Properties of the glucose phosphotransferase system of Clostridium acetobutylicum NCIB 8052.

The glucose phosphotransferase system (PTS) of Clostridium acetobutylicum was studied by using cell extracts. The system exhibited a Km for glucose of 34 microM, and glucose phosphorylation was inhibited competitively by mannose and 2-deoxyglucose. The analogs 3-O-methylglucoside and methyl alpha-glucoside did not inhibit glucose phosphorylation significantly. Activity showed no dependence on Mg2+ ions or on pH in the range 6.0 to 8.0. The PTS comprised both soluble and membrane-bound proteins, which interacted functionally with the PTSs of Clostridium pasteurianum, Bacillus subtilis, and Escherichia coli. In addition to a membrane-bound enzyme IIGlc, sugar phosphorylation assays in heterologous systems incorporating extracts of pts mutants of other organisms provided evidence for enzyme I, HPr, and IIIGlc components. The HPr was found in the soluble fraction of C. acetobutylicum extracts, whereas enzyme I, and probably also IIIGlc, was present in both the soluble and membrane fractions, suggesting a membrane location in the intact cell.     

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.     

Overproduction and rapid purification of the phosphoenolpyruvate:sugar phosphotransferase system proteins enzyme I, HPr, and Protein IIIGlc of Escherichia coli.

We present methods for the rapid, simple purification of Enzyme I, HPr, and Protein IIIGlc of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS) using plasmids overproducing gene products. The gene for HPr (ptsH) was cloned into the expression vector pKC30. A simple procedure was devised for the purification to homogeneity of this protein from extracts of heat-induced cells containing pKC30/ptsH recombinant clone. The genes for Enzyme I (ptsI) and Protein IIIGlc (crr) were cloned separately into the expression vector pRE1. Rapid purification procedures were developed for the isolation of homogeneous preparations of these two proteins from extracts of heat-induced cells containing pRE1/ptsI and pRE1/crr recombinants. From about 6 g of cells, these procedures yielded 100, 86, and 50 mg of Enzyme I, HPr, and Protein IIIGlc, respectively. The activity of the proteins purified by these methods was comparable to that of the proteins isolated by previously published less efficient procedures.     

Separation of four components of the phosphoenolpyruvate: glucose phosphotransferase system in Vibrio parahaemolyticus.

Four classes of Vibrio parahaemolyticus mutants defective in the phosphoenolpyruvate: glucose phosphotransferase system (PTS) are described. They were phenotypically different, and were defective in different PTS components. The components designated tentatively as II, I, III, and H were separated by gel filtration of a wild-type extract. Component II, which was specific for glucose and found in the particulate fraction, is probably membrane-bound, glucose-specific enzyme II. Both components I and H were soluble proteins, and the latter was relatively heat-stable. Component I was required for phosphorylation of glucose, trehalose, fructose, mannose, and mannitol. Component H was also required for phosphorylating all the above sugars except fructose. These and some additional findings strongly suggest that components I and H correspond to enzyme I and HPr, respectively. Component III, a soluble heat-stable protein, may be equivalent to the sugar-specific factor III found in other organisms, although it seems to participate in phosphorylating two sugars, glucose and trehalose. There were evidences that mutants defective in components I and III were deficient in cyclic adenosine 3',5'-monophosphate synthesis under certain conditions.     

The phosphoenolpyruvate:glucose phosphotransferase system of Salmonella typhimurium. The phosphorylated form of IIIGlc

Enzyme IIIGlc of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) of Salmonella typhimurium can occur in two forms: phosphorylated and nonphosphorylated. Phosphorylated IIIGlc (P-IIIGlc) has a slightly lower mobility during sodium dodecyl sulphate/polyacrylamide gel electrophoresis than IIIGlc. In bacterial extracts both phosphoenolpyruvate (the physiological phosphoryl donor of the PTS) as well as ATP can phosphorylate IIIGlc. The ATP-catalyzed reaction is dependent on phosphoenolpyruvate synthase, however, and is due to prior conversion of ATP to phosphoenolpyruvate. The phosphoryl group of phosphorylated IIIGlc is hydrolysed after boiling in sodium dodecyl sulfate but phosphorylated IIIGlc can be discriminated from IIIGlc if treated with this detergent at room temperature. We have used the different mobilities of IIIGlc and P-IIIGlc to estimate the proportion of these two forms in intact cells. Wild-type cells contain predominantly P-IIIGlc in the absence of PTS sugars. In an S. typhimurium mutant containing a leaky ptsI17 mutation (0.1% enzyme I activity remaining) both forms of IIIGlc occur in approximately equal amounts. Addition of PTS sugars such as glucose results, both in wild-type and mutant, in a dephosphorylation of P-IIIGlc. This correlates well with the observed inhibition of non-PTS uptake systems by PTS sugars via nonphosphorylated IIIGlc.     

