Hfr Strains and Interrupted Mating: Mapping Gene Order by Conjugation

In an Hfr (high frequency of recombination) strain, the F factor is integrated into the bacterial chromosome. During conjugation the Hfr cell transfers its chromosome to an F^- recipient in a fixed linear order, starting at the origin of transfer (oriT) inside the F factor. That order is what makes interrupted mating useful as a mapping method.

Cell types

Why the recipient usually stays F-

Transfer begins inside the F factor, so the F genes are at the far end of the transferred strand. The whole chromosome would take about 100 minutes in E. coli, and the mating bridge usually breaks before that, so the rear part of the F factor rarely arrives.

Interrupted mating

1. Mix Hfr and F^- cells.
2. At timed intervals, break the mating by blending or vortexing.
3. Plate the recipients on selective media and score which Hfr genes they received.

Genes that enter earlier are found in more recipients and appear at earlier times. The time of first appearance is the distance from oriT, in minutes.

The gene order from oriT is thr, azi, gal. The distance between thr and azi is 9 minutes, and between azi and gal is 8 minutes. Genes closer to oriT also give a higher frequency of recombinants because more of them were transferred before the bridge broke.

Different Hfr strains, different starting points

Different Hfr strains integrate F at different sites and in different orientations, so each starts at another gene and transfers in a different direction. Overlapping orders from several strains reveal that the E. coli chromosome is circular.

Transfer is not recombination until integration

Transferred DNA is a linear fragment. To be inherited it must recombine with the recipient chromosome by crossing over. Fragments that do not integrate are lost.

Common traps

• Thinking Hfr makes the recipient Hfr or F⁺. Usually it stays F^-.
• Mixing conjugation with transformation or transduction. Conjugation needs cell contact through the pilus. See the three-way comparison.
• Treating minutes as map units from eukaryotic crosses. They are a different scale.
• Forgetting that the first gene transferred is the one nearest oriT.

One-minute recap

Hfr cells carry an integrated F factor and transfer chromosomal genes in a fixed order from oriT. Interrupt the mating at different times and the order of first appearance gives gene order, with time giving distance. The F genes enter last, so the recipient usually stays F^-.