Bacterial Genetics
• Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKUext [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)
Introductio
n• 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:
1. Single circular chromosome,
one copy (haploid)
2. Extrachromosomal genetic
elements:
Plasmids (autonomously self- replicating)
Bacteriophages (bacterial viruses)
3. Maintained by polyamines, ex. spermine & spermidine
Human:
1. 23 chromosomes, two copi
es (diploid)
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
4. Proofreading
(DNA polymerase)
Bacterial DNA is the storehouse of information.
=> Essential to replicate DNA correctly => Daughter 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
Negative ctrl
- Repressor
- Inducer (Allolactose)
- Operator
Lac Operon:
- Lac metabolism
- Under pos & neg control
In presence of Lactose
Positive control
Activator:
CAP-cAMP
(catabolite gene-activator protein)
CAP RNA pol
Lactose Operon-Positive Control
In absence of Lactose
Tryptophan OperonNegative control- Repressor- Corepressor (Tryptophan)- Operator
Attenuation
Transcription termination signal
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
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 acidMissense 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 mutationsPhysical mutagens:
e.g., UV irradiation (heat, ionizing radiation)
Chemical mutagens
Base analog
Frameshift
intercalating agents
Base modification
Transposable elements
DNA Repair1. 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
Nucleotide excision repair
Base excision repair
Double-strand break repair(postreplication repair)
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
SOS repair in bacteria
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.
Natural transformation
Transformation
Artificial transformation(conventional method and electroporation)
Demonstration of
transformation
Avery, MacLeod, and McCarty (1944)
Gene exchange by Plasmids
PlasmidExtrachromosomal
Autonomously replicating
Circular or linear (rarely)
May encode drug resistance or toxins
Various copy numbers
Some are self-transmissible
Conjugationmediated by
self-transmissible plasmids
(e.g., F plasmid; R
plasmids)
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
Transductionphage-mediated genetic recombination
Generalized v.s. specialized transduction
TransposonsMobile genetic elements
May carry drug resistance genes
Sometimes insert into genes and inactivate them (insertional mutation)
Trans-Gram gene transfer
Spread of transposon throughout a bacterial population
Mechanisms of evolution of Vancomycin-resistant Staphylococcus Aureus
Cloning
Cloning vectors
plasmids
phages
Restriction enzymes
Ligase
In vitro phage packaging
Library construction
Genomic library
cDNA library
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)
Applications of genetic engineering
The End & Thank You
Take-Home Question: Mutations are good or bad for bacteria
Mechanism of Recombination
Homologous recombination Site-specific recombination
Transposition Illegitimate recombination
Intermolecular
Intramolecular
Double crossover
Homologous recombination
E Conjugational transposon
Trans-Gram gene transfer
Spread of transposon throughout a bacterial population
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, interf
eron, etc.) in bacteria, yeasts, insect and mam
malian cells
Recombinant vaccines (e.g. HBsAg)
Bacteriophage (bacterial virus)
Icosahedral tailess
Icosahedral tailed
Filamentous
Structure and genetic materials of phages
Coat (Capsid)
Nucleic acid
Lysogenic phaseLytic phase
Life cyclePhage as an example
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.
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