Phosphoenolpyruvate:glycose phosphotransferase system in species of Vibrio, a widely distributed marine bacterial genus.

The genus Vibrio is one of the most common and widely distributed groups of marine bacteria. Studies on the physiology of marine Vibrio species were initiated by examining 15 species for the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS). All species tested contained a PTS analogous to the glucose-specific (IIGlc) system in enteric bacteria. Crude extracts of the cells showed immunological cross-reactivity with antibodies to enzyme I, HPr, and IIIGlc from Salmonella typhimurium when assayed by the rocket-line method. Toluene-permeabilized cells of 11 species were tested and were active in phosphorylating methyl alpha-D-glucoside with phosphoenolpyruvate but not ATP as the phosphoryl donor. Membranes from 10 species were assayed, and they phosphorylated methyl alpha-D-glucoside when supplemented with a phospho-IIIGlc-generating system composed of homogeneous proteins from enteric bacteria. Toluene-permeabilized cells and membranes of seven species were assayed, as were phosphorylated fructose and 2-deoxyglucose. IIIGlc was isolated from Vibrio fluvialis and was active in phosphorylating methyl alpha-D-glucoside when supplemented with a phospho-HPr-generating system composed of homogeneous proteins from Escherichia coli and membranes from either E. coli or V. fluvialis. These results show that the bacterial PTS is widely distributed in the marine environment and that it is likely to have a significant role in marine bacterial physiology and in the marine ecosystem.     

Regulation of cyclic AMP synthesis by enzyme IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system in crp strains of Salmonella typhimurium.

We investigated the claim (J. Daniel, J. Bacteriol. 157:940-941, 1984) that nonphosphorylated enzyme IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system is required for full synthesis of bacterial cyclic AMP (cAMP). In crp strains of Salmonella typhimurium, cAMP synthesis by intact cells was regulated by the phosphorylation state of enzyme IIIGlc. Introduction of either a pstHI deletion mutation or a crr::Tn10 mutation resulted in a low level of cAMP synthesis. In contrast, crp strains containing a leaky pstI mutation exhibited a high level of cAMP synthesis which was inhibited by phosphotransferase system carbohydrates. From these results, we conclude that phosphorylated enzyme IIIGlc rather than nonphosphorylated enzyme IIIGlc is required for full cAMP synthesis.     

Structural comparison of phosphorylated and unphosphorylated forms of IIIGlc, a signal-transducing protein from Escherichia coli, using three-dimensional NMR techniques.

The 18.1-kDa protein IIIGlc from Escherichia coli acts as both a phosphocarrier protein in the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and as a signal-transducing protein with respect to the uptake of non-PTS sugars. Phosphorylation of IIIGlc at the N epsilon (N3) position of His-90 was effected through a regeneration system that included MgCl2, DTT, excess PEP, and catalytic amounts of Enzyme I and HPr. NH, 15N, and 13C alpha signal assignments for P-IIIGlc were made through comparison of 15N-1H correlation spectra (HSQC) of uniformly 15N-labeled preparations of phosphorylated and unphosphorylated protein and through analysis of three-dimensional triple-resonance HNCA spectra of P-IIIGlc uniformly labeled with both 15N and 13C. Backbone and side-chain 1H and 13C beta signals were assigned using 3D heteronuclear HCCH-COSY and HCCH-TOCSY spectra of P-IIIGlc. Using this approach, the assignments were made without reference to nuclear Overhauser effect data or assumptions regarding protein structure. The majority of NH, 15N, H alpha, and 13C alpha chemical shifts measured for P-IIIGlc were identical to those obtained for the unphosphorylated protein [Pelton, J. G., Torchia, D. A., Meadow, N. D., Wong, C.-Y., & Roseman, S. (1991) Biochemistry 30, 10043]. Those signals that exhibited shifts corresponded to residues within four segments (1) Leu-87-Gly-100, (2) Val-36-Val-46, (3) His-75-Ser-78, and (4) Ala-131-Val-138. These four segments are in close proximity to the active site residues His-75 and His-90 in the unphosphorylated protein [Worthylake, D., Meadow, N. D., Roseman, S., Liao, D., Hertzberg, O., & Remington, S.J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10382], and the chemical shift data provide strong evidence that if any structural changes accompany phosphorylation, they are confined to residues in these four segments. This conclusion is confirmed by comparing NOEs observed in 3D 15N/13C NOESY-HMQC spectra of the two forms of the protein. No NOE differences are seen for residues having the same chemical shifts in IIIGlc and P-IIIGlc. Furthermore, with the exception of residues Ala-76, Asp-94, and Val-96, the NOEs of residues (in the four segments) which exhibited chemical shift differences also had the same NOEs in IIIGlc and P-IIIGlc. In the case of residues Ala-76, Asp-94, and Val-96, minor differences in NOEs, corresponding to interproton distances changes of less than 1.5 A, were observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphoproteins and the phosphoenolpyruvate:sugar phosphotransferase system of Streptococcus salivarius. Detection of two different ATP-dependent phosphorylations of the phosphocarrier protein HPr

