Microbiology

Bacterial Conjugation and Genetics



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In 1946, Joshua Lederberg and Edward Tatum published an article in the Journal of Bacteriology entitled "Gene Recombination in the Bacterium Escherichia coli". This article not only described a new method of genetic transfer among bacteria, conjugation, it would also revolutionize the whole field of bacterial genetics.

Edward Tatum was born on December 14th, 1909. He obtained both his M.S. in microbiology and Ph.D. in biochemistry at the University of Wisconsin. From 1945-48 he was a faculty member at Yale University, where the research on conjugation was conducted (Edward Tatum- Biography). Joshua Lederberg was born on May 23, 1925. He received his B.A. in zoology, and enrolled in the Columbia University Medical School. He left Columbia in 1946 and went to the department of microbiology at Yale. Here he worked as Tatum's graduate student, and together they conducted the research behind the discovery of bacterial conjugation (Joshua Lederberg- Biography). Both men would go on to share the 1958 Nobel Prize in Medicine or Physiology with George Beadle.

Prior to the discovery of conjugation by Lederberg and Tatum, bacteria were deemed unsuitable for genetic analysis, as it was believed that all cells were essentially clones of the parent cell (The Joshua Lederberg Papers: The Development of Bacterial Genetics). Lederberg and Tatum believed that there had to be some kind of "mating" between bacteria, and that this mating process would lead to a transfer of genetic information. They set out to prove their hypothesis.

The experiment that Lederberg and Tatum performed that led to the discovery of the mating process, bacterial conjugation, was actually quite simple in design. A key to the understanding of the experiment is the concept of nutritionally mutant strains of bacteria. A mutant strain of E. coli is a strain that is deficient for a specific growth factor, such as an amino acid (e.g. arginine, histidine, etc.) or other factor, and will not grow on plated media unless that media has been enriched with that growth factor. Prior to joining Dr. Tatum at Yale, Lederberg had done previous research using single nutritional mutant strains of E. coli that had failed because the recovery of converted cells (cells that were able to synthesize the growth factors) was approximately the same as would be expected given normal spontaneous mutation rates. Lederberg had learned that Dr. Tatum had double mutant strains of a certain E. coli, strain K-12, and went to Yale to continue the research. These strains would prove to be of great value since the frequency of two spontaneous mutations was extremely low.

To conduct the experiment, Lederberg and Tatum used two different strains of double mutant (missing the capability of synthesizing two growth factors) E. coli K-12. They incubated 3 cultures, one of each strain alone, and one combining both strains in one culture. These cultures were incubated in a media solution containing the growth factors that both mutant strains required. After the incubation period, the cells were collected and washed. Each culture of an individual mutant strain was plated on a minimal media not containing the needed growth factors. As expected, there was no growth on these plates. When the third culture, containing the mixture of the two strains, was plated, they observed colony growth on the minimal media. The growth of colonies on this plate gave evidence of genetic recombination between the two strains. However, two more tests would have to be done before they could claim the discovery of conjugation.

The first test would be to rule out the possibility that one of the strains had cells that had been lysed, freeing the dna which could then be taken up by the other strain. The process by which cells take up free dna and incorporate that dna into their chromosome is known as transformation. To rule out transformation, Lederberg and Tatum filtered each culture, and then incubated the culture of one strain with the filtrate of the other strain. By filtering, they removed living cells from the filtrate, while allowing free dna to pass through. When these combinations were plated on the minimal agar plates, there was no growth. This proved that transformation was not involved in the process, and free dna was not responsible for the growth on the plates.

The second test they would perform would be to rule out the possibility that one strain of the E. coli was secreting metabolites that were being used by the other strain in order to grow. To show that this was not the case, they performed a U-tube experiment. In this experiment, the two liquid cultures are placed in a U- shaped tube, with a filter placed at the bottom of the U portion of the tube. This filter will keep the cells from contacting each other, but will let the metabolites, such as amino acids, pass through. After the two cultures were allowed to mix with each other, by applying pressure or suction to one end, both cultures were plated on the minimal agar plates. Both strains failed to grow on the plates, proving the metabolites were not the causative factor for growth.

These two tests showed that there had to be physical contact between living cells of the two strains in order for growth to appear on the plates. This showed that there had to be some kind of mating process between the two strains, which was termed conjugation. Later research by others would show that the "mating" process was controlled by a plasmid, called the fertility, or F, factor. In order for conjugation to occur, one bacterial cell had to be F+, and one F-. After conjugation, the F- cell would become F+, and able to conjugate with other F- cells.

The discovery of conjugation really gave new life to the study of bacterial genetics. One of the most important advances in bacterial genetics involving conjugation is the concept of gene mapping. By physically stopping the conjugation process (by blending, etc.) at different times, and subjecting the bacterial culture to differing conditions and checking for growth, the relative order and spacing of genes can be determined. A genetic map of the plasmid or chromosome may then be ascertained. Another useful tool is using conjugation to study the transfer of antimicrobial resistance genes between bacteria. Conjugation is known to be the primary mechanism for the spread of resistance genes among bacterial populations. Studying the process may lead to novel therapies to inhibit the transmission of antimicrobial resistance, which would help solve one of medicines largest looming concerns. There is also hope for the potential of using bacterial conjugation as a tool for genomic manipulation, including gene therapy (Llosa and de la Cruz, pg. 5).

References:
1. Edward Tatum- Biography
Retrieved February 1, 2007
http://nobelprize.org/nobel_prizes/medicine/laureates/1958/tatum-bio.html

2. Joshua Lederberg- Biography
Retrieved February 1, 2007
http://nobelprize.org/nobel_prizes/medicine/laureates/1958/lederberg-bio.html

3. Lederberg, J. and Tatum, E.. "Gene Recombination in Escherichia coli." Nature 158, (19 October 1946): 558.

4. Llosa, M. and de la Cruz, F. Bacterial conjugation: a potential tool for genomic engineering. Research in Microbiology 156(2005): 1-6

5. Tatum, E. and Lederberg, J.. "Gene Recombination in the Bacterium Escherichia coli." Journal of Bacteriology 53, 6 (June 1947): 673-684.

6. The Joshua Lederberg Papers: The Development of Bacterial Genetics
Retrieved February 3, 2007
http://profiles.nlm.nih.gov/BB/Views/Exhibit/narrative/bacgen1.html

7. Tortora, G.J., Funke, B.R., & Case, C.L. (1992). Microbiology: An Introduction (4th ed). Redwood City, CA: The Benjamin/Cummings Publishing Company

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