Inactivation of the Streptococcus mutans fxpC gene confers resistance to xylitol, a caries-preventive natural carbohydrate sweetener.

Xylitol is transported by Streptococcus mutans via a constitutive phosphoenolpyruvate:fructose phosphotransferase system (PTS) composed of a IIABC protein. Spontaneous xylitol-resistant strains are depleted in constitutive fructose-PTS activity, exhibit additional phenotypes, and are associated with the caries-preventive properties of xylitol. Polymerase chain-reactions and chromosome walking were used to clone the fxp operon that codes for the constitutive fructose/xylitol-PTS. The operon contained three open reading frames: fxpA, which coded for a putative regulatory protein of the deoxyribose repressor (DeoR) family, fxpB, which coded for a 1-phosphofructokinase, and fxpC, which coded for a IIABC protein of the fructose-PTS family. Northern blot analysis revealed that these genes were co-transcribed into a 4.4-kb mRNA even in the absence of fructose. Inactivation of the fxpC gene conferred resistance to xylitol, confirming its function. The fxp operon is also present in the genomes of other xylitol-sensitive streptococci, which could explain their sensitivity to xylitol.

Identification of mutans and other oral streptococci by random amplified polymorphic DNA analysis.

The identification and classification of the non-haemolytic or viridans group of streptococci have long been recognised as difficult and unsatisfactory. Phenotypic and genotypic heterogeneity have resulted in ambiguous speciation, particularly with mutans streptococci and other oral streptococci. This study was done to determine whether random amplified polymorphic DNA (RAPD) analysis is useful to identify and even classify oral and other streptococci. DNA was prepared and purified from 25 strains of mutans streptococci including 11 reference strains of Streptococcus mutans, seven of S. sobrinus, three of S. rattus and one each of the four other species of the mutans group, together with 20 other reference species, mostly streptococci, and from 49 fresh isolates of mutans streptococci and of S. mutans from human saliva and dental plaque. DNA amplification was primed with each of three arbitrarily selected primers nine or 10 nucleotides in length. The amplified DNA fragments (amplicons) obtained were compared by agarose gel electrophoresis. Species- and strain-specific RAPD fingerprints were obtained not only from pure genomic DNA, but also from the supernates of crude cellular or colony extracts. Pending the analysis of numerous other strains, the data suggest that RAPD may be of value: (i) to distinguish the species S. mutans and S. sobrinus from each other and potentially from other species of oral streptococci, (ii) to differentiate and possibly classify oral streptococci and (iii) as a valuable tool in mutans streptococci epidemiology and transmission studies, by virtue of its rapidity, efficiency and reproducibility in generating genetic fingerprints of streptococcal isolates.

Xylitol disturbs protein synthesis, including the expression of HSP-70 and HSP-60, in Streptococcus mutans.

Xylitol, a natural sugar alcohol and a caries-preventive carbohydrate sweetener, inhibits xylitol-sensitive wild-type Streptococcus mutans but also selects for its natural xylitol-resistant mutants. The aim of the work was to verify the influence of xylitol on heat shock proteins HSP-60 (GroEL-like) and HSP-70 (DnaK-like) in xylitol-sensitive and xylitol-resistant strains. Cells from fresh isolate S. mutans 123.1 were grown at 37 degrees C and constant pH 7.0. The cell culture was stressed by raising the temperature to 43 degrees C or adding xylitol (4% final). Cell proteins labeled with a cocktail of 14C-amino acids were analyzed by SDS-PAGE and autoradiography whereas HSP-60 and HSP-70 were visualized using Western immunoblotting. In both xylitol-sensitive and xylitol-resistant strains, heat stress was associated with an increase of both HSP-60 (63 kDa) and HSP-70 (71 kDa) and a decrease in the intensity of a number of other protein bands compared with cells maintained at 37 degrees C. Exposure to xylitol but not to other polyols induced a decrease of both these heat shock proteins in the xylitol-sensitive strain but did not modify them in the xylitol-resistant mutant. It also decreased all protein bands above 60 kDa together with a 53 kDa protein and increased the amount of 57-, 50- and 40-kDa proteins in the xylitol-sensitive strain whereas the proteins of the xylitol-resistant strain remained unchanged. The results suggest that xylitol is a strong metabolic inhibitor that disturbs protein synthesis and reduces the expression of HSP-70 and HSP-60 proteins in the wild-type xylitol-sensitive S. mutans but not in the xylitol-resistant natural mutant strain.

