Structures and conjugation apparatus
For the transfer of genetic material to take place, the donor bacterium must possess specialized structures whose synthesis is encoded in transmissible plasmids. The most important element of this apparatus is sex pili.
When the donor and recipient approach each other, these protein outgrowths form a specific bridge—the conjugation tube. It is through this physical channel that the DNA molecule is transported from one cytoplasm to another. Without establishing such direct contact, the exchange of genetic information is impossible.
Molecular mechanism of plasmid DNA transfer
The DNA transfer process itself is strictly controlled by proteins encoded by the plasmid itself. The procedure takes place in several strictly sequential steps:
- Initiation. A specialized protein synthesized thanks to the tra operon finds a unique sequence on the plasmid: the origin site.
- Nick formation. This same protein makes a single-stranded break in the DNA helix at the identified site. It then covalently attaches to the liberated 5' end of the cleaved strand.
- Transfer. The DNA strand is pulled out by its 5' end (along with the bound protein) and transported through the conjugation bridge directly into the recipient cell.
- Replication. At this moment, the cellular machinery of both bacteria operates. In the donor, a new complementary strand is synthesized based on the remaining strand. In the recipient, the second strand is also synthesized on the newly arrived single-stranded DNA.
- Termination. The protein located at the 5' end of the transferred strand performs its final function: it helps the linear DNA molecule circularize inside the recipient.
As a result of these molecular manipulations, both bacteria end up with fully functional double-stranded circular plasmids.
Role of the F factor and cell statuses
One of the most famous examples of a transmissible plasmid is the F factor (from the word fertility). This plasmid has two crucial characteristics: it can be transferred independently (transmissibility) and is capable of integrating directly into the bacterial chromosome.
The presence or absence of the F factor divides the bacterial population into two types:
- F⁺ cells are donors. They contain the F factor and possess everything necessary for DNA transfer.
- F⁻ cells are recipients. They lack the F plasmid and can only receive genetic material.
If the F plasmid is located autonomously in the donor cytoplasm (separate from the chromosome), then upon crossing F⁺ and F⁻ cells, the recipient receives a copy of the plasmid. As a result of this event, the F⁻ cell acquires donor properties and becomes F⁺ itself.
Integrated state: conjugation of Hfr strains
A completely different scenario unfolds if 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 called Hfr strains (High frequency of recombination) because they are capable of transferring their own chromosomal genes—rather than just the plasmid—to recipients at a very high frequency.
The transfer mechanism in Hfr strains has its own features:
- Initiation starts at the point where the F factor is integrated. The DNA unwinds, and one strand is directed into the bridge while complementary strand synthesis occurs in parallel.
- Directionality. Chromosomal gene transfer proceeds in a strict order and in the direction opposite to the integrated plasmid.
- Order. The F factor DNA itself is transferred last, serving as the closing element.
In practice, the conjugation tube is an extremely fragile structure and almost always breaks before the huge bacterial chromosome can pass through it entirely. Because of this, the plasmid, which goes last, rarely reaches the recipient cell.
As a result, the obtained DNA fragment is integrated into the recipient's genome via homologous recombination. The recipient acquires new traits but does not acquire donor properties (remains F⁻). Because genes in this type of conjugation are transferred in a strict temporal sequence, this process is actively used in genetics for genome mapping of bacteria.
Briefly: answers to topical questions
Which genes make up the tra operon of the F plasmid and what proteins do they encode?
The tra operon includes a set of transfer genes encoding the proteins of the conjugation apparatus:
- genes encoding the synthesis of sex pili, which form the conjugation tube (bridge) between the donor and recipient;
- gene(s) encoding a special protein that recognizes the origin site, introduces a single-stranded break, and covalently binds to the 5' end of the cleaved strand for its transfer.
To remember the recipient's status during an Hfr and F⁻ cross: "The F factor rides in the caboose." Due to the fragility of the conjugation bridge, this "caboose" almost never arrives, so the recipient bacterium receives new chromosomal genes but does not become a donor itself.
Class and exam questions
Why is the F plasmid called the fertility factor?
Its name comes from the word fertility, as it carries genes that allow the bacterium to form sex pili, conjugate, and transfer its genetic material to other cells.
Why is the protein at the 5' end of the transferred DNA strand needed?
This protein performs two functions: it acts as a "locomotive" by which the strand is pulled through the bridge, and after entering the recipient's cytoplasm, it facilitates the circularization of the linear strand back into a ring.
Why does the recipient not become a donor during conjugation of an Hfr cell with an F⁻ cell?
In such a cross, the F factor is integrated into the chromosome and transferred last. The conjugation tube usually breaks before the process is complete, so the plasmid itself does not manage to enter the recipient.
What else to study on this topic
- Structural features and protein functions of the tra operon during the transfer initiation stage.
- Enzymatic mechanisms of complementary DNA strand synthesis in donor and recipient cells.
- The process of homologous recombination of chromosome fragments in the recipient's cytoplasm.
- Methods of bacterial chromosome genetic mapping using Hfr strains.
- Differences between integrative and autonomous plasmids and the conditions for their transition from one state to another.
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