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Tina M. Henkin. Snyder and Champness Molecular Genetics of Bacteria
Table of Contents
List of Illustrations
Guide
Pages
About the Companion Website
Snyder & Champness Molecular Genetics of Bacteria
Preface
Acknowledgments
About the Authors
Introduction
The Biological Universe. The Bacteria
GRAM-NEGATIVE AND GRAM-POSITIVE BACTERIA
The Archaea
The Eukaryotes
MITOCHONDRIA AND CHLOROPLASTS AND THE ROLE OF ENDOSYMBIOSIS IN EVOLUTION
What Is Genetics?
Bacterial Genetics
Bacteria Are Haploid
Short Generation Times
Asexual Reproduction
Colony Growth on Agar Plates
Colony Purification
Serial Dilutions
Selections
Storing Stocks of Bacterial Strains
Genetic Exchange
Phage Genetics
Phages Are Haploid
Selections with Phages
Crosses with Phages
A Brief History of Bacterial Molecular Genetics
Inheritance in Bacteria
Transformation
Conjugation
Transduction
Recombination within Genes
Semiconservative DNA Replication
mRNA
The Genetic Code
The Operon Model
Enzymes for Molecular Biology
Synthetic Genomics
What Is Ahead
SUGGESTED READING
1 The Bacterial Chromosome: DNA Structure, Replication, and Segregation
DNA Structure
The Deoxyribonucleotides
The DNA Chain
The 5′ and 3′ Ends
Base Pairing
Antiparallel Construction
The Major and Minor Grooves
The Mechanism of DNA Replication
Deoxyribonucleotide Precursor Synthesis
Replication of the Bacterial Chromosome
DNA POLYMERASES
PRIMASES
NUCLEASES
DNA LIGASES
ACCESSORY PROTEINS
Replication of Double-Stranded DNA
SEPARATING THE TWO TEMPLATE DNA STRANDS
PROCESSING THE TWO TEMPLATE DNA STRANDS
COORDINATING REPLICATION OF THE TWO TEMPLATE STRANDS
THE GENES FOR REPLICATION PROTEINS
Replication Errors
Editing
RNA Primers and Editing
Impediments to DNA Replication
Damaged DNA and DNA Polymerase III
Mechanisms To Deal with Impediments on Template DNA Strands
Physical Blocks to Replication Forks
Replication of the Bacterial Chromosome and Cell Division
Structure of Bacterial Chromosomes
Replication of the Bacterial Chromosome
Initiation of Chromosome Replication
ORIGIN OF CHROMOSOMAL REPLICATION
INITIATION PROTEINS
RNA Priming of Initiation
Termination of Chromosome Replication
BOX 1.1. Structural Features of Bacterial Genomes
References
Chromosome Segregation
RESOLUTION OF DIMER CHROMOSOMES
DECATENATION
CONDENSATION
Condensins
Supercoiling
KEEPING NEW SISTER CHROMOSOMES SEPARATE INVOLVES COORDINATING MULTIPLE PROCESSES
CHROMOSOME PARTITIONING
The Par Proteins
Macrodomains
Coordinating Cell Division and Chromosome Partitioning in E. coli and B. subtilis
The Min Proteins
Nucleoid Occlusion
Coordination of Cell Division with Replication of the Chromosome
TIMING OF REPLICATION IN THE CELL CYCLE
Timing of Initiation of Replication
ROLE OF THE DnaA PROTEIN
Inactivation of DnaA by hydrolysis to DnaA-ADP
Reactivation of DnaA by nucleotide exchange to DnaA-ATP
SeqA-MEDIATED HEMIMETHYLATION AND SEQUESTRATION
SeqA activities outside of oriC
The Bacterial Nucleoid
Supercoiling in the Nucleoid
SUPERCOILING OF NATURAL DNAs
SUPERCOILING STRESS
Topoisomerases
TYPE I TOPOISOMERASES
TYPE II TOPOISOMERASES
BOX 1.2. Antibiotics That Affect Replication and DNA Structure
Antibiotics That Block Precursor Synthesis
Inhibition of dihydrofolate reductase
Inhibition of ribonucleotide reductase
Competition with deoxyuridine monophosphate
Antibiotics That Block Polymerization of Deoxynucleotides
Deoxynucleotide precursor mimics
Cross-linking
Antibiotics That Affect DNA Structure. Acridine dyes
Thymidine mimic
Antibiotics That Affect Gyrase
GyrA inhibition
GyrB inhibition
The Bacterial Genome
Summary
QUESTIONS FOR THOUGHT
SUGGESTED READING
2 Bacterial Gene Expression: Transcription, Translation, Protein Folding, and Localization
Overview
The Structure and Function of RNA
Types of RNA
RNA Precursors
RNA Structure
PRIMARY STRUCTURE
SECONDARY STRUCTURE
TERTIARY STRUCTURE
RNA Processing and Modification
Transcription
Structure of Bacterial RNA Polymerase
