Bacterial Genetics
• Pin Ling (凌 斌), Ph.D.
Department of Microbiology & Immunology, NCKU
ext 5632
lingpin@mail.ncku.edu.tw
• 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 Chromosome
E Coli:
Human:
1. Single circular chromosome,
1. 23 chromosomes, two copies
double-stranded; one copy (haploid)
2. Extrachromosomal genetic
elements:
Plasmids (autonomously selfreplicating)
(diploid)
2. Extrachromosomal genetic
elements:
- Mitochondrial DNA
- Virus genome
Phages (bacterial viruses)
Transposons (DNA sequences that
move within the same or between
two DNA molecules)
3. Structurally maintained by
polyamines, ex spermine &
spermidine
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 => b-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)
Lactose operon:
Lactose
metabolism
Under positive or
negative ctrl
Negative control
Repressor
Inducer
Operator
Lactose Operon: Positive Control
Positive control
Activator: CAP
(catabolite
gene-activator
protein)
CAP RNA
pol
Inducer
Transcriptional Regulation of gene
expression (Example II)
Negative control
Repressor
Corepressor
Operator
Tryptophan 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
Caused 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
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.
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
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.
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)
History of signaling transduction
Adopted from Nobelprize.org