Transcript Bacterial Genetics

Bacterial Genetics
• Pin Lin (凌 斌), Ph.D.
Departg ment of Microbiology & Immunology, NCKU
ext 5632
[email protected]
• References:
1. Chapters 5 in Medical Microbiology (Murray, P. R.
et al; 5th edition)
2. Chapter 25 in Biochemistry (Nelson, D. et al; 4th
edition)
Outline
• Introduction
• Replication of DNA
• Bacterial Transcription
• Other Genetic Regulation (Mutation,
Repair, & Recombination)
Introduction
• Gene:
a segment of DNA (or chromosome),
the fundamental unit of information in a cell
• Genome:
the collection of total genes in an organism
• Chromosome:
the large DNA molecule associated with proteins or
other components
Why do 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.
Bacterial vs Human Chromosome
E Coli:
Human:
1. Single circular chromosome,
1. 23 chromosomes, two
one copy (haploid)
copies (diploid)
2. Extrachromosomal genetic
elements:
Plasmids (autonomously selfreplicating)
Bacteriophages (bacterial
viruses)
3. Maintained by polyamines, ex.
spermine & spermidine
2. Extrachromosomal genetic
elements:
- Mitochondrial DNA
- Virus genome
3. Maintained by histones
Replication of Bacterial DNA-I
Features:
1.Semi-conservative
2. Multiple growing
forks
3. Bidirectional
Bacterial DNA is the storehouse of information.
4. Proofreading
=>polymerase)
Essential to replicate DNA correctly => Daughter
(DNA
cells
Discovery of DNA synthesis
Replication of Bacterial DNA-II
Replication of bacterial genome requires several enzymes:
- Helicase, unwind DNA at the replication origin (OriC)
- Primase, synthesize primers to start the process
- DNA polymerase, synthesize a copy of DNA, first found by
Arthur Kornberg
- DNA ligase, link two DNA fragements
- Topoisomerase, relieve the torsional strain during the
process, found by James Wang
Outline
• Introduction
• Replication of DNA
• Bacterial Transcription
• Other Genetic Regulation (Mutation,
Repair, & Recombination)
Transcriptional Regulation in Bacteria
1. Bacteria regulate 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
monosaccharide Glu & Gal
Y gene => lactose permease => pumping Lac into the cell
A gene => Acetylase
Lactose Operon-Negative Control
Lac Operon:
- Lac
metabolism
- Under pos &
neg control
Negative ctrl
- Repressor
- Inducer
(Allolactose)
- Operator
In presence of
Lactose
Lactose Operon-Positive Control
Positive control
Activator:
CAP-cAMP
(catabolite
gene-activator
protein)
CAP RNA pol
In absence of
Lactose
Tryptophan Operon
Negative control
- Repressor
- Corepressor
(Tryptophan)
- Operator
Attenuation
Couple Translation
w/ Transcription
Sequence 3:4 pair
-G-C rich stem loop
- Called attenuator
-Like
transcriptional
terminator
Sequence2: 3 pair
- weak loop won’t
block translation
Transcription
termination signal
Outline
• Introduction
• Replication of DNA
• Bacterial Transcription
• Other Genetic Regulation (Mutation,
Repair, & Recombination)
Types of mutations
1. Base substitutions
Silent mutation – No change of amino acid
Missense mutation – Switch to another amino acid
Nonsense mutation – Change to a stop codon
2. Deletion & Insertion - Change more base pairs in DNA
=> frameshift => truncated gene product
3. Rearrangements - Duplication, Inversion, Transposition
Induced mutations
Physical mutagens:
e.g., UV irradiation
(heat, ionizing radiation)
Chemical mutagens
Base analog
Frameshift
intercalating agents
Base modification
Transposable elements
DNA Repair
1. Direct DNA repair
(e.g., photoreactivation)
2. Excision repair
Base excision repair
Nucleotide excision repair
3. Post-replication or
Recombinational repair
4. SOS response: induce many
genes
5. Error-prone repair: fill in gaps
with random sequences
Thymine-thymine dimer
formed by UV radiation
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
Mechanisms of gene transfer
Transformation: uptake of naked exogenous DNA
by living cells.
Conjugation: mediated by self-transmissible
plasmids.
Transduction: phage-mediated genetic recombination.
Transposons: DNA sequences that move within the
same or between two DNA molecules
Importance of gene transfer to bacteria
• Gene transfer => a source of genetic variation =>
alters the genotype of bacteria.
• The new genetic information acquired allows the
bacteria to adapt to changing environmental
conditions through natural selection.
Drug resistance (R plasmids)
Pathogenicity (bacterial virulence)
• Transposons greatly expand the opportunity for
gene movement.
Transformation
Natural transformation
Artificial transformation
(conventional method
and electroporation)
Demonstration
of
transformation
Avery, MacLeod, and
McCarty (1944)
Gene exchange by Plasmids
Plasmid
Extrachromosomal
Autonomously replicating
Circular or linear (rarely)
May encode drug resistance
or toxins
Various copy numbers
Some are self-transmissible
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
Mobile genetic elements
Transposons
May carry drug resistance genes
Sometimes insert into genes and inactivate them
(insertional mutation)
Spread of transposon
throughout a bacterial
population
Trans-Gram
gene transfer
Mechanisms of evolution of Vancomycinresistant Staphylococcus Aureus
Cloning
Cloning vectors
plasmids
phages
Restriction enzymes
Ligase
In vitro phage packaging
Library
construction
Genomic library
cDNA library
Applications of genetic engineering
1. Construction of industrially important bacteria
2. Genetic engineering of plants and animals
3. Production of useful proteins (e.g. insulin, interferon,
etc.) in bacteria, yeasts, insect and mammalian cells
4. Recombinant vaccines (e.g. HBsAg)
Take-Home Question:
Mutations are good or bad for bacteria
The End & Thank You
Mechanism of Recombination
Homologous recombination
Site-specific recombination
Transposition
Illegitimate recombination
Intermolecular
Intramolecular
Double
crossover
Homologous recombination
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)
Bacteriophage (bacterial virus)
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.