Soluble sugar permeases of the phosphotransferase system in Escherichia coli: evidence for two physically distinct forms of the proteins in vivo

The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) consists of a set of cytoplasmic energy-coupling proteins and various integral membrane permeases/sugar phosphotransferases, each specific for a different sugar. We have conducted biochemical analyses of three PTS permeases (enzymes II), the glucose permease (IIGlc), the mannitol permease (IIMtl) and the mannose permease (IIMan). These enzymes each catalyse two vectorial/chemical reactions, sugar phosphorylation using phosphoenolpyruvate (PEP) as the phosphoryl donor, dependent on enzyme I, HPr and IIA as well as IIBC (the PEP reaction), and transphosphorylation using a sugar phosphate (glucose-6-P for IIGlc and IIMan; mannitol-1-P for IIMtl) as the phosphoryl donor, dependent only on IIBC (the TP reaction). When crude extracts of French-pressed or osmotically shocked Escherichia coli cells are centrifuged in an ultracentrifuge at high speed, 5-20% of the enzyme II activity remains in the high-speed supernatant, and passage through a gel filtration column gives two activity peaks, one in the void volume exhibiting high PEP-dependent and TP activities, and a second included peak with high PEP-dependent activity and high (IIMan), moderate (IIGlc) or negligible (IIMtl) TP activities. Both log and stationary phase cells exhibit comparable relative amounts of pelletable and soluble enzyme II activities, but long-term exposure of cells to chloramphenicol results in selective loss of the soluble fraction with retention of much of the pelleted activity concomitant with extensive protein degradation. Short-term exposure of cells to chloramphenicol results in increased activities in both fractions, possibly because of increased lipid association, with more activation in the soluble fraction than in the pelleted fraction. Western blot analyses show that the soluble IIGlc exhibits a subunit size of about 45 kDa, and all three soluble enzymes II elute from the gel filtration column with apparent molecular weights of 40-50 kDa. We propose that enzymes II of the PTS exist in two physically distinct forms in the E. coli cell, one tightly integrated into the membrane and one either soluble or loosely associated with the membrane. We also propose that the membrane-integrated enzymes II are largely dimeric, whereas the soluble enzymes II, retarded during passage through a gel filtration column, are largely monomeric.     

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.     

Glucose permease of Escherichia coli. Purification of the IIGlc subunit and functional characterization of its oligomeric forms

The membrane subunit (IIGlc) of the glucose permease has been purified from overproducing Escherichia coli. About 2 mg of pure protein was obtained from 10 g (wet weight) of cells. IIGlc of E. coli and Salmonella typhimurium are functionally indistinguishable. A small difference was revealed, however, by a monoclonal antibody which neutralizes glucose phosphorylation activity of IIGlc from S. typhimurium, but does not cross-react with IIGlc of E. coli. A dimeric form of purified IIGlc can be detected by chemical cross-linking and by zonal sedimentation at 4 degrees C. Upon mild oxidation a disulfide bond is formed between the subunits of the dimer. Oxidized IIGlc is more stable than the reduced form but is inactive because it cannot be phosphorylated by the cytoplasmic subunit (IIIGlc) of the glucose permease. Cys-421 could be identified as the oxidation-sensitive residue, using a novel assay to detect IIIGlc-dependent phosphorylation of nitrocellulose-bound IIGlc that has been purified by gel electrophoresis. No dimeric form of phosphorylated IIGlc could be detected. Because phosphorylated IIGlc is a catalytic intermediate it is concluded that catalytically active IIGlc is a monomer and that the dimeric form is an artefact observed only with purified resting IIGlc. That IIGlc is active as a monomer is further supported by the observation that monomeric IIGlc catalyzes phosphoryl exchange between glucose and glucose 6-phosphate at equilibrium and that an excess of inactive IIGlc with a serine replacing Cys-421 does not interfere with the activity of wild-type IIGlc as would be expected if interaction between the subunits in a dimer were essential for activity.     

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.     

