Conjugation

Question 1

What are the two origins of replication in conjugative plasmids?

Answer 1

oriV- where vegetative replication begins (used by the remaining strand of the plasmid that was not transferred by conjugation)
oriT- the origin of transfer (where the relaxase creates a nick in the strand that will be transferred by conjugation)

Question 2

Describe the F (fertility factor) plasmid.

Answer 2

The F plasmid is a narrow host-range plasmid isolated from E.coli, and therefore can only transfer between relatives of E.coli. A third of plasmid encodes genes for conjugal transfer. Has some homologous genes to the host and so can integrate by recombination.

Question 3

Describe the RP4 (resistance factor) plasmid.

Answer 3

It is a broad host-range plasmid first isolated from Pseudomonas aeruginosa (an opportunistic pathogen), and confers resistance to antibiotics (encodes 3 different antibiotic resistance genes). It is considered promiscuous (can spread between species and in the most efficient way).

Question 4

Describe the Ti (tumour inducing) plasmid

Answer 4

It is found in Agrobacterium tumefaciens and is involved in causing crown gall disease in plants i.e. the DNA can transfer from a bacterium to a plant cell in this conjugation system. Has both vir and tra conjugation systems -> in vir system only the T-region is transferred.

Question 5

What are the conjugal transfer genes referred to when conjugation is occurring between prokaryotes? And between a prokaryote and eukaryote (e.g. a plant cell)?

Answer 5

Referred to as tra/trb genes between prokaryotes, and vir genes between a prokaryote and eukaryote.

Question 6

What are the 4 main steps in conjugation?

Answer 6

1) Mating pair formation (Mpf), also referred to as pilus formation which is a type IV secretion system (T4SS)
2) Signalling event occurs that triggers DNA transfer
3) DNA transfer (Dtr), which involves relaxosome formation
4) Coupling protein synchronises Mpf with Dtr and is thought to ‘pump’ the DNA into the recipient cell

Question 7

Describe the mechanism of conjugation between 2 prokaryotic cells in more detail.

Answer 7

1) Cell to cell contact made by pilus
2) Pilus retracts bringing the cells closer together
3) DNA strand to be transferred is nicked at oriT by relaxase (acts as a nickase)
4) Relaxase also acts as a helicase and unwinds the DNA to be transferred
5) Rolling circle replication replaces DNA strand in the donor cell
6) Complementary strand is also made in the recipient, and so the recipient is now a donor

Question 8

What are the benefits of phospholipids being attached to the lumen of the F-pilus?

Answer 8

The alteration in electrostatic potential of the pilus when phospholipids are bound facilitates pilus insertion into host membranes, pilus retraction/depolymerisation, and helps the DNA to travel down the pilus and into the recipient.

Question 9

What is the relaxosome?

Answer 9

The relaxase enzyme and accessory proteins, which stabilise the relaxosome and are involved in its formation.

Question 10

What is the relaxase enzyme in the RP4 model? And the accessory proteins? Which of these are essential?

Answer 10

TraI is the relaxase and is essential
The accessory proteins are TraJ (essential) and TraH (acts as a ‘helper’ stabilising the complex- not essential but if it is mutated then there is a reduction in transfer)

Question 11

Describe the 4 stages of relaxosome formation.

Answer 11

1) TraJ binds to the inverted repeat closest to the oriT (the nic site)
2) The relaxase TraI binds to the TraJ-oriT complex
3) Relaxosome formation facilitated by the bent region in oriT that allows it to wrap around several TraK subunits
4) The TraI-TraJ-oriT complex is stabilised by TraH

Question 12

Why is the wrapping around TraK subunits in relaxosome formation beneficial?

Answer 12

It enhances the fraction of plasmids that can be captured and cleaved at the nic site, as it facilitates the binding of the proteins to the DNA and makes the DNA more accessible.

Question 13

Is the relaxase (TraI) transferred with the DNA to the recipient cell?

Answer 13

Yes.

Question 14

What 2 operons does the Ti plasmid system have?

Answer 14

virB operon - genes involved in Mpf

virD operon - genes involved in Dtr

Question 15

Describe virD4.

Answer 15

An energetic component of T4SS- a coupling protein that is not actually part of the system but acts as a ‘pilot’ between T4SS and the relaxosome by interacting directly with them.

