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 glycerol uptake by the phosphoenolpyruvate-sugar phosphotransferase system in Bacillus subtilis.

Enteric bacteria have been previously shown to regulate the uptake of certain carbohydrates (lactose, maltose, and glycerol) by an allosteric mechanism involving the catalytic activities of the phosphoenolpyruvate-sugar phosphotransferase system. In the present studies, a ptsI mutant of Bacillus subtilis, possessing a thermosensitive enzyme I of the phosphotransferase system, was used to gain evidence for a similar regulatory mechanism in a gram-positive bacterium. Thermoinactivation of enzyme I resulted in the loss of methyl alpha-glucoside uptake activity and enhanced sensitivity of glycerol uptake to inhibition by sugar substrates of the phosphotransferase system. The concentration of the inhibiting sugar which half maximally blocked glycerol uptake was directly related to residual enzyme I activity. Each sugar substrate of the phosphotransferase system inhibited glycerol uptake provided that the enzyme II specific for that sugar was induced to a sufficiently high level. The results support the conclusion that the phosphotransferase system regulates glycerol uptake in B. subtilis and perhaps in other gram-positive bacteria.     

Evidence for the evolutionary relatedness of the proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase system

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) found in enteric bacteria is a complex enzyme system consisting of a non-sugar-specific phosphotransfer protein called Enzyme I, two small non-sugar-specific phosphocarrier substrates of Enzyme I, designated HPr and FPr, and at least 11 sugar-specific Enzymes II or Enzyme II-III pairs which are phosphorylated at the expense of phospho-HPr or phospho-FPr. In this communication, evidence is presented which suggests that these proteins share a common evolutionary origin and that a fructose-specific phosphotransferase may have been the primordial ancestor of them all. The evidence results from an evaluation of 1) PTS protein sequence data; 2) structural analysis of operons encoding proteins of the PTS; 3) genetic regulatory mechanisms controlling expression of these operons; 4) enzymatic characteristics of the PTS systems; 5) immunological cross reactivities of these proteins; 6) comparative studies of phosphotransferase systems from evolutionarily divergent bacteria; 7) the nature of the phosphorylated protein intermediates; 8) molecular weight comparisons among the different Enzymes II and Enzyme II-III pairs; and 9) interaction studies involving different PTS protein constituents. The evidence leads to a unifying theory concerning the evolutionary origin of the system, explains many structural, functional, and regulatory properties of the phosphotransferase system, and leads to specific predictions which should guide future research concerned with genetic, biochemical, and physiological aspects of the system.     

Kinetic analyses of the sugar phosphate:sugar transphosphorylation reaction catalyzed by the glucose enzyme II complex of the bacterial phosphotransferase system.

The sugar phosphate:sugar transphosphorylation reaction catalyzed by the glucose Enzyme II complex of the phosphotransferase system has been analyzed kinetically. Initial rates of phosphoryl transfer from glucose-6-P to methyl alpha-glucopyranoside were determined with butanol/urea-extracted membranes from Salmonella typhimurium strains. The kinetic mechanism was shown to be Bi-Bi Sequential, indicating that the Enzyme II possesses nonoverlapping binding sites for sugar and sugar phosphate. Binding of the two substrates appears to occur in a positively cooperative fashion. A mutant with a defective glucose Enzyme II was isolated which transported methyl alpha-glucoside and glucose with reduced maximal velocities and higher Km values. In vitro kinetic studies of the transphosphorylation reaction catalyzed by the mutant enzyme showed a decrease in maximal velocity and increases in the Km values for both the sugar and sugar phosphate substrates. These results are consistent with the conclusion that a single Enzyme II complex catalyzes both transport and transphosphorylation of its sugar substrates.     