Bacterial phosphotransferase system. Solubilization and purification of the glucose-specific enzyme II from membranes of Salmonella typhimurium

The glucose-specific enzyme II (IIGlc) of the phosphoenolpyruvate-dependent phosphotransferase system of Salmonella typhimurium has been purified to homogeneity. Purification included the following steps: detergent solubilization of membranes in polydisperse octyloligooxyethylene, isoelectrofocusing, chromatofocusing, and either glycerol gradient centrifugation or gel filtration, all in the presence of the same detergent. Enzymatic activity was assayed as phosphoenolpyruvate-dependent phosphorylation of methyl-alpha-D-glucopyranoside. It could be measured after detergent dilution only and required the presence of phosphatidylglycerol in a sonicated suspension. An antiserum prepared against enzyme IIGlc specifically inhibited phosphorylation of methyl-alpha-D-glucopyranoside. In the solubilized state, purified enzyme IIGlc exists as a complex of molecular weight of 105,000 and a sedimentation coefficient of 3.8 S. In polyacrylamide gels in sodium dodecyl sulfate, it has an apparent molecular weight of about 40,000.     

In vitro interconversion of the soluble and membrane- integrated forms of the Escherichia coli glucose enzyme II of the phosphoenolpyruvate-dependent sugar-transporting phosphotransferase system

In previous publications, we have shown that integral membrane sugar permeases of the bacterial phosphotransferase system can exist in a 'soluble' (probably micellar) monomeric form (SII) as well as a membrane-integrated dimeric form (MII). We here show that the two forms of the his-tagged glucose permease of Escherichia coli can be interconverted in vitro. Conversion of MII to SII is promoted by (1) low protein concentration, (2) detergent, (3) high pH, and (4) phospholipase A(2) treatment. Conversion of SII to MII is promoted by: (1) high protein concentration, (2) adherence to and elution from an Ni(2+) column, (3) neutral pH, and (4) incorporation into phospholipid liposomes.     

Glucose permease of Escherichia coli. The effect of cysteine to serine mutations on the function, stability, and regulation of transport and phosphorylation

The glucose permease (IIGlc/IIIGlc complex) of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It contains 3 cysteine residues, of which Cys-204 and Cys-326 are localized in the hydrophobic part and Cys-421 in the hydrophilic part of the IIGlc subunit. The cysteines were replaced, one at a time, by serines, and the effect of these mutations on stability, regulation, and catalytic properties of IIGlc was investigated in vivo and in vitro. Cys-204 and Cys-326 are not required for catalytic function and are not involved in the membrane potential-dependent regulation of IIGlc activity (Robillard, G. T., and Konings, W. N. (1982) Eur. J. Biochem. 127, 597-604). Replacement of these cysteines by serines results, however, in reduced stability of IIGlc in vivo (C204S) and in vitro (C204S and C326S), indicating that these substitutions in a hydrophobic environment can destabilize the protein structure. Cys-421 is absolutely required for transport and phosphorylation of glucose. C421S can neither be phosphorylated by phospho-IIIGlc nor catalyze the phosphoryl exchange between [14C] glucose and glucose 6-phosphate at equilibrium. C421S does not interfere with the activity of simultaneously expressed wild-type IIGlc. Unexpectedly C421S and wild-type IIGlc support growth on maltose of Escherichia coli ZSC112L (Curtis, S. J., and Epstein, W. (1975) J. Bacteriol. 122, 1189-1199), a strain which otherwise does not grow on this disaccharide as the only carbon source. C421S appears to facilitate the efflux of a growth inhibiting intermediate (glucose?) of maltose. Wild-type IIGlc catalyzes the intracellular phosphorylation of glucose derived from maltose. It is concluded that the cytoplasmic domain of IIGlc interacts with IIIGlc, the cytoplasmic subunit of the glucose permease, and also participates in phosphorylation of glucose, and that phosphorylation occurs independently of transport, although transport of glucose by wild-type IIGlc cannot occur without concomitant phosphorylation.     

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.     

Analysis of mutations that uncouple transport from phosphorylation in enzyme IIGlc of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system.

