Topic F - Conjugation

Vertical and Horizontal Gene Transfer

Vertical Gene Transfer - Transfer of DNA from a parent cell to its progeny through cell division (ex; parent cell providing DNA to two daughter cells)

Horizontal Gene Transfer - Transfer of DNA between cells unrelated/distantly related (produces genetic diversity in a bacterial population)

Mechanisms of Horizontal Gene Transfer

1. Conjugation - Physical cell-to-cell contact required

-Donor cell creates pilus, grabbing onto recipient cell, transferring genetic information

2. Transformation - Exchange of genetic material via free-floating DNA material (donor cell dies releasing DNA, recipient cell uptakes the DNA)

3. Transduction - Transfer of genetic material between cells using bacteriophage

-Phage accidently package bacterial DNA, resulting in the transfer of bacterial DNA to foreign bacteria

Conjugation

Transfer of genetic material from one cell to another via direct cell-to-cell contact

-Pilus connecting donor/recipient cell is the “Conjugative Pilus”

History of Conjugation

First observed by Lederberg and Tatum in 1946, using 2 experiments

Experiment 1 - Conjugation

Mixing different auxotrophic strains and got prototrophs (WTs)

-Strain A - Auxotrophic for multiple nutrients when plated on MM = no colonies

-Strain B - Auxotrophic for a different set of nutrients when plated on MM = no colonies

Mixing Strain A + B and plating on MM = observed colonies

Experiment 2 - Conjugation

Separate cells to see if transfer occurs

-Needed to rule out factors in the medium that may have caused observed phenotypes (DNA, bacteriophage)

Two auxotrophic strains separated by filter allowing passage of nutrients, but NOT bacteria

-Vacuum applied to maintain movement of liquid

Observed no colonies on MM → Direct contact therefore needed for transfer of DNA

Bacterial Plasmids

Small, circular supercoiled dsDNA molecules

-Must contain origin of replication

Depends on host cell for most replication proteins (helicase, DNAP)

-Some encode special proteins for plasmid replication and segregation

Low Copy Number Plasmids

Plasmid replication coordinated with chromosome

-Once prior to cell division, segregated following division to ensure each daughter cell gets a copy

-Proteins used to move one copy to each end of thecell

High Copy Number Plasmids

Plasmid replication not linked to cell division

-Lots of replication to get lots of plasmid copies, regulation control maximum number of plasmids in the cell

Random segregation during division, high number ensures each daughter cell gets at least one copy

Plasmid Incompatibility

Inability of two plasmids to co-exist stably in the same cell, occurs if:

-Plasmids have the same replication mechanism; replication mechanism sees them as the same plasmid, making only 6 copies of the plasmid (rather than 6 copies of EACH plasmid), overtime one of the plasmids will be lost (ex; both plasmids having the same ori)

-Plasmids have the same partitioning mechanism

Role of Plasmids

Carry genes that are dispensable for growth (don’t have essential genes)

-may be lost (cured) with no negative effect

But provide selective advantage under some conditions

1. Ex; Tol Plasmid - Toluene degradation (energy source)

2. Ex; Ti plasmid - tumor inducing (bacteria infects plant-host cells, causing tumor via conjugation)

3. Ex; R plasmid - antibiotic resistance

4. Ex; F plasmid - sex pili + conjugation

Addiction Systems

Some plasmids contain addiction systems

-Encode a stable toxin and unstable anti-toxin

If plasmid is lost, the cell will die

-Allows plasmid to be maintained in a bacterial population, even in non-selective conditions

Mechanism of Addiction Systems

When the anti-toxin is expressed, it binds to the toxin preventing it from killing the bacterial cell

-If the bacteria cell divides and only one daughter cell receives the plasmid, the remaining unstable anti-toxin in cytoplasm will become degraded resulting in cell death

Plasmid and Conjugation

Some plasmids carry genes for plasmid transfer by conjugation between bacteria

-Includes “tra” genes or “mob” genes

F Plasmid of E.Coli

F (fertility) plasmid, has a low copy number (1 per cell), consists of:

1. OriV - Origin of replication

2. OriT - Origin of Transfer (for Conjugation)

3. Tra genes - Encodes proteins involved in conjugation

4. IS (Insertion Sequence) Elements - Transposons

Transfer of Plasmid DNA

Some plasmids are self-transmissible

-Encode all functions needed for conjugation from cell-to-cell

Donor Strain - Bacterium that carries a self-transmissible plasmid

Recipient Strain - Bacterium lacking self-transmissible plasmid, receives DNA by conjugation

