Inducible phosphoenolpyruvate-dependent hexose phosphotransferase activities in Escherichia coli

1. A method is described for measuring the rate of phosphoenolpyruvate-dependent phosphotransferase activity for a variety of hexoses in toluene-treated suspensions of Escherichia coli. 2. The specific activities of the phosphotransferases that catalyse the phosphorylation of hexoses are greatly affected by the carbon source for growth. 3. In all strains of E. coli tested, fructose phosphotransferase activity is induced by growth on fructose. 4. Strains of E. coli differ greatly in the rate at which they phosphorylate glucose, but all strains possess at least a low glucose phosphotransferase activity under any tested condition of growth. Glucose phosphotransferase activity is further induced by growth on glucose; this does not occur in a mutant that lacks the ability to take up methyl alpha-d-[(14)C]glucopyranoside and hence grows poorly on glucose. 5. When growing on fructose, two strains of E. coli synthesize the inducible glucose phosphotransferase system gratuitously, and to specific activities higher than observed during growth on glucose. A phosphotransferase catalysing the phosphorylation of mannose is similarly induced.     

Purification of the NusB gene product of Escherichia coli K12

Summary The nucleotide sequence of the entire nusB gene of Escherichia coli has recently been determined and the amino acid sequence of its product deduced (Ishii et al. 1984; Swindle et al. 1984). The NusB protein was purified by chromatography on Sephadex G-100, phosphocellulose and hydroxylapatite. Purification of the protein was monitored using ¹⁴C-labelled NusB protein, which was synthesized in a maxicell containing an nusB plasmid as a marker. The final product, which was at least 95% pure as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, had a molecular weight of about 16,000 and an isoelectric point of about 7.3. Analytical data on the amino acid composition of the purified protein agreed with that deduced from the DNA sequence and indicated that this protein was indeed the product of the nusB gene.Abbreviations SDS sodium dodecyl sulphate - kDa kilodaltons - bp base pair(s) - kbp kilobase pair(s)     

Dominant ptsH mutations of the gene coding for synthesis of the HPr protein of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli K-12 merodiploids

Abstract The Escherichia coli ptsI and ptsH genes code for the synthesis of two proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), namely enzyme I and protein HPr. A number of ptsI ⁺ ptsH ⁺/F' ptsI ⁺ ptsH merodiploids was obtained. It was shown in experiments in vivo that ptsH mutations in the transposition are dominant. Bacterial extracts from these merodiploids supported [¹⁴C]methyl glucoside (MG) phosphorylation at the expense of phosphoenolpyruvate only half as much as extracts from the pts ⁺ cells. ptsI ⁺ ptsH /F' ptsI ⁺ ptsH ⁺ merodiploids appeared to be non-viable; the reason for this lack of viability is discussed.     

Enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: protein-protein and protein-phospholipid interactions

The mannitol-specific enzyme II (EII), purified free of phospholipid, exhibits a concentration dependence in its specific activity with P-HPr and mannitol as the donor and acceptor substrates, respectively. This concentration dependence, previously observed only in the case of mannitol----mannitol phosphate exchange reaction, indicates that an oligomeric form of the enzyme is responsible for catalyzing the phosphorylation reaction (P-HPr + mannitol----mannitol-P + HPr) as well as the exchange reaction. Kinetic analysis revealed that the monomeric enzyme has a much lower specific activity than the associated species. The specific activity can be increased by raising the steady-state level of phosphorylation of EII and also by adding phospholipid, demonstrating that phosphorylation and the binding of phospholipid facilitate the association process. Kinetic measurements and fluorescence energy transfer measurements demonstrate a strong preference of EII for phospholipids with specific head group and fatty acid composition.     

The phosphoenolpyruvate-dependent fructose-specific phosphotransferase system in Rhodopseudomonas sphaeroides. Energetics of the phosphoryl group transfer from phosphoenolpyruvate to fructose

Energy coupling to fructose transport in Rhodopseudomonas sphaeroides is achieved by phosphorylation of the membrane-spanning fructose-specific carrier protein, EFruII. The phosphoryl group of phosphoenolpyruvate is transferred to EFruII via the cytoplasmic component SF (soluble factor). The standard free enthalpy of hydrolysis of the two phosphorylated proteins has been estimated from isotope exchange measurements in chemical equilibrium. The delta G degrees for SF-P is -60.5 kJ/mol. The standard free enthalpy for hydrolysis of EII-P is -37.9 kJ/mol, but -45.2 kJ/mol when SF is still complexed to it, as in the overall reaction. Therefore the standard free enthalpy of hydrolysis of SF X EII-P is 70% of the standard free enthalpy of hydrolysis of P-enolpyruvate. The measurements reveal two regulation sites in the system. First, the phosphorylation of SF is inhibited by pyruvate when the concentration ratio of pyruvate/P-enolpyruvate becomes too high. Second, a low concentration of internal fructose prevents the phosphorylation of the carrier by the internal fructose-1-P pool when the concentration of the latter becomes too high or the phosphorylation rate by P-enolpyruvate too slow. Furthermore comparison of the isotope exchange and the overall phosphotransferase reaction kinetics leads to the conclusion that binding of fructose to the carrier is a slow step relative to the phosphoryl group transfer from EFruII to fructose.     

