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enetic variability in eukaryotes involves processes occurring during sexual reproduction. Meiosis itself helps “shufﬂe” the G genes 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, in prokaryotic organisms, there is no sexual reproduction. Yet genetic variability occurs to maintain the evolutionary success within and between species. In prokaryotes, horizontal (lateral) gene transfer can occur between cells within the same generation. One mechanism driving genetic variability in bacteria is conjugation. This form of horizontal gene transfer involves the one-way transfer of genetic material from a donor cell to a recipient cell. Transfer requires a physical contact between the two cells through the formation of a conjugation bridge. Cells having an F factor represent donor cells because they have the ability to produce the conjugation bridge. If the F factor is extrachromosomal (a plasmid), the donor cells are designated as F. If the F factor becomes incorporated into the bacterial chromosome of the donor, the resulting cells are designated Hfr, for high-frequency recombinants. Cells missing the F factor are the recipients of the conjugation process and are designated as F4. In F+ cells, a copy of the plasmid usually is completely transferred to an F4 recipient. The result is that the F4 cell now has an F plasmid, so it has become an F+ cell. In Hfr cells, there is a transfer of chromosomal genes, but the conjugation bridge usually breaks before a complete copy of the donor chromosome can be transferred to an F4 cell. A few genes may be transferred, so the recipient is referred to as an F4 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) but requires the amino acids threonine (thr) and leucine (leu) to build pro- teins, and the vitamin thiamine (thi); and a third containing a mixture of the Hfr and F− strains. Following a short incubation period, isolation of only the F− recombinants will be performed by plating the mixed culture on a minimal medium containing streptomycin and thiamine. A genetic map indi- cating the origin and direction of transfer and the sites of the relevant marker genes is provided in Figure 28.1. 43038_CH28_0253.qxd 1/3/07 3:56 PM Page 254

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Origin and order of gene transfer THR LEU THI 0 80 HFR 10 STR-R 70 20

60 30

50 40 Time FIGURE 28.1 (minutes) The genetic map for the E. coli HFR for transfer strain

S 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

P rocedure

PURPOSE: to demonstrate 1. Obtain two 1-ml sterile pipettes. Use one pipette to aseptically transfer bacterial genetic recombina- 1 ml of the F− E. coli broth culture and the other pipette to aseptically trans- tion through conjugation and the formation of recombi- fer 0.3 ml of the Hfr E. coli broth culture into a sterile 13- × 100-mm test nant cells. tube. 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 add 0.1 ml of each E. coli strain to its appropriately labeled agar plate. With an alcohol-dipped and ﬂamed glass rod, spread the inoculum over the entire surface of the agar plate.

Only place the glass rod in 6. Following the 30-minute incubation, vigorously agitate the mixed culture the Bunsen burner flame to terminate the genetic transfer by breaking the conjugation bridge. long enough to catch the alcohol on fire. Longer 7. Obtain another minimal medium plus streptomycin and thiamine agar exposure to the flame will melt the glass or cause the plate. Label the bottom of the plate with your name and date. Label this plate rod to break. ! “Hfr × F−.” 43038_CH28_0253.qxd 1/3/07 3:56 PM Page 255

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(a) Dip the bent glass rod into the (b) Sterilize the glass rod by flaming beaker of 95% ethyl alcohol. with a Bunsen burner.

(c) Remove from Bunsen burner, allow (d) Spread the inoculum over the agar flame to extinguish, and cool the surface by rotating the plate. glass rod. FIGURE 28.2 The Spread-Plate Technique.

8. Aseptically add 0.1 ml of the mixed culture. Spread the inoculum over the 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; then refrigerate. 10. Observe the three plates for the presence of (+) or absence of (−) bacterial colonies. Enter your data in the Results section (Table 28.1). In your conclusions, write why you would expect to see colonies or see no colonies on the appropriate minimal medium plus streptomycin and thiamine agar plates. 43038_CH28_0253.qxd 1/3/07 3:56 PM Page 256

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Q uestions

1. Explain why you would not expect any growth on the agar plates containing 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 recipient cells. 4. Which plate or plates represented the controls in the conjugation experiment? 5. Describe how conjugation can be a mechanism for genetic variability in bacteria. 43038_CH28_0253.qxd 1/3/07 3:56 PM Page 257

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Name

Date Section

Exercise28 Results Bacterial Conjugation

Table 28.1. Conjugation in Bacteria Minimal 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 258