Transformation in bacteria was first studied by a scientist Frederick Griffith in the year 1928. According to Griffith, the DNA or gene transfer can occur either naturally or artificially from one type of bacterial cell to the other type of bacteria. For example, Transformation of Non-virulent strain to a virulent cell or vice versa.
To explain the transformation principle, Griffith performed certain experiments on the mice by taking pathogenic bacteria “Streptococcus pneumoniae”.
Transformation results in gene alteration in the recipient cell, by the incorporation of free DNA from its surrounding through the cell membrane. The process of transformation is widely used in gene cloning, DNA linkage, generation of cDNA libraries and protein expression etc.
Content: Transformation in Bacteria
- Definition of Transformation
- Streptococcus pneumoniae
- Strains used in Griffith Experiment
- Transformation Experiment
- Transforming Factor
- Stages of Transformation
Definition of Transformation
Transformation can define as the process of taking up of extracellular or free DNA strand from one bacterial cell (Donor’s cell) by the competent bacterial cell (Recipient’s cell). The taking up of the DNA strand is either by natural or artificial means. The transformation occurs mostly in closely related species.
- Kingdom: Bacteria
- Phylum: Firmicutes
- Class: Bacilli
- Order: Lactobacillales
- Family: Streptococcaceae
- Genus: Streptococcus
- Species: pneumoniae
Streptococcus pneumoniae are the gram-positive bacteria which are mostly diplococci, non-motile, non- spore formers. The cell wall of Streptococcus pneumoniae is encapsulated with polysaccharide which provides virulency to the bacteria.
Streptococcus pneumoniae is the strain of bacteria which was used to demonstrate the principle of transformation first by Griffith. This was also used to identify the transferring factor by the three scientists Avery, Macleod and McCarty.
Strains used in Griffith Experiment
To demonstrate the transformation principle, Frederick Griffith had taken the pathogenic bacteria Streptococcus pneumoniae. Then further, he observed two different strains of Streptococcus pneumoniae and named it as S-III and R-II strain.
S-III strain: It is the smooth strain of Streptococcus pneumoniae which is encapsulated with the polysaccharide. As the polysaccharide is a virulent factor, hence S-III strain will act as “Virulent or Wild strain”.
R-II strain: It is the rough strain of Streptococcus pneumoniae which lacks the polysaccharide covering. And, as the polysaccharide is absent, the R-II strain will act as “Mutant or Avirulent strain”.
To explain the theory of transformation principle, Frederick Griffith performed a series of experiments where he injected two different strains of Streptococcus pneumoniae into the mice and reported the effect of the particular strain onto the mice.
- In his first experiment, Griffith used a rough strain of Streptococcus pneumoniae, i.e. R-II and injected it into the mice. After doing this, he observed that the R-II strain of bacteria did not affect the mice and the mice lived. Therefore, Griffith named R-II strain as an “Avirulent strain”.
- In his second experiment, Griffith used a smooth strain of Streptococcus pneumoniae, i.e. S-III and injected it into the mice. After doing this, he observed that S-III strain of bacteria killed the mice. Therefore, Griffith named S-III strain as a “Virulent strain”.
- In the third experiment, Griffith used smooth and virulent strain S-III and subjected it to the heat to destroy the virulence. Then, he injected the heat killed S-III strain into the mice. After doing this, he observed that the heat killed S-III strain did not affect the mice and the mice lived. Therefore, Griffith concluded that the virulence of the S-III strain has lost and becomes ineffective when it subjects to the heat.
- In the fourth experiment, Griffith used rough R-II strain plus dead or heat-killed S-III strain which he injected into the mice. After doing this, he observed the death of mice. Then he concluded that the S-III strain has transferred something which transformed the R-II strain into the virulent strain (R-III) and caused the death of mice.
Therefore, Griffith in his experiment concluded that there is a transformation factor which has caused the transformation of the sensitive strain to virulent type. According to him, the transforming factor was a protein which he was not sure about.
