Genetic transformation, Conjugation and Transduction in bacteria,

Genetic transformation,  Conjugation and Transduction in bacteria,

Bacteria can exchange or transfer DNA between other bacteria in three different ways. In every case the source cells of the DNA are called the DONORS and the cells that receive the DNA are called the RECIPIENTS. In each case the donor DNA is incorporated into the recipients cell's DNA by recombination exchange (Figure ). If the exchange involves an allele of the recipient's gene, the recipient's genome and phenotype will have changed. The three forms of bacterial DNA exchange are (1) TRANSFORMATION, (2) CONJUGATION and (3) TRANSDUCTION.

Plasmids - Before DNA exchange can be discussed it is necessary to understand what PLASMIDS are? Plasmids are best thought of as MINI-CHROMOSOMES. Plasmids are composed of DNA which usually exists as a CIRCULAR MOLECULE, only much SMALLER than the genomic DNA (Figure 2.10). Plasmids vary in size, but most are between 1,000 to 25,000 base pairs vs. 4,000,000 bp in the genome. Plasmids REPLICATE AUTONOMOUSLY from the genomic chromosome. Often there are MANY PLASMID COPIES present in one cell (Figure ). 

Further, a cell may contain SEVERAL DIFFERENT PLASMIDS or it may contain NO PLASMIDS at all. Plasmids generally carry genes that are NOT ESSENTIAL for a cell's survival except under special circumstances. For example, many plasmids carry genes for ANTIBIOTIC RESISTANCE (Figure ). 

When these plasmids are present in a cell, it is unaffected by the appropriate antibiotic, but if the plasmid and its antibiotic resistant gene is lost, the host cell becomes sensitive to a given antibiotic. Some plasmids carry resistance genes to several antibiotics, making them very dangerous pathogens. In other cases plasmids, called VIRULENCE-PLASMIDS, carry VIRULENCE GENES that enhance a host's ability to cause a disease. That is, a bacterium carrying a plasmid containing the virulence gene is able to CAUSE A DISEASE (Figure ), 

but when the plasmid is missing that same bacterium is unable to produce that disease. One such plasmid-based disease of recent concern is the strain of E. coli - O157:H7 that produces a severe food-borne disease.

Other plasmids carry genes for protecting a cell against DELETERIOUS substances like mercury, copper or they may carry genes that make it possible for a cell to metabolize an UNUSUAL SUBSTRATE, such as gasoline, as a nutrient or energy source.

The question naturally arises as to the PURPOSE of these plasmids in the evolutionary scheme. The current explanation is that plasmids constitute an EXTRA POOL OF GENE ALLELES and thus enlarge the effective gene pool of the population. Remember that the genome of prokaryotes carries only enough information for between 1,000 to 5,000 genes. But, as we've already learned, the more variety the better a species' chances of survival are in a fickle universe. The phenomenon of ANTIBIOTIC RESISTANCE is a case in point. Antibiotics, being natural products of certain organisms, are never-the-less unlikely to be encountered very often in quantities that endanger susceptible sensitive strains, so there is no need to carry resistance genes against the hundreds of antibiotics that lurk in the nooks 'n crannies of the environment. Indeed, to do so would likely tie up all your genes just for this one purpose; clearly not a survival plus.

However, random mutation has produced antibiotic resistance genes that clearly can prove useful under the RIGHT CIRCUMSTANCES, but how do they remain available, without tying up huge quantities of LIMITED RESOURCES? The answer is PLASMIDS, of course (bet you saw that coming didn't you?). A RARE PLASMID, randomly carrying a RARE ANTIBIOTIC RESISTANT GENE to, for example, penicillin, happens to be in a patient suffering from an infection (e. g. - clap) which is treated by a shot in the you-bloody-well-know-where. All the resistant bacteria's mates, lacking the resistance plasmid, are quickly killed, but the lucky bacterium with its penicillin-resistant-plasmid survives and reproduces while swimming in a sea of penicillin. Naturally, all the subsequent daughter cells carry the resistance plasmid, because if they didn't they'd die very quickly. This is a classical example of SURVIVAL OF THE FITTEST & of evolution in action.

In the modern world we produce huge quantities of antibiotics, so the selective pressure on bacteria containing plasmids carrying antibiotic resistant genes is intense, particularly in places like hospitals. As a consequence of this evolutionary process, current antibiotics are losing their effectiveness. To compound the problem, most of the plasmids carrying the antibiotic resistant genes have the ability to move from one bacteria to another by conjugation. In effect, a single cell carrying an antibiotic- resistant plasmid can "INFECT" many other cells with this plasmid thereby spreading the resistance plasmid rapidly THROUGHOUT a bacterial population (sort of like us getting a flu shot). The survival logic of this ability is obvious, at least as far as the bacteria are concerned.

Plasmids have one other very significant role to play in this story. They serve as the VEHICLES for carrying genes between cells in the genetic engineering revolution.

Transformation - The discovery of transformation was previous described. Since its initial discovery transformation has been shown to occur throughout the bacterial world and it has become the most commonly used artificial way of moving genes from one bacterium to another. The basic procedure involves:

Breaking open the donor cells and removing DNA from them so as to obtain a CELL-FREE, usually purified, form of DNA (NAKED DNA) (Figure ). 

Transformation is used to move DNA between bacteria, plants and animals. In each case the methods used to get the DNA into the recipient cells are slightly different. In bacteria COMPETENCY (Figure) 

is an empirical matter; that is it can not be predicted what conditions will produce competency in a given strain of bacteria. However, the following treatment often induces competency in G- bacteria:

Young cells are incubated with a CALCIUM CHLORIDE SOLUTION for approximately 30 min on ice. In some cases magnesium is also present.

The cells are concentrated and suspended as a thick suspension in the calcium solution. The cells may be mixed with reagents like glycerol and stored at -80 ^oC for later use or they may be used immediately.

Cell-free DNA is then mixed with these competent cells (Figure) on ice for approximately 30 min followed by a brief mild heating. 

The transformed cells are incubated in a rich medium for approximately 1 to 1.5 hr. and then plated on medium containing materials that will detect the presence of the transformed genes.

A variety of other transformation techniques are used for eukaryotic cells. These include mixing certain salts with DNA. These salts bind the DNA and the salt-DNA-complex is then taken into the eukaryotic cells where the DNA is subsequently incorporated into the recipient cell's DNA. Plant cells are often covered with a thick cell wall that is difficult to penetrate. To get DNA into these cells tiny metal beads coated with the donor DNA are "shot" into the cytoplasm of the recipient cells using a "gas gun".

A strong jolt of electricity is also used to drive the DNA into recipient cells. Because of the similar chemical nature of DNA, DNA from any living form can, in theory, function in any other life form. Animals or plants that have been transformed with DNA from other species are called TRANSGENIC organisms. For example, we have transgenic pigs and cows containing functional "human genes". Transgenic plants containing "bacterial genes" that make a protein toxic to certain insect pathogens are currently growing around the world.