Lecture 50: Conjugation Flashcards
Monday 24th February 2025
What is conjugation?
Conjugation is the process of
moving genetic material
(often, but not always, plasmids)
via direct cell-to-cell contact
is conjugation one of the mechanisms of horizontal gene transfer?
Yes
Who carried out an experiment to demonstrate bacterial conjugation and when?
Lederberg and Tatum in 1946
Describe Lederberg and Tatum’s experiment
- They set up 2 bacterial strains /colonies (A and B)
- Strain A: met⁻ bio⁻ thr⁺ leu⁺ thi⁺
- Strain B: met⁺ bio⁺ thr⁻ leu⁻ thi⁻
- Each strain had mutations that prevented it from growing on minimal media alone.
- The colonies were grown separately, then mixed together.
- The mixed culture was then grown on minimal media and colonies appeared, indicating that some bacteria had acquired the ability to grow.
- This suggests that some bacteria formed wild-type prototrophic colonies (met⁺ bio⁺ thr⁺ leu⁺ thi⁺), meaning they regained the ability to grow on minimal medium.
- This suggest that genes were transferred between the bacterial strains by bacterial conjugation.
Who provided further evidence in support of bacterial conjugation, and when?
Davies, in 1950
Describe Davies experiment
- He put A and B in a semi-permeable U tube that prevents cells passing through to either side
- He blocked one end with cotton wool, and added pressure/suction to the other end
- Adding suction/pressure caused A and B to mix.
- Plating bacteria from either sides resulted in no growth on minimal media. They remained as auxotrophs.
- But removing the filter and allowing the 2 colonies to mix properly resulted in growth on minimal media .
- This experiment ruled out: cross feeding, transformation, and conduction.
- This is because cells can’t pass through the semi-permeable filter. So cells must be able to come into direct contact with each other in order for conjugation to occur . (Reversion to wt must require
cell-to-cell contact
)
What does conjugation require?
Plasmids
Describe plasmids
- Usually consist of double stranded DNA
- Most are circular, but can be linear
- Vary greatly in size (1 kb to >1Mbp
) - Replicate independently of chromosomal DNA
- Vary in copy numbers
- Can be incompatible
high copy number…
can be >100
low copy number…
1 or a few
How may plasmids be incompatible?
Related plasmids sharing common mechanisms of replication often cannot coexist
Is it true that cells can contain many non related plasmids?
Yes
Describe curing
Plasmid is lost from host
Happens spontaneously or in response to certain chemicals
What are episomes?
special plasmids that can integrate into host genome
what are the roles of plasmids?
Carries non-essential but often highly useful genes
Carries antibiotic resistance genes problematic from medical point of view useful from a biotech point of view
Can carry virulence factors i.e toxins that increase pathogenicity
- May contain bacteriocins (Proteins killing or inhibiting closely related species (not as broad spectrum as antibiotics)
)
Give an example of a bacteriocin
colicins (forms pores in membrane, degrades DNA etc.)
Is it true that some plasmids are conjugative and some are not?
Yes
What do conjugative plasmids do?
they themselves encode the genes (tra genes) that will allow transfer (of themselves) to other cells.
What is an example of a conjugative plasmid?
F plasmid in E. coli
How may a mating pair of plasmids be connected?
by an F pilus
Does an F pilus allow for unidirectional transfer of DNA from donor to recipient?
Yes
What does the ‘F’ in F pilus stand for?
Fertility factor
Is it true that the F plasmid can spread through F- strain quickly similar to an infectious agent?
Yes
What is meant by the fact that F is an intergrative plasmid?
F is integrative plasmid – can integrate in a number of
locations or exist as free plasmid
Describe how the F pilus creates contact
- The donor has the F plasmid. The F plasmid will start expressing the Tra genes. This will cause the creation of the pilus. The pilus will go in search of a recipient cell.
- Once the pilus comes into contact with the recipient cell, it will drag the donor and recipient cells closer together.
- The pilus will shorten and the cells will come closer together
- An opening will be created between the 2 cells. This mating bridge will allow the transfer of the plasmid from the donor to the recipient.
- The plasmid will be retained by the first cell, but will make a copy and put it into the second cell.
- Both cells will now have a plasmid and each cell will continue to make contact with other bacterial cells
How is the plasmid transferred from one bacterial cell to another?
- One of the strands of the DNA of the F+ plasmid is nicked.
- The nicked strand is ‘unrolled’ and transferred through the mating bridge to the donor cell.
- Now there will be single stranded DNA in both of the cells.
- The cells will then start the synthesis of the other strands of DNA. You will then end up with a double stranded plasmid in both cells.
-
Describe the transfer if the plasmid in more detail
(RCR)
- One strand is nicked at the
Double Stranded Origin
of replication - The 3’ end of the nicked strand will serve as a primer for replication.
- DNA polymerase will bind to the 3’ end and add bases.
- Once a full round has been
completed “old” strand is released as ssDNA. - DNA strajd in donor cell will be ligated.
- The old ssDNA will now be transferred into the recipient cell. It is the lagging strand
- In recipient cell, ssDNA is circularised and ligated.
- The SSO (single stranded origin of replication) will be recognised by an RNA polymerase and RNA polymerase will create an RNA primer.
- DNA polymerase will the nsynthesise the second strand of DNA.
- New strand will then be ligated to heal the nick.
(Another replication mechanism for viruses)
‘F plasmid can spread through an F- culture rapidly ensuring the cells are converted to F+
Whatever other genes are encoded by the plasmid will now be spread.’ Is this statement true?
Yes
How long does it take cells in culture to all obtain an F plasmid?
Process takes about 2 min at 37∘C. And sensitive to agitation
At what temperature do cells in culture obtain an f plasmid?
at 37∘C
What are High Frequency Recombination strains?
HFr strains can partially transfer part of their genome
What strain are High Frequency Recombination strains derived from?
The F+ strain
How can the F plasmid be integrated into the genome?
Through recombination
Is the integration of the F plasmid into the genome via recombination a rare event?
Yes
is the F plasmid an episome?
Yes
Is it true that high frequency recombination strains still produce the F pili, even though they have no plasmid to transfer?
Yes
How do high frequency recombination strains transfer their genome?
- The plasmid is still nicked at the origin of replication
- DNA polymerase will bind to the 3’ end.
- The replcaed strand of chromosomal DNA will be transferred to the recipient cell
- Usually DNA brakes before entire chromosome is transferred
- A complementary strand will be synthesised in the recipient cell.
- This complementary strand will be a free-floating linear form that can’t circularise, may be degraded, but occasionaly, recombination will take place
In high frequencty recombination, why is the new strain not F+ ?
Because not all of the genome is transferrerd to recipient cell, so new strain onyl has some new genes
Is it true that Gene transfer stops when mating pair breaks apart?
yes
Define Merodiploid
haploid stain that is diploid only in some genes
Time of entry mapping…
The further gene is away from origin the longer and less likely transfer is
Is it true that HFr strains can become F’ strains?
Yes
How can HFr strains become F’ strains?
F plasmid can excise from genome to become F plasmid again. Occasionally excision is imprecise and some chromosomal genes end up in the plasmid
What can the F’ strain mate with?
F- strain
Key messages
Plasmids exist outside of the chromosomal DNA + control their own replication and copy number
Conjugation requires cell-to-cell contact to transfer plasmid
HFr strains have high frequency of recombination as they can transfer part of their genome to another cell
