Phosphoenolpyruvate Phosphotransferase System Research Articles

Enhanced fermentation of mannitol and release of cytotoxin by Clostridium difficile in alkaline culture media

Clostridium difficile ATCC 43255 fermented less than 10% of the mannitol in a medium at pH 7; however, when the initial pH of the medium was adjusted to 8.5 or 9, about 80% of the mannitol was fermented. Cell extracts of C. difficile phosphorylated mannitol with phosphoenolpyruvate, not ATP, indicating a phosphoenolpyruvate phosphotransferase system transport phosphorylation of mannitol. The phosphorylation product was dehydrogenated by D-mannitol-1-phosphate:NAD oxidoreductase. Growth at an initial pH of 8.5 yielded cytotoxin titers of 10(7) to 10(8) in Trypticase-yeast extract-mannitol medium, wit a titer of 10(8) as early as 13 h.

Glucose transport by a mutant of Streptococcus mutans unable to accumulate sugars via the phosphoenolpyruvate phosphotransferase system

Streptococcus mutans transports glucose via the phosphoenolpyruvate (PEP)-dependent sugar phosphotransferase system (PTS). Earlier studies indicated that an alternate glucose transport system functions in this organism under conditions of high growth rates, low pH, or excess glucose. To identify this system, S. mutans BM71 was transformed with integration vector pDC-5 to generate a mutant, DC10, defective in the general PTS protein enzyme I (EI). This mutant expressed a defective EI that had been truncated by approximately 150 amino acids at the carboxyl terminus as revealed by Western blot (immunoblot) analysis with anti-EI antibody and Southern hybridizations with a fragment of the wild-type EI gene as a probe. Phosphotransfer assays utilizing 32P-PEP indicated that DC10 was incapable of phosphorylating HPr and EIIAMan, indicating a nonfunctional PTS. This was confirmed by the fact that DC10 was able to ferment glucose but not a variety of other PTS substrates and phosphorylated glucose with ATP and not PEP. Kinetic assays indicated that the non-PTS system exhibited an apparent Ks of 125 microM for glucose and a Vmax of 0.87 nmol mg (dry weight) of cells-1 min-1. Sugar competition experiments with DC10 indicated that the non-PTS transport system had high specificity for glucose since glucose transport was not significantly by a 100-fold molar excess of several competing sugar substrates, including 2-deoxyglucose and alpha-methylglucoside. These results demonstrate that S. mutans possesses a glucose transport system that can function independently of the PEP PTS.

Regulation of ATP-dependent P-(Ser)-HPr formation in Streptococcus mutans and Streptococcus salivarius

