Lecture 50: Conjugation

Question 1

What is conjugation?

Answer 1

Conjugation is the process of
moving genetic material
(often, but not always, plasmids)
via direct cell-to-cell contact

Question 2

is conjugation one of the mechanisms of horizontal gene transfer?

Answer 2

Yes

Question 3

Who carried out an experiment to demonstrate bacterial conjugation and when?

Answer 3

Lederberg and Tatum in 1946

Question 4

Describe Lederberg and Tatum’s experiment

Answer 4

• 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.

Question 5

Who provided further evidence in support of bacterial conjugation, and when?

Answer 5

Davies, in 1950

Question 6

Describe Davies experiment

Answer 6

• 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
  )

Question 7

What does conjugation require?

Answer 7

Plasmids

Question 8

Describe plasmids

Answer 8

• 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

Question 9

high copy number…

Answer 9

can be >100

Question 10

low copy number…

Answer 10

1 or a few

Question 11

How may plasmids be incompatible?

Answer 11

Related plasmids sharing common mechanisms of replication often cannot coexist

Question 12

Is it true that cells can contain many non related plasmids?

Answer 12

Yes

Question 13

Describe curing

Answer 13

Plasmid is lost from host

Happens spontaneously or in response to certain chemicals

Question 14

What are episomes?

Answer 14

special plasmids that can integrate into host genome

Question 15

what are the roles of plasmids?

Answer 15

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)
  )

Question 16

Give an example of a bacteriocin

Answer 16

colicins (forms pores in membrane, degrades DNA etc.)

Question 17

Is it true that some plasmids are conjugative and some are not?

Answer 17

Yes

Question 18

What do conjugative plasmids do?

Answer 18

they themselves encode the genes (tra genes) that will allow transfer (of themselves) to other cells.

Question 19

What is an example of a conjugative plasmid?

Answer 19

F plasmid in E. coli

Question 20

How may a mating pair of plasmids be connected?

Answer 20

by an F pilus

Question 21

Does an F pilus allow for unidirectional transfer of DNA from donor to recipient?

Answer 21

Yes

Question 22

What does the ‘F’ in F pilus stand for?

Answer 22

Fertility factor

Question 23

Is it true that the F plasmid can spread through F- strain quickly similar to an infectious agent?

Answer 23

Yes

Question 24

What is meant by the fact that F is an intergrative plasmid?

Answer 24

F is integrative plasmid – can integrate in a number of
locations or exist as free plasmid

Question 25

Describe how the F pilus creates contact

Answer 25

- 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

Question 26

How is the plasmid transferred from one bacterial cell to another?

Answer 26

- 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. - -

Question 27

Describe the transfer if the plasmid in more detail (RCR)

Answer 27

- 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)

Question 28

'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?

Answer 28

Yes

Question 29

How long does it take cells in culture to all obtain an F plasmid?

Answer 29

Process takes about 2 min at 37∘C. And sensitive to agitation

Question 30

At what temperature do cells in culture obtain an f plasmid?

Answer 30

at 37∘C

Question 31

What are High Frequency Recombination strains?

Answer 31

HFr strains can partially transfer part of their genome

Question 32

What strain are High Frequency Recombination strains derived from?

Answer 32

The F+ strain

Question 33

How can the F plasmid be integrated into the genome?

Answer 33

Through recombination

Question 34

Is the integration of the F plasmid into the genome via recombination a rare event?

Answer 34

Yes

Question 35

is the F plasmid an episome?

Answer 35

Yes

Question 36

Is it true that high frequency recombination strains still produce the F pili, even though they have no plasmid to transfer?

Answer 36

Yes

Question 37

How do high frequency recombination strains transfer their genome?

Answer 37

- 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

Question 38

In high frequencty recombination, why is the new strain not F+ ?

Answer 38

Because not all of the genome is transferrerd to recipient cell, so new strain onyl has some new genes

Question 39

Is it true that Gene transfer stops when mating pair breaks apart?

Answer 39

yes

Question 40

Define Merodiploid

Answer 40

haploid stain that is diploid only in some genes

Question 41

Time of entry mapping...

Answer 41

The further gene is away from origin the longer and less likely transfer is

Question 42

Is it true that HFr strains can become F’ strains?

Answer 42

Yes

Question 43

How can HFr strains become F' strains?

Answer 43

F plasmid can excise from genome to become F plasmid again. Occasionally excision is imprecise and some chromosomal genes end up in the plasmid

Question 44

What can the F’ strain mate with?

Answer 44

F- strain

Question 45

Key messages

Answer 45

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