Effect of growth rate and pH on intracellular levels and activities of the components of the phosphoenolpyruvate: sugar phosphotransferase system in Streptococcus mutans Ingbritt.

The growth of Streptococcus mutants Ingbritt in continuous culture at low pH or high growth rates repressed the biosynthesis of the components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The cellular concentrations of the soluble components HPr, enzyme I (EI), and EIII for mannose (IIIman) and EII activity for glucose, mannose, 2-deoxyglucose (2DG), and fructose were determined in membrane preparations from cells grown at pHs from 8.0 to 5.0 and at dilution (D) or growth rates from 0.1 to 1.0 h-1. The cellular levels of HPr and EI varied less than threefold under all of the growth conditions tested. On the other hand, EII activity in membranes from cells grown at D = 0.1 h-1 was repressed by growth at pHs below 8.0, with cells grown at pH 5.0 completely devoid of EII activity. In addition, cells grown at D = 0.5 and 1.0 h-1 exhibited little PTS activity for glucose, mannose, and 2DG and twofold-lower activity for fructose. These activities were stimulated by the addition of a membrane-free cytoplasmic fraction, and this activating activity was shown to be due to the presence of IIIman. Estimation of the cellular content of IIIman indicated that the synthesis of this factor was repressed by growth above and below pH 7.0 and was particularly sensitive to growth at high rates. These results indicate that with S. mutans Ingbritt, both pH and growth rate regulate the genes for the synthesis of EIIs involved in the phosphorylation of glucose, mannose, 2DG, and fructose and the gene for the formation of IIIman.     

Properties of Streptococcus mutans Ingbritt growing on limiting sucrose in a chemostat: repression of the phosphoenolpyruvate phosphotransferase transport system.

Growth of Streptococcus mutans Ingbritt on limiting sucrose in a chemostat at dilution rates of 0.05 to 0.4 h-1 (mean generation time, 14 to 1.7 h) resulted in a heterofermentative pattern of metabolic end products. During fast growth, lactic acid was the major end product, whereas at slower growth rates, acetic and formic acids, as well as ethanol, increased to be major end products. The patterns obtained were similar to those seen with the same organism growing on glucose. The glycolytic rate by washed cells was maximum at the lowest dilution rates and decreased as the cells were made to grow faster. Transport of sucrose, glucose, and fructose via the phosphoenolpyruvate phosphotransferase system (PTS) was repressed during growth on sucrose after growth on glucose. Uptake rates suggested that sucrose was transported in the PTS as the intact disaccharide. Comparison of the rate of sugar uptake in the chemostat with the rate of PTS activity in the cells at each growth rate indicated that the PTS was capable of supporting growth only at a dilution rate of 0.05 h-1. Growth on sucrose at faster growth rates required the activity of a second transport system, supporting our earlier observations with glucose that S. mutans contains at least two sugar transport systems.     

Characterization of a phosphoenolpyruvate-dependent sucrose phosphotransferase system in Streptococcus mutans.

A phosphoenolpyruvate-dependent sucrose phosphotransferase system has been identified in Streptococcus mutans. Sucrose phosphotransferase activity was inducible by sucrose and had an apparent Km for sucrose of 70 microM. The product of the sucrose phosphotransferase reaction was isolated and identified as sucrose phosphate. Additional analysis revealed that the phosphate group was on the glucose moiety. Mutants unable to grow in media containing low concentrations of sucrose were isolated and found to be missing either sucrose phosphotransferase activity or the ability to hydrolyze sucrose phosphate.     

Repeated DNA sequence involved in mutations affecting transport of sucrose into Streptococcus mutans V403 via the phosphoenolpyruvate phosphotransferase system.