Purification and characterization of the fructose-inducible HPr-like protein, FPr, and the fructose-specific enzyme III of the phosphoenolpyruvate: sugar phosphotransferase system of Salmonella typhimurium

The proteins comprising the fructose-specific phosphoenolpyruvate:sugar phosphotransferase system were investigated using a strain of Salmonella typhimurium which lacks the general phosphotransferase system proteins, HPr and Enzyme I, synthesizes the fructose phosphotransferase system proteins, FPr, Enzyme IIfru, Enzyme IIIfru, and fructose-1-phosphate kinase, constitutively, and expresses the Enzyme I-like protein Enzyme I. Enzyme I activity was found in the cytoplasmic fraction, Enzyme IIfru in the membrane fraction, and FPr and Enzyme IIIfru activities were distributed between the two fractions. Extraction of membranes with butanol and urea led to quantitative release of the membrane-associated Enzyme IIIfru and FPr activities, while Enzyme IIfru remained with the membranes. FPr was purified to homogeneity using ion exchange chromatography, gel filtration, and reversed phase high pressure liquid chromatography (HPLC), and its amino acid composition and N-terminal sequence were determined. A complex of FPr and Enzyme IIIfru (Mr 50,000) was also purified to near homogeneity using ion exchange chromatography, gel filtration, and chromatography on hydroxylapatite. When the purified complex was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was visualized as two protein bands with mobilities corresponding to molecular weights of about 40,000 (Enzyme IIIfru) and 9,000 (FPr). Neither the FPr and Enzyme IIIfru activities nor the proteins represented by these two bands separated during the above chromatography steps or using any of several other techniques, including reversed phase HPLC, indicating a very tight association. Active Enzyme IIIfru free of FPr was never isolated or observed. The proteins could be separated in denatured form by gel filtration in the presence of guanidine HCl or urea. Free FPr and the FPr-Enzyme IIIfru complex were characterized, and the properties of free and complexed FPr were compared to those of HPr.     

Regulation of carbohydrate permeases and adenylate cyclase in Escherichia coli. Studies with mutant strains in which enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system is thermolabile.

Carbohydrate uptake and cyclic adenosine 3':5'-monophosphate (cyclic AMP) synthesis were studied employing mutant strains of Escherichia coli in which Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system was heat-labile. Partial loss of Enzyme I activity, which resulted from incubation of cells at the nonpermissive temperature, depressed the rate and extent of methyl alpha-glucoside uptake. Temperature inactivation of Enzyme I also rendered cyclic AMP synthesis and the uptake of several carbohydrates (glycerol, maltose, melibiose, and lactose) hypersensitive to inhibition by methyl alpha-glucoside. Protein synthesis did not appear to be required for these effects. The parental strains and "revertant" strains in which Enzyme I was less sensitive to temperature did not exhibit heat-enhanced regulation. Inhibition was abolished by the crr mutation. The results suggest that Enzyme I functions as a catalytic component of the regulatory system. Simple positive selection procedures are described for the isolation of bacterial mutants which are deficient for either Enzyme I or the heat-stable protein of the phosphotransferase system.     

Purification of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The inducible, mannitol-specific Enzyme II of the phosphoenolpyruvate:sugar phosphotransferase system has been purified approximately 230-fold from Escherichia coli membranes. The enzyme, initially solubilized with deoxycholate, was first subjected to hydrophobic chromatography on hexyl agarose and then purified by several ion exchange steps in the presence of the nonionic detergent, Lubrol PX. The purified protein appears homogeneous by several criteria and probably consists of a single kind of polypeptide chain with a molecular weight of 60,000 (+/- 5%). In addition to catalyzing phosphoenolpyruvate-dependent phosphorylation of mannitol in the presence of the soluble enzymes of the phosphotransferase system, the purified Enzyme II also catalyzes mannitol 1-phosphate:mannitol transphosphorylation in the absence of these components. A number of other physical and catalytic properties of the enzyme are described. The availability of a stable, homogeneous Enzyme II should be invaluable for studying the mechanism of sugar translocation and phosphorylation catalyzed by the bacterial phosphotransferase system.     

Permease-specific mutations in Salmonella typhimurium and Escherichia coli that release the glycerol, maltose, melibiose, and lactose transport systems from regulation by the phosphoenolpyruvate:sugar phosphotransferase system.

