Transformation is a genetic transfer mechanism by which foreign genetic material is taken up by a cell. Other types of genetic transfer mechanisms include conjugation and transduction. The process results in a stable genetic change within the transformed cell. Transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane. In transformation, a bacterium takes up a piece of DNA, usually exogenous DNA molecules floating freely in its environment. This exogenous DNA molecule can usually come from bacterial cells or organisms that die but whose DNA or genetic material is still free-floating in the environment. Such exogenous DNA can be picked up and incorporated into the genome of susceptible bacteria or organism in the environment. This type of genetic transfer mechanism through which free DNA is incorporated by bacteria in its environment is known as transformation. In the laboratory, bacterial cells must be in a state of competence for transformation to take place effectively. Bacterial cells can be made competent through chemical methods (e.g., use of calcium chloride) and physical methods (e.g., use of electric charge in a process known as electroporation).
MECHANISM OF TRANSFORMATION
In transformation, a bacterium takes in DNA from its environment, often DNA that’s been shed by other bacteria (Figure 1). In a laboratory, the DNA may be introduced by scientists. If the DNA is in the form of a circular DNA called a plasmid, it can be copied in the receiving cell and passed on to its descendants or progeny. Plasmids are extrachromosomal DNA molecules that have the ability to replicate autonomously.
Why is transformation important? Transformation is an important genetic transfer mechanism that allows genetic factors to move from one organism to another; and this can occur in ways that can either be advantageous or disadvantageous to the recipient host or bacteria. Imagine that a harmless bacterium takes up DNA for a toxin gene from a pathogenic (disease-causing) species of bacterium. If the receiving cell incorporates the new DNA into its own chromosome (which can happen by a process called homologous recombination), it too may become pathogenic. More so, if a bacterium that is susceptible to chloramphenicol picks up gene or DNA that mediates antibiotic resistance (e.g., genes for chloramphenicol resistance) in its environment, the recipient bacterium will automatically become resistant to chloramphenicol even though it was formerly susceptible to this antibiotic (chloramphenicol). This is how transformation works.
- Transformation results in the genetic alteration of the recipient cell.
- Exogenous DNA is taken up into the recipient cell from its surroundings through the cell membrane (s).
- Transformation occurs naturally in some species of bacteria, but it can also be affected by artificial means in other cells.
- Transformation: Illustration of bacterial transformation. DNA from dead cells gets cut into fragments and exits the cell. The free-floating DNA can then be picked up by competent cells. The exogenous DNA is incorporated into the host cell’s chromosome through recombination. In the laboratory, bacterial cells can be made competent so that they can take up exogenous DNA into their genome. A bacterial cell can be made competent through: (1) electroporation (which is a physical process that uses electric charge) and through (2) chemical means, such as subjecting the cells to calcium chloride treatment. These processes of making a cell competent are important because they help to make the cell membrane of the recipient bacterial cell to be easily receptive to the exogenous DNA trying to gain access into the genome of the recipient bacterium.
Figure 1. Illustration of Transformation
TYPES OF TRANSFORMATION
- Transformation occurs naturally in some species of bacteria, but it can also be affected by artificial means in other cells. For transformation to happen, bacteria must be in a state of competence, which might occur as a time-limited response to environmental conditions such as starvation and cell density. Transformation is one of three processes by which exogenous genetic material may be introduced into a bacterial cell; the other two being conjugation (transfer of genetic material between two bacterial cells in direct cell to cell contact), and transduction (injection of foreign DNA by a bacteriophage virus into the host bacterium).
- Transformation may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because “transformation” has a special meaning in relation to animal cells, indicating progression to a cancerous state, the term should be avoided for animal cells when describing introduction of exogenous genetic material. The introduction of foreign DNA into eukaryotic cells is often called “transfection“.
- Bacterial transformation may be referred to as a stable genetic change, brought about by the uptake of naked DNA (DNA without associated cells or proteins). Competence refers to the state of being able to take up exogenous DNA from the environment. There are two forms of competence: natural and artificial.
- About 1% of bacterial species are capable of naturally taking up DNA under laboratory conditions; and more may be able to take it up in their natural environments. DNA material can be transferred between different strains of bacteria in a process that is called horizontal gene transfer.
- Some species, upon cell death, release their DNA to be taken up by other cells; however, transformation works best with DNA from closely-related species. These naturally-competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s). The transport of the exogenous DNA into the cells may require proteins that are involved in the assembly of type IV pili and type II secretion system, as well as DNA translocase complex at the cytoplasmic membrane.
DIFFERENCES IN DNA UPTAKE BY GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA
- Due to the differences in structure of the cell envelope between Gram-positive and Gram-negative bacteria, there are some differences in the mechanisms of DNA uptake in these cells. However, most of them share common features that involve related proteins. The DNA first binds to the surface of the competent cells on a DNA receptor, and passes through the cytoplasmic membrane through DNA translocase. Only single-stranded DNA may pass through, one strand is therefore degraded by nucleases in the process, and the translocated single-stranded DNA may then be integrated into the bacterial chromosomes by a RecA-dependent process.
- In Gram-negative cells, due to the presence of an extra membrane, the DNA requires the presence of a channel formed by secretins on the outer membrane. Pilin may be required for competence however, its role is uncertain. The uptake of DNA is generally non-sequence specific, although in some species the presence of specific DNA uptake sequences may facilitate efficient DNA uptake.
ARTIFICIAL TRANSFORMATION OR TRANSFER OF DNA
- Artificial competence can be induced in the laboratory procedures that involve making the cell passively permeable to DNA, by exposing it to conditions that do not normally occur in nature. Typically, the cells are incubated in a solution containing divalent cations (e.g., calcium chloride). The most commonly used solution with divalent cations is calcium chloride solution; band this is usually done under cold condition, which is then exposed to a pulse of mild heat shock to ease the inflow or uptake of the exogenous gene or DNA by the recipient cell. However, the mechanism of the uptake of DNA through chemically-induced competence in this calcium chloride transformation method is unclear.
- The surface of bacteria such as coli is negatively-charged due to phospholipids and lipopolysaccharides on its cell surface, and the DNA is also negatively-charged. One function of the divalent cation therefore, would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a DNA molecule to adhere to the cell surface. It is suggested that exposing the cells to divalent cations (as is obtainable in calcium chloride solution) in cold condition may also change or weaken the cell surface structure of the cells making it more permeable to DNA, i.e., more competent to take up exogenous DNA or gene. The heat-pulse is thought to create a thermal imbalance on either side of the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall.
- Electroporation is another method of promoting competence. Unlike the use of calcium chloride solution which is a chemical method of making bacterial cells more competent, electroporation is a physical method of making bacterial cells more competent to take up exogenous DNA or gene. Using this method, the cells are briefly shocked with an electric field of 10-20 kV/cm which is thought to create holes in the cell membrane of the bacterial cell through which the plasmid DNA may enter. After the electric shock, the holes are rapidly closed by the cell’s membrane-repair mechanisms.