Genetic transformation
For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.
[1] Transformation is one of three processes that lead to horizontal gene transfer, in which exogenous genetic material passes from one bacterium to another, the other two being conjugation (transfer of genetic material between two bacterial cells in direct contact) and transduction (injection of foreign DNA by a bacteriophage virus into the host bacterium).
[1] In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium.
In 1944 this "transforming principle" was identified as being genetic by Oswald Avery, Colin MacLeod, and Maclyn McCarty.
[5] It was originally thought that Escherichia coli, a commonly used laboratory organism, was refractory to transformation.
However, in 1970, Morton Mandel and Akiko Higa showed that E. coli may be induced to take up DNA from bacteriophage λ without the use of helper phage after treatment with calcium chloride solution.
[6] Two years later in 1972, Stanley Norman Cohen, Annie Chang and Leslie Hsu showed that CaCl2 treatment is also effective for transformation of plasmid DNA.
[12] Not all plant cells are susceptible to infection by A. tumefaciens, so other methods were developed, including electroporation and micro-injection.
[14][15][16] Transformation is one of three forms of horizontal gene transfer that occur in nature among bacteria, in which DNA encoding for a trait passes from one bacterium to another and is integrated into the recipient genome by homologous recombination; the other two are transduction, carried out by means of a bacteriophage, and conjugation, in which a gene is passed through direct contact between bacteria.
[1] In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium.
[19] The best studied Pseudomonadota with respect to transformation are the medically important human pathogens Neisseria gonorrhoeae, Haemophilus influenzae, and Helicobacter pylori.
[2] Naturally competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s).
In order for a bacterium to bind, take up and recombine exogenous DNA into its chromosome, it must become competent, that is, enter a special physiological state.
[19] Competence for transformation is typically induced by high cell density and/or nutritional limitation, conditions associated with the stationary phase of bacterial growth.
[28] Transformation in Streptococcus mutans, as well as in many other streptococci, occurs at high cell density and is associated with biofilm formation.
[29] Competence in B. subtilis is induced toward the end of logarithmic growth, especially under conditions of amino acid limitation.
[30] Similarly, in Micrococcus luteus (a representative of the less well studied Actinomycetota phylum), competence develops during the mid-late exponential growth phase and is also triggered by amino acids starvation.
[35] In Helicobacter pylori, ciprofloxacin, which interacts with DNA gyrase and introduces double-strand breaks, induces expression of competence genes, thus enhancing the frequency of transformation[36] Using Legionella pneumophila, Charpentier et al.[37] tested 64 toxic molecules to determine which of these induce competence.
Artificial competence can be induced in laboratory procedures that involve making the cell passively permeable to DNA by exposing it to conditions that do not normally occur in nature.
[44] The role of lipopolysaccharides here are verified from the observation that shorter O-side chains are more effectively transformed – perhaps because of improved DNA accessibility.
In this poly (HB) is envisioned to wrap around DNA (itself a polyphosphate), and is carried in a shield formed by Ca ions.
However, fungi have to be treated differently due to some of their microscopic and biochemical traits: As stated earlier, an array of methods used for plant transformation do also work in fungi: Introduction of DNA into animal cells is usually called transfection, and is discussed in the corresponding article.
Another method of selection is the use of certain auxotrophic markers that can compensate for an inability to metabolise certain amino acids, nucleotides, or sugars.
Both genes by themselves produce non-functional peptides, however, when expressed together, as when a plasmid containing lacZ-α is transformed into a lacZΔM15 cells, they form a functional β-galactosidase.
The presence of an active β-galactosidase may be detected when cells are grown in plates containing X-gal, forming characteristic blue colonies.
Successful ligation therefore disrupts the lacZα gene, and no functional β-galactosidase can form, resulting in white colonies.
Cells containing successfully ligated insert can then be easily identified by its white coloration from the unsuccessful blue ones.