Nucleotide sequence of bglC, the gene specifying enzymeIIbgl of the PEP:sugar phosphotransferase system in Escherichia coli K12, and overexpression of the gene product

The EnzymeIIbgl of the phosphoenolpyruvate- (PEP-) dependent phosphotransferase system catalyses the uptake and concomitant phosphorylation of beta-glucosides by Escherichia coli; it is specified by the gene bglC. The nucleotide sequence of a 3.6 kb HindIII restriction fragment spanning bglC, cloned on a plasmid, was determined. DNA analysis strongly suggests that the published order of this and other genes involved in beta-glucoside utilization, bgl C, S, B, is incorrect, and that the regulatory gene bglS may be located upstream of the structural genes bglC and bglB. From the deduced amino acid sequence it is predicted that the membrane protein specified by bglC consists of 625 amino acid residues (66.48 kDa). The protein has the hydropathic profile expected of an integral membrane protein (average hydropathy = 0.62). Comparisons between the amino acid sequences deduced for the EnzymeIIbgl and for the mannitol-specific EnzymeIImtl show that these proteins are related, and a little direct homology is apparent. A 2.3 kb AluI fragment spanning bglC was subcloned into an expression vector which carries the lambda PL promoter and then transformed into a host strain which produces thermolabile cI857 repressor and the anti-terminator N; thermoinduction resulted in the overproduction of a membrane protein and the appearance of Bgl activity.     

Kinetics and subunit interaction of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system

Purified mannitol-specific enzyme II (EIImtl), in the presence of the detergent Lubrol, catalyzes the phosphorylation of mannitol from P-HPr via a classical ping-pong mechanism involving the participation of a phosphorylated EIImtl intermediate. This intermediate has been demonstrated by using radioactive phosphoenolpyruvate. Upon addition of mannitol, at least 80% of the enzyme-bound phosphoryl groups can be converted to mannitol 1-phosphate. The EIImtl concentration dependence of the exchange reaction indicates that self-association is a prerequisite for catalytic activity. The self-association can be achieved by increasing the EIImtl concentration or at low concentrations of EIImtl by adding HPr or bovine serum albumin. The equilibrium is shifted toward the dissociated form by mannitol 1-phosphate, resulting in a mannitol 1-phosphate induced inhibition. Mannitol does not affect the association state of the enzyme. Both mannitol and mannitol 1-phosphate also act as classical substrate inhibitors. The apparent Ki of each compound, however, is approximately equal to its apparent Km, suggesting that mannitol and mannitol 1-phosphate bind at the same site on EIImtl. Due to strong inhibition provided by mannitol and mannitol 1-phosphate in the exchange reaction, the kinetics of this reaction cannot be used to determine whether the reaction proceeds via a ping-pong or an ordered reaction mechanism.     

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.     

Control of glucose metabolism by enzyme IIGlc of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli.

The quantitative effects of variations in the amount of enzyme IIGlc of the phosphoenolpyruvate:glucose phosphotransferase system (PTS) on glucose metabolism in Escherichia coli were studied. The level of enzyme IIGlc could be adjusted in vivo to between 20 and 600% of the wild-type chromosomal level by using the expression vector pTSG11. On this plasmid, expression of the structural gene for enzyme IIGlc, ptsG, is controlled by the tac promoter. As expected, the control coefficient (i.e., the relative increase in pathway flux, divided by the relative increase in amount of enzyme) of enzyme IIGlc decreased in magnitude if a more extensive pathway was considered. Thus, at the wild-type level of enzyme IIGlc activity, the control coefficient of this enzyme on the growth rate on glucose and on the rate of glucose oxidation was low, while the control coefficient on uptake and phosphorylation of methyl alpha-glucopyranoside (an enzyme IIGlc-specific, nonmetabolizable glucose analog) was relatively high (0.55 to 0.65). The implications of our findings for PTS-mediated regulation, i.e., inhibition of growth on non-PTS compounds by glucose, are discussed.     

The Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: observation of heterogeneity in the amino acid composition of HPr.

Resonances of the aromatic protons of tyrosine have been observed in the proton nuclear magnetic resonance (1H NMR) spectrum of purified HPr from Escherichia coli. Analysis of the NMR spectrum of native HPr suggests that the tyrosine is located in a single position in the secondary structure and that this position is on the interior of the molecule inaccessible to solvent. Previous reports suggested that E. coli HPr contained no tyrosine [Anderson, B., Weigel, N., Kundig, W., & Roseman, S. (1971) J. Biol. Chem. 246, 7023--7033]. In contrast, we find, by amino acid analysis and ultraviolet and NMR spectroscopy, that E. coli HPr does contain tyrosine but at a subintegral level of 0.5 +/- 0.1 mol of tyrosine per mol of HPr.     

The NMR side-chain assignments and solution structure of enzyme IIBcellobiose of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli.