Growth of xylitol-resistant versus xylitol-sensitive Streptococcus mutans strains in saliva.

Five Streptococcus mutans pairs (serotype c S. mutans 10449 and four clinical isolates of S. mutans: 123.1, LG1, OMFA, T10B) were used to find out if the xylitol-resistant (XR) natural mutants of the corresponding xylitol-sensitive (XS) S. mutans parental strains differ in their growth patterns in saliva. The isogenic X natural mutants of the parental S. mutans strains were selected after sequential cultivations in the presence of xylitol and glucose. The XR/XS strains pairs were grown in individual and pooled glucose-supplemented filter-sterilized salivas (one to five sequential cultivations). The two salivas used represented subjects with good or poor support of the growth of S. mutans in vivo. Protease and peptidase activities were determined from the saliva growth media and cell suspensions. Salivary protein profiles were analyzed using SDS-PAGE and native IEF before and after the cultivations. The growth properties of the XR/XS S. mutans pairs were similar in both individual and pooled salivas. Sequential cultivation of all strains did not show any differences in growth patterns. XS strains were inhibited by the presence of xylitol (2% w/v) in pooled saliva, as shown for other glucose-supplemented media. Protease and peptidase activities of the XR/XS S. mutans pairs were low and of similar magnitude. Also, the general hydrolytic properties of most XR/XS S. mutans pairs appeared similar as judged by the small growth-induced changes in salivary protein profiles. In conclusion, saliva, the source of nutrients for salivary microorganisms in vivo, favored neither the XR nor the XS strains of S. mutans.

Effects of xylitol, xylitol-sorbitol, and placebo chewing gums on the plaque of habitual xylitol consumers.

Xylitol reduces plaque but the reduction mechanism is largely unknown. The main aim of the present study was to determine whether the xylitol-induced reduction in the amount of plaque and the number of mutans streptococci could be demonstrated in subjects with (presumably) high levels of xylitol-resistant (XR; not inhibited by xylitol) mutans streptococci acquired following previous xylitol consumptions. 37 healthy dental students participated in the double-blind study. All subjects had been uncontrolled, habitual consumers of xylitol-containing products for at least 1 yr before the study. A 1-month washout period was followed by a 2-week test period during which either xylitol, xylitol-sorbitol or unsweetened chewing gum base was chewed 3-5 x a day. Plaque and saliva samples were collected at baseline and at the 2-week point for determination of the amount of plaque, microbiological variables, and hydrolytic enzymes. Mixtures of xylitol and sorbitol seemed to perform equally well with respect to reduction in the amount of plaque but not the number of mutans streptococci. Thus, polyols were the active ingredients of chewing gums able to modulate the amount of plaque and its microbial composition. Xylitol reduced plaque with a mechanism which appeared not to be associated with the study-induced changes in the proportion (%) of mutans streptococci in plaque, the number of salivary mutans streptococci, the proportion of XR strains in plaque or saliva, or the hydrolytic enzyme activities of plaque.

Emergence of multiple xylitol-resistant (fructose PTS-) mutants from human isolates of mutans streptococci during growth on dietary sugars in the presence of xylitol.

The growth inhibition of mutans streptococci is one of the proposed mechanisms of action of xylitol, a caries-preventive natural carbohydrate sweetener. Xylitol is taken up and accumulated as non-metabolizable, toxic xylitol phosphate via a constitutive fructose PTS, and selects, during in vitro growth at the expense of glucose, for natural xylitol-resistant mutants that lack constitutive fructose PTS activity. Since long-term xylitol consumption leads to the emergence of xylitol-resistant mutans populations in humans in an oral environment containing sugars of dietary origin, we wanted to test the hypothesis that xylitol-resistant cells could be selected from mutans streptococci strains during in vitro growth on fructose, sucrose, or lactose. Three laboratory strains and three fresh mutans streptococcal isolates were repeatedly transferred in trypticase-yeast extract medium supplemented with glucose, fructose, sucrose, or lactose in the presence and absence of xylitol. Depending on the growth sugar, the presence of xylitol resulted in the selection of xylitol-resistant populations for several of the six strains tested, but not necessarily in the presence of all four sugars. All six strains rapidly became xylitol-resistant when grown on glucose in the presence of xylitol. All three fresh isolates became xylitol-resistant after 9 to 16 transfers in the presence of fructose or sucrose plus xylitol, while none of the laboratory strains became xylitol-resistant after 16 transfers in the presence of these sugars. The growth rates of 12 xylitol-resistant mutants in the presence of eight sugars suggested the existence of various types of xylitol-resistant mutants. The data partially explain the occurrence of xylitol-resistant mutans populations in long-term xylitol consumers and suggest a mechanism consistent with a selection process. Since various preliminary results suggest that xylitol-resistant natural mutants may be less virulent and less cariogenic than their parent strains, this selection process may alter, for the better, the mutans streptococci population of the plaque and play a role in the caries-preventive action of xylitol.

