Effects of fluoride on carbohydrate metabolism by washed cells of Streptococcus mutans grown at various pH values in a chemostat.
Streptococcus mutans Ingbritt was grown anaerobically in a chemostat, at a rate (mean generation time, 13 h) similar to that in dental plaque, in a complex medium with excess glucose and at pH values of 6.5, 6.0, and 5.5. The yield of cells was constant at pH 6.5 and 6.0 (2.00 mg/ml) but fell to 1.25 at pH 5.5; Y(glucose) was relatively constant under all conditions. Lactic acid was the major end product. Amino acid analysis of the culture supernatants indicated that growth was probably limited by the availability of cysteine. Cells were harvested and monitored for their capacity to produce acid from endogenous polysaccharide and exogenous sugars in the presence and absence of NaF, as well as for their glucose phosphoenolpyruvate (PEP)-phosphotransferase activity. Surprisingly, cells grown at pH 5.5 possessed two to three times more glycolytic activity, as measured by the rate of acid production, than cells grown at pH 6.5 and 6.0 when incubated in a washed suspension at constant pH with a sugar source. Furthermore, the cells grown at pH 5.5 were about twice as resistant to the effect of NaF in reducing the rate of acid production in this system. Fluoride inhibition could be reversed by increasing the pH of the system. Cells grown at all three pH values showed significant acid production from endogenous reserves, despite the fact that the glucoamylase-specific glycogen content of the cells dropped from 33% of the total carbohydrate during pH 6.5 growth to only 3% after growth at pH 6.0 and 6.5. Incubation of washed cells for 18 h in phosphate buffer resulted in the loss of 62% of the total carbohydrate, indicating that nonglycogen cellular polysaccharide was metabolized. A comparison of the fluoride effect on endogenous and exogenous metabolism under pH fall conditions showed that, with pH 6.5- and 6.0-grown cells, the inhibitor was more effective in the presence of an exogenous carbon source than in its absence. This effect was not seen with pH 5.5-grown cells. The decreased sensitivity of the pH 5.5-grown cells to fluoride was probably associated with the decreased glucose PEP-phosphotransferase activity (11%) in these cells compared with the activity of those grown at pH 6.5. This evidence supports the hypothesis that S. mutans possesses at least two glucose transport systems, one of which is relatively fluoride insensitive.
Effects of fluoride on carbohydrate metabolism by washed cells of Streptococcus mutans grown at various pH values in a chemostat. Streptococcus mutans Ingbritt was grown anaerobically in a chemostat, at a rate (mean generation time, 13 h) similar to that in dental plaque, in a complex medium with excess glucose and at pH values of 6.5, 6.0, and 5.5. The yield of cells was constant at pH 6.5 and 6.0 (2.00 mg/ml) but fell to 1.25 at pH 5.5; Y(glucose) was relatively constant under all conditions. Lactic acid was the major end product. Amino acid analysis of the culture supernatants indicated that growth was probably limited by the availability of cysteine. Cells were harvested and monitored for their capacity to produce acid from endogenous polysaccharide and exogenous sugars in the presence and absence of NaF, as well as for their glucose phosphoenolpyruvate (PEP)-phosphotransferase activity. Surprisingly, cells grown at pH 5.5 possessed two to three times more glycolytic activity, as measured by the rate of acid production, than cells grown at pH 6.5 and 6.0 when incubated in a washed suspension at constant pH with a sugar source. Furthermore, the cells grown at pH 5.5 were about twice as resistant to the effect of NaF in reducing the rate of acid production in this system. Fluoride inhibition could be reversed by increasing the pH of the system. Cells grown at all three pH values showed significant acid production from endogenous reserves, despite the fact that the glucoamylase-specific glycogen content of the cells dropped from 33% of the total carbohydrate during pH 6.5 growth to only 3% after growth at pH 6.0 and 6.5. Incubation of washed cells for 18 h in phosphate buffer resulted in the loss of 62% of the total carbohydrate, indicating that nonglycogen cellular polysaccharide was metabolized. A comparison of the fluoride effect on endogenous and exogenous metabolism under pH fall conditions showed that, with pH 6.5- and 6.0-grown cells, the inhibitor was more effective in the presence of an exogenous carbon source than in its absence. This effect was not seen with pH 5.5-grown cells. The decreased sensitivity of the pH 5.5-grown cells to fluoride was probably associated with the decreased glucose PEP-phosphotransferase activity (11%) in these cells compared with the activity of those grown at pH 6.5. This evidence supports the hypothesis that S. mutans possesses at least two glucose transport systems, one of which is relatively fluoride insensitive.