Overview of Transcription
PROMOTERS
THE STEPS OF TRANSCRIPTION
Details of Transcription
PROMOTER RECOGNITION
ISOMERIZATION
INITIATION
ELONGATION
TERMINATION OF TRANSCRIPTION
Factor-Independent Termination
BOX 2.1. Antibiotic Inhibitors of Transcription
Inhibitors of rNTP Synthesis
Inhibitors of Transcription Initiation
Inhibitors of RNA Elongation and Termination
Inhibitors that Affect the DNA Template
Factor-Dependent Termination
rRNAs and tRNAs
BOX 2.2. Molecular Phylogeny
References
MODIFICATION OF RNA
RNA Degradation
RNases
MODULATION OF RNase ACTIVITY
The Structure and Function of Proteins
Protein Structure
PRIMARY STRUCTURE
SECONDARY STRUCTURE
TERTIARY STRUCTURE
QUATERNARY STRUCTURE
Translation
Structure of the Bacterial Ribosome
BOX 2.3. Antibiotic Inhibitors of Translation
Antibiotics that block translation
Inhibitors that Mimic tRNA
Inhibitors that Bind to the 23S rRNA
Inhibitors of Binding of aa-tRNA to the A Site
Inhibitors of Translocation
Overview of Translation
Details of Protein Synthesis
READING FRAMES
TRNA AMINOACYLATION
TRANSLATION INITIATION REGIONS
Initiation Codons
Shine-Dalgarno Sequences
INITIATOR tRNA
STEPS IN INITIATION OF TRANSLATION
Translation Initiation from Leaderless mRNAs
TRANSLATION INITIATION IN ARCHAEA AND EUKARYOTES
TRANSLATION ELONGATION
TRANSLATION TERMINATION
RELEASE FACTORS
REMOVAL OF THE FORMYL GROUP AND THE N-TERMINAL METHIONINE
BOX 2.4. Mimicry in Translation
References
trans-TRANSLATION (tmRNA)
The Genetic Code
REDUNDANCY
BOX 2.5. Exceptions to the Code
References
WOBBLE
TERMINATION CODONS
AMBIGUITY
CODON USAGE
Polycistronic mRNA
TRANSLATIONAL COUPLING
POLAR EFFECTS ON GENE EXPRESSION
Protein Folding and Degradation
Protein Chaperones
THE DnaK PROTEIN AND OTHER Hsp70 CHAPERONES
TRIGGER FACTOR AND OTHER CHAPERONES
CHAPERONINS
Protein Degradation
Protein Localization
The Translocase System
The Signal Sequence
The Targeting Factors
THE SecB PATHWAY
THE SRP PATHWAY
Sec Systems of Archaea and Eukaryotes
The Tat Secretion Pathway
STRUCTURE OF THE TAT SYSTEM
The Tat Signal Sequence
Tat Systems in Other Organisms
Disulfide Bonds
Protein Secretion and Export
Protein Secretion Systems in Bacteria with an Outer Membrane
TYPE I SECRETION SYSTEMS
TYPE II SECRETION SYSTEMS
TYPE III SECRETION SYSTEMS
TYPE IV SECRETION SYSTEMS
TYPE V SECRETION SYSTEMS: AUTOTRANSPORTERS
Chaperone-Usher Secretion
TYPE VI SECRETION SYSTEMS
Protein Secretion in Bacteria That Lack an Outer Membrane
INJECTOSOMES
Sortases
Regulation of Gene Expression
Transcriptional Regulation
Posttranscriptional Regulation
What You Need To Know
Open Reading Frames
Transcriptional and Translational Fusions
Summary
QUESTIONS FOR THOUGHT
SUGGESTED READING
3 Bacterial Genetic Analysis: Fundamentals and Current Approaches
Definitions
Terms Used in Genetics
MUTANT
PHENOTYPE
GENOTYPE
MUTATION
ALLELE
USE OF GENETIC DEFINITIONS
Genetic Names
NAMING MUTANT ORGANISMS
NAMING GENES
NAMING MUTATIONS
NAMING PHENOTYPES
Useful Phenotypes in Bacterial Genetics
Auxotrophic and Catabolic Mutants
ISOLATING AUXOTROPHIC MUTANTS
Conditional-Lethal Mutants
TEMPERATURE-SENSITIVE MUTANTS
Isolating Temperature-Sensitive Mutants
COLD-SENSITIVE MUTANTS
NONSENSE MUTATIONS
CONDITIONAL EXPRESSION OF THE WILD-TYPE ALLELE
Resistant Mutants
Inheritance in Bacteria
The Luria and Delbrück Experiment
Mutants Are Clonal
Esther and Joshua Lederberg’s Experiment
Mutation Rates
Calculating Mutation Rates
DETERMINING THE NUMBER OF CELL DIVISIONS
DETERMINING THE NUMBER OF MUTATIONS THAT HAVE OCCURRED IN A CULTURE
Calculating the Mutation Rate from the Data of Luria and Delbrück
Calculating the Mutation Rate from the Number of Clones of Mutants
Calculating the Mutation Rate from the Rate of Increase in the Proportion of Mutants
PRACTICAL IMPLICATIONS OF POPULATION GENETICS
Types of Mutations
Properties of Mutations
Base Pair Changes
CAUSES OF BASE PAIR CHANGES
Base Pair Changes Due to Mispairing during Replication
