Enzyme III stimulation of cyclic AMP synthesis in an Escherichia coli crp mutant.

Cyclic AMP (cAMP) synthesis in Escherichia coli is altered in cAMP receptor protein mutants and in phosphoenolpyruvate:sugar phosphotransferase transport system mutants. The stimulation of cAMP synthesis observed in cAMP receptor protein-deficient mutants is largely dependent upon enzyme III of the phosphoenolpyruvate:sugar phosphotransferase transport system. The phosphoenolpyruvate:sugar phosphotransferase transport system enzyme I is not required for elevated cAMP synthesis. These results suggest that enzyme III plays an important role in regulating adenylate cyclase activity.     

Regulation of carbohydrate uptake in gram-positive bacteria.

Uptake of glycerol and other carbohydrates by Staphylococcus aureus cells is sensitive to regulation by sugar substrates of the phosphoenolpyruvate:sugar phosphotransferase system. Inhibition requires an intact phosphotransferase system. In contrast to results obtained with Gram-negative bacteria, it appears that intracellular sugar phosphate is the inhibiting species.     

Interaction between IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system and glycerol kinase of Salmonella typhimurium.

Purified IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system of Salmonella typhimurium inhibits glycerol kinase. Phosphorylation of IIIGlc via phosphoenolpyruvate, enzyme I, and HPr abolishes this inhibition. The glycerol facilitator is not inhibited by IIIGlc. It is proposed that regulation of glycerol metabolism by the phosphoenolpyruvate:sugar phosphotransferase system is at the level of glycerol kinase.     

The Transport of Carbohydrates by a Bacterial Phosphotransferase System

The components and properties of a phosphoenolpyruvate: glucose phosphotransferase system are reviewed, along with the evidence implicating this system in sugar transport across bacterial membranes. Some possible physiological implications of sugar transport mediated by the phosphotransferase system are also considered.     

Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation.

In Salmonella typhimurium, glucose, mannose, and fructose are normally transported and phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system. We have investigated the transport of these sugars and their non-metabolizable analogs in mutant strains lacking the phospho-carrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system, the enzymes I and HPr, to determine whether the sugar-specific, membrane-bound components of the phosphonenolpyruvate: sugar phosphotransferase system, the enzymes II, can catalyze the uptake of these sugars in the absence of phosphorylation. This process does not occur. We have also isolated mutant strains which lack enzyme I and HPr, but have regained the ability to grow on mannose or fructose. These mutants contained elevated levels of mannokinase (fructokinase). In addition, growth on mannose required constitutive synthesis of the galactose permease. When strains were constructed which lacked the galactose permease, they were unable to grow even on high concentrations of mannose, although elevated levels of mannokinase (fructokinase) were present. These results substantiate the conclusion that the enzymes II of the phosphoenolpyruvate:sugar phosphotransferase system are unable to carry out facilitated diffusion.     

Pyruvate formation during the catabolism of simple hexose sugars by Escherichia coli: studies with pyruvate kinase-negative mutants.

Escherichia coli K-12 mutants lacking the adenosine 5'-monophosphate-activated pyruvate kinase have been isolated accidentally and used to prepare further mutants additionally devoid of the fructose bisphosphate-activated pyruvate kinase. Such double mutants totally devoid of pyruvate kinase activity still grow well under aerobic conditions on sugars that are catabolized by the phosphoenolpyruvate (PEP):sugar phosphotransferase system, but they grow poorly on non-phosphotransferase system sugars. This suggests that although pyruvate kinase plays a major role in the formation of pyruvate from PEP during growth on non-phosphotransferase system sugars, the operation of the PEP:sugar phosphotransferase system can contribute significantly to pyruvate production from PEP. In the absence of pyruvate kinase and an active PEP:sugar phosphotransferase system the methylglyoxal glycolytic bypass may also function to some extent for the formation of pyruvate during the catabolism of simple hexose sugars. No unique physiological role can yet be ascribed to the adenosine 5'-monophosphate-activated pyruvate kinase as a result of these studies.     