Sugar transport via the phosphoenolpyruvate (PEP) phosphotransferase system involves PEP-dependent phosphorylation of the general phosphotransferase system protein, HPr, at histidine 15. However, gram-positive bacteria can also carry out ATP-dependent phosphorylation of HPr at serine 46 by means of (Ser)HPr kinase. In this study, we demonstrate that (Ser)HPr kinase in crude preparations of Streptococcus mutans Ingbritt and Streptococcus salivarius ATCC 25975 is membrane associated, with pH optima of 7.0 and 7.5, respectively. The latter organism possessed 7- to 27-fold-higher activity than S. mutans NCTC 10449, GS-5, and Ingbritt strains. The enzyme in S. salivarius was activated by fructose-1,6-bisphosphate (FBP) twofold with 0.05 mM ATP, but this intermediate was slightly inhibitory with 1.0 mM ATP at FBP concentrations up to 10 mM. Similar inhibition was observed with the enzyme from S. mutans Ingbritt. A variety of other glycolytic intermediates had no effect on kinase activity under these conditions. The activity and regulation of (Ser)HPr kinase were assessed in vivo by monitoring P-(Ser)-HPr formation in steady-state cells of S. mutans Ingbritt grown in continuous culture with limiting glucose (10 and 50 mM) and with excess glucose (100 and 200 mM). All four forms of HPr [free HPr, P approximately (His)-HPr, P-(Ser)-HPr, and P approximately (His)-P-(Ser)-HPr] could be detected in the cells; however, significant differences in the intracellular levels of the forms were apparent during growth at different glucose concentrations. The total HPr pool increased with increasing concentrations of glucose in the medium, with significant increases in the P-(Ser)-HPr and P approximately HHis)-P-(Ser)-HPr concentrations. For example, while total PEP-dependent phosphorylation [P approximately(His)-HPr plus P approximately (His)-P-(Ser)-HPr] varied only from 21.5 to 52.5 microgram mg of cell protein (-1) in cells grown at the four glucose concentrations, the total ATP-dependent phosphorylation [P-(Ser)-HPr plus P approximately (His)-P-(Ser)-HPr] increased 12-fold from the 10 mM glucose-grown cells (9.1 microgram mg of cell protein (-1) to 106 and 105 microgram mg(-1) in the 100 and 200 mM glucose-grown cultures, respectively. (Ser)HPr kinase activity in membrane preparations of the cells varied little between the 10, 50, and 100 mM glucose-grown cells but increased threefold in the 200 mM glucose-grown cells. The intracellular levels of ATP, glucose-6-phosphate, and FBP increased with external glucose concentration, with the level of FBP being 3.8-fold higher for cells grown with 200 mM glucose than for those grown with 10 mM glucose. However, the variation in the intracellular levels of FBP, particularly between cells grown with 100 and 200 mM glucose, did not correlate with the extent of P-(Ser)-HPr formation, suggesting that the activity of (Ser)HPr kinase is not critically dependent on the availability of intracellular FBP.

Current research on the genetics of lactic acid production in lactic acid bacteria

Abstract Lactic acid derived from lactose is a major by-product of energy production in lactic acid bacteria. The uptake of lactose by these organisms is mediated either by the lactose phosphoenolpyruvate-phosphotransferase system (lactose PEP-PTS), or by a lactose-proton symport system. The disaccharide is then converted to lactate with the concomitant production of ATP. In Lactococcus lactis the genes encoding the lactose PEP-PTS, phospho-β-galactosidase and the tagatose 6-phosphate pathway enzymes are plasmid encoded, while other genes required for lactate synthesis, including those of the Embden-Meyerhof pathway, are on the chromosome. We have compiled a current list of genes required for lactate synthesis in the lactic acid bacteria that have been cloned and characterized and discuss the present status of genetic research in this area. The analyses of the L. lactis lac operon have yielded one of the most detailed pictures of genetic regulation in this bacterium. The operon has been fully sequenced, the regulatory protein LacR which represses lac operon transcription has been purified and its properties determined, and the operon promoters and operators have been identified. Investigations of chromosomally encoded L. lactis genes have resulted in the identification and characterization of pfk, pyk, ldh, tpi and gap , which encode phosphofructokinase, pyruvate kinase, l -(+)-lactate dehydrogenase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, respectively. All of these enzymes (except triosephosphate isomerase) are known from previous studies to be important in metabolite level regulation of the pathway, pfk, pyk and ldh are organized into a tricistronic operon (the las operon), while tpi and gap are in monocistronic units. The las operon is so far unique to L. lactis . A number of investigators have studied the effect of gene dosage on glycolytic flux in lactic acid bacteria and their results are reviewed. We have introduced multiple copies of pfk, pyk, Idh and the las operon into L. lactis and report the effect of the increase in gene dosage on enzyme levels and the rate of lactic acid production.

Nutrient transport by ruminal bacteria: a review.