Question 16

Describe virB4.

Answer 16

An energetic component of T4SS- involved in energising the assembly/activity of the channel.

Question 17

Describe virB11.

Answer 17

An energetic component of T4SS- form as homohexamer rings and contain a central cavity.

Question 18

Where are the proteins that hydrolyse ATP found?

Answer 18

In the cytoplasm but they are also embedded in the membrane.

Question 19

How many inner membrane channel/scaffold proteins are there in T4SS?

Answer 19

4: VirB3, VirB6, VirB8, and VirB10

Question 20

Describe VirB3.

Answer 20

An inner membrane channel/scaffold protein of T4SS- interacts with B4 and B2, and involved in the pilus assembly pathway and substrate translocation.

Question 21

Describe VirB6.

Answer 21

An inner membrane channel/scaffold protein of T4SS- interacts with the DNA substrate and mediates its transfer. Interacts with components of the channel to allow its formation.

Question 22

Describe VirB8.

Answer 22

An inner membrane channel/scaffold protein of T4SS- interacts with channel proteins, and is involved in the positioning of the other proteins - if don’t have B8 then the proteins don’t know where to go.

Question 23

Describe VirB10.

Answer 23

An inner membrane channel/scaffold protein of T4SS- interacts with channel proteins and with several other components, linking proteins in the inner and outer membranes as it spans the periplasm (makes a scaffold).

Question 24

How many periplasmic/outer membrane channel subunits are there?

Answer 24

5: VirB1, VirB7, VirB9, VirB2, and VirB5

Question 25

Describe VirB1.

Answer 25

A periplasmic/outer membrane channel subunit in T4SS- a hydrolase that degrades peptidoglycan to allow the assembly of the pilus

Question 26

Describe VirB7.

Answer 26

Is localised in the outer membrane and is a lipoprotein that stabilises several VirB subunits. B9 and B7 form a structure that the pilus grows through.

Question 27

Describe VirB9.

Answer 27

A periplasmic/outer membrane channel subunit in T4SS- required for channel assembly and pilus biogenesis. B9 and B7 form a structure that the pilus grows through.

Question 28

Describe VirB2.

Answer 28

This is the pilin and is processed to form cyclic polypeptides that are the building blocks for pilus polymerisation.

Question 29

Describe VirB5.

Answer 29

Is found on the end of the pilus and is exported to the periplasm. Is important for DNA transfer.

Question 30

Explain how the vir genes are only stimulated to be transcribed in response to the plant being wounded.

Answer 30

When a plant is wounded it secretes a signalling molecule that binds to a protein in the membrane called virA. VirA autophosphorylates, activating it, and then transfers the phosphate to a transcriptional regulator called virG. VirG binds to the promoter upstream of the vir genes on the plasmid, which initiates their transcription. The translated vir proteins cut the border of the T-DNA.

Question 31

What is the relaxase in the Ti plasmid system?

Answer 31

VirD2, which is transferred along with the DNA through the T4SS.

Question 32

What is the use of virE2?

Answer 32

It coats the T-DNA that is exposed during conjugation to protect it against plant nucleases. It is exported independently of the DNA.

Question 33

What is the use of virE3?

Answer 33

It facilitates the movement of T-DNA into the nucleus, and is exported independently of the DNA.

Question 34

What does entry exclusion do?

Answer 34

It reduces the conjugal transfer between cells that contain the same plasmid (cells in the same incompatibility group).

Question 35

In the F-system, what 2 proteins are important in entry exclusion?

Answer 35

TraS and TraT are important for the blocking. TraS is an inner membrane protein that blocks entry after the Mpf has formed, and TraT is an outer membrane lipoprotein that reduces the number of mating pairs that can form in the first place (blocking therefore occurs due to TraT initially).

Question 36

Why is entry exclusion important? (theories)

Answer 36

• Limits the damage of lethal zygosis (bacterial cell death produced by excessive rounds of conjugation)
• Avoids competition in host among identical plasmid backbones

Question 37

What does the spread of antibiotic resistance tend to be due to?

Answer 37

Due to conjugative plasmids, and if it has a broad host range too then it can spread easily to lots of different species e.g. the IncP-1 (RP4) plasmid.