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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 ICE Bs 1 236 Tn 916 237 Tn GBS 1 and Tn GBS 2 240 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