Fine control of adenylate cyclase by the phosphoenolpyruvate:sugar phosphotransferase systems in Escherichia coli and Salmonella typhimurium.

Inhibition of cellular adenylate cyclase activity by sugar substrates of the phosphoenolpyruvate-dependent phosphotransferase system was reliant on the activities of the protein components of this enzyme system and on a gene designated crrA. In bacterial strains containing very low enzyme I activity, inhibition could be elicited by nanomolar concentrations of sugar. An antagonistic effect between methyl alpha-glucoside and phosphoenolpyruvate was observed in permeabilized Escherichia coli cells containing normal activities of the phosphotransferase system enzymes. In contrast, phosphoenolpyruvate could not overcome the inhibitory effect of this sugar in strains deficient for enzyme I or HPr. Although the in vivo sensitivity of adenylate cyclase to inhibition correlated with sensitivity of carbohydrate permease function to inhibition in most strains studied, a few mutant strains were isolated in which sensitivity of carbohydrate uptake to inhibition was lost and sensitivity of adenylate cyclase to regulation was retained. These results are consistent with the conclusions that adenylate cyclase and the carbohydrate permeases were regulated by a common mechanism involving phosphorylation of a cellular constituent by the phosphotransferase system, but that bacterial cells possess mechanisms for selectively uncoupling carbohydrate transport from regulation.     

Coordinate regulation of adenylate cyclase and carbohydrate permeases by the phosphoenolpyruvate:sugar phosphotransferase system in Salmonella typhimurium.

Adenylate cyclase (EC 4.6.1.1) and several carbohydrate permeases are inhibited by D-glucose and other substrates of the phosphoenolpyruvate:sugar phosphotransferase system. These activities are coordinately altered by sugar substrates of the phosphotransferase system in a variety of bacterial strains which contain differing cellular levels of the protein components of the phosphotransferase system: Enzyme I, a small heat-stable protein, and Enzyme II. It is suggested that the activities of adenylate cyclase and the permease proteins are subject to allosteric regulation and that the allosteric effector is a regulatory protein which can be phosphorylated by the phosphotransferase system.     

Triclosan inhibition of membrane enzymes and glycolysis of Streptococcus mutans in suspensions and biofilms

Triclosan was found to be a potent inhibitor of the F(H+)-ATPase of the oral pathogen Streptococcus mutans and to increase proton permeabilities of intact cells. Moreover, it acted additively with weak-acid transmembrane proton carriers, such as fluoride or sorbate, to sensitize glycolysis to acid inhibition. Even at neutral pH, triclosan could inhibit glycolysis more directly as an irreversible inhibitor of the glycolytic enzymes pyruvate kinase, lactic dehydro genase, aldolase, and the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Cell glycolysis in suspensions or biofilms was inhibited in a pH-dependent manner by triclosan at a concentration of about 0.1 mmol/L at pH 7, approximately the lethal concentration for S. mutans cells in suspensions. Cells in intact biofilms were almost as sensitive to triclosan inhibition of glycolysis as were cells in suspensions but were more resistant to killing. Targets for irreversible inhibition of glycolysis included the PTS and cytoplasmic enzymes, specifically pyruvate kinase, lactic dehydrogenase, and to a lesser extent, aldolase. General conclusions are that triclosan is a multi-target inhibitor for mutans streptococci, which lack a triclosan-sensitive FabI enoyl-ACP reductase, and that inhibition of glycolysis in dental plaque biofilms, in which triclosan is retained after initial or repeated exposure, would reduce cariogenicity.     

Phosphoenolpyruvate:sugar phosphotransferase system in Ancalomicrobium adetum.

Ancalomicrobium adetum possesses a membrane-associated phosphoenolpyruvate:sugar phosphotransferase system, the components of which exhibited enzymatic cross-reactivity with those from Salmonella typhimurium.     

