Nucleotide sequence of the uhp region of Escherichia coli.

The Escherichia coli uhp region encodes the transport system that mediates the uptake of a number of sugar phosphates as well as the regulatory components that are responsible for induction of this transport system by external glucose 6-phosphate. Four uhp genes have been identified by analysis of the complementation behavior and polypeptide coding capacity of plasmids carrying subcloned regions or transposon insertions. The nucleotide sequence of a 6.5-kilobase segment that contains the 3' end of the ilvBN operon and the entire uhp region was determined. Four open reading frames were identified in the locations expected for the various uhp genes; all were oriented in the same direction, counterclockwise relative to the genetic map. The properties of the polypeptides predicted from the nucleotide sequence were consistent with their observed features. The 196-amino-acid UhpA polypeptide has the composition characteristic of a soluble protein and bears homology to the DNA-binding regions of many regulatory activators and repressors. The 518-amino-acid UhpB and the 199-amino-acid UhpC regulatory proteins contain substantial segments of hydrophobic character. Similarly, the 463-amino-acid UhpT transporter is a hydrophobic protein with numerous potential transmembrane segments. The UhpC regulatory protein has substantial sequence homology to part of UhpT, suggesting that this regulatory protein might have evolved by duplication of the gene for the transporter and that its role in transmembrane signaling may involve sugar-phosphate-binding sites and transmembrane orientations similar to those of the transport protein.     

Properties of the glucose-6-phosphate transporter from Chlamydia pneumoniae (HPTcp) and the glucose-6-phosphate sensor from Escherichia coli (UhpC)

The amino acid sequence of the proposed glucose-6-phosphate (Glc6P) transporter from Chlamydia pneumoniae (HPTcp; hexose phosphate transporter [Chlamydia pneumoniae]) exhibits a higher degree of similarity to the Escherichia coli Glc6P sensor (UhpC) than to the E. coli Glc6P transporter (UhpT). Overexpression of His-UhpC in a UhpT-deficient E. coli strain revealed that the sensor protein is also able to transport Glc6P and exhibits an apparent K(m) ((Glc6P)) of 25 microM, whereas His-HPTcp exhibits an apparent K(m)( (Glc6P)) of 98 microM. His-HPTcp showed a four-times-lower specific activity than His-UhpT but a 56-times-higher specific activity than His-UhpC. Like His-UhpT and His-UhpC, the carrier His-HPTcp performs a sugar-phosphate/inorganic-phosphate antiporter mode of transport. Surprisingly, while physiological concentrations of inorganic phosphate competitively inhibited transport mediated by the E. coli proteins His-UhpT and His-UhpC, transport mediated by His-HPTcp was not inhibited. Interestingly, C(3)-organophosphates stimulated His-HPTcp activity but not His-UhpT- or His-UhpC-catalyzed Glc6P transport. In contrast to His-UhpC, the His-HPTcp protein does not act as a Glc6P sensor in the uhp regulon.     

Genetic control of the hexose phosphate transport system of Escherichia coli: mapping of deletion and insertion mutations in the uhp region.

The Escherichia coli transport system responsible for the accumulation of a number of sugar phosphates is encoded by the uhp region and is induced by external, but not intracellular, glucose 6-phosphate. To delineate the genetic organization of the uhp region, a total of 225 independent point, deletion, and transposon Tn10 insertion mutations were collected. Mutations conferring the Uhp-phenotype were obtained on the basis of their resistance to fosfomycin and their inability to use sugar phosphates as carbon source. Deletions of uhp sequences were obtained as a consequence of imprecise excision of Tn10 insertions located on either side of uhp. Conjugal crosses between these deletions and the point of insertion mutations allowed determination of the relative order of the uhp alleles and of the deletion endpoints. Specialized lambda transducing phages carrying a uhpT-lac operon fusion and various amounts of adjacent uhp material were isolated and used as genetic donors. Results from these crosses corroborated those obtained in the conjugal crosses. The locations of the mutant alleles were compared with the regulatory properties of Uhp+ revertants of these alleles. This comparison suggested the existence of at least three genes in which mutation yields the Uhp-phenotype. Mapping experiments were consistent with the gene order pyrE-gltS-uhpTRA-ilvB, where uhpT encodes the transport system and uhpR and uhpA are regulatory genes whose products are necessary for proper uhp regulation.     

