The protonmotive force is required for maintaining ATP homeostasis and viability of hypoxic, nonreplicating Mycobacterium tuberculosis.

The persistence of Mycobacterium tuberculosis despite prolonged chemotherapy represents a major obstacle for the control of tuberculosis. The mechanisms used by Mtb to persist in a quiescent state are largely unknown. Chemical genetic and genetic approaches were used here to study the physiology of hypoxic nonreplicating mycobacteria. We found that the intracellular concentration of ATP is five to six times lower in hypoxic nonreplicating Mtb cells compared with aerobic replicating bacteria, making them exquisitely sensitive to any further depletion. We show that de novo ATP synthesis is essential for the viability of hypoxic nonreplicating mycobacteria, requiring the cytoplasmic membrane to be fully energized. In addition, the anaerobic electron transport chain was demonstrated to be necessary for the generation of the protonmotive force. Surprisingly, the alternate ndh-2, but not -1, was shown to be the electron donor to the electron transport chain and to be essential to replenish the [NAD(+)] pool in hypoxic nonreplicating Mtb. Finally, we describe here the high bactericidal activity of the F(0)F(1) ATP synthase inhibitor R207910 on hypoxic nonreplicating bacteria, supporting the potential of this drug candidate for shortening the time of tuberculosis therapy.