Phosphoproteins which arise from incubation of Streptococcus salivarius ATCC25975 crude extracts with [32P]phosphoenolpyruvate and [gamma-32P]ATP, were separated and detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. These procedures were carried out using the methodology that has been developed to allow for the detection of phosphoproteins containing 1-P-histidinyl and 3-P-histidinyl residues, and also to distinguish between these and phosphoproteins containing acid-stable phosphoamino acids such as phosphoserine, phosphothreonine, and phosphotyrosine. Extracts of cells which had been grown with various sugars as carbon sources were investigated to determine both constitutive and inducible phosphoproteins. No evidence was found for phosphoproteins specifically induced by a sugar, and in particular no evidence was found for any IIIsugar phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Incubation with [gamma-32P]ATP showed that histidine-containing phosphocarrier protein (HPr) of the PTS could be phosphorylated to give both acid-stable and acid-labile phosphoamino acid residues. The acid-labile ATP-dependent phosphorylation activity was activated by glucose-6-P and appeared to produce a 3-P-histidinyl residue in HPr.     

Sugar transport by the bacterial phosphotransferase system. Reconstitution of inducer exclusion in Salmonella typhimurium membrane vesicles

The accompanying articles (Saffen, D.W., Presper, K.A., Doering, T.L., and Roseman, S. (1987) J. Biol. Chem. 262, 16241-16253; Mitchell, W.J., Saffen, D. W., and Roseman, S. (1987) J. Biol. Chem. 262, 16254-16260) show that "inducer exclusion" in intact cells of Escherichia coli is regulated by IIIGlc, a protein encoded by the crr gene of the phosphoenolpyruvate:glycose phosphotransferase system (PTS). The present studies attempt to show a direct effect of IIIGlc on non-PTS transport systems. Inner membrane vesicles prepared from a wild type strain of Salmonella typhimurium (pts+), carrying the E. coli lactose operon on an episome, showed respiration-dependent accumulation of methyl-beta-D-thiogalactopyranoside (TMG) via the lactose permease. In the presence of methyl-alpha-D-glucopyranoside or other PTS sugars, TMG uptake was reduced by an amount which was dependent on the relative concentrations of IIIGlc and lactose permease in the vesicles. The endogenous IIIGlc concentration in these vesicles was in the range 5-10 microM, similar to that found in whole cells. Methyl-alpha-glucoside had no effect on lactose permease activity in vesicles prepared from a deletion mutant strain lacking the soluble PTS proteins Enzyme I, HPr, and IIIGlc. One or more of the pure proteins could be inserted into the mutant vesicles; when one of the two electrophoretically distinguishable forms of the phosphocarrier protein, IIIGlc Slow, was inserted, both the initial rate and steady state level of TMG accumulation were reduced by up to 40%. The second electrophoretic form, IIIGlc Fast, had much less effect. A direct relationship was observed between the intravesicular concentration of IIIGlc Slow and the extent of inhibition of the lactose permease. No inhibition was observed when IIIGlc Slow was added to the outside of the vesicles, indicating that the site of interaction with the lactose permease is accessible only from the inner face of the membrane. In addition to the lactose permease, IIIGlc Slow was found to inhibit both the galactose and the melibiose permeases. Uptake of proline, on the other hand, was unaffected. The results are therefore consistent with an hypothesis that dephosphorylated IIIGlc Slow is an inhibitor of certain non-PTS permeases.     

Molecular cloning, sequencing, and expression of the crr gene: the structural gene for IIIGlc of the bacterial PEP:glucose phosphotransferase system.

