOD) 551

Mini-Paper: A Focus on the Research ENHANCED BIOLOGICAL REMOVAL OF PHOSPHORUS 553

Toolbox 16.2 MOST PROBABLE NUMBER (MPN) METHOD 556

17 Microbial Symbionts 562

17.1 Types of Microbe-Host Interactions 564

17.2 Symbionts of Plants 566

17.3 Symbionts of Humans 571

Toolbox 17.1 GERM-FREE AND GNOTOBIOTIC ANIMALS 575

Perspective 17.1 FOOD PROBIOTICS-DO THEY WORK? 577

Mini-Paper: A Focus on the Research FECAL BACTERIOTHERAPY: REPOOPULATION OF THE GUT 580

17.4 Symbionts of Herbivores 582

Perspective 17.2 COWS CONTRIBUTE TO CLIMATE CHANGE 588

17.5 Symbionts of Invertebrates 588

Perspective 17.3 MIDICHLORIANS-NOT JUST FOR JEDI 592

Perspective 17.4 DEATH OF CORAL REEFS 595

PART IV MICROBES AND DISEASE

18 Introduction to Infectious Diseases 600

18.1 Pathogenic Microbes 603

Toolbox 18.1 MEASURING THE VIRULENCE OF PATHOGENS 605

18.2 Microbial Virulence Strategies 607

Perspective 18.1 GENOME EDITING: A POWERFUL AND CONTROVERSIAL NEW TECHNIQUE 613

18.3 The Transmission of Infectious Diseases 614

Mini-Paper: A Focus on the Research EPIDEMIOLOGY OF AN INFECTIOUS DISEASE 622

18.4 Proving Cause and Effect in Microbial Infections 624

Perspective 18.2 THE ARMADILLO-AN IDEAL ANIMAL MODEL? 628

18.5 The Evolution of Pathogens 629

19 Innate Host Defenses Against Microbial Invasion 638

19.1 Immunity 640

19.2 Barriers to Infection 641

Perspective 19.1 MESSY MUCUS 644

19.3 The Inflammatory Response 645

19.4 The Molecules of the Innate System 646

Mini-Paper: A Focus on the Research MAMMALIAN CELLS CAN RECOGNIZE BACTERIAL DNA 650

Toolbox 19.1 THE COMPLEMENT FIXATION TEST 654

19.5 The Cells of Innate Immunity 657

19.6 Invertebrate Defenses 665

Toolbox 19.2 THE LIMULUS AMOEBOCYTE ASSAY FOR LPS 667

20 Adaptive Immunity 672

20.1 Features of Adaptive Immunity 674

20.2 T Cells 677

20.3 Antigen Processing 682

20.4 Antigen-Presenting Cells 684

20.5 Humoral and Cell-Mediated Immune Responses 688

Mini-Paper: A Focus on the Research ATTEMPTING TO ENGINEER A VIRUS TO IMPROVE IMMUNOCONTRACEPTION 690

Perspective 20.1 TOO MUCH OF A GOOD THING? 691

20.6 B Cells and the Production of Antibody 692

Perspective 20.2 VACCINES AGAINST T-INDEPENDENT ANTIGENS 696

Toolbox 20.1 MONOCLONAL ANTIBODY (mAb) PRODUCTION 698

Perspective 20.3 TURNING ANTIBODY UPSIDE DOWN 703

Toolbox 20.2 ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) 704

21 Bacterial Pathogenesis 710

21.1 Bacterial Virulence Factors 712

Mini-Paper: A Focus on the Research ESCHERICHIA COLI INJECTS ITS OWN RECEPTOR 718

Perspective 21.1 IRON, VAMPIRES, FASHION, AND THE WHITE PLAGUE 722

21.2 Bacterial Virulence Factors-Toxins 723

Toolbox 21.1 SEROTYPING 724

Perspective 21.2 THE GOOD, THE BAD, AND THE UGLY SIDE OF BOTULINUM TOXIN 732

Perspective 21.3 SUPERABSORBENT TAMPONS AND SUPERANTIGENS 737

21.3 Survival in the Host: Strategies and Consequences 738

Toolbox 21.2 THE TUBERCULIN TEST FOR TUBERCULOSIS 746

21.4 Evolution of Bacterial Pathogens 746

Perspective 21.4 ANTIBIOTICS TRIGGER TOXINS? 750

22 Viral Pathogenesis 756

22.1 Recurring Themes in Viral Pathogenesis 758

Perspective 22.1 VERTICAL TRANSMISSION OF HIV 763

22.2 Interactions with the Host: Strategies and Consequences 765

Perspective 22.2 VIRAL INDUCTION OF APOPTOSIS 767

22.3 Viral Infections and Cancer 770

Toolbox 22.1 IMMUNOPRECIPITATION 773

Mini-Paper: A Focus on the Research VIRUSES THAT CAUSE CANCER BY AFFECTING CELLULAR PROLIFERATION 774

Perspective 22.3 SV40 AND HUMAN CANCERS 776

22.4 Evolution of Viral Pathogens 780

Perspective 22.4 ETHICAL CONCERNS ABOUT AVIAN FLU RESEARCH 784

23 Eukaryal Pathogenesis 788

23.1 Mechanisms of Eukaryal Pathogenesis 790

Mini-Paper: A Focus on the Research AN EXPERIMENTAL SYSTEM FOR THE GENOMIC STUDY OF DUTCH ELM DISEASE 794

Perspective 23.1 PNEUMOCYSTIS JIROVECII OR CARINII : THE EVOLVING FIELD OF TAXONOMY 797

Perspective 23.2 MAGIC MUSHROOMS 802

23.2 Pathogen Study: Plasmodium Falciparum 803

23.3 Macroscopic Eukaryal Pathogens 807

Toolbox 23.1 TESTING FOR MALARIA 808

23.4 Evolution of Eukaryal Pathogens 811

Perspective 23.3 CHYTRID FUNGUS: AN EMERGING FUNGAL PATHOGEN 812

24 Control of Infectious Diseases 818

24.1 Historical Aspects of Infectious Disease Treatment and Control 820

24.2 Antimicrobial Drugs 821

24.3 Antimicrobial Drug Resistance 834

Toolbox 24.1 DRUG SUSCEPTIBILITY TESTING AND MIC 838

Mini-Paper: A Focus on the Research SOIL MICROORGANISMS POSSESS EXTENSIVE RESISTANCE TO ANTIBIOTICS 840

Perspective 24.1 THE PURSUIT OF NEW ANTIBIOTICS: WHY BOTHER? 844

Perspective 24.2 HEALTH CARE-ASSOCIATED INFECTIONS: A RECIPE FOR RESISTANCE 845

24.4 Predicting and Controlling Epidemics 846

24.5 Immunization and Vaccines 848

Perspective 24.3 VARIOLATION: DELIBERATE INFECTION WITH SMALLPOX VIRUS 849

Perspective 24.4 THE WAR AGAINST VACCINES 854

Appendix A Reading and Understanding the Primary Literature A-1

Appendix B Microscopy A-9

Appendix C Taxonomys A-13

Appendix D Origin of Blood Cells A-15

GLOSSARY G-1

INDEX I-1

ONLINE APPENDICES

Appendix E Classification of Archaea

Appendix F Classification of Viruses

Appendix G Origin of Blood Cells