pH Regulation by Streptococcus mutans S.G. DASHPER and E.C. REYNOLDS Biochemistry and Molecular Biology Unit, School of Dental Science, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 711 Elizabeth Street, Melbourne, Victoria, 3000, Australia The intracellular pH (pH) optimum for glycolysis in Streptococcus mutans Ingbritt was determined to be 7.0 by use of the ionophore gramicidin for manipulation of pH1. Glycolytic activity decreased to zero as the pH was lowered from 7.0 to 5.0. In contrast, glycolysis had an extracellular pH (pH0) optimum of 6.0 with a much broader profile. The relative insensitivity of glycolysis to the lowering of pH0 was attributed to the ability of S. mutans to maintain a transmembrane pH gradient (ApH, inside more alkaline) at low pH0. At a pH0 of 5.0, glycolyzing cells of S. mutans maintained a ApH of 1.37 ± 0.09 units. The maintenance ofthis ApH was dependent on the concentration of potassium ions in the extracellular medium. Potassium was rapidly taken up by glycolyzing cells ofS. mutans at a rate of 70 nmol/mg dry weight/min. This uptake was dependent onthe presence ofbothATP and a proton motive-force (Ap). The addition of N-N'dicyclohexylcarbodiimide (DCCD) to glycolyzing cells of S. mutans caused a partial collapse ofthe ApH. Growth ofS. mutans at pH. 5.5 in continuous culture resulted in the maintenance of a ApH larger than that produced by cells grown at pHO 7.0. These results suggest the presence of a proton-translocating F1F0-ATPase in S. mutans whose activity is regulated by the intracellular pH and transmembrane electrical potential (Ai). The production of an artificial Ap of 124 mV across the cell membrane of S. mutans did not result in proton movement through the F1F0-ATPase coupled to ATP synthesis. This suggests that ATP synthesis is not driven by the low Ap values obtained under physiological conditions in S. mutans. Therefore, the major role of the F1F0-ATPase is likely to be in pH. regulation. The extracellular concentration of sodium had no effect on ApH maintenance in glycolyzing cells, whereas the addition of 500 jtmol/L fluoride caused a significant fall in pHi. S. mutans was able to grow in the absence of a Ap, indicating that the transmembrane circulation of protons is not obligatory for growth ofthis organism. J Dent Res 71(5):1159-1165, May, 1992
Introduction. The oral bacterium Streptococcus mutans and dietary sugar have been implicated in the development of dental caries (Hamada and Slade, 1980; Loesche, 1986). Acidogenicity and acidurance, the ability to generate acid and to function at low pH, appear to be the main physiological traits associated with the cariogenicity of this organism (Loesche, 1986). A narrow range of pH values around neutralitylimits the activity ofmanyproteins (Padan and Schuldiner, 1986), and at least one of the glycolytic enzymes (enolase) of S. mutans has been demonstrated to be extremely pH-sensitive (Bunick and Kashket, 1981). In order for glycolytic activity and energy production to be maximized, S. mutans must therefore be able to maintain a transmembrane pH gradient (ApH) with its intracellular pH nearer neutrality when the extracellular pH is low. Kobayashi (1985) concluded that a proton-translocatingATPase (F1Fo-ATPase) was responsible for the maintenance of a ApH in
Enterococcus faecalis. Bender et al. (1986) have demonstrated the presence of an ATPase in the membrane fraction ofS. mutans which may be involved in pH regulation. The activity of the ATPase increased four-fold as the pH of the growth medium was lowered from 8.0 to 5.0 (Hamilton and Buckley, 1991). Potassium movement into the cell has also been shown to be important for ApH maintenance in bacteria (Harold et al., 1970; Booth, 1985; Bakker, 1990). The formation of a large ApH in S. mutans is dependent on the presence of potassium in the medium (Nojietal., 1988).
Luoma
andTuompo (1975) have linked potassium
movement to metabolism and carbohydrate uptake in S. mutans. The aim of the present study was to investigate the extent to which S. mutans Ingbritt was able to regulate its intracellular pH, especially at the low pH values associated with caries initiation.
