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This lecture deals about
- What is genetic recombination?
- How recombination occurs in bacteria?
- Different types of gene transfers
- What is transformation?
Recombination
Recombination
If a DNA molecule enters to the bacterial DNA, its faces three possible fates including, a. degradation by restriction enzymes; b. Replication by themselves (if they have their own origin of replication Ex. Plasmids and phages) or c. Recombination with host chromosomal DNA.
Recombination is the physical exchange of DNA between genetic elements. Most of the bacterial recombinations are referred as homologous recombination, a process that results in genetic exchange between homologous DNA sequences from two different sources. Homologous DNA sequences are those that have nearly the same sequence; therefore, bases can pair over an extended length of the two DNA molecules. This type of recombination is involved in the process referred to as “crossing over” in classical genetics.
These videos describe how recombination (cross over) occur. It may be for eukaryotic or prokaryotic cell
Molecular mechanism of genetic recombination
Molecular mechanism of genetic recombination
The RecA protein is the key to homologous recombination. RecA is essential in nearly every homologous recombination pathway. The molecular mechanism of homologous recombination as event-wise as follows:
- An enzyme that cuts DNA in the middle of a strand, known as an endonuclease, begins the process by nicking one strand of the first DNA molecule.
- This nicked strand is separated from the other strand by proteins with helicase activity.
- Single-strand binding protein then binds to the resulting ssDNA and the RecA protein binds to it.
- This complex promotes base pairing with the complementary sequence in the second DNA molecule.
- This in turn displaces the other strand of the second DNA molecule and is therefore called strand invasion.
- The base pairing of one strand from each of the two DNA molecules over long stretches generates recombination intermediates, where each strand has originated from a different chromosome which is referred as heteroduplex region.
- Finally, the linked molecules are separated or “resolved” by resolvases that cut and rejoin the second (previously unbroken) strands of both original DNA molecules.
Recombinants and their selection
Recombinants and their selection
For homologous recombination to generate new genotypes (known as recombinant), the two homologous sequences must be related but genetically distinct. In prokaryotes, only part of a chromosome is transferred; therefore, if recombination does not occur, the DNA fragment will be lost because it cannot replicate independently. Thus, in prokaryotes, DNA transfer is just the first step in generating recombinant organisms. To detect physical exchange of DNA segments, the cells resulting from recombination must be phenotypically different from both parents. Genetic crosses in bacteria usually depend on using recipient strains that lack some selectable character that the recombinants will gain. Like mutation, there are several phenotypic characters including antibiotic resistance, virulence, phage resistance, colony morphology, nutritional characters (auxotrophs) can be used to identify the recombinants from their parents, donor and recipient.
Three mechanisms of Gene Transfer
Three mechanisms of Gene Transfer
As discussed earlier, transfer of DNA from donor to recipient is the first step of homologous recombination. Three mechanisms of genetic exchange are known in prokaryotes:
(1) transformation, in which free DNA released from one cell is taken up by another;
(2) transduction, in which DNA transfer is mediated by a virus; and (3) conjugation, in which DNA transfer involves cell-to-cell contact and a conjugative (F) plasmid in the donor cell. The two DNA molecules from donor and recipient brought together in three different ways but after this, the homologous recombination is equivalent in all the cases.
In prokaryotes, only part of a chromosome is transferred; therefore, if recombination does not occur, the DNA fragment will be lost because it cannot replicate independently. Thus, in prokaryotes, transfer is just the first step in generating recombinant organisms.
Transformation
Transformation
Transformation is a genetic transfer process by which free DNA is incorporated into a recipient cell and brings about genetic change. Several prokaryotes are naturally transformable, including certain species of both gram-negative and gram-positive Bacteria and also some species of Archaea. As the bacterial DNA is a large single molecule, when the cell is gently lysed, the DNA pours out. Because of their extreme length (1700 µm in Bacillus subtilis), bacterial chromosomes break easily and serve as source DNA for transformation. The average size of DNA as transformable element is about 10 kbp. A single cell usually incorporates only one or a few DNA fragments, so only a small proportion of the genes of one cell can be transferred to another by a single transformation event.
