Mycobacterium smegmatis L-alanine dehydrogenase (Ald) is required for proficient utilization of alanine as a sole nitrogen source and sustained anaerobic growth.
ABSTRACT NAD(H)-dependent L-alanine dehydrogenase (EC 126.96.36.199) (Ald) catalyzes the oxidative deamination of L-alanine and the reductive amination of pyruvate. To assess the physiological role of Ald in Mycobacterium smegmatis, we cloned the ald gene, identified its promoter, determined the protein expression levels, and analyzed the combined effects of nutrient supplementation, oxygen availability, and growth stage on enzyme activity. High Ald activities were observed in cells grown in the presence of L- or D-alanine regardless of the oxygen availability and growth stage. In exponentially growing cells under aerobic conditions, supplementation with alanine resulted in a 25- to 50-fold increase in the enzyme activity. In the absence of alanine supplementation, 23-fold-higher Ald activities were observed in cells grown exponentially under anaerobic conditions. Furthermore, M. smegmatis ald null mutants were constructed by targeted disruption and were shown to lack any detectable Ald activity. In contrast, the glycine dehydrogenase (EC 188.8.131.52) (Gdh) activity in mutant cells remained at wild-type levels, indicating that another enzyme protein is responsible for the physiologically relevant reductive amination of glyoxylate. The ald mutants grew poorly in minimal medium with L-alanine as the sole nitrogen source, reaching a saturation density 100-fold less than that of the wild-type strain. Likewise, mutants grew to a saturation density 10-fold less than that of the wild-type strain under anaerobic conditions. In summary, the phenotypes displayed by the M. smegmatis ald mutants suggest that Ald plays an important role in both alanine utilization and anaerobic growth.
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ABSTRACT: In vitro cultures. To characterize nitrate reduction during aerobic growth and hypoxic shiftdown to non-replicating persistence of Mycobacterium tuberculosis cultures. The rates of reduction of nitrate to nitrite were measured in cultures of M. tuberculosis growing aerobically or undergoing hypoxic shiftdown. 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. 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.Tubercle and Lung Disease 02/1998; 79(2):127-32.