Several carbohydrate permease systems in Salmonella typhimurium and Escherichia coli are sensitive to regulation by the phosphoenolpyruvate:sugar phosphotransferase system. Mutant Salmonella strains were isolated in which individual transport systems had been rendered insensitive to regulation by sugar substrates of the phosphotransferase system. In one such strain, glycerol uptake was insensitive to regulation; in another, the maltose transport system was resistant to inhibition; and in a third, the regulatory mutation specifically rendered the melibiose permease insensitive to regulation. An analogous mutation in E. coli abolished inhibition of the transport of beta-galactosides via the lactose permease system. The mutations were mapped near the genes which code for the affected transport proteins. The regulatory mutations rendered utilization of the particular carbohydrates resistant to inhibition and synthesis of the corresponding catabolic enzymes partially insensitive to repressive control by sugar substrates of the phosphotransferase system. Studies of repression of beta-galactosidase synthesis in E. coli were conducted with both lactose and isopropyl beta-thiogalactoside as exogenous sources of inducer. Employing high concentrations of isopropyl beta-thiogalactoside, repression of beta-galactosidase synthesis was not altered by the lactose-specific transport regulation-resistant mutation. By contrast, the more severe repression observed with lactose as the exogenous source of inducer was partially abolished by this regulatory mutation. The results support the conclusions that several transport systems, including the lactose permease system, are subject to allosteric regulation and that inhibition of inducer uptake is a primary cause of the repression of catabolic enzyme synthesis.     

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.     

Genetic Engineering of the Phosphocarrier Protein NPr of the Escherichia coli Phosphotransferase System Selectively Improves Sugar Uptake Activity

The Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS) in prokaryotes mediates the uptake and phosphorylation of its numerous substrates through a phosphoryl transfer chain where a phosphoryl transfer protein, HPr, transfers its phosphoryl group to any of several sugar-specific Enzyme IIA proteins in preparation for sugar transport. A phosphoryl transfer protein of the PTS, NPr, homologous to HPr, functions to regulate nitrogen metabolism and shows virtually no enzymatic cross-reactivity with HPr. Here we describe the genetic engineering of a "chimeric" HPr/NPr protein, termed CPr14 because 14 amino acid residues of the interface were replaced. CPr14 shows decreased activity with most PTS permeases relative to HPr, but increases activity with the broad specificity mannose permease. The results lead to the proposal that HPr is not optimal for most PTS permeases but instead represents a compromise with suboptimal activity for most PTS permeases. The evolutionary implications are discussed.     

Cooperative binding of the sugar substrates and allosteric regulatory protein (enzyme IIIGlc of the phosphotransferase system) to the lactose and melibiose permeases in Escherichia coli and Salmonella typhimurium

An Escherichia coli strain which overproduces the lactose permease was used to investigate the mechanism of allosteric regulation of this permease and those specific for melibiose, glycerol, and maltose by the phosphoenolpyruvate-sugar phosphotransferase system (PTS). Thio-beta-digalactoside, a high affinity substrate of the lactose permease, released the glycerol and maltose permeases from inhibition by methyl-alpha-d-glucoside. Resumption of glycerol uptake occurred immediately upon addition of the galactoside. The effect was not observed in a strain which lacked or contained normal levels of the lactose permease, but growth of wild-type E. coli in the presence of isopropyl-beta-thiogalactoside plus cyclic AMP resulted in enhanced synthesis of the lactose permease so that galactosides relieved inhibition of glycerol uptake. Thiodigalactoside also relieved the inhibition of glycerol uptake caused by the presence of other PTS substrates such as fructose, mannitol, glucose, 2-deoxyglucose, and 5-thioglucose. Inhibition of adenylate cyclase activity by methyl-alpha-glucoside was also relieved by thiodigalactoside in E. coli T52RT provided that the lactose permease protein was induced to high levels. Cooperative binding of sugar and enzyme III(Glc) to the melibiose permease in Salmonella typhimurium was demonstrated, but no cooperativity was noted with the glycerol and maltose permeases. These results are consistent with a mechanism of PTS-mediated regulation of the lactose and melibiose permeases involving a fixed number of allosteric regulatory proteins (enzyme III(Glc)) which may be titrated by the increased number of substrate-activated permease proteins. This work suggests that the cooperativity in the binding of sugar substrate and enzyme III(Glc) to the permease, demonstrated previously in in vitro experiments, has mechanistic significance in vivo. It substantiates the conclusion that PTS-mediated regulation of non-PTS permease activities involves direct allosteric interaction between the permeases and enzyme III(Glc), the postulated regulatory protein of the PTS.     

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.     

