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Griffith's experiment, reported in 1928 by Frederick Griffith,[1]

 was one of the first experiments

suggesting that bacteria are capable of transferring genetic information through a process known

as transformation.[2][3]

 

Griffith used two strains of pneumococcus (Streptococcus pneumoniae) bacteria which infect

mice  – 

 a type III-S (smooth) and type II-R (rough) strain. The III-S strain covers itself with a polysaccharide capsule that protects it from the host's immune system, resulting in the death of

the host, while the II-R strain doesn't have that protective capsule and is defeated by the host's

immune system. A German bacteriologist, Fred Neufeld, had discovered the three pneumococcaltypes (Types I, II, and III) and discovered the Quellung reaction to identify them in vitro.

[4] Until

Griffith's experiment, bacteriologists believed that the types were fixed and unchangeable, from

one generation to another.

In this experiment,  bacteria from the III-S strain were killed by heat, and their remains were

added to II-R strain bacteria. While neither alone harmed the mice, the combination was able to

kill its host. Griffith was also able to isolate both live II-R and live III-S strains of pneumococcus

from the blood of these dead mice. Griffith concluded that the type II-R had been "transformed"into the lethal III-S strain by a "transforming principle" that was somehow part of the dead III-S

strain bacteria.

Today, we know that the "transforming principle" Griffith observed was the DNA of the III-S

strain bacteria. While the bacteria had been killed, the DNA had survived the heating process andwas taken up by the II-R strain bacteria. The III-S strain DNA contains the genes that form the

 protective polysaccharide capsule. Equipped with this gene, the former II-R strain bacteria were

now protected from the host's immune system and could kill the host. The exact nature of the

transforming principle (DNA) was verified in the experiments done by Avery, McLeod andMcCarty and by Hershey and Chase. 

DNA Genetic Material Griffith Effect

The fact that DNA is genetic material came from the experiments using bacteria and viruses.

The first series of experiments were performed by a British bacteriologist F. Griffith in 1928,

using the bacterium Diplococcus pneumoniae which causes pneumonia in mammals.

Griffith noticed that this bacterium had two types of strains.

 S-type, which was capsulated and produced a smooth colony on a synthetic medium.

  R-type, which was non-capsulated and produced rough colony on a synthetic medium.

When S-type of bacteria was injected into healthy mice, the mice developed pneumonia anddied. So S-type was named as virulent or pathogenic.

However, R-type of bacteria was non-pathogenic.

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If heat killed S-type of bacteria were injected into healthy mice, they did not cause disease and

the mice remained healthy.

When heat killed S-type of bacteria were mixed with R-type living bacteria and the mixture

injected into healthy mice, the mice developed pneumonia and died.

When bacteria were isolated from the dead mice, they were of living S-type and R-type.

Griffith announced that it was because of a phenomenon other than mutation, which he called

transformation.

In 1944, O. T. Avery, McCleod and McCarty repeated the experiments of Griffith and found thatwhen living R cells were mixed with the capsule of heat killed S type and infected into mice,there was no disease.

But when they injected a mixture of R cells and the chromosome of S-bacteria into mice, themice developed pneumonia and died.

This led to the conclusion that the chromosome of S-bacteria causes the transformation and not

the capsule.

So they announced that bacterial transformation involves transfer of a part of DNA from the

dead bacterium (donor) to the active living bacterium (recipient), which expresses the characterof the donor cell, and so is called a recombinant.

Mixture Result

R-Type Bacteria + Carbohydrates of S-Type Bacteria R-Type Bacteria

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 Mixture Result

R-Type Bacteria + Protein of S-Type Bacteria R-Type Bacteria

R-Type Bacteria + DNA of S-Type Bacteria S-Type Bacteria

R-Type Bacteria + DNA of S-Type Bacteria

+Deoxyribonuclease

R-Type Bacteria

Summary of the Experiment

Experiment of Avery and others showin

Transformation is one of three basic mechanisms for genetic exchange in bacteria.

Transformation may be either a natural process — that is, one that has evolved in certain bacteria — or it may be an artificial process whereby the recipient cells are forced to take up DNA

 by a physical, chemical, or enzymatic treatment. In both cases, exogenous DNA (DNA that is

outside the host cell), is taken into a recipient cell where it is incorporated into the recipient

genome, changing the genetic makeup of the bacterium.

 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

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 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 thatexist on the entering DNA.

Artificial Transformation

While a wide variety of bacteria can transform naturally, many species cannot take up DNA froman outside source. In some cases DNA can be forced into these cells by chemical, physical, orenzymatic 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 precipitatethe DNA on the surface of the cell, where the DNA is forced into the recipient.

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In transformation, bacteria pick up DNA from theirenvironment. This illustration shows Frederick Griffith's classic experiment which first

demonstrated transformation. The live nonvirulent bacteria absorb DNA from the deadnonvirulent bacteria, and become virulent themselves. Adapted from Curtis and Barnes, 1994.

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. TheDNA 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 proteincoat. 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

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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 toanother, 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 smoothstrains of the bacterium could be reisolated from the infected mice. However, when he infectedthe 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 lesscapable 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 preventdisease. 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 Griffithinjected heated extracts of both smooth and rough strains into mice, neither caused disease. Thissuggested 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 convertedthe 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. 

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,

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CA: Brooks/Cole Publishing, 1999.

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

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

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Transformation from Macmillan Science Library: Genetics. Copyright © 2001-2006 by

Macmillan Reference USA, an imprint of the Gale Group. All rights reserved.

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g that DNA is the Transforming Ag

ent