What is the Transformation Experiment in Bacteria?

In the discovery of genetic material, the experiment conducted by Frederick Griffith on Streptococcus pneumonia proved to be a stepping stone.

Process of Transformation

• The transformation experiment established the concept that bacteria transfer genetic information.
• Two strains of Pneumococcus bacteria were used, R(rough) strain and S(smooth) strain. The smooth strain was virulent, and the rough strain was non-virulent.
• The S strain could protect itself from the host immune system due to the production of a polysaccharide capsule surrounding the bacterial cell.
• In the R bacteria, this polysaccharide capsule was absent. 
• The two strains were cultured, and the bacteria showed two different growth patterns. A smooth, shiny colony was present on one plate, referred to as the S strain colony. The other colony appeared rough and is said to be the R strain colony.
• These two different bacterial colonies were seen due to the production of mucous coat by the S strain bacteria.
• In the R strain bacteria, the mucous coat is absent. Therefore, these bacteria are considered non-virulent. 
• R strain: When the R strain was grown in the petri dish, it produced irregular rough colonies. These non-virulent bacteria were injected into mice, and the mice survived.
• S strain: When S strain was grown in the petri dish, the colonies formed by these bacteria were round and smooth. The mice injected with the S strain developed pneumonia and died.
• In the second process, Griffith prepared a heat-killed S strain and injected the same into the mice. The mice survived this time.
• Now, the heat-killed S strain was introduced into the mice along with the R strain. Consequently, the mice died, and the virulent S strain was isolated from the dead mice.

Based on this experiment, Griffith concluded that the R strain was transformed virulent by the S strain. The R strain inherited some transforming substances from the heat-killed S strain, due to which it became virulent. Later, Griffith considered the transforming substance/molecule as the hereditary material.

DNA (Deoxyribonucleic Acid) as the Genetic Material 

• The Griffith experiment provided a clue about the genetic material but failed to explain its nature.
• Three scientists, Oswald Avery, Colin MacLeod, and McCarty, did the continuation of the Griffith experiment in search of the hereditary material's biochemical nature.
• They cultured the heat-killed S cells and performed various biochemical tests. Protein purification, DNA, and RNA (ribonucleic acid) extraction were performed in the heat-killed S strain.
• It was discovered that DNA was responsible for the transformation of R strain bacteria. Several shreds of evidence prove that DNA is the genetic material. The substance purified from the R strain was negative to protein tests but positive to tests that detect DNA. The composition of purified substances resembled the DNA composition. The nitrogen-phosphorus ratio obtained from the purified substance was similar to the nitrogen-phosphorus ratio of DNA.
• Proteases that perform proteolysis and RNAse that perform RNA degradation didn't inhibit transformation but were inhibited by DNAse. All these results showed that DNA is likely to be the transforming molecule. Hershey and Chase also performed legendary experiments to show DNA is the hereditary material.

Transformation

The first horizontal gene transfer mechanism was discovered in bacteria, known as transformation. Frederick Griffith discovered this mode of gene transfer in 1928. During transformation, the naked DNA molecule is taken from the surrounding by a cell and is further incorporated into the recipient's chromosome.

The process of bacterial transformation can be natural or artificial. Natural bacterial transformation is very rare and is seen in both Gram-negative as well as Gram-positive bacteria. Competence is defined as the ability of the recipient bacterium to take up the DNA molecule from the surrounding environment and get transformed. It is an inheritable characteristic. The competent bacteria encode the competence factor. With the help of this competence factor, the DNA fragments bind to the cell surface and are taken into the cytoplasm.

In S. pneumoniae, the mechanism of transformation has been studied widely. It is a Gram-positive bacterium that becomes competent during the exponential growth phase. The double-stranded DNA fragments present in the environment bind to the surface of the competent bacterial cells. A membrane-associated exonuclease hydrolyzes the uptake of one strand, and the other strand moves into the cytoplasm.

Transformation in Haemophilus influenzae is different from that in Gram-positive bacteria such as S. pneumoniae.

Process of Transformation

During transformation, the bacteria actively take in the DNA fragments from the environment. After a bacterium dies, the DNA is released into the environment. When the free DNA comes in contact with any competent bacteria, it sticks to the surface of the recipient cell. The exonuclease acts upon the double-stranded DNA strand such that only one strand enters the cytoplasm with its 3'end leading while the other strand is degraded. By homologous recombination, the translocated DNA integrates with the chromosome. The transformed bacteria are those recipient cells that acquire a new character or phenotype by transformation.

An exogenote is a part of donor DNA that plays a role in the mating of prokaryotic organisms. In contrast, an endogenote is defined as a homologous part of the fertility factor.

Integration of DNA into bacterial chromosomes occurs by the process of homologous recombination. The heteroduplex is a double-stranded molecule in which the exogenote contains an allele of an endogenote. Therefore the resulting recombinant double helix would contain one or more mismatched pairs. Several DNA repair enzymes repair this mismatch.

Transduction

A vector bacteriophage is used during transduction, which transfers DNA from a donor bacterium to a recipient bacterium. Lederberg and Zinder discovered this process of gene transfer in Salmonella typhimurium. 

The two kinds of transduction are generalized and specialized transduction. 

1. During generalized transduction, any part of the donor bacterial chromosome may enter the transducing phage. The phage enters the donor cell and takes over its cellular machinery, directing the host bacterium to produce viral particles. The viral enzymes also break down the donor chromosome. At the time of viral packaging, some part of the donor chromosome gets incorporated within a phage progeny. When this phage lyses the donor cell and enters another bacterial (recipient) cell, it introduces the donor chromosome into the recipient cell. The donor chromosome binds with the recipient chromosome through homologous recombination. 
2. During specialized transduction, the temperate phage integrates its DNA into the donor chromosome and remains dormant. A certain stimulus such as ultraviolet rays activates the integrated phage and stimulates donor cell lysis. A part of donor DNA lying at the sides of the integrated phage DNA also gets cleaved from the donor chromosome. When this phage carrying a part of the donor DNA enters a recipient cell, the specific part of the donor DNA gets incorporated into the recipient chromosome.

Context and Application

This topic is important in various entrance exams, graduate and post-graduate courses.

• Bachelors of Science in Botany/Zoology
• Masters of Science in Botany/Zoology