Bacterial Genetics
• Pin Ling ( 凌 斌 ), Ph.D.
Department of Microbiology & Immunology, NCKU
ext 5632
• Reference: Murray, P. et al., Medical Microbiology (5th edition)
Outline
• Introduction
• Replication of DNA
• Bacterial Transcription
• Other Genetic Regulation
(Mutation, Repair, &
Recombination)
Introduction
• DNA:the genetic material
• Gene: a segment of DNA (or chromosome), the fundamental unit of information in a cell
• Genome: the collection of genes
• Chromosome: the large DNA molecule associated with proteins or other components
Why we study Bacterial Genetics?
• Bacterial genetics is the foundation of the modern Genetic Engineering & Molecular Biology.
• The best way to conquer bacterial disease is to understand bacteria first.
Human vs Bacterial ChromosomeE Coli:
1. Single circular chromosome,
double-stranded; one copy (haploid)
2. Extrachromosomal genetic
elements:
Plasmids (autonomously self- replicating)
Phages (bacterial viruses)
Transposons (DNA sequences that move within the same or between two DNA molecules)
3. Structurally maintained by polyamines, ex spermine & spermidine
Human:
1. 23 chromosomes, two copies (d
iploid)
2. Extrachromosomal genetic ele
ments:
- Mitochondrial DNA
- Virus genome
3. Maintained by histones
Replication of Bacterial DNA
1. Bacterial DNA is the storehouse of information. => It is essential to replicate DNA correctly and pass into the daughter cells.
2. Replication of bacterial genome requires several enzymes:- Replication origin (oriC), a specific sequence in the
chromosome - Helicase, unwind DNA at the origin- Primase, synthesize primers to start the process- DNA polymerase, synthesize a copy of DNA- Topoisomerase, relieve the torsional strain during the
process
Replication of Bacterial DNA
Features:
1. Semiconservative
2. Bidirectional
3. Proofreading (DNA polymerase)
Transcriptional Regulation in Bacteria
1. Bacteria regulates expression of a set of genes coordinately & quickly in response to environmental changes.
2. Operon: the organization of a set of genes in a biochemical pathway.
3. Transcription of the gene is regulated directly by RNA polymerase and “repressors” or “inducers” .
4. The Ribosome bind to the mRNA while it is being transcribed from the DNA.
Lactose Operon
1. E Coli can use either Glucose or other sugars (ex: lactose) as the source of carbon & energy.
2. In Glu-medium, the activity of the enzymes need to metabolize Lactose is very low.
3. Switching to the Lac-medium, the Lac-metabolizing enzymes become increased for this change .
4. These enzymes encoded by Lac operon: Z gene -galactosidase => split disaccharide Lac into
monosaccgaride Glu & Gal Y gene => lactose permease => pumping Lac into the cell A gene => Acetylase
Transcriptional regulation of gene expression (Example I)
Negative control
Repressor
Inducer
Operator
Lactose operon:
Lactose metabolism
Under positive or negative ctrl
Positive control
Activator: CAP (catabolite gene-activator protein)
CAP RNA pol
Inducer
Lactose Operon: Positive Control
Tryptophan operon
Transcriptional Regulation of gene expression (Example II)
Negative controlRepressorCorepressorOperator
MutationTypes of mutations1. Base substitutions
Silent vs. neutral; missense vs. nonsense2. Deletions3. Insertions4. Rearrangements: duplication, inversion, transposition
May cause frameshift or null mutation
Spontaneous mutations
Caused by tautomeric shift of the nucleotides; replication errors
Induced mutationsPhysical mutagens:
e.g., UV irradiation (heat, ionizing radiation)
Chemical mutagens
Base analog
Frameshift
intercalating agents
Base modification
Transposable elements
Mutator strains
DNA Repair
1. Direct DNA repair
(e.g., photoreactivation)
2. Excision repair
Base excision repair
Nucleotide excision repair
3. Mismatch repair
4. SOS response
5. Error-prone repair
Thymine-thymine dimer formed by UV radiation
SOS repair in bacteria
1. Inducible system used only when error-free
mechanisms of repair cannot cope with
damage
2. Insert random nucleotides in place of the
damaged ones
3. Error-prone
Gene exchange in bacteriaMediated by plasmids and phages
PlasmidExtrachromosomal
Autonomously replicating
Circular or linear (rarely)
May encode drug resistance or toxins
Various copy numbers
Some are self-transmissible
Bacteriophage (bacterial viruse)
Icosahedral tailess
Icosahedral tailed
Filamentous
Structure and genetic materials of phages
Coat (Capsid)
Nucleic acid
Virulent phages: undergo only lytic cycle
Temperate phages: undergo both lytic and lysogenic cycles
Plaques: a hollow formed on a bacterial lawn resulting from infection of the bacterial cells by phages.
Mechanisms of gene transfer
Transformation: uptake of naked exogenous DNA by living cells.
Conjugation: mediated by self-transmissible plasmids.
Transduction: phage-mediated genetic recombination.
Natural transformation
Transformation
Artificial transformation(conventional method and electroporation)
F’ plasmid
Hfr strain
F plasmid
F plasmid can integrate into bacterial chromosome to generate Hfr (high frequency of recombination) donors
Excision of F plasmid can produce a recombinant F plasmid (F’) which contains a fragment of bacterial chromosomal DNA
F plasmid
--an episome
Mechanism of Recombination
Homologous recombination Site-specific recombination
Transposition Illegitimate recombination
Intermolecular
Intramolecular
Double crossover
Homologous recombination
Importance of gene transfer to bacteria
• Gene transfer provide a source of genetic variation in addition to mutation that alters the genotype of bacteria. The new genetic information acquired allows the bacteria to adapt to changing environmental conditions through the process of natural selection.
Drug resistance (R plasmids)
Pathogenicity (bacterial virulence)
• Transposons greatly expand the opportunity for gene movement.
TransposonsMobile genetic elements
May carry drug resistance genes
Sometimes insert into genes and inactivate them (insertional mutation)
Applications of genetic engineering
Construction of industrially important bacteria
Genetic engineering of plants and animals
Production of useful proteins (e.g. insulin, interf
eron, etc.) in bacteria, yeasts, insect and mam
malian cells
Recombinant vaccines (e.g. HBsAg)