Molecular Genetics of Bacteria Fourth Edition

Molecular Genetics
 
Author:
Larry Snyder, Joseph E. Peters, Tina M. Henkin & Wendy Champness
Publisher: ASM Press
ISBN No: 978-1-55581-716-9
Release at: 2013
Pages: 732
Edition:
Fourth Edition
File Size: 99 MB
File Type: pdf
Language: English



Description of Molecular Genetics of Bacteria

the fourth edition of the textbook Molecular Genetics of Bacteria has been substantially revised and some new sections have been added. We tried to do this without increasing the length of the book, which, at more than 700 pages, was already quite long. While the book retains the same number and order of chapters, many topics have been moved or integrated more completely into the text to reflect a more modern perspective. 

The purpose was to convey more accurately how one approaches questions in modern bacterial genetics, using the full repertoire of methods now available. Also, to make room for the new material, we made the philosophical decision to condense or eliminate descriptions of methods where they seemed unnecessarily detailed for a textbook.

Content of Molecular Genetics of Bacteria



Introduction 1

The Biological Universe 3
The Bacteria 3
The Archaea 3
The Eukaryotes 5
Speculations on the Origin of the Three Domains of Life 5
What Is Genetics? 6
Bacterial Genetics 6
Bacteria Are Haploid 7
Short Generation Times 7
Asexual Reproduction 7
Colony Growth on Agar Plates 7
Colony Purification 7
Serial Dilutions 7
Selections 8
Storing Stocks of Bacterial Strains 8
Genetic Exchange 8
Phage Genetics 8
Phages Are Haploid 8
Selections with Phages 9
Crosses with Phages 9

A Brief History of Bacterial Molecular Genetics 9

Inheritance in Bacteria 9
Transformation 9
Conjugation 10
Transduction 10
Recombination within Genes 10
Semiconservative DNA Replication 10
mRNA 10
The Genetic Code 10
The Operon Model 10
Enzymes for Molecular Biology 10
What Is Ahead 11
SUGGESTED READING 11

CHAPTER 1
The Bacterial Chromosome: DNA Structure, Replication, and Segregation 13

DNA Structure 13
The Deoxyribonucleotides 13
The DNA Chain 14
The 5′ and 3′ Ends 14
Base Pairing 16
Antiparallel Construction 17
The Major and Minor Grooves 17
The Mechanism of DNA Replication 17
Deoxyribonucleotide Precursor Synthesis 17
Replication of the Bacterial Chromosome 19
Replication of Double-Stranded DNA 23
Replication Errors 26
Editing 26
RNA Primers and Editing 27
Impediments to DNA Replication 28
Damaged DNA and DNA Polymerase III 28
Mechanisms To Deal with Impediments on Template DNA Strands 28
Physical Blocks to Replication Forks 30
Replication of the Bacterial Chromosome and Cell Division 31
Structure of the Bacterial Chromosome 31
Replication of the Bacterial Chromosome 31
Initiation of Chromosome Replication 32
RNA Priming of Initiation 33
Termination of Chromosome Replication 33
Chromosome Segregation 34
Coordination of Cell Division with Replication of the Chromosome 43
Timing of Initiation of Replication 45
The Bacterial Nucleoid 47
Supercoiling in the Nucleoid 47
Topoisomerases 49
The Bacterial Genome 50
Antibiotics That Affect Replication and DNA Structure 51
Antibiotics That Block Precursor Synthesis 51
Antibiotics That Block Polymerization of Deoxynucleotides 52
Antibiotics That Affect DNA Structure 52
Antibiotics That Affect Gyrase 52
Molecular Biology Manipulations with DNA 53
Restriction Endonucleases 53
Hybridizations 56
Applications of the Enzymes Used in DNA Replication 58
Polymerase Chain Reaction 58
BOX 1.1 Structural Features of Bacterial Genomes 37
BOX 1.2 Advanced Genome-Sequencing
Techniques 59
SUMMARY 62
QUESTIONS FOR THOUGHT 64
PROBLEMS 64
SUGGESTED READING 65

CHAPTER  2
Bacterial Gene Expression: Transcription, Translation, and Protein Folding 67