The assignment of the side-chain NMR resonances and the determination of the three-dimensional solution structure of the C10S mutant of enzyme IIBcellobiose (IIBcel) of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli are presented. The side-chain resonances were assigned nearly completely using a variety of mostly heteronuclear NMR experiments, including HCCH-TOCSY, HCCH-COSY, and COCCH-TOCSY experiments as well as CBCACOHA, CBCA(CO)NH, and HBHA(CBCA)(CO)NH experiments. In order to obtain the three-dimensional structure, NOE data were collected from 15N-NOESY-HSQC, 13C-HSQC-NOESY, and 2D NOE experiments. The distance restraints derived from these NOE data were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In an iterative procedure, additional NOE assignments were derived from the calculated structures and new structures were calculated. The final set of structures, calculated with approximately 2000 unambiguous and ambiguous distance restraints, has an rms deviation of 1.1 A on C alpha atoms. IIBcel consists of a four stranded parallel beta-sheet, in the order 2134. The sheet is flanked with two and three alpha-helices on either side. Residue 10, a cysteine in the wild-type enzyme, which is phosphorylated during the catalytic cycle, is located at the end of the first beta-strand. A loop that is proposed to be involved in the binding of the phosphoryl-group follows the cysteine. The loop appears to be disordered in the unphosphorylated state.     

The phosphoenolpyruvate-dependent fructose-specific phosphotransferase system in Rhodopseudomonas sphaeroides. Distribution of EIIFru over the membranes of phototrophically grown Rps. sphaeroides

The distribution of the fructose carrier over the membranes of Rhodopseudomonas sphaeroides was studied in cells grown under light saturation and light limitation. Three types of membranes were isolated after disruption of the cells in a French press. All three types were present in the cells grown either under the high or low light intensity, but they were present in different quantities. The cytoplasmic membrane could be separated from the photosynthetic membranes by Sephacryl S-1000 chromatography. The cytoplasmic membrane has the highest specific density and fructose carrier content and does not contain the light-harvesting pigments. The photosynthetic membranes could be resolved into two types by sucrose density gradient centrifugation. Type A predominates when cells are grown under light saturation, whereas type B, the chromatophores, is synthesized abundantly under light limitation. The properties of type A are in between the properties of the cytoplasmic membrane and the chromatophores. It has a slightly lower specific density and contains four times less fructose carrier than the cytoplasmic membrane, but contains half of the light-harvesting bacteriochlorophyll of the chromatophore membrane. The fructose carrier content in the type B membranes, the chromatophores, is very low.     

Mechanistic coupling of transport and phosphorylation activity by enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system

Mannitol bound to enzyme IImtl could be trapped specifically by rapid phosphorylation with P-HPr. The assay was used to demonstrate transport of mannitol across the cytoplasmic membrane with and without phosphorylation of mannitol. The latter was 2-3 orders of magnitude slower. The fraction of bound mannitol molecules that was actually phosphorylated, the efficiency of the trap, was less than 50%. The efficiency was not very different for enzyme IImtl embedded in the membrane of vesicles with an inside-out orientation or solubilized in detergent. Subsequently, it is argued that the fraction of the bound mannitol molecules that was not phosphorylated dissociated into the cytoplasmic space. A model for the catalytic mechanism of enzyme IImtl is proposed on the basis of interpretations of the present experiments. The main features of the model are the following: (i) mechanistically, the coupling between transport and phosphorylation is less than 50%; (ii) in the physiological steady state of mannitol transport and metabolism, the coupling is 100%; (iii) phosphorylated enzyme IImtl catalyzes facilitated diffusion at a high rate; (iv) the state of phosphorylation of the cytoplasmic domain modulates the activity of the translocator domain; (v) the enzyme catalyzes phosphorylation of free cytoplasmic mannitol at least as fast as it catalyzes transport plus phosphorylation of free periplasmic mannitol.     

Mechanics of solute translocation catalyzed by enzyme IImtl of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli.

The kinetics of binding of mannitol to enzyme IImtl embedded in the membrane of vesicles with an inside-out or a right-side-out orientation were analyzed at 4 degrees C in the absence of the phosphoryl group donor, P-HPr. The binding to the right-side-out oriented vesicles equilibrated too fast to be monitored by the flow dialysis technique. On the other hand, with the inside-out oriented membrane vesicles two conformational changes of the enzyme could be detected kinetically. One change involved a recruitment of binding sites from a state of the enzyme where the binding sites were inaccessible from the cytoplasmic volume. The second change involved a conformational change of the enzyme that followed upon the initial binding to the cytoplasmic-facing binding site leading to a state with a higher affinity for mannitol. Equilibrium binding to the inside-out and right-side-out oriented membrane vesicles at 4 degrees C indicated that the two transitions did not represent the translocation of the binding site, free and with mannitol bound to it, to the other side of the membrane. Instead, a model is proposed in which the conformational changes represent transitions from states with the binding pocket opened to the cytoplasmic side of the membrane to occluded states of the enzyme in which the binding sites, with or without mannitol bound, are not accessible to either side of the membrane.