FIG. 4.

Requirements for metabolism of NEM by E. coli cells. (a) Strain NK6033 (NadA⁻) was grown and starved for nicotinamide as described in Materials and Methods. Samples were taken and incubated with 10 μM ¹⁴C-NEM (specific activity, 6.5 Ci · mol⁻¹). Symbols: ▵, control, no starvation; ○, after 6 h of starvation for nicotinamide; ● NK6033 starved of nicotinamide for 24 h. (b) Cells of strain MJF274 were grown to mid-exponential phase in K₁₀ medium, filtered, and washed with glucose-free medium and suspended to a final OD₆₅₀ of 0.1. One aliquot was incubated with glucose (0.2% [wt/vol]) and assayed immediately at 37°C for ¹⁴C-NEM metabolism (●) as described for Fig. 1a. An identical aliquot was incubated without glucose for 3 h at 37°C and then assayed for NEM metabolism (▵). After 15 min the culture was split into two identical portions, and glucose was added to one culture (▴), and the incubation was continued. (c) Cells of MJF407 (GshA⁻ Ggt⁻) were prepared as described in Materials and Methods and incubated with 500 μM ³H-GSH (2 Ci · mol⁻¹) (○). After 20 min (arrow A), the cells were harvested by centrifugation and resuspended in prewarmed medium lacking ³H-GSH; the culture was then split, and 500 μM NEM was added to one aliquot of cells (●) (arrow B) while the other was used as a control (○). The radioactivity in the cells determined as described for Fig. 1a. All experiments were repeated at least three times, and the data shown are representative of the phenomena observed on each occasion.