Nitrate reduction as a marker for hypoxic shiftdown of Mycobacterium tuberculosis.

SETTING In vitro cultures. OBJECTIVE To characterize nitrate reduction during aerobic growth and hypoxic shiftdown to non-replicating persistence of Mycobacterium tuberculosis cultures. DESIGN The rates of reduction of nitrate to nitrite were measured in cultures of M. tuberculosis growing aerobically or undergoing hypoxic shiftdown. RESULTS Tubercle bacilli growing aerobically in the presence of nitrate reduce nitrate at a rate proportional to the substrate concentration, continuing until the substrate is exhausted. When the bacilli in an oxygen restricted model enter microaerophilic non-replicating persistence (NRP) stage 1, they exhibit a marked increase in rate of nitrate reduction that is independent of substrate concentration, and terminates by feedback inhibition when the concentration of nitrite produced approaches 2.5 mM. When bacilli in the oxygen restricted model are not supplemented with nitrate until they enter microaerophilic NRP stage 1, they exhibit an induction period before the rapid nitrate reduction starts. When the nitrate is not added until the bacilli have entered the anaerobic NRP stage 2, reduction of the substrate starts immediately. Nitrite is not reduced by M. tuberculosis in any stage of its growth or NRP. CONCLUSION The hypoxically induced nitrate reduction probably serves a respiratory function in supporting hypoxic shiftdown of M. tuberculosis from aerobic growth to non-replication persistence and represents a useful new marker for monitoring that shiftdown. This response may help the bacilli survive in oxygen depleted regions of inflammatory or necrotic tissue, where nitrate can occur as a degradation product of nitric oxide.

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