Methylglyoxal-mediated growth inhibition in an Escherichia coli cAMP receptor protein mutant

Under certain growth conditions, some strains of Escherichia coli accumulate toxic levels of methylglyoxal. This report characterizes a strain which synthesizes a mutant cAMP receptor protein in an adenylate cyclase deletion background. When cultured in glucose 6-phosphate minimal medium, this strain (222) was prematurely growth arrested due to methylglyoxal production; growth inhibition did not occur when the strain was grown in glucose minimal medium. A comparison of a variety of enzyme and cofactor levels in the related strains 222 (mutant) and 225 (wild-type) grown on either glucose or glucose 6-phosphate medium was carried out. The only difference found that might explain an increase in methylglyoxal accumulation was an elevated level of phosphofructokinase in strain 222 grown on glucose 6-phosphate. Since this enzyme activity probably limits hexose phosphate metabolism, it is suggested that growth inhibition in strain 222 may be due to increased production of triose phosphate, some of which is converted to methylglyoxal.     

UhpT, the sugar phosphate antiporter of Escherichia coli, functions as a monomer

We have characterized the minimal functioning unit of UhpT, the secondary carrier that mediates exchange of phosphate and glucose 6-phosphate in Escherichia coli. Membranes of a UhpT overproducing strain were solubilized with 1.25% octyl beta-D-glucopyranoside, in the presence of 0.1% E. coli phospholipid and with 20% glycerol as the osmolyte stabilant. That soluble UhpT could bind its natural substrates was indicated by the protections afforded by sugar phosphates against thermal inactivation or chemical modification with pyridoxal 5'-phosphate. Moreover, the degree of protection correlated with the strength of interaction between UhpT and the test substrate (2-deoxyglucose 6-phosphate = glucose 6-phosphate greater than galactose 6-phosphate = glucose 1-phosphate much greater than glucose 6-sulfate). Other experiments demonstrated that soluble UhpT existed as a monomer. For example, during both high performance liquid chromatography and conventional gel permeation chromatography, the elution pattern of UhpT activity was measured directly by a rapid reconstitution technique. In both cases, and in the presence and absence of substrate, UhpT activity traveled as a single component of Mr 53,000, corresponding closely to the sequence prediction of 50,600. Finally, reconstitution was studied at protein to lipid ratios low enough to achieve between 0.075 and 1.5 UhpT monomers/proteoliposome. Specific activity was constant throughout this range, a finding consistent with the idea of a functional monomer. Mitochondria and chloroplasts provide the only other anion exchange carriers described at this level of biochemical resolution, and these organelle antiporters function as dimers. By contrast, work summarized here places their bacterial counterpart, UhpT, in the same class as the lactose carrier of E. coli and the glucose carrier of the human erythrocyte, both of which function as monomers. Consideration of this pattern in conjunction with the known hydropathy profiles of these proteins suggests a novel scheme for the classification of all secondary carriers, with implications for both the structure and origin of these transport proteins.     

Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio.

During batch growth of Lactococcus lactis subsp. lactis NCDO 2118 on various sugars, the shift from homolactic to mixed-acid metabolism was directly dependent on the sugar consumption rate. This orientation of pyruvate metabolism was related to the flux-controlling activity of glyceraldehyde-3-phosphate dehydrogenase under conditions of high glycolytic flux on glucose due to the NADH/NAD+ ratio. The flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase led to an increase in the pool concentrations of both glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate and inhibition of pyruvate formate lyase activity. Under such conditions, metabolism was homolactic. Lactose and to a lesser extent galactose supported less rapid growth, with a diminished flux through glycolysis, and a lower NADH/NAD+ ratio. Under such conditions, the major pathway bottleneck was most probably at the level of sugar transport rather than glyceraldehyde-3-phosphate dehydrogenase. Consequently, the pool concentrations of phosphorylated glycolytic intermediates upstream of glyceraldehyde-3-phosphate dehydrogenase decreased. However, the intracellular concentration of fructose-1,6-bisphosphate remained sufficiently high to ensure full activation of lactate dehydrogenase and had no in vivo role in controlling pyruvate metabolism, contrary to the generally accepted opinion. Regulation of pyruvate formate lyase activity by triose phosphates was relaxed, and mixed-acid fermentation occurred (no significant production of lactate on lactose) due mostly to the strong inhibition of lactate dehydrogenase by the in vivo NADH/NAD+ ratio.     

