Lecture 8

Question 1

Why is E.coli so often used, paarticularly with relation to conjugation?

Answer 1

Escheria coli is a non-pathogenic, rapid growth bacteria with simple nutritional requirements. It supports the growth of a range of bacterial viruses chosen by groups of physicists and biologists (the “phage group”) to study the problem of replication. It was also the first bacterium that sexual recombination was discovered in, meaning real genetics was possible e.g genetic crosses.

Question 2

What was Lederberg and Tatum’s experiment?

Answer 2

Lederberg and Tatum’s experiment was to mix a series of mutants which couldn’t grow without certain nutrients with mutants which couldn’t grow without other mutants but could grow without the first set of mutant’s nutrients, the resulting mix sprouted colonies on a petri dish without any of the nutrients. This showed that conjugation had occured between the bacteria.

Question 3

Why were recombinants bad for Lederberg and how did he stop this?

Answer 3

The detection of genetic exchange could be shrouded via the appearance of revertants, hence using doubly marked strains (10^-6 * 10^-6 = 10^-12 odds of revertants instead of 10^-6). This made it very likely that the surviving colonies had to be recombinants.

Question 4

Why was Lederberg’s approach considered brilliant?

Answer 4

Lederberg’s approach was considered brilliant because:

1. It was the first use of conditional mutants to select against the parental type.
2. the mutants were double mutants
3. The prototrophic recovery technique had enormous sensitivity.

Question 5

How do we know conjugation needs distance?

Answer 5

The Davis U-Tube experiment showed physical contact was necessary for conjugation by blocking physical access but keeping the cells close.

Question 6

What does conjugation rely on?

Answer 6

Conjugation relies on a plasmid called the F(fertility) factor, the donor is F+ (male), the recipient is F- (female, no F factor), F factor can transfer from F+ cell to F- cell at high frequency by rolling circle mechanism, F- becomes F+ and F+ stays F+. F+ does not mate with any other F+ due to surface exclusion preventing pili from attaching to the surface.

Question 7

How does conjugation occur?

Answer 7

Conjugation occurs via:

1. contact between donor and recipient cells, the DNA relaxase nicks at oriT (one half of this spot will be the first part transfered, one half will be the last part transfered) and covalently binds to 5’.
2. Pilus retracts, bringing the donor and recipient into close proximity and Tra proteins form a pore complex that spans the membrane.
3. Rolling circle DNA replication initiates at 3’OH and proceeds 5’ to 3’ (check slide)
4. DNA replication pushes ssDNA into recipient cell
5. lagging strand DNA replication in recipient cell converts the ssDNA to dsDNA.
6. Upon complete replication of plasmid, the old and new otiT sites “colide and nicking between the oriT sites occurs.
7. ds plasmid DNA in donor and recipient are religated and the mating complex collapses, the cells then seperate.

Question 8

What does having an integrated F mean?

Answer 8

In a small proportion of cells in a F+ population the F exists as a plasmid integrated into the bacterial chromosome, as opposed to free in the cytoplasm. This can still transfer to another cell, as it is part of the chromosome it will transfer the whole chromosome (or at least attempt to, as the bridge is likely to break, meaning only a fraction of the chromosome will transfer). This chromosomal DNA must be incorporated via recombination or it will be lost.

Question 9

What are Hfr strains?

Answer 9

Hfr strains are when every cell in the population has F integrated, hence meaning they all transfer chromosomal genes and therefore give a high frequency of recombination.