Galactose metabolism by Streptococcus mutans

The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-beta-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.     

Galactose Metabolism by Streptococcus mutans

The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-β-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.     

Uptake patterns and transport enhancements in cultures of hamster cells deprived of carbohydrates.

Dense cell cultures of the hamster lines, NIL, and polyoma transformed NIL were exposed to culture media containing various sugars (or no sugar). Various responses to these culture conditions were observed as changes in the uptake of galactose and its subsequent metabolism. Cells deprived of sugar have higher uptake rates for galactose and markedly different accumulation products from identical cells treated with sugar. A persistent increase in the transport of the amino acid, cycloleucine, was also observed as a response to culture conditions devoid of sugar     

A genomic view of sugar transport in Mycobacterium smegmatis and Mycobacterium tuberculosis

We present a comprehensive analysis of carbohydrate uptake systems of the soil bacterium Mycobacterium smegmatis and the human pathogen Mycobacterium tuberculosis. Our results show that M. smegmatis has 28 putative carbohydrate transporters. The majority of sugar transport systems (19/28) in M. smegmatis belong to the ATP-binding cassette (ABC) transporter family. In contrast to previous reports, we identified genes encoding all components of the phosphotransferase system (PTS), including permeases for fructose, glucose, and dihydroxyacetone, in M. smegmatis. It is anticipated that the PTS of M. smegmatis plays an important role in the global control of carbon metabolism similar to those of other bacteria. M. smegmatis further possesses one putative glycerol facilitator of the major intrinsic protein family, four sugar permeases of the major facilitator superfamily, one of which was assigned as a glucose transporter, and one galactose permease of the sodium solute superfamily. Our predictions were validated by gene expression, growth, and sugar transport analyses. Strikingly, we detected only five sugar permeases in the slow-growing species M. tuberculosis, two of which occur in M. smegmatis. Genes for a PTS are missing in M. tuberculosis. Our analysis thus brings the diversity of carbohydrate uptake systems of fast- and a slow-growing mycobacteria to light, which reflects the lifestyles of M. smegmatis and M. tuberculosis in their natural habitats, the soil and the human body, respectively.     

Regulation of methyl beta-galactoside permease activity in pts and crr mutants of Salmonella typhimurium

Summary We have studied the regulation of the synthesis and activity of a major galactose transport system, that of methyl -galactoside (MglP), in mutants of Salmonella typhimurium. Two classes of mutation that result in a (partially) defective phosphoenolpyruvate: sugar phosphotransferase system (PTS) interfere with MglP synthesis. pts mutations, which eliminate the general proteins of the PTS Enzyme I and/or HPr and crr mutations, which result in a defective glucose-specific factor III^Gle of the PTS, lead to a low MglP activity, as measured by methyl -galactoside transport. In both ptsH,I, and crr mutants the amount of galactose binding protein, one of the components of MglP, is only 5%–20% of that in wild-type cells, as measured with a specific antibody. We conclude that synthesis of MglP is inhibited in pts and crr mutants. Once the transport system is synthesized, its transport activity is not sensitive to PTS sugars (i.e., no inducer exclusion occurs). The defect in pts and crr mutants with respect to MglP synthesis can be relieved in two ways: by externally added cyclic adenosine 3, 5-monophosphate (cAMP) or by a mutation in the cAMP binding protein. The conclusion that MglP synthesis is dependent on cAMP is supported by the finding that its synthesis is also defective in mutants that lack adenylate cyclase. pts and crr mutations do not affect growth of S. typhimurium on galactose, however, since the synthesis and activity of the other major galactose transport system, the galactose permease (GalP), is not sensitive to these mutations. If the galactose permease is eliminated by mutation, growth of pts and crr mutants on low concentrations of galactose becomes very slow due to inhibited MglP synthesis. Residual growth observed at high galactose concentrations is the result of yet another transport system with low affinity for galactose.     

