Conjugation

Conjugation

Lederberg and Tatum in 1946 first demonstrated the recombination in bacteria. Conjugation and its evidence came from the experiment they perform. The genetics of plants and animals depends on upon the regular cycle of sexual reproduction in these organisms; there is an opportunity for different mutants of a species to mate with each other and produce. New individual wit new combinations of mutations, i.e. to recombine with each other, or to produce recombinations. For example, a plant that produces smooth, yellow peas can be bred with one that produces wrinkled, green peas, some of the next generations will be plants that produced the parental types- recombinant types smooth and green of wrinkled and yellow. Only by performing such crosses and observing the progeny can genetic work be done. The first demonstration of recombination in bacteria was achieved by Lederberg and Tatum in 1946 in a brilliant and remarkable experiment that opened the door to a whole new world of microbiology. Cederberg the Tatum knew the conjunction in bacteria must be quite rare, since no had found it in spite of many attempts, and so they determine to select the few possible recombinants out of the large population. They combine two different auxotrophic strains of E.coli and gave them an opportunity to mate. Then they plated the combined cultures on a minimal medium, where only phototrophs could grow ; then they found phototrophic colonies growing there, they knew that these must have been the result of a recombination between the auxotrophs. The principle of their experiments in simplified form. when Lederberg and Tatum did their experiments, they used poly auxotrophs ( mutants with more than one nutritional requirements) so that back mutation or spontaneous reversion to the wild type would not occur to confused their results.

Some terms related to conjugation:

# F^- cell→ bacteria that lack F-factor

# F⁺ cell→ bacteria that have F-factor

# HFr cell→ high frequently recombination cell

Bacteria in which F-factor is integrated into chromosomal DNA in linear form.

# F’ cell: bacteria in which F-factor carries some frequently of chromosomal DNA.

Conjugation processes

1. Conjugation between F⁺ and F^- cell: First of all physical contact of the cell is necessary for conjugation. The conjugation is mediated by the F-plasmids. In E. Coli the physical contact between two cell is established by F-pilus. The cells of bacteria having F-plasmids are called as the donor and those lacking F- plasmids are called recipient by F⁺ and F^- cells respectively. So, the F-pilus comes in close contact with recipient cell and it retracts bringing the F^-cell closer. The cells are fused forming a conjugation tube. F-plasmids nicked at the site called origin of replication in one strand by the endonuclease enzyme. The nicked strand is transferred through conjugation tube with its 5’ end passing first.

As the transfers occur F-plasmids in simultaneously replicated in F⁺ cells by rolling circle mechanism and transfer strand is replaced. In F^- cell the synthesis of the complementary strand for transferred single is started. Completion of DNA transfer and synthesis in F^- cell occurs and then recircularization of transferred plasmid occurs. Finally, the cells are separated. In this process F^- cell is converted into F⁺ cell. Here transfer of F-plasmids is 100% but the rate of recombination is only one in 10⁴-10⁵ cells. ie. these transferred plasmids rarely combine with chromosomal DNA of F^- cell.

2. HFr conjugation (HFr Ñ… F^-)

The cell in which F-plasmid is integrated into the chromosome in linear form is called HFr cells. These are donor cells. The F^- cells are the recipient cells. When integrated, the F-plasmid can still direct the synthesis of pili, carried out rolling circle replication and transfer genetic materials to F^- cells. At first conjugation, the tube is formed between HFr cells and F^- cell. A nick is produced on one strand of F-factor at its replication origin. DNA transfer begins with its 5’ end entering first but during transfer, only a part of F-factor is transferred and the large segment of chromosomal DNA is transferred . Finally, the cells are separated.

Since only part of the F-factor is transferred, F^- doesn’t become F⁺ cell. Transfer of entire chromosome along with integrated F-factor requires a long time. For eg. it takes 100 minutes in E. Coli but in the natural environment, conjugation tube is always broken before 100 minutes. So the chance of transfer of whole F-factor is very rare. A small segment of chromosomal DNA is always transferred. So the frequency of recombination is very high. Hence such bacterial cells are called HFr cells. Finally in this conjugation frequency of recombination is high and transfer of F-factor is low.

3. Conjugation between F’ and F^- cells

The cells in which F-factor carries some fragment of chromosomal DNA as it is detached from its integrated form into chromosome are called F’ donor cells. The F^- cells are the recipient cells. F’ cell directs the synthesis of the pilus, conjugation tube is formed, the nick is produced on one strand of F-factor then the transfer of genetic materials occurred with its 5’ end passing first. Rolling circle replication replaces the transferred strand. Finally, cells are separated. In this conjugation, the F-factor is transferred very effectively together with added bacterial chromosomal genes. The F^- cell becomes F’ cell i.e.secondary f’ cell. This process where bacterial genes are transmitted from donor to recipient as a part of the F-factor has been termed as seduction.

Significance of conjugation:

1. It is used in gene mapping.
2. It is the frequent method of gene transfer
3. Whether the genes carried by F-plasmid are dominant or recessive can be distinguished.

References

Arvind, Keshari K. and Kamal K Adhikari. A Textbook of Biology. Vidyarthi Pustak Bhander.

Michael J.Pleczar JR, Chan E.C.S. and Noel R. Krieg. Microbiology. Tata Mc GrawHill, 1993.

Powar. and Daginawala. General Microbiology.

Rangaswami and Bagyaraj D.J. Agricultural Microbiology.