The phosphoenolpyruvate:glucose phosphotransferase system (PTS) of Salmonella typhimurium is involved both in glucose transport and in the regulation and synthesis of adenylate cyclase and several transport systems. The crr gene has been implicated in this regulating mechanism. A 9.6-kb segment of the S. typhimurium chromosome containing the crr gene was cloned in pAT153. The cloned fragment also complemented cysA mutations but did not contain a functional pts operon which is closely linked to the crr gene and codes for two enzymes of the PTS. Although cysA and crr have been reported to be located on opposite sides of ptsHI, our results suggest that the correct gene order is cysK-ptsHI-crr-cysA. Expression of crr plasmids in a maxicell system yielded two proteins which reacted with specific anti-serum against IIIGlc. The apparent mol. wts. in SDS-polyacrylamide gels were 20 000 and 21 000, the former corresponding to the major band of purified IIIGlc. Both forms were also observed in bacterial extracts and purified IIIGlc. The crr gene was localized on a 1-kb EcoRI-EcoRV fragment of the 9.6-kb insert and sequenced. It codes for a single protein (18 556 D) containing 169 amino acid residues and identified as IIIGlc.     

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.     

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.     

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.     

The Escherichia coli adenylate cyclase complex. Stimulation by potassium and phosphate

In Escherichia coli, adenylate cyclase activity in toluene-treated cells can be inhibited by glucose while the activity in a broken cell preparation cannot. Adenylate cyclase activity in the permeabilized but not in broken cells is stimulated somewhat specifically and additively by potassium and phosphate. Kinetic studies show sigmoid substrate-velocity curves for the toluene-treated cells but hyperbolic curves for the broken cells. The stimulatory effects of potassium and phosphate on adenylate cyclase activity in tolulene-treated cells are associated with increases in the Vmax and Km for ATP. While the enzyme activity in toluene-treated cells shows a preference for magnesium over manganese, the reverse is observed in broken cells. Stimulation of adenylate cyclase activity in toluene-treated cells requires the presence of the proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS proteins can be phosphorylated in a P-enolpyruvate-dependent reaction. The stimulatory effects of ions will not occur if the PTS proteins are not phosphorylated. Since potassium phosphate stimulates both adenylate cyclase and PTS activities in toluene-treated cells, it is proposed that the effect of potassium phosphate on adenylate cyclase activity is mediated through an effect on the PTS. A model for dual regulation by glucose of adenylate cyclase activity is proposed. This model involves regulation of both the condition of the PTS proteins as well as the cellular concentration of phosphate.     

The dihydroxyacetone kinase of Escherichia coli utilizes a phosphoprotein instead of ATP as phosphoryl donor

The dihydroxyacetone kinase (DhaK) of Escherichia coli consists of three soluble protein subunits. DhaK (YcgT; 39.5 kDa) and DhaL (YcgS; 22.6 kDa) are similar to the N- and C-terminal halves of the ATP-dependent DhaK ubiquitous in bacteria, animals and plants. The homodimeric DhaM (YcgC; 51.6 kDa) consists of three domains. The N-terminal dimerization domain has the same fold as the IIA domain (PDB code 1PDO) of the mannose transporter of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS). The middle domain is similar to HPr and the C-terminus is similar to the N-terminal domain of enzyme I (EI) of the PTS. DhaM is phosphorylated three times by phosphoenolpyruvate in an EI- and HPr-dependent reaction. DhaK and DhaL are not phosphorylated. The IIA domain of DhaM, instead of ATP, is the phosphoryl donor to dihydroxyacetone (Dha). Unlike the carbohydrate-specific transporters of the PTS, DhaK, DhaL and DhaM have no transport activity.     

Glucose-specific permease of the bacterial phosphotransferase system: phosphorylation and oligomeric structure of the glucose-specific IIGlc-IIIGlc complex of Salmonella typhimurium

The glucose-specific membrane permease (IIGlc) of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates active transport and concomitant phosphorylation of glucose. The purified permease has been phosphorylated in vitro and has been isolated (P-IIGlc). A phosphate to protein stoichiometry of between 0.6 and 0.8 has been measured. Phosphoryl transfer from P-IIGlc to glucose has been demonstrated. This process is, however, slow and accompanied by hydrolysis of the phosphoprotein unless IIIGlc, the cytoplasmic phosphoryl carrier protein specific to the glucose permease (IIGlc) of the PTS, is added. Addition of unphosphorylated IIIGlc resulted in rapid formation of glucose 6-phosphate with almost no hydrolysis of P-IIGlc accompanying the process. A complex of IIGlc and IIIGlc could be precipitated from bacterial cell lysates with monoclonal anti-IIGlc immunoglobulin. The molar ratio of IIGlc:IIIGlc in the immunoprecipitate was approximately 1:2. Analytical equilibrium centrifugation as well as chemical cross-linking showed that purified IIGlc itself is a dimer (106 kDa), consisting of two identical subunits. These results suggest that the functional glucose-specific permease complex comprises a membrane-spanning homodimer of IIGlc to which four molecules of IIIGlc are bound on the cytoplasmic face.     

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.     

