Rhodobacter capsulatus Gene Transfer Agent Transduction Assay

Materials and Reagents

1. RcGTA donor strain (e.g. R. capsulatus Y262 or Rif^R strain)
   
2. Recipient strain (e.g. R. capsulatus DW5 [photosynthesis mutant] or Rif^S strain to test the transfer of the Rif^R allele)
   
3. Potassium phosphate buffer
   
4. L-malic acid (Sigma-Aldrich, catalog number: 240176 )
   
5. Thiamine hydrochloride (Sigma-Aldrich, catalog number: T1270 )
6. G-buffer (filter sterilize) (see Recipes)
   
7. RCV broth/agar¹ (see Recipes)
   
8. YPS broth/agar³ (autoclaved) (see Recipes)
   

Equipment

1. Variable temperature shaker for test tubes (30-35 °C)
   
2. Anaerobic jars for photosynthetic growth (if using photosynthesis mutant for recipient)
   
3. Light box for photosynthetic growth
   
4. 30 °C incubator
   
5. Test tubes
   
6. 17 ml glass screwcap tubes
   
7. 10 ml plastic culture tubes
   
8. Glass beads
   
9. 0.45 μm pore-size filter
   
10. 10 ml syringe
    

Procedure

Day 1

1. Streak Rhodobacter capsulatus Y262 containing plasmid containing gene to be transferred (or other gene donor), and DW5 (or other desired recipient strain) on RCV agar plates with appropriate antibiotics.
   
2. Incubate plates aerobically at 30 °C for 2 days (or until colonies form)
   
   

Day 3

3. In a 17 ml screwcap tube (should contain sterile glass beads to help with mixing the cells during growth) inoculate 17 ml of YPS broth (NO antibiotic) with Y262 (containing plasmid) from a single colony.
   
4. Grow photosynthetically at 30 °C until culture is well into the stationary phase (cell density should be around 450-500 Klett units).
   
   

Day 4

5. In test tubes inoculate 7 ml RCV broth with R. capsulatus DW5 or recipient strain(s) from a single colony.
   
6. Incubate at 30 °C, shaking at 250 RPM for 2 days (cell density should be around 300-350 Klett units).
   
   

Day 6

7. Spin down 7 ml of DW5 and recipient cultures at ~20,000 x g (5,000 rpm in a Beckman JA-20 rotor) for 5 min.
   
8. Decant and resuspend cell pellets in 3.5 ml G-buffer.
   
9. Spin down 17 ml of Y262 (plasmid) donor strain at ~20,000 x g for 5 min to pellet most of the cells.
   
10. Filter the remaining culture supernatant with a 0.45 μm pore-size filter into a sterile plastic (polystyrene) tube (not glass because it is suspected that RcGTA particles stick to glass).
    
11. Prepare the transduction mix in a 10 ml plastic culture tube:
    0.1 ml filtrate (donor)
    0.2 ml recipient
    0.4 ml G-buffer
    0.7 ml total volume
    (Negative controls: 1) lacking the recipient and 2) lacking the RcGTA donor; positive control: DW5 with photosynthetically capable donor RcGTA).
    
12. Incubate the transduction mix for 1-1.5 h at 30-35 °C with slow shaking (150 rpm).
    
13. Add 0.9 ml RCV broth to each tube and incubate for 3-4 h at 30-35 °C with slow shaking (~150 rpm).
    
14. Transfer transduced culture to a 1.7 ml microcentrifuge tube and pellet cells at maximum rpm for 30 sec.
    
15. Decant the majority of the supernatant leaving about 50 μl.
    
16. Resuspend cells in remaining supernatant and spread cells on an agar plate appropriate for selection (for DW5 positive control may use YPS medium).
    
17. Incubate plates in conditions appropriate for selection for 3-4 days (for positive control, select for anaerobic photosynthetic growth).
    
18. Test colonies for desired insertion.
    

Recipes

1. G-buffer (filter sterilize)
   10 mMTris-HCl (pH 7.8)
   1 mMMgCl₂
   1 mMCaCl₂
   1 mMNaCl
   0.5 mg/mlBSA
   
2. RCV broth/agar¹ (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
   pH adjusted to 6.8 with NaOH before autoclaving
   (for agar add 1.5 % agar)
   Trace element solution (in 250 ml dH₂O)
   0.7 g H₃BO₃
   398 mg MnSO₄^.H₂O
   188 mg Na₂MoO_4^.2H₂O
   60 mg ZnSO₄^.7H₂O
   10 mg Cu(NO₃)^.3H₂O
   
3. YPS broth/agar³ (autoclaved)
   0.3% Difco yeast extract
   0.3% Difco Bactopeptone
   2 mM CaCl₂
   2 mM MgSO₄
   (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. This protocol was adapted from the assay developed by Solioz et al. (1975).

References

1. Beatty, J. T. and H. Gest (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):759-774.
   
3. Solioz, M., Yen, H. C. and Marris, B. (1975). Release and uptake of gene transfer agent by Rhodopseudomonas capsulata. J Bacteriol 123(2): 651-657.
4. Wall, J. D., Weaver, P. F. and Gest, H. (1975). Gene transfer agents, bacteriophages, and bacteriocins of Rhodopseudomonas capsulata. Arch Microbiol 105(3): 217-224.