Fermentation pathways have been elucidated for predominant ruminal bacteria, but information is limited concerning the specific transport mechanisms used by these microorganisms for C, energy, and N sources. In addition, it is possible that changes in ruminal environmental conditions could affect transport activity. Five carrier-mediated soluble nutrient transport mechanisms have been identified in bacteria: 1) facilitated diffusion, 2) shock sensitive systems, 3) proton symport, 4) Na+ symport, and the 5) phosphoenolpyruvate phosphotransferase system (PEP-PTS). Several regulatory mechanisms are also involved at the cell membrane to coordinate utilization of different sugars. Recent research has shown that predominant ruminal bacteria are capable of transporting soluble nutrients by several of the mechanisms outlined above. Megasphaera elsdenii, Selenomonas ruminantium, and Streptococcus bovis transport glucose by the PEP-PTS, and S. ruminantium and S. bovis also possess PEP-PTS activity for disaccharides. Glucose PTS activity in S. bovis was highest at a growth pH of 5.0, low glucose concentrations, and a dilution rate of .10 h-1. The cellulolytic ruminal bacterium Fibrobacter succinogenes uses a Na+ symport mechanism for glucose transport that is sensitive to low extracellular pH and ionophores. Sodium also stimulated cellobiose transport by F. succinogenes, and there is evidence for a proton symport in the transport of both arabinose and xylose by S. ruminantium. A chemical gradient of Na+ seems to play an important role in AA transport in several ruminal bacteria. Studying nutrient transport mechanisms in ruminal bacteria will lead to a better understanding of the ruminal fermentation.

Vesicles prepared from Streptococcus mutans demonstrate the presence of a second glucose transport system.

Streptococcus mutans, an important aetiological agent of dental caries, is known to transport glucose via the phosphoenolpyruvate (PEP) phosphotransferase system (PTS). An alternative non-PTS glucose transport system in S. mutans Ingbritt was suggested by the increased ATP-dependent phosphorylation of glucose and the presence of higher cellular concentrations of free glucose in cells grown in continuous culture under PTS-repressed conditions compared to those resulting in optimal PTS activity. A method was developed for the preparation of membrane vesicles in order to study this system in the absence of PTS activity. These vesicles had very low activity of the cytoplasmic enzymes, glucokinase, pyruvate kinase and lactate dehydrogenase. This, coupled with the lack of glycolytic activity and the inability to transport glucose, suggested that the vesicles would also be deficient in PTS activity because of the absence of the general soluble PTS proteins, Enzyme I and HPr, required for the transport of all PTS sugars. Freeze-fracture electron microscopy and membrane H(+)-ATPase analysis indicated that over 90% of the vesicles had a right-side-out orientation. Vesicles from cells grown in continuous culture under PTS-dominant and PTS-repressed conditions both exhibited glucose counterflow. This indicates the presence of a constitutive non-PTS carrier in the organism capable of transporting glucose and utilizing ATP for glucose phosphorylation. Analysis of growth yields of cells grown under PTS-repressed and PTS-optimal conditions suggests that ATP, or an equivalent high energy molecule, must be involved in the actual transport process. This analysis is consistent with an ATP-binding protein model such as the Msm transport system reported by R. R. B. Russell and coworkers (J Biol Chem 267, 4631-4637), but it does not exclude the possibility of a separate permease for glucose.

Genetic regulation of fructosyltransferase in Streptococcus mutans

Streptococcus mutans possesses several extracellular sucrose-metabolizing enzymes which have been implicated as important virulence factors in dental caries. This study was initiated to investigate the genetic regulation of one of these enzymes, the extracellular fructosyltransferase (Ftf). Fusions were constructed with the region upstream of the S. mutans GS5 Ftf gene (ftf) and a promoterless chloramphenicol acetyltransferase (CAT) gene. The fusions were integrated at a remote site in the chromosome, and transcriptional activity in response to the addition of various carbohydrates to the growth medium was measured. A significant increase in CAT activity was observed when glucose-grown cells were shifted to sucrose-containing medium. Sucrose-induced expression was repressed immediately upon addition of phosphoenolpyruvate phosphotransferase system sugars to the growth media. Deletion analysis of the ftf upstream region revealed that an inverted repeat structure was involved in the control of ftf expression in response to carbohydrate. However, the control of the level of ftf transcription appeared to involve a region distinct from that mediating carbohydrate regulation. CAT gene fusions also were constructed with the ftf upstream region from S. mutans V403, a fructan-hyperproducing strain which synthesizes increased levels of Ftf. Sequence analysis of the upstream ftf region in this strain revealed several nucleotide sequence changes which were associated with high-level ftf expression. Comparison of the GS5 and V403 ftf expression patterns suggested the presence of a trans-acting factor(s) involved in modulation of ftf expression in response to carbohydrate. This factor(s) was either absent or altered in V403, resulting in the inability of this organism to respond to the presence of carbohydrate. The sequences of the ftf regions from three additional fructan-hyperproducing strains were determined and compared with that of V403. Only one strain displayed nucleotide changes similar to those of V403. Two additional strains did not have these changes, suggesting that several mechanisms for up-regulation of ftf expression exist.

