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PubMed2002

Analysis of carbon metabolism in Escherichia coli strains with an inactive phosphotransferase system by (13)C labeling and NMR spectroscopy.

We have developed Escherichia coli strains that internalize glucose utilizing the GalP permease instead of the phosphoenolpyruvate:carbohydrate phosphotransferase system. It has been demonstrated that a strain with these modifications (PTS(-)Glc(+)) can direct more carbon flux into the aromatic pathway than the wild-type parental strain (N. Flores et al., 1996, Nat. Biotechnol. 14, 620-623; G. Gosset et al., 1996, J. Ind. Microbiol. 17, 47-52; J. L. Baéz et al., 2001, Biotechnol. Bioeng. 73, 530-535). In this study, we have determined and compared the carbon fluxes of a wild-type strain (JM101), a PTS(-)Glc(-) strain, and two isogenic PTS(-)Glc(+) derivatives named PB12 and PB13 by combining genetic, biochemical, and NMR approaches. It was determined that in these strains a functional glk gene in the chromosome is required for rapid glucose consumption; furthermore, glucokinase-specific activities were higher than in the wild-type strain. (13)C labeling and NMR analysis allowed the determination of differences in vivo which include higher glycolytic fluxes of 93.1 and 89.2% compared with the 76.6% obtained for the wild-type E. coli. In PB12 and PB13 we found a flux through the malic enzymes of 4 and 10%, respectively, compared to zero in the wild-type strain. While flux through the Pck enzyme was absent in PB12 and PB13, in the wild type it was 7.7%. Finally, it was found that in the JM101 and PB12 strains both the oxidative and the nonoxidative branches of the pentose phosphate pathway contributed to ribose 5-phosphate synthesis, whereas in PB13 this pentose was synthesized almost exclusively through the oxidative branch. The determined carbon fluxes correlate with biochemical and genetic characterizations.

Biomedical Engineering↗

PubMed1993

Purification of polyphosphate and ATP glucose phosphotransferase from Mycobacterium tuberculosis H37Ra: evidence that poly(P) and ATP glucokinase activities are catalyzed by the same enzyme.

Polyphosphate [poly(P)n]:D-(+)-glucose-6-phosphotransferase (EC 2.7.1.63) from Mycobacterium tuberculosis H37Ra was purified to homogeneity using an improved method which yielded a 634-fold purification with higher recovery. The purified enzyme migrated as a single band with M(r) 33 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The native enzyme was shown to be a dimer by gel filtration using high-performance liquid chromatography (HPLC). The purified enzyme fractionated as a single peak on a C8 reverse-phase HPLC column and was found to display both polyphosphate- and ATP-dependent glucokinase activities. Further evidence that a single protein was responsible for both activities was shown by nondenaturing PAGE, in which the two activities (as determined by an activity stain in dual experiments) were found to comigrate. The C-terminal analysis yielded a single sequence while the N-terminus which was blocked also yielded a single sequence after deblocking. The two activities were found to have the same temperature optimum of 50 degrees C. The pH optima were 9.5 and 8.6-9.5 with poly(P)32 and ATP as the phosphoryl donors, respectively. The apparent Km for poly(P)32 was 18.4 microM while the Km for ATP was 1.46 mM. In addition, the nucleotide analogue, Reactive Blue 4, was found to be a competitive inhibitor with ATP in the ATP-dependent glucokinase reaction, while it displayed noncompetitive inhibition patterns with poly(P) in the poly(P)-dependent glucokinase reaction. It is concluded that the poly(P) and ATP glucokinase activities are catalyzed by the same enzyme but that the two substrates may have different binding sites.

Amino Acid Sequence↗

PubMed1995

Overproduction and purification of the Mycoplasma capricolum phosphocarrier protein, HPr, of the phosphoenolpyruvate: sugar phosphotransferase system.