To explain the transformation factor, whether it was a protein or some other component, Avery, Macleod and McCarty performed certain experiments.
To identify the transformation of R-II to virulent type Avery, Macleod and McCarty performed sequential steps which are as follows:
- First, they extracted different components like protein, polysaccharide, lipid, RNA and DNA from the heat killed S-III strain.
- After that, they added R-II strain individually into each test tubes.
- In the third step, they used specific enzymes for the digestion of specific components.
- Then, they injected it to the mice.
After doing this experiment, they observed the death of four mice except for the last one. They concluded that the DNA is the transformation factor which has transformed the R-II strain to the virulent type. Therefore, the DNA is the heritable material which has transferred the virulence from the dead or heat-killed S-III strain to the R-II strain.
As the DNA of S-III or virulent strain is destroyed by the enzyme DNase, there will not be any transformation between the heat killed S-III strain and the R-II strain, and thus there will be no effect on the mice. They also concluded that even though the polysaccharide is a virulent factor, but still it is not involved in the transformation as it is not heritable.
Stages of Transformation
There are three stages of transformation which are as follows:
Competence is the first stage where a cell must be competent to take up the DNA. To develop competence, the cell responses to the environmental signal which allow the binding and penetration of DNA.
The DNA binding is the second stage of transformation in which the exogenous DNA first binds to the recipient cell wall as a result of developed competence. This stage occurs at the time of incubation of bacterial cell culture on ice. The DNA will bind to the recipient cell wall of bacteria by forming calcium chloride + DNA complex.
DNA integration is the incorporation of the exogenous DNA that has entered to the recipient cell cytoplasm. Therefore, the insertion of foreign DNA into the chromosome of the recipient cell will cause transformation.
To carry out the process of transformation, the bacteria should be competent to take up the free DNA.
Competence can define as the physiological state, where a recipient cell is in a state where it responds to the environmental conditions such as starvation and cell density. Therefore, when a cell becomes competent it can take up the exogenous DNA from the donor’s cell.
In the process of transformation, competence is of two types:
- Natural competence
- Artificial competence
It is a type, where a transformation occurs naturally in response to environmental signals and extreme conditions. There are about 1% of bacteria which can develop competence naturally. A set of genes are carried by the naturally competent bacteria which helps in the migration of DNA across the cell membrane naturally and incorporation into the recipient’s cell.
It is a type where transformation is induced artificially by some chemical or physical method. In this, the transformation process is forced, did not occur naturally. Artificial competence can be achieved by both chemical and physical methods:
The artificial competence can be achieved by the chemical method through the Divalent cation method and physical method through the Electroporation.
Divalent cation method: It was first introduced by the two scientists Mandel and Higa in the year 1970. In the divalent cation method, the E.coli culture is taken which is in the log phase. Then the E.coli culture is centrifuged. From the E.coli culture, the pellet of bacteria is resuspended in the divalent ion solution like calcium chloride. After that, the culture is kept in the cold conditions which will weaken the cell surface of the bacteria and will allow the binding of free DNA molecule.
Then, the bacterial suspension is suddenly subjected to the high temperature (42 Degrees Celsius) for 30 seconds in the boiling water bath will refer as Heat shock. This will create the thermal imbalance in the bacterial cell and will force the binding of free DNA into the cell.
Electroporation: It is an alternative method of chemical transformation. In electroporation, the bacterial cell is subjected to high voltage of 15KV/cm for a 5µ sec, by applying an electric field will refer as Electroshock. The electric shock enhances the ability to take up the free DNA strand. In 1982, a technique of introducing free DNA into the mice was by a scientist Neumann where he treated it with the short pulses at high voltage.
Therefore for transformation, the non-competent cell has to be competent. The competence is developed by the environmental signals like temperature, pH, heat etc. which makes the cell competent by enhancing the ability to take up the free DNA.