The properties of citrate transport in membrane vesicles from Bacillus subtilis

The uptake system for citrate is induced in Bacillus subtilis W23 by growth in the presence of citrate and only membrane vesicles isolated from these cells show energy-dependent citrate uptake. Citrate transport in membrane vesicles is strictly dependent on the presence of divalent cations such as Mg2+, Mn2+, Zn2+, Ba2+, Be2+, Ca2+, Cu2+, Co2+ or Ni2+. The initial rate of citrate transport increases with the divalent cation concentration up to a maximum. The maximum initial rate of citrate uptake is reached with 2 mM Mg2+. The cations form stable chelates with citrate. The metal citrate complex is the transported solute. This is demonstrated for citrate uptake in the presence of Ca2+. Membrane vesicles from citrate-grown cells accumulate Ca2+ and citrate only if both solutes are present. Citrate and Ca2+ are accumulated in equimolar quantities. The uptake of Ca2+ but not of citrate is inhibited by Mg2+. Uptake of the metal-citrate complex is inhibited by the uncoupler carbonylcyanide p-trifluoromethoxyphenyl-hydrazone and in the presence of K+ ions by valinomycin and nigericin. The inhibitory effects correlate with the effects observed on the components of the proton-motive force, indicating that the proton-motive force is a driving force for metal-citrate transport. The number of protons (n) symported with the metal-citrate complex has been determined under different experimental conditions from the steady state levels of citrate accumulation, the electrical potential and pH gradient. This number varies from 1 at pH 4.7 to 2 at pH 8.0.