Reduction of Acidurance of Streptococcal Growth and Glycolysis by Fluoride and Gramicidin

Abstract

The acidurance of glycolysis by intact cells of Streptococcus mutans GS-5, Streptococcus salivarius ATCC 25925, and Streptococcus sanguis NCTC 10904 was found to be highly dependent on membrane functions affected by gramicidin, which increases the proton permeability of cell membranes. Plots of % glucose utilized during two hours against suspension pH values for cells suspended in 100 mM phosphate buffer plus 1 mM MgCl ₂ plus 13.9 mM glucose indicated, for 50% glucose utilization, pH values of 5.0 for S. mutans, 5.7 for S. salivarius, and 6.2 for S. sanguis. Gramicidin treatment shifted these values to 6.0, 6.3, and 6.9, respectively.

Growth of S. mutans and S. salivarius in complex media proved to be more acid-sensitive than was glycolysis, and in batch cultures, there was a well-defined, post-growth phase of glycolysis. Minimum pH values for growth and for glycolysis in medium with excess glucose were approximately 4.8 and 4.4, respectively, for S. mutans, and 4.9 and 4.3 for S. salivarius. S. sanguis was less aciduric and showed little differential acid sensitivity, with minimum pH values of about 5.2 for both growth and glycolysis.

Fluoride acted to eliminate the differences in acidurance of growth and glycolysis for S. mutans or S. salivarius and to render both processes more acid-sensitive. Thus, glycolysis was more fluoride-sensitive than was growth. Growth was found to be acid-limited in media with initial glucose levels greater than 0.2, 0.3, and 0.5% (weight/volume) for S. sanguis, S. mutans, and S. salivarius, respectively, and to be glucose-limited at lower levels. When 1.0 mM NaF was added to the media, the acid-sensitizing effect of fluoride was evident in major shifts in the initial glucose levels for transition from glucose-limited to acid-limited growth to 0.1 and 0.2% for S. mutans and S. salivarius, respectively. S. sanguis was less severely affected by fluoride.

Fluoride treatment of glycolyzing cells of S. mutans at a pH value of 5 resulted in little change in intracellular levels of phosphoenolpyruvate but increased levels of 2-phosphoglycerate, indicative of inhibition of intracellular enolase. However, there were also large decreases in levels of early glycolytic intermediates, to about the levels found in starved cells, presumably due to fluoride inhibition of glucose uptake.