The three modes of recombination in bacteria
Fog 1: Modes of Bacterial recombination.

In this post, we discuss the conjugation.

Conjugate literally means to join together especially in pairs or couples (Merriam-webster dictionary). Bacterial conjugation was discovered by Joshua Lederberg and Edward Tatum in 1946.

This mode of recombination occurs between two bacterial cells, joined with each other through a conjugation tube also known as conjugation pilus. As the genetic material is transferred from one bacterial cell to another from the same generation and not from mother cell to the daughter cells, this is known as horizontal gene transfer.

The genes transferred is actually present on a plasmid. Conjugation is considered to be similar to sexual mating in eukaryotes, this plasmid is known as sex factor or F-plasmid, where F stands for fertility. This plasmid is also known as a conjugative plasmid.

Separation of Plasma and Serum from Whole Blood

In the process of conjugation, the F-plasmid is transferred from a bacterial cell having an F-plasmid, known as a donor, to the one having no F-plasmid, known as the recipient. The donor is also known as F plus cell (F+) and the recipient as an F-minus cell (F-).

The F plasmid is a circular DNA molecule about 100 kb in size. This plasmid is capable of self-replication and is inherited by the daughter cells after the cell division. F plasmid have different regions;

Ori V: the origin at which replication of the F factor begins is designated as “ori V” abbreviation for Origin of vegetative replication.

Ori T: This is the ‘origin of transfer‘ of F-plasmid. This region is the first region of the F-plasmid to be transferred into the recipient cell.

IS: The F-plasmid contains a region having insertion sequence elements or IS elements, that provide the sites for the integration of the plasmid into the host chromosome. through homologous recombination. Plasmids having the ability to integrate into the host genome is known as an episome.

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Fig 2; F plasmid.Tra genes: The transfer operon (tra) covers a large region (33 kb) of the F plasmid. The tra operon contains more than 25 genes that are required for the synthesis of pili (singular: pilus) and the transfer of DNA.

Around half of the tra genes are involved in the assembly of the pili. The pili, known as F-pili is the initial physical contact between the two cells. Then the pili retract to bring the two cells close to each other. F-pili has a different symmetry than the other bacterial pili. The central inner diameter of the F-pili is ∼ 30 Å, through which the single-stranded DNA can pass easily.

• Process of Conjugation:

In Gram-negative bacteria, conjugation involves a form of conjugation bridge between the donor and the recipient cell. The conjugation in these bacteria take place in the following steps:

1. The conjugation takes place when the F+ and F- cells are present in the population. The F+ plasmid has all the genes necessary for the synthesis of the F pilus (fig 3.1).

2. The F-pilus by the donor cell binds the recipient cell, then the pilus restraints bringing the two cells close to each other (fig 3.2).

3. The pilus retracts and brings the two cells close to each other. The point of contact is known as a conjugational junction which is similar to the tight junctions in eukaryotic cells. A pore through the cell envelope is formed which connects the cytoplasm of the two cells. The F plasmid can pass through this pore (fig 3.3).

4. A nick is formed at the ori T (origin of transfer). A protein called the mobilization protein, Mob gets covalently attached to the 5′ end of the nicked DNA strand and attaches it to the transport pore.

DNA is replicated simultaneously by the rolling-circle method, synthesizing a single-strand copy of the F-plasmid, which is transported through the pore to the recipient cell. Hence, a closed single–a stranded copy of the plasmid is transferred into the cytoplasm of the recipient.

5. The complementary strand is then synthesized in the recipient cell forming a double-stranded circular plasmid (fig 3.5). The recipient cell on receiving the F plasmid can act as a donor.

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Sometimes the F plasmid undergoes homologous recombination with the bacterial genome and gets integrated into the bacterial genome. Such an integrated F-plasmid is known as High-frequency recombination or Hfr.

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Fig 4: Integration of F plasmid into bacterial genome and formation of Hfr.Even after the integration into the bacterial genome, the F-plasmid can initiate conjugation. The integrated F-factor can direct the processes like the synthesis of pili, rolling-circle replication, nick formation at oriT and subsequent transfer F-factor in its integrated form into a recipient cell.

In a conjugation between Hfr and F- cell, the entire integrated F plasmid may not be transferred, due to interruptions or breaking apart of the pairing. However the bacterial genes closer to the oriT get transferred and these genes may get incorporated into the genome of the recipient cell, forming to a recombinant. Hence the frequency of the recombinants is very high in such cases.

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Fig 5: video-enhanced microscopy of bacterial conjugation in E Coli. (a) Donor cell (D) (b) & (c) The close contact between donor and recipient (R) cells (d) Disaggregation. Bar marker 1 µm.In the case of Gram-positive bacteria, pili or any pairing bridge is not formed between the mating partners. In some Gram-positive SPS, like enterococci and related SPS, the recipient cells release clumping agents (CIAs), known as sex pheromones. These CIAs cause the donor cell to produce surface proteins that promote cell clumping. The clumping of cells brings the cells in close contact and further processes required for the transfer of the plasmid take place.

In some Gram-positive bacteria like streptococci and staphylococci, conjugative plasmid transfer occurs in bacteria, when the donor and recipient, come in direct contact on solid surfaces. These also include species of, Clostridium, Lactobacillus, Listeria, Bacillus, etc.

• Applications:

– Transfer of certain genes can be done using the F plasmid, e.g. Antibiotic resistance gene.

– The map of the bacterial genome can be constructed on the basis of time (in mins) required for the transfer of marker(s) in Hfr.


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