Regulation of the ald Gene Encoding Alanine Dehydrogenase by AldR in Mycobacterium smegmatis

Abstract

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...

Regulation Mechanism of the ald Gene Encoding Alanine Dehydrogenase in Mycobacterium smegmatis and Mycobacterium tuberculosis by the Lrp/AsnC Family Regulator AldR

“…Other studies reported that expression of the ald gene was upregulated in Mycobacterium marinum during persistence within its host granulomas and in M. tuberculosis under nutrient starvation conditions (5, 15). Expression of the ald gene was demonstrated to be strongly induced in M. smegmatis and M. tuberculosis grown in the presence of alanine (2,3,14).…”

“…Other studies reported that expression of the ald gene was upregulated in Mycobacterium marinum during persistence within its host granulomas and in M. tuberculosis under nutrient starvation conditions (5, 15). Expression of the ald gene was demonstrated to be strongly induced in M. smegmatis and M. tuberculosis grown in the presence of alanine (2,3,14).Expression of the ald gene in M. smegmatis is under the control of the AldR transcriptional regulator that belongs to the Lrp/AsnC (leucine-responsive regulatory protein/asparagine synthase C) family. AldR is a direct sensor of alanine and serves as both an activator of ald expression in the presence of alanine and a repressor in the absence of alanine (14).…”

“…The enzyme forms a hexamer of identical subunits with the N-terminal catalytic domain and the C-terminal NAD(H)-binding domain (6, 7). Ald was shown to be one of the major antigens found in culture filtrates of Mycobacterium tuberculosis (8,9).Expression of the ald gene as well as the synthesis and activity of Ald were observed to be increased under oxygen-limiting conditions in M. tuberculosis and Mycobacterium smegmatis (2)(3)(4)(10)(11)(12)(13)(14). Other studies reported that expression of the ald gene was upregulated in Mycobacterium marinum during persistence within its host granulomas and in M. tuberculosis under nutrient starvation conditions (5, 15).…”

“…Expression of the ald gene was demonstrated to be strongly induced in M. smegmatis and M. tuberculosis grown in the presence of alanine (2,3,14).…”

Cloning, overexpression, purification and preliminary X-ray analysis of a feast/famine regulatory protein (Rv2779c) fromMycobacterium tuberculosisH37Rv

“…Incidentally, the latter is listed in one study as among the top three targets against persistence (Hasan et al, 2006). Recently, it has been reported that in a hypoxic model of M. smegmatis the expression of alanine dehydrogenase increases and is regulated by aldR (Jeong et al, 2013), a gene whose product is homologous to the current protein. We have purified and crystallized the protein in order to understand the molecular mechanisms that are fundamental to its regulatory activity.…”