Bacterial Transformation- definition, principle, steps, examples
Bacterial transformation is a process of horizontal gene transfer by which some bacteria take up foreign genetic material (naked DNA) from the environment. The foreign DNA can be derived from another bacterium that has died and released its DNA, or from a plasmid (a small circular piece of DNA) that has been artificially introduced into the environment. The uptake of foreign DNA can result in changes in the phenotype and genotype of the recipient bacterium, such as acquiring new traits or abilities.
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. Competence is the ability of a cell to incorporate naked DNA in the process of transformation.
Organisms that are naturally transformable spontaneously release their DNA in the late stationary phase via autolysis. 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. Electroporation or heat shock increases the competence by increasing the permeability of the cell wall, which allows the entry of the donor DNA.
Similarly, transformants can be selected if the transformed DNA contains a selectable marker, such as antimicrobial resistance, or if the DNA encodes for utilization of a growth factor, such as an amino acid. In most of the naturally competent bacteria, the free DNA binds to the bacteria, and the DNA is integrated into the chromosomal DNA. Sometimes, the free DNA is inserted into a plasmid which is capable of replicating autonomously from the chromosome, and thus, the insert doesn’t have to be integrated into the chromosome. Plasmid encodes some enzymes and antibiotic-resistant markers which are later expressed in the transformant after transformation.
In this process of transformation, the donor DNA is first inserted into the plasmid. The plasmid containing the donor DNA is then inserted into the competent host bacteria. After the transformation is completed, the bacteria containing the plasmid can be detected either by using a growth media supplemented with a particular antibiotic.
- Development of competence
- Binding of DNA to the cell surface
- Processing and uptake of free DNA (usually in a 3’ to 5’ direction)
- 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 a short time. This creates a thermal imbalance across the cell membrane, which induces the formation of pores that allow the entry of the donor DNA.
- For electroporation, the mixture is transferred to an electroporator and is exposed to a brief pulse of a high-voltage electric field. This creates transient holes in the cell membrane, through which the donor DNA can pass.
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 some cases, the donor DNA is inserted into a plasmid, which is a circular piece of DNA that can replicate independently from the chromosome. The plasmid may encode some enzymes or antibiotic resistance markers that are later expressed in the transformant after transformation.
The cells that have successfully incorporated the donor DNA can be detected by using a selective medium that contains an antibiotic or a growth factor that only allows the growth of transformants.
Bacterial transformation can be classified into two types based on the source and nature of the DNA that is transferred into the recipient cell: natural and artificial transformation.
Natural transformation is the process by which bacteria can take up DNA from their environment without any external intervention. This process occurs naturally in some bacterial species, such as Streptococcus pneumoniae, Neisseria gonorrhoeae, Haemophilus influenzae, and Bacillus subtilis. These bacteria are called naturally competent because they have the ability to develop competence, which is the state of being able to take up DNA. Natural competence is regulated by various factors, such as cell density, nutrient availability, stress conditions, and DNA concentration.
Natural transformation involves four steps:
- Development of competence: The competent cells express specific proteins that allow them to bind and uptake DNA. These proteins include DNA-binding proteins, membrane transporters, endonucleases, and recombinases.
- Binding of DNA to the cell surface: The competent cells recognize and bind to extracellular DNA fragments that are released by other bacteria through lysis or secretion. The DNA fragments are usually double-stranded and have specific sequences that are recognized by the DNA-binding proteins.
- Processing and uptake of free DNA: The bound DNA is cut into smaller pieces by endonucleases and one strand is degraded while the other strand is transported into the cell through a membrane-spanning channel. The uptake of DNA is usually directional, from 3` to 5` end.
- Integration of the DNA into the chromosome by recombination: The imported DNA strand can integrate into the recipient chromosome by homologous recombination, which requires a high degree of sequence similarity between the donor and recipient DNA. Alternatively, the imported DNA can remain as an extrachromosomal element or be degraded by nucleases.
Natural transformation is an important mechanism for genetic exchange and diversity among bacteria. It can also confer new traits to the recipient cells, such as antibiotic resistance, virulence factors, metabolic capabilities, or adaptation to environmental changes.
Artificial transformation is the process by which bacteria are induced to take up DNA by external means in a laboratory setting. This process is widely used for cloning and genetic engineering purposes. Most bacteria are not naturally competent and need to be treated with chemicals or physical methods to increase their permeability and ability to take up DNA. The most common methods of artificial transformation are:
- Chemical transformation: The bacteria are treated with chemicals, such as calcium chloride or rubidium chloride, that create pores in their cell membranes and allow the entry of DNA. The chemical treatment is followed by a heat shock, which induces a thermal gradient that drives the DNA into the cells.
- Electroporation: The bacteria are exposed to a brief pulse of a high-voltage electric field that creates transient pores in their cell membranes and allows the entry of DNA. Electroporation can achieve higher transformation efficiencies than chemical transformation but requires specialized equipment and conditions.
Artificial transformation also involves four steps:
- Preparation of competent cells: The bacteria are grown in optimal conditions and treated with chemicals or electric pulses to make them competent.
- Transformation of cells: The competent cells are mixed with plasmid DNA or linear DNA fragments that contain the gene of interest and a selectable marker, such as an antibiotic resistance gene.
- Cell recovery: The transformed cells are incubated in a nutrient-rich medium without antibiotics to allow them to recover from the stress and express the selectable marker.
- Cell plating: The transformed cells are plated on a medium containing antibiotics to select for those that have taken up the plasmid or integrated the DNA into their chromosome.
Artificial transformation is a powerful tool for introducing foreign genes into bacteria for various applications, such as protein production, gene expression analysis, gene knockout or knock-in, or metabolic engineering.
Bacterial transformation is a process of gene transfer that has many applications in biotechnology and medicine. Here are some examples of bacterial transformation:
- The first and most prominent example of bacterial transformation is the transformation of DNA from smooth capsule-positive colonies of Streptococcus pneumoniae to the rough capsule-negative colonies. This was the first mechanism of bacterial genetic exchange to be recognized by Frederick Griffith in 1928. Griffith observed that when he mixed heat-killed smooth bacteria (which were virulent) with live rough bacteria (which were non-virulent), some of the rough bacteria became smooth and acquired the ability to cause pneumonia in mice. This phenomenon was later explained by Avery, MacLeod and McCarty in 1944, who showed that the transforming principle was DNA.
- Another example of bacterial transformation is the transfer of antibiotic resistance genes between different strains or species of bacteria. This can occur naturally or artificially, and can pose a serious threat to public health. For instance, Neisseria gonorrhoeae and Haemophilus influenzae can take up DNA from their own species or from other bacteria, which can confer resistance to antibiotics such as penicillin. Similarly, Escherichia coli can acquire plasmids that carry genes for resistance to multiple antibiotics, such as ampicillin, tetracycline, chloramphenicol, and kanamycin.
- Bacterial transformation is also used as a tool for DNA cloning and protein production in biotechnology. By inserting a foreign gene into a plasmid and introducing it into a competent bacterium, researchers can make multiple copies of the gene or express it in the bacterial cell. For example, human insulin, which is used to treat diabetes, is produced by recombinant E. coli that carry a plasmid with the human insulin gene. Other examples of proteins produced by bacterial transformation include human growth hormone, interferon, vaccines, and enzymes.
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