Transport of Dicarboxylic Acids in Bacillus subtilis

The uptake of several dicarboxylic acids by Bacillus subtilis has been surveyed. Gratuitous conditions for induction of l-malate uptake were achieved under which the addition of l-malate to the medium resulted in increased uptake of l-malate by the cells without significantly influencing the growth rate. The specificity of induction of l-malate uptake was investigated using other dicarboxylate anions of the tricarboxylic acid cycle. Fumarate induced l-malate uptake with about ⅓ the efficiency of l-malate. No induction of l-malate uptake was observed in a succinic dehydrogenase (EC 1.3.99.1) negative mutant grown in the presence of succinate, although this strain could be fully induced for l-malate uptake by the addition of l-malate to the growth medium. l-Malate uptake in a B. subtilis mutant lacking malic dehydrogenase (EC 1.1.1.37) activity appeared constitutive, probably owing to endogenous induction caused by the accumulation of l-malate inside the cells. These data suggested that l-malate was the true inducer of the l-malate transport system, but confirmation of this notion must come from a system in which l-malate is strictly nonmetabolizable. The l-malate transport system could be catabolite repressed by d-glucose. l-Malate uptake followed simple Michaelis-Menten kinetics; its apparent Km was 0.4 mm and Vmax approached 400 µmoles per min per g dry weight of cells. The system was shown to be highly specific by using competitive inhibitors, of which l(+)citramalate, mesotartrate,3-fluoro-dl-malate, and d(+)-malate were most effective. 2,4-Dinitrophenol, azide, and classical uncouplers of oxidative phosphorylation strongly inhibited l-malate uptake in B. subtilis. The l-malate transport system was different from the previously reported citrate uptake system: the two systems could be separately induced. Furthermore, mutants deficient in citrate uptake largely retained the ability to be induced for l-malate uptake. Fumarate uptake in B. subtilis was inducible to fairly high activities but seemed dependent on the continual removal of fumarate via the tricarboxylic acid cycle in order to exhibit maximal uptake rates. The Km for the process was 0.7 mm. Similarly, α-ketoglutarate was transported by an inducible system; the process had a high Km (6.7 mm). We were unable to demonstrate induction of a system for succinate uptake, and saturation kinetics were not observed over the 50-fold concentration range tested (0.1 to 5.0 mm). dl-Tartrate uptake could be strongly inhibited by the addition of l-malate or l(+)tartrate to the medium during growth.