Bacterial Conjugation

Genetic recombination Genetic variability – ESSENTIAL!!! Survival of the species Diploid Eukaryotes Crossing over Meiosis Haploid Prokaryotes Transduction Transformation Conjugation Image 1 - http://www.nature.com/nrmicro/journal/v4/n1/images/nrmicro1325-f2.jpg

Transduction Genetic material exchanged via Viruses E.g. bacteriophage lambda No physical contact b/w bacteria Promotes drug resistance Horizontal gene transfer Image 2 - http://schaechter.asmblog.org/.a/6a00d8341c5e1453ef0133ee699fe4970b-250wi

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!

Conjugation Exchange of genetic material b/w bacteria Physical contact required DNA exchanged via Pilli Drug resistance!! Horizontal Gene Transfer Image 3 - http://upload.wikimedia.org/wikipedia/commons/8/8e/Conjugative_plasmids.png

Bacterial Conjugation Overview

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

1. Cell – Cell Contact Attachment Via Pilli Formation of “sex pilus” or F-bridge between cells Image 4 - http://withfriendship.com/images/i/40591/Bacterial-conjugation-picture.jpg Image 5 - http://science.kennesaw.edu/~jdirnber/Bio2108/Lecture/LecBiodiversity/27_Labeled_Images/27_12BactConjugation-L.jpg

Genetic Transfer of ssDNA 2. Mobilization Target DNA is replicated in F+ Genetic transfer of ssDNA to F- Image 6 - http://www.sciencephoto.com/image/147959/350wm/C0085040-Bacterial_conjugation,_artwork-SPL.jpg Image 7 - http://www.sciencephoto.com/image/151994/350wm/C0089308-Bacterial_conjugation,_artwork-SPL.jpg

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 - http://www.scientificcomputing.com/sites/scientificcomputing.com/files/legacyimages/Images/01_2012/Conjugation_hm5.jpg Image 9 - http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/conjugation.gif

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

Bacterial Conjugation in E. coli Experiment 3

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

Media M9ST Agar Minimal Media 9 Streptomycin Thiamine Antibiotic formation Inhibition of F- growth Thiamine Inhibition of F+ growth Promotion of HFR growth Image - http://biology.clc.uc.edu/fankhauser/labs/microbiology/bacterial_inhibition/images/07_Poured_seeded_agar_P7231209.jpg

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

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-

Bacterial Conjugation http://www.youtube.com/watch?v=O-EdX4MaMFE http://www.youtube.com/v/O-EdX4MaMFE

References http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/plasmids/conjugation-mech.html BIOL 3116 Advanced Microbiology Lab Manual