Isolation, characterization, and nucleotide sequence of the Streptococcus mutans mannitol-phosphate dehydrogenase gene and the mannitol-specific factor III gene of the phosphoenolpyruvate phosphotransferase system.

Streptococcus mutans, the causative agent of dental caries, utilizes carbohydrates by means of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The PTS facilitates vectorial translocation of metabolizable carbohydrates to form the corresponding sugar-phosphates, which are subsequently converted to glycolytic intermediates. The PTS consists of both sugar-specific and sugar-independent components. Complementation of an Escherichia coli mtlD mutation with a streptococcal recombinant DNA library allowed isolation of the mannitol-1-phosphate dehydrogenase gene (mtlD) and the adjacent sugar-specific mannitol factor III gene (mtlF) from S. mutans. Subsequent transposon mutagenesis of the complementing DNA fragment with Tn5seq1 defined the region that encodes the mtlD-complementing activity, the streptococcal mtlD gene. Nucleotide sequence analysis of this region revealed two complete open reading frames (ORFs) from within the streptococcal mannitol PTS operon. One ORF encodes the mtlD gene product, a 43.0-kDa protein which exhibits similarity to the E. coli and Enterococcus faecalis mannitol-1-phosphate dehydrogenases. The second ORF encodes a 15.8-kDa protein which exhibits similarity to mannitol factor III proteins from several bacterial species. In vitro transcription-translation assays were used to produce proteins of the sizes predicted by the streptococcal ORFs. These data indicate that the S. mutans mannitol PTS utilizes an enzyme II-factor III complex similar to the mannitol system found in other gram-positive organisms, as opposed to that of E. coli, which utilizes an independent enzyme II system.

Mode of sugar phosphorylation inClostridium thermocellum

Clostridium thermocellum uses cellobiose as a favored carbon source. After growth in cellobiose, cells cannot grow on glucose or fructose until they mutate to glucose or fructose utilization (1). This usually requires 180–200 h. One explanation for the initial lack of cell growth on monosaccharides is that cellobiose uptake and metabolism occur by a different phosphorylative mechanism than that for glucose or fructose, and the cells require a mutation to activate the new type of system. Our results show that there is no difference in the mode of phosphorylation of the three sugars. They all use an adenosine triphosphate-dependent mechanism rather than the phosphoenolpyruvate-phosphotransferase system.

Mutational analysis of the enzyme III Glc of the phosphoenolpyruvate phosphotransferase system in Escherichia coli

The phosphoenolpyruvate phosphotransferase system (PTS) component EIII Glc is responsible for transport and phosphorylation of glucose via EII Glc. 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 EIII Glc was performed in order to identify crucial residues mediating the interactions between EIII Glc and its target proteins. Such mutations were isolated by in vitro hydroxylamine mutagenesis of the cloned EIII Glc gene, crr. Five mutated EIII Glc impaired in the function of inducer exclusion were obtained. However, these mutations did not abolish the function of EIII Glc 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: Gly 47 → Ser 47 for the HA2 and HA5 mutations, Ala 76 → Thr 76 for HA4 mutation and Ser 78 → Phe 78 for HA3 mutation, indicative of quaternary interactions between the corresponding region of EIII Glc and its target protein(s).

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 (p I = 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°C for 20 min or storage at −40°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. A single form was detected in L casei, E faecalis, S aureus, and B subtilis. It thus appears that the presence of 2 forms of HPr is restricted to the genera Streptococcus and Lactococcus.

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.

