Conjugation Workshop Flashcards
Vertical evolution and horizontal gene transfer
Bacteria typically evolve by vertical evolution (accumulation of mutations in different lineages)
Occasionally they evolve via horizontal gene transfer resulting in rapid evolution e.g. to gain antibiotic resistance
Means of gene transmission
Transformation: some bacteria can take up genes from their surroundings - E. Coli can be artificially induced to do this
Transduction: occurs when phages (virus) infect bacteria then incorporate bacterial DNA into their head region and inject it into another bacterium to be recombined and integrated into its chromosome
Conjugation: plasmids that can copy their DNA into new cells. Pilus structure allows them to form a bridge between the two cells to copy their plasmid into the next cell
Bacterial plasmids
-bacterial plasmids - naturally occurring circular double stranded DNA molecules that replicate independently from the chromosome (extrachromosomal)
- range in size from 2-100 kilobases
- some plasmids have only 1-2 copies per cell others >100 copies
- usually confer a new function to the host cell, giving it an advantage over other bacteria e.g. antibiotic or heavy metal resistance
- many have the capacity to transfer themselves horizontally (ie between individuals)
- they have the ability to make a sex pilus
Discovery of bacterial conjugation
in 1946 Joshua Lederberg and Edward Tatum discovered bacteria of strain A and B cannot grow on minimal media (mm)
(Mm is a basic growth medium containing salt, some trace elements and sugar to grow - bacteria can make most other nutrients from these carbon sources)
Strain A and B were mutants hence they were deficient in certain genes essential for growth.
Strain A lacked genes for Met and need biotin but could produce Thr, Leu and Thy.
Strain B could synth Met and biotin but was unable to synth the 3 aas that strain A could.
So Lederberg and Tatum grew these bacteria, washed them and plated them onto mm.
Strain A couldn’t grow
(without Met and biotin)
Strain B couldn’t grow
(without Thr, Leu and Thy)
However when Strain A and B were mixed and plated after a few hours colonies of both strains began to grow - without any supplementation of the mm.
This suggests that something happened between the two strains allowing them to exchange genes by transfer and add them to their own genome by recombination.
This was the first indication that gene transfer could occur between bacterial species and earned them a Nobel prize.
A problem with their experiment was that there was a potential that it was actually metabolic nutrients being transferred between the 2 strains that allowed them to grow rather than genetic info transfer
Symbols used in bacterial genetics
Bio- : requires biotin supplement to mm
Arg- : “ Arg “ “ “
Met- :” Met “ “ “
Lac- : cannot use lactose as C source
Gal- : - “ galactose “ “ “
Str^r : resistant to antibiotic streptomycin
Str^s: sensitive to “ “ “
Bernard Davis 1950
Utilised a u tube with a fine filter in the middle dividing it into two sections with one bacterial strain in each compartment. He used the same strains A and B. The filter allowed nutrients and medium but not bacteria to pass through so the bacteria could not come into physical contact.
After several hours of incubation both strains were plated but no colonies grew on mm.
This demonstrating that physical contact between the bacterial strains is necessary showing that it is not nutrients transfer that allows the two strains to grow but genetic transfer and recombination as proposed by Lederberg and Tatum
William Hayes 1953
Determined that genetic transfer is unidirectional on these crosses.
Transfer is not reciprocal, one cell is donor (termed male) and one cell is reciprocal (termed female)
Hayes discovered that a variant of his donor strain that had lost the ability to transfer genetic material had become ‘sterile’ and realised that ‘fertility’ the ability to donate gene, could be lost/gained.
This suggested some sort of fertility factor (F). Strains carrying F (F+) donate to strains lacking F (F-) that are recipients.
F factor conjugation
F factor eventually found to be a plasmid that can transfer itself between bacterial cells
It does this by conjugation, allowing a copy to be transferred to another cell through cell to cell contact via a pilus.
Bacteria that have incorporated DNA from another bacterium by conjugation are known as transconjugants.