Mutants of Streptococcus mutans V403 defective in the intracellular sucrose-6-phosphate hydrolase (product of the scrB gene) are sensitive to sucrose because of the intracellular accumulation of the phosphorylated sugar. Using a scrB mutant prepared by allelic exchange, we have isolated and characterized a number of sucrose-resistant revertants. One such mutant was found to lack the ability to transport sucrose into the cell via the phosphoenolpyruvate-dependent sucrose phosphotransferase system (PTS). Genetic analysis of this strain revealed this lesion to be linked to the scrB gene. This was corroborated by the physical demonstration of an insertion mutation very near scrB. Taken together with DNA sequence information (Y. Sato, F. Poy, G. R. Jacobson, and H. K. Kuramitsu, J. Bacteriol. 171:263-271, 1989), our results indicated that all of the mutations characterized were located in the adjoining scrA gene which encodes the membrane-associated, sugar-specific enzyme II (EIIsucrose) component of the sucrose PTS in S. mutans. Biochemically, such a genetic lesion disables the sucrose PTS and prevents sucrose from entering the cell by this system. In this paper, we detail the nature of two independent insertion mutations and conclude them to be the result of duplicative transposition events into the scrA gene. This region of the chromosome was amplified and purified in large quantities by using the polymerase chain reaction. Examination of the amplified DNA revealed that the two independent insertion mutations were composed of sequences that were indistinguishable by size and by restriction site endonuclease maps. Their insertion points in the scrA gene were approximately 200 bp apart. The amplified DNA fragment was also used as a probe to demonstrate the presence of five copies of this element on the S. mutans V403 chromosome. A second strain, S. mutans V310, also was found to carry similarly arranged, multiple copies of this sequence on its chromosome, suggesting a clonal origin of V403 and V310. The small size of this sequence, its presence in multiple copies on the V403 chromosome, and its ability to duplicate itself semiconservatively into remote sites argue compellingly that it is an insertion sequence element. One such insertion mutant, with a defective sucrose PTS, was tested for virulence in rats and was found to cause caries at levels similar to those of the wild-type strain.     

Effect of growth conditions on sucrose phosphotransferase activity of Streptococcus mutans.

Sucrose and glucose phosphoenolpyruvate-dependent phosphotransferase (PTS) activities were studied in growing cultures of Streptococcus mutans serotype c and d/g cells adapted to either glucose or sucrose. Both acid production and optical absorbance were used to monitor growth in pH-controlled defined growth medium. The sucrose PTS activity appeared to be significant only under conditions of substrate limitation or slow growth as a result of low environmental pH. However, under environmental conditions which permitted rapid growth sucrose PTS activity appeared to be repressed, and only when the cells approached substrate-limited stationary phase after growth on high sucrose-supplemented medium was significant sucrose PTS activity again observed. A mutant apparently defective in sucrose PTS activity grew rapidly and produced acid under conditions of high environmental sucrose level but showed no sucrose PTS activity when the culture approached stationary phase. The mutant, however, after adaptation to glucose, demonstrated significant glucose PTS once the culture had attained the stationary growth phase. During diauxie growth in the presence of glucose and sucrose, there were sequential apparent inductions and repressions of glucose and sucrose PTS activities corresponding to decreases and increases of growth rate on the two substrates. Thus, S. mutans possesses at least two transport mechanisms for each substrate studied. One system (PTS) functions under conditions permitting slow growth and another functions under conditions permitting rapid growth.     

Effect of Bdellovibrio bacteriovorus infection on the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli: evidence for activation of cytoplasmic proteolysis.