Phosphoproteins and the phosphoenolpyruvate: sugar phosphotransferase system in Salmonella typhimurium and Escherichia coli: evidence for IIImannose, IIIfructose, IIIglucitol, and the phosphorylation of enzyme IImannitol and enzyme IIN-acetylglucosamine

Phosphoproteins produced by the incubation of crude extracts of Salmonella typhimurium and Escherichia coli with either [32P]phosphoenolpyruvate or [gamma 32P]ATP have been resolved and detected using sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Simple techniques were found such that distinctions could be made between phosphoproteins containing acid-labile or stable phosphoamino acids and between N1-P-histidine and N3-P-histidine. Phosphoproteins were found to be primarily formed from phosphoenolpyruvate, but because of an efficient phosphoexchange, ATP also led to the formation of the major phosphoenolpyruvate-dependent phosphoproteins. These proteins had the following apparent subunit molecular weights: 65,000, 65,000, 62,000, 48,000, 40,000, 33,000, 25,000, 20,000, 14,000, 13,000, 9,000, 8,000. Major ATP-dependent phosphoproteins were detected with apparent subunit molecular weights of 75,000, 46,000, 30,000, and 15,000. Other minor phosphoproteins were detected. The phosphorylation of the 48,000- and 25,000-MW proteins by phosphoenolpyruvate was independent of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS phosphoproteins were identified as enzyme I (soluble; MW = 65,000); enzyme IIN-acetylglucosamine (membrane bound; MW = 65,000); enzyme IImannitol (membrane bound; MW = 62,000); IIIfructose (soluble; MW = 40,000); IIImannose (partially membrane associated; MW = 33,000); IIIglucose (soluble; MW = 20,000); IIIglucitol (soluble; MW = 13-14,000); HPr (soluble; MW = 9,000); FPr (fructose induced HPr-like protein (soluble; MW = 8,000). HPr and FPr are phosphorylated on the N-1 position of a histidyl residue while all the others appear to be phosphorylated on an N-3 position of a histidyl residue. These studies identify some previously unknown proteins of the PTS and show the phosphorylation of others, which although previously known, had not been shown to be phosphoproteins.     

Carbohydrate Metabolism in Lactic Streptococci: Fate of Galactose Supplied in Free or Disaccharide Form

Phosphorylation of free galactose by lactic streptococci was mediated by an adenosine triphosphate (ATP)-dependent kinase. The phosphoenolpyruvate (PEP) phosphotransferase system (PTS) was involved to a limited extent in transport of the sugar. The conversion of free galactose to glucose also was demonstrated, and uridine diphosphogalactose-4-epimerase was demonstrated to account for this change. Galactose, supplied as lactose, was phosphorylated during transport by means of the PTS with PEP as the phosphate donor. Data also indicated that galactose derived from lactose was catabolized by the glycolytic pathway. Results showed the participation of ATP or PEP, or both, in the phosphorylation of five growth sugars for lactic streptococci, namely, galactose, glucose, lactose, maltose, and mannose. Free galactose was phosphorylated exclusively by ATP except when cells were grown on galactose; in this case, slight involvement of PEP in phosphorylation also was noted. Lactose phosphorylation was much more effective with PEP except when cells were grown on lactose, in which case ATP was equally effective. Glucose was phosphorylated to about the same degree by either ATP or PEP.     

Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated IIIGlc, a signal-transducing protein from Escherichia coli, using two-dimensional heteronuclear NMR techniques.

IIIGlc is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system from Escherichia coli. The 1H, 15N, and 13C histidine ring NMR signals of both the phosphorylated and unphosphorylated forms of IIIGlc have been assigned using two-dimensional 1H-15N and 1H-13C heteronuclear multiple-quantum coherence (HMQC) experiments and a two-dimensional 13C-13C-1H correlation spectroscopy via JCC coupling experiment. The data were acquired on uniformly 15N-labeled and uniformly 15N/13C-labeled protein samples. The experiments rely on one-bond and two-bond J couplings that allowed for assignment of the signals without the need for the analysis of through-space (nuclear Overhauser effect spectroscopy) correlations. The 15N and 13C chemical shifts were used to determine that His-75 exists predominantly in the N epsilon 2-H tautomeric state in both the phosphorylated and unphosphorylated forms of IIIGlc, and that His-90 exists primarily in the N delta 1-H state in the unphosphorylated protein. Upon phosphorylation of the N epsilon 2 nitrogen of His-90, the N delta 1 nitrogen remains protonated, resulting in the formation of a charged phospho-His-90 moiety. The 1H, 15N, and 13C signals of the phosphorylated and unphosphorylated proteins showed only minor shifts in the pH range from 6.0 to 9.0. These data indicate that the pK alpha values for both His-75 and His-90 in IIIGlc and His-75 in phospho-IIIGlc are less than 5.0, and that the pK alpha value for phospho-His-90 is greater than 10. The results are presented in relation to previously obtained structural data on IIIGlc, and implications for proposed mechanisms of phosphoryl transfer are discussed.     