Inverse correlation between the proportion of salivary bacteria inhibiting Streptococcus mutans and the percentage of untreated carious teeth.

To evaluate the role of inhibitory substances produced by bacteria in the oral cavity, we estimated, by a deferred test on Todd-Hewitt agar enriched with hemin and vitamin K, the proportion of bacteria that inhibited or stimulated the growth of Streptococcus mutans and Porphyromonas gingivalis, from the saliva of 109 patients (54 males and 55 females) attending our dental clinics. The patients, aged from 8 to 75 years old (mean: 31 +/- 18 years), were randomly selected whatever the reason for their visit. The results, evaluated with the Spearman rank test, indicated that there was no statistically significant (P > 0.05) correlation between the proportion of salivary bacteria inhibiting or stimulating P. gingivalis with the Community Periodontal Index of Treatment Needs (CPITN), the number of carious, missing and filled teeth, or with the decayed, missing and filled teeth index (DMFT). Also, no statistically significant correlation was observed between the proportion of salivary bacteria stimulating the growth of S. mutans and the above mentioned health indexes. However, a statistically significant (P < 0.005) negative correlation was found between the percentage of cultivated bacteria that inhibit S. mutans and the percentage of untreated carious teeth as well as with the CPITN. The results thus indicate a possible role for inhibitory substances produced by bacteria in the maintenance of oral health.

Xylitol: a review of its action on mutans streptococci and dental plaque--its clinical significance.

Many mechanisms have been proposed to explain the caries preventive effect of xylitol as a total or partial dietary sugar substitute. This article reviews the current knowledge of the effect of xylitol on the microbial population of dental plaque, particularly on mutans streptococci, in the light of an ecological concept of the oral environment and of the potential clinical significance. A noncariogenic commensal plaque flora constitutes the biotic component of a balanced ecosystem compatible with dental health. Dietary sugars, particularly sucrose, and sugar substitutes are abiotic environmental factors that can shift the delicate balance of the ecosystem towards a more or less cariogenic microbiota. Most dietary sugars are fermented by plaque microorganisms, favour the establishment of a cariogenic microflora and contribute to bacterial virulence. The vast majority of plaque bacteria, however, are incapable of fermenting xylitol into cariogenic acid end-products. There is no evidence that the plaque microbiota can adapt to metabolise xylitol or can be enriched with xylitol-metabolising cells even after long exposure to xylitol. Accumulated intracellularly as a non-metabolisable metabolite by mutans streptococci, xylitol inhibits its growth in vitro and reduces the amount of plaque and the number of mutans streptococci in both the plaque and saliva of xylitol consumers. When present in the oral environment xylitol not only prevents a shift of the bacterial community towards a more cariogenic microflora but also selects for a mutants population that was shown to have weakened virulence factors in preliminary in vitro experiments and in rats. Further research is needed to fully understand the clinical importance in the prevention of caries of this xylitol-selected population.

The effects of carrier gas composition on the performance of the Tec 6 desflurane vaporizer.