Intact cells of Bdellovibrio bacteriovorus strain 109J were found to be incapable of taking up 14C-methyl alpha-glucoside, mannitol or fructose, and extracts derived from these cells exhibited negligible activities of the protein components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Escherichia coli strain ML35 cells exhibited high in vivo sugar uptake activities that were progressively lost over a period of 2 h at 30 degrees C following the entry of B. bacteriovorus into the periplasm of E. coli. In vitro complementation assays revealed that the E. coli PTS enzymes, enzyme I, HPr, and the glucose- and mannitol-specific enzymes II, were all lost almost in parallel with the disappearance of uptake activity. Thus, loss of activity in vivo was not due to membrane leakiness, energy depletion, or preferential inhibition or inactivation of any one protein component of the PTS. Instead, loss of PTS activity was attributed to digestion of the protein constituents of the system by proteases present in the cytoplasm of the host cell after bdellovibrio entry. Both ethylenediaminetetraacetate and phenylmethylsulphonyl fluoride partially protected against inactivation in vitro, and the two inhibitors together gave full protection, suggesting that both metallo- and seryl-proteases were responsible for the inactivation. Protease activity increased progressively with time following bdellovibrio entry and appeared to degrade the E. coli PTS enzymes in vivo. Preliminary evidence suggested that the proteases responsible for PTS enzyme degradation may be encoded by the B. bacteriovorus chromosome.     

Starvation-induced stimulation of sugar uptake in Streptococcus mutans is due to an effect on the activities of preexisting proteins of the phosphotransferase system.

We examined the effects of sugar concentration in the medium on sugar uptake and phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) activities in Streptococcus mutants GS-5. Kinetic analyses of sucrose uptake in cells harvested under conditions of sucrose excess or sucrose limitation showed that increased uptake under the latter condition was almost completely due to an increase in the Vmax of the high-affinity PTS. In a series of experiments in which cells growing under conditions of sucrose or glucose excess were shifted to a medium lacking sugar, starvation resulted in a stimulation of sugar uptake and a parallel increase in PTS activity. These starvation-induced increases in PTS-mediated uptake were not affected by the presence of either chloramphenicol or rifampin during the starvation period, indicating that neither protein nor RNA synthesis was necessary for the stimulation. In vivo labeling experiments with 32Pi revealed that uptake stimulation during starvation was accompanied by a loss of acid-stable phosphate covalently bound to the phosphocarrier protein HPr of the PTS. We conclude, therefore, that stimulation of PTS-mediated uptake of sucrose and glucose during sugar limitation in S. mutans GS-5 is at least partially the result of increased activities of preexisting PTS proteins and that this may be due, at least in part, to dephosphorylation of a previously identified site in S. mutans HPr that can be phosphorylated by an ATP-dependent kinase.     

Phosphoenolpyruvate-dependent sucrose phosphotransferase activity in five serotypes of Streptococcus mutans.

An inducible phosphoenolpyruvate-dependent sucrose phosphotransferase system has been demonstrated in decryptified cell suspensions of the various common serotypes of the cariogenic microorganism Streptococcus mutans.     

Transport of glucose and mannose by a common phosphoenolpyruvate-dependent phosphotransferase system in Streptococcus mutans GS5.

Decryptified cells of Streptococcus mutans GS5 transport glucose, mannose, and fructose by constitutive phosphoenolpyruvate-dependent phosphotransferase systems (PTSs). Although the non-metabolizable glucose analog 2-deoxyglucose is transported by a PTS, alpha-methylglucose is not taken up by strain GS5. The transport of [14C]mannose and [14C]glucose was almost totally blocked by the heterologous sugars, indicating that these substrates may share a common PTS permease. [14C]fructose transport, however, was not inhibited by large excesses of glucose, indicating the existence of a separate fructose PTS. All "tight" glucose PTS- mutant clones studied were also unable to transport mannose, whereas some "leaky" glucose PTS- clones also were leaky for mannose phosphorylation. Fructose transport in most of these mutant strains was unimpaired, indicating that genetic lesions did not involve soluble (cytoplasmic) PTS components.     

