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