Five-Step Process of Plasmid Transfer

1. Formation of Mating Pairs

2. Preparation for DNA Transfer

3. DNA Transfer

4. Restoration of Double-Stranded plasmid in donor

5. Formation of replicative plasmid in recipient

Formation of Mating Pairs

Requires cell-to-cell contact between donor and recipient;

-Mediated by the conjugative pilus (F pilus or sex pilus); polymer of a single protein called “Pilin”

a) Pilus tip binds to receptor on recipient cell surface to initiate mating pair formation

b) Pilus contracts to pull the two cells into close contact

c) Conjugation Bridge forms between cells, consists of pore complex formed by proteins

-Passageway to allow DNA transfer

Preparation for DNA Transfer

Requires relaxase enzyme, F Plasmid-encoded protein

Recognizes unique DNA sequence of the oriT

-Nicks one strand of plasmid DNA at oriT

DNA Transfer

Cut strand of plasmid is transferred to recipient via conjugation bridge

1. Transfer starts at 5’ end of nick at oriT

2. Transferred strand is linear/single-stranded

3. Enabled by protein complex (relaxosome) that binds at oriT

4. Helicase separates two strands of plasmid

-Other DNA strand stays in donor cell as intact circle

Restoration of Double-Stranded Plasmid in Donor

ssDNA remaining in donor is replicated to regenerate double-stranded plasmid

-Primed by 3’ end of nicked strand of plasmid

Synthesis occurs during plasmid transfer

Formation of Replicative Plasmid in Recipient

ssDNA in recipient recircularizes:

-Nick is sealed by relaxase, transferred into recipient with plasmid DNA

Second strand produced by replication to give double-stranded plasmid

-Primase enzyme (from recipient cell) synthesizes RNA primer to initiate DNA replication

Both cells are now F+

-DNA synthesis only occurs after DNA transfer is complete

Diagram of Transfer

tra Genes

Conjugation functions are encoded by tra genes

-Two main types of genes:

1. MPF - Encode mating pair formation proteins

2. DTR - Encode DNA transfer and replication

Are trans-acting; act on another plasmid in the same cell, as long as it has an oriT

-oriT is “Cis-acting”; plasmid must have an oriT in its sequence to transfer

-Plasmid MUST have an oriT in its sequence to transfer (oriT in one plasmid, can’t act on another plasmid)

Self-Transmissible Plasmid

Transfer between cells without help

-has MPF and DTR genes

MPF Gene - Pilin subunit, assembly of pilus, stabilization of mating pairs, inhibition of mating between two F+ cells

DTR Gene - Helicase, initiation of plasmid transfer, Relaxase

Conjugative Plasmid

Can form pilus and mating pair, but can’t be transferred by conjugation

-Has MPF genes only, no DTR genes or oriT

Mobilizable Plasmid

Two types:

1. Has DTR + oriT, no MPF

• When in cell with conjugative/self-transmissible plasmid, it can be transferred by conjugation

2. Only has oriT, no MPF/DTR

• Can be transferred by conjugation only when in a cell with self-transmissible plasmid (has MPF and DTR)

Plasmid Interaction Example

Two Plasmids:

-F Plasmid - Self-transmissible

-Other Plasmid - Mobilizable

Genes encoding proteins for transfer are encoded on a different plasmid than the one being transferred

-recipient does not become F+

Hfr Strains

“High Frequency of Recombination” strains

-Formed when F-plasmid integrates into the bacterial chromosome

-Can enable transfer of chromosomal DNA to a recipient by conjugation

Two sites on the F plasmid and chromosome are homologous to each other (IS elements), resulting in homologous recombination for integration

Hfr Integration

Integration occurs via recombination between homologous IS (insertion sequence) elements

-Found on both the F plasmid and chromosome, have conserved sequences

F-Plasmid Integration

Can integrate in one of two ways, and at any one of more than 20 sites in the E.coli chromosome

-only ONE F-plasmid is allowed per genome since cell becomes F+ (no additional conjugation an occur)

Mechanism of Hfr Integration

Homologous sequence on plasmid will align in the direction of the homologous sequence in the chromosome

-Different direction of integration produces a different direction of transfer during conjugation

-Transfer occurs as if arrow is being shot into the recipient cell

DNA behind the oriT (the “arrow”) will be transferred first during conjugation