Characterization of factor IIIGLc in catabolite repression-resistant (crr) mutants of Salmonella typhimurium.

crr mutants of Salmonella typhimurium are thought to be defective in the regulation of adenylate cyclase and a number of transport systems by the phosphoenolpyruvate-dependent sugar phosphotransferase system, crr mutants are also defective in the enzymatic activity of factor IIIGlc (IIIGlc), a protein component of the phosphotransferase system involved in glucose transport. Therefore, it has been proposed that IIIGlc is the primary effector of phosphotransferase system-mediated regulation of cell metabolism. We characterized crr mutants with respect to the presence and function of IIIGlc by using an immunochemical approach. All of the crr mutants tested had low (0 to 30%) levels of IIIGlc compared with wild-type cells, as determined by rocket immunoelectrophoresis. The IIIGlc isolated from one crr mutant was investigated in more detail and showed abnormal aggregation behavior, which indicated a structural change in the protein. These results supported the hypothesis that a crr mutation directly affects IIIGlc, probably by altering the structural gene of IIIGlc. Several crr strains which appeared to be devoid of IIIGlc in immunoprecipitation assays were still capable of in vitro phosphorylation and transport of methyl alpha-glucoside. This phosphorylation activity was sensitive to specific anti-IIIGlc serum. Moreover, the membranes of crr mutants, as well as those of wild-type cells, contained a protein that reacted strongly with our anti-IIIGlc serum. We propose that S. typhimurium contains a membrane-bound form of IIIGlc which may be involved in phosphotransferase system activity.     

Sugar transport by the bacterial phosphotransferase system. In vivo regulation of lactose transport in Escherichia coli by IIIGlc, a protein of the phosphoenolpyruvate:glycose phosphotransferase system

Escherichia coli and Salmonella typhimurium preferentially utilize sugar substrates of the phosphoenol-pyruvate:glycose phosphotransferase system (PTS) when the growth medium also contains other sugars. This phenomenon, diauxic growth, is regulated by the crr gene, which encodes the PTS protein IIIGlc (Saffen, D.W., Presper, K.A., Doering, T.L., and Roseman, S. (1987) J. Biol. Chem. 16241-16253). We have proposed that non-PTS permeases are regulated by their interaction with IIIGlc, and in vitro studies from other laboratories have provided support for this model, but the in vivo effects of excess IIIGlc are not known. In the present studies, transformed cells that overproduced IIIGlc 2- and 10-fold, respectively, were constructed from a pts+ strain of E. coli and plasmids containing the crr gene. In the 2-fold overproducer, fermentation of, and growth on the non-PTS carbohydrates glycerol, lactose, maltose, and melibiose was generally more sensitive to the glucose analogue methyl-alpha-D-glucopyranoside than in a control strain containing normal levels of IIIGlc. In addition, inhibition of lactose permease activity by methyl-alpha-glucoside (inducer exclusion) was more effective in the 2-fold overproducer than in the control strain, particularly when the permease activity was high. The 10-fold IIIGlc overproducing strain had a requirement for the amino acids methionine, isoleucine, leucine, and valine that may or may not be related to the increased concentration of IIIGlc. Fermentation of non-PTS carbohydrates was also poor in the latter strain. Finally, lactose permease activity was 50% of that in control cells containing the same levels of beta-galactosidase, and the lactose permease activity in the IIIGlc overproducer was reduced to an extremely low level in the presence of methyl alpha-glucoside. Thus there is an inverse relationship between the cellular concentration of IIIGlc and the ability to metabolize non-PTS substrates. The results are consistent with the model where inducer exclusion is affected by a direct interaction between IIIGlc and a non-PTS transport system.     

Suppression of IIIGlc-defects by Enzymes IINag and IIBgl of the PEP:carbohydrate phosphotransferase system

The Enzymes II of the PEP:carbohydrate phosphotransferase system (PTS) specific for N-acetylglucosamine (IINag) and beta-glucosides (IIBgl) contain C-terminal domains that show homology with Enzyme IIIGlc of the PTS. We investigated whether one or both of the Enzymes II could substitute functionally for IIIGlc. The following results were obtained: (i) Enzyme IINag, synthesized from either a chromosomal or a plasmid-encoded nagE+ gene could replace IIIGlc in glucose, methyl alpha-glucoside and sucrose transport via the corresponding Enzymes II. An Enzyme IINag with a large deletion in the N-terminal domain but with an intact C-terminal domain could also replace IIIGlc in IIGlc-dependent glucose transport. (ii) After decryptification of the Escherichia coli bgl operon, Enzyme IIBgl could substitute for IIIGlc. (iii) Phospho-HPr-dependent phosphorylation of methyl alpha-glucoside via IINag/IIGlc is inhibited by antiserum against IIIGlc as is N-acetylglucosamine phosphorylation via IINag. (iv) In strains that contained the plasmid which coded for IINag, a protein band with a molecular weight of 62,000 D could be detected with antiserum against IIIGlc. We conclude from these results that the IIIGlc-like domain of Enzyme IINag and IIBgl can replace IIIGlc in IIIGlc-dependent carbohydrate transport and phosphorylation.     

