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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
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g that DNA is the Transforming Ag
ent