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Regulation of glucose metabolism in oral streptococci through independent pathways of glucose 6-phosphate and glucose 1-phosphate formation
No full textIn vivo rates of glucose uptake and acid production by oral streptococci grown in glucose- or nitrogen-limited continuous culture and batch culture were compared with the glucose phosphorylation activities of harvested, decryptified cells. The strains examined contained significant phosphoenolpyruvate-phosphotransferase system (PTS) activity, measured by a glucose 6-phosphate (G6P) dehydrogenase-linked assay procedure, but this activity was insufficient to account for the in vivo glucose uptake rates. However, ATP was a superior phosphoryl donor to phosphoenolpyruvate, and unlike the PTS, phosphoryl transfer with ATP was insensitive to bacteriostatic concentrations of chlorhexidine, suggesting glucokinase-mediated G6P formation. Again, G6P formation from the PTS and glucokinase reactions was not commensurate with some of the glucose uptake rates observed, implying that other phosphorylation reactions must be occurring. Two novel reactions involving carbamyl phosphate and acetyl phosphate were identified in some of the strains. No G6P formation was detected with these potential phosphoryl donors, but in the presence of phosphoglucomutase, glucose 1-phosphate (G1P) formation was evident, which was insensitive to chlorhexidine. G1P is a precursor of glycogen, and good correlation was obtained between G1P formation activity and endogenous metabolism of washed cells measured either as a rate of acid production at a constant pH 7 or as a decrease in pH with time in the absence of titrant. A 'league table' of abilities to synthesize G1P and produce acid from endogenous metabolism was complied for oral streptococci grown in batch culture. This indicated that Streptococcus mutans Ingbritt and Streptococcus sanguis Challis, were unable to form G1P or produce much acid endogenously, whereas increasing activities were obtained with Streptococcus salivarius, Streptococcus sanguis, and Streptococcus mitis. In particular, S. mitis had the highest G1P formation activities and was able to decrease the pH to less than 5 in 15 min by endogenuous metabolism alone. The data are consistent with the intracellular accumulation of free glucose driven by proton motive force when PTS activities are low and the subsequent phosphorylation to either G6P for metabolism via glycolysis or G1P for glycogen biosynthesis. The accumulation of acetyl phosphate during glucose-limited growth and the availability of arginine for catabolism to carbamyl phosphate provide an explanation as to why some glucose-limited oral streptococci continue to synthesize glycogen under these conditions, which might prevail in plaque.</p
Relationship of bioenergetic processes to the pathogenic properties of oral bacteria
No full textThe energized membrane has been shown to affect properties (sugar transport, acid production, intracellular polysaccharide formation, and glycosyltransferase secretion) related to the pathogenicity of oral bacteria. The activity of the energized membrane was susceptible to modulation by environmental conditions likely to be encountered by bacteria in dental plaque.</p
Inhibition by the antimicrobial agent chlorhexidine of acid production and sugar transport in oral streptococcal bacteria
No full textOral streptococci transport sugars via the phosphoenolpyruvate-phosphotransfer-ase (PEP-PTS) system. In a specific assay of this system, low concentrations of chlorhexidine abolished the activity of the glucose and sucrose PTS in batch-grown cells of Streptococcus mutans Irgbritt and B13, Strep. sanguis NCTC 7865, Strep. mitis ATCC 903, Strep. milleri NCTC 10709 and Strep. salivarius NCTC 8606. Intact cells and cells made permeable to the assay reagents with toluene were used. Toluenized cells were more sensitive to chlorhexidine than intact cells (0.09 and 0.25 mM, respectively). This PTS-inhibitory concentration of chlorhexidine reduced acid production from glucose in pH fall experiments to values higher than are obtained solely from endogenous metabolism. The effect of chlorhexidine on rates of acid production was determined at pH 7.0 using cells washed with either 135 mM NaCl or 135 mM KCl. In general, faster rates of acid production from the metabolism of glucose and sucrose were obtained with potassium-treated cells. Addition of the PTS-inhibitory concentration of chlorhexidine markedly reduced or totally abolished acid production by NaCl-treated cells; a greater residual-activity was detected in the same cells washed with KCl (except with Strep. mutans B13 and Strep. mitis ATCC 903). The PTS-inhibitory concentration of chlorhexidine had little or no effect on the viability of cells. The results confirm the existence of sugar uptake systems in oral streptococci additional to the PTS and provide an explanation for the additive anti-caries effect of mouth-rinses containing both fluoride and chlorhexidine.</p