Overview 67
The Structure and Function of RNA 68
Types of RNA 69
RNA Precursors 69
RNA Structure 69
RNA Processing and Modification 70
Transcription 70
Structure of Bacterial RNA Polymerase 70
Overview of Transcription 71
Details of Transcription 75
rRNAs and tRNAs 81
RNA Degradation 82
RNases 83
The Structure and Function of Proteins 84
Protein Structure 85
Translation 86
Structure of the Bacterial Ribosome 86
Overview of Translation 88
Details of Protein Synthesis 90
The Genetic Code 97
Protein Folding and Degradation 105
Protein Chaperones 105
Protein Degradation 107
Membrane Proteins and Protein
Export 108
Regulation of Gene Expression 108
Transcriptional Regulation 108
Posttranscriptional Regulation 109
Genomes and Genomics 109
Annotation and Comparative Genomics 110
What You Need To Know 110
Open Reading Frames 116
Transcriptional and Translational Fusions 116
Antibiotics That Block Transcription and Translation 116
Antibiotic Inhibitors of Transcription 117
Antibiotic Inhibitors of Translation 118
BOX 2.1 Molecular Phylogeny 82
BOX 2.2 Mimicry in Translation 96
BOX 2.3 Exceptions to the Code 99
BOX 2.4 Selfish DNAs: RNA Introns and Protein Inteins 102
BOX 2.5 Annotation and Comparative Genomics 110
SUMMARY 120
QUESTIONS FOR THOUGHT 122
PROBLEMS 122
SUGGESTED READING 123

C HAPTER 3
Bacterial Genetic Analysis: Fundamentals and Current Approaches 125

Definitions 125
Terms Used in Genetics 125
Genetic Names 126
Auxotrophic and Catabolic Mutants 127
Conditional-Lethal Mutants 128
Resistant Mutants 130
Inheritance in Bacteria 130
The Luria and Delbrück Experiment 131
Mutants Are Clonal 132
The Lederbergs’ Experiment 133
Mutation Rates 133
Calculating Mutation Rates 135
Calculating the Mutation Rate from the Rate of Increase in the Proportion of Mutants 136
Types of Mutations 137
Properties of Mutations 138
Base Pair Changes 138
Frameshift Mutations 142
Deletion Mutations 144
Tandem-Duplication Mutations 145
Inversion Mutations 147
Insertion Mutations 148
Reversion versus Suppression 149
Intragenic Suppressors 149
Intergenic Suppressors 150
Genetic Analysis in Bacteria 153
Isolating Mutants 153
Genetic Characterization of Mutants 157
Complementation Tests 161
Genetic Crosses in Bacteria 167
Mapping of Bacterial Markers by Transduction and Transformation 168
Other Uses of Transformation and Transduction 172
Genetic Mapping by Hfr Crosses 173
Isolation of Tandem Duplications of the his Operon in Salmonella 1764
Lengths of Tandem Duplications 178
Frequency of Spontaneous Duplications 179
BOX 3.1 Statistical Analysis of the Number of Mutants per Culture 134
BOX 3.2 Inversions and the Genetic Map 148
SUMMARY 179
QUESTIONS FOR THOUGHT 181
PROBLEMS 181
SUGGESTED READING 182

CHAPTER  4
Plasmids 183

What Is a Plasmid? 183
Naming Plasmids 184
Functions Encoded by Plasmids 184
Plasmid Structure 185
Properties of Plasmids 186
Replication 186
Functions of the ori Region 189
Plasmid Replication Control Mechanisms 194
Mechanisms To Prevent Curing of Plasmids 203
The Par Systems of Plasmids 205
Plasmid Cloning Vectors 209
Examples of Plasmid Cloning Vectors 210
Broad-Host-Range Cloning Vectors 213
BOX 4.1 Linear Chromosomes and Plasmids in Bacteria 190
BOX 4.2 Toxin-Antitoxin Systems and Plasmid Maintenance 204
SUMMARY 216
QUESTIONS FOR THOUGHT 217
PROBLEMS 217
SUGGESTED READING 217

CHAPTER 5
Conjugation 219

Overview 219
Classification of Self-Transmissible Plasmids 220
The Fertility Plasmid 220
Mechanism of DNA Transfer during Conjugation in Gram-Negative Bacteria 221
Transfer (tra) Genes 221
The oriT Sequence 225
Male-Specific Phages 226
Efficiency of Transfer 227
Interspecies Transfer of Plasmids 227
Conjugation and Type IV Protein Secretion 228
Mobilizable Plasmids 232
Chromosome Transfer by Plasmids 235
Formation of Hfr Strains 235
Transfer of Chromosomal DNA by Integrated Plasmids 236
Chromosome Mobilization 236
Prime Factors 236
Transfer Systems of Gram-Positive Bacteria 237
Plasmid-Attracting Pheromones 237
Integrating Conjugative Elements 240
BOX 5.1 Gene Exchange between Domains 230
SUMMARY 242
QUESTIONS FOR THOUGHT 243
PROBLEMS 243
SUGGESTED READING 244

