bacterial conjugation

Waring blenders, gene transfer & recombination.

Chromosomal transfer begins at a break in the chromosomal DNA adjacent to inserted F-plasmid.

The entire chromosome can be transferred from the male to the female cell, and the F-plasmid region is the last part to be transferred.

At 37°C complete transfer of the chromosome take 100 minutes.

An amazing answer. Knowing that 1 base pair of DNA is 0.34 nanometers long, the E. coli genome is ~4,640,000 base pairs in length, and that it take ~100 minutes to transfer a complete copy of the genome from donor to recipient cell.

How fast does DNA move during conjugation?
Is energy required to transfer DNA during conjugation? Explain your answer.

Complete transfer of the chromosome from one cell to the other is rare. The fragile link between Hfr-male and F- female cells usually breaks before transfer is complete.

Elie Wollman and Francois Jacob exploited this fragility to define the order of genes on the bacterial chromosome.

They mixed Hfr (male) and female cells and at a various times used a short pulse with a Waring blender to shear conjugating cells apart.

Because every cell of a particular Hfr strain has the F-plasmid integrated at the same site, there is a distinct, and reproducible time from the moment of conjugation to the time when specific genes are transferred to the recipient.

After the transfer, the donor genes are recombined into the recipient chromosome, replacing the original alleles.

The key to using conjugation and recombination to map the position of genes along the chromosome is that the process generates new combinations of phenotypes at high frequency.

While these 'recombinant' phenotypes could arise by multiple mutations in the recipient strain, this is extremely unlikely

How unlikely is it?: Assume the mutation frequency is 10^-6 per cell for each locus,

What is the probability of obtaining three mutations spontaneously?

Bacteria are effectively haploid, expect for the short time following chromosome transfer and before recombination, when they are partially diploid. Why is it possible to detect the transfer of both recessive and dominant mutant phenotypes following a conjugation event?

Mapping lac-/rob- mutations

With our lac- and rob- mutations in hand, we need to answer a few questions.

1. Where do these mutations map on the E. coli chromosome?
in one position (or locus) or all over the place?

2. Do lac- and rob- mutation map to the same or different regions of the chromosome?

To do this we use genetic markers.

An important characteristic of genetic markers is that they mutations in nonessential genes whose phenotypes can be readily identified using either a selection or screen.

revised 9 July 2003