Snyder and Champness Molecular Genetics of Bacteria

The single most comprehensive and authoritative textbook on bacterial molecular genetics

Snyder & Champness Molecular Genetics of Bacteria is a new edition of a classic text, updated to address the massive advances in the field of bacterial molecular genetics and retitled as homage to the founding authors.

In an era experiencing an avalanche of new genetic sequence information, this updated edition presents important experiments and advanced material relevant to current applications of molecular genetics, including conclusions from and applications of genomics; the relationships among recombination, replication, and repair and the importance of organizing sequences in DNA; the mechanisms of regulation of gene expression; the newest advances in bacterial cell biology; and the coordination of cellular processes during the bacterial cell cycle. The topics are integrated throughout with biochemical, genomic, and structural information, allowing readers to gain a deeper understanding of modern bacterial molecular genetics and its relationship to other fields of modern biology.

Although the text is centered on the most-studied bacteria, Escherichia coli and Bacillus subtilis, many examples are drawn from other bacteria of experimental, medical, ecological, and biotechnological importance. The book's many useful features include

• Text boxes to help students make connections to relevant topics related to other organisms, including humans
• A summary of main points at the end of each chapter
• Questions for discussion and independent thought
• A list of suggested readings for background and further investigation in each chapter
• Fully illustrated with detailed diagrams and photos in full color
• A glossary of terms highlighted in the text

While intended as an undergraduate or beginning graduate textbook, Molecular Genetics of Bacteria is an invaluable reference for anyone working in the fields of microbiology, genetics, biochemistry, bioengineering, medicine, molecular biology, and biotechnology.

"This is a marvelous textbook that is completely up-to-date and comprehensive, but not overwhelming. The clear prose and excellent figures make it ideal for use in teaching bacterial molecular genetics."
—Caroline Harwood, University of Washington

Watch an interview with the authors as they discuss their book further: https://www.youtube.com/watch?v=NEl-dfatWUU

Tina M. Henkin is Professor of Microbiology and Robert W. and Estelle S. Bingham Professor of Biological Sciences at Ohio State University, where she has been teaching since 1995. Dr. Henkin received a PhD in genetics at the University of Wisconsin.

Joseph E. Peters is Professor of Microbiology and Director of the Graduate Program in Microbiology at Cornell University, where he has been teaching since 2002. Dr. Peters received a PhD in microbiology at the University of Maryland.

