Glycine and alanine dehydrogenase activities are catalyzed by the same protein inMycobacterium smegmatis: upregulation of both activities under microaerophilic adaptation

Microaerophilic adaptation has been described as one of the in vitro dormancy models for tuberculosis. Studies on Mycobacterium tuberculosis adapted to low oxygen levels showed an enhancement of glycine dehydrogenase (deaminating) activity. We studied the physiology of the fast-growing, nonpathogenic strain of Mycobacterium smegmatis ATCC 607 under low oxygen by shifting the actively growing M. smegmatis cells to static microaerophilic growth conditions. This shifting of M. smegmatis culture resulted in a similar phenomenon as seen with M. tuberculosis, i.e., elevated glycine dehydrogenase activity. Further purification of glycine dehydrogenase from M. smegmatis demonstrated glyoxylate amination, but failed to demonstrate glycine deamination, even in the purified fraction. Moreover, the purified protein showed pyruvate amination as well as L-alanine deamination activities. By activity staining, the protein band positive for glyoxylate amination demonstrated only pyruvate amination in the presence of NAD. Absence of glycine deamination activity strongly suggested that alanine dehydrogenase of M. smegmatis was responsible for glyoxylate amination in the cell lysate. This was further confirmed by demonstrating the similar level of upregulation of both glyoxylate and pyruvate amination activities in the cell lysate of the adapted culture.Key words: Mycobacterium smegmatis, glycine dehydrogenase, microaerophilic adaptation, alanine dehydrogenase.

Cerulenin inhibition of lipid synthesis and its reversal by exogenous fatty acids in Mycobacterium smegmatis ATCC 607

Cerulenin inhibited the lipid synthesis of Mycobacterium smegmatis ATCC 607 over the range of 0.5–1.8 μg/mL with complete inhibition at 1.8 μg/mL, as monitored by [14C]glycerol incorporation into lipids. Exogenous fatty acids failed to restore the lipid synthesis at 1.8 μg/mL; however, the addition of palmitic acid to the growth medium partially restored the lipid synthesis when cerulenin concentration was decreased to 1.6 μg/mL. Fatty acid analysis of cerulenin plus palmitic acid supplemented cultures revealed that exogenously supplied fatty acid was incorporated into cellular phospholipids. Further investigations with 1.6 μg/mL of cerulenin and [14C]acetate and [32P]orthophosphoric acid showed that cerulenin inhibited the synthesis of saturated plus unsaturated fatty acids and phospholipids. Pulse–chase studies with [14C]acetate revealed decreased synthesis and degradation of each of the phospholipid components.