CHAPTER  6
Transformation 247

Natural Transformation 248
Discovery of Transformation 248
Competence 248
DNA Processing after Uptake 252
Experimental Evidence for Models of Natural Transformation 252
Plasmid Transformation and Phage Transfection of Naturally Competent Bacteria 254
Regulation of Natural Competence 255
Role of Natural Transformation 257
Importance of Natural Transformation for Forward and Reverse Genetics 259
Congression 259
Artificially Induced Competence 260
Chemical Induction 260
Electroporation 261
Protoplast Transformation 261
BOX 6.1 Antigenic Variation in Neisseria gonorrhoeae 259
SUMMARY 262
QUESTIONS FOR THOUGHT 262
PROBLEMS 262
SUGGESTED READING 263

CHAPTER 7
Bacteriophages: Lytic Development, Genetics, and Generalized Transduction 265

Regulation of Gene Expression during Lytic Development 270
Phages That Encode Their Own RNA Polymerases 272
T7 Phage-Based Expression Vectors 273
Making Riboprobes and RNA-Processing Substrates 273
Phage T4: Transcriptional Activators, a New Sigma Factor, and Replication-Coupled Transcription 275
Phage DNA Genome Replication and Packaging 279
Phages with Single-Stranded Circular DNA 279
Replication and DNA Packaging: Linear Genomes 285
Phage T7: Linear DNA That Forms Concatemers 285
Phage T4: Another Phage That Forms Concatemers 286
Phage Lysis 289
Single-Protein Lysis 289
Timed Lysis 290
Timing of Lysis by Holins 290
Phage Display 292
Genetic Analysis of Phages 298
Infection of Cells 298
Phage Crosses 299
Recombination and Complementation Tests with Phages 299
Genetic Experiments with the r II Genes of Phage T4 301
Constructing the Genetic-Linkage Map of a Phage 307
Phage Defense Mechanisms 309
Restriction-Modification Systems 310
Abi Systems 310
CRISPR Loci 311
Generalized Transduction 314
What Makes a Transducing Phage? 315
Shuttle Phasmids 316
Role of Transduction in Bacterial
Evolution 317
BOX 7.1 Phage Genomics 268
BOX 7.2 RNA Phages 271
BOX 7.3 Protein Priming 286
BOX 7.4 Phage Display 294
SUMMARY 318
QUESTIONS FOR THOUGHT 319
PROBLEMS 319
SUGGESTED READING 320

CHAPTER 8
Lysogeny: the λ Paradigm and the Role of Lysogenic Conversion in Bacterial Pathogenesis 323

Phage λ 324
λ Lytic Development 324
Replication of λ DNA 331
Lysogeny by Phage λ 333
The Lytic-versus-Lysogen Decision: the Roles of cI, cII,
and cIII Gene Products 333
Phage λ Integration 334
Maintenance of λ Lysogeny 335
Immunity to Superinfection 337
Induction of λ 338
Summary of the Lytic and Lysogenic Cycles 340
Specialized Transduction 340
Selection of HFT Particles 342
Other Lysogen-Forming Phages 343
Phage P2 343
Phage P4: a Satellite Virus 343
Prophages That Replicate as Plasmids 345
Phage Mu: a Transposon Masquerading as a Phage 345
Lysogenic Conversion and Bacterial Pathogenesis 345
E. coli and Dysentery: Shiga Toxins 346
Diphtheria 347
Cholera 347 
S. aureus and Toxic Shock Syndrome 349
Synopsis 350
Uses of Lysogeny in Genetic Analysis and Biotechnology 350
Complementation and Gene Expression Studies 350
Use of Phage Display and Frequency of Mixed Dilysogens To Detect Protein-Protein Interactions 350
Genetic Experiments with Phage λ 351
Genetic Analysis of λ Lysogen Formation 351
Genetics of the CI Repressor: Evidence for the Domain Structure of Proteins 353
Identification of λ nut Sites Involved in Progressive Transcription Antitermination 354
Isolation of Host nus Mutations: E. coli Functions Involved in Transcription Elongation-Termination 356
BOX 8.1 Effects of Prophage Insertion on the Host 336
SUMMARY 357
QUESTIONS FOR THOUGHT 358
PROBLEMS 358
SUGGESTED READING 359

CHAPTER 9
Transposition, Site-Specific Recombination, and Families of Recombinases 361