Preface xv

Acknowledgments xix

About the Authors 1

Introduction 3

The Biological Universe 5

The Bacteria 5

The Archaea 7

The Eukaryotes 7

What is Genetics? 8

Bacterial Genetics 8

Bacteria Are Haploid 9

Short Generation Times 9

Asexual Reproduction 9

Colony Growth on Agar Plates 9

Colony Purification 9

Serial Dilutions 9

Selections 10

Storing Stocks of Bacterial Strains 10

Genetic Exchange 10

Phage Genetics 10

Phages Are Haploid 11

Selections

with Phages 11

Crosses with Phages 11

A Brief History of Bacterial Molecular Genetics 11

Inheritance in Bacteria 11

Transformation 11

Conjugation 12

Transduction 12

Recombination within Genes 12

Semiconservative DNA Replication 12

mRNA 12

The Genetic Code 12

The Operon Model 12

Enzymes for Molecular Biology 12

Synthetic Genomics 13

What is Ahead 13

1 The Bacterial Chromosome: DNA Structure, Replication, and Segregation 17

DNA Structure 17

The Deoxyribonucleotides 17

The DNA Chain 18

The 5’ and 3’ Ends 18

Base Pairing 20

Antiparallel Construction 20

The Major and Minor Grooves 21

The Mechanism of DNA Replication 21

Deoxyribonucleotide Precursor Synthesis 21

Replication of the Bacterial Chromosome 21

Replication of Double- Stranded DNA 26

Replication Errors 30

Editing 30

RNA Primers and Editing 31

Impediments to DNA Replication 31

Damaged DNA and DNA Polymerase III 31

Mechanisms To Deal with Impediments on Template DNA Strands 32

Physical Blocks to Replication Forks 32

Replication of the Bacterial Chromosome and Cell Division 32

Structure of Bacterial Chromosomes 34

Replication of the Bacterial Chromosome 34

Initiation of Chromosome Replication 34

RNA Priming of Initiation 35

Termination of Chromosome Replication 35

Chromosome Segregation 37

Coordination of Cell Division with Replication of the Chromosome 47

Timing of Initiation of Replication 49

The Bacterial Nucleoid 51

Supercoiling in the Nucleoid 51

Topoisomerases 52

The Bacterial Genome 55

Box 1.1 Structural Features of Bacterial Genomes 37

Box 1.2 Antibiotics That Affect Replication and DNA Structure 54

2 Bacterial Gene Expression: Transcription, Translation, Protein Folding, and Localization 61

Overview 61

The Structure and Function of RNA 62

Types of RNA 62

RNA Precursors 62

RNA Structure 62

RNA Processing and Modification 64

Transcription 64

Structure of Bacterial RNA Polymerase 64

Overview of Transcription 65

Details of Transcription 67

rRNAs and tRNAs 74

RNA Degradation 77

RNases 77

The Structure and Function of Proteins 78

Protein Structure 78

Translation 80

Structure of the Bacterial Ribosome 80

Overview of Translation 83

Details of Protein Synthesis 84

The Genetic Code 92

Polycistronic mRNA 96

Protein Folding and Degradation 98

Protein Chaperones 98

Protein Degradation 101

Protein Localization 101

The Translocase System 101

The Signal Sequence 103

The Targeting Factors 103

The Tat Secretion Pathway 104

Disulfide Bonds 105

Protein Secretion and Export 105

Protein Secretion Systems in Bacteria with an Outer Membrane 106

Protein Secretion in Bacteria That Lack an Outer Membrane 110

Sortases 110

Regulation of Gene Expression 111

Transcriptional Regulation 112

Posttranscriptional Regulation 113

What You Need To Know 114

Open Reading Frames 115

Transcriptional and Translational Fusions 115

Box 2.1 Antibiotic Inhibitors of Transcription 72

Box 2.2 Molecular Phylogeny 75

Box 2.3 Antibiotic Inhibitors of Translation 81

Box 2.4 Mimicry in Translation 91

Box 2.5 Exceptions to the Code 94

3 Bacterial Genetic Analysis: Fundamentals and Current Approaches 123

Definitions 123

Terms Used in Genetics 123

Genetic Names 124

Auxotrophic and Catabolic Mutants 125

Conditional- Lethal Mutants 126

Resistant Mutants 128

Inheritance in Bacteria 128

The Luria and Delbrück Experiment 129

Mutants Are Clonal 130

Esther and Joshua Lederberg’s Experiment 130

Mutation Rates 132

Calculating Mutation Rates 133

Calculating the Mutation Rate from the Rate of Increase in the Proportion of Mutants 135