Xylose uptake by the ruminal bacterium Selenomonas ruminantium

Selenomonas ruminantium HD4 does not use the phosphoenolpyruvate phosphotransferase system to transport xylose (S. A. Martin and J. B. Russell, J. Gen. Microbiol. 134:819-827, 1988). Xylose uptake by whole cells of S. ruminantium HD4 was inducible. Uptake was unaffected by monensin or lasalocid, while oxygen, o-phenanthroline, and HgCl2 were potent inhibitors. Menadione, antimycin A, and KCN had little effect on uptake, and acriflavine inhibited uptake by 23%. Sodium fluoride decreased xylose uptake by 10%, while N,N'-dicyclohexylcarbodiimide decreased uptake by 31%. Sodium arsenate was a strong inhibitor (83%), and these results suggest the involvement of a high-energy phosphate compound and possibly a binding protein in xylose uptake. The protonophores carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, and SF6847 inhibited xylose uptake by 88, 82, and 43%, respectively. The cations Na+ and K+ did not stimulate xylose uptake. The kinetics of xylose uptake were nonlinear, and it appeared that more than one uptake mechanism may be involved or that two proteins (i.e., a binding protein and permease protein) with different affinities for xylose were present. Excess (10 mM) glucose, sucrose, or maltose decreased xylose uptake less than 40%. Uptake was unaffected at extracellular pH values between 6.0 and 8.0, while pH values of 5.0 and 4.0 decreased uptake 28 and 24%, respectively. The phenolic monomers p-coumaric acid and vanillin inhibited growth on xylose and xylose uptake more than ferulic acid did. The predominant end products resulting from the fermentation of xylose were lactate (7.5 mM), acetate (4.4 mM), and propionate (5.1 nM), and the Yxylose was 24.1 g/mol.

Prospects for the genetic manipulation of lactobacilli

Efforts are underway in a number of laboratories around the world to develop methods for the improvement of Lactobacillus strains by recombinant DNA techniques. Research has centered on: (i) characterization and construction of chimeric shuttle vectors based on endogenous Lactobacillus plasmids which are capable of replicating in lactobacilli; (ii) molecular cloning of genes and operons from lactobacilli encoding important pathways such as the lactose : phospho enolpyruvate phosphotransferase system, phosphogalactoside-β- d-galactohydrolase, and β-galactosidase; and (iii) methods for introduction of genes in vivo and in vitro through conjugation, transfection and transformation. The lack of natural gene exchange systems has prompted research efforts to devise a protoplast transformation system. Initial early successes in gene cloning, vector development, transfection, conjugation, protoplast fusion and, recently, transformation, have laid the ground-work for rapid development of gene exchange systems for the genus.

Prospects for the genetic manipulation of lactobacilli

Efforts are underway in a number of laboratories around the world to develop methods for the improvement of Lactobacillus strains by recombinant DNA techniques. Research has centered on: (i) characterization and construction of chimeric shuttle vectors based on endogenous Lactobacillus plasmids which are capable of replicating in lactobacilli; (ii) molecular cloning of genes and operons from lactobacilli encoding important pathways such as the lactose : phosphoenolpyruvate phosphotransferase system, phosphogalactoside-β-d-galactohydrolase, and β-galactosidase; and (iii) methods for introduction of genes in vivo and in vitro through conjugation, transfection and transformation. The lack of natural gene exchange systems has prompted research efforts to devise a protoplast transformation system. Initial early successes in gene cloning, vector development, transfection, conjugation, protoplast fusion and, recently, transformation, have laid the ground-work for rapid development of gene exchange systems for the genus.

Preferential Utilization of Cellobiose by Thermomonospora curvata

Thermomonospora curvata was cultivated on mineral salts medium containing glucose and cellobiose under conditions that increasingly favored the uptake of glucose. In each case cellobiose was utilized in preference to glucose and induced beta-glucosidase and endoglucanase activity. [C]glucose metabolism studies indicated that cellobiose was not cleaved by extracellular beta-glucosidase and transported as glucose. No evidence of cellobiose phosphorylase or a cellobiose-specific phosphoenolpyruvate-phosphotransferase system was observed.

Enterotoxin A synthesis in Staphylococcus aureus: inhibition by glycerol and maltose.