The new Tec 6 desflurane vaporizer is an electrically heated, pressurized, electromechanically coupled dual-circuit blender. We hypothesized that carrier gas viscosity should affect the electromechanical coupling of the fresh gas and vapor circuits, and that desflurane output should vary with different carrier gases. In the first portion of the study, the performance of eight vaporizers was evaluated using a constant dial setting of 10% desflurane with four different carrier gases and three different fresh gas flow rates. In the second portion of the study, the carrier gas flow rate was maintained at 1, 5, or 10 L/min, and vaporizer output was analyzed at all integer dial settings from 1% to 18%. Vaporizer output was highest when oxygen was the carrier gas and lowest when nitrous oxide was the carrier gas. This effect was accentuated at low fresh gas flow rates and correlated with carrier gas viscosity. At a flow rate of 1.0 L/min with a constant dial setting of 10%, the averaged output from vaporizers was 10.3 +/- 0.66, 9.4 +/- 0.58, 8.7 +/- 0.52, and 8.1 +/- 0.44 vol% for 100% oxygen, air, 30% oxygen plus 70% nitrous oxide, and 100% nitrous oxide, respectively. With 100% nitrous oxide as the carrier gas at a flow rate of 1.0 L/min, the vaporizer delivered 2 vol% less than the dial setting at dial settings in excess of 12%. Differences between the analyzed concentration and the dial setting were most pronounced with high concentrations of nitrous oxide at low fresh gas flow rates.

Positive selection for resistance to 2-deoxyglucose gives rise, in Streptococcus salivarius, to seven classes of pleiotropic mutants, including ptsH and ptsI missense mutants.

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)

Amino-terminal methionine processing of the protein HPr in Streptococcus salivarius grown in continuous culture.

HPr is a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Streptococci possess two forms of HPr which differ by the presence or the absence of the N-terminal methionine (Met). These forms are called HPr-1 (without Met) and HPr-2 (with Met). In order to determine whether the ratio of these two forms varies with growth conditions, we measured the amount of HPr-1 and HPr-2 present in Streptococcus salivarius grown in continuous culture at pH 7.5. The results indicated that the HPr-1/HPr-2 ratio: 1) was not related to the cellular amount of total HPr; 2) was highest (10.2 +/- 3.5) under glucose (a PTS sugar) limitation (10 mM) and low dilution rate (D = 0.1 h-1; g = 6.9 h); 3) was decreased 2.4- to 5.7-fold when the amount of glucose and/or D was increased; 4) was not influenced by D when cells were cultured on galactose (a non-PTS sugar) but was two-fold higher under conditions of galactose excess (200 mM). We suggest that the cleavage of the N-terminal HPr Met is not a stochastic phenomenon but is dictated by growth conditions.

Alterations in the cellular envelope of spontaneous IIIManL-defective mutants of Streptococcus salivarius.

In Streptococcus salivarius, the phosphoenolpyruvate: mannose phosphotransferase system (PTSMan) transports and concomitantly phosphorylates mannose, glucose, fructose and 2-deoxyglucose. PTSMan consists of a membrane Enzyme II and two forms of Enzyme III (IIIMan) having molecular masses of 38.9 kDa (IIIManH) and 35.2 kDa (IIIManL) respectively. We have previously reported the isolation of spontaneous mutants lacking IIIManL, and showed that they exhibited abnormal growth when cultured in mixtures of sugars containing glucose. The mutants also synthesize several cytoplasmic glucose-repressible proteins during growth on glucose and some of them constitutively express a fructose PTS which is induced by fructose in the parental strain. We have now investigated the properties and composition of the cellular envelope of three S. salivarius IIIManL-defective mutants (strains A37, B31 and G29) after growth on glucose. The mutants have altered sensitivity to various toxic compounds that interfere with cell-envelope functions. The mutants also exhibited altered membrane-protein profiles when analysed by two-dimensional PAGE and modified total lipid and phosphorus contents and lipid/protein ratio. In one mutant (strain G29), the proportion of the phospholipids separated by TLC was different from the parental strain. Electron microscopy indicated that one mutant (strain A37) possessed more fimbriae than the parental strain. The results suggested that these IIIManL-defective mutants were affected in a global regulatory gene controlling several cellular or physiological functions, many of these being related to the cellular envelope.

Effect of xylitol consumption on the plaque-saliva distribution of mutans streptococci and the occurrence and long-term survival of xylitol-resistant strains.

