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 3 1.1.3 Hydrophobic interactions 3 1.1.4 Different forms of the double helix 5 1.1.5 Supercoiling 6 1.1.6 Denaturation and hybridization 10 1.1.7 Orientation of nucleic acid strands 11 1.2 Replication of DNA 12 1.2.1 Unwinding and rewinding 13 1.2.2 Fidelity of replication: proof-reading 13 1.3 Chromosome replication and cell division 14 1.4 DNA repair 19 1.4.1 Mismatch repair 19 1.4.2 Excision repair 19 1.4.3 Recombination (post-replication) repair 19 1.4.4 SOS repair 20 1.5 Gene expression 21 1.5.1 Transcription 22 1.5.2 Translation 26 1.5.3 Post-translational events 32 1.6 Gene organization 34 2 Mutation and Variation 37 2.1 Variation and evolution 37 2.1.1 Fluctuation test 38 2.1.2 Directed mutation in bacteria? 40 2.2 Types of mutations 41 2.2.1 Point mutations 41 2.2.2 Conditional mutants 42 2.2.3 Variation due to larger scale DNA alterations 44 2.2.4 Extrachromosomal agents and horizontal gene transfer 44 2.3 Phenotypes 45 Molecular Genetics of Bacteria, 4th Edition by Jeremy Dale and Simon F. Park # 2004 John Wiley & Sons, Ltd ISBN 0 470 85084 1 (cased) ISBN 0 470 85085 X (pbk) 2.4 Restoration of phenotype 47 2.4.1 Reversion and suppression 47 2.4.2 Complementation 49 2.5 Recombination 49 2.6 Mechanisms of mutation 50 2.6.1 Spontaneous mutation 50 2.6.2 Chemical mutagens 52 2.6.3 Ultraviolet irradiation 54 2.7 Isolation and identification of mutants 58 2.7.1 Mutation and selection 58 2.7.2 Replica plating 59 2.7.3 Penicillin enrichment 61 2.7.4 Isolation of other mutants 62 2.7.5 Molecular methods 62 3 Regulation of Gene Expression 67 3.1 Gene copy number 69 3.2 Transcriptional control 70 3.2.1 Promoters 70 3.2.2 Terminators, attenuators and anti-terminators 77 3.2.3 Induction and repression: regulatory proteins 79 3.2.4 Attenuation: trp operon 87 3.2.5 Two-component regulatory systems 92 3.2.6 Global regulatory systems 94 3.2.7 Feast or famine and the RpoS regulon 95 3.2.8 Quorum sensing 95 3.3 Translational control 99 3.3.1 Ribosome binding 99 3.3.2 Codon usage 101 3.3.3 Stringent response 101 3.3.4 Regulatory RNA 102 3.3.5 Phase variation 102 4 Genetics of Bacteriophages 103 4.1 Single-stranded DNA bacteriophages 106 4.1.1 fX174 106 4.1.2 M13 109 4.2 RNA-containing phages: MS2 109 4.3 Double-stranded DNA phages 110 4.3.1 Bacteriophage T4 110 4.3.2 Bacteriophage lambda 113 4.3.3 Lytic and lysogenic regulation of bacteriophage lambda 118 4.4 Restriction and modification 125 4.5 Complementation and recombination 128 4.6 Why are bacteriophages important? 130 4.6.1 Phage typing 131 vi CONTENTS 4.6.2 Phage therapy 131 4.6.3 Phage display 132 4.6.4 Bacterial virulence and phage conversion 133 5 Plasmids 137 5.1 Some bacterial characteristics are determined by plasmids 137 5.1.1 Antibiotic resistance 137 5.1.2 Colicins and bacteriocins 138 5.1.3 Virulence determinants 138 5.1.4 Plasmids in plant-associated bacteria 139 5.1.5 Metabolic activities 139 5.2 Molecular properties of plasmids 141 5.2.1 Plasmid replication and control 143 5.3 Plasmid stability 154 5.3.1 Plasmid integrity 155 5.3.2 Partitioning 157 5.3.3 Differential growth rate 160 5.4 Methods for studying plasmids 161 5.4.1 Associating a plasmid with a phenotype 161 5.4.2 Classification of plasmids 163 6 Gene Transfer 165 6.1 Transformation 166 6.2 Conjugation 167 6.2.1 Mechanism of conjugation 168 6.2.2 The F plasmid 173 6.2.3 Conjugation in other bacteria 174 6.3 Transduction 178 6.3.1 Specialized