BacterialConjugation

enetic variability in eukaryotes involves processes occurringduring sexual reproduction. Meiosis itself helps “shuffle” thegenes donated to an egg or female gamete, while the process

of crossing over (exchange of genetic material between homologous chro-mosomes) during meiosis I further mixes up the genes. However, inprokaryotic organisms, there is no sexual reproduction. Yet genetic vari-ability occurs to maintain the evolutionary success within and betweenspecies. In prokaryotes, horizontal (lateral) gene transfer can occur betweencells within the same generation.

One mechanism driving genetic variability in bacteria is conjuga-tion. This form of horizontal gene transfer involves the one-way transferof genetic material from a donor cell to a recipient cell. Transfer requiresa physical contact between the two cells through the formation of a con-jugation bridge. Cells having an F factor represent donor cells becausethey have the ability to produce the conjugation bridge. If the F factor isextrachromosomal (a plasmid), the donor cells are designated as F�. If theF factor becomes incorporated into the bacterial chromosome of the donor,the resulting cells are designated Hfr, for high-frequency recombi-nants. Cells missing the F factor are the recipients of the conjugationprocess and are designated as F4. In F+ cells, a copy of the plasmid usuallyis completely transferred to an F4 recipient. The result is that the F4 cellnow has an F plasmid, so it has become an F+ cell. In Hfr cells, there is atransfer of chromosomal genes, but the conjugation bridge usually breaksbefore a complete copy of the donor chromosome can be transferred to anF4 cell. A few genes may be transferred, so the recipient is referred to as anF4 recombinant.

In this experiment, you will prepare three cultures of Escherichia coli:one containing an Hfr strain sensitive to the antibiotic streptomycin (Str-s);another containing an F4 strain that is resistant to streptomycin (Str-r) butrequires the amino acids threonine (thr) and leucine (leu) to build pro-teins, and the vitamin thiamine (thi); and a third containing a mixture of theHfr and F− strains. Following a short incubation period, isolation of only theF− recombinants will be performed by plating the mixed culture on a min-imal medium containing streptomycin and thiamine. A genetic map indi-cating the origin and direction of transfer and the sites of the relevantmarker genes is provided in Figure 28.1.

28B A C T E R I A L C O N J U G A T I O N 28 253

G

43038_CH28_0253.qxd 1/3/07 3:56 PM Page 253

pecial Materials

• 12-hour nutrient broth cultures of F− E. coli strain [thr−, leu−, thi−, and Str-r](ATCC e23724)

• 12-hour nutrient broth cultures of Hfr E. coli strain [thr+, leu+, thi+, and Str-s](ATCC e 23740)

• Minimal medium agar plates with streptomycin and thiamine

• Beakers with 95% ethyl alcohol

• Bent (L-shaped) glass rods

• 1-ml sterile pipettes and mechanical pipetting devices

• Sterile 13- × 100-mm test tubes

• Waterbath at 37°C

rocedure

1. Obtain two 1-ml sterile pipettes. Use one pipette to aseptically transfer 1 ml of the F− E. coli broth culture and the other pipette to aseptically trans-fer 0.3 ml of the Hfr E. coli broth culture into a sterile 13- × 100-mm testtube.

2. Mix by gently rotating the culture between the palms of your hands.

3. Incubate the culture for 30 minutes at 37°C waterbath.

4. Obtain two minimal medium plus streptomycin and thiamine agar plates.Label the bottom of both plates with your name and date. Label one plate“Hfr” and the other “F−.”

5. Using the spread-plate technique illustrated in Figure 28.2, aseptically add0.1 ml of each E. coli strain to its appropriately labeled agar plate. With analcohol-dipped and flamed glass rod, spread the inoculum over the entiresurface of the agar plate.

6. Following the 30-minute incubation, vigorously agitate the mixed cultureto terminate the genetic transfer by breaking the conjugation bridge.

7. Obtain another minimal medium plus streptomycin and thiamine agarplate. Label the bottom of the plate with your name and date. Label this plate“Hfr × F−.”

P

S

254 28 B A C T E R I A L C O N J U G A T I O N

THI

20

10

30

4050

60

70

800

HFRSTR-R

LEUTHR

Origin and order of gene transfer

Time(minutes)

for transfer

F I G U R E 2 8 . 1The genetic map for the E. coli HFRstrain

PURPOSE: to demonstratebacterial genetic recombina-tion through conjugation andthe formation of recombi-nant cells.

!Only place the glass rod inthe Bunsen burner flamelong enough to catch thealcohol on fire. Longerexposure to the flame willmelt the glass or cause therod to break.

43038_CH28_0253.qxd 1/3/07 3:56 PM Page 254

B A C T E R I A L C O N J U G A T I O N 28 255

(a) Dip the bent glass rod into the beaker of 95% ethyl alcohol.

(b) Sterilize the glass rod by flaming with a Bunsen burner.

(c) Remove from Bunsen burner, allow flame to extinguish, and cool the glass rod.

(d) Spread the inoculum over the agar surface by rotating the plate.

F I G U R E 2 8 . 2The Spread-Plate Technique.

8. Aseptically add 0.1 ml of the mixed culture. Spread the inoculum overthe entire surface with a sterile glass rod as described in step 5.

9. Incubate all three plates in an inverted position for 48 hours at 37°C; thenrefrigerate.

10. Observe the three plates for the presence of (+) or absence of (−) bacte-rial colonies. Enter your data in the Results section (Table 28.1). In yourconclusions, write why you would expect to see colonies or see no colonieson the appropriate minimal medium plus streptomycin and thiamine agarplates.

43038_CH28_0253.qxd 1/3/07 3:56 PM Page 255

uestions

1. Explain why you would not expect any growth on the agar plates con-taining only donor or recipient cells.

2. What specific genes were transferred from donor to recipient cells?

3. Assess the importance of streptomycin as a marker in the donor and recip-ient cells.

4. Which plate or plates represented the controls in the conjugation experi-ment?

5. Describe how conjugation can be a mechanism for genetic variability inbacteria.

Q

256 28 B A C T E R I A L C O N J U G A T I O N

43038_CH28_0253.qxd 1/3/07 3:56 PM Page 256

Name

Date Section

Exercise Results

Bacterial Conjugation

28

B A C T E R I A L C O N J U G A T I O N 28 257

Table 28.1. Conjugation in BacteriaMinimal Medium Plus Streptomycin and Thiamine Agar Plate

“Hfr” “F_” “Hfr � F

_”

Colonies present(+) or absent (-)

Observations and Conclusions:

43038_CH28_0253.qxd 1/3/07 3:56 PM Page 257

43038_CH28_0253.qxd 1/3/07 3:56 PM Page 258