Density-dependent changes in hexose transport, glycolytic enzyme levels, and glycolytic rates, in uninfected and murine sarcoma virus-transformed rat kidney cells

In normal rat kidney (NRK) cell cultures, increased cell density results in a decrease in the rates of hexose transport, glucose utilization, and lactate production and an increase in the level of hexokinase activity. A murine sarcoma virus (Kirsten)-transformed cell line (KNRK) showed little or no density-dependent variation in sugar uptake, glucose consumption, or lactate production. On the other hand, hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase activities were elevated in dense transformed cultures as compared to sparse or uninfected cultures. In another virus-transformed cell line (ts339/NRK) exhibiting temperature-dependent morphology, growth pattern, and transport of 2-deoxy- -glucose, the levels of glycolytic enzyme activity were related to cell density but not to the culture temperature. The lack of correlation between glycolytic enzyme activity and lactate production by either uninfected or murine sarcoma virus-transformed cultures supports the suggestion that enhanced growth and/or hexose transport capacity rather than elevated glycolytic enzyme activity are responsible for the increased rate of lactate production by virus-transformed NRK cells.     

Inhibition of the glycolytic pathway by methylglyoxal in human platelets

The incubation of human platelets with methylglyoxal and glucose produces a rapid transformation of the ketoaldehyde to D-lactate by the glyoxalase system and a partial reduction in GSH. Glucose utilization is affected at the level of the glycolytic pathway. No effect of the ketoaldehyde on glycogenolysis and glucose oxidation through the hexose monophosphate shunt was demonstrated. Phosphofructokinase, fructose 1,6 diphosphate (F1, 6DP) aldolase, glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate mutase were mostly inhibited by methylglyoxal. A decrease in lactate and pyruvate formation and an accumulation of some glycolytic intermediates (fructose 1,6 diphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate) was observed. Moreover methylglyoxal induced a fall in the metabolic ATP concentration. Since methylglyoxal is an intermediate of the glycolytic bypass system from dihydroxyacetone phosphate to D-lactate, it may be assumed that ketoaldehyde exerts a regulating effect on triose metabolism.     

The mechanism of glucose 6-phosphate transport by Escherichia coli

To evaluate anion exchange as the mechanistic basis of sugar phosphate transport, natural and artificial membranes were used in studies of glucose 6-phosphate (Glc-6-P) and inorganic phosphate (Pi) accumulation by the uhpT-encoded protein (UhpT) of Escherichia coli. Experiments with intact cells demonstrated that UhpT catalyzed the neutral exchange of internal and external Pi, and work with everted as well as right-side-out membrane vesicles showed further that UhpT mediated the heterologous exchange of Pi and Glc-6-P. When loaded with Pi, but not when loaded with morpholinopropanesulfonate (MOPS), everted vesicles took up Glc-6-P to levels 100-fold above medium concentration in a reaction unaffected by the ionophores valinomycin, valinomycin plus nigericin, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Similarly, right-side-out vesicles were capable of Glc-6-P transport, but only if a suitable internal countersubstrate was available. Thus, in MOPS-loaded vesicles, oxidative metabolism established a proton-motive force that supported proline or Pi accumulation, but transport of Glc-6-P was found only if vesicles could accumulate Pi during a preincubation. After reconstitution of UhpT into proteoliposomes it was possible to show as well that the level of accumulation of Glc-6-P (17 to 560 nmol/mg of protein) was related directly to the internal concentration of Pi. These results are most easily understood if the transport of glucose 6-phosphate in E. coli occurs by anion exchange rather than by nH+/anion support.     

Altered substrate selectivity in a mutant of an intrahelical salt bridge in UhpT, the sugar phosphate carrier of Escherichia coli

