Regulation of Dextransucrase Secretion by Proton Motive Force in Leuconostoc Mesenteroides.

Abstract

The relationship between proton motive force and the secretion of the enzyme dextransucrase (E.C. 2.4.1.5) in Leuconostoc mesenteroides was investigated. The transmembrane pH gradient was determined by measurement of the uptake of radiolabeled benzoate or methylamine while the membrane potential was determined by measurement of the uptake of radiolabeled tetraphenylphosphonium bromide. Leuconostoc mesenteroides was able to maintain a constant proton motive force of $-130$ mV when grown in fermenters at constant pH while a value of $-140$ mV was determined from concentrated cell suspensions. The contribution of the membrane potential and transmembrane pH gradient to the proton motive force varied depending on the cation concentration and pH of the medium. The differential rate of dextransucrase secretion was relatively constant at 1040 $\Delta$mU/$\Delta$mg dry weight when cells were grown at pH 6.0 to pH 6.7. At this pH range, the cell interior was alkaline with respect to the growth medium. Above pH 7.0, dextransucrase secretion was severely inhibited. This inhibition was accompanied by an inversion of the pH gradient across the cytoplasmic membrane as the cell interior became acidic with respect to the growth medium. Treatment of cells with the lipophilic cation methyltriphenylphosphonium bromide had no effect on the rate of dextransucrase secretion at pH 5.5 but inhibited secretion by 95% at pH 7.0. The inhibition of secretion by methyltriphenylphosphonium bromide was correlated to the dissipation of the proton motive force by this compound. Values of proton motive force greater than $-90$ mV were required for maximal rates of dextransucrase secretion. The results of this study strongly indicate that dextransucrase secretion Leuconostoc mesenteroides is dependent on the presence of a proton gradient across the cytoplasmic membrane. The results further suggest that dextransucrase secretion is coupled to proton influx into the cell.