Regulation of E. coli glycogen phosphorylase activity by HPr

Bacteria sense continuous changes in their environment and adapt metabolically to effectively compete with other organisms for limiting nutrients. One system which plays an important part in this adaptation response is the phosphoenol-pyruvate:sugar phosphotransferase system (PTS). Many proteins interact with and are regulated by PTS components in bacteria. Here we review the interaction with and allosteric regulation of Escherichia coli glycogen phosphorylase (GP) activity by the histidine phosphocarrier protein HPr, which acts as part of a phosphoryl shuttle between enzyme I and sugar-specific proteins of the PTS. HPr mediates crosstalk between PTS sugar uptake and glycogen breakdown. The evolution of the allosteric regulation of E. coli GP by HPr is compared to that of other phosphorylases.     

HPr/HPr-P phosphoryl exchange reaction catalyzed by the mannitol specific enzyme II of the bacterial phosphotransferase system

The mannitol specific Enzyme II of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli catalyzes an exchange reaction in which a phosphoryl moiety is transferred from one molecule of the heat stable phosphocarrier protein HPr to another. An assay was developed for measuring this reaction. Unlabeled phospho-HPr and 125I-labeled free HPr were incubated together in the presence of Enzyme IImtl, and production of 125I-labeled phospho-HPr was measured. The reaction was concentration-dependent with respect to Enzyme IImtl and did not occur in its absence. The reaction occurred in the absence of Mg2+ in the presence of 10 mM EDTA. Treatment of Enzyme IImtl with the histidyl reagent diethylpyrocarbonate inactivated it with respect to the exchange reaction. Levels of N-ethylmaleimide which inactivate Enzyme IImtl with respect to both P-enolpyruvate-dependent phosphorylation of mannitol and mannitol/mannitol-1-P transphosphorylation did not affect its activity in the exchange reaction; however, treatment with another sulfhydryl reagent, p-chloromercuribenzoate, resulted in partial inactivation. The pH optimum for the Enzyme IImtl-catalyzed exchange reaction was about 7.5. Enzyme I and the glucose specific Enzyme III, two other E. coli phosphotransferase system proteins which, like Enzyme IImtl, interact directly with HPr, were also shown to catalyze 125I-HPr/HPr-P phosphoryl exchange.     

The phosphoenolpyruvate : methyl-alpha-d-glucoside phosphotransferase system in Bacillus subtilis Marburg : kinetic studies of enzyme ii and evidence for a phosphoryl enzyme ii intermediate.

The Enzyme II complex catalyzing the phosphoryl transfer from P-HPr to sugar in the inducible methyl-alpha-D-glucoside : phosphotransferase system in Bacillus subtilis acts according to a ping-pong mechanism, implying a phosphorylated Enzyme II intermediate. This result is supported by the demonstration of a specific transphosphorylation between [14C] alphaMG and glucose-6-phosphate in the presence of an induced Enzyme II preparation.     

Involvement of the glucose enzymes II of the sugar phosphotransferase system in the regulation of adenylate cyclase by glucose in Escherichia coli.

The nature of the interaction of glucose with toluene-treated cells of Escherichia coli leading to inhibition of adenylate cyclase was examined by the use of analogues. Those analogues with variations of the substituents about carbon atoms 1 or 2 (e.g. alpha-methylglucoside or 2-deoxyglucose) are inhibitory, and they are also substrates of the phosphoenolpyruvate-dependent sugar phosphotransferase system. Analogues with changes in other parts of the molecule (e.g. 3-O-methylglucose or galactose), L-glucose and several disaccharides and pentoses, do not inhibit adenylate cyclase and are not substrates of the phosphotransferase system. This correlation suggests some functional relationship between the adenylate cyclase and phosphotransferase systems. Further studies were done with mutants defective in glucose enzymes II of the phosphotransferase system (designated GPT and MPT); these two activities are measured by phosphorylation of alpha-methyl-glucoside and 2-deoxyglucose, respectively. The wild-type parent phosphorylates both analogues, and both inhibit adenylate cyclase. In the GPT- mutant, alpha-methylglucoside does not inhibit adenylate cyclase and is not phosphorylated, while 2-deoxyglucose is inhibitory and phosphorylated. In the GPT- MPT- double mutant, adenylate cyclase activity is present, but neither alpha-methylglucoside nor 2-deoxyglucose inhibits adenylate cyclase, and neither sugar is phosphorylated. These studies demonstrate that glucose inhibition of adenylate cyclase in toluene-treated cells requires an interaction of this sugar with either the GPT or mpt enzyme II of the phosphotransferase system.     

