A lanine dehydrogenase (EC 1.4.1.1; Ald) catalyzes the reversible oxidative deamination of L-alanine to pyruvate with the concomitant reduction of oxidized nicotinamide adenine dinucleotide (NAD ϩ ) to nicotinamide adenine dinucleotide phosphate (NADH). Its forward reaction appears to be necessary for the aerobic utilization of alanine as a nitrogen source in Mycobacterium tuberculosis, Mycobacterium smegmatis, and Mycobacterium bovis BCG (1-3). The reverse reaction of Ald was proposed to play a role in recycling NADH under respiration-inhibitory conditions, such as hypoxia, by oxidizing NADH to NAD ϩ (2, 4, 5). Ald proteins from M. tuberculosis and M. smegmatis have glyoxylate-reductive aminase activity, which converts glyoxylate to glycine, but do not catalyze the reverse reaction, in which glycine is converted to glyoxylate by oxidative deamination (3, 6). The quaternary structure of Ald is a homohexamer consisting of three dimers, and each subunit is composed of an N-terminal catalytic domain and C-terminal NADH (NAD ϩ ) binding domain (7,8). It was reported that expression of the ald genes encoding Ald was upregulated in M. tuberculosis under nutrient starvation and energy-limiting conditions, as well as in Mycobacterium marinum during long-term granulomatous infection in its host (5, 9, 10). The synthesis and activity of Ald as well as expression of ald were shown to be induced when M. tuberculosis and M. smegmatis were shifted from aerobic to hypoxic growth conditions (4, 6, 11-13). Furthermore, the addition of alanine to aerobic cultures of M. tuberculosis and M. smegmatis led to a strong induction of ald expression (2, 3). Although the induction conditions of the ald gene were well known, the regulatory mechanism, which underlies upregulation of the gene under the different conditions mentioned above, remained unsolved. Here, we report that expression of the ald gene of M. smegmatis is under the control of its upstream gene product (AldR), which acts as both activator and repressor depending on the presence or absence of alanine, and that the hypoxic induction of ald is a result of increased levels of alanine in M. smegmatis cells grown under hypoxic conditions. MATERIALS AND METHODSBacterial strains, plasmids, and culture conditions. The bacterial strains and plasmids used in this study are listed in Table 1. M. smegmatis strains were grown in Middlebrook 7H9 medium (Difco, Sparks, MD) supplemented with 0.2% (wt/vol) glucose as a carbon source and 0.02% (vol/ vol) Tween 80 as an anticlumping agent at 37°C. M. smegmatis strains were grown aerobically or hypoxically as described previously (14). For various stress conditions, except hypoxic conditions, M. smegmatis strains were grown to an optical density at 600 nm (OD 600 ) of 0.5 to 0.6 on a gyratory shaker (200 rpm). Following the addition of chemicals to the cultures, the strains were further grown for 1 h. The treatment concentrations of the chemicals were 25 mM L-amino acids, 5 mM diamide, 15 mM hydrogen peroxide, and 5 mM sodium nitroprusside (SNP...