| Author: |
Jeremy W. Dale, Simon F. Park
|
| Release at: | 2010 |
| Pages: | 403 |
| Edition: |
Fifth Edition
|
| File Size: | 8 MB |
| File Type: | |
| Language: | English |
Description of Molecular Genetics of Bacteria
In the preface to the fourth edition (published in 2004) we referred to the revolution in bacterial genetics that was started by gene cloning and sequencing, coupled with related techniques such as the polymerase chain reaction (PCR) and microarrays, and culminated in the knowledge of genome sequences of a rapidly expanding range of bacteria. This posed a dilemma. How could we accommodate these new techniques, and the wealth of exciting new information they provided, while not losing sight of classical bacterial genetics? This dilemma has become even more acute.
True, many of the older methods are now no longer used and could be relegated to the pages of history. But there is a danger of throwing out the baby with the bathwater. Not only is there a need to maintain some sense of how the subject has got to the stage we are now at, but also a discussion of some of these methods is useful in establishing an understanding of how bacterial genetics operates in natural environments. Genetics is not just about how we find out about bacteria: it is about how bacteria have evolved, and continue to evolve and continue to adapt to changing environments. Molecular genetics, in isolation, is essentially reductionist. Even genome sequencing, and global analysis of gene expression, by themselves merely provide catalogs of genes. Ultimately, those lists have to be related to the behavior of the whole organism, and from there to how organisms interact with one another and with their environment.
True, many of the older methods are now no longer used and could be relegated to the pages of history. But there is a danger of throwing out the baby with the bathwater. Not only is there a need to maintain some sense of how the subject has got to the stage we are now at, but also a discussion of some of these methods is useful in establishing an understanding of how bacterial genetics operates in natural environments. Genetics is not just about how we find out about bacteria: it is about how bacteria have evolved, and continue to evolve and continue to adapt to changing environments. Molecular genetics, in isolation, is essentially reductionist. Even genome sequencing, and global analysis of gene expression, by themselves merely provide catalogs of genes. Ultimately, those lists have to be related to the behavior of the whole organism, and from there to how organisms interact with one another and with their environment.
Content of Molecular Genetics of Bacteria
1 Nucleic Acid Structure and Function 1
1.1 Structure of nucleic acids 1
1.1.1 DNA 1
1.1.2 RNA 2
1.1.3 Hydrophobic interactions 3
1.1.4 Different forms of the double helix 4
1.1.5 Supercoiling 5
1.1.6 Denaturation and hybridization 8
1.1.7 Orientation of nucleic acid strands 10
1.2 Replication of DNA 11
1.2.1 Unwinding and rewinding 12
1.2.2 Fidelity of replication; proofreading 12
1.3 Chromosome replication and cell division 14
1.4 DNA repair 16
1.4.1 Mismatch repair 16
1.4.2 Excision repair 18
1.4.3 Recombination (post-replication) repair 19
1.4.4 SOS repair 21
1.5 Gene expression 21
1.5.1 Transcription 21
1.5.2 Translation 25
1.5.3 Post-translational events 31
1.6 Gene organization 35
2 Mutation and Variation 37
2.1 Variation and evolution 37
2.1.1 Fluctuation test 39
2.1.2 Replica plating 41
2.1.3 Directed mutation in bacteria? 41
2.2 Types of mutation 43
2.2.1 Point mutations 43
2.2.2 Conditional mutants 44
2.2.3 Variation due to larger-scale DNA alterations 45
2.2.4 Extrachromosomal agents and horizontal gene transfer 46
2.3 Recombination 47
2.3.1 A model of the general (homologous)
recombination process 48
2.3.2 Enzymes involved in recombination 49
2.4 Phenotypes 50
2.4.1 Restoration of phenotype 52
2.5 Mechanisms of mutation 55
2.5.1 Spontaneous mutation 55
2.5.2 Chemical mutagens 57
2.5.3 Ultraviolet irradiation 60
2.6 Isolation and identification of mutants 63
2.6.1 Mutation and selection 63
2.6.2 Replica plating 64
2.6.3 Isolation of other mutants 65
2.6.4 Molecular methods 66
3 Regulation of Gene Expression 71
3.1 Gene copy number 73
3.2 Transcriptional control 73
3.2.1 Promoters 73
3.2.2 Terminators, attenuators and anti-terminators 84
3.2.3 Induction and repression: regulatory proteins 84
3.2.4 Two-component regulatory systems 93
3.2.5 Global regulatory systems 97
3.2.6 Quorum sensing 98
3.3 Translational control 102
3.3.1 Ribosome binding 102
3.3.2 Codon usage 104
3.3.3 Stringent response 104
3.3.4 Regulatory RNA 105
3.4 Phase variation of 114
4 Genetics of Bacteriophages 115
4.1 Bacteriophage structure 116
4.2 Single-strand DNA bacteriophages 118
4.2.1 φX174 118
4.2.2 M13 121
4.3 RNA-containing phages: MS2 121
4.4 Double-stranded DNA phages 121
4.4.1 Bacteriophage T4 122
4.4.2 Bacteriophage λ 124
4.4.3 Lytic and lysogenic regulation of bacteriophage λ 128
