Topic 16, Genetic recombination and
mapping in bacteria and bacteriophages

There are three mechanisms for recombination in bacteria: transformation, conjugation, and transduction. 
All three mechanisms involve the unidirectional transfer of genetic information from a donor to a recipient.

  1. Transformation
    A. We have already discussed this in the Avery, MacLeod, and Mcarty study
          of the transformation of Pneumococcus which first demonstrated that
          DNA is the genetic material.
    B. DNA is taken up directly by a recipient bacterial cell
        1. One of the two strands is degraded
    C. Recombination takes place between the donor DNA and the
        homologous region in the recipient's bacterial chromosome
    D. It has recently become possible to carry out transformation in higher plants and animals
        This has been carried out to produce transgenic plants and animals such as
        1. Roundup-ready soybeans
        2. Bt corn

  2. Conjugation
    A. Discovery of conjugation in E. coli by Lederberg and Tatum, 1946
        1. Prior to 1946, all genetics texts said bacterial can only produce asexually
        2. Their experiment is described in Figure 7.3 in the text
            a. Strain A, which is met- and bio-, produces no colonies when plated onto minimal medium
            b. Strain B, which is thr-, leu-, and thi-, produces no colonies when plated onto minimal medium
            c. A mixture of strains A and B is allowed to grow for a few cell divisions in complete medium
                and then plated on minimal medium, 1/10,000,000 cells grow into colonies; therefore,
                a recombinational process is taking place
            d. Cells in the colonies which grew were prototrophs (wild-type)
    B. Davis' U-tube experiment (1950)
        1. Cells of strain A were placed on one side of a U-tube and cells of strain B
            were placed in the other side.  A filter with pore size that allowed liquid but
            not bacterial cells to pass separated the two sides of the tube
        2. The medium was pumped back and forth by air pressure/vacuum
        3. Cells from both sides were plated on minimal medium and none grew
        4. He concluded that cell-to-cell contact is necessary for genetic recombination to occur
    C. F+ and F- strains of E. coli
        1. F+ cells have a F (fertility) factor and are the genetic donor (act as males)
        2. F- cells do not have a F factor and are genetic recipients (act as females)
        3. The F factor is a plasmid (a large circular piece of DNA that is present
            in addition to the bacterial chromosome)
        4. It contains ~100,000 base pairs, is ~2% the size of the E coli chromosome
            and contains 19 genes
        5. Mating (conjugation) between F+ and F- bacteria, described in figure 7.6
            a. A conjugation tube forms between the two cells and a copy of the F plasmid moves
                from the F+ cell into the F- cell, so both of the resultant cells become F+
            b. Occasionally, a piece of the bacterial DNA from the F+ cell also moves
                into the F- cell to produce the recombinants
            c. After the cells have separated, they are referred to as exconjugates
    D. Hfr bacteria and chromosome mapping
        1. F+ bacteria were treated with a chemical mutagen, and a type of male (Hfr) was
            recovered which produced about 1000x as many recombinants as F+ bacteria
            (a much sexier male).
            a. Also, different genes were recombined at greatly differing frequencies in the cross
            b. In the Hfr, the F plasmid was integrated into the E. coli chromosome
        2. Mating between Hfr and F- cells, described in figure 7.10.
            a. Hfr and F- cells come into contact and a conjugation tube forms
            b. One strand of the bacterial chromosome breaks next to the integrated
                F factor in the Hfr cell and a single strand of DNA moves into the recipient cell
                1) The cell is now referred to as a "merioygote" = partial diploid
            c. Recombination occurs between the DNA from the Hfr cell and the homologous
                region on F- chromosome
            d. Usually, the conjugation terminates before the entire chromosome moves across
        3. The interrupted mating technique
            a. The chromosome moves over from a fixed starting point in the Hfr cell
            b. The longer the cells are conjugating, the more of the chromosome moves over
            c. Cells are placed in a blender at various times after mating is initiated (e.g., 10, 15, 20,
                etc. minutes), and this breaks the conjugation bridge and the F- and Hfr cells separate
            d. We can see how long it takes each genetic marker to move over
            e. This information can be used to construct a genetic map where the map distances
                are measured in minutes (the entire genetic map is approximately 60 minutes long)
            f. The F factor would be the last to move over and it never enters the F- cell, so the
                F- cell does not become F+ or Hfr
            g. The F factor can be inserted at different positions in different bacterial chromosomes,
                so the genes move over in the same order but from different starting points in different strains.
                Also, the F factor can be present in the reverse orientation, so the order with which the genes
                would move over would be reversed in these strains.
        4. The F' state and merizygotes
            a. The F factor can excise from the chromosome and include a part of the bacterial chromosome
                to become a F'
            b. When a F' is mated with a F-, both resultant cells become F' and both cells also contain
                the bacterial chromosomal segment that was present in the F'

  3. Transduction = viral mediated bacterial DNA transfer
    A. Bacterial DNA is transported from one cell to another cell by a virus particle (phage)
    B. Discovery of transduction by Zinder and Lederberg, 1952 in Salmonella typhanurium
        (see Fig. 7.15)
           1. They plated two auxotrophic strains (LA-2 and LA-22) individually on minimal medium,
            no cells grew
        2. They plated a mixture of the two auxotrophic strains on minimal medium, cells grew into colonies
        3. Thus, genetic exchange was taking place between the two cell types
    C. They did a U-tube experiment (described above) and found that part of the cells on one side
        of the U-tube were protrophs (could grow on minimal medium).  They concluded that cell-to-cell
        contact was not necessary for this type of recombination, and therefore it was different from
        conjugation
    D. See figure 7.18 for a visual representation of the transduction process
        1. A viral particle (phage) infects a bacterial host cell.
        2. Most of the progeny phages contain phage DNA, but a few contain some bacterial DNA
        3. When the latter type infects another bacterial cell, a chromosomal segment from the first
            cells is transferred into the second cell where it may become integrated in the genome

  4. Bacteriophage genetics
    A. There is insufficient time to discuss this topic, but please read over this topic in the book

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Updated 12/03/00