Malate dehydrogenase catalyzes the interconversion of malate and oxaloacetate. It participates as a member of the tricarboxylic acid cycle and the branched noncyclic pathways under aerobic and anaerobic cell growth conditions, respectively. To investigate how the mdh gene is expressed under these different conditions, an mdh-lacZ operon fusion was constructed and analyzed in vivo. The mdh-lacZ fusion was expressed about twofold higher under aerobic conditions than under anaerobic cell growth conditions on most media tested. This anaerobic response is modulated by the ArcA protein, which functions as a repressor of mdh gene expression under both aerobic and anaerobic conditions. In contrast, mutations in the fnr gene did not affect mdh gene expression. Interestingly, cells grown anaerobically with glycerol and trimethylamine N-oxide or fumarate showed higher levels of mdh expression than did cells that were grown aerobically. Depending on the type of carbon compound used for cell growth, mdh expression varied by 11-fold and 5-fold under aerobic and anaerobic conditions, respectively. While mdh transcription was shown to be inversely proportional to the cell growth rate, cellular heme limitation stimulated a fivefold increase in mdh gene expression. The mdh gene appears to be highly regulated to adapt to changing conditions of aerobic and anaerobic cell growth with various types of carbon substrates.Malate dehydrogenase (EC 1.1.1.37) (MDH) is an essential enzyme in the tricarboxylic acid (TCA) cycle as well as the noncyclic anaplerotic pathway of Escherichia coli (15). It catalyzes the interconversion of malate and oxaloacetate with a concomitant reduction of NAD or oxidation of NADH. The reaction carried out by MDH under aerobic conditions in which the TCA cycle is operative is from malate to oxaloacetate (i.e., oxidation); the reaction proceeds in the opposite direction for participation in the branched noncyclic pathway. This pathway is considered to operate predominantly under anaerobic cell growth conditions (15). However, the effects of different environmental conditions on the regulation of mdh gene expression in E. coli or in other microorganisms have not been examined.The mdh gene in E. coli, which encodes MDH, is located at 70.6 min on the chromosome (8), and it has recently been cloned and sequenced (11,22). The E. coli enzyme is a dimeric enzyme with a molecular weight of 60,000 (13), like the MDHs isolated from most other organisms (14). It has a remarkable homology to the mitochondrial MDH (e.g., 59% amino acid identity to porcine mitochondrial MDH), especially in the nucleotide binding site located near the amino terminus of the protein. The E. coli enzyme shows a lower degree of sequence similarity to the alternative eucaryotic MDH isozyme that is cytoplasmically located (ca. 20% similarity to the porcine cytoplasmic MDH). The level of the E. coli MDH enzyme in the cell is reduced by anaerobiosis and glucose (6, 21) and induced by acetate and malate (21). In addition, MDH activity is elevated in ar...