Bacterial Conjugation

Question 1

Conjugation: DEFINITION & HISTORY

Answer 1

• Transfer of genetic material between cells by
direct cell-to-cell contact
• Discovered in 1946 by Lederberg and Tatum
• Plasmids or transposons can transfer from one
organism to another by conjugation
• Occurs in the Bacteria and Archaea

Question 2

Conjugation -

Answer 2

• Most common mechanism of genetic exchange between prokaryotes
• Leads to acquisition of traits e.g. antibiotic and metal resistance, pathogenicity etc.
• Important mechanism in evolution of prokaryotes

Question 3

Conjugative plasmids

Answer 3

• Two origins of replication (oriV & oriT)
• oriV – vegetative replication
• oriT – origin of transfer – essential for conjugal
transfer
• Conjugative or transfer genes establish a stable mating pair and trigger DNA transport from donor to recipient via a specialised transfer pore/channel

Question 4

Most well studied conjugation systems –

Answer 4

F, RP4 and Ti (Tumour-inducing) plasmid

Question 5

F (…) is a

Answer 5

(Fertility factor)

narrow-host-range plasmid isolated from Escherichia coli

Question 6

RP4 (…) is a

Answer 6

(Resistance factor–confers resistance to antibiotics) is a broad-host-range plasmid isolated from Pseudomonas aeruginosa – Burns unit in Birmingham hospital

Question 7

Ti (…) is a

Answer 7

(tumour inducing)

plasmid that is found in Agrobacterium tumefaciens and is involved in causing crown gall disease in plants

Question 8

Conjugal transfer genes referred to as …. when conjugation occurs between prokaryotes
and …. when conjugation occurs between prokaryotic and eukaryotic (e.g. plant) cell

Answer 8

tra/trb genes

vir genes

Question 9

Ti plasmid has two conjugation systems –

Answer 9

vir which involves transfer of DNA from bacterium to plant cells and tra involved in transfer of DNA from one bacterium to another
• Vir system is most well studied

Question 10

F plasmid transfers by …

Answer 10

conjugation and can integrate into host chromosome by recombination and in doing so can also transfer host genes (described later)

Question 11

Main steps in conjugation

Answer 11

• Mating-pair formation (Mpf) – pilus formation which is a type IV secretion system (T4SS)
• Signalling event that triggers DNA transfer
• DNA transfer (Dtr) which involves relaxosome
formation
• Coupling protein – synchronises Mpf with Dtr and is thought to “pump” the DNA into recipient cell
F+ is a donor, F- recipient

Question 12

Mechanism of conjugation between two prokaryotic cells

Answer 12

1) Cell to cell contact made by pilus
2) Pilus retracts bringing cells closer together
3) DNA strand to be transferred is nicked at origin of transfer (oriT) by relaxase (also known as nickase)
4) Relaxase also acts as a helicase unwinding DNA to be
transferred
5) Rolling circle replication replaces DNA strand in the donor cell
6) Complimentary DNA strand is made in the recipient –
recipient is now a donor

Question 13

Sex Pili

Answer 13

• F pilus:
– 1-20 μm long (usually ~ 2 μm) (average E. coli cell
is ~ 2-3 μm)
– 8 nm diameter
– composed of identical 7.2 kDa pilin subunits
– pore 2 nm – enough space to accommodate single
stranded DNA and protein (i.e. the relaxase)

Question 14

Relaxosome =

Answer 14

relaxase and accessory proteins

Accessory proteins involved in relaxosome formation vary in different transfer systems but all contain the relaxase

Question 15

RP4 as the model for relaxosome formation

Answer 15

• Relaxase - TraI (essential)
• Accessory proteins – TraJ (essential) and TraH
(acts as a “helper” stabilizing the complex)
• Key enzyme is TraI which nicks one DNA strand and unwinds DNA (i.e. acting as a DNA helicase) = single stranded DNA molecule ready for transfer

Question 16

Stages of relaxosome formation (using RP4 as the model)

Answer 16

1. TraJ binds to inverted repeat closest to the site of DNA cleavage (i.e. nic)
2. TraI binds to TraJ-oriT complex
3. Relaxosome formation is facilitated by the intrinsically bent region in oriT that allows it to wrap around a core of several subunits of TraK – enhances the fraction of plasmids that can be captured and cleaved at nic
4. TraI-TraJ-oriT complex is stabilised by TraH

• TraI – relaxase - cleaves a specific phosphodiester bond in the oriT initiating conjugative transfer
• Relaxase is covalently bound to the 5΄ end of the T-strand piloting the DNA through the conjugative channel – transfer occurs from 5΄ to 3΄

Question 17

Ti plasmid system

Answer 17

– virB operon – virB1 – virB11 encodes proteins
involved in Mpf
– virD operon – virD1 – virD5 – virD1 and virD2 essential for Dtr and virD4 is coupling protein
– Conjugation systems also export proteins independently of DNA (e.g. VirE2, VirE3 and VirF

Question 18

T4SS – Type IV Secretion System

Answer 18

• Mediates translocation of macromolecules across cell envelopes of bacteria
• Conjugation Mpf system is a large subfamily of T4SS – translocation of DNA and proteins
• T4SS can also function in the uptake of nucleic acids or proteins from the extracellular environment