The mechanisms of this intracellular pH regulation were investigated, as were the effects of intracellular and extracellular pH on glycolysis. The effect of fluoride on intracellular pH and cellular metabolism was also studied.
Materials and methods. Bacterial strains and culture conditions.-Streptococcus mutans Ingbritt (Krasse, 1966; Linzer, 1976) was kindly supplied by B. Krasse (Karolinska Institute, Stockholm). Streptococcus sanguis NCTC 7863 was obtained from the culture collection of the Department of Preventive and Community Dentistry, University of Melbourne, Parkville. The bacteria were stored as lyophilized cultures in sealed ampoules or in 30% glycerol broth at -200C. The strains were checked regularly according to the criteria of Hardie and Bowden (1976). Bacteria were grown in batch culture at 370C in TYE growth medium and harvested at mid-exponential phase, as described previously (Dashper and Reynolds, 1990). For growth studies in the presence of the ionophore gramicidin D (Sigma Chemical Co., St. Louis, MO), the concentration of potassium in the medium was increased to 200 mmol/L by the addition of extra KCl. Gramicidin was added as a 10 mmol/L solution in 95% ethanol to give a final concentration of 10 pmol'L. A control had an equivalent amount of ethanol added, which had no effect on growth. Continuous cultures were grown as previously described (Dashper and Reynolds, 1990). The temperature was maintained at 37TC and the pH controlled at 7.0 or 5.5 as specified, by the automatic addition of 5 molIL KOH. Cultures were gassed with 5% CO2 in nitrogen. The dilution rate was set at D = 0.1 h-1, which is equivalent to a mean generation time of 6.9 h, and cultures were allowed to grow for ten generations before being sampled. Culture purity was checked regularly by microscopic examination and by the criteria of Hardie and Bowden (1976). Chemostat cells were collected from the overflow at 40C for less than one hour. Chemostat- and batch-grown cells were harvested by centrifugation at 1000 g for 15 min at 4VC.
Measurement ofglycolytic activity.-Batch- or chemostat-grown cells were washed twice with fermentation minimal medium (FMM, Received for publication August 14, 1991 Dashper and Reynolds, 1990) and then suspended in FMM to give Accepted for publication November 26, 1991 a cell density of 1.25 mg dry weight cells/mL. Glycolytic activity was Abbreviations: CCCP, carbonyl cyanide chlorophenylhydrazone; F1FO- determined as described previously (Dashper and Reynolds, 1990). ATPase; DCCD, bacterial membrane-bound, proton-translocating ATPase, was maintained at the desired pH by the The cell suspension intracelluN-N-dicyclohexylcarbodiimide; PEP, phosphoenolpyruvate; pH, titrant (usually 0.1 mol/L KOH). standard of a volumetric addition lar pH; pH,, extracellular pH; Ap, transmembrane electrochemical proton potential or proton motive force; PTS, phosphotransferase system; Ai, In cells glycolyzing at pH values below the pre-incubation pH of 7.0, transmembrane electrical potential; and ApH, transmembrane pH gradient. the sugar solution was added at pH 7.0, and the cells were allowed Downloaded from jdr.sagepub.com at MICHIGAN STATE UNIV LIBRARIES on February 13, 2015 For personal use only. No other uses without permission.
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Introduction. The oral bacterium Streptococcus mutans and dietary sugar have been implicated in the development of dental caries (Hamada and Slade, 1980; Loesche, 1986). Acidogenicity and acidurance, the ability to generate acid and to function at low pH, appear to be the main physiological traits associated with the cariogenicity of this organism (Loesche, 1986). A narrow range of pH values around neutralitylimits the activity ofmanyproteins (Padan and Schuldiner, 1986), and at least one of the glycolytic enzymes (enolase) of S. mutans has been demonstrated to be extremely pH-sensitive (Bunick and Kashket, 1981). In order for glycolytic activity and energy production to be maximized, S. mutans must therefore be able to maintain a transmembrane pH gradient (ApH) with its intracellular pH nearer neutrality when the extracellular pH is low. Kobayashi (1985) concluded that a proton-translocatingATPase (F1Fo-ATPase) was responsible for the maintenance of a ApH in
Enterococcus faecalis. Bender et al. (1986) have demonstrated the presence of an ATPase in the membrane fraction ofS. mutans which may be involved in pH regulation. The activity of the ATPase increased four-fold as the pH of the growth medium was lowered from 8.0 to 5.0 (Hamilton and Buckley, 1991). Potassium movement into the cell has also been shown to be important for ApH maintenance in bacteria (Harold et al., 1970; Booth, 1985; Bakker, 1990). The formation of a large ApH in S. mutans is dependent on the presence of potassium in the medium (Nojietal., 1988).
