Bacterial Transformation Biotechnology Notes
Bacterial Transformation- recombination is a process in which genetic recombination occurs in the bacterial cell. The three modes of transfer of genetic material are; transformation, conjugation, and transduction (fig 1).
In these processes, the genetic material is transferred from one bacteria to another belonging to the same generation, sometimes different species or even kingdom, and is known as horizontal gene transfer or lateral gene transfer. In this post, we are going to discuss Transformation.
Transformation is the process wherein the bacteria take up naked DNA. The bacterial cell which takes up the DNA is said to be competent, which is ready to take up the exogenous DNA fragment. The incoming DNA can transform the recipient bacterium only if the DNA is homologous to the recipient’s DNA and the homologous recombination occurs.
The transformation was discovered by Frederick Griffith in Streptococcus pneumoniae in 1928. It was later demonstrated by Oswald Avery, Colin MacLeod, and Maclyn McCarty in 1944 that the ‘transforming principle’ was DNA.
Transformation can be said to be of two types; Natural transformation & Artificial transformation
1. Natural transformation:
Naturally, the transformation has been observed to be associated with DNA damage, growth rate decrease or competition in bacteria. For e.g. in Bacillus subtilis, a small subset of cells become competent when cell density increases or as the cells approach stationary phase. It is viewed as a way to acquire new genes for survival and is said to play an important role in evolution.
The bacteria having all the genes, whose products are involved in this process, are capable of carrying out the natural transformation. This has been reported to occur in both the Gram-positive and the Gram-negative bacteria, albeit with for few differences.
• Gram-positive Bacteria:
Usually, in natural environments, the DNA fragments are derived from the dying cells (fig 2.1 &2.2).
In gram-positive bacterium, soluble competence factor is involved in the process, which induces the competent state in the bacteria. On exposure to the competence factor, the cell wall projects the receptors for DNA (fig 2.3) to which the DNA fragments bind. This receptor is associated with a nuclease, which creates a nick in the double-stranded DNA. One strand of the DNA is hydrolyzed to form a single single-stranded DNA usually about 10 kb long (fig 2.4). The cytoplasmic proteins bind and protect the single-stranded DNA as it passes through the cytoplasm. The single-stranded DNA then replaces the homologous DNA in the recipient genome, resulting in the formation of a heteroduplex (fig 2.5). The mismatches are corrected and a recombinant cell is formed (fig 2.6).
Fig 2. Different steps in the transformation.
The Gram-positive bacteria are not specific about the sequence of DNA fragments and can take in fragments of different sequence, even if it may not recombine with the recipient genome.
• Gram-negative bacteria:
Natural transformation in Gram-negative bacteria is similar to the Gram-positive bacteria but has few differences. Firstly, in the gram-negative bacteria transformation process, the competence factor has not been detected. Unlike Gram-positive bacteria, Gram-negative are specific about the fragment of DNA they accept. These cells have receptors that bind DNA with a specific sequence. For e.g., H. influenza uses a specific 11-bp sequence to recognize DNA as its own DNA.
The DNA in single-stranded form has been not detected in Gram-negative bacteria and is thought to be closely bound with the membrane receptor.
2. Artificial Transformation:
The bacteria which do not undergo natural transformation can be made competent artificially. Such transformations, known as artificial transformation, are brought about in the laboratory using different procedures. These methods can be used to increase the frequency of transformation of naturally transforming bacteria.
The DNA is usually in the form of a plasmid, capable of independent replication. These plasmids can, hence, exist in the cell even if homologous recombination does not occur. Therefore the plasmid usually has an origin for replication, along with restriction sites for insertion of the gene of interest, and marker genes usually an antibody resistance gene (for selection of transformed/ recombinant cell).
To make the cells competent the permeability of the cell is increased using various treatments, for the exogenous DNA to enter. Different treatment includes subjecting cells to various chemical like dimethyl sulfoxide (DMSO), divalent cations, or polyethylene glycol (PEG).
E.g. Freezing and thawing the Agrobacterium tumefaciens cells in polyethylene glycol.
Divalent ions like calcium ions increase the permeability of the bacterial cells to DNA, especially when combined with brief exposure to an elevated temperature (heat shock).
Electroporation treatment of the bacteria makes the cell competent as electric pulses create pores in the cell membrane, facilitating the entry of the DNA into the cell. Once the electric field is turned off the pores close up and the DNA gets entrapped within the cell.
• Application of Bacterial Transformation:
– It is used for genetic modification of the bacteria.
– It can be used for linkage studies.
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