Since the exposure of mutans streptococci to xylitol is known to select for xylitol-resistant (XR) natural mutants, the occurrence and long-term survival of such xylitol-resistant strains was evaluated in a cross-sectional sampling of participants of the Ylivieska xylitol study four years after the original two-year experimental period. Paraffin-stimulated whole saliva was first collected, and then plaque was collected and pooled. The salivary and dental plaque mutans streptococci were enumerated after growth on TSY20B agar. The proportion of XR strains was determined by autoradiography with 14C-xylitol. A strong and significant correlation (r = 0.645 and p = 0.005) between the number of mutans streptococci in saliva and in dental plaque was observed in non-consumers of xylitol. Such a correlation totally disappeared (r = 0.098 and p = 0.612) in xylitol-exposed consumers (habitual and former xylitol-consumers). The proportion of the salivary XR mutants (35%) in non-consumers (n = 16) was significantly lower than in the xylitol-exposed consumers (79%) (n = 27), (p = 0.0001) or in former consumers (75%) (n = 13), (p = 0.0008) or in the habitual consumers (83%) (n = 14), (p = 0.004). The proportion of XR mutants in dental plaque was, on the average, much lower than in the corresponding saliva. The proportion of XR in the plaque of xylitol non-consumers was half of that of the xylitol-exposed group, but the difference was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

A multivariate model to predict caries increment in Montreal children aged 5 years.

A study was carried out in Montreal (Canada) to predict caries development over the period of one year in primary teeth of kindergarten children (mean age 5 years 8 months +/- 4 months) living in a non-fluoridated area. The 302 children were examined at school on two occasions, one year apart. At the first examination selected predictors were collected: caries experience, salivary S. mutans and lactobacilli, buffer capacity, debris index, parents' education, fluoride consumption and family structure (one or two parents). Regression analysis was performed to select the significant factors. A total of 143 children developed new caries over the study period; the mean increment for the whole group was 2.1 dmfs. Sensitivity (Sn) and specificity (Sp) were calculated for each predictor and for the final model. The best model comprised only two factors, caries experience and lactobacillus. This could identify 81.8 per cent of children who would develop new caries during the next 12 months (Sn) and 77.4 per cent of those who would not (Sp). Among the single predictors caries experience alone reached 78.3 per cent for sensitivity and 77.4 per cent for specificity. None of the other predictors, except parents' education, was very good at predicting caries increment over one year.

Factors affecting the ingestion of fluoride dentifrice by children.

Several factors affecting the amount of fluoride ingested during toothbrushing by 2- to 7-year-old children were investigated. The specific purpose of this study was to determine the contribution of age, the amount of dentifrice used, and rinsing after brushing to the variation in the ingestion of fluoride dentifrice. Four hundred and five children brushed their teeth in front of a portable sink. The tubes of dentifrice in gel (0.24% NaF) were weighed before and after use to determine the amount of toothpaste used. The fluoride content of the collected liquids was determined with a fluoride-ion-specific electrode. The amount of fluoride ingested was derived by determining the difference between the amounts used and recovered. The amount of dentifrice used, the age, and the rinsing habits, entered in a multiple regression model, explained up to 66 percent of the total variation in the amount of fluoride ingested. The amount of dentifrice used accounted by itself for 60 percent of the total variation. Therefore, these results indicate that the quantity of dentifrice used was the most important factor affecting the ingestion of fluoride through toothbrushing by young children.

Intracellular xylitol-phosphate hydrolysis and efflux of xylitol in Streptococcus sobrinus.

The parental strain Streptococcus sobrinus (Streptococcus mutans ATCC 27352), which is known to transport, phosphorylate and accumulate xylitol intracellularly as nonmetabolizable xylitol-phosphate (xylitol-sensitive (XS) strain) and its xylitol-resistant (XR) spontaneous mutant were used to further investigate the inhibitory action of xylitol on oral streptococci. Fructose-grown XR cells did not accumulate xylitol-phosphate, indicating that the inducible fructose PTS is incapable of transporting the pentitol. The intracellularly accumulated pentitol-phosphate by the XS cells did not prevent the subsequent uptake and degradation of glucose or fructose, despite a drop in the PEP pool and a 50% inhibition of the glucose but not the fructose catabolism. Intracellular dephosphorylation of the pentitol-phosphate and release of xylitol in the extracellular medium resulted in a rapid decrease of the intracellular level of this nonmetabolizable product. A Mg(++)- or Mn(++)-independent sugar-phosphate hydrolysing activity capable of splitting xylitol-phosphate was demonstrated in both XS and XR strains. Preincubation in the presence of N1-ethylmaleimide (NEM) and xylitol or NEM and fructose resulted in the subsequent inhibition of both xylitol uptake and efflux. The efflux kinetic at various temperatures is compatible with a facilitated diffusion by the phosphotransferase system EIIfru without, however, excluding the existence of an additional exit route, but it excludes a simple diffusion exit process. The results are consistent with the existence of a xylitol futile cycle contributing to the growth inhibition of S. sobrinus by the pentitol without excluding a toxic effect of xylitol-phosphate. Discrepancies in the literature on the action of xylitol on S. mutans could be explained in the light of the evidence presented.