The gene (ptsH) for the phosphocarrier protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system from Mycoplasma capricolum was previously cloned and sequenced. We present here the results of experiments in which the ptsH gene was cloned into a vector for high level expression in Escherichia coli of the phosphocarrier protein. Conditions were developed for overproduction and purification of HPr by a two-column procedure. The purified protein, analyzed by Edman degradation and mass spectrometry, was found to have been processed by removal of the N-terminal methionine residue. Examination of the purified protein by gel electrophoresis under isoelectric focusing conditions revealed that it has an unusually high isoelectric point.

Amino Acid Sequence↗

PubMed1992

Expression in transgenic tobacco of the bacterial neomycin phosphotransferase gene modified by intron insertions of various sizes.

A plant selectable marker gene consisting of cauliflower mosaic virus expression signals and the protein-coding sequence of bacterial neomycin phosphotransferase was modified by insertion of an intron sequence from a storage protein gene, phaseolin. Correct and efficient splicing of the resulting mosaic RNA was observed in transgenic tobacco plants. The insertion of various linkers or gradual increase of intron size by addition in both orientations of internal intron sequences from another plant gene (parsley, 4-coumarate ligase) had little or no effect on the precision of slicing. The gene activity measured by selectability assay in the protoplast transformation showed that only introns enlarged to 1161 bases and longer caused decreased selectability. The suitability of such mosaic marker genes for studies of RNA splicing, DNA recombination and early events after infection of plants with Agrobacterium is discussed.

Base Sequence↗

PubMed1990

Occurrence of aminoglycoside phosphotransferase subclass I and II structural genes among Enterobacteriaceae spp. isolated from meat samples.

3'-Aminoglycoside phosphotransferase [APH(3')] enzymes are a group responsible for resistance to the antibiotics kanamycin (Km) and neomycin (Nm) in bacteria. Escherichia coli ECT24, originally isolated from a meat sample, harboured an 83-kb conjugative R-plasmid (pRPJ24) that carries transferable resistance to Km and Nm. Plasmid pRPJ24 was transferred by conjugation to Enterobacter cloacae 94R, which was used as the source of plasmid DNA in development of a probe for the Km-resistance determinant. Random cloning of BamHI and HindIII double-digest restriction fragments of pRPJ24 in the pUC18 vector plasmid produced clones resistant to both Nm and Km carrying a 1.9-kb DNA insert. Southern hybridization of pRPJ24 cloned chimeric plasmid DNA (pKPJ94) showed homology with the APH(3')II gene from transposon Tn5. A PstI digest of pKPJ94 produced a 920-bp fragment which hybridized with the APH(3')II structural gene, and was used as a DNA probe for the APH(3')II subclass gene. A 980-bp BamHI fragment from plasmid pGH54 carrying the APH(3')I gene from transposon Tn903 was used as a subclass I probe. Total DNA from 206 randomly screened Km-resistant Enterobacteriaceae isolates from raw ground beef and chicken meat samples were examined for the occurrence of APH(3') subclass I and II using non-radioactively-labelled DNA probes. Thirty-six percent and 60% of the isolates examined carried subclass I and II resistances, respectively, in the isolates from chicken meat samples. The corresponding values for bacterial strains from raw ground beef samples were 51% and 72%, respectively. Four percent of the resistant bacterial isolates from chicken samples did not display homology to either probe.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Non-PTS uptake and subsequent metabolism of glucose in Pediococcus halophilus as demonstrated with a double mutant defective in phosphoenolpyruvate:mannose phosphotransferase system and in phosphofructokinase.