Transposition 361
Overview of Transposition 362
Structure of Bacterial Transposons 362
Types of Bacterial Transposons 364
Assays of Transposition 366
Mechanisms of Transposition 368
Genetic Requirements for Transposition of Tn3 368
A Molecular Model for Transposition of Tn3 372
Transposition by Tn10 and Tn5 373
Details of Transposition by the DDE Transposons 376
Details of the Mechanism of Transposition by Tn5 and Tn7 376
Rolling-Circle Transposons 378
Y and S Transposons 378
General Properties of Transposons 379
Target Site Specificity 379
Effects on Genes Adjacent to the Insertion Site 380
Regulation of Transposition 380
Target Immunity 381
Transposon Mutagenesis 382
Transposon Mutagenesis In Vivo 382
Transposon Mutagenesis In Vitro 382
Transposon Mutagenesis of Plasmids 385
Transposon Mutagenesis of the Bacterial Chromosome 386
Transposon Mutagenesis of All Bacteria 386
Using Transposon Mutagenesis To Make Random Gene Fusions 387
Site-Specific Recombination 387
Integrases 387
Resolvases 390
DNA Invertases 391
Y and S Recombinases 392
Y Recombinases: Mechanism 392
S Recombinases: Mechanism 397
Importance of Transposition and Site-Specific Recombination in Bacterial Adaptation 398
BOX 9.1 Transposons and Genomics 383
SUMMARY 399
QUESTIONS FOR THOUGHT 400
PROBLEMS 400
SUGGESTED READING 401

CHAPTER 10
Molecular Mechanisms of Homologous Recombination 403

Homologous Recombination and DNA
Replication in Bacteria 404
Early Evidence for the Interdependence of Homologous Recombination and DNA Replication 404
The Molecular Basis for Recombination in E. coli 405
Chi (χ) Sites and the RecBCD Complex 405
The RecF Pathway 409
Synapse Formation and the RecA Protein 411
The Ruv and RecG Proteins and the Migration and Cutting of Holliday Junctions 414
Recombination between Different DNAs in Bacteria 416
How Are Linear DNA Fragments Recombined into the E. coli Chromosome? 417
Phage Recombination Pathways 417
Rec Proteins of Phages T4 and T7 417
The RecE Pathway of the rac Prophage 417
The Phage λ Red System 419
Recombineering: Gene Replacements in E. coli with Phage λ Recombination Functions 419
Genetic Analysis of Recombination in Bacteria 422
Isolating Rec − Mutants of E. coli 422
Isolating Mutants with Mutations in Other Recombination Genes 423
Gene Conversion and Other Manifestations of Heteroduplex Formation during Recombination 426
BOX 10.1 Other Types of Double-Strand Break Repair in Bacteria 410
BOX 10.2 Breaking and Entering: Introns and Inteins Move by Double-Strand Break Repair or Retrohoming 425
SUMMARY 429
QUESTIONS FOR THOUGHT 430
PROBLEMS 430
SUGGESTED READING 431

CHAPTER 11
DNA Repair and Mutagenesis 433

Evidence for DNA Repair 434
Specific Repair Pathways 435
Deamination of Bases 435
Damage Due to Reactive Oxygen 438
Damage Due to Alkylating Agents 441
Damage Due to UV Irradiation 443
General Repair Mechanisms 445
Base Analogs 445
Frameshift Mutagens 445
Methyl-Directed Mismatch Repair 445
Nucleotide Excision Repair 452
DNA Damage Tolerance Mechanisms 453
Homologous Recombination and DNA
Replication 454
SOS-Inducible Repair 458
Mechanism of TLS by the Pol V Mutasome 463
Other Specialized Polymerases and Their Regulation 464
Summary of Repair Pathways in E. coli 466
Bacteriophage Repair Pathways 466
BOX 11.1 The Role of Reactive Oxygen Species in Cancer and Degenerative Diseases 439
BOX 11.2 DNA Repair and Cancer 449
BOX 11.3 The Ames Test 465
SUMMARY 468
QUESTIONS FOR THOUGHT 469
PROBLEMS 469
SUGGESTED READING 469

