Transformation, Conjugation, Sexduction, Transduction

• A bacterial chromosome is sometimes called a genophore.
• Transformation is genetic change via the uptake of exogenous DNA from the environment. This is like Griffith's experiments discussed in a previous chapter.
• Conjugation is a way of transferring DNA involving the direct coupling of two bacterial cells and the one way transfer of sequential DNA.
• Sexduction is related to conjugation, but specialized around a certain type of phenomenon.
• Transduction is genetic exchange mediated by viruses, but involves the exchange of genetic materials between different bacteria.

In bacteria, the cells take-up DNA from the environment and integrate it into the chromosome, if homologous regions exist between the chromosomal DNA and the DNA taken up. See Figure 16.6.

• To occur, 2 things must exist.
  • One, the DNA must be competent.
  • Two, the cell must be competent.
    • Competent DNA is it is large enough and it is double-stranded.
    • A competent cell is one that has a competence factor, a cell-surface protein that is used to transport the DNA into the cell. See Figure 16.7.

How does it occur?

• Extracellular DNA is bound by CF protein.
• One of the strands is hydrolyzed to provide energy for transport.
• The other single-stranded DNA molecule is transported into the cell.
• Crossing over between homologous regions occurs. It is not reciprocal and only involves replacement of DNA on the existing chromosomal strand.
• Of the bacteria that take up the DNA, only some will be transformed. You find them by selecting for them. See Figure 16.8.

You can map genes using transformation analysis.

One Method:

1. Take strain A (e.g., his-, met-) and collect DNA (kill the cells).
2. Take strain B (e.g., his+, met+), grow, treat with above, grow.
3. Plate 2 above onto a plate with pen and on minimal medium.
4. After a short wait, wash away the pen and plate onto complete medium.
5. All survivors must have received at least one of the minus genes. (Mutants occur at a lower frequency than transformants.)

In general:

• All transformants must have had two crossovers. One outside both genes and the other either between or on the other side of both (so it would get both).
• Distance is reflected in the ratio of single to (single plus double transformants). The higher the ratio, the further away the genes are located.

E.g., if 50 met+ his-, 50 met- his+, and 100 met- met-; then (50+50)/(50+50+100) =.5

• F Factor is a fertility factor possessed by the donor strain. It is necessary to have this in order to transfer genetic material.
  • In its simplest form, the F factor is a plasmid, a small circular DNA molecule, separate from the large circular chromosome containing most genes. Plasmids are autonomously replicating genetic elements.
  • F Factors are also episomes. Episomes can on occasion integrate into the main chromosome.
  • When normal F⁺ is crossed to F^-, the only genetic material usually transferred is the F factor itself.
  • F⁺ types are able to synthesize a special "sex" pilus. See Figure 16.10.
• Special F⁺ strains were found that transferred chromosomal DNA from the F⁺ to the F^-, but only rarely transferred the F factor itself. See Figure 16.11.
  • It was found that the F factor plasmid had integrated into the chromosome of the F⁺ strain forming what was called a Hfr for high frequency type.
  • Using a method called interrupted mating, it was possible to map crudely the bacterial genome. It was found to be circular.
    • Treat Hfr strain with a agent that will kill it, but not before it has a chance to conjugate with an F^- strain.
    • Take aliquots every few minutes and stick the bacteria into a food blender to "break-up" the relationship. See Figure 16.14.
    • Then screen the bacteria to determine its genotype.
    • Repeat with other Hfr strains whose insertion point is different.
    • The collected data look like this: See Figure 16.15.

The rule for mapping is again that genes that are close together tend to stay together more frequently than genes that are father apart. The resulting map looks like this: See Figures 16.16 and "How we got it" 16.12.

Sexduction (F-duction)

• F-Factor can not only integrate into the main chromosome but can also excise.
  • Sometimes when it does, it takes another piece with it.
  • This piece can transfer during conjugation, and when it does, it re-integrates with high frequency, because there are homologous regions. See Figure 16.13.

This has been used for mapping. What do you expect? CTSTFAS

First Lysogeny:

• Sometimes the phage does not replicate immediately, but becomes dormant. To do this the phage must integrate.
  • Host is called lysogenic. Phage is called temperate.
  • Integrated phage is called a prophage.
  • Prophage can become virulent by induction. It excises, reproduces using host machinery, then lyses the host.
  • Induction involves switching the genetic program of the prophage to a lytic phase.
  • Sometimes when the prophage excises, it caries host genes with it. If two genes are very close they might get moved together. This has been used for mapping.
• This is called specialized transduction mapping. See Figure 16.20, 16.21, 16.22.
• Generalized Transduction:
  • Transduction is when a virus transfers genetic material of bacterial origin to other bacteria.
    • There are two kinds of transduction, specific and generalized.
    • Generalized occurs with Salmonella typhimurium and P22, for example.
    • Any locus can be transduced using this system. It is not dependent on the integration site. There is no integration.
    • What happens is that the host DNA is randomly and occasionally incorporated into the viral particle heads.
    • The resulting particle is called a transducing particle.
    • About 1/100,000 phages carry a random 2 - 2.5 % of the bacterial genome.
    • This makes mapping of close genes possible. See Figure 16.19.

Example:

• Grow phage on A+, B+, C+ bacteria.
• Then treat A-, B-, C- bacteria with the resulting phage.
• A+ B+ 30
• A+ C+ 0
• B+ C+ 25
• A+ B+ C+ 0
• What is the order of the genes?
• This is useful for genes not too close, not too far. What about ordering close genes?

Three point analysis:

• Again, grow phage on A+, B+, C+; then treat A-, B-, C- with the phage.
• Transfer the infected bacteria to a dish with no A substance (with B and C). Only A+ bacteria can grow. Replica plate the bacteria onto plates with no B or with no C and class the bacteria.

You expect four different classes:

A+, B+, C+

A+, B-, C-

A+, B-, C+

A+, B+, C-

• If there is four classes, the rarest, the one with the fewest, has one gene different from the donor; this gene is in the middle. The genes can then be ordered.
• What if there are only three groups observed? Can you still order the genes?
• Usually, with mapping, the fewer the crossover, the closer the genes. Here, the greater the co-transduction rate, the closer the genes.
• But, still, genes that are close together tend to stay…