Studies indicated that prior growth of Staphylococcus aureus 196E on glycerol or maltose led to cells with repressed ability to produce staphylococcal enterotoxin A (SEA). A PTS- mutant (196E-MA) lacking the phosphoenolpyruvate phosphotransferase system (PTS), derived from strain 196E, showed considerably less repression of SEA synthesis when cells were grown in glycerol or maltose. Since SEA synthesis is not repressed in the PTS- mutant, repression of toxin synthesis by glycerol, maltose or glucose in S. aureus 196E appears to be related to the presence of a functional PTS irrespective of whether the carbohydrate requires the PTS for cell entry. With lactose as an inducer, glucose, glycerol, maltose or 2-deoxyglucose repressed the synthesis of beta-galactosidase in S. aureus 196E. It is postulated that these compounds repress enzyme synthesis by an inducer exclusion mechanism involving phosphorylated sugar intermediates. However, inducer exclusion probably does not explain the mechanism of repression of SEA synthesis by carbohydrates.

Protonmotive force driven 6-deoxyglucose uptake by the oral pathogen, Streptococcus mutans Ingbritt.

Streptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (3H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (delta p) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference (delta psi) dissipator, KSCN. The membrane ATPase inhibitor, N,N1-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (delta pH) and delta psi. ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to delta pH.

Enterotoxin A production in Staphylococcus aureus: inhibition by glucose.

In this study, we investigated the relationship between carbohydrate metabolism and repression of staphylococcus enterotoxin A (SEA) in Staphylococcus aureus 196E and a pleiotrophic mutant derived from strain 196E. The mutant, designated at strain 196E-MA, lacked a functional phosphoenolpyruvate phosphotransferase system (PTS). The mutant produced acid, under aerobic conditions, from only glucose and glycerol. The parent strain contained an active PTS, and aerobically produced acid from a large number of carbohydrates. Prior growth in glucose led to repression of SEA synthesis in the parent strain; addition to the casamino acids enterotoxin production medium (CAS) led to more severe repression of toxin synthesis. The repression was not related to pH decreases produced by glucose metabolism. When S. aureus 196E was grown in the absence of glucose, there was inhibition of toxin production as glucose level was increased in CAS. The inhibition was related to pH decrease and was unlike the repression observed with glucose-grown strain 196E. The inhibition of SEA synthesis in mutant strain 196E-MA was approximately the same in cells grown with or without glucose and was pH related. Repression of SEA synthesis similar to that seen with glucose-grown S. aureus 196E could not be demonstrated in the mutant. In addition, glucose-grown S. aureus 196E neither synthesized beta-galactosidase nor showed respiratory activity with certain tricarboxylic acid (TCA) cycle compounds. Glucose-grown strain 196E-MA, however, did not show suppressed respiration of TCA cycle compounds; beta-galactosidase was not synthesized because the mutant lacked a functional PTS.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence of a quinoprotein glucose dehydrogenase apoenzyme in several strains ofEscherichia coli

When grown on glucose in K+-limited chemostat culture, or in batch culture with or without 2,4-dinitrophenol, several strains of Escherichia coli (including the type strain) were found to synthesize a quinoprotein glucose dehydrogenase apoenzyme. The pyridine nucleotides, NAD+ and NADP+, would not serve as cofactor, but activity could be demonstrated upon addition of 2,7,9-tricarboxy-1 H-pyrrolo(2,3-f)quinoline-4,5-dione (PQQ). Thus, in the presence of PQQ, but not in its absence, glucose was oxidized to gluconic acid. A mutant of E. coli PC 1000 was isolated that lacked Enzyme I of the phosphoenolpyruvate phosphotransferase system (PTS) but still synthesized the glucose dehydrogenase apoenzyme. Whereas this mutant would not grow on glucose in the absence of PQQ, it would do so in the presence of low concentrations (1 μM) of this cofactor. On the basis of these observations, it is concluded that the protein (apoenzyme) formed is a genuine glucose dehydrogenase, but that it is not functional in growing cells due to their inability to synthesize the appropriate cofactor (PQQ), at least under these conditions.