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.     

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

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

Sugar transport by the bacterial phosphotransferase system. Regulation of other transport systems (lactose and melibiose)

The role of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in the phenomenon of inducer exclusion was examined in whole cells of Salmonella typhimurium which carried the genes of the Escherichia coli lactose operon on an episome. In the presence of the PTS substrate methyl alpha-D-glucopyranoside, the extent of accumulation of the lactose analog methyl beta-D-thiogalactopyranoside was reduced. A strain carrying a mutation in the gene for Enzyme I was hypersensitive to the PTS effect, while a crr mutant strain was completely resistant. Influx, efflux, and exchange of galactosides via the lactose "permease" were inhibited by methyl alpha-glucoside. This inhibition occurred in the presence of metabolic energy poisons, and therefore does not involve either the generation of metabolic energy or energy-coupling to the lactose transport system. When the cellular content of the lactose permease was increased by induction with isopropyl beta-D-thiogalactopyranoside, cells gradually became less sensitive to inducer exclusion. The extent of inhibition of methyl beta-thiogalactoside accumulation by methyl alpha-glucoside was shown to be dependent on the relative cellular content of the PTS and lactose system. The data were consistent with an hypothesis involving partial inactivation of galactoside transport due to interaction between a component of the PTS and the lactose permease. By examination of the effects of the PTS and lactose uptake and melibiose permease-mediated uptake of methyl beta-thiogalactoside, it was further shown that the manner in which inducer exclusion is expressed is independent on the routes available to the non-PTS sugar for exit from the cell.     

Phosphoenolpyruvate-dependent phosphotransferase system enzyme III and plasmid-encoded sucrose transport in Escherichia coli K-12.

The phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system enzyme IISCR, specific for and regulated by sucrose, was analyzed in derivatives of Escherichia coli K-12 carrying the sucrose plasmid pUR404. Enzyme IIScr, coded for by gene scrA of the plasmid, depended for its transport and phosphorylation activity directly on the phosphotransferase system enzyme IIIGlc, Scr, coded for by the chromosomal gene crr.     

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.     

Isolation of IIIGlc of the phosphoenolpyruvate-dependent glucose phosphotransferase system of Salmonella typhimurium.

We report a procedure for the isolation of IIIglc of Salmonella typhimurium, a protein component of the phosphoenolpyruvate-dependent sugar phosphotransferase system. IIIGlc is a soluble protein with a molecular weight of 21,000, as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein is involved in the phosphoenolpyruvate-dependent phosphorylation of methyl alpha-glucoside in vitro. Its affinity for octyl-Sepharose may be an indication of the partial hydrophobic nature of IIIGlc. A specific antiserum against purified IIIGlc was prepared. Growth on different carbon sources did not affect the synthesis of IIIGlc, as determined by quantitative immunoelectrophoresis. Mutations which lower the adenosine 3',5'-phosphate level, such as cya and pts, do not alter the IIIGlc level. The closely related enteric bacteria Escherichia coli and Klebsiella aerogenes contain a protein factor which is closely related to IIIGlc of S. typhimurium, whereas Staphylococcus aureus does not.     

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.     

Sugar transport by the bacterial phosphotransferase system. Molecular cloning and structural analysis of the Escherichia coli ptsH, ptsI, and crr genes

Specialized lambda-transducing phages that carry the Escherichia coli genes ptsH, ptsI, crr, cysM, and cysA have been isolated, and the genes were subcloned in plasmid pBR322. Subcloning and restriction mapping data gave the following clockwise order of genes located at about 52 min on the E. coli genetic map: lig, cysK, ptsH, ptsI, crr, cysM, cysA. The nucleotide sequences of ptsH, ptsI, and crr and the corresponding flanking regions have been determined. These genes encode three cytoplasmic proteins of the phosphoenol-pyruvate:glycose phosphotransferase system: HPr, Enzyme I, and IIIGlc, respectively. The deduced amino acid sequences are consistent with amino acid composition and Edman degradation analyses obtained with the purified proteins. The calculated subunit molecular weight values (9,109 for HPr, 63,489 for Enzyme I, and 18,099 for IIIGlc) also agree well with values obtained with the proteins. Results of gamma delta-transposon insertional studies provided definitive evidence that IIIGlc is the gene product of crr, and therefore that IIIGlc plays a critical role in regulating the metabolism and uptake of certain non-PTS sugars (see accompanying papers: Mitchell, W.J., Saffen, D.W., and Roseman, S. (1987) J. Biol. Chem. 16254-16260; Misko, T.P., Mitchell, W.J., Meadow, N.D., and Roseman, S. (1987) J. Biol. Chem. 16261-16266). The gamma delta transposon studies also suggest that crr is transcribed from an independent promoter located within the ptsI gene. Putative regulatory sequence features include a catabolite gene activator protein-cAMP-binding site and two regions of 2-fold rotational symmetry adjacent to the potential promoter upstream from the HPr structural gene, several ribosome-binding sites, and a rho-independent RNA polymerase termination site downstream from crr. In addition, the ptsI gene contains two highly conserved direct repeats. The significance of these sequence features is discussed with respect to possible multiple forms of pts regulation.     