The phosphotransferase system (PTS) of Streptomyces coelicolor identification and biochemical analysis of a histidine phosphocarrier protein HPr encoded by the gene ptsH.

HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS) controls sugar uptake and carbon utilization in low-GC Gram-positive bacteria and in Gram-negative bacteria. We have purified HPr from Streptomyces coelicolor cell extracts. The N-terminal sequence matched the product of an S. coelicolor orf, designated ptsH, sequenced as part of the S. coelicolor genome sequencing project. The ptsH gene appears to form a monocistronic operon. Determination of the evolutionary relationship revealed that S. coelicolor HPr is equally distant to all known HPr and HPr-like proteins. The presumptive phosphorylation site around histidine 15 is perfectly conserved while a second possible phosphorylation site at serine 47 is not well-conserved. HPr was overproduced in Escherichia coli in its native form and as a histidine-tagged fusion protein. Histidine-tagged HPr was purified to homogeneity. HPr was phosphorylated by its own enzyme I (EI) and heterologously phosphorylated by EI of Bacillus subtilis and Staphylococcus aureus, respectively. This phosphoenolpyruvate-dependent phosphorylation was absent in an HPr mutant in which histidine 15 was replaced by alanine. Reconstitution of the fructose-specific PTS demonstrated that HPr could efficiently phosphorylate enzyme IIFructose. HPr-P could also phosphorylate enzyme IIGlucose of B. subtilis, enzyme IILactose of S. aureus, and IIAMannitol of E. coli. ATP-dependent phosphorylation was detected with HPr kinase/phosphatase of B. subtilis. These results present the first identification of a gene of the PTS complement of S. coelicolor, providing the basis to elucidate the role(s) of HPr and the PTS in this class of bacteria.     

Bacterial phosphoenolpyruvate-dependent phosphotransferase system. Mechanism of the transmembrane sugar translocation and phosphorylation

The phosphoryl-group transfer from PHPr to glucose or alpha-methylglucose and from glucose 6-phosphate to these same sugars catalyzed by membrane-bound EIIBGlc of the bacterial phosphoenolpyruvate-dependent phosphotransferase system has been studied in vitro. Kinetic measurements revealed that both the phosphorylation reaction and the exchange reaction proceed according to a ping-pong mechanism in which a phosphorylated membrane-bound enzyme II acts as an obligatory intermediate. The occurrence of a phospho-IIBGlc/IIIGlc has been physically demonstrated by the production of a glucose 6-phosphate burst from membranes phosphorylated by phosphoenolpyruvate, HPr, and EI. The observation of similar second-order rate constants for the production of sugar phosphate starting with different phosphoryl-group donors confirms the catalytic relevance of the phosphoenzyme IIBGlc intermediate. The in vitro results, together with data published by other investigators, have led to a model describing sugar phosphorylation and transport in vivo.     

Fractionation and characterization of the phosphoenolpyruvate: fructose 1-phosphotransferase system from Pseudomonas aeruginosa

The initial reactions involved in the catabolism of fructose in Pseudomonas aeruginosa include the participation of a phosphoenolpyruvate:fructose 1-phosphotransferase system (F-PTS). Fractionation of crude extracts of fructose-grown cells revealed that both membrane-associated and soluble components were essential for F-PTS activity. Further resolution of the soluble fraction by both size exclusion and ion-exchange chromatography revealed the presence of only one component, functionally analogous to enzyme I. Enzyme I exhibited a relative molecular weight of 72,000, catalyzed the pyruvate-stimulated hydrolysis of phosphoenolpyruvate to pyruvate, and mediated the phosphorylation of fructose when combined with a source of enzyme II (washed membranes). No evidence for the requirement of a phosphate carrier protein, such as HPr, could be demonstrated. Thus, the F-PTS requires a minimum of two components, a soluble enzyme I and a membrane-associated enzyme II complex, and both were shown to be inducible. Reconstituted F-PTS activity was specific for phosphoenolpyruvate as a phosphate donor (Km, approximately -0.6 mM) and fructose as the sugar substrate (Km, approximately 18 microM). Components of the Pseudomonas F-PTS did not restore activity to extracts of deletion mutants of Salmonella typhimurium deficient in individual proteins of the PTS or to fractionated membrane and soluble components of the F-PTS of Escherichia coli. Similarly, membrane and soluble components of E. coli and S. typhimurium would not cross-complement the F-PTS components from P. aeruginosa.     

