Lucie Gauthier

Transport and Phosphorylation of Xylitol by a Fructose Phosphotransferase System in Streptococcus mutans

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 XR and LG1 XR, 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 XR. 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 XR and LG1 XR 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.

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‐metabolizabie glucose/ mannose analogue 2‐deoxygiucose to isolate a comprehensive collection of mutants of the phosphoenoipyruvate: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, meli‐biose, galactose or fructose. We found that the frequency at which 2‐deoxygiucose‐resistant mutants Were isolated varied according to the growth substrate. The highest frequency was obtained with the combination galactose and 2‐deoxygiucose and was 15‐fold higher than the rate observed with the mixture melibiose and 2‐deoxygiucose, the combination that gave the lowest frequency. By combining results from: (i) Western biol 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. Class 1 was composed of strains devoid of IIIManL, a low‐molecular‐weight form of IIIManL (35200), class 2 was composed of strains exhibiting a reduced level of IIIManL, class 3 was composed of strains devoid of both forms of IIIMan (IIIManL as well as IIIManH, the high‐molecular‐weight form of IIIMan (38900)), class 4 was composed of mutants bearing a mutation in ptsH, the gene encoding HPr, class 5 was composed of mutants bearing a mutation in ptsl, the gene encoding EI, class 6 was composed of 2‐deoxygiucose‐resistant strains without any apparent defect in PTS components, and class 7 was composed of strains possessing both forms of IIIMan but abnormal levels of HPr and/or EI without any mutation in the ptsH and/or the ptsI genes. Preliminary characterization of representative strains of each class is reported.

The presence of two forms of the phosphocarrier protein HPr of the phosphoenolpyruvate: sugar phosphotransferase system in streptococci

The protein, HPr, a necessary component of the phosphoenolpyruvate phosphotransferase system (PTS) in bacteria, was purified from Streptococcus salivarius by column chromatography. The purified preparation gave only one band when analyzed by sodium dodecylsulfate gel electrophoresis or by isoelectric focusing in polyacrylamide gel (pI = 4.85). However, electrophoresis in Tris-containing buffers under non-denaturing conditions revealed 2 bands that could be phosphorylated by PEP in the presence of enzyme I of the PTS or by ATP with the HPr kinase. Homogeneous preparations of these 2 forms could be obtained by preparative electrophoresis. Each preparation exhibited only 1 band when analyzed by electrophoresis under non-denaturing conditions, indicating that the doublet observed before preparative electrophoresis was not an electrophoretic artefact. The electrophoretic mobility of each protein was not modified following heat-treatment at 100 degrees C for 20 min or storage at -40 degrees C for several months. Both HPr proteins catalyzed in vitro the PEP-dependent phosphorylation of glucose, but at a rate slightly lower than that observed with a preparation of HPr containing both forms of the protein. Both forms were also able to transfer the phosphate group from PEP to the other specific PTS proteins known in S salivarius. Rabbit polyclonal antibodies directed against each form reacted with both proteins. The presence of the 2 forms of HPr was detected in fresh cellular extracts of S salivarius; however, their intracellular ratio varied according to growth conditions. A doublet was also found in many other streptococcal species tested (S mutans, S sobrinus, S sanguis, S thermophilus, S bovis, S rattus) and also in L lactis.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of ptsH and ptsl gene expression in Streptococcus salivarius ATCC 25975

The transcriptional regulation of the Streptococcus salivarius ptsH and ptsl genes coding for the general energy‐coupling proteins HPr and enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system were investigated. These genes form an operon with the gene order ptsH–ptsl. Three distinct mRNA species were detected: a 0.5 kb transcript specific for ptsH, and two long transcripts (2.2 arid 2.4 kb) covering the whole pts operon. Transcription of all these mRNAs initiated at the same nucleotide located 9 bp downstream from a promoter located immediately upstream from the ptsH gene. The presence of a high‐energy stem–loop structure (T0) located at the begining of ptsl was responsible for the premature transcrrption termination generating the 0.5 kb ptsH‐specific transcript. The long transcripts ended in the poly(U) region of two rho‐independent‐like terminators (T1 and T2) at the 3′ end of ptsl. Studies with a 2‐deoxyglucose‐resistant spontaneous mutant of S. salivarius (L26) that produces an HPr–El fusion protein suggest that the regulation of HPr and El expression involves transcriptional as well as translational mechanisms.

HPr polymorphism in oral streptococci is caused by the partial removal of the N-terminal methionine

HPr is a small phosphocarrier protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system involved in the transport and phosphorylation of sugars. It has recently been reported that streptococci possess two forms of HPr having identical biochemical properties. In this communication, we show by N-terminal amino-acid sequencing and by ion-spray mass spectroscopy that these two forms differ by the presence or the absence of the N-terminal methionine.