Base Pair Changes Due to Spontaneous Deamination
Base Pair Changes Due to Oxidation of Bases
CONSEQUENCES OF BASE PAIR CHANGES
Missense Mutations
Nonsense Mutations
PROPERTIES OF BASE PAIR CHANGE MUTATIONS
Frameshift Mutations
CAUSES OF FRAMESHIFT MUTATIONS
PROPERTIES OF FRAMESHIFT MUTATIONS
Deletion Mutations
CAUSES OF DELETIONS
PROPERTIES OF DELETION MUTATIONS
Isolating Deletion Mutants
NAMING DELETION MUTATIONS
Tandem-Duplication Mutations
CAUSES OF TANDEM DUPLICATIONS
PROPERTIES OF TANDEM-DUPLICATION MUTATIONS
Identifying Mutants with Tandem-Duplication Mutations
ROLE OF TANDEM-DUPLICATION MUTATIONS IN EVOLUTION
Inversion Mutations
CAUSES OF INVERSIONS
PROPERTIES OF INVERSION MUTATIONS
Identification of Inversion Mutations
NAMING INVERSIONS
Insertion Mutations
PROPERTIES OF INSERTION MUTATIONS
BOX 3.1. Inversions and the Genetic Map
References
IDENTIFICATION OF INSERTION MUTATIONS
NAMING INSERTION MUTATIONS
Reversion versus Suppression
Intragenic Suppressors
Intergenic Suppressors
NONSENSE SUPPRESSORS
Types of Nonsense Suppressors
Consequences of Nonsense Suppression
Genetic Analysis in Bacteria
Isolating Mutants
MUTAGENESIS
INDEPENDENT MUTATIONS
Regional and Site-Specific Mutagenesis
IDENTIFICATION OF MUTANTS
Selection of Mutants
Screening for Mutants without a Selection
Genetic Characterization of Mutants
LOCATING MUTATIONS BY RECOMBINATION
Consequences of Recombination in Bacteria
Genetic Markers
Marker rescue
Complementation Tests
RECESSIVE OR DOMINANT
cis/trans TESTS
ALLELISM TESTS
Intragenic Complementation
Polarity
CLONING BY MARKER RESCUE AND COMPLEMENTATION
Genetic Crosses in Bacteria
SELECTED AND UNSELECTED MARKERS
Mapping of Bacterial Markers by Transduction and Transformation
TRANSFORMATION
TRANSDUCTION
ANALYZING DATA OBTAINED BY TRANSFORMATION AND TRANSDUCTION
MAPPING BY COTRANSDUCTION FREQUENCIES
Ordering Mutations by Three-Factor Crosses
Other Uses of Transformation and Transduction
STRAIN CONSTRUCTION
REVERSION VERSUS SUPPRESSION
Genetic Mapping by Hfr Crosses
MAPPING BY GRADIENT OF TRANSFER
Perspective
Summary
QUESTIONS FOR THOUGHT
SUGGESTED READING
4 Plasmids
What Is a Plasmid?
Naming Plasmids
Functions Encoded by Plasmids
Plasmid Structure
PURIFYING PLASMIDS
Properties of Plasmids. Replication
THETA REPLICATION
ROLLING-CIRCLE REPLICATION
REPLICATION OF LINEAR PLASMIDS
Functions of the ori Region
HOST RANGE
BOX 4.1. Linear Chromosomes and Plasmids in Bacteria
References
Determining the Host Range
REGULATION OF COPY NUMBER
INCOMPATIBILITY
Incompatibility Due to Shared Replication Control
BOX 4.2. Determining the Inc Group
Incompatibility Due to Partitioning
Plasmid Replication Control Mechanisms
ColE1-DERIVED PLASMIDS: REGULATION OF PROCESSING OF PRIMER BY COMPLEMENTARY RNA
R1 AND Col1B-P9 PLASMIDS: REGULATION OF TRANSLATION OF Rep PROTEIN BY COMPLEMENTARY RNA
The R1 Plasmid
The Collb-P9 Plasmid
THE pT181 PLASMID: REGULATION OF TRANSCRIPTION OF THE rep GENE BY A SMALL COMPLEMENTARY RNA
THE ITERON PLASMIDS: REGULATION BY PLASMID COUPLING
HOST FUNCTIONS INVOLVED IN REGULATING PLASMID REPLICATION
Mechanisms To Prevent Curing of Plasmids
RESOLUTION OF MULTIMERIC PLASMIDS
BOX 4.3. Toxin-Antitoxin Systems and Plasmid Maintenance
References
PARTITIONING
The Par Systems of Plasmids
THE R1 PLASMID PAR SYSTEM
THE P1 AND F PLASMID Par SYSTEMS
INCOMPATIBILITY DUE TO PLASMID PARTITIONING
Plasmid Cloning Vectors
ANATOMY OF A PLASMID CLONING VECTOR
ORIGINS OF REPLICATION
SELECTABLE GENES
UNIQUE RESTRICTION SITES
Examples of Plasmid Cloning Vectors
pUC PLASMIDS
CONDITIONAL VECTORS
BACTERIAL ARTIFICIAL CHROMOSOME VECTORS
Broad-Host-Range Cloning Vectors
SHUTTLE VECTORS
Summary
QUESTIONS FOR THOUGHT
SUGGESTED READING
5 Conjugation
Overview
Classification of Self-Transmissible Plasmids and Integrating Elements
The Fertility Plasmid
Mechanism of DNA Transfer during Conjugation in Proteobacteria
Transfer (tra) Genes
THE Mpf COMPONENT
Coupling Proteins
BOX 5.1. Pilus-Specific Phages