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.     

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.     

Properties of a Streptococcus salivarius spontaneous mutant in which the methionine at position 48 in the protein HPr has been replaced by a valine.

HPr is a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) that participates in the concomitant transport and phosphorylation of sugars in bacteria. In gram-positive bacteria, HPr is also reversibly phosphorylated at a seryl residue at position 46 (Ser-46) by a metabolite-activated ATP-dependent kinase and a Pi-dependent HPr(Ser-P) phosphatase. We report in this article the isolation of a spontaneous mutant (mutant A66) from a streptococcus (Streptococcus salivarius) in which the methionine at position 48 (Met-48) in the protein HPr has been replaced by a valine (Val). The mutation inhibited the phosphorylation of HPr on Ser-46 by the ATP-dependent kinase but did not prevent phosphorylation of HPr by enzyme I or the phosphorylation of enzyme II complexes by HPr(His-P). The results, however, suggested that replacement of Met-48 by Val decreased the affinity of enzyme I for HPr or the affinity of enzyme II proteins for HPr(His-P) or both. Characterization of mutant A66 demonstrated that it has pleiotropic properties, including the lack of IIILman, a specific protein of the mannose PTS; decreased levels of HPr; derepression of some cytoplasmic proteins; reduced growth on PTS as well as on non-PTS sugars; and aberrant growth in medium containing a mixture of sugars.     

Identification of a phosphoenolpyruvate:fructose phosphotransferase system (fructose-1-phosphate forming) in Listeria monocytogenes.

Listeria monocytogenes is a gram-positive bacterium whose carbohydrate metabolic pathways are poorly understood. We provide evidence for an inducible phosphoenolpyruvate (PEP):fructose phosphotransferase system (PTS) in this pathogen. The system consists of enzyme I, HPr, and a fructose-specific enzyme II complex which generates fructose-1-phosphate as the cytoplasmic product of the PTS-catalyzed vectorial phosphorylation reaction. Fructose-1-phosphate kinase then converts the product of the PTS reaction to fructose-1,6-bisphosphate. HPr was shown to be phosphorylated by [32P]PEP and enzyme I as well as by [32P]ATP and a fructose-1,6-bisphosphate-activated HPr kinase like those found in other gram-positive bacteria. Enzyme I, HPr, and the enzyme II complex of the Listeria PTS exhibit enzymatic cross-reactivity with PTS enzyme constituents from Bacillus subtilis and Staphylococcus aureus.     

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.     

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.     

The glucose permease of Bacillus subtilis is a single polypeptide chain that functions to energize the sucrose permease

Biochemical, immunological, and sequence analyses demonstrated that the glucose permease of Bacillus subtilis, the glucose-specific Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system, is a single polypeptide chain with a C-terminal Enzyme III-like domain. A flexible hydrophilic linker, similar in length and amino acid composition to linkers previously identified in other regulatory or sensory transducing proteins, functions to tether the Enzyme IIIGlc-like domain of the protein to the membrane-embedded Enzyme IIGlc. Evidence is presented demonstrating that the Enzyme IIIGlc-like domain of the glucose permease plays a dual role and functions in the transport and phosphorylation of both glucose and sucrose. The sucrose permease appears to lack a sucrose-specific Enzyme III-like domain or a separate, soluble IIIScr protein. Enzyme IIScr was capable of utilizing the IIIGlc-like domain of the glucose permease regardless of whether the IIIGlc polypeptide was provided as a purified, soluble protein, as a membrane-bound protein within the same membrane as Enzyme IIScr, or as a membrane-bound protein within membrane fragments different from those bearing Enzyme IIScr. These observations suggest that the IIIGlc-like domain is an autonomous structural unit that assumes a conformation independent of the hydrophobic, N-terminal intramembranal domain of Enzyme IIGlc. Preferential uptake and phosphorylation of glucose over sucrose has been demonstrated by both in vivo transport studies and in vitro phosphorylation assays. Addition of the purified IIIGlc-like domain strongly stimulated the phosphorylation of sucrose, but not that of glucose, in phosphorylation assays that contained the two sugars simultaneously. The results suggest that the preferential uptake of glucose over sucrose is determined by competition of the corresponding sugar-specific permeases for the common P approximately IIIGlc/Scr domain.     

