8-3 DNA transfer by conjugation
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Although bacteria do not undergo true sexual reproduction as seen for eukaryotic organisms, there are mechanisms for gene transfer and genetic recombination in bacteria. The means by which genes are transferred between cells are divided into three general categories. You can read more about these phenomena in the chapter on Bacterial Genetics.
In all three mechanisms, the DNA is transferred from a donor cell to a recipient cell. In most instances, only a small fraction of a chromosome is transferred. Once inside the recipient cell, the donor DNA has two pathways. If the DNA is capable of autonomous replication, it can exist indefinitely in the host cell. Any DNA entering the cell to position itself alongside the homologous portion of the recipient chromosome. Recombination may occur where, due to enzymatic action, a segment of the recipient DNA is excised and replaced by the integration of the homologous segment of donor DNA. In either case, the recombinant cell contains DNA derived from both the donor and recipient cells. Acquisition of donor genes may confer to the recipient an advantage such as being able to grow in a particular habitat or medium where growth was before impossible.
In Escherichia coli and presumably many other species, distinct mating types may be found. For E. coli, the F+ and F- mating types are distinguished by the presence of a special plasmid, the F factor, in the former. This plasmid includes genes involved in the transfer by conjugation of DNA into compatible recipient cells; included are genes responsible for the synthesis of the F pilus. When the F plasmid is transferred to an F- cell, the donor F+ cell retains a complete copy of the plasmid while the recipient F- cell becomes an F+ donor cell. If the F plasmid becomes integrated into the chromosome of a F+ cell, an Hfr cell results. A cell of this mating type will be a donor, able to transfer part of its regular chromosome during conjugation (with part of the F factor on the leading end). However, an Hfr donor rarely transfers its mating ability to a recipient, since it would require complete transfer of the entire chromosome. Recombination involving any part of the transferred Hfr chromosome may subsequently occur in the recipient cell as discussed above.
In order for us to demonstrate conjugation and recombination effectively in this experiment, we must choose parent cells which are not only different mating types but are also different in certain phenotypic properties such that we may detect the result of recombination easily. We could not demonstrate the end result of these processes with phenotypically-identical mating types! For this experiment, we have chosen an Hfr strain which is a methionine auxotroph and an F- strain which is a threonine auxotroph. Neither should grow on a minimal medium. However, after mixing the two strains and allowing for conjugation and subsequent recombination to occur, the appearance of any colony on the minimal medium can be attributed to an F- cell which acquired the ability, from an Hfr cell (via conjugation and recombination), to synthesize threonine and thus all of its amino acids.
The real starting point in this experiment is the mixture of Hfr and F- cells where we hope to have conjugation proceed. Knowing the concentration of each mating type in the mixture, we can calculate the percentage of F- cells which acquire the ability to synthesize threonine and therefore produce colonies on the minimal medium. This percentage we call the recombination frequency.
The growth characteristics of relevant E. coli strains can be summarized as follows:
Broth culture of E. coli: Hfr (donor) mating type; Thr+ Met-; approximately 108 cells/ml
Broth culture of E. coli: F- (recipient) mating type; Thr- Met+; approximately 108 cells/ml
2 plates of Tryptone Yeast Extract Glucose Agar (TYEG)
(Alternatively, any all-purpose medium can be substituted; Minimal Medium plus threonine and methionine may also be used.)
6 plates of Minimal Medium (MM)
1 empty, sterile test tube
3 dilution blanks (9 ml)
Micropipettes and sterile tips
2 sterile swabs
Procedure I (quantitative)
Figure 8-3 Diagram of mating protocol
A diagram of the steps for performing the protocol. Whenever a procedure seems complex or confusing, it often helps to draw it out.
Procedure II (qualitative)
Figure 8-4 Control plates
Typical growth observed on the TYEG (rich medium) and minimal medium plates. (A) F- strain on Minimal Medium; (B) F- strain on Rich Medium (TYEG); (C) Hfr strain on Minimal Medium; (D) Hfr strain on Rich Medium (TYEG);
Figure 8-5 Growth on Minimal Medium
Number of colony forming units formed from the mating of the donor and recipient. The cells growing here have to be both Thr+ and Met+. The labels refer to the dilution spread on each plate. Use these plates to count the number colonies and calculate the recombination frequency. To get a better view, click on the picture for an enlargement.
Figure 8-6 Cross-streak
The cross-streak of donor and recipient on Minimal Medium. Note how significant numbers of colonies only grow after the donor and recipient meet.
Figure 8-7 Calculating %recombinants
The percent of recombinants that successfully mating with the Hfr strain and recombined the thr region into the chromosome. The number of recipient cells per ml of the mixture was 5 x 107.