F plasmid codes f pilus which recognises and binds to specific receptor sites on recipient cell
Rolling circle replication
This is how plasmids are replicated.
Specific origin of replication (OriT) where a specific nuclease enzyme nicks the outer strand resulting in a 5’ phosphate (5’P) and 3’ hydroxyl (3’OH)
3’ OH can be used to begin DNA synthesis, replicating from this point by using the template strand as a copy and displacing the 5’ phosphate strand.
The strand extends from the 3’ OH inserting nucleotides and displacing the other strand producing rolling circle replication.
5’ P tail is inserted into the recipient (F-) cell as ssDNA, once in the cell RNA priming begins synthesising RNA primers and synthesising the second strand resulting in a new dsDNA plasmid.
Replication continues around the circle and multiple plasmid units aka concatamers can be generated
Formation of Hfr strains
1956 Luca Cavalli-Sforza discovered a derivative of an F+ strain produced 1000x as many recombinants as a normal F+ would.
He named it Hfr for high frequency of recombination.
Due to the fact that F plasmids can occasionally integrate into the host chromosome. Some plasmid can now transfer the chromosome to a new bacteria creating a Hfr strain
Chromosome transfer
During conjugation between an Hfr and a F- cell a part of the chromosome is transferred with F.
Random breakage usually interrupts the transfer before the whole chromosome is transferred (as the chromosome is very large)
Chromosomal fragments can recombine with the recipient chromosome.
Chromosomal DNA transfer by the embedded conjugative plasmid in Hfr strains allow gene transfer between species that are not naturally transformable (allows rapid evolution)
DNA is replicated and transferred by rolling circle replication
1957 Elie Wolman and Francois Jacob: crossed an Hfr with an F-
Crossed
Hfr str^s azi+ ton+ lac+ gal+
With
F- str^r azi- ton- lac- gal-
At specific time intervals they removed samples and placed them in a kitchen blender for a few seconds to disrupt the mating cell pairs then played them onto medium containing streptomycin to kill the Hfr donor cells.
The procedure is called interrupted mating.
Recipient str^r cells were tested for presence of marker alleles from the donor - genes were found to be transferred in a particular order
Wollman and Jacob constructed linking maps using as a measure of distance the times at which the donor alleles first appeared after mating.
Units of distance in this case minutes e.g. if lac+ begins to enter F- cell 10 minutes after azi+ begins to enter them lac+ and azi+ are 10 units apart.
The whole E. coli chromosome takes 100 minutes to transfer.
Diversity of Hfr strains - genetic mapping
Through use of different Hfr strains that have a fertility factor inserted into the chromosome at different points and different directions, interrupted mating experiments were used to map the position of genes on the E. Coli chromosome and reveal that its chromosome was circular
Conjugation practical
Mate an F+ bacteria with an F-
- donor (F+) is TC^rStr^s
- recipient is TC^sStr^r
1) make up 3 plates one of donor, one of recipient and one of D+R and allow to grow for 2hrs at 37°C in the dark
Take samples of these 3 plates and add to autoclave tubes along with a square of filter paper and 1.2ml saline.
2) create a series of 10 fold dilutions
E.g. take 100microlitres from D+R autoclaved sample equal to 10⁰ dilution and add to 900 microlitres of saline to give 10-¹ repeat to get to 10-⁵ dilution.
3) plating bacteria,
Spread bacteria on all plates using aseptic technique and incubate at 37°c overnight
1st set plated on Tet (T) only medium
Expect to see donor strain and F+ recipient growth - large no. of colonies
2nd set with Tet (T) and Str(S) medium
Expect only recipient’s of F+ to grow (must be resistant to both) few colonies
D 10⁰ and R10⁰ plate for control
Aseptic handling: use sterile tweezers (sterilised by burning ethanol by Bunsen) and sterilised filters sterilised by autoclave at 121°C for 15 mins. Spread plates using burning ethanol sterilised glass spreader make sure it has cooled before applying bacteria to avoid killing them