THE Dtr COMPONENT
Relaxase
The Relaxosome
The oriT Sequence
PRIMASE
Efficiency of Transfer
REGULATION OF THE tra GENES
BOX 5.2. Delivery of Conditional Plasmids by Conjugation
Processing Events in the Donor Cell (Panel A)
Processing Events in the Recipient Cell (Panel B)
References
EXAMPLE: REGULATION OF tra GENES IN F PLASMIDS
Interspecies Transfer of Plasmids
Conjugation and Type IV Secretion Systems Capable of Translocating Proteins
BOX 5.3. Gene Exchange between Domains
Agrobacterium tumefaciens and Crown Gall Tumors in Plants
References
Mobilizable Plasmids
Chromosome Transfer by Plasmids
Formation of Hfr Strains of E. coli
Transfer of Chromosomal DNA by Integrated Plasmids
Chromosome Mobilization
Prime Factors
GENERATION OF PRIME FACTORS
COMPLEMENTATION TESTS USING PRIME FACTORS
BOX 5.4. Conjugation and Synthetic Genomics
References
ROLE OF PRIME FACTORS IN EVOLUTION
Diversity in Transfer Systems
Integrating Conjugative Elements
SXT/R391 ICE
EXAMPLE: SURFACE EXCLUSION IN ICE WITH SXT/R391 ELEMENTS
EXAMPLE: SELF-TRANSMISSIBLE AND MOBILIZABLE ICE WITH SXT/R391 ELEMENTS
ICEBs1
EXAMPLE: AN ICE REPLICATION SYSTEM
EXAMPLE: TARGETING CONJUGATION IN ICE WITH ICEBs1
cis-MOBILIZABLE ELEMENTS: EVOLUTION BY ACCRETION-MOBILIZATION
Tn916
EXAMPLE: SITE-SPECIFIC RECOMBINATION WITHOUT USING A SPECIFIC SITE
TnGBS1 and TnGBS2
Summary
QUESTIONS FOR THOUGHT
SUGGESTED READING
6 Transformation
Natural Transformation
Discovery of Transformation
Overview of Natural Transformation
DNA Uptake Mechanisms
DNA UPTAKE IN FIRMICUTES
BOX 6.1. Experimental Measurements of DNA Uptake
DNA UPTAKE IN PROTEOBACTERIA
COMPETENCE SYSTEMS BASED ON TYPE IV SECRETION SYSTEMS
Specificity of DNA Uptake
BOX 6.2. Genetic Evidence for Single-Stranded DNA Uptake
DNA Processing after Uptake
Natural Transformation as a Tool
PLASMID TRANSFORMATION AND PHAGE TRANSFECTION OF NATURALLY COMPETENT BACTERIA
IMPORTANCE OF NATURAL TRANSFORMATION FOR FORWARD AND REVERSE GENETICS
CONGRESSION
Regulation of Natural Competence
COMPETENCE REGULATION IN B. SUBTILIS
Competence Pheromones
Relationship between Competence, Sporulation, and Other Cellular States
REGULATION OF COMPETENCE IN S. PNEUMONIAE
COMPETENCE REGULATION IN H. INFLUENZAE
Identification of Competence in Other Organisms
Role of Natural Transformation
NUTRITION
RECOMBINATIONAL REPAIR
GENETIC REASSORTMENT
BOX 6.3. Role of Natural Transformation in Pathogens
References
Artificially Induced Competence
Chemical Induction
TRANSFORMATION BY PLASMIDS
TRANSFECTION BY PHAGE DNA
TRANSFORMATION OF CELLS WITH CHROMOSOMAL GENES
Electroporation
Protoplast Transformation
Summary
QUESTIONS FOR THOUGHT
SUGGESTED READING
7 Bacteriophages and Transduction
BOX 7.1. Phage Genomics
References
Lytic Development
The Lytic Cycle
Transcriptional Regulation of Phage Gene Expression
PHAGES THAT ENCODE THEIR OWN RNA POLYMERASES
T7: A New RNA Polymerase for the Late Genes
N4: An RNA Polymerase Packaged in the Virion
REPROGRAMMING OF HOST RNA POLYMERASE BY SIGMA FACTOR REPLACEMENT
SPO1: Cascade of Sigma Factors
MODIFICATION OF RNA POLYMERASE ACTIVITY WITH TRANSCRIPTIONAL REGULATORY PROTEINS
BOX 7.2. Phage T7-Based Tools
Phage T4: Transcriptional Activators, a New Sigma Factor, and Replication-Coupled Transcription
ANTITERMINATION MECHANISMS
λ N-mediated antitermination
λ Q-mediated Antitermination
HK022 put-Mediated Antitermination
Phage Genome Replication and Packaging
PHAGES WITH SINGLE-STRANDED CIRCULAR DNA
Replication of Single-Stranded Phage DNA
PHAGES WITH CIRCULAR DOUBLE-STRANDED DNA GENOMES
Circle-to-Circle, or θ, Replication of λ DNA
Rolling-Circle Replication of λ DNA
Genetic Requirements for λ DNA Replication
REPLICATION AND DNA PACKAGING OF DOUBLE-STRANDED DNA LINEAR GENOMES
The Primer Problem
Phage T7: Linear DNA That Forms Concatemers
BOX 7.3. Protein Priming
References
Phage T4: Another Phage That Forms Concatemers
RNA PHAGES
Host Cell Lysis
SINGLE-PROTEIN LYSIS
TIMED LYSIS