Pediococcus halophilus possesses phosphoenolpyruvate:mannose phosphotransferase system (man:PTS) as a main glucose transporter. A man:PTS defective (man:PTSd) strain X-160 could, however, utilize glucose. A possible glucose-transport mechanism other than PTS was studied with the strain X-160 and its derivative, man:PTSd phosphofructokinase defective (PFK-) strain M-13. Glucose uptake by X-160 at pH 5.5 was inhibited by any of carbonylcyanide m-chlorophenylhydrazone, nigericin, N,N'-dicyclohexylcarbodiimide, or iodoacetic acid. The double mutant M-13 could still transport glucose and accumulated intracellularly a large amount of hexose-phosphates (ca. 8 mM glucose 6-phosphate and ca. 2 mM fructose 6-phosphate). Protonophores also inhibited the glucose transport at pH 5.5, as determined by the amounts of accumulated hexose-phosphates (less than 4 mM). These showed involvement of proton motive force (delta P) in the non-PTS glucose transport. It was concluded that the non-PTS glucose transporter operated in concert with hexokinase or glucokinase for the metabolism of glucose in the man:PTSd strain.

Biological Transport, Active↗

PubMed1991

Release of glucose-mediated catabolite repression due to a defect in the membrane fraction of phosphoenolpyruvate: mannose phosphotransferase system in Pediococcus halophilus.

A spontaneous mutant 9R-4 resistant to 2-deoxyglucose (2DG) was derived from a wild-type strain Pediococcus halophilus I-13. Phosphoenolpyruvate (PEP)-dependent glucose-6-phosphate formation by the permeabilized 9R-4 cells was less than 5% of that observed with the parent I-13. In vitro complementation of PEP-dependent 2DG-6-phosphate formation was assayed with combination of the cytoplasmic and membrane fractions prepared from the I-13 and the mutants (9R-4, and X-160 isolated from nature), which were defective in PEP:mannose phosphotransferase system (man:PTS). The defects in man:PTS of both the strain 9R-4 and X-160 were restricted to the membrane fraction (e.g. EIIman), not to the cytoplasmic one. Kinetic studies on the glucose transport with intact cells and iodoacetate-treated cells also supported the presence of two distinct transport systems in this bacterium as follows: (i) The wild-type I-13 possessed a high-affinity man:PTS (Km = 11 microM) and a low-affinity proton motive force driven glucose permease (GP) (Km = 170 microM). (ii) Both 9R-4 and X-160 had only the low-affinity system (Km = 181 microM for 9R-4, 278 microM for X-160). In conclusion, a 2DG-induced selective defect in the membrane component (EIIman) of the man:PTS could partially release glucose-mediated catabolite repression but not fructose-mediated catabolite repression in soy pediococci.

Biological Transport↗

PubMed1981

The phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system enzymes II as chemoreceptors in chemotaxis of Escherichia coli K 12.

In Escherichia coli K12, eight substrate-specific, membrane-bound enzymes II of the PEP-dependent carbohydrate: phosphotransferase system (PTS), specific for hexoses, hexosamines and hexitols, have been characterised in a series of isogenic and constitutive strains. In such mutants, lacking all but one enzyme II, the transport and vectorial phosphorylation activities as well as the chemotactical response in capillary tube assays have been compared. According to the data obtained, all enzymes II not only are directly involved in the transport and vectorial phosphorylation of their substrates, but they have also a primary role as the chemoreceptors for these substrates: (1) Metabolism of the attractant beyond the phosphorylation step is not a pre-requisite to eliciting positive chemotaxis. (2) Mutants, having only one enzyme II react in the capillary tube assay only to substrates of this enzyme II, but not to substrates of the missing enzymes II. This holds for enzymes II consisting of one membrane-bound protein as well as for systems containing a soluble factor III (FIII). (3) The substrate specificities or affinities, whether tested by transport and chemotaxis assays in vivo or by phosphorylation tests in vitro, are in correspondence. (4) The activities of enzymes II, regulated in a complex way at the level of enzyme synthesis and activity and tested as above, are also in agreement, (5) Mutants lacking the soluble proteins enzyme I or HPr of the PTS no longer respond chemotactically to any substrate taken up and phosphorylated by enzymes II. It is concluded that in PTS enzymes II some functions required for transport and chemotaxis are identical. It is suggested furthermore, that the alternation of intrinsic membrane-bound proteins between a phosphorylated and a dephosphorylated state, rather than binding of the substrate to the enzyme II, is the decisive stimulus in the chemotaxis toward carbohydrates taken up by these transport systems.