The phosphoenolpyruvate:sugar phosphotransferase system in gram-positive bacteria: properties, mechanism, and regulation

This review consists of three major sections. The first and largest section reviews the protein constituents and known properties of the phosphotransferase systems present in well-studied Gram-positive bacteria. These bacteria include species of the following genera: (1) Staphylococcus, (2) Streptococcus, (3) Bacillus, (4) Lactobacillus, (5) Clostridium, (6) Arthrobacter, and (7) Brochothrix. The properties of the different systems are compared. The second major section deals with the regulation of carbohydrate uptake. There are four parts: (1) inhibition by intracellular sugar phosphates in Staphylococcus aureus, (2) PTS-mediated regulation of glycerol uptake in Bacillus subtilis, (3) competition for phospho-HPr in Streptococcus mutans, and (4) the possible involvement of protein kinases in the regulation of sugar uptake via the phosphotransferase system. The third section deals with the phenomenon of inducer expulsion. The first part is concerned with the physiological characterization of the phenomenon; then the consequences of unregulated uptake and expulsion, a futile cycle of energy expenditure, are considered. Finally, the biochemistry of the protein kinase and the protein phosphate phosphatase system, which appears to regulate sugar transport via the phosphotransferase system, is defined. The review, therefore, concentrates on the phosphotransferase system, its functions in carbohydrate transport and phosphorylation, the mechanisms of its regulation, and the mechanism by which it participates in the regulation of other physiological processes in the bacterial cell.     

Effect of endogenous phosphoenolpyruvate potential on fluoride inhibition of glucose uptake by Streptococcus mutans.

The fluoride sensitivity of glucose uptake by whole cell suspensions of Streptococcus mutans was studied. Preincubation of the organism with up to 1 mM glucose markedly reduced the fluoride sensitivity of subsequent glucose uptake at pH 7.0 and 5.5. Glucose preincubation was shown to result in the establishment of a stable pool of three-carbon glycolytic intermediates. On the basis of inhibition studies and thin-layer chromatography of cell extracts, we suggest that 3- and 2-phosphoglycerate are the principal constituents of the pool. Increased concentrations of glucose used in preincubation mixtures was associated with increased pool sizes of the glycolytic intermediates and increased fluoride resistance. Transport of 2-deoxy-D-glucose by permeabilized cells was inhibited by fluoride when 2-phoshoglycerate served as the energy source. Increased concentrations of 2-phosphoglycerate were shown to overcome the fluoride inhibition of transport. The data suggest that establishment of a stable pool of glycolytic intermediates that includes 2-phosphoglycerate (or its progenitors) may contribute significantly to the apparent refractoriness of plaque microbes to fluoride in vivo.     

Evidence that a low-affinity sucrose phosphotransferase activity in Streptococcus mutans GS-5 is a high-affinity trehalose uptake system.

High-affinity sucrose uptake in the oral pathogen Streptococcus mutans is mediated by the phosphoenolpyruvate-dependent phosphotransferase system. In this report, we provide evidence that a lower-affinity sucrose phosphotransferase system in S. mutans GS-5, previously described by others, is in fact a high-affinity trehalose uptake system that also recognizes sucrose as a substrate.     

Involvement of phosphoenolpyruvate in the catabolism of caries-conducive disaccharides by Streptococcus mutans: lactose transport.

