Structures and Conjugation Machinery
For genetic transfer to take place, the donor bacterium must possess specialized structures whose synthesis is encoded by transmissible plasmids. A critical element of this machinery is the sex pilus (pl. pili).
When the donor and recipient approach each other, these proteinaceous extensions form a bridge called the conjugation tube. This physical channel serves as the conduit for transporting the DNA molecule from one cytoplasm to the other. Without this direct contact, genetic exchange cannot occur.
Molecular Mechanism of Plasmid DNA Transfer
The DNA transfer process is tightly regulated by proteins encoded by the plasmid itself. The procedure proceeds through several sequential steps:
- Initiation. A specialized relaxosome protein synthesized via the tra operon recognizes a unique sequence on the plasmid—the origin site (oriT).
- Nick generation. This protein makes a single-stranded cut (nick) in the DNA duplex at the oriT site. It then covalently attaches to the newly liberated 5' end of the cleaved strand.
- Transfer. The DNA strand is pulled by its 5' end (along with the attached protein) and transported through the conjugation bridge directly into the recipient cell.
- Replication. Cellular machineries of both bacteria become active at this stage. In the donor, a complementary strand is synthesized using the remaining strand as a template. In the recipient, a second strand is synthesized along the newly arrived single-stranded DNA.
- Termination. The protein at the 5' end of the transferred strand performs its final function by helping the linear DNA molecule circularize back into a plasmid inside the recipient.
As a result of these molecular events, both bacteria end up with functional double-stranded circular plasmids.
Role of the F-Factor and Cell States
One of the most classic examples of a transmissible plasmid is the F-factor (fertility factor). This plasmid possesses two critical features: it is autonomously transmissible, and it can integrate directly into the bacterial chromosome (integrativity).
The presence or absence of the F-factor divides a bacterial population into two states:
- F⁺ cells are donors. They contain the F-factor and possess all necessary machinery for DNA transfer.
- F⁻ cells are recipients. They lack the F-plasmid and can only receive genetic material.
If the F-plasmid resides autonomously in the donor cytoplasm (separate from the chromosome), mating between F⁺ and F⁻ cells results in the recipient receiving a copy of the plasmid. Consequently, the F⁻ cell acquires donor properties and becomes F⁺.
Integrated State: Conjugation of Hfr Strains
A completely different scenario unfolds when the F-factor integrates into the bacterial chromosome. In this case, the plasmid and chromosome merge into a single transmissible replicon. Bacteria with this status are termed Hfr strains (High frequency of recombination) because they can transfer their own chromosomal genes—rather than just the plasmid—to recipients at a high frequency.
Hfr transfer has distinct features:
- Initiation begins at the site where the F-factor is integrated. The DNA unwinds, and one strand is directed into the bridge while complementary strand synthesis occurs simultaneously.
- Polarity. Chromosomal genes are transferred in a strict order and in a direction opposite to the integrated plasmid.
- Sequence. The F-factor DNA itself is transferred last, serving as the trailing element.
In practice, the conjugation tube is extremely fragile and almost always breaks before the massive bacterial chromosome can fully pass through it. Consequently, the plasmid DNA—entering last—rarely reaches the recipient cell.
The resulting transferred DNA fragment integrates into the recipient's genome via homologous recombination. The recipient acquires new traits but does not become a donor (it remains F⁻). Because genes are transferred in a strict temporal sequence during this type of conjugation, the process is widely used in bacterial genome mapping.