Explanation to Bacterial Transduction Transformation and Conjugation

QUESTION

Explain with diagram

  1. Transduction
  2. Transformation
  3. Conjugation

ANSWER

  1. Transduction

Transduction  is the process by which foreign DNA is introduced into a cell by a virus or viral vector. An example is the viral transfer of DNA from one bacterium to another and hence an example of horizontal gene transfer. Transduction does not require physical contact between the cell donating the DNA and the cell receiving the DNA (which occurs in conjugation), and it is DNase resistant (transformation is susceptible to DNase ). It is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell’s genome (both bacterial and mammalian cells).

It happens through either the lytic cycle or the lysogenic cycle. When bacteriophages (viruses that infect bacteria) that are lytic infect bacterial cells, they harness the replicational, transcriptional,  and translation machinery of the host bacterial cell to make new viral particles (virions). The new phage particles are then released by lysis of the host. In the lysogenic cycle, the phage chromosome is integrated  as a prophage into the bacterial chromosome, where it can stay dormant for extended periods of time. If the prophage is induced (by UV light for example), the phage genome is excised from the bacterial chromosome and initiates the lytic cycle, which culminates in lysis of the cell and the release of phage particles. Generalized transduction occurs in both cycles during the lytic stage, while specialized transduction occurs when a prophage is excised in the lysogenic cycle.

 

Generalized transduction

It occurs when random pieces of bacterial DNA are packaged into a phage. It happens when a phage is in the lytic stage, at the moment that the viral DNA is packaged into phage heads. If the virus replicates using ‘headful packaging’, it attempts to fill the head with genetic material. If the viral genome results in spare capacity, viral packaging mechanisms may incorporate bacterial genetic material into the new virion. Alternatively, generalized transduction may occur via recombination.

The new virus capsule that contains part bacterial DNA then infects another bacterial cell. When the bacterial DNA packaged into the virus is inserted into the recipient cell three things can happen to it: the DNA is recycled for spare parts, if the DNA was originally a plasmid, it will re-circularize inside the new cell and become a plasmid again and if the new DNA matches with a homologous region of the recipient cell’s chromosome, it will exchange DNA material similar to the actions in bacterial recombination.

Specialized transduction

Specialized transduction is the process by which a restricted set of bacterial genes is transferred to another bacterium. The genes that get transferred (donor genes) flank where the prophage is located on the chromosome. Specialized transduction occurs when a prophage excises imprecisely from the chromosome so that bacterial genes lying adjacent to it are included in the excised DNA. The excised DNA is then packaged into a new virus particle, which then delivers the DNA to a new bacterium. Here, the donor genes can be inserted into the recipient chromosome or remain in the cytoplasm, depending on the nature of the bacteriophage.

When the partially encapsulated phage material infects another cell and becomes a prophage, the partially coded prophage DNA is called a “heterogenote”.

An example of specialized transduction is λ phage in Escherichia coli.

Lateral transduction is the process by which very long fragments of bacterial DNA are transferred to another bacterium. So far, this form of transduction has been only described in Staphylococcus aureus, but it can transfer more genes and at higher frequencies than generalized and specialized transduction.

  • Transformation

Bacterial transformation is the transfer of free DNA released from a donor bacterium into the extra-cellular environment that results in assimilation and usually an expression of the newly acquired trait in a recipient bacterium.  This process doesn’t require a living donor cell and only requires free DNA in the environment. The recipient that successfully propagates the new DNA is called the transformant. During extreme environmental conditions, some bacterial genera spontaneously release DNA from the cells into the environment free to be taken up by the competent cells. The competent cells also respond to the changes in the environment and control the level of gene acquisition through a natural transformation process. 

Transformation is adopted as the most common method of gene transfer as it is the best way for the transfer of artificially altered DNA into recipient cells. Bacterial transformation is based on the natural ability of bacteria to release DNA which is then taken up by another competent bacterium. The success of transformation depends on the competence of the host cell. Several bacteria, including Escherichia coli, can be artificially treated in the laboratory to increase their transformability by chemicals, such as calcium, or by applying a strong electric field (electroporation) or by using a heat shock. 

