Lecture 10 - Bacterial Genetics Flashcards
What is the difference in recombination between eukaryotes and prokaryotes?
Evolution requires the generation of genetic diversity via genetic exchange (recombination) between individuals carrying different alleles (mutations)
* Eukaryotes use the sexual cycle and meiosis to effect recombination
* Prokaryotes divide by binary fission; no sexual cycle. No meiosis
Prokaryotes achieve genetic exchange via
* Transformation - Uptake of naked DNA
* Conjunction - utilises plasmids
* Transduction - uses bacteriophages
Describe what may be present in the bacterial geneome.
In addition to essential chromosome bacteria may contain additional extrachromosomal, small, circular DNA molecules - plasmids - Nonessential by t may confer advantages to host cells:
*Sex plasmids
*R plasmids
*Col plasmids
What are sex plasmids?
Sex plasmids
e.g. F(fertility) plasmid of E.coli
* Approx. 35% of sequence encode functions permitting transfer between individual bacteria.
* Mediate transfer of bacterial genes by conjugation
F-plasmid is an episome - may exist as free circular plasmid, or may be integrated into chromosome.
What are R plasmids?
R Plasmids
* Self-mobile. Can transfer between unrelated bacterial species.
* Encode resistance to one or more antimicrobial drugs
* Evolved particularly in the last 60 years, in parallel with the widespread use of antibiotics.
R plasmid spread through environment and between unrelated bacterial species. Poses a threat to treatment of infectious disease
What are Col plasmids.
Col Plasmids
* Small plasmids
* Encode biological factors (e.g. colicin)
* Does not encode functions permitting transfer between individual bacteria.
* May be transferred if F or R plasmids present in same cell encoding functions required for contact and transfer
Col plasmids have been extensively manipulated by molecular biologists to generate useful vectors for DNA cloning.
How was F plasmid mediated conjucation discovered in E.coli?
Discovery of F plasmid mediated conjugation in E.coli
Unidirectional transfer of genetic information from donor to recipient cells
Lederberg and Tatum - 1946
* Mixed two auxotrophic strains of bacteria - each had separate mutations meaning they couldn’t grow in minimal media.
* Observed some prototrophic colonies when mixture was plated on minimal media (Makeup for each other’s mutations).
* Colonies resulted from genetic exchange between original strains
* U-tube experiment confirmed physical contact between stains is required.
Descrive Bacterial conjugation.
- An F+ cell contacts an F- cell - initial connection established by a long tubular F-pilus
- F+ pilus contracts forming a bridge
- Genetic material transferred through cytoplasmic bridge.
- F plasmid carries tra (transfer) genes for contact and mobilisation functions - encodes pilin protein to build pilus.
Mechanism for F-plasmid transfer - F plasmid - tra genes encode contact and DNA transfer functions
- Transfer initiated by introducing a nick in DNA at OriT (origin of Transfer)
- 5’ end of ssDNA transferred to recipient
- Rolling circle replication forms ssDNA from F plasmid
- DNA synthesis in F- recipient restores second strand
Transfer of F plasmid by conjugation in E.coli
Transfer of the F factor from donor F+ cell to recipient F- cell during F+ x F- mating
If all F plasmid is transferred and replicated in recipient, DNA is circularised and original F- cell becomes F+ cell.
How is the F plasmid integrated into the bacterial chromosome.
Integration of the F plasmid into the bacterial chromosome - Hfr strains.
In F+ x F- crosses only the F factor is transferred. Transfer of bacterial chromosome first requires F factor to become integrated into bacterial chromosomes.
Chromosomal integration of F plasmid
* Hfr strains discovered by Hayes and Cavalli-Sforza
* Rare event - F plasmid integrated into chromosome Hfr strain
* F plasmid can be integrated in either orientation according to orientation of recombining sequences.
* F plasmid integrated into chromosome via recombination between insertion sequences on F plasmid and chromosome
* F plasmid integration is reversible
Hfr transfer (High frequency transfer of bacterial genes)
1. F plasmid integrated into chromosome
2. Encodes transfer functions
3. Integrated F plasmid oriT is nicked
4. F factor initiates transfer to recipient
5. F plasmid transferred following bacterial chromosome
Transferred chromosomal DNA recombines with recipient chromosome.
What is Hfr conjugation.
Hfr conjugation
Hfr strains can conjugate with F- strains
* Unidirectional, ordered transfer of chromosomal genes form donor to recipient.
* Part of F transferred first - bridge fragile and breaks - very rare more than a fraction of chromosome exchanged
* Conjunction not species specific which allows horizontal gene transfer
* Success is dependent on DNA homology - as recombination required to integrate transferred fragment.
Conjugation can be used to map bacterial genes
Mix bacteria at various times after mating commences, withdraw samples, break mating cells apart and plate bacteria on selective media to determine which genes have been transferred from Hfr to F-.
Interrupted mating experiments with a variety of Hfr strains show that the E.coli generic map is circular.
How does imprecise excision of F plasmid from the bacterial chromsome generate F’ plasmids carrying bacterial genes?
Imprecise excision of F plasmid from bacterial chromosome generates F’ plasmids carrying bacterial genes.
1. F plasmid integrated into bacterial chromosome e.g. adjacent to lac+ region
2. F factor loops out incorrectly, including a piece of the chromosome
3. Single crossover generates F’lac and F factor including a Lac+ region
4. F’lac+ can transfer to recipient. As Circular DNA can be maintained without integration into recipient chromosome. Recipient becomes Lac+ partial diploid. Used in genetic analysis of gene regulation.
How can bacteriophages mediate gene transfer.
Bacteriophage P1 can mediate gene transfer of any part of bacterial chromosome by generalised transduction
1. Infection of donor bacterium with P1
2. Phage reproduction
3. Assembly of progeny wild-type and transducing phage’s, Some progeny phage’s package bacterial genes in heads.
4. Release of progeny phases by cell lysis. Some phage’s are transuding phage’s - they carry a piece of donor bacterial DNA.
5. Infection of recipient bacterium with a transducing phage
6. Genetic exchange of donor gene with recipient gene by a double crossover
7. Stable transduced bacterium produced by recombination. Linear fragments degraded by cellular nucleases.
How can cotransduction frequency be used to map genes.
Cotransduction frequency ban be used to map genes
* Genes that are closer together are co-transduced more frequently than gens that are further apart
* Frequency with which genes are co-transduced allows determination of gene order
Limit of cotransduction approx. 100 kbp.