Luoma
andTuompo (1975) have linked potassium
movement to metabolism and carbohydrate uptake in S. mutans. The aim of the present study was to investigate the extent to which S. mutans Ingbritt was able to regulate its intracellular pH, especially at the low pH values associated with caries initiation.
The mechanisms of this intracellular pH regulation were investigated, as were the effects of intracellular and extracellular pH on glycolysis. The effect of fluoride on intracellular pH and cellular metabolism was also studied.
Materials and methods. Bacterial strains and culture conditions.-Streptococcus mutans Ingbritt (Krasse, 1966; Linzer, 1976) was kindly supplied by B. Krasse (Karolinska Institute, Stockholm). Streptococcus sanguis NCTC 7863 was obtained from the culture collection of the Department of Preventive and Community Dentistry, University of Melbourne, Parkville. The bacteria were stored as lyophilized cultures in sealed ampoules or in 30% glycerol broth at -200C. The strains were checked regularly according to the criteria of Hardie and Bowden (1976). Bacteria were grown in batch culture at 370C in TYE growth medium and harvested at mid-exponential phase, as described previously (Dashper and Reynolds, 1990). For growth studies in the presence of the ionophore gramicidin D (Sigma Chemical Co., St. Louis, MO), the concentration of potassium in the medium was increased to 200 mmol/L by the addition of extra KCl. Gramicidin was added as a 10 mmol/L solution in 95% ethanol to give a final concentration of 10 pmol'L. A control had an equivalent amount of ethanol added, which had no effect on growth. Continuous cultures were grown as previously described (Dashper and Reynolds, 1990). The temperature was maintained at 37TC and the pH controlled at 7.0 or 5.5 as specified, by the automatic addition of 5 molIL KOH. Cultures were gassed with 5% CO2 in nitrogen. The dilution rate was set at D = 0.1 h-1, which is equivalent to a mean generation time of 6.9 h, and cultures were allowed to grow for ten generations before being sampled. Culture purity was checked regularly by microscopic examination and by the criteria of Hardie and Bowden (1976). Chemostat cells were collected from the overflow at 40C for less than one hour. Chemostat- and batch-grown cells were harvested by centrifugation at 1000 g for 15 min at 4VC.
Measurement ofglycolytic activity.-Batch- or chemostat-grown cells were washed twice with fermentation minimal medium (FMM, Received for publication August 14, 1991 Dashper and Reynolds, 1990) and then suspended in FMM to give Accepted for publication November 26, 1991 a cell density of 1.25 mg dry weight cells/mL. Glycolytic activity was Abbreviations: CCCP, carbonyl cyanide chlorophenylhydrazone; F1FO- determined as described previously (Dashper and Reynolds, 1990). ATPase; DCCD, bacterial membrane-bound, proton-translocating ATPase, was maintained at the desired pH by the The cell suspension intracelluN-N-dicyclohexylcarbodiimide; PEP, phosphoenolpyruvate; pH, titrant (usually 0.1 mol/L KOH). standard of a volumetric addition lar pH; pH,, extracellular pH; Ap, transmembrane electrochemical proton potential or proton motive force; PTS, phosphotransferase system; Ai, In cells glycolyzing at pH values below the pre-incubation pH of 7.0, transmembrane electrical potential; and ApH, transmembrane pH gradient. the sugar solution was added at pH 7.0, and the cells were allowed Downloaded from jdr.sagepub.com at MICHIGAN STATE UNIV LIBRARIES on February 13, 2015 For personal use only. No other uses without permission.
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