Bacterial Proteins↗

PubMed1993

Cloning and characterization of the scrA gene encoding the sucrose-specific Enzyme II of the phosphotransferase system from Staphylococcus xylosus.

By insertional mutagenesis with the staphylococcal transposon Tn551, mutants of Staphylococcus xylosus were isolated that were unable to utilize sucrose. One of these was found to be deficient in sucrose uptake. The genomic region containing this sucrose uptake gene of Staphylococcus xylosus (scrA) was cloned in Staphylococcus carnosus. The scrA gene was further localized to a 4.4 kb DNA fragment by complementation of the sucrose transport-deficient S. xylosus mutant. The DNA sequence analysis of the scrA region revealed three open reading frames, one of which encodes a protein of 480 amino acids (51.335 kDa) with significant similarity to sucrose-specific Enzymes II of phosphoenolpyruvate-dependent carbohydrate phosphotransferase systems (PTS). A protein with an apparent molecular weight of 50 kDa was obtained in Escherichia coli by expression of scrA with the bacteriophage T7 RNA polymerase promoter system. Transcriptional start sites of the scrA gene were localized by primer extension analysis to positions 46 and 49 nucleotides upstream of the scrA start codon. No additional sucrose utilization genes are encoded close to scrA on the S. xylosus chromosome.

Amino Acid Sequence↗

PubMed1995

Molecular analysis of the phosphoenolpyruvate-dependent L-sorbose: phosphotransferase system from Klebsiella pneumoniae and of its multidomain structure.

We have cloned a 3.4 kb DNA fragment from the chromosome of Klebsiella pneumoniae that codes for a phosphoenolpyruvate-dependent L-sorbose: phosphotransferase system (PTS). The cloned fragment was sequenced and four open reading frames coding for 135 (sorF), 164 (sorB), 266 (sorA) and 274 (sorM) amino acids, respectively, were found. The corresponding proteins could be detected in a T7 overexpression system, which yielded molecular masses of about 14,000 for SorF, 19,000 for SorB, 25,000 for SorA and 27,000 for SorM. SorF and SorB have all the characteristics of soluble and intracellular proteins in accordance with their functions as EIIASor and EIIBSor domains of the L-sorbose PTS. SorA and SorM, by contrast, are strongly hydrophobic, membrane-bound proteins with two to five putative transmembrane helices that alternate with a series of hydrophilic loops. They correspond to domains EIICSor and EIIDSor. The four proteins of the L-sorbose PTS resemble closely (27%-60%) the four subunits of a D-fructose PTS (EIIALev, EIIBLev, EIICLev, and EIIDLev) from Bacillus subtilis and the three subunits of the D-mannose PTS (EIIA,BMan, EIICMan, and EIIDMan) from Escherichia coli K-12. The three systems constitute a new PTS family, and sequence comparisons revealed highly conserved structures for the membrane-bound proteins. A consensus sequence for the membrane proteins was used to postulate a model for their integration into the membrane.

Amino Acid Sequence↗

PubMed1989

The repressor of the PEP:fructose phosphotransferase system is required for the transcription of the pps gene of Escherichia coli.

We have cloned the pps gene, coding for PEP synthase, of Escherichia coli. PEP synthase catalyses the ATP-dependent conversion of pyruvate into phosphoenol-pyruvate and is required for gluconeogenesis. The pps gene was cloned by an in vivo cloning method using a mini-Mulac bacteriophage containing a plasmid replicon. Upon expression of the cloned pps gene in the maxicell system a protein with an apparent molecular weight of 84 kDa was synthesized. The position of the pps gene of the plasmid was localized by restriction analysis of isolated transposon insertions and the determination of the PEP synthase activities of the different clones. An operon fusion between the pps gene and the galK gene was constructed. Measurements of the galactokinase activity in Salmonella typhimurium galK and galK fruR mutants showed that the transcription of the pps gene requires the presence of FruR, the repressor of the PEP: fructose phosphotransferase system (PTS) in E. coli and S. typhimurium. To test whether the components of the Fructose PTS, in particular FPr, are involved in the expression of the pps gene, we investigated a S. typhimurium galK strain, containing the fusion plasmid, in which the chromosomal fru operon was inactivated by a transposon insertion. Measurements of the galactokinase activity showed that the absence of the Fructose PTS proteins has no significant influence on the regulation of the pps gene.

