transformation
- 2. DNA exchange among bacteriaDNA exchange among bacteria • DNA can be exchanged among bacteria in three ways: 1. Conjugation The transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. 2. Transduction A phage carries DNA from one bacterium to another. 3. Transformation Cells take up free DNA directly from their environment.
- 3. TransformationTransformation • Incorporation of naked DNA from extracellular environment. • Cells that can be used for transformation are called competent.
- 4. HistoryHistory • In 1928, Fred Griffith found that one form of the pathogenic pneumococci (now called Streptococcus pneumoniae) could be mysteriously “transformed” into another form. • Griffith made a conclusion that the dead pathogenic bacteria gave off a “transforming principle” that changed the live nonpathogenic rough-colony-forming bacteria into the pathogenic smooth-colony form.
- 5. CompetenceCompetence • The ability of some bacteria to take up naked DNA from their environment. • It is genetically programmed. Generally, more than a dozen genes are involved, encoding both regulatory and structural components. • The general steps that occur in natural transformation differ in Gram-negative and positive bacteria.
- 7. Types of TransformationTypes of Transformation There are two types of transformation: 1.Natural transformation 2.Artificial transformation
- 8. Natural TransformationNatural Transformation • In this case DNA take-up occurs without outside help. • Naturally competent bacterium – They can take up DNA from the environment without requiring special treatment. • About 40 species have been found to be naturally competent or transformable. • Examples: Bacillus subtilis, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria gonorrhoeae, Helicobacter pylori, Acinetobacter baylyi, and some species of marine cyanobacteria.
- 9. Steps involved in naturalSteps involved in natural transformationtransformation Following proteins are involved in this process: •ComEA: binds directly extracellular double-stranded DNA. •The comF genes encode proteins that translocate the DNA into the cell. • ComFA provides the energy for translocation of DNA through the membrane. •ComEA, ComEC, and ComFA form a sort of ATP- DNA into the cell. •The genes in the comG operon encode proteins that might form a “pseudopilus” which helps move DNA through the ComEC channel. They might bind to extracellular DNA, perhaps acting through the ComEA DNA-binding protein, and then retract, drawing the DNA into the cell.
- 10. Conti…Conti… • The comE, comF and comG operons are all under the transcriptional control of ComK. • Some of genes involved in the transformation process are not designated as com, because such genes were first discovered on the basis of their involvement in other processes. 1. The nucA gene product makes double-strand breaks in extracellular DNA. 2. Other examples are single-stranded-DNA binding protein (SSB), and RecA functions in the recombination of transforming DNA with chromosome DNA.
- 12. Conti….Conti…. • The lengths of single-stranded DNA incorporated into the recipient chromosome are about 8.5 to 12 kb on cotransformation of genetic markers • incorporation takes only few minutes to be completed.
- 13. Natural transformation of GramNatural transformation of Gram positive bacteriapositive bacteria
- 14. Natural transformation in gramNatural transformation in gram negative bacterianegative bacteria
- 15. Artificially induced competenceArtificially induced competence • Bacteria can be sometimes be made competent by certain chemical treatments or DNA can be forced into bacteria by a strong electric field in a process called electroporation. 1. Chemical Treatment (with calcium ions). • Chemically induced transformation is usually inefficient, and only a small percentage of the cells are ever transformed. • The cells must be plated under conditions, selective for the transformed cells. • Therefore, the DNA used for the transformation should contain a selectable gene such as encoding resistance to an antibiotic.
- 16. Artificially induced competenceArtificially induced competence 2. Electroporation • The bacteria are mixed with DNA and briefly exposed to a strong electric field. • The bacteria first be washed extensively in buffer with very low ionic strength such as distilled water. • The brief electric field across the cellular membranes might create artificial pore of H2O lined by phospholipid head groups. DNA can pass through these temporary hydrophilic pores. • Electroporation requires specialized equipment.
- 18. Applications of TransformationApplications of Transformation Bacterial transformation is used: •To make multiple copies of DNA, called DNA cloning. •To make large amounts of specific human proteins, for example, human insulin, which can be used to treat people with Type I diabetes. •To genetically modify a bacterium or other cell.
Editor's Notes
- #10: ComEA encoded by the first gene of the comE operon, ComFA is an ATPase
- #11: ComK, a transcriptional factor that is itself regulated by ComA. The free DNA ends become the substrates for the competence proteins.
- #14: ComEA binds directly extracellular double- stranded DNA. The comF genes encode proteins that translocate the DNA into the cell. ComEA, ComEC, and ComFA form a sort of ATP-binding cassette (ABC) transporter. The genes in the comG operon encode proteins that might form a “pseudopilus” which helps move DNA through the ComEC channel, and the ComECs retract,drawing the DNA into the cell.
- #15: PilQ (secretion proteins): 12 ~ 14 copies making the pore through the outer membrane. . ComA protein of Neisseria is an ortholog of ComEC of B. subtilis. 2. The DNA is shown running through the cell wall alonside the pseudopilus (ComG in B. subtilis; PilE in G – systems). 3. In most G – bacteria specific sequences are required for the binding of DNA, so that these species usually take up DNA only of the same species.
- #17: . the buffer usually also contains a nonionic solute such glycerol to prevent osmotic shock.