Biochemical and functional properties of a pyruvate formate-lyase (PFL)-activating system in Streptococcus mutans.

Streptococcus mutans has an oxygen-sensitive enzyme, pyruvate formate-lyase (PFL), which is a key enzyme in anaerobic sugar fermentation. We have shown that S. mutans has an activating system, including a PFL-activating enzyme (PFL-activase) and an electron transport system; the latter transfers an electron from NADPH to PFL-activase, as occurs in Escherichia coli. NADPH was a physiological electron donor for the electron transport system and as little as 0.02 mM NADPH activated over 80% of PFL of S. mutans. The optimum pH of the PFL-activating system was around 6.8, whereas the optimum of the E. coli system is at alkaline pH. In addition, small dialyzable molecules in cell-free extracts participated in keeping PFL active in S. mutans. These results suggest that, in dental plaque under anaerobic conditions where sugar supply is often limited or pH frequently falls below neutrality, S. mutans always keeps PFL active through the activating system.

Inhibitory effect of sorbitol on sugar metabolism of Streptococcus mutans in vitro and on acid production in dental plaque in vivo.

This study was conducted to find out whether sorbitol inhibits the sugar metabolism of Streptococcus mutans in vitro and the acid production in dental plaque in vivo. S. mutans NCIB 11723 was anaerobically grown in sorbitol-containing medium. The rate of acid production from sugars was estimated with a pH stat. The rate of acid production from glucose or sucrose was not changed at various concentrations of oxygen. By the addition of sorbitol to sugar, however, the acid production was decreased with increasing levels of oxygen. Intracellular NADH/NAD+ ratio and (dihydroxyacetone-phosphate+glyceraldehyde-phosphate)/3-phosphoglycerate ratio were high whenever the acid production was inhibited by sorbitol. Sorbitol also inhibited the acid production in dental plaque in vivo. These results suggest that the increased NADH/NAD+ ratio during sorbitol metabolism through the inactivation of pyruvate formate-lyase by oxygen inhibited glyceraldehyde-phosphate dehydrogenase and then the acid production of S. mutans and the one in dental plaque.

The time-course of acid excretion, levels of fluorescence dependent on cellular nicotinamide adenine nucleotide and glycolytic intermediates of Streptococcus mutans cells exposed and not exposed to air in the presence of glucose and sorbitol.

The aim of this study was to examine glucose and sorbitol metabolism in Streptococcus mutans cells exposed and not exposed to air at the coexistence of these compounds by measuring acid excretion, levels of fluorescence dependent on cellular NADH and glycolytic intermediates. An aliquot of bacterial cells grown under strictly anaerobic conditions (anaerobic cells) was exposed temporarily to air (aerobic cells). When glucose was added to the anaerobic cells metabolizing sorbitol, the acid excretion was increased. The level of NADH decreased initially and then increased to the higher plateau level than that during glucose metabolism. The aerobic cells neither metabolized sorbitol nor contained glycolytic intermediates. However, 2 min after glucose was added in the presence of sorbitol, the acid excretion was started slowly and the intermediates appeared. The level of NADH was decreased at first and then increased. These results suggested that the anaerobic S. mutans cells metabolized glucose and sorbitol simultaneously, and that in the presence of sorbitol the aerobic cells could start to metabolize glucose 2 min after glucose was added, as the intermediates (phosphoenopyruvate potential) for the glucose transport were accumulated.

Characterization of the Streptococcus mutans pyruvate formate-lyase (PFL)-activating enzyme gene by complementary reconstitution of the In vitro PFL-reactivating system.

The act gene was identified and an act mutant as well as the pfl mutant was constructed in Streptococcus mutans. Pyruvate formate-lyase (PFL) activity was regenerated with the mixture of the respective cell extracts from these mutants by complementary reconstitution of the in vitro reactivating system. The S. mutans act gene encoded the sole enzyme able to activate the PFL protein in this organism.

Rate-limiting steps of glucose and sorbitol metabolism in Streptococcus mutans cells exposed to air.

It has been supposed that rate of sorbitol metabolism in the air-exposed streptococcal cells could be limited by the low capacity to regenerate nicotinamide adenine dinucleotide (NAD) from reduced NAD (NADH) following inactivation of pyruvate formate-lyase by oxygen. The rate-limiting steps, however, have not been identified. The aim of this study was to examine the effect of temporary exposure of the streptococcal cells to air on the intracellular flux of glucose and sorbitol metabolism by measuring acid excretion, fluorescence dependent on cellular level of NADH, glycolytic intermediates and enzyme activities. The exposure of cells to air decreased the acid excretions during glucose and sorbitol metabolism. The analysis of the glycolytic intermediates and the fluorescence suggested that the reduced level of acid excretion in the air-exposed glucose metabolizing cells resulted from the decrease in pyruvate catabolism. In the presence of sorbitol, the decreased acid production resulted from the reduced rates of the reactions catalyzed by sorbitol-phosphoenolpyruvate phosphotransferase and sorbitol 6-phosphate dehydrogenase because of shortage of substrates for these enzymes in addition to the decrease in pyruvate catabolism.

Cloning and sequence analysis of the pfl gene encoding pyruvate formate-lyase from Streptococcus mutans.