The mechanisms for transport and hydrolysis of lactose were investigated in five cariogenic strains (HS6, AHT, FA1, NCTC 10449, and SL1) representing the four serogenetic groups of Streptococcus mutans. The systems for transport and hydrolysis of lactose had the characteristics of a phosphoenolpyruvate (PEP)-dependent lactose (Lac) phosphotransferase (PT) system and phospho-beta-galactosidase (P-beta-gal), respectively, in all strains tested, except strain HS6. Decryptified cells required PEP and Mg(2+) for transport of the non-metabolizable model beta-galactosides o-nitrophenyl-beta-d-galactopyranoside (ONPG) and thiomethyl-beta-d-galactopyranoside (TMG). Substitution of 2-phosphoglycerate (2-PG) for PEP also stimulated the Lac PT system. Other potential high-energy phosphate donors (adenosine tri-, di-, and monophosphates and guanosine triphosphate) did not stimulate the Lac PT system. Sodium fluoride had no effect upon the PEP-dependent Lac PT system in decryptified cells with PEP as the energy source; however, when 2-PG was used as the energy source, F(-) inhibited ONPG phosphorylation. With intact cells which must generate PEP endogenously, the presence of F(-) in concentration >/= 10 mM completely inhibited the Lac PT system, presumably through inhibition of 2-PG hydrolyase (EC 4.2.1.11; enolase). Both intact and decryptified cells accumulated a phosphorylated derivative of TMG that behaved chromatographically as TMG-phosphate. After alkaline phosphatase treatment, the derivative had an R(f) identical to that of TMG. No beta-galactosidase (beta-gal) activity was detected with ONPG as the substrate; hydrolysis occurred only when ONPG-6-phosphate was supplied as the substrate. Strain HS6 apparently transported lactose by an active transport-type system in which the accumulated intracellular product was the free disaccharide based on the following criteria: (i) ONPG transport and hydrolysis in decryptified cells was not stimulated by PEP; (ii) ONPG hydrolysis occurred in the absence of PEP; and (iii) ONPG-6-phosphate was not hydrolyzed. These data indicate that, in all strains tested except strain HS6, lactose transport was mediated by a PEP-dependent Lac PT system, resulting in accumulation of lactose-phosphate that was hydrolyzed by an enzyme similar to the P-beta-gal of group N streptococci and Staphylococcus aureus; conversely, strain HS6 transported and hydrolyzed lactose by a PEP-independent transport system and beta-gal, respectively.     

Lactose transport in Streptococcus mutans: isolation and characterization of factor IIIlac, a specific protein component of the phosphoenolpyruvate-lactose phosphotransferase system.

The transport of lactose in Streptococcus mutans is mediated via an inducible phosphoenolpyruvate-lactose phosphotransferase system. This system requires for catalytic activity a membrane fraction (enzyme II), two general proteins called enzyme I and HPr, and a soluble specific protein termed factor IIIlac. This protein factor was purified from S. mutans ATCC 27352 by chromatographies on DEAE-cellulose, hydroxylapatite, Ultrogel AcA 34, and phosphocellulose. The purified protein migrated as a single band with a molecular weight of 10,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea. The molecular weight calculated from the amino acid composition was 10,541. Gel filtration of the native protein gave a molecular weight of 41,500. Its isoelectric point was ca. 4.70. A specific antiserum was prepared against purified factor IIIlac. Immunodiffusion experiments revealed that only cellular extracts from lactose-grown cells contained factor IIIlac. A cross-reaction was observed with all of the S. mutans strains tested as well as with Streptococcus sanguis 10556, Streptococcus lactis 11454, and Staphylococcus aureus 6538. No precipitin band was observed with extracts of Streptococcus salivarius, Streptococcus faecalis, Lactobacillus casei, and Bacillus subtilis.     

Resolution of the phosphotransferase enzymes of Streptococcus mutans: purification and preliminary characterization of a heat-stable phosphocarrier protein.

The sucrose phosphotransferase system of Streptococcus mutans catalyzes the phosphorylation of sucrose to sucrose-6-phosphate with concomitant translocation of this disaccharide across the cytoplasmic membrane in reactions requiring intracellular phosphoenolpyruvate. Soluble proteins released by vigorous homogenization of cells with glass beads are shown to be necessary for the phosphoenolpyruvate-dependent phosphorylation of sucrose in combination with one or more proteins that remain tightly associated with the membrane fraction. We have partially purified phosphotransferase enzyme I and have purified a heat-stable phosphocarrier protein (HPr) to apparent homogeneity, by gel filtration and ion-exchange chromatography from the soluble fraction. HPr from S. mutans has an apparent molecular weight larger than that of Escherichia coli HPr but has properties similar to those of Staphylococcus aureus HPr. Furthermore, it appears to be partially complexed with a heat-stable enzyme III-like protein in cell-free fractions from S. mutans, and we also report the purification of this complex. Enzyme I from S. mutans is a protein (native Mr greater than 100,000) that cross-complements enzyme I from S. aureus. Preliminary characterizations of homogeneous HPr and its complex with the putative enzyme III are also presented.     