The chitin disaccharide, N,N'-diacetylchitobiose, is catabolized by Escherichia coli and is transported/phosphorylated by the phosphoenolpyruvate:glycose phosphotransferase system

We have previously reported that wild type strains of Escherichia coli grow on the chitin disaccharide N,N'-diacetylchitobiose, (GlcNAc)(2), as the sole source of carbon (Keyhani, N. O., and Roseman, S. (1997) Proc. Natl. Acad. Sci., U. S. A. 94, 14367-14371). A nonhydrolyzable analogue of (GlcNAc)(2,) methyl beta-N, N'-[(3)H]diacetylthiochitobioside ([(3)H]Me-TCB), was used to characterize the disaccharide transport process, which was found to be mediated by the phosphoenolpyruvate:glycose phosphotransferase system (PTS). Here and in the accompanying papers (Keyhani, N. O., Boudker, O., and Roseman, S. (2000) J. Biol. Chem. 275, 33091-33101; Keyhani, N. O., Bacia, K., and Roseman, S. (2000) J. Biol. Chem. 275, 33102-33109; Keyhani, N. O., Rodgers, M., Demeler, B., Hansen, J., and Roseman, S. (2000) J. Biol. Chem. 275, 33110-33115), we report that transport of [(3)H]Me-TCB and (GlcNAc)(2) involves a specific PTS Enzyme II complex, requires Enzyme I and HPr of the PTS, and results in the accumulation of the sugar derivative as a phosphate ester. The phosphoryl group is linked to the C-6 position of the GlcNAc residue at the nonreducing end of the disaccharide. The [(3)H]Me-TCB uptake system was induced only by (GlcNAc)(n), n = 2 or 3. The apparent K(m) of transport was 50-100 micrometer, and effective inhibitors of uptake included (GlcNAc)(n), n = 2 or 3, cellobiose, and other PTS sugars, i.e. glucose and GlcNAc. Presumably the PTS sugars inhibit by competing for PTS components. Kinetic properties of the transport system are described.     

Single‐cell characterization of metabolic switching in the sugar phosphotransferase system of Escherichia coli

The utilization of several sugars in Escherichia coli is regulated by the Phosphotransferase System (PTS), in which diverse sugar utilization modules compete for phosphoryl flux from the general PTS proteins. Existing theoretical work predicts a winner‐take‐all outcome when this flux limits carbon uptake. To date, no experimental work has interrogated competing PTS uptake modules with single‐cell resolution. Using time‐lapse microscopy in perfused microchannels, we analyzed the competition between N‐acetyl‐glucosamine and sorbitol, as representative PTS sugars, by measuring both the expression of their utilization systems and the concomitant impact of sugar utilization on growth rates. We find two distinct regimes: hierarchical usage of the carbohydrates, and co‐expression of the genes for both systems. Simulations of a mathematical model incorporating asymmetric sugar quality reproduce our metabolic phase diagram, indicating that under conditions of nonlimiting phosphate flux, co‐expression is due to uncoupling of both sugar utilization systems. Our model reproduces hierarchical winner‐take‐all behaviour and stochastic co‐expression, and predicts the switching between both strategies as a function of available phosphate flux. Hence, experiments and theory both suggest that PTS sugar utilization involves not only switching between the sugars utilized but also switching of utilization strategies to accommodate prevailing environmental conditions.     

Improvement of <Emphasis Type="Italic">Escherichia coli</Emphasis> production strains by modification of the phosphoenolpyruvate:sugar phosphotransferase system

The application of metabolic engineering in Escherichia coli has resulted in the generation of strains with the capacity to produce metabolites of commercial interest. Biotechnological processes with these engineered strains frequently employ culture media containing glucose as the carbon and energy source. In E. coli, the phosphoenolpyruvate:sugar phosphotransferase system (PTS) transports glucose when this sugar is present at concentrations like those used in production fermentations. This protein system is involved in phosphoenolpyruvate-dependent sugar transport, therefore, its activity has an important impact on carbon flux distribution in the phosphoenolpyruvate and pyruvate nodes. Furthermore, PTS has a very important role in carbon catabolite repression. The properties of PTS impose metabolic and regulatory constraints that can hinder strain productivity. For this reason, PTS has been a target for modification with the purpose of strain improvement. In this review, PTS characteristics most relevant to strain performance and the different strategies of PTS modification for strain improvement are discussed. Functional replacement of PTS by alternative phosphoenolpyruvate-independent uptake and phosphorylation activities has resulted in significant improvements in product yield from glucose and productivity for several classes of metabolites. In addition, inactivation of PTS components has been applied successfully as a strategy to abolish carbon catabolite repression, resulting in E. coli strains that use more efficiently sugar mixtures, such as those obtained from lignocellulosic hydrolysates.     

Effect of anaerobiosis, dinitrophenol and fluoride on the active intestinal transport of galactose in snail.

