Figure 1.

The toxin MbcT depletes NAD⁺ and is bactericidal in Mycobacterium smegmatis. (a–c) Cultures of M. smegmatis strain mc² 155 transformed with plasmids producing MbcT under the control of the indicated promoter were diluted at time 0 in fresh medium supplemented with streptomycin alone (blue) or streptomycin and 200 ng.mL⁻¹ anhydro-tetracycline (Atc) (red) and bacterial growth was followed over up to 24 h. (d) Samples of the M. smegmatis cultures were harvested after 8 h of treatment and relative content of NAD⁺ was measured in cell extracts. Values are representative of two independent replicates of the same experiment. (e) Samples of the cultures of M. smegmatis mc² 155/pGMCS-TetR-P606-mbcT were harvested at the indicated times, diluted, and plated on L agar. Colonies were counted after 3 days of growth at 37 °C. (f–h) Samples of the cultures of mc² 155/pGMCS-TetR-P606-mbcT were harvested after 8 or 24 h of treatment with Atc, labeled with the LIVE/DEAD BacLight dyes (Syto9 and Propidium Iodide (PI)) and analyzed by flow cytometry (FACS). (i) Cells grown without Atc were heat-killed at 98 °C for 30 min before LIVE/DEAD labeling and FACS analysis. Data are representative of two experiments with similar results.