Lecture 9 - bacterial conjugation Flashcards
What are 3 different means of genetic transfer?
Transformation
Transduction
Conjugation
Transformation
Some bacteria can take up DNA from their surroundings
Transduction
Incorporation of bacterial DNA into a phage for transfer
Conjugation
Plasmids that copy their DNA into new cells
Plasmids
Circular double stranded DNA molecules that replicate independently of the chromosome
Confer a new function upon the host cell, giving it an advantage over other bacteria
Joshua Lederberg & Edward Tatum
Bacteria strain A and strain B could not grow on minimal media. A & B carry mutations that cause an inability to synthesise constituents needed for growth.
When A & B are mixed for a few hours colonies begin to grow due to the exchange of genetic material
Minimal medium
The basic synthetic medium for bacterial growth without nutrient supplements
bio-
arg-
met-
Required biotin/arginine/methionine added as a supplement to minimal medium
lac-
gal-
Cannot use galactose/lactose as a carbon source
Str^r
Str^s
Resistant to antibiotic streptomycin
Sensitive to the antibiotic streptomycin
Bernard Davis
Created an experiment to demonstrate that physical contact between bacterial cells is needed for genetic recombination to occur.
A suspension of strain A which is unable to synthesise certain nutrients was placed in one arm of a U tube and strain B, which was unable to synthesise other metabolites was placed in the other arm. After several hours of incubation no colonies grew.
(The medium could move in either direction but the bacteria could not)
William Hayes
Determined that genetic transfer occurred in one direction and that transfer is not reciprocal - one cell acts as the donor while the other is the recipient.
F+
F-
F+ strains are those that carry the F factor and can create a pilus
F- strains are those that do not carry the F factor so cannot create a pilus
F factor
A plasmid that can transfer itself between bacterial cells by conjugation which allows a copy of the plasmid to move from one cell to the next
Transconjugants
Bacteria that have incorporated DNA from another bacterium by conjugation.
What are the steps in bacterial conjugation?
F+ donor cells contain the F plasmid that allows them to make the F pilus and act as a genetic donor.
Contact between the donor and recipient as the F pilus recognises and binds receptor sites on the cell wall.
A relaxosome cleaves the DNA at the origin of transfer. The relaxosome associates with the T-DNA strand
A single F plasmid, beginning at the origin of transfer, enters the F- cell.
Does gene transfer require direct contact between cells?
Yes, there must be direct contact between donor and recipient cells
What are the steps to rolling circle replication?
One DNA strand is nicked at oriTand the 3’ OH is extended by DNA polymerase.
The 3’ end on the circle is lengthened while the growing point rolls around the circular template.
The 5’ end is displaced and forms a tail of single stranded DNA that extends from the circle.
The single stranded DNA is converted to double stranded DNA through the use od RNA primers.
The 5’ end will enter the recipient cell as a single strand.
Hfr
High frequency of recombination
Strain that produces 1000X more recombinants than a normal F+ strain
The F plasmid is cut at oriT by an exonuclease and integrated into the host cell genome.
How do Hfr strains form?
As plasmids are being transferred random breakage can occur which interrupts the transfer before the entire chromosome is transferred. This fragment may then recombine with the recipient chromosome.
In Hfr how is the donor DNA incorporated into the recipients chromosome?
Homologous recombination
In Hfr what happens to linear DNA that is not incorporated into the chromosome?
It is degraded
How did Wollman and Jacob discover linkage maps could be constructed?
Use interrupted mating as a measure of time at which a donor allele begins to appear after mating.
e.g. if lac+ begins to enter F- cell 10 minutes after azi^r then these genes are 10 units apart