CHAPTER 12
Regulation of Gene Expression: Genes and Operons 471

Transcriptional Regulation in Bacteria 472
Genetic Evidence for Negative and Positive Regulation 474
Negative Regulation of Transcription Initiation 474
Negative Inducible Systems 474
Negative Repressible Systems 484
Molecular Mechanisms of Transcriptional
Repression 486
Positive Regulation of Transcription Initiation 487
Positive Inducible Systems 487
Positive Repressible Systems 496
Molecular Mechanisms of Transcriptional Activation 496
Regulation by Transcription Attenuation 497
Modulation of RNA Structure 497
Changes in Processivity of RNA Polymerase 506
Regulation of mRNA Degradation 507
Protein- Dependent Effects on RNA Stability 507
RNA- Dependent Effects on RNA Stability 508
Regulation of Translation 508
Regulation of Translation Initiation 509
Translational Regulation in the Exit Channel of the Ribosome 511
Regulation of Translation Termination 512
Posttranslational Regulation 514
Posttranslational Protein Modification 514
Regulation of Protein Turnover 514
Feedback Inhibition of Enzyme Activity 515
Why Are There So Many Mechanisms of Gene Regulation? 520
Operon Analysis for Sequenced Genomes 521
BOX 12.1 The Helix-Turn-Helix Motif of DNA- Binding Proteins 473
BOX 12.2 Families of Regulators 488
BOX 12.3 Special Problems in Genetic Analysis of Operons 516
SUMMARY 521
QUESTIONS FOR THOUGHT 522
PROBLEMS 522
SUGGESTED READING 523

CHAPTER 13
Global Regulation: Regulons and Stimulons 525

Carbon Catabolite Regulation 526
Catabolite Regulation in E. coli: Catabolite Activator Protein (CAP) and cAMP 526
Carbon Catabolite Regulation in B. subtilis: CcpA and Hpr 535
Regulation of Nitrogen Assimilation 536
Pathways for Nitrogen Assimilation 536
Regulation of Nitrogen Assimilation Pathways in E. coli by the Ntr System 537
Regulation of Nitrogen Assimilation in B. subtilis 547
Regulation of Ribosome and tRNA Synthesis 547
Ribosomal Protein Gene Regulation 548
Regulation of rRNA and tRNA Synthesis 550
The Stringent Response 551
Stress Responses in Bacteria 554
Heat Shock Regulation 555
General Stress Response in Gram- Negative Bacteria 558
General Stress Response in Gram- Positive Bacteria 559
Extracytoplasmic (Envelope) Stress Responses 563
Iron Regulation in E. coli 568
The Fur Regulon 568
The RyhB sRNA 569
The Aconitase Translational Repressor 570
Regulation of Virulence Genes in Pathogenic Bacteria 571
Diphtheria 572
Cholera and Quorum Sensing 572
Whooping Cough 578
From Genes to Regulons to Networks 579
BOX 13.1 cAMP-Independent Carbon Catabolite Regulation in E. coli 529
BOX 13.2 A Bacterial Two- Hybrid System Based on Adenylate Cyclase 531
BOX 13.3 Nitrogen Fixation 537
BOX 13.4 Signal Transduction Systems in Bacteria 539
BOX 13.5 Sigma Factors 542
BOX 13.6 Regulatory RNAs 560
BOX 13.7 Tools for Studying Global-Regulation 571
SUMMARY 580
QUESTIONS FOR THOUGHT 582
PROBLEMS 582
SUGGESTED READING 582

CHAPTER  14 
Bacterial Cell Biology and Development 585

Membrane Proteins and Protein Export 585
The Translocase System 586
The Signal Sequence 586
The Targeting Factors 588
The Tat Secretion Pathway 590
Disulfide Bonds 590
Use of mal- lac Fusions To Study Protein Transport in E. coli 591
Genetic Analysis of Transmembrane Domains of Inner Membrane Proteins in Gram-Negative Bacteria 594
Identification of Genes for Inner Membrane Proteins by Random phoA Fusions 595
Protein Secretion 595
Protein Secretion Systems in Gram-Negative Bacteria 595
Protein Secretion in Gram-Positive Bacteria 602
Sortases 603
Bacterial Cell Biology and the Cell Cycle 605
The Bacterial Cell Wall 606
Septum Formation 615
The FtsZ Protein and the Septal Ring 616
Regulation of FtsZ Ring Formation in C. crescentus 619
Genetic Analysis of Sporulation in B. subtilis 621
Identification of Genes That Regulate Sporulation 622
Regulation of Initiation of Sporulation 623
Compartmentalized Regulation of Sporulation Genes 627
Analysis of the Role of Sigma Factors in Sporulation Regulation 627
Intercompartmental Regulation during Development 630
Finding Sporulation Genes: Mutant Hunts, Suppressor Analysis, and Functional Genomics 635
BOX 14.1 Secretion Systems and Motility 599
BOX 14.2 Example of a Sortase- Dependent Pathway: Sporulation in S. coelicolor 605
BOX 14.3 Evolutionary Origin of the Eukaryotic Cytoskeleton 612
BOX 14.4 Phosphorelay Activation of the Transcription Factor Spo0A 625
SUMMARY 636
QUESTIONS FOR THOUGHT 637
PROBLEMS 637
SUGGESTED READING 638
Answers to Problems and Questions for Thought 641

Glossary 655

Figure and Table Credits 685

Index 689

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