Galactose transport systems in Streptococcus lactis

Galactose-grown cells of Streptococcus lactis ML3 have the capacity to transport the growth sugar by two separate systems: (i) the phosphoenolpyruvate-dependent phosphotransferase system and (ii) an adenosine 5'-triphosphate-energized permease system. Proton-conducting uncouplers (tetrachlorosalicylanilide and carbonyl cyanide-m-chlorophenyl hydrazone) inhibited galactose uptake by the permease system, but had no effect on phosphotransferase activity. Inhibition and efflux experiments conducted using beta-galactoside analogs showed that the galactose permease had a high affinity for galactose, methyl-beta-D-thiogalactopyranoside, and methyl-beta-D-galactopyranoside, but possessed little or no affinity for glucose and lactose. The spatial configurations of hydroxyl groups at C-2, C-4, and C-6 were structurally important in facilitating interaction between the carrier and the sugar analog. Iodoacetate had no inhibitory effect on accumulation of galactose, methyl-beta-D-thiogalactopyranoside, or lactose via the phosphotransferase system. However, after exposure of the cells to p-chloromercuribenzoate, phosphoenolpyruvate-dependent uptake of lactose and methyl-beta-D-thiogalactopyranoside were reduced by 75 and 100%, respectively, whereas galactose phosphotransferase activity remained unchanged. The independent kinetic analysis of each transport system was achieved by the selective generation of the appropriate energy source (adenosine 5'-triphosphate or phosphoenolpyruvate) in vivo. The maximum rates of galactose transport by the two systems were similar, but the permease system exhibited a 10-fold greater affinity for sugar than did the phosphotransferase system.     

Cyclic AMP-dependent synthesis of fimbriae in Salmonella typhimurium: effects of cya and pts mutations.

Synthesis of bacterial fimbriae (group 1, subtype 1) was shown to be dependent on cyclic AMP and was subject to catabolite repression by many carbohydrates. Mutations in the genes coding for the energy-coupling protein constituents of the phosphoenolpyruvate:sugar phosphotransferase system prevented repression of fimbrial production by the sugar substrates of this enzyme system.     

Regulation of carbohydrate transport activities in Salmonella typhimurium: use of the phosphoglycerate transport system to energize solute uptake.

The phosphoglycerate transport system was employed to supply energy-depleted, lysozyme-treated Salmonella typhimurium cells with a continuous intracellular source of phosphoenolpyruvate. When the cells had been induced to high levels of the phosphoglycerate transport system, a low extracellular concentration of phosphoenolpyruvate (0.1 mM) half maximally stimulated uptake of methyl alpha-glucoside via the phosphoenolpyruvate:sugar phosphotransferase system. If the phosphoglycerate transport system was not induced before energy depletion, 100 times this concentration of phosphoenolpyruvate was required for half-maximal stimulation. Phosphoenolpyruvate could not be replaced by other energy sources if potassium fluoride (an inhibitor of enolase) was present. Inhibition of [14C]-glycerol uptake into energy-depleted cells by methyl alpha-glucoside was demonstrated. A concentration of phosphoenolpyruvate which stimulated methyl alpha-glucoside accumulation counteracted the inhibitory effect of the glucoside. In the presence of potassium fluoride, phosphoenolpyruvate could not be replaced by other energy sources. Inhibition of glycerol uptake by methyl alpha-glucoside in intact untreated cells was also counteracted by phosphoenolpyruvate, but several energy sources were equally effective; potassium fluoride was without effect. These and other results were interpreted in terms of a mechanism in which the relative proportions of the phosphorylated and nonphosphorylated forms of a cell constituent influence the activity of the glycerol transport system.     

Physiological desensitization of carbohydrate permeases and adenylate cyclase to regulation by the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli and Salmonella typhimurium. Involvement of adenosine cyclic 3',5'-phosphate and inducer