Role of the phosphoenolpyruvate-dependent glucose phosphotransferase system of Streptococcus mutans GS5 in the regulation of lactose uptake

When Streptococcus mutans GS5 was grown in equimolar (5 mM) amounts of glucose and lactose, a classical diauxic growth curve was obtained. Glucose was taken up during the first growth phase, followed by a 60-min lag, and then lactose was transported. Synthesis of lactose phosphotransferase system (PTS) enzymes was repressed until the complete exhaustion of glucose, indicative of an inducer exclusion mechanism of repression. The enzyme phospho-beta-galactosidase, however, was found in small amounts even in the presence of glucose. Repression was not observed when GS5 was grown in equimolar amounts of fructose and lactose. Although fructose was taken up preferentially, synthesis of the lactose PTS occurred from the onset of growth in these sugars. It is proposed that a component of the glucose PTS may be a regulatory factor in lactose transport. Glucose PTS- mutants did not display diauxic growth in glucoselactose mixtures and, in fact, transported the disaccharide preferentially.     

Mutational analysis of the enzyme IIIGlc of the phosphoenolpyruvate phosphotransferase system in Escherichia coli.

The phosphoenolpyruvate phosphotransferase system (PTS) component EIIIGlc is responsible for transport and phosphorylation of glucose via EIIGlc. It also regulates the catabolism of other carbon sources, such as lactose and maltose, by modulating both the intracellular concentrations of the corresponding inducers and of cAMP. Mutational analysis of EIIIGlc was performed in order to identify crucial residues mediating the interactions between EIIIGlc and its target proteins. Such mutations were isolated by in vitro hydroxylamine mutagenesis of the cloned EIIIGlc gene, crr. Five mutated EIIIGlc impaired in the function of inducer exclusion were obtained. However, these mutations did not abolish the function of EIIIGlc in the transport and phosphorylation of glucose, nor in activation of adenylate cyclase. A single amino acid change was found for each mutation, which is located in a restricted area of the polypeptide chain: Gly47-->Ser47 for the HA2 and HA5 mutations, Ala76-->Thr76 for HA4 mutation and Ser78-->Phe78 for HA3 mutation, indicative of quaternary interactions between the corresponding region of EIIIGlc and its target protein(s).     

Effects of oxygen on pyruvate formate-lyase in situ and sugar metabolism of Streptococcus mutans and Streptococcus sanguis.

The strictly anaerobic metabolism of sugar in strains of Streptococcus mutans and Streptococcus sanguis was studied because deep layers of dental plaque are strictly anaerobic. Galactose-grown cells of these streptococcal strains had higher pyruvate formate-lyase activity than did glucose-grown cells. Among these strains, two strains of S. mutans had a significantly higher pyruvate formate-lyase activity than did the others. This enzyme is extremely sensitive to oxygen, and even in situ the enzyme was inactivated by exposure of the cells to air. Lactate was less than 50% of the total end product of the strictly anaerobic incubation of the galactose-grown cells of S. mutans with excess glucose, and a significant amount of formate, acetate, and ethanol was produced through the catalysis of pyruvate formate-lyase. But the cells exclusively produced lactate when exposed to air for 2 min before the anaerobic incubation. The metabolism of sorbitol by S. mutans was seriously impaired by the exposure of the cells to oxygen, and the metabolic rate was reduced to less than 1/20 of that found under strictly anaerobic conditions because of the inactivation of pyruvate formate-lyase. S. sanguis produced a smaller amount of the volatile products from glucose than did S. mutans because of the low level of pyruvate formate-lyase. However, the pyruvate formate-lyase in situ in S. sanguis was less sensitive to oxygen than was that in S. mutans. Because of this low sensitivity, S. sanguis metabolized glucose more rapidly under aerobic conditions, whereas the rates of the aerobic and anaerobic metabolism of glucose by S. mutans were similar, which suggests that S. mutans rather than S. sanguis can sustain the rapid sugar metabolism in the deep layers of dental plaque.