Timing of Lysis by Holins
T4 Phage Lysis
λ Phage Lysis
Activation of SAR Endolysins
Lysogenic Development
The λ System
THE LYSIS-LYSOGENY DECISION
Role of the CII Protein, a Transcriptional Activator
Role of the CIII Protein: A Protease Inhibitor
PHAGE λ INTEGRATION
MAINTENANCE OF λ LYSOGENY
THE CI REPRESSOR
Regulation of CI Repressor Synthesis
IMMUNITY TO SUPERINFECTION
THE Cro PROTEIN
INDUCTION OF λ
EXCISION OF λ
SUMMARY OF THE λ LYTIC AND LYSOGENIC CYCLES
Other Lysogenic Systems
PHAGE P2
PHAGE P4: A SATELLITE VIRUS
PROPHAGES THAT REPLICATE AS PLASMIDS
Phage P1
Phage N15
PHAGE Mu: A TRANSPOSON MASQUERADING AS A PHAGE
Phi3T: SMALL MOLECULE CONTROL OF LYSOGENY
Genetic Analysis of Phages
Infection of Cells
MULTIPLICITY OF INFECTION
Phage Crosses
Recombination and Complementation Tests with Phages
RECOMBINATION TESTS
Recombination Frequency
COMPLEMENTATION TESTS
The Genetic-Linkage Map of a Phage
FEATURES OF THE GENETIC MAPS OF SOME PHAGES
Phage λ
Phage T4
Phage P22
Phage P1
Phage-Mediated Genetic Transfer
Generalized Transduction
WHAT MAKES A GENERALIZED TRANSDUCING PHAGE?
ROLE OF GENERALIZED TRANSDUCTION IN BACTERIAL EVOLUTION
Specialized Transduction
SELECTION OF HFT PARTICLES
Lysogenic Conversion and Bacterial Pathogenesis
E. COLI AND DYSENTERY: SHIGA TOXINS
DIPHTHERIA TOXIN
CHOLERA TOXIN
STAPHYLOCOCCUS AUREUS AND TOXIC SHOCK SYNDROME
Host Defenses Against Phage Infection
Restriction-Modification Systems
Abi Systems
CRISPR/Cas Systems
Small Molecules and Phage Defense
Phage versus Phage
Phages as Tools
Cloning Vectors
Phage Display
Phage Therapy
Summary
QUESTIONS FOR THOUGHT
SUGGESTED READING
8. Transposition, Site-Specific Recombination, and Families of Recombinases
Transposition
Overview of Transposition
Structure of Bacterial DNA Transposons
Types of Bacterial DNA Transposons
INSERTION SEQUENCE ELEMENTS
COMPOSITE TRANSPOSONS
Assembly of Plasmids by IS Elements
NONCOMPOSITE TRANSPOSONS
NONAUTONOMOUS ELEMENTS
Assays of Transposition
SUICIDE VECTORS
Phage Suicide Vectors
Plasmid Suicide Vectors
THE MATING-OUT ASSAY FOR TRANSPOSITION
Mechanisms of Transposition
DDE Transposons
DETAILS OF THE MECHANISM OF TRANSPOSITION BY Tn5: CUT-OUT AND PASTE-IN TRANSPOSITION
DETAILS OF THE MECHANISM OF TRANSPOSITION BY Tn3: COPY-IN OR REPLICATIVE TRANSPOSITION
DETAILS OF THE MECHANISM OF TRANSPOSITION BY Tn7: CUT-OUT AND PASTE-IN TRANSPOSITION USING HETEROMERIC TRANSPOSASE
DETAILS OF THE MECHANISM OF TRANSPOSITION BY IS911: COPY-OUT AND PASTE-IN TRANSPOSITION
HUH Transposons
DETAILS OF THE MECHANISM OF TRANSPOSITION BY IS608: SINGLE-STRAND TRANSPOSITION
ROLLING-CIRCLE TRANSPOSONS
BOX 8.1. Mobile Elements and DNA Replication
References
General Properties of Transposons
Transposition Regulation
Target Site Specificity
Effects on Genes Adjacent to the Insertion Site
Target Immunity
Transposon Mutagenesis
BOX 8.2. Transposons and Genomics
References
Transposon Mutagenesis In Vivo
Transposon Mutagenesis In Vitro
Transposon Mutagenesis of Plasmids
Transposon Mutagenesis of the Bacterial Chromosome
Transposon Mutagenesis of All Bacteria
CLONING GENES MUTATED WITH A TRANSPOSON INSERTION
Using Transposon Mutagenesis To Make Random Gene Fusions
Site-Specific Recombination
Integrases
PHAGE INTEGRASES
INTEGRASES OF TRANSPOSON INTEGRONS
Resolvases
DNA Invertases
PHASE VARIATION IN SALMONELLA SPECIES
OTHER INVERTIBLE SEQUENCES
Y and S Recombinases
Y Recombinases: Mechanism
S Recombinases: Mechanism
Group II Mobile Introns: Elements that Move Using an RNA Intermediate
Importance of Transposition and Site-Specific Recombination in Bacterial Adaptation
Summary
QUESTIONS FOR THOUGHT
SUGGESTED READING
9 Molecular Mechanisms of Homologous Recombination
Homologous Recombination and DNA Replication in Bacteria
Early Evidence for the Interdependence of Homologous Recombination and DNA Replication
The Molecular Basis for Recombination in E. coli
chi (χ) Sites and the RecBCD Complex
HOW RecBCD WORKS
WHY χ?