We have isolated a sorbitol-negative mutant of Streptococcus mutans GS-5 following random mutagenesis with plasmid pVA891 clone banks. This mutant did not metabolize sorbitol anaerobically but did so aerobically. A 10-kb chromosomal DNA fragment flanking the pVA891 insertion was deleted in this mutant. The corresponding region from the parental strain GS-5 was then recovered by a marker rescue method with Escherichia coli. The pyruvate formate-lyase gene, pfl, was identified within a 3-kb PstI-XbaI fragment located in the middle of the deleted region of the chromosome, and its inactivation in S. mutans produced the same sorbitol-negative phenotype. Nucleotide sequence analysis of the pfl gene revealed a 2.3-kb open reading frame (ORF) preceded by potential ribosome-binding and promoter-like sequences. The ORF specified a putative protein of 775 amino acid residues with a calculated molecular weight of 87,533. The amino acid sequence deduced from the ORF exhibited significant similarity to that of the E. coli pfl gene.

Acid production by streptococci growing at low pH in a chemostat under anaerobic conditions.

Streptococcus mutans and other oral streptococci were grown in continuous culture under strictly anaerobic conditions. When the cultural pH was kept at 7.0, the main acid products were formate and acetate, as reported previously. However, more lactate was produced at pH values of 5.5 or 6.0, with a concomitant decrease in formate and acetate production. This change in fermentation products could partly be ascribed to a change in intracellular pH and difference in the pH optima between pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH). At extracellular pH values of 7.0 and 5.5, the intracellular pH values of S. mutans NCIB 11723 were 7.5 and 6.6, respectively. The pH optima of PFL and LDH were 7.8 and 5.5-6.3, respectively. The cells had also a larger amount of LDH during growth at pH 5.5 than at pH 7.0.

Oxygen and the sugar metabolism in oral streptococci.

Streptococci have several ways of adapting themselves to the constantly changing environment of the human oral cavity. This paper discusses the adaptation of sugar metabolism to variations in oxygen levels. In all streptococci the Embden-Meyerhof pathway of glycolysis works under aerobic as well as anaerobic conditions, but pyruvate is converted into different metabolic end products depending on the oxygen levels. Under anaerobic conditions all streptococci form formate, acetate, and ethanol by a pyruvate formate-lyase pathway. If sugar is in excess, they also form lactate using a lactate dehydrogenase. Under aerobic conditions pyruvate formate-lyase is inactivated. This enzyme is then replaced by a pyruvate oxidase in some streptococci and by a pyruvate dehydrogenase in others. The characteristics of these enzymes help streptococci like S. sanguis, S. oralis, S. gordonii, and S. mitis to compete successfully with other bacteria in those sites of the oral cavity that are freely exposed to saliva, while mutans streptococci have to colonize anaerobic sites such as those in-between the teeth and in the occlusal fissures of the teeth.

Oxygen sensitivity of sugar metabolism and interconversion of pyruvate formate-lyase in intact cells of Streptococcus mutans and Streptococcus sanguis.

Pyruvate formate-lyase (PFL) (formate acetyltransferase; EC 2.3.1.54) of oral streptococci is essential for metabolizing sugar into volatile compounds (formate, acetate, and ethanol). This enzyme is extremely sensitive to oxygen, and its activity is irreversibly inactivated by oxygen. When Streptococcus sanguis was anaerobically starved, a part of the active form of PFL was converted into a reversible inactive form that was tolerant of oxygen. This reversible inactive enzyme could be reactivated to the active enzyme by anaerobic sugar metabolism, with the recovery of volatile compound production. The PFL in Streptococcus mutans was not converted into an oxygen-tolerant inactive form by anaerobic starvation, and after exposure of the cells to oxygen the PFL could not be reactivated. These findings suggest that S. mutans can produce acids rapidly under anaerobic conditions because of its capacity to keep PFL active and that S. sanguis can protect its sugar metabolism from oxygen impairment because of its interconversion of PFL.

Role of NADH oxidase in the oxidative inactivation of Streptococcus salivarius fructosyltransferase.

A cell-associated fructosyltransferase produced by Streptococcus salivarius was irreversibly inactivated in a time-dependent manner when resting or permeabilized cell suspensions were incubated with low concentrations (less than 1.0 microM) of copper. In addition to copper, the inactivation was dependent on oxygen and on a fermentable carbon source (endogenous intracellular polysaccharide or glucose). In starved, permeabilized cell suspensions, the fermentable carbon source could be replaced by NADH but not by NADPH or ATP. Of several other S. salivarius enzymes tested, only fructosyltransferase was inactivated under these conditions. The available evidence indicated that NADH oxidase is the enzyme responsible for fructosyltransferase inactivation. Results from oxygen radical scavenger studies implicated one or more species of oxygen radicals and hydrogen peroxide in the inactivation reaction.

Anaerobic and aerobic metabolism of sorbitol in Streptococcus sanguis and Streptococcus mitior.

Sorbitol-fermenting strains of Streptococcus sanguis and Streptococcus mitior were grown both anaerobically and in the presence of oxygen in a sorbitol-containing complex medium. Washed-cell suspensions were incubated with an excess of sorbitol, and the production of lactate, formate, ethanol, and acetate was analyzed. Moreover, we determined the lactate dehydrogenase and pyruvate formate-lyase activities in cell-free extracts of anaerobically grown cells. The anaerobically grown cells produced lactate, formate, ethanol, and acetate under anaerobic conditions. When these cells were exposed to air, the amounts of formate, ethanol, and acetate were reduced in comparison with those of the strictly anaerobic cells. Cells grown in the presence of oxygen only produced detectable levels of lactate and acetate. Anaerobically grown cells possessed lactate dehydrogenase and pyruvate formate-lyase activities under strictly anaerobic conditions. The level of pyruvate formate-lyase was dramatically reduced when cells were exposed to air, while the level of lactate dehydrogenase was about the same as that under strictly anaerobic conditions. Thus, the results indicate that S. sanguis and S. mitior both metabolize sorbitol differently under anaerobic and aerobic conditions. This difference may depend on the oxygen-sensitivity of the pyruvate formatelyase of these micro-organisms.

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.