Materials and Reagents

R. capsulatus recipient strain
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)
Plasmid to be conjugated into R. capsulatus (e.g. pXCA601; Tetracycline resistance)
Appropriate antibiotic (resistance specified by plasmids and bacterial strains)
Thiamine hydrochloride
H₃BO₃
MnSO₄·H₂O
Na₂MoO₄·2H₂O
ZnSO₄·7H₂O
Cu(NO₃)·3H₂O
D, L-malic acid
Na₂EDTA
MgSO₄·7H₂O
CaCl₂·2H₂O
FeSO₄·7H₂O
10 mM potassium phosphate buffer
0.3% Difco yeast extract
0.3% Difco bactopeptone
Bacto-tryptone
Yeast extract
Trace element solution (see Recipes)
RCV broth (see Recipes)
RCV agar (see Recipes)
LB broth (see Recipes)
LB agar (see Recipes)
YPS agar (see Recipes)

Equipment

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

Procedure

Streak recipient R. capsulatus strain on RCV agar plate (with appropriate antibiotics) and incubate at 30 °C for 2-3 days.
Streak donor E. coli strain (and helper E. coli) on LB agar plate with appropriate antibiotics and incubate at 37 °C overnight.
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.
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.
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.
Spin microcentrifuge tubes containing cultures at 3,500 x g for 1 min. in bench-top centrifuge.
Decant all supernatant from microcentrifuge tubes.
Resuspend cell pellets in 500 μl RCV broth per microcentrifuge tube to wash away residual antibiotics and LB broth.
Spin microcentrifuge tubes containing resuspended cultures at 4,000 x g for 1 min in bench-top centrifuge.
Decant all supernatant from microcentrifuge tubes.
Resuspend donor E. coli strain cell pellet in 50 μl RCV broth.
Transfer all of the resuspended donor E. coli strain to helper E. coli strain cell pellet and resuspend (if applicable).
Transfer all of the resuspended donor E. coli strain (and helper E. coli strain) to the recipient R. capsulatus strain and resuspend.
Aliquot 10 μl drops of donor-helper-recipient mix onto a dry RCV agar plate (no antibiotics) and allow for the drops to dry.
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.
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.
Incubate streaked plate at 30 °C for 3-4 days or until you see colonies.
Optional (this will also be done in step 20): Test the R. capsulatus colonies for plasmid using your choice method, such as colony PCR.
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.
Test the “non-contaminated” R. capsulatus colonies for the conjugated plasmid by colony PCR.

Recipes

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
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)
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)
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

Beatty J. T. and Gest H. (1981). Generation of succinyl-coenzyme A in photosynthetic bacteria. Arch Microbiol 129(5): 335-340.
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.
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.
Wall J.D., Weaver P.F., et al. (1975). Gene transfer agents, bacteriophages, and bacteriocins of Rhodopseudomonas capsulata. Arch Microbiol 105(3): 217-224.
Willetts,N., and Wilkins, B. (1984). Processing of plasmid DNA during bacterial conjugation. Microbiol Rev 48: 24-41.