Materials and Reagents

1. Sinorhizobium meliloti (S. meliloti 1021, Galibert et al., 2001)
   Note: The protocol can be also applied to other bacteria.
   
2. Bio-Rad Protein Assay Kit I (Sigma-Aldrich, catalog number: 500-0001 )
   
3. 50 mM Tris-HCl buffer (pH 7.5) (5 ml per sample)
   
4. 1x BugBuster reagent (Novagen, catalog number: 70921 )
   
5. 0.5 M EDTA (pH 8.0) (2.5 µl per sample)
   
6. GTP (Guanosine 5’-triphosphate sodium salt hydrate) (Sigma-Aldrich, catalog number: G8877 )
   
7. HPLC Standard: 6,7-Dimethylpterin (Schircks Laboratories, catalog number: 11.503 )
   
8. Biotin
   
9. YMB medium (Somerville and Kahn, 1983) (see Recipes) (1 plate per sample)
   
10. MMNH₄ medium (Somerville and Kahn, 1983) (see Recipes) (13 ml per sample)
    
11. Lysis buffer (see Recipes) (1 ml per sample)
    
12. Desalting buffer (see Recipes) (3 ml per sample)
    
13. RibA assay buffer (see Recipes) (2.5 µl per sample)
    
14. Derivatization reagent (see Recipes) (50 µl per sample)
    
15. HPLC mobile phase (see Recipes)
    Note: Except as otherwise noted, all other chemicals were obtained from Sigma-Aldrich.
    

Equipment

1. 14 ml Falcon tubes (BD Biosciences, catalog number: 352059 )
   
2. Eppendorf 1.7 ml tubes (Thermo Fisher Scientific, catalog number: 14-222-168 )
   
3. 0.22 µm syringe filter (Microsolv Technology, catalog number: 58022-N04-C )
   
4. 2 ml Zeba Spin Desalting Columns (Thermo Fisher Scientific, catalog number: 87768 )
   
5. Mini-centrifuge
   
6. Centrifuge
   
7. Shaker
   
8. HPLC: Waters Alliance 2695 HPLC system linked to a 2475 fluorescence detector
   
9. XTerra MS C18 column (4.6 x 100 mm, 5 µm) (Waters, part number: 186000486 )
   

Procedure