Evidence that glucose and sucrose uptake in oral streptococcal bacteria involves independent phosphotransferase and proton-motive force-mediated mechanisms
No full textSugar transport and glycolysis in Streptococcus sanguis NCTC 7865, Streptococcus mitis ATCC 903, Streptococcus salivarius NCTC 8606 and several strains of Streptococcus mutans were investigated by following the rate of acid production by washed bacteria at a constant pH of 7.0. The phosphoenolpyruvate-phosphotransferase system (PTS) was inhibited by low concentrations of chlorhexidine. When this PTS-inhibitory concentration of chlorhexidine was added to cells washed and re-suspended in KCl, glucose uptake and glycolysis continued at a greatly-reduced rate. Chlorhexidine abolished glucose and sucrose uptake and metabolism in bacteria washed and incubated in saline. The Na+-inhibition was reproduced in KCl-washed bacteria using the cyclic peptide ionophores, valinomycin and gramicidin, to dissipate K+ and H+ gradients across the cell membrane. Glucose metabolism by Strep, mutans B13 was more resistant to chlorhexidine than that of Strep, mutans NCTC 10449 or Strep, sanguis but was more sensitive to the ionophores. Valinomycin had a greater inhibitory effect on strain B13 than the other two. That ion gradients are important in the chlorhexidine-resistant glucose-uptake mechanism was confirmed using the classical uncoupling agents, carbonylcyanide-m-chlorophenylhydrazone, 2,4-dinitrophenol and KSCN. Glucose metabolism was inhibited in the presence of both the uncouplers and the PTS-inhibitory concentration of chlorhexidine and significant inhibition was also observed in the absence of the PTS inhibitor. Lactate or the ATPase inhibitor, dicyclohexyl carbodiimide (DCCD), had similar inhibitory effects on the non-PTS uptake system. The results are consistent with the existence of an alternative (non-PTS) sugar-uptake system driven by proton-motive force (pmf) which is relatively insensitive to chlorhexidine but is inhibited by Na+, lactate, ionophores and uncouplers. The partial inhibition by DCCD in the absence of chlorhexidine indicates a role for ATP in the pmf-driven system but not in the PTS, and is probably required by the intracellular glucokinase for glucose phosphorylation. Comparison of published molar growth yields with theoretical yields predicted from the metabolic pathways in oral streptococci suggested that energy derived from substrate-level phosphorylation could not account for the high yields obtained but that pmf-driven processes could satisfy the discrepancies. Hexose uptake by oral streptococci is apparently driven by pmf when PTS activities are low. Such conditions are likely to occur in dental plaque.</p
Effect of environmental phosphate concentration on protein export by Bacillus licheniformis NCIB 6346
No full textProtonmotive force driven 6-deoxyglucose uptake by the oral pathogen, Streptococcus mutans Ingbritt
No full textStreptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (3H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (Δp) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference (ΔΨ) dissipator, KSCN. The membrane ATPase inhibitor, N,N1-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (ΔpH) and ΔΨ. ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to ΔpH.</p
Influence of sodium and potassium ions on acid production by washed cells of Streptococcus mutans Ingbritt and Streptococcus sanguis NCTC 7865 grown in a chemostat