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

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

Defective enzyme II-BGlc of the phosphoenolpyruvate:sugar phosphotransferase system leading to uncoupling of transport and phosphorylation in Salmonella typhimurium.

Transport and phosphorylation of glucose via enzymes II-A/II-B and II-BGlc of the phosphoenolpyruvate:sugar phosphotransferase system are tightly coupled in Salmonella typhimurium. Mutant strains (pts) that lack the phosphorylating proteins of this system, enzyme I and HPr, are unable to transport or to grow on glucose. From ptsHI deletion strains of S. typhimurium, mutants were isolated that regained growth on and transport of glucose. Several lines of evidence suggest that these Glc+ mutants have an altered enzyme II-BGlc as follows. (i) Insertion of a ptsG::Tn10 mutation (resulting in a defective II-BGlc) abolished growth on and transport of glucose in these Glc+ strains. Introduction of a ptsM mutation, on the other hand, which abolishes II-A/II-B activity, had no effect. (ii) Methyl alpha-glucoside transport and phosphorylation (specific for II-BGlc) was lowered or absent in ptsH+,I+ transductants of these Glc+ strains. Transport and phosphorylation of other phosphoenolpyurate:sugar phosphotransferase system sugars were normal. (iii) Membranes isolated from these Glc+ mutants were unable to catalyze transphosphorylation of methyl alpha-glucoside by glucose 6-phosphate, but transphosphorylation of mannose by glucose 6-phosphate was normal. (iv) The mutation was in the ptsG gene or closely linked to it. We conclude that the altered enzyme II-BGlc has acquired the capacity to transport glucose in the absence of phosphoenolpyruvate:sugar phosphotransferase system-mediated phosphorylation. However, the affinity for glucose decreased at least 1,000-fold as compared to the wild-type strain. At the same time the mutated enzyme II-BGlc lost the ability to catalyze the phosphorylation of its substrates via IIIGlc.     

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.     

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.     

Bacterial phosphotransferase system: regulation of the glucose and mannose enzymes II by sulfhydryl oxidation

We have investigated the effect of oxidizing agents on methyl alpha-glucoside phosphorylation by the Escherichia coli phosphotransferase system (PTS). Oxidizing agents inhibited methyl alpha-glucoside phosphorylation at low methyl alpha-glucoside concentrations, and the degree of inhibition was shown to decrease with increasing concentrations of methyl alpha-glucoside. Results of studies with mutant bacteria and substrate analogues of the glucose and mannose enzymes II showed that contrary to the interpretation of Robillard and Konings [Robillard, G. T., & Konings, W. N. (1981) Biochemistry 20, 5025-5032] the apparent change in the Km value for methyl alpha-glucoside phosphorylation induced by sulfhydryl oxidation is not due to the formation of a low-affinity, oxidized form of the glucose enzyme II. Rather, the results are explained by the presence of two phosphotransferase systems that phosphorylate methyl alpha-glucoside with different affinities and that are differentially sensitive to oxidizing agents. The low Km system corresponds to the glucose enzyme II, which is strongly inhibited by potassium ferricyanide, phenazine methosulfate, and plumbagin. The high Km system corresponds to the mannose enzyme II, which is less sensitive to inhibition by these oxidizing agents. This differential sensitivity to inhibition by oxidizing agents can account for the apparent Km change for methyl alpha-glucoside phosphorylation reported by Robillard and Konings. The physiological significance of sulfhydryl oxidation in the enzymes II of the PTS has yet to be ascertained.     

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.     

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.     

Autoregulation of lactose uptake through the LacY permease by enzyme IIAGlc of the PTS in Escherichia coli K-12

Bacterial growth on one or more carbon sources requires careful control of the uptake and metabolism of these carbon sources. In Escherichia coli, the phosphorylation state of enzyme IIAGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is involved in this control in two ways. The unphosphorylated form of IIAGlc causes 'inducer exclusion', the inhibition of uptake of a number of non-PTS carbon sources, including lactose uptake by the lactose permease. The phosphorylated form of enzyme IIAGlc probably activates adenylate cyclase. In cells growing on lactose, enzyme IIAGlc was approximately 50% dephosphorylated, suggesting that lactose could inhibit its own uptake. This inhibition could be demonstrated by comparing lactose uptake rates in the wild-type strain and in a mutant in which the lactose carrier was insensitive to inducer exclusion. In this deregulated mutant strain, lactose was consumed much faster, and large amounts of glucose were excreted. It was shown that enzyme IIAGlc was dephosphorylated more strongly and that the cAMP level was lower in the mutant, most probably causing the observed decrease in lac expression level. When the lac expression level in the mutant strain was increased to that of the parent strain by adding exogenous cAMP, growth on lactose was slower, suggesting that enzyme IIAGlc-mediated inhibition of lactose uptake and downregulation of the lac expression level protected the cells against excessive lactose influx. An even stronger increase in the lac expression level in a mutant lacking enzyme IIAGlc caused complete growth arrest. We conclude that the autoregulatory mechanism that controls lactose uptake is an important mechanism for the cells in adjusting the uptake rate to their metabolic capacity.     

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.