Identification of catalytic residues in the beta-glucoside permease of Escherichia coli by site-specific mutagenesis and demonstration of interdomain cross-reactivity between the beta-glucoside and glucose systems

beta-Glucoside Enzyme II (IIBgl) of the Escherichia coli phosphotransferase system transports and phosphorylates beta-glucosides, whereas the glucose Enzyme II-III pair (IIGlc-IIIGlc) transports and phosphorylates glucose as well as certain aliphatic alpha- and beta-glucosides. Comparisons of their respective amino acid sequences previously revealed that both systems are homologous and must be evolutionarily related. To gain more insight into the details of the transport mechanism, we made use of the observed homologies among phosphotransferase system permeases to design a suitable set of site-specific mutants within the gene encoding IIBgl. This set was used to study in vivo fermentation and to analyze in vitro P-enolpyruvate-dependent sugar phosphorylation as well as sugar phosphate-dependent sugar transphosphorylation. The following results were obtained. (i) IIBgl transports and phosphorylates glucose as well as aryl- and alkyl-beta-glucosides; (ii) histidyl 547 is essential for the phosphorylation of IIBgl by the histidine-containing phosphoryl carrier protein of the phosphotransferase system (HPr) (first phosphorylation site); (iii) both cysteyl 24 and histidyl 306 are essential for the transfer of the phosphoryl group to the sugar; (iv) replacement of Cys-24 by serine leads to uncoupling of sugar transport from phosphorylation; and (v) histidyl 183 is important for substrate specificity. Our studies also revealed heterologous phosphoryl transfer between the beta-glucoside and glucose permease components which probably occurs as follows: 1) HPr-P----IIBgl (His-547)----IIGlc----alkyl-alpha- or -beta-glucosides or glucose (but not aryl-beta-glucosides) and 2) HPr-P----IIIGlc----IIBgl (Cys-24 or His-306)----alkyl- or aryl-beta-glucosides or glucose (but not methyl-alpha-glucoside). In addition to the essential residues noted above, several residues in IIBgl were identified which when mutated reduced the in vitro catalytic efficiency of the enzyme more than 10-fold. Thus, aspartyl 551 and arginyl 625 appeared to function together with histidyl 547 in phosphoryl transfer involving the first phosphorylation site in the permease, whereas histidyl 183 appeared to function together with cysteyl 24 and histidyl 306 in phosphoryl transfer involving the second phosphorylation site in the permease.     

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.     

The Escherichia coli adenylate cyclase complex. Regulation by enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system.

The activity of adenylate cyclase of Escherichia coli measured in toluene-treated cells under standard conditions is subject to control by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Sugars such as glucose, which are transported by the PTS, will inhibit adenylate cyclase provided the PTS is functional. An analysis was made of the properties of E. coli strains carrying mutations in PTS proteins. Leaky mutants in the PTS protein HPr are similar to wild-type strains with respect to cAMp regulation; adenylate cyclase activity in toluene-treated cells and intracellular cAMP levels are in the normal range. Furthermore, adenylate cyclase in toluene-treated cells of leaky HPr mutants is inhibited by glucose. In contrast, mutations in the PTS protein Enzyme I result in abnormalities in cAMP regulation. Enzyme I mutants generally have low intracellular cAMP levels. Leaky Enzyme I mutants show an unusual phosphoenolpyruvate-dependent activation of adenylate cyclase that is not seen in Enzyme I⁺ revertants or in Enzyme I deletions. A leaky Enzyme I mutant exhibits changes in the temperature-activity profile for adenylate cyclase, indicating that adenylate cyclase activity is controlled by Enzyme I. Temperature-shift studies suggest a functional complex between adenylate cyclase and a regulator protein at 30 °C that can be reversibly dissociated at 40 °C. These studies further support the model for adenylate cyclase activation that involves phosphoenolpyruvate-dependent phosphorylation of a PTS protein complexed to adenylate cyclase.