Transformation

Natural Transformation

Natural transformation is a physiological process that is genetically encoded in a wide range of bacteria. Most bacteria must shift their physiology in order to transform DNA; that is, they must become "competent" for taking up exogenous DNA. There appear to be two basic mechanisms by which bacteria can become competent for transformation. In some bacteria, including Streptococcus pneumoniae and Bacillus subtilis, competence is externally regulated. These bacteria produce and secrete a small protein called competence factor that accumulates in the growth medium.

When the bacterial culture reaches a sufficient density, the concentration of competence factor reaches a level high enough to bind receptors on the outside of the cell. This event causes an internal signal to turn on the expression of the genes needed for transformation. Thus, competence development is controlled by cell density. There are a number of other bacterial functions that are similarly regulated, and these processes are collectively called quorum sensing mechanisms. In other bacteria, including Haemophilus influenzae and Pseudomonas stutzeri, competence development is internally regulated. When there is a shift in the growth dynamics of the bacterium, an internal signal triggers competence development.

Once competence is induced, three additional steps are required for natural transformation. After induction of competence, double-stranded DNA is bound to specific receptors on the surface of the competent cells. These receptors are lacking in noncompetent cells. The double-stranded DNA is nicked and one strand is degraded while the other strand enters the cell. This process is called DNA uptake. Finally, the recombination enzymes of the recipient cell will bind the single-strand DNA that has entered it, align it with its homologous DNA on the recipient chromosome, and recombine the new DNA into the chromosome, incorporating any genetic differences that exist on the entering DNA.

Artificial Transformation

While a wide variety of bacteria can transform naturally, many species cannot take up DNA from an outside source. In some cases DNA can be forced into these cells by chemical, physical, or enzymatic treatment. This is especially important in genetic engineering, as artificial transformation is essential for the introduction of genetically altered sequences into recipient cells. One of the two most common methods is a chemical process where cells are heat-shocked, then treated with the DNA and a high concentration of calcium ions. The calcium ions precipitate the DNA on the surface of the cell, where the DNA is forced into the recipient.

More recently a new method, called electroporation, has been used to introduce DNA by artificial transformation. In this process a suspension of recipient bacteria and transforming DNA is placed in a container with metal sides. A high-voltage electrical current is passed through the sample, temporarily creating small pores, or channels, in the membranes of the bacteria. The DNA enters the cells and the pores close. Thus, exogenous (outside) DNA is introduced into the recipient.

Because exogenous DNA is not enclosed within cell walls, it is susceptible to enzymes that degrade DNA, called DNases. A hallmark of transformation is that it is sensitive to DNase, while the other two processes of genetic exchange, transduction and conjugation, are DNase resistant. Transduction is DNase resistant because the DNA is protected inside a viral protein coat. Conjugation is DNase resistant because fusion occurs between donor and recipient cells, meaning the DNA is never exposed to the outside environment or to enzymes.

Discovery of Transformation

The first report of transformation was an example of natural transformation. Dr. Frederick Griffith was a public health microbiologist studying bacterial pneumonia during the 1920s. He discovered that when he first isolated bacteria from the lungs of animals with pneumonia, the bacterial colonies that grew on the agar plates were of reasonable size and had a glistening, mucoid appearance. When he transferred these colonies repeatedly from one agar plate to another, however, mutant colonies would appear that were much smaller and were chalky in appearance. He designated the original strains as "smooth" strains, and the mutants as "rough" strains. When Griffith injected mice with smooth strains they contracted pneumonia, and smooth strains of the bacterium could be reisolated from the infected mice. However, when he infected the mice with rough strains they did not develop the disease. The smooth strains were capable of causing disease, or were "virulent," while the rough strains did not cause disease, or were "aviruluent."

Griffith questioned whether the ability to cause disease was a direct result of whatever product was making the bacterial colonies smooth, or whether rough strains of the bacterium were less capable of establishing disease for some other reason. To investigate this idea, he prepared cultures of both bacterial types. He pasteurized (killed) each of these cultures by heating them for an hour and then injected the heat-treated extracts into mice. His hypothesis was that if the bacteria had to be living to cause disease, heat-treating that killed the bacteria would prevent disease. If, on the other hand, the smooth material was itself a toxin, heating would not destroy it, meaning heated extracts of smooth strains would continue to cause disease. When Griffith injected heated extracts of both smooth and rough strains into mice, neither caused disease. This suggested to him that only living smooth cells could cause disease.

In his next experiment he coinjected unheated, live rough bacteria with heat-treated, dead smooth bacteria into mice. All of the mice developed disease, and when bacteria were isolated from the lungs of the diseased mice, all the isolates were smooth. This led Griffith to propose that there was some "transforming principle" in the heated smooth extract that converted the rough strains back to smooth ones capable of causing diseases. Griffith was not able to determine the nature of this transforming principle, but his experiments suggested that some "inheritable" material present in the heated extract could genetically convert strains from one colony type to another.

Approximately ten years later, another research team, that of Oswald Avery, Colin Munro MacLeod, and Maclyn McCarty, followed up on Griffith's experiments by enzymatically and biochemically characterizing the heated transforming extracts that Griffith had produced. Their studies indicated that the transforming principle was deoxyribonucleic acid (DNA), providing the first definitive evidence that DNA was the inheritable material.

SEE ALSO CONJUGATION; NATURE OF THE GENE, HISTORY; RECOMBINANT DNA; TRANSDUCTION.

Gregory Stewart

Bibliography

Curtis, Helen, and N. Susan Barnes. Invitation to Biology, 5th ed. New York: WorthPublishers, 1994.

Ingraham, John, and Catherine Ingraham. Introduction to Microbiology, 2nd ed. PacificGrove, CA: Brooks/Cole Publishing, 1999.

Madigan, Michael T., John Martinko, and Jack Parker. Brock Biology of Microorganisms, 10th ed. Upper Saddle River, NJ: Prentice Hall, 2000.

Streips, Uldis N., and Ronald E. Yasbin. Modern Microbial Genetics, 2nd ed. Hoboken, NJ: John Wiley & Sons, 2002.