transduction 180 6.4 Recombination 181 6.4.1 General (homologous) recombination 181 6.4.2 Site-specific and non-homologous (illegitimate) recombination 186 6.5 Mosaic genes and chromosome plasticity 187 7 Genomic Plasticity: Movable Genes and Phase Variation 189 7.1 Insertion sequences 189 7.1.1 Structure of insertion sequences 189 7.1.2 Occurrence of insertion sequences 190 7.2 Transposons 192 7.2.1 Structure of transposons 194 7.2.2 Integrons 196 7.3 Mechanisms of transposition 197 7.3.1 Replicative transposition 197 7.3.2 Non-replicative (conservative) transposition 200 7.3.3 Regulation of transposition 201 7.3.4 Activation of genes by transposable elements 203 CONTENTS vii 7.3.5 Mu: a transposable bacteriophage 204 7.3.6 Conjugative transposons and other transposable elements 205 7.4 Phase variation 205 7.4.1 Variation mediated by simple DNA inversion. 207 7.4.2 Variation mediated by nested DNA inversion 208 7.4.3 Antigenic variation in the gonococcus 208 7.4.4 Phase variation by slipped strand mispairing 211 7.4.5 Phase variation mediated by differential DNA methylation 214 8 Genetic Modification: Exploiting the Potential of Bacteria 215 8.1 Strain development 215 8.1.1 Generation of variation 215 8.1.2 Selection of desired variants 216 8.2 Overproduction of primary metabolites 216 8.2.1 Simple pathways 217 8.2.2 Branched pathways 218 8.3 Overproduction of secondary metabolites 220 8.4 Gene cloning 221 8.4.1 Cutting and joining DNA 222 8.4.2 Plasmid vectors 223 8.4.3 Transformation 225 8.4.4 Bacteriophage lambda vectors 225 8.4.5 Cloning larger fragments 227 8.4.6 Bacteriophage M13 vectors 229 8.5 Gene libraries 229 8.5.1 Construction of genomic libraries 229 8.5.2 Screening a gene library 231 8.5.3 Construction of a cDNA library 233 8.6 Products from cloned genes 234 8.6.1 Expression vectors 234 8.6.2 Making new genes 236 8.6.3 Other bacterial hosts 239 8.6.4 Novel vaccines 241 8.7 Other uses of gene technology 242 9 Genetic Methods for Investigating Bacteria 245 9.1 Metabolic pathways 245 9.1.1 Complementation 246 9.1.2 Cross-feeding 246 9.2 Microbial physiology 247 9.2.1 Reporter genes 249 9.2.2 Lysogeny 250 9.2.3 Cell division 251 9.2.4 Motility and chemotaxis 252 9.2.5 Cell differentiation 253 viii CONTENTS 9.3 Bacterial virulence 257 9.3.1 Wide range mechanisms of bacterial pathogenesis 257 9.3.2 Detection of virulence genes 259 9.4 Specific mutagenesis 262 9.4.1 Gene replacement 262 9.4.2 Antisense RNA 264 9.5 Taxonomy, evolution and epidemiology 264 9.5.1 Molecular taxonomy 264 9.5.2 Diagnostic use of PCR 267 9.5.3 Molecular epidemiology 267 10 Gene Mapping to Genomics 273 10.1 Gene mapping 273 10.1.1 Conjugational analysis 273 10.1.2 Co-transformation and co-transduction 276 10.1.3 Molecular techniques for gene mapping 277 10.2 Gene sequencing 279 10.2.1 DNA sequence determination 281 10.2.2 Genome sequencing 282 10.2.3 Comparative genomics 285 10.2.4 Bioinformatics 288 10.3 Physical and genetic maps 289 10.3.1 Deletions and insertions 290 10.3.2 Transposon mutagenesis 290 10.3.3 Gene replacement 292 10.3.4 Site-directed mutagenesis 292 10.4 Analysis of gene expression 292 10.4.1 Transcriptional analysis 293 10.4.2 Translational analysis 296 10.4.3 Systematic analysis of gene function 300 10.5 Conclusion 300 Appendix A Further Reading 301 Appendix B Abbreviations 305 Appendix C Glossary 309 Appendix D Enzymes 323 Appendix E Genes 327 Appendix F Standard Genetic Code 331 Appendix G Bacterial Species 333 Index 337