Mutations that uncouple glucose transport from phosphorylation were isolated in plasmid-encoded Escherichia coli enzyme IIGlc of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The uncoupled enzymes IIGlc were able to transport glucose in the absence of the general phosphoryl-carrying proteins of the PTS, enzyme I and HPr, although with relatively low affinity. Km values of the uncoupled enzymes IIGlc for glucose ranged from 0.5 to 2.5 mM, 2 orders of magnitude higher than the value of normal IIGlc. Most of the mutant proteins were still able to phosphorylate glucose and methyl alpha-glucoside (a non-metabolizable glucose analog specific for IIGlc), indicating that transport and phosphorylation are separable functions of the enzyme. Some of the uncoupled enzymes IIGlc transported glucose with a higher rate and lower apparent Km in a pts+ strain than in a delta ptsHI strain lacking the general proteins enzyme I and HPr. Since the properties of these uncoupled enzymes IIGlc in the presence of PTS-mediated phosphoryl transfer resembled those of wild-type IIGlc, these mutants appeared to be conditionally uncoupled. Sequencing of the mutated ptsG genes revealed that all amino acid substitutions occurred in a hydrophilic segment within the hydrophobic N-terminal part of IIGlc. These results suggest that this hydrophilic loop is involved in binding and translocation of the sugar substrate.     

Mannitol-specific enzyme II of the bacterial phosphotransferase system. I. Properties of the purified permease

The integral membrane protein responsible for the transport and phosphorylation of D-mannitol in Escherichia coli, the mannitol-specific Enzyme II of the phosphotransferase system (Mr = 60,000), has been purified to apparent homogeneity using a modification of a previously published procedure (Jacobson, G. R., Lee, C. A., and Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 249-252). The purified enzyme was dependent on Lubrol PX and phospholipid for maximal activity. It catalyzed both the phosphoenolpyruvate- and the mannitol 1-phosphate-dependent phosphorylation of D-mannitol with high specificity for the accepting sugar and the phosphoryl donor. Both mannitol and mannitol 1-phosphate gave strong substrate inhibition at neutral pH in the transphosphorylation reaction catalyzed by the purified mannitol Enzyme II, while no substrate inhibition by mannitol was observed for the phosphoenolpyruvate-dependent reaction. The purified enzyme did not catalyze hydrolysis of mannitol 1-phosphate, a product of both reactions. Antibody directed against the mannitol Enzyme II inhibited the phosphoenolpyruvate-dependent activity to a greater extent than the transphosphorylation activity. Limited proteolysis with trypsin rapidly inactivated both purified and membrane-bound mannitol Enzyme II, and the purified protein was concomitantly cleaved into fragments with apparent molecular weights of about 29,000. These results show that although the mannitol Enzyme II is an integral membrane protein, a considerable portion of its polypeptide chain must also extend into a hydrophilic environment, presumably the cytoplasm.     

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.     

Galactose transport systems in Streptococcus lactis

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

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.     

Biophysical studies of the membrane-embedded and cytoplasmic forms of the glucose-specific Enzyme II of the E. coli phosphotransferase system (PTS)

The glucose Enzyme II transporter complex of the Escherichia coli phosphotransferase system (PTS) exists in at least two physically distinct forms: a membrane-integrated dimeric form, and a cytoplasmic monomeric form, but little is known about the physical states of these enzyme forms. Six approaches were used to evaluate protein-protein and protein-lipid interactions in this system. Fluorescence energy transfer (FRET) using MBP-II(Glc)-YFP and MBP-II(Glc)-CFP revealed that the homodimeric Enzyme II complex in cell membranes is stable (FRET(-)) but can be dissociated and reassociated to the heterodimer only in the presence of Triton X100 (FRET(+)). The monomeric species could form a heterodimeric species (FRET(+)) by incubation and purification without detergent exposure. Formaldehyde cross linking studies, conducted both in vivo and in vitro, revealed that the dimeric MBP-II(Glc) activity decreased dramatically with increasing formaldehyde concentrations due to both aggregation and activity loss, but that the monomeric MBP-II(Glc) retained activity more effectively in response to the same formaldehyde treatments, and little or no aggregation was observed. Electron microscopy of MBP-II(Glc) indicated that the dimeric form is larger than the monomeric form. Dynamic light scattering confirmed this conclusion and provided quantitation. NMR analyses provided strong evidence that the dimeric form is present primarily in a lipid bilayer while the monomeric form is present as micelles. Finally, lipid analyses of the different fractions revealed that the three lipid species (PE, PG and CL) are present in all fractions, but the monomeric micellar structure contains a higher percentage of anionic lipids (PG & CL) while the dimeric bilayer form has a higher percentage of zwitterion lipids (PE). Additionally, evidence for a minor dimeric micellar species, possibly an intermediate between the monomeric micellar and the dimeric bilayer forms, is presented. These results provide convincing evidence for interconvertible physical forms of Enzyme-II(Glc).     

