The time-course of acid excretion, levels of fluorescence dependent on cellular nicotinamide adenine nucleotide and glycolytic intermediates of Streptococcus mutans cells exposed and not exposed to air in the presence of glucose and sorbitol

The aim of this study was to examine glucose and sorbitol metabolism in Streptococcus mutans cells exposed and not exposed to air at the coexistence of these compounds by measuring acid excretion, levels of fluorescence dependent on cellular NADH and glycolytic intermediates. An aliquot of bacterial cells grown under strictly anaerobic conditions (anaerobic cells) was exposed temporarily to air (aerobic cells). When glucose was added to the anaerobic cells metabolizing sorbitol, the acid excretion was increased. The level of NADH decreased initially and then increased to the higher plateau level than that during glucose metabolism. The aerobic cells neither metabolized sorbitol nor contained glycolytic intermediates. However, 2 min after glucose was added in the presence of sorbitol, the acid excretion was started slowly and the intermediates appeared. The level of NADH was decreased at first and then increased. These results suggested that the anaerobic S. mutans cells metabolized glucose and sorbitol simultaneously, and that in the presence of sorbitol the aerobic cells could start to metabolize glucose 2 min after glucose was added, as the intermediates (phosphoenopyruvate potential) for the glucose transport were accumulated.     

Rate‐limiting steps of glucose and sorbitol metabolism in Streptococcus mutans cells exposed to air

It has been supposed that rate of sorbitol metabolism in the air‐exposed streptococcal cells could be limited by the low capacity to regenerate nicotinamide adenine dinucleotide (NAD) from reduced NAD (NADH) following inactivation of pyruvate formate‐lyase by oxygen. The rate‐limiting steps, however, have not been identified. The aim of this study was to examine the effect of temporary exposure of the streptococcal cells to air on the intracellular flux of glucose and sorbitol metabolism by measuring acid excretion, fluorescence dependent on cellular level of NADH, glycolytic intermediates and enzyme activities. The exposure of cells to air decreased the acid excretions during glucose and sorbitol metabolism. The analysis of the glycolytic intermediates and the fluorescence suggested that the reduced level of acid excretion in the air‐exposed glucose metabolizing cells resulted from the decrease in pyruvate catabolism. In the presence of sorbitol, the decreased acid production resulted from the reduced rates of the reactions catalyzed by sorbitol‐phosphoenolpyruvate phosphotransferase and sorbitol 6‐phosphate dehydrogenase because of shortage of substrates for these enzymes in addition to the decrease in pyruvate catabolism.     

Intracellular flux of glucose metabolism in streptococcal cells by simultaneous monitoring of fluorescence dependent on reduced nicotinamide adenine nucleotide and acid excretion under strictly anaerobic conditions

Reduced nicotinamide adenine nucleotide (NADH)-dependent fluorescence and acid excretion during glucose pulse to washed Streptococcus mutans cells were monitored simultaneously at pH 7.0 with a fluorescence spectrophotometer and a pH-stat. Acid excretion started with addition of glucose. At the same time, the fluorescence dropped quickly to a minimum level and increased to a plateau level, suggesting that pyruvate metabolism started immediately after addition of glucose, then the rate of the pyruvate metabolism became almost equal to the rate of glycolysis. When the acid excretion stopped, the fluorescence increased rapidly from the plateau to the maximum level, suggesting that the pyruvate metabolism stopped first, and then began to decrease to the original level. The system used in this study for simultaneously monitoring the level of NADH and acid excretion gives us a crucial tool to clarify a biochemical mechanism of the control of sugar metabolism by streptococci.     

Inhibitory effect of sorbitol on sugar metabolism of Streptococcus mutans in vitro and on acid production in dental plaque in vivo

This study was conducted to find out whether sorbitol inhibits the sugar metabolism of Streptococcus mutans in vitro and the acid production in dental plaque in vivo. S. mutans NCIB 11723 was anaerobically grown in sorbitol-containing medium. The rate of acid production from sugars was estimated with a pH stat. The rate of acid production from glucose or sucrose was not changed at various concentrations of oxygen. By the addition of sorbitol to sugar, however, the acid production was decreased with increasing levels of oxygen. Intracellular NADH/NAD+ ratio and (dihydroxyacetone-phosphate+glyceraldehyde-phosphate)/3-phosphoglycerate ratio were high whenever the acid production was inhibited by sorbitol. Sorbitol also inhibited the acid production in dental plaque in vivo. These results suggest that the increased NADH/NAD+ ratio during sorbitol metabolism through the inactivation of pyruvate formate-lyase by oxygen inhibited glyceraldehyde-phosphate dehydrogenase and then the acid production of S. mutans and the one in dental plaque.