Variability in the ingestion of toothpaste by preschool children.

The amount of dentifrice used and ingested on three occasions by a group of 48 children aged between 3 and 5 years was measured. The purpose of the present study was to investigate the variability in the amount of dentifrice used and ingested. The quantity of toothpaste ingested was derived from the differences between the amounts used and rejected. On average, the difference in the amount used between any two brushings was less than 0.250 g for 66% of the subjects, and the difference in the amount ingested was less than 0.100 g for 69% of the children. There were no statistically significant differences in the quantities used and ingested between the three brushings.

The ingestion of fluoride dentifrice by young children.

Through the fluoridation of drinking water, fluorides are becoming an increasing part of the human environment in industrialized countries. Fluoridated toothpastes are widely used in the United States and Canada. The younger a subject is, the greater proportion of toothpaste he or she tends to swallow.

Preparation and Purification of Xylitol-5-Phosphate from a Cell Extract of Lactobacillus casei Cl-16.

A simple procedure which yields pure xylitol-5-phosphate is described. A cell extract of Lactobacillus casei Cl-16 from a 6-liter culture was used to synthesize up to 70 mg of xylitol-5-phosphate overnight from xylitol and phosphoenolpyruvate via a xylitol phosphoenolpyruvate:phosphotransferase system with a 53% yield. Centrifugation, filtration, precipitation as a barium salt, and ion-exchange batch chromatography permitted recovery of nearly 90% of the phosphorylated product synthesized. Thin-layer chromatography and enzymatic analysis indicated a purity level of more than 99%. The method was used to synthesize [U-C]xylitol-5-phosphate, and it is suitable for the synthesis of many other nonmetabolizable sugar phosphates.

Effect of nutritional constraints on the biosynthesis of the components of the phosphoenolpyruvate: sugar phosphotransferase system in a fresh isolate of Streptococcus mutans.

A procedure for the purification of enzyme I (EI) and the protein HPr, the general components of the phosphoenolpyruvate:sugar phosphotransferase system, from Streptococcus mutans serotype c is presented. The method was also applied successfully to the purification of EI and HPr from Streptococcus salivarius, Streptococcus sobrinus, and Streptococcus sanguis. Using specific antibodies obtained against the proteins purified from S. mutans DR0001, we determined quantitatively by rocket electrophoresis the cellular levels of EI and HPr in a freshly isolated strain of S. mutans grown under various conditions in continuous culture. The activity of a few specific EIIs was also determined by an in vitro phosphorylation test. Results indicated that maximum EII activities for glucose, mannose, and 2-deoxyglucose were obtained under conditions of glucose limitation, at pH 7.0 and low dilution rate (D = 0.057/h). Increasing the amount of glucose or the dilution rate (D = 0.40/h) or decreasing the pH from 7.0 to 5.5 resulted in a 1.4- to 24-fold decrease in these activities. The EII activity for fructose was not influenced by the growth conditions in the same way as the other EIIs. The fructose EII was highest at pH 5.5 and at high dilution rate under conditions of glucose or nitrogen limitation and was always repressed at pH 7.0 and at low dilution rates. The intracellular levels of EI were also dependent on the growth conditions. The highest concentration (0.65 nmol/mg of protein) was observed in cells grown under glucose limitation at pH 7.0 and high dilution rate, and the lowest concentration (0.12 nmol/mg of protein) was found in cells grown under glucose excess at pH 7.0 and high dilution rate. The other general component of the phosphoenolpyruvate:sugar phosphotransferase system, the protein HPr, was not influenced significantly by varying growth conditions.