Properties of a Tn5 insertion mutant defective in the structural gene (fruA) of the fructose-specific phosphotransferase system of Rhodobacter capsulatus and cloning of the fru regulon.

In photosynthetic bacteria such as members of the genera Rhodospirillum, Rhodopseudomonas, and Rhodobacter a single sugar, fructose, is transported by the phosphotransferase system-catalyzed group translocation mechanism. Previous studies indicated that syntheses of the three fructose catabolic enzymes, the integral membrane enzyme II, the peripheral membrane enzyme I, and the soluble fructose-1-phosphate kinase, are coordinately induced. To characterize the genetic apparatus encoding these enzymes, a Tn5 insertion mutation specifically resulting in a fructose-negative, glucose-positive phenotype was isolated in Rhodobacter capsulatus. The mutant was totally lacking in fructose fermentation, fructose uptake in vivo, phosphoenolpyruvate-dependent fructose phosphorylation in vitro, and fructose 1-phosphate-dependent fructose transphosphorylation in vitro. Extraction of the membrane fraction of wild-type cells with butanol and urea resulted in the preparation of active enzyme II free of contaminating enzyme I activity. This preparation was used to show that the activity of enzyme I was entirely membrane associated in the parent but largely soluble in the mutant, suggesting the presence of an enzyme I-enzyme II complex in the membranes of wild-type cells. The uninduced mutant exhibited measurable activities of both enzyme I and fructose-1-phosphate kinase, which were increased threefold when it was grown in the presence of fructose. Both activities were about 100-fold inducible in the parental strain. Although the Tn5 insertion mutation was polar on enzyme I expression, fructose-1-phosphate kinase activity was enhanced, relative to the parental strain. ATP-dependent fructokinase activity was low, but twofold inducible and comparable in the two strains. A second fru::Tn5 mutant and a chemically induced mutant selected on the basis of xylitol resistance showed pleiotropic loss of enzyme I, enzyme II, and fructose-1-phosphate kinase. These mutants were used to clone the fru regulon by complementing the negative phenotype with a wild-type cosmid bank.     

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.     

Outer Membrane Proteins of Escherichia coli III. Evidence that the Major Protein of Escherichia coli O111 Outer Membrane Consists of Four Distinct Polypeptide Species

Previous studies have shown that the outer membrane of Escherichia coli O111 gives a single, major, 42,000-dalton protein peak when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis at neutral pH. Further studies have shown that this peak consists of more than a single polypeptide species, and on alkaline SDS-gel electrophoresis this single peak is resolved into three subcomponents designated as proteins 1, 2, and 3. By chromatography of solubilized, outer membrane protein on diethylaminoethyl-cellulose followed by chromatography on Sephadex G-200 in the presence of SDS, it was possible to separate the 42,000-dalton major protein into four distinct protein fractions. Comparison of cyanogen bromide peptides derived from these fractions indicated that they represented at least four distinct polypeptide species. Two of these proteins migrated as proteins 1 and 2 on alkaline gels. The other two proteins migrated as protein 3 on alkaline gels and cannot be separated by SDS-polyacrylamide gel electrophoresis. In purified form, these major proteins do not contain bound lipopolysaccharide, phospholipid, or phosphate. These proteins may contain a small amount of carbohydrate, as evidenced by the labeling of these proteins by glucosamine, and to a lesser extent by glucose, under conditions where the metabolism of these sugars to amino acids and lipids is blocked. All of the proteins were labeled to the same extent by these sugars. Thus, it was concluded that there are at least four distinct polypeptide species with apparent molecular masses of about 42,000 daltons in the outer membrane of E. coli O111.     

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.