BOX 9.1. Discovery of χ Sites
References
BOX 9.2. Other Types of Double-Strand Break Repair in Bacteria
References
HELICASE-NUCLEASE PROCESSING IN OTHER BACTERIA: AddAB
The RecF Pathway
Synapse Formation and the RecA Protein
HOLLIDAY JUNCTIONS
The Ruv and RecG Proteins and the Migration and Cutting of Holliday Junctions
RuvABC
RecG
Recombination between Different DNAs in Bacteria
How Are Linear DNA Fragments Recombined into the E. coli Chromosome?
Recombination during Natural Transformation
Phage Recombination Pathways
Rec Proteins of Phages T4 and T7
The RecE Pathway of the rac Prophage
The Phage λ Red System
Recombineering: Gene Replacements in E. coli with Phage λ Recombination Functions
Gene Conversion and Other Manifestations of Heteroduplex Formation during Recombination
GENE CONVERSION
MANIFESTATIONS OF MISMATCH REPAIR IN HETERODUPLEXES IN PHAGES AND BACTERIA
Map Expansion
Marker Effects
Summary
QUESTIONS FOR THOUGHT
SUGGESTED READING
10 DNA Repair and Mutagenesis
Evidence for DNA Repair
Specific Repair Pathways
Deamination of Bases
DEAMINATING AGENTS
Hydroxylamine
Nitrous Acid
REPAIR OF DEAMINATED BASES
VERY-SHORT-PATCH REPAIR OF DEAMINATED 5-METHYLCYTOSINE
Damage Due to Reactive Oxygen
8-oxoG
MutM, MutY, and MutT
MutM
MutY
BOX 10.1. The Role of Reactive Oxygen Species in Cancer and Degenerative Diseases
References
MutT
GENETICS OF 8-oxoG MUTAGENESIS
Damage Due to Alkylating Agents
SPECIFIC N-GLYCOSYLASES
METHYLTRANSFERASES
AlkB AND AidB
THE ADAPTIVE RESPONSE
Regulation of the Adaptive Response
Damage Due to UV Irradiation
PHOTOREACTIVATION OF CYCLOBUTANE DIMERS
N-GLYCOSYLASES SPECIFIC TO PYRIMIDINE DIMERS
General Repair Mechanisms
Base Analogs
Frameshift Mutagens
Mismatch Repair
GENETIC EVIDENCE FOR METHYL-DIRECTED MISMATCH REPAIR
Isolation of mut Mutants
BOX 10.2. DNA Repair and Cancer
References
ROLE OF THE MISMATCH REPAIR SYSTEM IN PREVENTING HOMEOLOGOUS AND ECTOPIC RECOMBINATION
Nucleotide Excision Repair
MECHANISM OF NUCLEOTIDE EXCISION REPAIR
TRANSCRIPTION-COUPLED REPAIR
Mfd-dependent transcription coupled repair
INDUCTION OF NUCLEOTIDE EXCISION REPAIR
DNA Damage Tolerance Mechanisms
Homologous Recombination and DNA Replication
LAGGING-STRAND DAMAGE
LEADING-STRAND DAMAGE
BREAKING THE CHROMOSOME TO REPAIR THE CHROMOSOME
REPAIR OF INTERSTRAND CROSS-LINKS IN DNA
SOS-lnducible Repair
THE SOS RESPONSE
GENETICS OF SOS-INDUCIBLE MUTAGENESIS
Determining Which Repair Pathway Is Mutagenic
Isolation of umuC and umuD Mutants
Experiments Showing that Only umuC and umuD Must Be Induced for SOS Mutagenesis
Experiments Show that RecA Has a Role in UV Mutagenesis in Addition to Its Role as a Coprotease
Mechanism of TLS by the Pol V Mutasome
Other Specialized Polymerases and Their Regulation
DNA Pol II
DNA Pol IV (DlnB)
BOX 10.3. The Ames Test
Reference
Summary of Repair Pathways in E. coli
Bacteriophage Repair Pathways
Summary
QUESTIONS FOR THOUGHT
SUGGESTED READING
11 Regulation of Gene Expression: Genes and Operons
Transcriptional Regulation in Bacteria
BOX 11.1. The Helix-Turn-Helix Motif of DNA-Binding Proteins
References
Genetic Evidence for Negative and Positive Regulation
Negative Regulation of Transcription Initiation
Negative Inducible Systems
THE E. COLI iac OPERON
Mutations of the lac Operon
Complementation Tests with lac Mutations
The Jacob and Monod Operon Model
Refinements to the Regulation of the lac Operon
Catabolite Regulation of the lac Operon
Structure of the lac Control Region
Locations of lacI Mutations in the Three-Dimensional Structure of the LacI Repressor
Experimental Uses of the lac Operon
THE E. COLI gal OPERON
Two gal Repressors: GalR and GalS
Two gal Operators
Two gal Promoters and Catabolite Regulation of the gal Operon
Negative Repressible Systems
THE E. COLI trp OPERON
Isolation of trpR Mutants
Other Types of Regulation of the E. coli trp Operon