The active transport of galactose across the intestinal wall (everted sacs) of the snail Cryptomphalus hortensis Müller has been studied in vitro, under several metabolic conditions. Anaerobiosis does not change the serosal/mucosal galactose gradients which are developed in oxygen atmosphere. Dinitrophenol (10(-4) M) greatly increased the O2 uptake by the tissue and clearly inhibits the sugar transport. At 5 times 10(-4) M concentration, DNP totally prevents the uphill transport while the O2 uptake is normal. The inhibition produced by DNP does not increase by anaerobiosis. Fluoride inhibits the galactose transport and also the O2 uptake. It is deduced that in snail intestine the energy for the active transport of galactose can be supplied by aerobic as much as by anaerobic metabolism. The inhibition by dinitrophenol seems to be independent of its uncoupling action on the oxidative phosphorylation. The inhibitory effect of NaF may be due both to glycolisis inhibition and to alteration of the digestive epithelium.     

Interactions in vivo between IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system and the glycerol and maltose uptake systems of Salmonella typhimurium

Our previous studies indicated that the ability of phosphoenolpyruvate:sugar phosphotransferase system (PTS) substrates to inhibit the uptake of glycerol or maltose in Salmonella typhimurium is dependent on the relative cellular content of the PTS-sensitive uptake system and of the PTS protein IIIGlc. Our present study confirms and extends those observations. The maltose and glycerol uptake systems are rendered (wholly or partially) insensitive to PTS inhibition by the presence of a second PTS-sensitive uptake system (respectively that for glycerol or maltose) and its substrate. Both the second PTS-sensitive uptake system and its substrate were needed for this protective effect. Galactose and the galactose permease (a PTS-insensitive transport system) did not have any effect on PTS-mediated inhibition of the maltose uptake system. The protective effect of the second PTS-sensitive uptake system and its substrate is counteracted by increasing the cellular levels of IIIGlc. Overproduction of IIIGlc in crr-plasmid-containing strains renders the glycerol and maltose uptake systems hypersensitive to inhibition by PTS substrates. We interpret our results on the basis of a stoichiometric interaction between IIIGlc and a PTS-sensitive uptake system, in which the IIIGlc--transport-system complex is inactive. Competition between two PTS-sensitive transport systems for formation of inactive complex with IIIGlc lowers the free intracellular concentration of IIIGlc resulting in a mutual protective effect against inhibition by IIIGlc.     

Role of the phosphoenolpyruvate-dependent fructose phosphotransferase system in the utilization of mannose by Escherichia coli.

Mutants of Escherichia coli devoid of the membrane-spanning proteins PtsG and PtsMP, which are components of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and which normally effect the transport into the cells of glucose and mannose, do not grow upon or take up either sugar. Pseudorevertants are described that take up, and grow upon, mannose at rates strongly dependent on the mannose concentration in the medium (apparent Km > 5 mM); such mutants do not grow upon glucose but are derepressed for the components of the fructose operon. Evidence is presented that mannose is now taken up via the fructose-PTS to form mannose 6-phosphate, which is further utilized for growth via fructose 6-phosphate and fructose 1,6-bisphosphate.     

How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria.

The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.     

Transport and phosphorylation of D-galactose in renal cortical cells.

An improved analytical procedure for the extraction and determination of total, free and phosphorylated tissue sugar is described. This method, employing ZnSO4 plus Ba(OH)2 for the precipitation of sugar phosphates, yields values identical with those obtained by the more laborious separation of free and phosphorylated sugar by ion-exchange chromatography. Erroneous values for free sugar due to the action of a Zn2+ -activated phosphatase and/or the lability to acids of some sugar phosphates, are avoided. Using this technique for the sudy of transport and phosphorylation of D-galactose in rabbit renal cortical slices and tissue extracts, it was found: 1. The cellular uptake of D-galactose was associated with the appearance of both free and phosphorylated sugar whether or not external Na+ was present. At 1 mM sugar, galactose was accumulated in the cells against a modest concentration gradient of 1.445 +/- 0.097 (n = 17). Galactose phosphate appeared in the cells considerably faster than free sugar under conditions of net uptake as well as of steady-state exchange (pulse-labelling). 2. Increasing saline pH (6-8) increased the cellular levels of sugar phosphate without affecting the steady-state values of free sugar. With tissue extracts, increasing pH also stimulated the activity of galactokinase and the dephosphorylation of galactose 1-phosphate by a Zn2+ -activated phosphatase. 3. 0.5 mM phlorizin inhibited the tissue uptake of galactose and its subsequent oxidation to CO2 only to a minor degree (30 and 10%, respectively). The absence of external Na+ further depressed the phlorizin effect. Preincubation of the tissue with phlorizin and subsequent washing in part abolished the inhibitory effect. The data suggest that a major portion of the galactose uptake by the tissue proceeds by a mechanism with a low affinity for phlorizin. 4. Efflux studies showed that the wash-out of free galactose from slices was associated with a net decrease of both free and phosphorylated tissue sugar. 5. The above results suggest the possibility that phosphorylation may represent a step in the Na+ -independent, phloretin-sensitive transfer of D-galactose across the antiluminal cell membrane. The participation of intracellular galactokinase and a Zn2+ -activated alkaline phosphatase in the maintenance of the steady state of free and phosphorylated galactose in the cells has been demonstrated.     