There are four steps in transformation:

  • development of competence,
  • binding of DNA to the cell surface,
  • processing and uptake of free DNA (usually in a 3’ to 5’ direction), and
  • integration of the DNA into the chromosome by recombination.
  • The artificial development of competence can be achieved either through electroporation or through heat shock treatment. The choice depends on the transformation efficiency required, experimental goals, and available resources.
  • For heat shock, the cell-DNA mixture is kept on ice (0°C) and then exposed to 42°C.
  • For electroporation, the mixture is transferred to an electroporator and is exposed to a brief pulse of a high-voltage electric field.
  • The double-stranded DNA released from lysed cells binds noncovalently to cell surface receptors. There is no DNA sequence-specific recognition; thus, these organisms can potentially incorporate DNA from outside their species.
  • The bound double-stranded DNA is nicked and cleaved into smaller fragments by membrane-bound endonucleases, allowing the single strand to enter the cell through a membrane-spanning DNA translocation channel.
  • The transformed DNA integrates into the chromosome and replaces the chromosomal DNA fragment by recombination. This integration, however, requires significant nucleotide sequence homology between the donating DNA fragment and the fragment in the chromosome.
  • In the case of plasmid, the plasmid with the donor DNA is inserted during the heat shock or electroporation.  The cells with the plasmid can be detected by growing these cells is a growth media supplemented with a specific antibiotic

Types of Bacterial Transformation

There are two forms of transformation:

Natural Transformation

In natural transformation, bacteria naturally have the ability to incorporate DNA from the environment directly.

Artificial Transformation

In the case of artificial transformation, the competence of the host cell has to be developed artificially through different techniques.

3. Conjugation

Bacterial conjugation is a way by which a bacterial cell transfers genetic material to another bacterial cell. The genetic material that is transferred through bacterial conjugation is a small plasmid, known as F-plasmid (F for fertility factor), that carries genetic information different from that which is already present in the chromosomes of the bacterial cell. In fact, the F-plasmid can replicate in the cytoplasm separately from the bacterial chromosome.

A cell that already has a copy of the F-plasmid is called an F-positive, F-plus or F+ cell, and is considered a donor cell, while a cell that does not have a copy of the F-plasmid is called an F-negative, F-minus or F– cell, and is considered a recipient cell. The transfer of the F-plasmid takes place through a horizontal connection by which the donor cell and the recipient cell directly contact each other or form a bridge between the two through which the genetic material is transferred. In cases where the F-plasmid of a donor cell has been integrated in the cell’s genome (i.e., in the chromosome), a part of the chromosomal DNA may also be transferred to the recipient cell together with the F-plasmid.

Bacterial Conjugation Steps

In order to transfer the F-plasmid, a donor cell and a recipient cell must first establish contact. At this point, when the cells establish contact, the F-plasmid in the donor cell is a double-stranded DNA molecule that forms a circular structure. The following steps allow the transfer of the F-plasmid from one bacterial cell to another:

Step 1

The F+ (donor) cell produces the pilus, which is a structure that projects out of the cell and begins contact with an F– (recipient) cell.

Step 2

The pilus enables direct contact between the donor and the recipient cells.

Step 3

Because the F-plasmid consists of a double-stranded DNA molecule forming a circular structure, i.e., it is attached on both ends, an enzyme (relaxase, or relaxosome when it forms a complex with other proteins) nicks one of the two DNA strands of the F-plasmid and this strand (also called T-strand) is transferred to the recipient cell.

Step 4

In the last step, the donor cell and the recipient cell, both containing single-stranded DNA, replicate this DNA and thus end up forming a double-stranded F-plasmid identical to the original F-plasmid. Given that the F-plasmid contains information to synthesize pili and other proteins (see below), the old recipient cell is now a donor cell with the F-plasmid and the ability to form pili, just as the original donor cell was. Now both cells are donors or F+.

The four steps mentioned above can be seen in this figure:

The advantages of bacterial conjugation make this method of gene transfer a widely used technique in bioengineering. Some of the advantages include the ability to transfer relatively large sequences of DNA and not harming the host’s cellular envelope. Furthermore, conjugation has been achieved in laboratories not only between bacteria, but also between bacteria and types of cells such as plant cells, mammalian cells and yeast.

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