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.

We have cloned and sequenced the Lactobacillus casei ptsH and ptsI genes, which encode enzyme I and HPr, respectively, the general components of the phosphoenolpyruvate–carbohydrate phosphotransferase system (PTS). Northern blot analysis revealed that these two genes are organized in a single‐transcriptional unit whose expression is partially induced. The PTS plays an important role in sugar transport in L. casei, as was confirmed by constructing enzyme I‐deficient L. casei mutants, which were unable to ferment a large number of carbohydrates (fructose, mannose, mannitol, sorbose, sorbitol, amygdaline, arbutine, salicine, cellobiose, lactose, tagatose, trehalose and turanose). Phosphorylation of HPr at Ser‐46 is assumed to be important for the regulation of sugar metabolism in Gram‐positive bacteria. L. casei ptsH mutants were constructed in which phosphorylation of HPr at Ser‐46 was either prevented or diminished (replacement of Ser‐46 of HPr with Ala or Thr respectively). In a third mutant, Ile‐47 of HPr was replaced with a threonine, which was assumed to reduce the affinity of P–Ser–HPr for its target protein CcpA. The ptsH mutants exhibited a less pronounced lag phase during diauxic growth in a mixture of glucose and lactose, two PTS sugars, and diauxie was abolished when cells were cultured in a mixture of glucose and the non‐PTS sugars ribose or maltose. The ptsH mutants synthesizing Ser‐46–Ala or Ile‐47–Thr mutant HPr were partly or completely relieved from carbon catabolite repression (CCR), suggesting that the P–Ser–HPr/CcpA‐mediated mechanism of CCR is common to most low G+C Gram‐positive bacteria. In addition, in the three constructed ptsH mutants, glucose had lost its inhibitory effect on maltose transport, providing for the first time in vivo evidence that P–Ser–HPr participates also in inducer exclusion.

The presence of three distinct enzymes II that catalysed the phosphoenolpyruvate-dependent phosphorylation of glucose, fructose, and mannose was established in membranes of glucose-grown cells of Streptococcus salivarius 25975 and various strains of Streptococcus mutans. The enzyme II mannose phosphorylated mainly mannose, glucose, and 2-deoxyglucose, and the enzyme II glucose phosphorylated glucose, alpha-methylglucoside, and 2-deoxyglucose. The phosphoenolpyruvate-dependent phosphorylation of glucose and alpha-methylglucoside by isolated membrane of wild-type or EII mannose negative mutant cells did not require the presence of any soluble protein other than enzyme I and the phosphocarrier protein HPr. This result suggested that oral streptococci do not possess a soluble factor III glucose. The enzyme II activities varied as a function of the growth sugar but were not coordinately regulated. The variation elicited by specific sugars was not identical for all the strains tested. Nevertheless, in the case of the S. mutans strains, growth at the expense of lactose always caused a significant decrease in the level of enzyme II activities. Finally, experiments conducted with EII mannose negative mutants and also with a pseudorevertant isolated from one of these mutants indicated that the preferential utilization of glucose over lactose by cells growing in mixtures depended on the presence of the EII mannose, but not on glucose-derived metabolites.

Streptococcus salivarius is a lactose-and galactose-positive bacterium that is phylogenetically closely related to Streptococcus thermophilus, a bacterium that metabolizes lactose but not galactose. In this paper, we report a comparative characterization of the S. salivarius and S. thermophilus gal-lac gene clusters. The clusters have the same organization with the order galR (codes for a transcriptional regulator and is transcribed in the opposite direction), galK (galactokinase), galT (galactose-1-P uridylyltransferase), galE (UDP-glucose 4-epimerase), galM (galactose mutarotase), lacS (lactose transporter), and lacZ (␤-galactosidase). An analysis of the nucleotide sequence as well as Northern blotting and primer extension experiments revealed the presence of four promoters located upstream from galR, the gal operon, galM, and the lac operon of S. salivarius. Putative promoters with virtually identical nucleotide sequences were found at the same positions in the S. thermophilus gal-lac gene cluster. An additional putative internal promoter at the 3 end of galT was found in S. thermophilus but not in S. salivarius. The results clearly indicated that the gal-lac gene cluster was efficiently transcribed in both species. The Shine-Dalgarno sequences of galT and galE were identical in both species, whereas the ribosome binding site of S. thermophilus galK differed from that of S. salivarius by two nucleotides, suggesting that the S. thermophilus galK gene might be poorly translated. This was confirmed by measurements of enzyme activities.Streptococcus salivarius is an oral bacterium that is phylogenetically closely related to Streptococcus thermophilus, which is used in food fermentation (16,17,25,29). Both species were initially placed in the S. salivarius taxon as S. salivarius subsp. salivarius and S. salivarius subsp. thermophilus (7) but were regarded as distinct species by Schleifer et al. (28) on the basis of both genetic and phenetic criteria. These two species, together with Streptococcus vestibularis, form a distinct cluster within the streptococcal phylogenetic tree (13,16,25). Lactose, the principal energy source used by S. thermophilus for growth in milk, is transported into the cell by a permease (LacS) belonging to the glycoside-pentoside-hexuronide-cation symporter family (23). Lactose is hydrolyzed within the cell into glucose and galactose by ␤-galactosidase. Glucose is metabolized to lactic acid via the glycolytic, Embden-Meyerhof-Parnas pathway, whereas in most strains galactose cannot be metabolized and is expelled into the external medium (11,14). The organization of the galactose operon coding for the Leloir pathway enzymes in S. thermophilus has recently been elucidated (5, 24, 36), indicating that the inability of S. thermophilus to metabolize galactose is not caused by the absence of the genetic information required for the synthesis of suitable metabolic pathways. Moreover, the activities of the enzymes involved in the Leloir pathway (galactokinase, galactose-1-P uridylyltransferase, and...

No abstract