Adenylate cyclase and a number of carbohydrate transport systems are subject to regulation by the phosphoenolpyruvate:sugar phosphotransferase system. These sensitive carbohydrate transport systems are desensitized to regulation by the phosphotransferase system, and adenylate cyclase is deactivated when cells are grown in medium containing cyclic AMP. These effects are specific for cyclic AMP and are potentiated by the genetic loss of cyclic AMP phosphodiesterase. Inclusion in the growth medium of an inducer of a sensitive transport system also promotes desensitization of that particular transport system. Inducer-promoted desensitization is specific for the particular target transport system, while cyclic AMP-promoted desensitization is general and affects several systems. Desensitization of the permeases to regulation, and inactivation of adenylate cyclase, are slow processes which are blocked by chloramphenicol and are therefore presumably dependent on protein synthesis. Several sugar substrates of the phosphotransferase system are capable of regulating the sensitive carbohydrate transport systems. The evidence suggests that desensitization to this regulation does not result from a direct effect on the functioning of Enzyme I, a small heat-stable protein of the phosphotransferase system, HPr, or an Enzyme II of the phosphotransferase system, but specifically uncouples the permease systems from regulation.     

Regulation of genes coding for enzyme constituents of the bacterial phosphotransferase system.

Regulation of the synthesis of the proteins of the phosphoenolpyruvate:sugar phosphotransferase system was systematically studied in wild-type and mutant strains of Salmonella typhimurium and Escherichia coli. The results suggest that enzyme I and HPr as well as the glucose-specific and the mannose-specific enzymes II are synthesized by a mechanism which depends on (i) cyclic adenosine monophosphate and its receptor protein; (ii) extracellular inducer; (iii) the sugar-specific enzyme II complex which recognizes the inducing sugar; and (iv) the general energy-coupling proteins of the phosphotransferase system, enzyme I and HPr.     

Phosphorylation of hexoses in Streptomyces aureofaciens: Evidence that the phosphoenolpyruvate: Sugar phosphotransferase system is not operative

Abstract Sugar phosphates are formed in cell-free extracts of Streptomyces aureofaciens RIA57 from glucose or fructose in the presence of phosphoenolpyruvate. In contrast to the phosphorylation by adenosine 5'-triphosphate the kinetics of formation of glucose 6-phosphate via phosphoenolpyruvate (PEP) is nonlinear. The product of fructose phosphorylation (only fructose 6-phosphate was determined by paper chromatography) and the absence of 1-phosphofructokinase indicate that fructose metabolism in S. aureofaciens does not proceed via the phosphoenolpyruvate:sugar phosphotransferase system (PTS).     

Uptake of glucose and maltose by Bacillus popillae.

Results of experiments with glucose and its analog, methyl alpha-D-glucopyranoside, indicated that when glucose was present at low concentrations, it was transported into Bacillus popilliae NRRL B-2309MC cells as glucose 6-phosphate by a phosphoenolpyruvate:sugar phosphotransferase system. An additional mode(s) of entry may be operative at higher glucose concentrations. Maltose appeared to enter the cells by a nonphosphorylative process and was hydrolyzed intracellularly to glucose. No phosphoryl donor was necessary for this hydrolysis.     

Uptake of glucose and maltose by Bacillus popillae.

Results of experiments with glucose and its analog, methyl alpha-D-glucopyranoside, indicated that when glucose was present at low concentrations, it was transported into Bacillus popilliae NRRL B-2309MC cells as glucose 6-phosphate by a phosphoenolpyruvate:sugar phosphotransferase system. An additional mode(s) of entry may be operative at higher glucose concentrations. Maltose appeared to enter the cells by a nonphosphorylative process and was hydrolyzed intracellularly to glucose. No phosphoryl donor was necessary for this hydrolysis.     

Phosphoenolpyruvate-dependent phosphorylation of alpha-methylglucoside in Streptococcus sanguis ATCC 10556

Spontaneous mutants defective in some undefined membrane components of the phosphoenolpyruvate:glucose phosphotransferase system were isolated by plating cells of Streptococcus sanguis ATCC 10556 onto an agar containing lactose and 10 mM 2-deoxyglucose. Toluenized cells of these mutants were defective in their ability to catalyse the phosphoenolpyruvate-dependent phosphorylation of 2-deoxyglucose but were still able to phosphorylate alpha-methylglucoside. The phosphorylation of alpha-methylglucoside was essentially dependent on phosphoenolpyruvate and required the presence of both soluble and membrane components. It was concluded that S. sanguis possessed two different phosphoenolpyruvate:glucose phosphotransferase systems.