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.     

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.     

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.     

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.     

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.     

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.     

Deletion mapping of the genes coding for HPr and enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system in Salmonella typhimurium

Sugars transported by a bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) require two soluble proteins: HPr, a low-molecular-weight phosphate-carrier protein, and enzyme I. The structural genes coding for HPr (ptsH) and Enzyme I (ptsI) are shown to be cotransducible in Salmonella typhimurium. The gene order of this region of the Salmonella chromosome is cysA-trzA-ptsH-ptsI...(crr). A method for the isolation of trzA-pts deletion is described. One class of pts deletions extends through ptsH and into ptsI; a second class includes both ptsH and ptsI and extends into or through the crr gene. The crr gene either codes for or regulates the synthesis of a third PTS protein (factor III) which is sugar-specific. A hypothesis is presented for a mechanism of deletion formation.     

Analysis of the ptsH-ptsI-crr region in Escherichia coli K-12: nucleotide sequence of the ptsH gene

The nucleotide sequence of an Escherichia coli DNA segment containing the ptsH gene and the first 162 nucleotides of the ptsI gene encoding, respectively, Hpr and enzyme I of the phosphoenolpyruvate-dependent glycose phosphotransferase system (PTS), was determined. The ptsH promoter was localized using the S1 mapping technique. A nucleotide sequence very similar to the consensus binding site for cAMP receptor protein was found in the -35 region of the ptsH promoter. The ptsH gene is transcribed in the same direction as the ptsI gene and the crr gene (encoding enzyme IIIGlc of the PTS). Analysis of the nucleotide sequence substantiates the notion that the ptsH-ptsI-crr genes constitute a polycistronic operon.     

Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene.

The genes ptsI and ptsH, which encode, respectively, enzyme I and Hpr, cytoplasmic proteins involved in the phosphoenolpyruvate:sugar phosphotransferase system, were cloned from Bacillus subtilis. A plasmid containing a 4.1-kilobase DNA fragment was shown to complement Escherichia coli mutations affecting the ptsH and ptsI genes. In minicells this plasmid expressed two proteins with the molecular weights expected for Hpr and enzyme I. Therefore, ptsH and ptsI are adjacent in B. subtilis, as in E. coli. In E. coli a third gene (crr), involved in glucose translocation and also in catabolite repression, is located downstream from the ptsHI operon. The 4.1-kilobase fragment from B. subtilis was shown to contain a gene that enables an E. coli crr mutant to use glucose. This gene, unlike the E. coli crr gene, was located to the left of ptsH.     

Interactions in vivo between IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system and the glycerol and maltose uptake systems of Salmonella typhimurium

Our previous studies indicated that the ability of phosphoenolpyruvate:sugar phosphotransferase system (PTS) substrates to inhibit the uptake of glycerol or maltose in Salmonella typhimurium is dependent on the relative cellular content of the PTS-sensitive uptake system and of the PTS protein IIIGlc. Our present study confirms and extends those observations. The maltose and glycerol uptake systems are rendered (wholly or partially) insensitive to PTS inhibition by the presence of a second PTS-sensitive uptake system (respectively that for glycerol or maltose) and its substrate. Both the second PTS-sensitive uptake system and its substrate were needed for this protective effect. Galactose and the galactose permease (a PTS-insensitive transport system) did not have any effect on PTS-mediated inhibition of the maltose uptake system. The protective effect of the second PTS-sensitive uptake system and its substrate is counteracted by increasing the cellular levels of IIIGlc. Overproduction of IIIGlc in crr-plasmid-containing strains renders the glycerol and maltose uptake systems hypersensitive to inhibition by PTS substrates. We interpret our results on the basis of a stoichiometric interaction between IIIGlc and a PTS-sensitive uptake system, in which the IIIGlc--transport-system complex is inactive. Competition between two PTS-sensitive transport systems for formation of inactive complex with IIIGlc lowers the free intracellular concentration of IIIGlc resulting in a mutual protective effect against inhibition by IIIGlc.     

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.     

Role of IIIGlc of the phosphoenolpyruvate-glucose phosphotransferase system in inducer exclusion in Escherichia coli.

The phosphoenolpyruvate-D-glucose phosphotransferase system of Enterobacteriaceae is thought to regulate the synthesis and activity of a number of catabolite uptake systems, including those for maltose, lactose, and glycerol, via the phosphorylation state of one of its components, IIIGlc. We have investigated the proposal by Kornberg and co-workers (FEBS Lett. 117(Suppl.):K28-K36, 1980) that not IIIGlc, but an unknown protein, the product of the iex gene, is responsible for the exclusion of the above-mentioned compounds from the cell. The iex mutant HK738 of Escherichia coli contains normal amounts of IIIGlc as measured by specific antibodies, in contrast to crr mutants that lack IIIGlc. The IIIGlc of the iex strain functions normally in glucose and methyl alpha-glucoside transport, and the specific activity in in vitro phosphorylation is approximately 60% of that of the parent. The IIIGlc activity of the iex strain is, however, heat labile, in contrast to the parental IIIGlc, suggesting that the mutant contains an altered IIIGlc. This is supported by the observation that IIIGlc from the iex strain cannot bind to the lactose carrier. Thus it cannot inhibit the carrier, and this explains why the uptake of non-phosphotransferase system compounds in an iex strain is resistant to phosphotransferase system sugars. The introduction of a plasmid containing a wild-type crr+ allele into the iex strain restores the iex phenotype to that of the iex+ parent. The IIIGlc produced from the plasmid in the iex strain is heat stable and binds normally to the lactose carrier. These results lead to the conclusion that the iex mutation is most likely allelic with crr and results in an altered, temperature-sensitive IIIGlc that is still able to function D-glucose and methyl alpha-glucoside uptake and phosphorylation and in the activation of adenylate cyclase, but is unable to bind to and inhibit the lactose carrier.     

Nucleotide sequence of the sucrase gene of Bacillus subtilis

The sucrase gene (sacA) and part of the sacP locus, which corresponds to a membrane component of the phosphotransferase system (PTS) of sucrose transport of Bacillus subtilis, were previously cloned on a 2.1-kb EcoRI DNA fragment. Genes sacA and sacP were localized on this DNA fragment and the nucleotide sequence of the 2.1-kb DNA fragment was determined. A 1440-bp open reading frame (480 codons) was identified coding for a deduced polypeptide of Mr54827, which corresponds to that of purified sucrase. The amino acid sequence shares homology with that of yeast invertase (SUC2 gene product). The sacA gene and the preceding sacP gene seem to belong to the same operon.     

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.     

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.