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.     

Two Uptake Systems for Fructose in Lactococcus lactis subsp. cremoris FD1 Produce Glycolytic and Gluconeogenic Fructose Phosphates and Induce Oscillations in Growth and Lactic Acid Formation

Fructose transport in lactococci is mediated by two phosphotransferase systems (PTS). The constitutive mannose PTS has a broad specificity and may be used for uptake of fructose with a fructose saturation constant (K_Fru) of 0.89 mM, giving intracellular fructose 6-phosphate. The inducible fructose PTS has a very small saturation constant (K_Fru, <17 μM), and the fructose 1-phosphate produced enters the Embden-Meyerhof-Parnas (EMP) pathway as fructose 1,6-diphosphate. Growth in batch cultures of Lactococcus lactis subsp. cremoris FD1 in a yeast extract medium with fructose as the only sugar is poor both with respect to specific growth rate and biomass yield, whereas the specific lactic acid production rate is higher than those in similar fermentations on other sugars metabolized via the EMP pathway, e.g., glucose. In fructose-limited chemostat cultures, the biomass concentration exhibits a strong correlation with the dilution rate, and starting a continuous culture at the end of a batch fermentation leads to large and persistent oscillations in the biomass concentration and specific lactic acid production rate. Two proposed mechanisms underlying this strange growth pattern follow. (i) Fructose transported via the fructose PTS cannot be converted into essential biomass precursors (glucose 6-phosphate or fructose 6-phosphate), because L. lactis subsp. cremoris FD1 is devoid of fructose 1,6-diphosphatase activity. (ii) The fructose PTS apparently produces a metabolite (presumably fructose 1-phosphate) which exerts catabolite repression of both mannose PTS and lactose PTS. Since the repressed mannose PTS and lactose PTS are shown to have identical maximum molar transport rates, the results indicate that it is the general PTS proteins which are repressed.     

Transport and phosphorylation of 2-deoxy-D-galactase in renal cortical cells.

The transport and phosphorylation of 2-deoxy-D-[3H]galactose in rabbit renal cortical cells was studied. 1. The uptake of 2-deoxy-galactose by cortical slices is associated with an appearance of both free and phosphorylated sugar in the cells. At 1 mM external sugar the cells establish a steady-state gradient of free 2-deoxy-galactose of 3.97 +/- 0.15 (23 animals). 2. The acid-labile sugar phosphate accumulated in the tissue has been identified by a combination of paper and radio-chromatography, as well as on the basis of some of its chemical properties, as 2-deoxy-D-galactose 1-phosphate. Ice-cold trichloroacetic acid produces a decomposition of this compound. 3. Increasing external pH (6-8) brings about a decrease in the steady-state levels of both free and phosphorylated sugar in slices. On the other hand, increasing pH activates the phosphorylation of 2-deoxy-D-galactose by a crude kinase in a tissue extract. 4. Sugar phosphate accumulated in the cells is dephosphorylated by the action of a Zn2+ -activated phosphatase. 5. The efflux of 2-deoxy-D-galactose from the cells is rather slow compared with that found for D-galactose. The efflux is associated with some dephosphorylation of cellular sugar phosphate, and some loss of 2-deoxy-galactose phosphate into the wash-out medium takes place. 6. An inhibition analysis of the uptake of 2-deoxy-D-galactose by the slices indicates that the transport site is shared by D-galactose. The following points of interaction between the sugar molecule and the carrier are identified: C1-OH, C3-OH and C4-OH (both axial) and C6-OH. A (pyranose) ring structure is also essential. A close packing between the substrate and the carrier in the vicinity of C2 is indicated. 7. The data suggest that the above transport system is localized predominantly at the antiluminal (basolateral) face of the renal tubular cells. While the detailed mechanism of the actual transport step (i.e. active transport of the free sugar, or by the action of a phosphotransferase) is still unclear, the data present evidence that both galactokinase and a Zn2+ -activated phosphatase participate in the maintenance of an intracellular steady state of the transported sugar.     

