The purpose of this work was to explain how the caries-preventive agent xylitol interferes with the growth of Streptococcus mutans. It was found that the xylitol-sensitive strain of S. mutans 27352 (serotype g) and LG1 (serotype c) took up ¹⁴C-xylitol when the labelled pentitol was added to cells growing at the expense of glucose. Uptake of xylitol by growing cells of S. mutans 27352 X^R and LG1 X^R, two xylitol-insensitive spontaneous mutants, and of S. mutans GS5-2, which was also insensitive to xylitol, was practically inexistent under the same conditions. Alkaline phosphatase treatment followed by enzymatic analysis and thin-layer chromatography revealed that the accumulated product was xylitol phosphate. Intracellular concentrations of 5–7 mM for resting cells and of up to 60 mM for growing cells were calculated. Xylitol was phosphorylated at the expense of phosphoenolpyruvate by toluenized cells of S. mutans LG1, but not by toluenized cells of GS5–2 and S. mutans LG1 X^R. The phosphorylation of xylitol was dependent on phosphoenolpyruvate and required the presence of both soluble and membrane cellular fractions in the reaction mixture. This indicated that xylitol was transported and phosphorylated by a phosphoenolpyruvate: sugar phosphotransferase system. The phosphoenolpyruvate-dependent phosphorylation by isolated membranes of S. mutans LG1 in the presence of the soluble fraction was inhibited by fructose but not by glucose, mannose or galactose. Measurement of phosphoenolpyruvate: phosphotransferase activities in isolated membrane revealed that strain 27352 and LG1 had activities for fructose and xylitol; membrane from 27352 X^R and LG1 X^R had very little activity for fructose and xylitol. It was concluded that xylitol was transported and phosphorylated by a constitutive phosphoenolpyruvate:fructose phosphotransferase system in S. mutans. The data suggested that xylitol toxicity in S. mutans is caused by the intracellular accumulation of xylitol phosphate.

The effect of xylitol on the growth of Streptococcus mutans LG-1 was investigated under various conditions. Concentrations of xylitol ranging from 0.5 to 2% increased the time usually needed by the cells to reach the stationary phase in the presence of 0.2% glucose, mannose, lactose, mannitol, or sorbitol. Xylitol had no effect in the presence of fructose or sucrose. The xylitol-mediated inhibition was not modified by temperature or pH variations or by the presence or absence of oxygen. Repeated culturing in the presence of xylitol plus one of the above-mentioned sugars enabled the bacterium to tolerate the presence of xylitol. The cells, however, were still unable to grow at the expense of xylitol. The results indicate that this adaptive process arose from a mutational event.

The physiological and biochemical characterization of Streptococcus salivarius mutants isolated by positive selection for resistance to 0.5 mM 2-deoxyglucose in the presence of lactose are reported. We found 2 classes of mutants following a series of experiments that included: growth rate determinations, uptake studies, measurement of phosphotransferase system (PTS) activities and detection of the IIIman proteins by Western blotting and analysis of [32P]PEP-phosphorylated proteins. Class 1 mutants did not possess the low-molecular-weight form of IIIman. They did not grow on mannose and were unable to transport 2-deoxyglucose. On the other hand, class 2 mutants possessed the 2 forms of IIIman, grew readily on mannose and transported 2-deoxyglucose, albeit at a lower rate than the parental strain. Both classes of mutants exhibited abnormal growth in media containing mixtures of sugars. Moreover, derepression of genes coding for catabolic enzymes was observed in all the mutant strains. Our data suggested that the role of the mannose PTS in the control of sugar utilization in S. salivarius is complex and may involve the participation of several components.

A double-spontaneous mutant resistant to the growth inhibitory effect of alpha-methylglucoside and 2-deoxyglucose was isolated from Streptococcus salivarius. This mutant strain, called alpha S3L11, did not grow on mannose and grew poorly on 5 mM fructose and 5 mM glucose. Isolated membranes of strain alpha S3L11 were unable to catalyse the phosphoenolpyruvate-dependent phosphorylation of mannose in the presence of purified enzyme I and HPr. Addition of dialysed membrane-free cellular extract of the wild-type strain to the reaction medium restored the activity. The factor that restored the phosphoenolpyruvate-mannose phosphotransferase activity to membranes of strain alpha S3L11 was called IIIman. This factor was partially purified from the wild-type strain by DEAE-cellulose chromatography, DEAE-TSK chromatography, and molecular seiving on a column of Ultrogel AcA 34. This partially purified preparation also enhanced the phosphoenolpyruvate-dependent phosphorylation of glucose, fructose, and 2-deoxyglucose in strain alpha S3L11.