Stereochemical course of the reactions catalyzed by the bacterial phosphoenolpyruvate:glucose phosphotransferase system

The overall stereochemical course of the reactions leading to the phosphorylation of methyl alpha-D-glucopyranoside by the glucose-specific enzyme II (enzyme IIGlc) of the Escherichia coli phosphotransferase system has been investigated. With [(R)-16O,17O,18O]phosphoenolpyruvate as the phosphoryl donor and in the presence of enzyme I, HPr, and enzyme IIIGlc of the phosphotransferase system, membranes from E. coli containing enzyme IIGlc catalyzed the formation of methyl alpha-D-glucopyranoside 6-phosphate with overall inversion of the configuration at phosphorus (with respect to phosphoenolpyruvate). It has previously been shown that sequential covalent transfer of the phosphoryl group of phosphoenolpyruvate to enzyme I, to HPr, and to enzyme IIIGlc occurs before the final transfer from phospho-enzyme IIIGlc to the sugar, catalyzed by enzyme IIGlc. Because overall inversion of the configuration of the chiral phospho group of phosphoenolpyruvate implies an odd number of transfer steps, the phospho group has been transferred at least five times, and transfer from phospho-enzyme IIIGlc to the sugar must occur in two steps (or a multiple thereof). On the basis that no membrane protein other than enzyme IIGlc is directly involved in the final phospho transfer steps, our results imply that a covalent phospho-enzyme IIGlc is an intermediate during transport and phosphorylation of glucose by the E. coli phosphotransferase system.     

Catabolite control of Escherichia coli regulatory protein BglG activity by antagonistically acting phosphorylations.

In bacteria various sugars are taken up and concomitantly phosphorylated by sugar-specific enzymes II (EII) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The phosphoryl groups are donated by the phosphocarrier protein HPr. BglG, the positively acting regulatory protein of the Escherichia coli bgl (beta-glucoside utilization) operon, is known to be negatively regulated by reversible phosphorylation catalyzed by the membrane spanning beta-glucoside-specific EIIBgl. Here we present evidence that in addition BglG must be phosphorylated by HPr at a distinct site to gain activity. Our data suggest that this second, shortcut route of phosphorylation is used to monitor the state of the various PTS sugar availabilities in order to hierarchically tune expression of the bgl operon in a physiologically meaningful way. Thus, the PTS may represent a highly integrated signal transduction network in carbon catabolite control.     

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.     

The glucose permease of the phosphotransferase system of Bacillus subtilis: evidence for IIGlc and IIIGlc domains

Glucose is taken up in Bacillus subtilis via the phosphoenolpyruvate:glucose phosphotransferase system (glucose PTS). Two genes, orfG and ptsX, have been implied in the glucose-specific part of this PTS, encoding an Enzyme IIGlc and an Enzyme IIIGlc, respectively. We now show that the glucose permease consists of a single, membrane-bound, polypeptide with an apparent molecular weight of 80,000, encoded by a single gene which will be designated ptsG. The glucose permease contains domains that are 40-50% identical to the IIGlc and IIIGlc proteins of Escherichia coli. The B. subtilis IIIGlc domain can replace IIIGlc in E. coli crr mutants in supporting growth on glucose and transport of methyl alpha-glucoside. Mutations in the IIGlc and IIIGlc domains of the B. subtilis ptsG gene impaired growth on glucose and in some cases on sucrose. ptsG mutants lost all methyl alpha-glucoside transport but retained part of the glucose-transport capacity. Residual growth on glucose and transport of glucose in these ptsG mutants suggested that yet another uptake system for glucose existed, which is either another PT system or regulated by the PTS. The glucose PTS did not seem to be involved in the regulation of the uptake or metabolism of non-PTS compounds like glycerol. In contrast to ptsl mutants in members of the Enterobacteriaceae, the defective growth of B. subtilis ptsl mutants on glycerol was not restored by an insertion in the ptsG gene which eliminated IIGlc. Growth of B. subtilis ptsG mutants, lacking IIGlc, was not impaired on glycerol. From this we concluded that neither non-phosphorylated nor phosphorylated IIGlc was acting as an inhibitor or an activator, respectively, of glycerol uptake and metabolism.