Bacterial Proteins↗

PubMed1989

The PEP: fructose phosphotransferase system in Salmonella typhimurium: FPr combines enzyme IIIFru and pseudo-HPr activities.

We have cloned the fru operon of Salmonella typhimurium, coding for the enzymes of the phosphoenolpyruvate: fructose phosphotransferase system (Fructose PTS). The fruFKA operon consists of three genes: fruF coding for FPr, fruK for fructose 1-phosphate kinase and fruA for Enzyme IIFru. Insertions of Tn5 in the different genes were isolated and the activities of the gene products were measured. Expression of the plasmid-encoded fru operon in the maxicell system resulted in the synthesis of three proteins with molecular weights of 47 kDa (fruA), 39 kDa (fruF) and 32 kDa (fruK). We have sequenced the fruF gene and the regulatory region of the fru operon. In contrast to previously published results, we have found that the fruF gene codes for a 39 kDa protein, FPr, that combines Enzyme IIIFru and pseudo-HPr activities. The N-terminal part of FPr is homologous to the cytoplasmic domain of the Escherichia coli Enzyme IIMtl, as well as several Enzymes IIIMtl from gram-positive bacteria. The C-terminal domain shows homology to HPr of E. coli and several gram-positive organisms. The fru operon is regulated by a repressor, FruR. We have constructed an operon fusion between fru and the galK gene and shown that regulation of the fru operon by FruR takes place at the level of transcription.

Amino Acid Sequence↗

PubMed1975

Identification of the structural gene of the PEP-phosphotransferase enzyme I in Bacillus subtilis Marburg.

A thermosensitive mutation pt8I1 in the gene coding for the Enzyme I of the PEP-phosphotransferase system pathway has been isolated. The mutant enzyme was shown to be sensitive to high temperature, but this effect is dependent on the ionic strength. The ptsI1 mutation was shown to belong to the previously described ctr locus. Following Lin (1970) it is proposed to retain the symbole ptsI for this locus.

Bacillus subtilis↗

PubMed1984

Role of 2-ketobutyrate as an alarmone in E. coli K12: inhibition of adenylate cyclase activity mediated by the phosphoenolpyruvate: glycose phosphotransferase transport system.

2-ketobutyrate and its analogues were found to inhibit strongly and transiently the rate of beta-galactosidase synthesis in Escherichia coli K12. This effect was ascribed to a strong and transient inhibition of the adenylate cyclase activity. By using pts mutants, we showed, in agreement with our previous results (Daniel et al. 1983), that the likely target of 2-ketobutyrate and its analogues is the phosphoenolpyruvate: glycose phosphotransferase transport system (PTS). Furthermore, evidence for such a cascade effect caused by 2-ketobutyrate and its analogues allowed us to corroborate our previous proposal (Daniel et al. 1983) that 2-ketobutyrate, a precursor of isoleucine, acts as an E. coli alarmone monitoring the passage from anaerobic to aerobic growth conditions.

Adenylyl Cyclase Inhibitors↗

PubMed1980

Influence of growth environment on the phosphoenolpyruvate: glucose phosphotransferase activities of Escherichia coli and Klebsiella aerogenes: a comparative study.

A consistent difference was found between glucose-limited cultures of Escherichia coli and Klebsiella aerogenes strains in the manner which their apparent cellular content of glucose:phosphoenolpyruvate phosphotransferase (glucose-PTS) varied with growth rate. With the former strains, activity increased as a function of growth rate; in the latter it decreased. However, under glucose-sufficient conditions (potassium- or ammonia-limitation) both species behaved similarly; the glucose-PTS activity was lower and bore no obvious relationship to the rate of glucose consumption expressed by the growing culture. These results are discussed in relation to the role of glucose as a regulator of glucose-PTS synthesis, and to the likely contribution which the glucose-PTS makes to the overall rate of glucose uptake, particularly by cells gorwing in glucose-sufficient environments.

