1. Bacterial Conjugation

2. Genetic recombination
Genetic variability – ESSENTIAL!!!
Survival of the species
Diploid Eukaryotes
Crossing over
Meiosis
Haploid Prokaryotes
Transduction
Transformation
Conjugation
Image 1 -

3. Transduction Genetic material exchanged via Viruses
E.g. bacteriophage lambda
No physical contact b/w bacteria
Promotes drug resistance
Horizontal gene transfer
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4. Transformation Acquisition of exogenous DNA fragments from environment
E.g. Plasmids
Antibiotic resistance
Pesticide resistance in crops
E.g. Agrobacterium tumefaciens
Horizontal gene transfer
Crops picture!

5. Conjugation Exchange of genetic material b/w bacteria
Physical contact required
DNA exchanged via Pilli
Drug resistance!!
Horizontal Gene Transfer
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6. Bacterial Conjugation
Overview

7. Terminology The Donor The Recipient The Conjugate F+
Fertility factor
Contains genetic material for gene exchange
“male” cell
The Recipient F-
No fertility factor
Receives genetic material
“Female” cell
The Conjugate
HFR
High frequency recombinant
Resulting genetic recombinant
Transfers entire genomic DNA when mixed with F- cells

8. 1. Cell – Cell Contact
Attachment Via Pilli
Formation of “sex pilus” or F-bridge between cells
Image 4 -
Image 5 -

9. Genetic Transfer of ssDNA
2. Mobilization
Target DNA is replicated in F+
Genetic transfer of ssDNA to F-
Image 6 -
Image 7 -

10. Synthesis of Complementary DNA
3. DNA Synthesis
Synthesis of Complementary DNA
Complementary strands of DNA are replicated in both F- and F+
Target DNA is incorporated into F- genome
Image 8 -
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11. Separation and Formation of Two F+ cells
Cells separate
Form two F+ cells with completed recombinant plasmids
Both serve as donor cells for further conjugation

12. Bacterial Conjugation in E. coli
Experiment 3

13. E. coli Strains HFR (F+) F- Mixed Culture Able to produce amino acids
Thr, Leu, Thi
Sensitive to Streptomycin
HFR strain used for experimental purposes
Requires amino acids:
Thr, Leu, Thi
Resistant to Streptomycin
Able to produce Thr and Leu
Thi not produced
Resistant to Strepromycin

14. Media M9ST Agar Minimal Media 9 Streptomycin Thiamine
Antibiotic formation
Inhibition of F- growth
Thiamine
Inhibition of F+ growth
Promotion of HFR growth
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15. Procedure Mix F- and HFR (F+) strains
Incubation in 37o water bath for proper conjugation
30 minutes*
Vortex to terminate genetic transfer
Centrifugation – DNA Pellet formation
Removal of supernatant (TSB broth)*
Resuspension with M9 Broth
Plated on M9ST Agar plates
Incubation at 37o for 48hrs

16. Results & Conclusion HFR (F+) No growth Sensitivity to Antibiotics F -
Absence of amino acids
Mixed Culture
Growth
Produced Thr and Leu
Antibiotic Resistance
HFR F-

17. Bacterial Conjugation

18. References
BIOL 3116 Advanced Microbiology Lab Manual