Griffith’s Experiment:
The discovery of transformation was one of the key events in biology, as it led to experiments demonstrating that DNA was the genetic material. This discovery became a cornerstone of molecular biology and modern genetics. The British scientist Frederick Griffith obtained the first evidence of bacterial transformation in the late 1920s. Streptococcus pneumoniae (pneumococcus), invade the body of mammals due to their presence of a polysaccharide capsule and cause disease. Mutants that lack this capsule cannot cause disease. Such mutants are called R strains because their colonies appear rough on agar, in contrast to the smooth appearance of encapsulated strains, called S strains. A mouse infected with only a few cells of an S strain succumbs in a day or two to a massive pneumococcus infection. By contrast, even large numbers of R cells do not cause death when injected. Griffith showed that if heat-killed S cells were injected along with living R cells, the mouse developed a fatal infection and the bacteria isolated from the dead mouse were of the S type. Because the S cells isolated in such an experiment always had the capsule type of the heat-killed S cells, Griffith concluded that the R cells had been transformed into a new type. This process set the stage for the discovery of DNA. (Refer lecture 2 and related video)
Griffith's experiment in animation (Youtube)
Competency
Competency
Even within transformable genera, only certain strains or species are transformable. A cell that is able to take up DNA and be transformed is said to be competent, and this capacity is genetically determined. Competence in most naturally transformable bacteria is regulated, and special proteins play a role in the uptake and processing of DNA. These competence-specific proteins include a membrane-associated DNA-binding protein, a cell wall autolysin, and various nucleases.
In Bacillus, roughly 20% of the cells in a culture become competent and stay for several hours. However, in Streptococcus, 100% of the cells can become competent, but only for a brief period during the growth cycle. High-efficiency, natural transformation is rare among Bacteria. For example, Acinetobacter, Bacillus, Streptococcus, Haemophilus, Neisseria and Thermus are naturally competent and easy to transform. However, if cells of E. coli are treated with high concentrations of calcium ions and then chilled for several minutes, they become adequately competent. Cells of E. coli treated in this manner take up double-stranded DNA, and therefore transformation of this organism by plasmid DNA is relatively efficient.
Electroporation is a physical technique that is used to get DNA into organisms that are difficult to transform, especially those with thick cell walls. In electroporation, cells are mixed with DNA and then exposed to brief high-voltage electrical pulses. This makes the cell envelope permeable and allows entry of the DNA. Electroporation is a quick process and works for most types of cells, including E. coli, most other Bacteria, some members of the Archaea, and even yeast and certain plant cells.
Molecular mechanism of Transformation
Molecular mechanism of Transformation
- During natural transformation, competent bacteria reversibly bind DNA. Soon, however, the binding becomes irreversible. Competent cells bind 1000 times more DNA than non-competent cells.
- Transforming DNA is bound at the cell surface by a DNA-binding protein.
- Either the entire double-stranded fragment is taken up, or a nuclease degrades one strand and the remaining strand is taken up, depending on the organism.
- After uptake, the DNA is bound by a competence-specific protein. This protects the DNA from nuclease attack until it reaches the chromosome, where the RecA protein takes over.
- The DNA is integrated into the genome of the recipient by recombination (as described earlier).
- If single-stranded DNA is integrated, a heteroduplex DNA is formed. During the next round of chromosomal replication, one parental and one recombinant DNA molecule are generated and subsequently the recombinants.
- On segregation at cell division, the recombinant molecule is present in the transformed cell, which is now genetically altered compared to its parent.
Transfection: Bacteria can be transformed with the DNA extracted from a bacteriophage rather than from another bacterium. This process is called transfection.
Transformation in bacteria in natural conditions
Detailed description of bacterial transformation
How transformation occurs in bacteria (Competent cells) under laboratory conditions (Youtube video)
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