Molecular Mechanisms of Transcriptional Repression
Positive Regulation of Transcription Initiation
Positive Inducible Systems
THE E. COLI ara OPERON
Genetic Evidence for Positive Regulation of the ara Operon
Isolating Constitutive Mutations of the ara Operon
A Model for Positive Regulation of the ara Operon
BOX 11.2. Families of Regulators
References
AraC Is Not Just an Activator
Face-of-the-Helix Dependence
Autoregulation of araC
Catabolite Regulation of the ara Operon
Uses of the ara Operon
Positive Repressible Systems
THE E. COLI fab OPERON
Molecular Mechanisms of Transcriptional Activation
INTERACTIONS OF ACTIVATORS AND REPRESSORS
Regulation by Transcription Attenuation
Modulation of RNA Structure
REGULATION OF THE E. COLI trp OPERON BY LEADER PEPTIDE TRANSLATION
Model for Regulation of the E. coli trp Operon by Attenuation
Genetic Evidence for the trp Attenuation Model
REGULATION OF THE B. SUBTILIS trp OPERON BY AN RNA-BINDING PROTEIN
REGULATION OF THE E. COLI bgl OPERON BY AN RNA-BINDING PROTEIN
RIBOSWITCH RNAs DIRECTLY SENSE METABOLIC SIGNALS
The T Box Mechanism: tRNA-Sensing Riboswitches
Metabolite-Binding Riboswitches
Changes in Processivity of RNA Polymerase
PHAGE λ N AND Q PROTEINS
BACTERIAL PROCESSIVE ANTITERMINATION SYSTEMS
The RfaH System
Regulation of mRNA Degradation
Protein-Dependent Effects on RNA Stability
RNA-Dependent Effects on RNA Stability
REGULATION OF mRNA DEGRADATION BY sRNAs
THE glmS RIBOZYME
Regulation of Translation
Regulation of Translation Initiation
RNA THERMOSENSORS: REGULATION BY MELTING SECONDARY STRUCTURE IN THE mRNA
The E. coli rpoH Heat Shock Thermosensor
RNA Thermosensors That Control Virulence
RIBOSWITCH REGULATION OF TRANSLATION INITIATION
TRANSLATIONAL REGULATION BY RNA-BINDING PROTEINS
TRANSLATIONAL REGULATION BY sRNAs
TRANSLATIONAL AUTOREGULATION OF INITIATION FACTOR IF3
Translational Regulation in the Exit Channel of the Ribosome
Regulation of Translation Termination
Posttranslational Regulation
Posttranslational Protein Modification
Regulation of Protein Turnover
REGULATION OF THE RpoS SIGMA FACTOR BY ADAPTORS AND ANTIADAPTORS
Feedback Inhibition of Enzyme Activity
FEEDBACK INHIBITION OF THE trp OPERON
Why Are There So Many Mechanisms of Gene Regulation?
Summary
QUESTIONS FOR THOUGHT
SUGGESTED READING
12 Global Regulation: Regulons and Stimulons
Carbon Catabolite Regulation
Carbon Catabolite Regulation in E. coli: Catabolite Activator Protein (CAP) and cAMP
REGULATION OF cAMP SYNTHESIS
BOX 12.1. cAMP-lndependent Carbon Catabolite Regulation in E. coli
Reference
CATABOLITE ACTIVATOR PROTEIN
REGULATION BY CAP-cAMP
RELATIONSHIP OF CATABOLITE REGULATION TO INDUCTION
GENETIC ANALYSIS OF CATABOLITE REGULATION IN E. COLI
Isolation of cya and crp Mutations
Promoter Mutations That Affect Activation by CAP-cAMP
Carbon Catabolite Regulation in Bacillus subtilis: CcpA and Hpr
Regulation of Nitrogen Assimilation
BOX 12.2. Nitrogen Fixation
References
Pathways for Nitrogen Assimilation
Regulation of Nitrogen Assimilation Pathways in E. coli by the Ntr System
REGULATION OF THE glnA-ntrB-ntrC OPERON BY A SIGNAL TRANSDUCTION PATHWAY
Regulation of Other Ntr Operons
TRANSCRIPTION OF THE glnA-ntrB-ntrC OPERON BY THE NITROGEN SIGMA FACTOR, σΝ
BOX 12.3. Signal Transduction Systems in Bacteria
References
The Transcription Activator NtrC
BOX 12.4. Sigma Factors
References
ADENYLYLATION OF GLUTAMINE SYNTHETASE
COORDINATION OF CATABOLITE REPRESSION, THE Ntr SYSTEM, AND THE REGULATION OF AMINO ACID-DEGRADATIVE OPERONS
Regulation of Nitrogen Assimilation in B. subtilis
THE TnrA PROTEIN REGULATES NITROGEN METABOLISM IN B. SUBTILIS
THE CodY GLOBAL REGULATOR
Regulation of Ribosome Components and tRNA Synthesis
Ribosomal Protein Gene Regulation
MAPPING OF RIBOSOMAL PROTEIN GENES
REGULATION OF THE SYNTHESIS OF RIBOSOMAL PROTEINS
Regulation of rRNA and tRNA Synthesis