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

SUMMARY

The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.     

Transport and metabolism of glucose and arabinose in Bifidobacterium breve

Glucose was required for the transport of arabinose into Bifidobacterium breve. The non-metabolisable glucose analogue 2-deoxy-d-glucose (2-DG) did not facilitate assimilation of arabinose. Studies using d-[U-¹⁴C]-labelled arabinose showed that it was fermented to pyruvate, formate, lactate and acetate, whereas the principal metabolic products of d-[U-¹⁴C]-labelled glucose were acetate and formate. In contrast to glucose, arabinose was not incorporated into cellular macromolecules. A variety of metabolic inhibitors and inhibitors of sugar transport (proton ionophores, metal ionophores, compounds associated with electron transport) were used to investigate the mechanisms of sugar uptake. Only NaF, an inhibitor of substrate level phosphorylation, and 2-DG inhibited glucose assimilation. 2-DG had no effect on arabinose uptake, but NaF was stimulatory. High levels of phosphorylation of glucose and 2-DG by PEP and to a lesser degree, ATP were seen in phosphoenolpyruvate: phosphotransferase (PEP:PTS) assays. These data together with strong inhibition of glucose uptake by NaF suggest a role for phosphorylation in the transport process. Arabinose uptake in B. breve was not directly dependent on phosphorylation or any other energy-linked form of transport but may be assimilated by glucose-dependent facilitated diffusion.Abbreviations (2,4-DNP) 2,4-dinitrophenol - (2,4-DNP) carbonylcyanide m-chlorophenylhydrazone - (CCCP) (phosphoenolpyruvate phosphotransferase system) - PEP: PTS trichloroacetic acid - (TCA) 2-deoxy-d-glucose - (2-DG) 2-deoxy-d-glucose     

Energetics of glucose uptake in a Salmonella typhimurium mutant containing uncoupled enzyme IIGlc

Uncoupled enzyme II^Glc of the phosphoenolpyruvate (PEP): glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. We have studied the energetics of glucose uptake catalyzed by this uncoupled enzyme II^Glc. The molar growth yields on glucose of two strains cultured anaerobically in glucose-limited chemostat-and batch cultures were compared. Strain PP 799 transported and phosphorylated glucose via an intact PTS, while strain PP 952 took up glucose exclusively via uncoupled enzyme II^Glc, followed by ATP-dependent phosphorylation by glucokinase. Thus the strains were isogenic except for the mode of uptake and phosphorylation of the growth substrate. PP 799 and PP 952 exhibited similar Y _Glc values. Assuming equal Y _ATP values for both strains this result indicated that there were no energetic demands for glucose uptake via uncoupled enzyme II^Glc.Abbreviations PTS phosphoenolpyruvate: carbohydrate phosphotransferase system - HPr histidine-containing phosphocarrier protein - GalP galactose permease     

Transport of trehalose in Salmonella typhimurium.

We have studied trehalose uptake in Salmonella typhimurium and the possible involvement of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in this process. Two transport systems could recognize and transport trehalose, the mannose PTS and the galactose permease. Uptake of trehalose via the latter system required that it be expressed constitutively (due to a galR or galC mutation). Introduction of a ptsM mutation, resulting in a defective IIMan/IIIMan system, in S. typhimurium strains that grew on trehalose abolished growth on trehalose. A ptsG mutation, eliminating IIGlc of the glucose PTS, had no effect. In contrast, a crr mutation that resulted in the absence of IIIGlc of the glucose PTS prevented growth on trehalose. The inability of crr and also cya mutants to grow on trehalose was due to lowered intracellular cyclic AMP synthesis, since addition of extracellular cyclic AMP restored growth. Subsequent trehalose metabolism could be via a trehalose phosphate hydrolase, if trehalose phosphate was formed via the PTS, or trehalase. Trehalose-grown cells contained trehalase activity, but we could not detect phosphoenolpyruvate-dependent phosphorylation of trehalose in toluenized cells.