Chemotaxis of Salmonella typhimurium to amino acids and some sugars.

Patterns of chemotaxis by Salmonella typhimurium strain LT-2 to l-amino acids and to several sugars were quantitated by the Adler capillary procedure. Competition experiments indicated that LT-2 possesses three predominant receptors, or interacting sets of receptors, for amino acids. These were termed the aspartate, serine, and alanine classes, respectively. Studies with strains carrying point and deletion mutations affecting components of the phosphoenolpyruvate: glycose phosphotransferase system (PTS) made unlikely a role in primary reception of d-glucose by the three soluble PTS components, namely HPr, enzyme I, and factor III. A ptsG mutant defective in membrane-bound enzyme IIB' of the high-affinity glucose transport system was shown to exhibit normal chemotaxis providing pleiotropic effects of the mutation were eliminated by its genotypic combination with other pts mutations or, phenotypically, by addition of cyclic AMP and substrate. A correlation was demonstrated between chemotaxis to glucose and activity of the low-affinity glucose transport complex, membrane-bound enzymes IIB:IIA, and an enzyme IIB:IIA mutant was shown to have a preponderant defect in chemotaxis to glucose and mannose. Of four systems capable of galactose transport, only the beta-methylgalactoside transport system was implicated in chemotaxis to galactose. Some properties of a mutant possibly defective in processing of signals for chemotaxis to sugars is described.     

Phosphoenolpyruvate-dependent fructose phosphotransferase system of Rhodopseudomonas sphaeroides: purification and physicochemical and immunochemical characterization of a membrane-associated enzyme I

The phosphotransferase system (PTS) of the phototrophic bacterium Rhodopseudomonas sphaeroides consists of a component located in the cytoplasmic membrane and a membrane-associated enzyme called "soluble factor" (SF) [Saier, M. H., Feucht, B. U., & Roseman, S. (1971) J. Biol. Chem. 246, 7819--7821]. SF has been partially purified by a combination of hydrophobic interaction and ion-exchange and gel-permeation chromatography. SF is similar to Escherichia coli enzyme I in its molecular characteristics and enzymatic properties. It has a molecular weight of 85 000 and readily dimerizes. Phosphoenolpyruvate and Mg2+ stabilize the dimer. The enzyme catalyzes the conversion of phosphoenolpyruvate into pyruvate and becomes phosphorylated in the process. The phosphoryl group is subsequently transferred to fructose in the presence of R. sphaeroides membranes. SF substitutes for E. coli enzyme I in fructose or glucose phosphorylation with E. coli enzyme II and HPr. The activities of SF with the R. sphaeroides PTS and the E. coli PTS reside on structurally distinct molecules as shown by their response to limited proteolytic digestion and by immunochemical studies. The activity of SF with the E. coli PTS arises during the isolation procedure and is most likely due to the removal of HPr-like protein from SF.     

Three-dimensional structures of protein-protein complexes in the E. coli PTS

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) includes a collection of proteins that accomplish phosphoryl transfer from phosphoenolpyruvate (PEP) to a sugar in the course of transport. The soluble proteins of the glucose transport pathway also function as regulators of diverse systems. The mechanism of interaction of the phosphoryl carrier proteins with each other as well as with their regulation targets has been amenable to study by nuclear magnetic resonance (NMR) spectroscopy. The three-dimensional solution structures of the complexes between the N-terminal domain of enzyme I and HPr and between HPr and enzyme IIA(Glc) have been elucidated. An analysis of the binding interfaces of HPr with enzyme I, IIA(Glc) and glycogen phosphorylase revealed that a common surface on HPr is involved in all these interactions. Similarly, a common surface on IIA(Glc) interacts with HPr, IIB(Glc) and glycerol kinase. Thus, there is a common motif for the protein-protein interactions characteristic of the PTS.