REGULATION OF rRNA TRANSCRIPTION
GROWTH RATE REGULATION OF rRNA AND tRNA TRANSCRIPTION
ANTITERMINATION OF rRNA OPERONS
Stringent Response
SYNTHESIS OF ppGpp DURING THE STRINGENT RESPONSE
SYNTHESIS OF ppGpp BY SpoT
ROLE OF ppGpp IN GROWTH RATE REGULATION, AFTER STRESS, AND IN STATIONARY PHASE
DksA: A PARTNER IN ppGpp ACTION
STRINGENT RESPONSE IN OTHER BACTERIA
Stress Responses in Bacteria
Heat Shock Regulation
HEAT SHOCK REGULATION IN E. COLI
Genetic Analysis of Heat Shock in E. coli
Regulation of σΗ Synthesis
rpoH mRNA: An RNA Thermosensor
DnaK: The E. coli Cellular Thermometer
Returning to Normal: Turning Off the Response
HEAT SHOCK REGULATION IN OTHER BACTERIA
General Stress Response in Enteric Bacteria
BOX 12.5. Regulatory RNAs
References
General Stress Response in Firmicutes
Extracytoplasmic (Envelope) Stress Responses
REGULATION OF PORIN SYNTHESIS
GENETIC ANALYSIS OF PORIN REGULATION
EnvZ and OmpR: A Sensor Kinase and Response Regulator Partnership
The Affinity Model for Regulation of ompC and ompF
REGULATION OF ompF BY THE MicF sRNA
REGULATION OF THE ENVELOPE STRESS RESPONSE BY THE CpxA-CpxR TWO-COMPONENT SYSTEM
THE EXTRACYTOPLASMIC SIGMA FACTOR σΕ
Iron Regulation in E. coli
The Fur Regulon
The RyhB sRNA
The Aconitase Translational Repressor
Regulation of Virulence Genes in Pathogenic Bacteria
Diphtheria
DIPHTHERIA TOXIN
Regulation of the tox Gene of C. diphtheriae
Cholera and Quorum Sensing
CHOLERA TOXIN
Regulation of the Synthesis of Cholera Toxin and Other Virulence Determinants
QUORUM SENSING
Quorum Sensing in V. cholerae
Whooping Cough
REGULATION OF B. PERTUSSIS VIRULENCE GENES
Developmental Regulation: Sporulation in B. subtilis
Identification of Genes That Regulate Sporulation
Regulation of Sporulation Initiation
REGULATION OF THE Spo0A PHOSPHORELAY SYSTEM
Compartmentalized Regulation of Sporulation Genes
The Role of Sigma Factors in Sporulation Regulation
TEMPORAL PATTERNS OF REGULATION
TRANSCRIPTION FACTOR DEPENDENCE PATTERNS OF EXPRESSION
CELLULAR LOCALIZATION
Intercompartmental Regulation during Development
TEMPORAL REGULATION AND COMPARTMENTALIZATION OF σΕ AND σF
Regulation of σF
Regulation of σΕ
σG, A SECOND FORESPORE-SPECIFIC SIGMA FACTOR
σK, A MOTHER CELL SIGMA
Other Sporulation Systems
Summary
QUESTIONS FOR THOUGHT
SUGGESTED READING
13 Genomes and Genomic Analysis
The Bacterial Genome
DNA Sequencing
BOX 13.1. Annotation and Comparative Genomics
References
BOX 13.2. Special Problems in Genetic Analysis of Operons
References
Advanced Genome-Sequencing Techniques
Polymerase Chain Reaction
PCR MUTAGENESIS
Barriers to Horizontal Transfer: Genome Gatekeepers and the Molecular Biologist’s Toolkit
Restriction Endonucleases
RESTRICTION ENDONUCLEASES THAT LIMIT GENE EXCHANGE IN E. COLI
OTHER DEFENSE SYSTEMS USING NUCLEASES AND METYHLATION
TYPE II RESTRICTION ENZYMES AND CLONING
Using Restriction Endonucleases To Generate Recombinant DNAs
Cloning and Cloning Vectors
DNA Libraries
Techniques for Nontraditional Cloning and Assembly
TA CLONING OF PCR-AMPLIFIED FRAGMENTS
Topo I CLONING
CLONING WITH λ SITE-SPECIFIC RECOMBINATION: GATEWAY CLONING
LIGATION-INDEPENDENT CLONING USING LONG OVERHANGS: URACIL-N-GLYCOSYLASE-MEDIATED AND T4 DNA POLYMERASE-MEDIATED CLONING
GOLDEN GATE CLONING
GIBSON ASSEMBLY
IN VIVO CLONING AND ASSEMBLY TECHNIQUES
CRISPR/Cas Systems
BOX 13.3. Synthesizing and Cloning Complete Bacterial Genomes
References
SPACER ACQUISITION, crRNA PROCESSING, AND INTERFERENCE
CLASSES AND TYPES OF CRISPR/Cas SYSTEMS
PROTOSPACER-ADJACENT MOTIFS (PAMs)
SPACER ACQUISITION WITH Cas1-Cas2
TYPE I CRISPR/Cas SYSTEMS
TYPE II Cas9 CRISPR/Cas SYSTEMS
OTHER TYPES OF CRISPR/Cas SYSTEMS
DEFENSE AND COUNTERDEFENSE WITH CRISPR/Cas SYSTEMS
Final Thoughts
Summary
QUESTIONS FOR THOUGHT
SUGGESTED READING
Glossary
Index
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