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Griffith was working with the micro-organism Diplococcus pneumonia also called Pneumococcus, a bacterium causing pneumonia. Some strains of this bacterium have a smooth polysaccharide capsule which causes disease and are designated as virulent S type strains. In a mutant strain the bacteria have no capsule, and when grown on the surface of an agar plate, they form small rough colonies and represent avirulent R strain.
The pathogenicity of the bacterium (ability to produce pneumonia) lies in the capsule. Colonies which appear rough due to lack of capsule are nonpathogenic or avirulent. The presence or absence of the capsule and its chemical composition are heritable features of the bacterium.
Capsules of different compositions are classified as type II and type III. In the same decade, mutations were being studied as stable heritable changes in genes. It was found that one in a million IIS bacterium could mutate to an avirulent, non-encapsulated strain of RII type.
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Similarly, and with the same frequency, a IIIS strain could mutate to a type designated IIIR. Very rarely, a IIR bacterium could mutate back to IIS, and a IIIR to IIIS. It is noteworthy that a IIR could mutate back only to IIS, never a IIIS; a IIIR to HIS, but never IIS.
Griffith found that when living S type bacteria are injected into mice they show symptoms of pneumonia and die; if living R type is injected, the mice continue to live. It was also found that if S type of bacteria are killed by heating to 65°C they become avirulent. Griffith mixed heat-killed IIIS cells with living IIR and injected them into a mouse.
Contrary to expectations, the animals did get pneumonia. When blood of affected mice was sampled, living IIIS Pneumococcus was found in it (Fig. 13.1). These IIIS cells could multiply and maintain their characteristics for many generations. How did this happen?
If mutation had occurred, living IIR could mutate to living IIS, not IIIS type. The temperature at which IIIS cells were heat-killed turned out to be important. When very high or very low temperatures were used for killing IIIS, the avirulent strain failed to show features of the virulent strain.
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Evidently some heat-stable component present in dead bacteria of type IIIS could confer characteristics of this strain on living IIR cells. The component was hypothesized by Griffith to be a transforming principle which was released by heat-killed cells of type IIIS and taken up by avirulent IIR cells, thus transforming their hereditary properties into those of type IIIS.
By further experiments an in vitro method was devised for detecting transformed cells of Pneumococcus. When serum containing antibodies specific for IIR cells is added to a tube, the IIR cells agglutinate and clump at the bottom of the tube. The transformed cells are not agglutinated; instead they grow and produce a cloudy suspension.
Stated briefly, transformation occurs when:
(a) Bacteria of one genotype (for example R strain of Pneumococcus) are recipients of DNA extracted from bacteria of a different genotype (example S type of Pneumococcus);
(b) Transformed cells undergo stable alteration in genotype and new phenotype is expressed through many generations. Although Griffith did not identify the transforming principle, yet his experiments set the stage for the discovery of the hereditary role of DNA.
In 1944 three scientists, O. T. Avery, C.M. Macleod and M. McCarty isolated and identified the chemical nature of Griffith’s transforming principle. They could show experimentally that highly purified DNA from heat-killed type IIIS cells could transform IIR cells into IIIS type in the in vitro system.
They could also demonstrate that enzymes that degrade DNA (the deoxyribonucleases) could destroy the transforming factor. Addition of the enzyme ribonuclease (which degrades RNA) or proteolytic (protein digesting) enzymes had no effect on transforming ability.
The DNA induced transformation was a permanent and heritable characteristic. This was the first conclusive evidence that DNA indeed was the transforming principle, and consequently also the genetic material involved in hereditary mechanism in bacteria.
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Later on other bacteria like Hemophilus influenzae, Bacillus subtilis and Escherichia coli were also shown to undergo transformation. Furthermore, bacterial strains which are sensitive to antibiotics like penicillin and streptomycin can acquire permanent resistance to these antibiotics by transformation with DNA from resistant strains.
