Cells were grown anaerobically, in LB (MR-1, I–C and
JS872) or M9 minimal medium (Maniatis et al., 1982) plus
10 mM NaNO3 (DM7), in the presence of 50 lM DNAN. The cells
were harvested at late exponential phase, washed twice in sterile
double-distilled water (ddH2O), and resuspended at an optical density
(at 600 nm) of 3.3 in ddH2O containing 200 lM DNAN. The
reactions were performed in triplicate in serum bottles that were
sealed and made anaerobic by briefly degassing (1 min) and then
purging with argon for 10 min at 5 psi. The bottles were incubated
at 25 C in the dark. Samples were collected at selected times (1–
22 h) for analysis of DNAN and its products as described below.