Fructose transport in Streptococcus mutans LG-1 is mediated by at least two distinct phosphoenolpyruvate fructose phosphotransferase systems. One system is constitutive and consists of membrane components enzyme II as well as enzyme I and heat-stable protein. The other system is inducible and requires, in addition to enzyme I and heat-stable protein, a soluble substrate-specific protein for catalytic activity. This protein factor, designated IIIfru, was purified by DEAE-cellulose chromatography, hydroxylapatite chromatography, molecular sieving on Sephadex G-75, and preparative electrophoresis. The purified preparation showed only one protein band, with a molecular weight of 12,600, on sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, on gel electrophoresis with the discontinuous buffer Tris-glycine, and after electrofocusing in gel (pI congruent to 3.7). The molecular weight of the native protein determined by gel filtration at 4 degrees C was 51,000. Immunodiffusion experiments performed with immunoglobulins prepared against the purified IIIfru from S. mutans LG-1 suggested that other S. mutans strains possessed a IIIfru. No precipitin bands, however, were detected with extracts from S. salivarius, S. sanguis, S. lactis, S. faecalis, Staphylococcus aureus, Bacillus subtilis, Lactobacillus casei, and Escherichia coli.

We have used the toxic non-metabolizable glucose/mannose analogue 2-deoxyglucose to isolate a comprehensive collection of mutants of the phosphoenolpyruvate:sugar phosphotransferase system from Streptococcus salivarius. To increase the range of possible mutations, we isolated spontaneous mutants on different media containing 2-deoxyglucose and various metabolizable sugars, either lactose, melibiose, galactose or fructose. We found that the frequency at which 2-deoxyglucose-resistant mutants were isolated varied according to the growth substrate. The highest frequency was obtained with the combination galactose and 2-deoxyglucose and was 15-fold higher than the rate observed with the mixture melibiose and 2-deoxyglucose, the combination that gave the lowest frequency. By combining results from: (i) Western blot analysis of IIIMan, a specific component of the phosphoenolpyruvate:mannose phosphotransferase system in S. salivarius; (ii) rocket immunoelectrophoresis of HPr and EI, the two general energy-coupling proteins of the phosphotransferase system; and (iii) from gene sequencing, mutants could be assigned to seven classes.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth of Streptococcus mutans Ingbritt in continuous culture (pH 7.0, dilution rate of 0.1 h-') at medium glucose concentrations above 2.6 mM resulted in repression of the sugar-specific membrane components, enzyme 1IGIc (EIIGic) and EIIMan, of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). In one experiment, significant repression (27-fold) was observed with 73 mM glucose when the glycolytic capacity of the cells was reduced by only 2-fold and when the culture was still glucose limited. In a more comprehensive experiment in which cells were grown in continuous culture at eight glucose concentrations from 2.6 to 304 mM, in addition to repression of specific EII activities for glucose, mannose, 2-deoxyglucose, and fructose, synthesis of the general protein, EI, was repressed at all glucose levels above 2.6 mM to a maximum of 4-fold at 304 mM glucose when the culture was growing with excess glucose (i.e., nitrogen limited). The other PTS general protein, HPr, was less sensitive to the exogenous glucose level but was nevertheless repressed fourfold under glucose-excess conditions. The Km for glucose for EIIGIC increased from 0.22 mM during growth at 3.6 mM glucose (glucose limited) to 0.48 mM at 271 mM glucose (glucose excess). The shift from heterofermentation to homofermentation during growth with increasing glucose levels suggests the involvement of glycolytic intermediates, ATP, or another high-energy phosphate metabolite in regulation of the synthesis of the PTS components in S. mutans.Streptococcus mutans, one of the principal etiological agents in dental caries (17), transports a variety of sugars by the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) (2,9,12,24,25). The PTS in oral streptococci, like many other bacteria (20,23), is a group translocation process that utilizes PEP as an energy source and substrate and catalyzes the sequential phosphorylation of the general, non-sugar-specific proteins enzyme I (El) and HPr, which are required for the uptake of all PTS sugars. In many cases, phospho-HPr (HPr-P), generated from phospho-EI, transfers the phosphoryl group directly to carbohydrate-specific, membrane-bound enzyme II (EII), which in turn phosphorylates the incoming sugar. In other cases, HPr-P transfers the phosphoryl group to a third soluble protein, called enzyme III (EIII), before interaction with EIl (7,15,16,27,(30)(31)(32)(33)35). Current evidence suggests that the PTS in the oral streptococci is supplemented by additional transport activity coupled to proton motive force (9,13,14); however, data obtained with S. mutans Ingbritt have indicated that the glucose-PTS is the high-affinity system, whereas the proton motive force-associated transport activity is the low-affinity system (13).Continuous-culture studies with S. mutans Ingbritt have indicated that the growth environment regulates transport via the two systems (8, 9), since earlier experiments with decryptified cells had demonstrated that the glucose-PTS was repressed by growth at low pH (11), at high gro...

No abstract