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. Flavin standards:
   1. Riboflavin (Sigma-Aldrich, catalog number: 95170 )
      
   2. FMN (Sigma-Aldrich, catalog number: F2253 )
      
   3. FAD (Sigma-Aldrich, catalog number: F6625 )
      
   4. Lumichrome (Acros, catalog number: 146930010 )
      
   Note: FMN and FAD were further purified as described previously (Sandov et al., 2008).
   
4. Ammonium formate
   
5. Formic acid
   
6. Methanol
   
7. YMB medium (Somerville and Kahn, 1983) (see Recipes) (1 plate per sample)
   
8. MMNH₄ medium (Somerville and Kahn, 1983) (see Recipes) (6 ml per sample)
   
9. Extraction buffer (see Recipes)
   
10. HPLC mobile phase (see Recipes)
    Note: Except as otherwise noted, all other chemicals were obtained from Sigma-Aldrich.
    

Equipment

1. Aluminum foil
   
2. Eppendorf 1.7 ml tubes (Thermo Fisher Scientific, catalog number: 14-222-168 )
   
3. 14 ml Falcon tubes (BD Biosciences, catalog number: 352059 )
   
4. 0.22 µm syringe filter for HPLC sample preparation (Microsolv Technology, catalog number: 58022-N04-C )
   
5. Scale (Ohaus Corporation, model: E10640 )
   
6. Mini-centrifuge
   
7. Shaker
   
8. HPLC: Waters Alliance 2695 HPLC system linked to a 2475 fluorescence detector
   
9. SunFire C18 reverse-phase column (4.6 x 150 mm, 3.5 µm)
   

Procedure