Determining whether or not cells can transfer genetic material by direct contact. Lederberg and Tatum began with 2 mutants of E. coli: (A) was Bio^-Met^-, (B) was Thr^-Leu^-. When these cells are plated on Bio^-Met^-Leu^-Thr^- agar, there will be no growth. When at least 10⁸ of these cells are mixed, and then plated on Bio^-Met^-Leu^-Thr^- agar, then there is growth. This could be due to transformation, though, and further experimentation is required. Conjugation is much less efficient than transformation, and you could not use boiled lysates and direct cell-cell contact still might not be required. To test this, Lederberg and Tatum constructed a U-Tube with media and in the middle was a cell-proof filter that was permeable to macromolecules. When these are plated on Bio^-Met^-Leu^-Thr^- agar, there is no growth. When the filter is removed, there is growth. As a result, direct cell-cell contact is required.

Conjugation summary

• Cell-cell contact
• Transfer is 1-way (donor to recipient)
• Not all cells are donors
• Recipients become donors
• Xsmal genes are rarel transferred

The hypothesis is that donor cells have a fertility factor transferred to recipient cells. The fertility factor is a non-xsm genetic element called the F-factor (renamed F-plasmid).

There is a donor, which shall be called F+. This is the first discovery of a plasmid. The F-plasmid carries genes for pillus formation (tra) and DNA transfer, and an origin of transfer. There are a huge number of naturally occurring plasmids, and they can carry various traits, including: drug resistance, metal ion resistance, virulence factors and metabolic pathways. Figure 10.20 shows the R-100 plasmid.

Hfr has a high frequency of recombination and is integrated into the host xsm. It is constantly being expressed.

The Interrupted Mating Experiment uses conjugation to determine the order of genes.

Transformation is the uptake of genetic information from the environment. This usually occurs when a competent cell (a cell capable of being transformed) uptakes a piece of DNA (typically a plasmid) and hence is transformed. The term transformed is used because the uptake of DNA usually introduces new genes which transform the cell and give it new capabilities.

Griffith discovered transformation using smooth (lethal) and rough (non-lethal) strains of S. pneumoniae. He boiled the smooth and rough strains, thereby killing the cells and creating a solution of their intracellular contents. He then mixed the solution with a live rough strain culture. He infected a patient with this mixture, and the patient died. He isolated cells from the infected patient, and found that the rough strain had become smooth. His negative control was to infect a patient with a boiled smooth strain culture, which did not result in death. Based on these results, he formulated the following hypothesis:

Genetic material is transferred from smooth cells to rough cells.

After formulating this hypothesis, Griffith performed an experiment to find out what is the genetic material. He purified macromolecules from smooth cells and mixed them with rough cells. He then assayed if the rough cells had become pathogenic. He found that polysaccaride, protein, lipid and RNA have no effect; only DNA is capable of transforming the rough cells. Hence, Griffith showed that cells are able to uptake naked DNA from their environment.

The Interrupted Mating Experiment basically uses the following steps:

1. Cells are mixed, then shaken to break their pili and stop conjugation.
2. Streptomycin is added to stop cell division.
3. Cells gain certain traits depending on which genes have been transferred.
4. By stopping conjugation at different times, gene order of a plasmid and its inserts can be determined.