Types of Mutations 136

Properties of Mutations 136

Base Pair Changes 136

Frameshift Mutations 140

Deletion Mutations 141

Tandem- Duplication Mutations 143

Inversion Mutations 144

Insertion Mutations 145

Reversion versus Suppression 147

Intragenic Suppressors 147

Intergenic Suppressors 147

Genetic Analysis in Bacteria 151

Isolating Mutants 151

Genetic Characterization of Mutants 155

Complementation Tests 160

Genetic Crosses in Bacteria 166

Mapping of Bacterial Markers by Transduction and Transformation 168

Other Uses of Transformation and Transduction 171

Genetic Mapping by Hfr Crosses 172

Perspective 176

Box 3.1 Inversions and the Genetic Map 146

4 Plasmids 181

What is a Plasmid? 181

Naming Plasmids 182

Functions Encoded by Plasmids 182

Plasmid Structure 183

Properties of Plasmids 184

Replication 184

Functions of the ori Region 187

Plasmid Replication Control Mechanisms 193

Mechanisms To Prevent Curing of Plasmids 200

The Par Systems of Plasmids 203

Plasmid Cloning Vectors 206

Examples of Plasmid Cloning Vectors 208

Broad- Host- Range Cloning Vectors 210

Box 4.1 Linear Chromosomes and Plasmids in Bacteria 188

Box 4.2 Determining the Inc Group 191

Box 4.3 Toxin- Antitoxin Systems and Plasmid Maintenance 201

5 Conjugation 215

Overview 215

Classification of Self- Transmissible Plasmids and Integrating Elements 217

The Fertility Plasmid 217

Mechanism of DNA Transfer during Conjugation in Proteobacteria 218

Transfer (tra) Genes 218

The oriT Sequence 221

Efficiency of Transfer 222

Interspecies Transfer of Plasmids 225

Conjugation and Type IV Secretion Systems Capable of Translocating Proteins 225

Mobilizable Plasmids 229

Chromosome Transfer by Plasmids 230

Formation of Hfr Strains of E. coli 230

Transfer of Chromosomal DNA by Integrated Plasmids 230

Chromosome Mobilization 231

Prime Factors 231

Diversity in Transfer Systems 233

Integrating Conjugative Elements 234

SXT/R391 ICE 234

ICEBs1 236

Tn916 237

TnGBS1 and TnGBS2 240

Box 5.1 Pilus- Specific Phages 220

Box 5.2 Delivery of Conditional Plasmids by Conjugation 223

Box 5.3 Gene Exchange between Domains 226

Box 5.4 Conjugation and Synthetic Genomics 232

6 Transformation 245

Natural Transformation 246

Discovery of Transformation 246

Overview of Natural Transformation 247

DNA Uptake Mechanisms 247

Specificity of DNA Uptake 251

DNA Pro cessing after Uptake 253

Natural Transformation as a Tool 253

Regulation of Natural Competence 254

Identification of Competence in Other Organisms 258

Role of Natural Transformation 258

Artificially Induced Competence 260

Chemical Induction 260

Electroporation 261

Protoplast Transformation 261

Box 6.1 Experimental Measurements of DNA Uptake 248

Box 6.2 Genetic Evidence for Single- Stranded DNA Uptake 252

Box 6.3 Role of Natural Transformation in Pathogens 260

7 Bacteriophages and Transduction 265

Lytic Development 268

The Lytic Cycle 268

Transcriptional Regulation of Phage Gene Expression 268

Phage Genome Replication and Packaging 279

Host Cell Lysis 289

Lysogenic Development 292

The λ System 292

Other Lysogenic Systems 299

Genetic Analysis of Phages 302

Infection of Cells 302

Phage Crosses 303

Recombination and Complementation Tests with Phages 303

The Genetic- Linkage Map of a Phage 305

Phage- Mediated Genetic Transfer 306

Generalized Transduction 306

Specialized Transduction 308

Lysogenic Conversion and Bacterial Pathogenesis 310

Host Defenses Against Phage Infection 313

Restriction- Modification Systems 313

Abi Systems 313

CRISPR/Cas Systems 314

Small Molecules and Phage Defense 314

Phage versus Phage 314

Phages as Tools 315

Cloning Vectors 315

Phage Display 315

Phage Therapy 317

Box 7.1 Phage Genomics 266

Box 7.2 Phage T7- Based Tools 271

Box 7.3 Protein Priming 285

8 Transposition, Site- Specific Recombination, and Families of Recombinases 321

Transposition 321

Overview of Transposition 322

Structure of Bacterial DNA Transposons 322

Types of Bacterial DNA Transposons 323

Assays of Transposition 326

Mechanisms of Transposition 328

DDE Transposons 328

HUH Transposons 332

General Properties of Transposons 334

Transposition Regulation 334

Target Site Specificity 335

Effects on Genes Adjacent to the Insertion Site 337

Target Immunity 337

Transposon Mutagenesis 337

Transposon Mutagenesis In Vivo 339

Transposon Mutagenesis In Vitro 340

Transposon Mutagenesis of Plasmids 341

Transposon Mutagenesis of the Bacterial Chromosome 341

Transposon Mutagenesis of All Bacteria 342

Using Transposon Mutagenesis To Make Random Gene Fusions 342

Site- Specific Recombination 343

Integrases 343

Resolvases 345

DNA Invertases 345

Y and S Recombinases 347

Y Recombinases: Mechanism 347

S Recombinases: Mechanism 351

Group II Mobile Introns: Elements That Move Using an RNA Intermediate 352

Importance of Transposition and Site- Specific Recombination in Bacterial Adaptation 354

Box 8.1 Mobile Elements and DNA Replication 333

Box 8.2 Transposons and Genomics 338

9 Molecular Mechanisms of Homologous Recombination 359

Homologous Recombination and DNA Replication in Bacteria 360

Early Evidence for the Interdependence of Homologous Recombination and DNA Replication 361

The Molecular Basis for Recombination in E. coli 361

chi (χ) Sites and the RecBCD Complex 361

The RecF Pathway 367

Synapse Formation and the RecA Protein 368

The Ruv and RecG Proteins and the Migration and Cutting of Holliday Junctions 371

Recombination between Different DNAs in Bacteria 373

How Are Linear DNA Fragments Recombined into the E. coli Chromosome? 373

Recombination during Natural Transformation 375

Phage Recombination Pathways 375

Rec Proteins of Phages T4 and T7 375

The RecE Pathway of the rac Prophage 375

The Phage λ Red System 375

Recombineering: Gene Replacements in E. coli with Phage λ Recombination Functions 376

Gene Conversion and Other Manifestations of Heteroduplex Formation during Recombination 379

