Microbial Genetics
Replication of chromosomal DNA
Transcriptional control
Mutation, repair, recombination
Gene exchange in bacteria
Genetic engineering
微免所
何漣漪
Eukaryotic microbes:
fungi, yeasts
Eukaryotic genome
Chromosomal DNA
Mitochondrial DNA
Plasmids in yeast
Prokaryotic microbes: bacteria
Prokaryotic genome
Chromosomal DNA: doublestranded; circular; haploid.
Extrachromosomal genetic
elements
Plasmids (autonomously selfreplicating)
Phages (bacterial viruses)
Transposons (DNA sequences
that move within the same or
between two DNA molecules)
Replication of chromosomal DNA
Replication of
bacterial genome
requires:
Replication origin
(oriC)
DNA polymerase
Primase
Helicase
Topoisomerase
Semiconservative
Bidirectional
Regulation of gene expression at
transcriptional level (Example I)
Operon
Negative
control
Repressor
Inducer
Operator
Lactose (Lac) operon
Positive
control
Activator
Inducer
Regulation of gene expression at
transcriptional level (Example II)
Negative control
Repressor
Corepressor
Operator
Tryptophan (Trp) operon
Attenuation
Transcription
termination signal
Mutation
Types of mutations
1. Base substitutions
Silent vs. neutral; missense vs. nonsense
2. Deletions
3. Insertions May cause frameshift or null mutation
4. Rearrangements: duplication, inversion, transposition
Spontaneous
mutations
Cuased by tautomeric
shift of the nucleotides;
replication errors
Induced mutations
Physical 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
Excision
repair
Base excision
repair
Nucleotide
excision
repair
Base excision
repair
Nucleotide
excision
repair
Double-strand
break repair
(postreplication
repair)
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
End-joining
(error-prone)
Translocation
Short deletion at
the joining point
Gene exchange in bacteria
Mediated by plasmids and phages
Plasmid
Extrachromosomal
Autonomously replicating
Circular or linear (rarely)
May encode drug resistance
or toxins
Various copy numbers
Some are self-transmissible
Mechanisms of gene transfer
Transformation: uptake of naked exogenous DNA by
living cells.
Conjugation: mediated by self-transmissible plasmids.
Transduction: phage-mediated genetic recombination.
Transformation
Natural transformation
Artificial transformation
(conventional method
and electroporation)
Demonstration
of
transformation
Avery, MacLeod, and
McCarty (1944)
Conjugation
mediated by
self-transmissible plasmids
(e.g., F plasmid; R plasmids)
F plasmid
F plasmid
--an episome
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
Hfr strain
F’ plasmid
Transduction
phage-mediated genetic recombination
Generalized v.s. specialized transduction
Bacteriophage (bacterial viruse)
Structure and genetic materials of phages
Coat (Capsid)
Nucleic acid
Icosahedral
tailess
Icosahedral
tailed
Filamentous
Life cycle
Phage l as an example
Lytic phase
Lysogenic phase
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.
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 provides 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.
Mobile genetic elements
Transposons
May carry drug resistance genes
Sometimes insert into genes and inactivate them
(insertional mutation)
E Conjugational transposon
Spread of transposon
throughout a bacterial
population
Trans-Gram
gene transfer
Cloning
Cloning vectors
plasmids
phages
Restriction enzymes
Ligase
In vitro phage packaging
Library
construction
Genomic library
cDNA library
Applications of genetic engineering
Construction of industrially important bacteria
Genetic engineering of plants and animals
Production of useful proteins (e.g. insulin,
interferon, etc.) in bacteria, yeasts, insect and
mammalian cells
Recombinant vaccines (e.g. HBsAg)