Transformation in Bacteria
Griffith laid the foundation for the identification of DNA as the genetic material in 1928 with his experiments on transformation in the bacterium pneumococcus, now known as Streptococcus pneumoniae. The wild-type organism is a spherical cell surrounded by a mucous coated capsule. The cells form large, glistening colonies, characterized as smooth. These cells are virulent- capable of causing lethal infections upon injection into mice. A certain mutant strain of S. pneumoniae has lost the ability to form a capsule. As a result, it grows as small, rough colonies. More importantly it is avirulent since it has no protective coat, it is engulfed by the host's white blood cells before it can proliferate enough to do any damage. The key finding of Griffith’s work was that heat-killed virulent colonies of S.pneumoniae could transform avirulent cells to virulent ones. Neither the heat-killed virulent bacteria nor the live avirulent ones by themselves could cause a lethal infection. Together, however they were deadly. Somehow the virulent trait passed from the dead cells to the live, avirulent ones. Transformation was not transient; the ability to make a capsule and therefore to kill host animals, once conferred upon the avirulent bacteria, was passed to their descendents as a heritable trait. In other words, the gene for virulence, missing in the avirulent cells, was somehow gained during transformation. This meant that the transforming substance in the heat-killed bacteria was probably the gene for virulence itself. The missing piece of the puzzle was the chemical nature of the transforming substance.
Avery, MacLeod and McCarty supplied the missing piece in 1944. They used a transformation test similar to the one that Griffith has introduced. First, they removed the protein from the extract with organic solvents and found that the extract still transformed. Next, they subjected it to digestion with various enzymes. Trypsin and Chymotrypsin, which destroy protein, had no effect on transformation. Neither did ribonuclease, which degrades RNA. These experiments ruled out protein or RNA as the transforming material. On the other hand, Avery and his coworkers found that the enzyme deoxyribonuclease (DNase), which breaks down DNA, destroyed the transforming ability of the virulent cell extract. These results suggested that the transforming substance was in fact DNA. Direct physical-chemical analysis supported the hypothesis that the purified transforming substance was DNA. The analytical tools Avery and his colleagues used were as following:
- Ultracentrifugation: They spun the transforming substance in an ultracentrifuge (a very high-speed centrifuge) to estimate its size. The material with transforming activity sedimented rapidly (moved rapidly toward the bottom of the centrifuge tube), suggesting a very high molecular weight, characteristic of DNA.
- Electrophoresis: They placed the transforming agent in an electric field to see how rapidly it moved. The transforming activity had a relatively high mobility, also characteristic of DNA because of its high charge/mass ratio.
- Ultraviolet absorption spectrophotometry: They placed a solution of the transforming substance in an spectrophotometer to see what kind of ultraviolet light is absorbed most strongly. Its absorption spectrum matched the DNA. That is, the light it absorbed most strongly had a wavelength of about 260 nm, in contrast to protein, which absorbs maximally at 280 nm.
- Elementary chemical analysis: This yielded an average nitrogen/phosphorus ratio of 1.67, about what one would expect for DNA, which is rich in both elements, but vastly lower that the value expected for protein, which is rich in nitrogen but poor in phosphorus. Even a slight protein contamination would have raised the nitrogen/phosphorus ratio.