No full textA comparison was made of acid production by cells of Streptococcus mutans Ingbritt and S. sanguis NCTC 7865 that had been washed twice and incubated in different concentrations of sodium and potassium ions. Organisms were grown under defined conditions in a chemostat under both glucose limitation and glucose excess conditions at a dilution rate of 0.1 h-1 (mean generation time, 6.9 h). Acid production after a pulse of glucose, sucrose, and fructose was measured by pH fall experiments and as a rate at pH 7.0. S. mutans produced more acid than S. sanguis as measured by either criterion, although statistically faster rates of acid production and lower terminal pH values were obtained when cells of both species were suspended in KCl rather than in NaCl, with 200 mM KCl resulting in the lowest terminal pH in pH fall experiments. Sodium ions inhibited acid production: 183 mM NaCl reduced the glycolytic rates of S. mutans and S. sanguis metabolizing glucose at pH 7.0 in 135 mM KCl by 39 and 33%, respectively. The most pronounced stimulatory effect of potassium on acid production was by washed cells of S. sanguis that had been grown under arginine and under phosphate limitation. The pH fell by a further 0.86 and 1.21 pH units, respectively, and to below the critical pH for enamel demineralization when these cells were metabolizing glucose in 135 mM KCl compared with the same concentration of NaCl. This enhancement of acid production was not due to potassium translocation, as had been suggested previously, because no movement of potassium ions across the cell membrane could be detected. An alternative explanation is proposed in which sodium ions are excluded from the cell at the expense of membrane energy, i.e., the proton motive force, which could otherwise be used for the transport of sugars.</p
Environmental regulation of carbohydrate metabolism by Streptococcus sanguis NCTC 7865 grown in a chemostat
No full textCarbohydrate metabolism by the oral bacterium Streptococcus sanguis NCTC 7865 was studied using cells grown in a chemostat at pH 7·0 under glucose or amino acid limitation (glucose excess) over a range of growth rates (D = 0·05 h-1-0·4 h-1). A mixed pattern of fermentation products was always produced although higher concentrations of lactate were formed under amino acid limitation. Analysis of culture filtrates showed that arginine was depleted from the medium under all conditions of growth; a further supplement of 10 mm-arginine was also consumed but did not affect cell yields, suggesting that it was not limiting growth. Except at the slowest growth rate (D = 0·05 h-1) under glucose limitation, the activity of the glucose phosphotransferase (PTS) system was insufficient to account for the glucose consumed during growth, emphasizing the importance of an alternative method of hexose transport in the metabolism of oral streptococci. The PTS for a number of sugars was constitutive in S. sanguis NCTC 7865 and, even though the cells were grown in the presence of glucose, the activity of the sucrose-PTS was highest. The glycolytic activity of cells harvested from the chemostat was affected by the substrate, the pH of the environment, and their original conditions of growth. Glucose-limited cells produced more acid than those grown under conditions of glucose excess; at slow growth rates, in particular, greater activities were obtained with sucrose compared with glucose or fructose. Maximum rates of glycolytic activity were obtained at pH 8·0 (except for cells grown at D = 0·4 h-1 where values were highest at pH 7·0), while slow-growing, amino acid-limited cells could not metabolize at pH 5·0. These results are discussed in terms of their possible significance in the ecology of dental plaque and the possible involvement of these bacteria in the initiation but not the clinical progression of a carious lesion.</p
The effect of chlorhexidine on the phosphoenolpyruvate-phosphotransferase (PEP-PTS) sugar uptake system and on the rate of acid production of oral streptococci
No full textAdditive inhibitory effects of combinations of fluoride and chlorhexidine on acid production by Streptococcus mutans and Streptococcus sanguis
No full textThe effect of 0.07 or 0.15 mMchlorhexidine (CHX) and 4.0 or 8.0 mM potassium fluoride (F), added singly and in combinations, on acid production by Streptococcus mutans and Streptococcus sanguis was studied. Cells were grown in a chemostat under different environmental conditions and acid production from glucose or sucrose was measured as a rate at pH 7.0 and by pH-fall experiments. CHX had a greater inhibitory action on S. mutans while S. sanguis was more sensitive to F. Growth conditions affected the sensitivity of both strains to the two inhibitors and, in general, cells grown glucose-limited were the most sensitive. Combinations of F and CHX showed additive inhibitory effects on acid production, irrespective of the method of measurement.</p
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