Bacterial Conjugation in Rhodobacter capsulatus   

Materials and Reagents

1. R. capsulatus recipient strain
   
2. Escherichia coli donor strain (containing plasmid to be conjugated) capable of conjugation (e.g. S17-1) or E. coli donor strain containing plasmid to be conjugated and a helper strain containing the tra genes [e.g. HB101 (pRK2013)]. For a review on conjugation and tra genes see Willetts et al. (1984)
   
3. Plasmid to be conjugated into R. capsulatus (e.g. pXCA601; Tetracycline resistance)
   
4. Appropriate antibiotic (resistance specified by plasmids and bacterial strains)
   
5. Thiamine hydrochloride
   
6. H₃BO₃
   
7. MnSO₄·H₂O
   
8. Na₂MoO₄·2H₂O
   
9. ZnSO₄·7H₂O
   
10. Cu(NO₃)·3H₂O
    
11. D, L-malic acid
    
12. Na₂EDTA
    
13. MgSO₄·7H₂O
    
14. CaCl₂·2H₂O
    
15. FeSO₄·7H₂O
    
16. 10 mM potassium phosphate buffer
    
17. 0.3% Difco yeast extract
    
18. 0.3% Difco bactopeptone
    
19. Bacto-tryptone
    
20. Yeast extract
    
21. Trace element solution (see Recipes)
    
22. RCV broth (see Recipes)
    
23. RCV agar (see Recipes)
    
24. LB broth (see Recipes)
    
25. LB agar (see Recipes)
    
26. YPS agar (see Recipes)

Equipment

1. 30 °C and 37 °C shakers
   
2. 30 °C and 37 °C incubator
   
3. Test tubes
   
4. Petri plates
   
5. Sterile 1.7 ml microcentrifuge tubes
   
6. Inoculation loop
   
7. Pipetmen (10 μl to 1 ml range) and appropriate tips
   
8. Graduated pipette (5 ml range) and aspiration bulb
   
9. Bench-top microcentrifuge with rotor for 1.7 ml microcentrifuge tubes
   

Procedure

1. Streak recipient R. capsulatus strain on RCV agar plate (with appropriate antibiotics) and incubate at 30 °C for 2-3 days.
   
2. Streak donor E. coli strain (and helper E. coli) on LB agar plate with appropriate antibiotics and incubate at 37 °C overnight.
   
3. Inoculate 4 ml of RCV broth (with appropriate antibiotics) with a single colony of the recipient R. capsulatus strain and incubate at 30 °C in a 200-250 rpm shaker for 2 days.
   
4. One day later inoculate 4 ml of LB broth (with appropriate antibiotics) with donor E. coli strain, and 4 ml of LB broth with helper E. coli strain if applicable (see Materials and Reagents for examples), and incubate at 37 °C in a 200-250 rpm shaker overnight.
   
5. In separate sterile microcentrifuge tubes transfer 100 μl of donor E. coli strain, 100 μl of helper E. coli strain (if applicable), and 200 μl of recipient R. capsulatus strain. Each strain should be in mid- to late- log phase.
   
6. Spin microcentrifuge tubes containing cultures at 3,500 x g for 1 min. in bench-top centrifuge.
   
7. Decant all supernatant from microcentrifuge tubes.
   
8. Resuspend cell pellets in 500 μl RCV broth per microcentrifuge tube to wash away residual antibiotics and LB broth.
   
9. Spin microcentrifuge tubes containing resuspended cultures at 4,000 x g for 1 min in bench-top centrifuge.
   
10. Decant all supernatant from microcentrifuge tubes.
    
11. Resuspend donor E. coli strain cell pellet in 50 μl RCV broth.
    
12. Transfer all of the resuspended donor E. coli strain to helper E. coli strain cell pellet and resuspend (if applicable).
    
13. Transfer all of the resuspended donor E. coli strain (and helper E. coli strain) to the recipient R. capsulatus strain and resuspend.
    
