Bacteriophages as natural vectors (Transduction)
Under natural conditions, bacterial viruses—bacteriophages—act as carriers of genetic material between cells. This process is called transduction. The mechanism begins when a lysed donor cell releases new phage particles. During assembly inside the donor, some phages may accidentally package a fragment of bacterial DNA instead of their own.
When such a virus infects a new recipient cell, it injects the previous host's genetic material into it. Thus, the bacteriophage acts as a natural vector, transferring new traits to the recipient. Commonly transferred traits include:
- Antibiotic resistance.
- Acquisition of motility.
- Ability to synthesize specific substances (e.g., in E. coli, the gal gene responsible for galactose fermentation is transferred).
Sometimes transduction radically alters the clinical picture: upon infection with a specific phage, a strain of Clostridium botulinum (the causative agent of botulism) can transform into a Clostridium strain that causes gas gangrene.
Temperate and defective phages in genetic engineering
For targeted gene transfer, researchers use specific types of viruses. Of great interest are defective phages—viruses that have lost the ability to form complete viral particles. They cannot complete their developmental cycle either in the natural environment or upon artificial induction, making them a convenient vehicle for gene delivery.
In addition, the genome of certain temperate phages (such as phage P1) possesses a unique feature: upon penetrating the bacterium, it does not integrate into the host chromosome. Instead, the viral DNA persists in the cytoplasm in a completely autonomous manner, behaving like a bacterial plasmid. It is precisely these phages that are actively used as vectors in genetic engineering technologies for transferring genetic information.
Retroviruses and the reverse flow of information
Another clinically significant type of viral vector is based on the mechanisms of retroviruses (which include oncoviruses and HIV). In these viruses, genetic information is stored as RNA, and the process of reverse transcription is implemented for its insertion into the cell.
A special viral enzyme—reverse transcriptase (revertase, or RNA-dependent DNA polymerase)—synthesizes a DNA copy from a viral RNA template. The scheme of information flow looks as follows:
- Viral genomic RNA.
- Synthesis of complementary (proviral) DNA.
- Integration into the host cell genome.
- mRNA transcription.
- Protein translation.
In nature, such integration can lead to oncogene expression and malignant cell transformation. In genetic engineering, this principle allows the insertion of designated genes directly into the recipient's chromosome.
Vector preparation: restriction-ligation technologies
For a viral vector to deliver the desired gene, this gene must first be inserted into it. Enzymes such as restriction endonucleases (restriction enzymes) and DNA ligases are used to create such recombinant DNA molecules.
Restriction enzymes recognize short, specific nucleotide sequences (restriction sites, typically 4–6 nucleotide pairs in length, less commonly 8–12) and cleave both DNA strands. The cut can be straight (forming blunt ends) or staggered (forming sticky ends with complementary single-stranded tails).
For example, the EcoRI enzyme cleaves the GAATTC site, leaving AATT sticky tails. This makes it possible to join a viral vector DNA fragment with a foreign gene of any origin based on the principle of complementarity. Then, the DNA ligase enzyme seals these fragments into a single molecule. Single-stranded DNA fragments of a given sequence (up to 100 nucleotides long) required for insertion can be obtained artificially using chemical synthesis on automated synthesizers.
To remember the essence of transduction: PHG — Phage Gently Hauls. The bacteriophage acts as a "truck" that accidentally captures a fragment of donor DNA and transports it into the recipient cell.
Class and exam questions
How does transduction differ from transformation and conjugation?
During transduction, gene transfer is carried out by a intermediary virus (bacteriophage). Transformation is the uptake of free, isolated DNA from the environment by a bacterium, whereas conjugation requires direct physical contact between cells via a conjugation tube (pili).
What properties can a bacterium acquire through a viral vector?
A cell may acquire antibiotic resistance, motility, the ability to synthesize new substances (e.g., fermenting galactose), or even alter pathogenic properties, as in the case of the botulism pathogen transforming into a gas gangrene strain.
How do retroviruses integrate their information into the genome?
They use the enzyme revertase (RNA-dependent DNA polymerase) to generate proviral DNA from their viral RNA template. The resulting DNA is then integrated into the host cell chromosome.
What else to study on this topic
- Mechanisms of transformation and free DNA uptake
- Bacterial conjugation and the role of the F-factor (fertility factor)
- Restriction-ligation technologies and the creation of recombinant DNAs
- Plasmids as alternative vectors in genetic engineering
- Pharmacological inhibition of reverse transcription
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