Ammonia↗

PubMed1986

Relationship between pseudo-HPr and the PEP: fructose phosphotransferase system in Salmonella typhimurium and Escherichia coli.

We have studied in Salmonella typhimurium and Escherichia coli the properties of pseudo-HPr suppressor mutations. These mutations suppressed the defects in a ptsH mutant which lacks HPr, one of the enzymes of the phosphoenolpyruvate: carbohydrate phosphotransferase system. The suppressor mutation was mapped in S. typhimurium at 3 min, closely linked to leu. The corresponding chromosomal fragment of 1.7 kb from S. typhimurium and E. coli (extending clockwise from ilvH) was cloned. In a maxicell system a protein with an approximate molecular weight of 36,000 was synthesized. Pseudo-HPr suppressor mutations (fruR) and a deletion extending clockwise from leu resulted in the constitutive expression of the fru operon containing the genes for IIFru (fruA), IIIFru (fruB), fructose 1-phosphate kinase (fruK) and pseudo-HPr (fruF). fruR probably codes for a repressor of the fru operon. Tn10 mutagenesis revealed the following order of genes in the fru operon: fruB-(fruK, fruF)-fruA. Pseudo-HPr activity could replace HPr in PEP-dependent phosphorylation of PTS carbohydrates. IIIFru could be phosphorylated both via HPr and pseudo-HPr, since mutants lacking pseudo-HPr activity were still able to phosphorylate fructose in the presence of added HPr. Both the pseudo-HPr suppressor mutations at 3 min and the deletion extending from leu had an additional phenotype. Introduction of these mutations or deletions was always accompanied by disappearance of PEP synthase activity. Complementation of such a mutant with the cloned fragments reversed both phenotypes at the same time. Possibly, the fruR gene product acts as an activator of the gene coding for PEP synthase.

Cloning, Molecular↗

PubMed1993

The Escherichia coli mannitol permease as a model for transport via the bacterial phosphotransferase system.

The bacterial phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) consists of several proteins whose primary functions are to transport and phosphorylate their substrates. The complexity of the PTS undoubtedly reflects its additional roles in chemotaxis to PTS substrates and in regulation of other metabolic processes in the cell. The PTS permeases (Enzymes II) are the membrane-associated proteins of the PTS that sequentially recognize, transport, and phosphorylate their specific substrates in separate steps, and the Escherichia coli mannitol permease is one of the best studied of these proteins. It consists of two cytoplasmic domains (EIIA and EIIB) involved in mannitol phosphorylation and an integral membrane domain (EIIC) which is sufficient to bind mannitol, but which transports mannitol at a rate that is dependent on phosphorylation of the EIIA and EIIB domains. Recent results show that several residues in a hydrophilic, 85-residue segment of the EIIC domain are important for the binding, transport, and phosphorylation of mannitol. This segment may be at least partially exposed to the cytoplasm of the cell. A model is proposed in which this region of the EIIC domain is crucial in coupling phosphorylation of the EIIB domain to transport through the EIIC domain of the mannitol permease.

Amino Acid Sequence↗

PubMed1978

Partial purification and some properties of a nucleoside phosphotransferase of chick embryos.

A nucleoside phosphotransferase purified about 40fold from chick embryos utilizes efficiently as phosphate donors deoxyribonucleoside and pyrimidine ribonucleoside monophosphates, whereas the pyrimidine deoxyribonucleoside appear to be the preferred acceptors of phosphate. The enzyme is very unstable to heat, dilution and dialysis. A marked enhancement in the stability is caused by nucleotides and it seems associated with the formation of an aggregated state of the protein.

Animals↗