Inducible Transport of Citrate in a Gram-positive Bacterium, Bacillus subtilis

Abstract

Abstract An inducible system for the active transport of citrate in Bacillus subtilis has been studied. Gratuitous conditions are used under which the addition of citrate to the medium results in an increased uptake of citrate into the bacteria without influencing the growth rate. Wild type cells of B. subtilis metabolize citrate quickly after it is taken up into the cells, thereby rendering transport kinetics difficult to interpret in quantitative terms. To avoid this difficulty, we investigated citrate transport in B. subtilis 60871 which carries a genetic block in aconitase activity. This strain can accumulate citrate inside the cells more than 100-fold over the citrate level in the external medium. Under these conditions citrate catabolism is insignificantly low. Citrate is transported in B. subtilis 60871 with a Vmax of 145 ± 25 µmoles per min per g, dry weight, of cells and an apparent Km of 2.3 ± 0.4 mm at 37°. These bacteria exhibit an apparent constitutivity for citrate uptake when compared with induced wild type cells. This phenomenon is probably due to endogenous induction caused by citrate accumulation as consequence of the aconitase block. Citrate uptake in B. subtilis is also regulated by catabolite repression. The steady state uptake rate of citrate into cells of the aconitase mutant previously loaded with citrate is about the same as the initial uptake rate into induced wild type cells. The outflow rate of citrate from cells of strain 60871 previously loaded with citrate to steady state was found to be only 22% of the initial uptake rate. High concentrations of citrate in the medium slightly stimulate the outflow rate. The citrate uptake system in B. subtilis is inhibited by sodium cyanide, dinitrophenol, iodoacetamide, and N-ethylmaleimide.