Box 9.1 Discovery of χ sites 364

Box 9.2 Other Types of Double- Strand Break Repair in Bacteria 365

10 DNA Repair and Mutagenesis 385

Evidence for DNA Repair 386

Specific Repair Pathways 387

Deamination of Bases 387

Damage Due to Reactive Oxygen 389

Damage Due to Alkylating Agents 393

Damage Due to UV Irradiation 395

General Repair Mechanisms 396

Base Analogs 396

Frameshift Mutagens 397

Mismatch Repair 398

Nucleotide Excision Repair 403

DNA Damage Tolerance Mechanisms 405

Homologous Recombination and DNA Replication 405

SOS- Inducible Repair 409

Mechanism of TLS by the Pol V Mutasome 416

Other Specialized Polymerases and Their Regulation 417

Summary of Repair Pathways in E. coli 418

Bacteriophage Repair Pathways 418

Box 10.1 The Role of Reactive Oxygen Species in Cancer and Degenerative Diseases 391

Box 10.2 DNA Repair and Cancer 401

Box 10.3 The Ames Test 417

11 Regulation of Gene Expression: Genes and Operons 425

Transcriptional Regulation in Bacteria 426

Genetic Evidence for Negative and Positive Regulation 427

Negative Regulation of Transcription Initiation 428

Negative Inducible Systems 428

Negative Repressible Systems 437

Molecular Mechanisms of Transcriptional Repression 439

Positive Regulation of Transcription Initiation 439

Positive Inducible Systems 440

Positive Repressible Systems 447

Molecular Mechanisms of Transcriptional Activation 447

Regulation by Transcription Attenuation 449

Modulation of RNA Structure 449

Changes in Processivity of RNA Polymerase 459

Regulation of mRNA Degradation 460

Protein- Dependent Effects on RNA Stability 460

RNA- Dependent Effects on RNA Stability 461

Regulation of Translation 461

Regulation of Translation Initiation 462

Translational Regulation in the Exit Channel of the Ribosome 464

Regulation of Translation Termination 465

Posttranslational Regulation 467

Posttranslational Protein Modification 467

Regulation of Protein Turnover 467

Feedback Inhibition of Enzyme Activity 468

Why Are There So Many Mechanisms of Gene Regulation? 469

Box 11.1 The Helix- Turn- Helix Motif of DNA- Binding Proteins 427

Box 11.2 Families of Regulators 442

12 Global Regulation: Regulons and Stimulons 473

Carbon Catabolite Regulation 474

Carbon Catabolite Regulation in E. coli: Catabolite Activator Protein (CAP) and cAMP 474

Carbon Catabolite Regulation in B. subtilis: CcpA and Hpr 481

Regulation of Nitrogen Assimilation 482

Pathways for Nitrogen Assimilation 483

Regulation of Nitrogen Assimilation Pathways in E. coli by the Ntr System 484

Regulation of Nitrogen Assimilation in B. subtilis 491

Regulation of Ribosome Components and tRNA Synthesis 491

Ribosomal Protein Gene Regulation 492

Regulation of rRNA and tRNA Synthesis 493

Stringent Response 494

Stress Responses in Bacteria 498

Heat Shock Regulation 498

General Stress Response in Enteric Bacteria 501

General Stress Response in Firmicutes 505

Extracytoplasmic (Envelope) Stress Responses 506

Iron Regulation in E. coli 510

The Fur Regulon 510

The RyhB sRNA 512

The Aconitase Translational Repressor 512

Regulation of Virulence Genes in Pathogenic Bacteria 513

Diphtheria 513

Cholera and Quorum Sensing 514

Whooping Cough 519

Developmental Regulation: Sporulation in B. subtilis 520

Identification of Genes That Regulate Sporulation 522

Regulation of Sporulation Initiation 522

Compartmentalized Regulation of Sporulation Genes 524

The Role of Sigma Factors in Sporulation Regulation 524

Intercompartmental Regulation during Development 525

Other Sporulation Systems 529

Box 12.1 cAMP-Independent Carbon Catabolite Regulation in E. coli 477

Box 12.2 Nitrogen Fixation 483

Box 12.3 Signal Transduction Systems in Bacteria 486

Box 12.4 Sigma Factors 488

Box 12.5 Regulatory RNAs 503

13 Genomes and Genomic Analysis 535

The Bacterial Genome 535

DNA Sequencing 537

Advanced Genome-Sequencing Techniques 545

Polymerase Chain Reaction 547

Barriers to Horizontal Transfer: Genome Gatekeepers and Molecular Biologist’s Toolkit 549

Restriction Endonucleases 549

Techniques for Nontraditional Cloning and Assembly 553

CRISPR/Cas Systems 559

Final Thoughts 568

Box 13.1 Annotation and Comparative Genomics 538

Box 13.2 Special Problems in Genetic Analysis of Operons 542

Box 13.3 Synthesizing and Cloning Complete Bacterial Genomes 560

Glossary 573

Index 599

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