4.5 Restriction and modification 136
4.6 Bacterial resistance to phage attack 138
4.7 Complementation and recombination 138
4.8 Why are bacteriophages important? 140
4.8.1 Phage typing 141
4.8.2 Phage therapy 141
4.8.3 Phage display 142
4.8.4 Phages in the natural environment 142
4.8.5 Bacterial virulence and phage conversion 144
5 Plasmids 147
5.1 Some bacterial characteristics are determined by plasmids 148
5.1.1 Antibiotic resistance 148
5.1.2 Colicins and bacteriocins 148
5.1.3 Virulence determinants 149
5.1.4 Plasmids in plant-associated bacteria 149
5.1.5 Metabolic activities 150
5.2 Molecular properties of plasmids 152
5.2.1 Plasmid replication and control 154
5.2.2 Partitioning 164
5.2.3 Host range 164
5.2.4 Plasmid incompatibility 167
5.3 Plasmid stability 168
5.3.1 Plasmid integrity 168
5.3.2 Partitioning 169
5.3.3 Differential growth rate of 174
5.4 Associating a plasmid with a phenotype 174
6 Gene Transfer 177
6.1 Transformation 178
6.2 Conjugation 180
6.2.1 Mechanism of conjugation 180
6.2.2 The F plasmid 185
6.2.3 Conjugation in other bacteria 186
6.3 Transduction 190
6.3.1 Specialized transduction 192
6.4 Recombination 192
6.4.1 Consequences of recombination 193
6.4.2 Site-specific and non-homologous (illegitimate) recombination 194
6.5 Mosaic genes and chromosome plasticity 195
7 Genomic Plasticity: Movable Genes and Phase Variation 199
7.1 Insertion sequences 199
7.1.1 Structure of insertion sequences 199
7.1.2 Occurrence of insertion sequences 200
7.2 Transposons 202
7.2.1 Structure of transposons 204
7.2.2 Integrons 206
7.2.3 ISCR elements 208
7.3 Mechanisms of transposition 209
7.3.1 Replicative transposition 209
7.3.2 Non-replicative (conservative) transposition 212
7.3.3 Regulation of transposition 213
7.3.4 Activation of genes by transposable elements 214
7.3.5 Mu: A transposable bacteriophage 214
7.3.6 Conjugative transposons 215
7.4 Phase variation of 215
7.4.1 Variation mediated by simple DNA inversion 217
7.4.2 Variation mediated by nested DNA inversion 218
7.4.3 Antigenic variation in the gonococcus 218
7.4.4 Phase variation by slipped-strand mispairing 222
7.4.5 Phase variation mediated by differential DNA methylation 223
7.5 Clustered regularly interspersed short palindromic repeats 224
8 Genetic Modification: Exploiting the Potential of Bacteria 227
8.1 Strain development 228
8.1.1 Generation of variation 228
8.1.2 Selection of desired variants 228
8.2 Overproduction of primary metabolites 229
8.2.1 Simple pathways 229
8.2.2 Branched pathways 230
8.3 Overproduction of secondary metabolites 232
8.4 Gene cloning 233
8.4.1 Cutting and joining DNA 234
8.4.2 Plasmid vectors 234
8.4.3 Bacteriophage λ vectors 238
8.4.4 Cloning larger fragments 240
8.4.5 Bacteriophage M13 vectors 242
8.5 Gene libraries 242
8.5.1 Construction of genomic libraries 242
8.5.2 Screening a gene library 244
8.5.3 Cloning PCR products 246
8.5.4 Construction of a cDNA library 247
8.6 Products from cloned genes 248
8.6.1 Expression vectors 248
8.6.2 Making new genes 250
8.6.3 Other bacterial hosts 253
8.6.4 Novel vaccines 255
8.7 Other uses of gene technology 256
9 Genetic Methods for Investigating Bacteria 257
9.1 Metabolic pathways 257
9.1.1 Complementation 257
9.1.2 Cross-feeding 258
9.2 Microbial physiology 259
9.2.1 Reporter genes 261
9.2.2 Chromatin immunoprecipitation 262
9.2.3 Cell division 264
9.2.4 Motility and chemotaxis 266
9.2.5 Cell differentiation 267
9.3 Bacterial virulence 271
9.3.1 Wide-range mechanisms of bacterial pathogenesis 271
9.3.2 Detection of virulence genes 272
9.4 Specific mutagenesis 280
9.4.1 Gene replacement 280
9.4.2 Antisense RNA 282
9.5 Taxonomy, evolution and epidemiology 282
9.5.1 Molecular taxonomy 282
9.5.2 GC content 283
9.5.3 16 S rRNA 283
9.5.4 Denaturing-gradient gel electrophoresis and temperature-gradient gel electrophoresis 286
9.5.5 Diagnostic use of PCR 286
9.5.6 Molecular epidemiology 287
10 Gene Mapping to Genomics and Beyond 295
10.1 Gene mapping 295
10.1.1 Conjugational analysis 295
10.1.2 Gene libraries 298
10.1.3 Restriction mapping and pulsed-field gel electrophoresis 298
10.2 DNA sequence determination 301
10.2.1 Sanger sequencing 301
10.2.2 Dye terminator sequencing 304
10.2.3 Pyrosequencing 304
10.2.4 Massively parallel sequencing 305
10.3 Genome sequencing 307
10.3.1 Genome-sequencing strategies 308
10.3.2 Relating sequence to function 309
10.3.3 Metagenomics 314
10.4 Comparative genomics 315
10.4.1 Microarrays 318
10.5 Analysis of gene expression 320
10.5.1 Transcriptional analysis 320
10.5.2 Translational analysis 325
10.6 Metabolomics 329
10.7 Systems biology and synthetic genomics 329
10.7.1 Systems biology 329
10.7.2 Synthetic genomics 329
10.8 Conclusion 330
Appendix A Further Reading 333
Appendix B Abbreviations Used 337
Appendix C Glossary 341
Appendix D Enzymes and other Proteins 365
Appendix E Genes 369
Appendix F Standard Genetic Code 373
Appendix G Bacterial Species 375
Index 379
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