14. Aliquot 10 μl drops of donor-helper-recipient mix onto a dry RCV agar plate (no antibiotics) and allow for the drops to dry.
    
15. Incubate plate upside down at 30 °C for 1-2 days. R. capsulatus but not E. coli will grow on the RCV agar plate and the cells are ready when the conjugation spots have a red ring around it. The middle of the spot will likely be pale pink.
    
16. Streak the conjugation spots onto RCV agar plates containing appropriate antibiotic to select for the cell containing the plasmid (see Materials and Reagents for plasmid example). Do this by scraping the red ring around the conjugation spot up with an inoculation loop.
    
17. Incubate streaked plate at 30 °C for 3-4 days or until you see colonies.
    
18. Optional (this will also be done in step 20): Test the R. capsulatus colonies for plasmid using your choice method, such as colony PCR.
    
19. Restreak colony on YPS agar plate containing appropriate antibiotics and incubate at 30 °C for 2-3 days to ensure that it is “clean” of E. coli cells. Although E. coli does not grow on RCV, it can survive. E. coli will grow on YPS agar plates. This YPS agar plate will isolate R. capsulatus cells containing the conjugated plasmid from the E. coli survivors as individual colonies. You can visually identify single R. capsulatus colonies on this YPS agar plate. It will be pink/maroon in colour compared to the cream colored E. coli colonies.
    
20. Test the “non-contaminated” R. capsulatus colonies for the conjugated plasmid by colony PCR.
    

Recipes

1. Trace element solution (in 250 ml dH₂O)
   0.7 g H₃BO₃
   398 mg MnSO₄·H₂O
   188 mg Na₂MoO₄·2H₂O
   60 mg ZnSO₄·7H₂O
   10 mg Cu(NO₃)·3H₂O
   
2. RCV broth/agar (Beatty et al., 1981) (in 1 L; autoclaved)
   4 g D, L-malic acid
   1 g (NH₄)₂SO₄
   10 mM potassium phosphate buffer
   200 mg MgSO₄·7H₂O
   75 mg CaCl₂·2H₂O
   12 mg FeSO₄·7H₂O
   20 mg Na₂EDTA
   1 ml trace element solution
   1 mg thiamine hydrochloride
   Adjust pH to 6.8 with NaOH before autoclaving
   (for agar add 1.5% Agar)
   
3. YPS broth/agar (Wall et al., 1975) (autoclaved)
   0.3% Difco yeast extract
   0.3% Difco bactopeptone
   2 mM CaCl₂
   2 mM MgSO₄
   (for agar add 1.5% agar)
4. LB Broth (Sambrook et al., 1989) (in 1 L; autoclaved)
   10 g Bacto-tryptone
   5 g Yeast extract
   10 g NaCl
   Adjust pH to 7.5 with NaOH before to autoclaving
   (for agar add 1.5% Agar)
   

Acknowledgments

The development of this protocol was funded by a grant to J.T.B. from the Canadian Institutes of Health Research.

References

1. Beatty J. T. and Gest H. (1981). Generation of succinyl-coenzyme A in photosynthetic bacteria. Arch Microbiol 129(5): 335-340.
   
2. Leung M.M., Brimacombe C.A., Spiegelman G.B., and Beatty, J.T. (2012). The GtaR protein negatively regulates transcription of the gtaRI operon and modulates gene transfer agent (RcGTA) expression in Rhodobacter capsulatus. Mol Microbiol 83(4):759-74.
   
3. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular cloning: a laboratory manual (2^nd edn). Plainview: New York: Cold Spring Harbor Laboratory Press.
   
4. Wall J.D., Weaver P.F., et al. (1975). Gene transfer agents, bacteriophages, and bacteriocins of Rhodopseudomonas capsulata. Arch Microbiol 105(3): 217-224.
   
5. Willetts,N., and Wilkins, B. (1984). Processing of plasmid DNA during bacterial conjugation. Microbiol Rev 48: 24-41.