Site-directed and second site suppressor mutagenesis identify an intrahelical salt bridge in the eleventh transmembrane segment of UhpT, the sugar phosphate carrier of Escherichia coli. Glucose 6-phosphate (G6P) transport by UhpT is inactivated if cysteine replaces either Asp388 or Lys391 but not if both are replaced. This suggests that Asp388 and Lys391 are involved in an intrahelical salt bridge and that neither is required for normal UhpT function. This interpretation is strengthened by the finding that mutations at Lys391 (K391N, K391Q, and K391T) are recovered as revertants of the inactive D388C variant. Further work shows that although the D388C variant is null for G6P transport, movement of 32Pi by homologous Pi/Pi exchange is unaffected. This raises the possibility that this derivative may have latent function, a possibility confirmed by showing that D388C is a gain-of-function mutation in which phosphoenolpyruvate (PEP) is the preferred substrate. Added study of the Pi/Pi exchange shows that in wild type UhpT this partial reaction is readily blocked by G6P but not PEP. By contrast, in the D388C variant, Pi/Pi exchange is unaffected by G6P but is inhibited by both PEP and 3-phosphoglycerate. These latter substrates are used by PgtP, a related Pi-linked antiporter, which lacks the Asp388-Lys391 salt bridge but has instead an uncompensated arginine at position 391. For this reason, we conclude that in both UhpT and PgtP position 391 can serve as a determinant of substrate selectivity by acting as a receptor for the anionic carboxyl brought into the translocation pathway by PEP.     

Physiological Consequences of Multiple-Target Regulation by the Small RNA SgrS in Escherichia coli

Cells use complex mechanisms to regulate glucose transport and metabolism to achieve optimal energy and biomass production while avoiding accumulation of toxic metabolites. Glucose transport and glycolytic metabolism carry the risk of the buildup of phosphosugars, which can inhibit growth at high concentrations. Many enteric bacteria cope with phosphosugar accumulation and associated stress (i.e., sugar-phosphate stress) by producing a small RNA (sRNA) regulator, SgrS, which decreases phosphosugar accumulation in part by repressing translation of sugar transporter mRNAs (ptsG and manXYZ) and enhancing translation of a sugar phosphatase mRNA (yigL). Despite a molecular understanding of individual target regulation by SgrS, previously little was known about how coordinated regulation of these multiple targets contributes to the rescue of cell growth during sugar-phosphate stress. This study examines how SgrS regulation of different targets impacts growth under different nutritional conditions when sugar-phosphate stress is induced. The severity of stress-associated growth inhibition depended on nutrient availability. Stress in nutrient-rich media necessitated SgrS regulation of only sugar transporter mRNAs (ptsG or manXYZ). However, repression of transporter mRNAs was insufficient for growth rescue during stress in nutrient-poor media; here SgrS regulation of the phosphatase (yigL) and as-yet-undefined targets also contributed to growth rescue. The results of this study imply that regulation of only a subset of an sRNA''s targets may be important in a given environment. Further, the results suggest that SgrS and perhaps other sRNAs are flexible regulators that modulate expression of multigene regulons to allow cells to adapt to an array of stress conditions.     

Energy coupling in the uptake of hexose phosphates by Escherichia coli.

Several methods were used to study the source of energy in the uptake of hexose phosphates by Escherichia coli K12. The uptake was sensitive to inhibition by agents that affect electron transport, such as lack of oxygen, cyanide, and heptylhydroxyquinoline-N-oxide, and by agents that affect ATP utilization, such as dicyclohexylcarbodiimide and arsenate. It was also sensitive to uncouplers in the presence of absence of oxygen. The strain of E. coli used extruded protons during respiration. Uncer anaerobic conditions, the uptake of approximately 1 eg to H+ per glucose 6-phosphate. These observations are consistent with a chemiosmotic mechanism of genergized glucose 6-phosphate uptake. The rate of glucose 6-phosphate uptake was maximal in KC1, but was also stimulated by MgC12 or CaC12. Inhibition by A217, a nigericin-like antibiotic, was prevented by K+ whereas valinomycin and gramicidin inhibited in the presence or absence of K+.     

Transmembrane segment 11 of UhpT, the sugar phosphate carrier of Escherichia coli, is an alpha-helix that carries determinants of substrate selectivity

In Escherichia coli, transport of hexose 6-phosphates is mediated by the P(i)-linked antiport carrier, UhpT, a member of the major facilitator superfamily. We showed earlier that Lys(391), a member of an intrahelical salt bridge (Asp(388)/Lys(391)) in the eleventh transmembrane segment (TM11) of this transporter, can function as a determinant of substrate selectivity (Hall, J. A., Fann, M.-C., and Maloney, P. C. (1999) J. Biol. Chem. 274, 6148-6153). Here, we examine in detail the role of TM11 in setting substrate preference. Derivatives having an uncompensated cationic charge at either position 388 or 391 (the D388C, D388V, or D388K/K391C variants) are gain-of-function mutants in which phosphoenolpyruvate, not sugar 6-phosphate, is the preferred organic substrate. By contrast, when an uncompensated anionic charge is placed at position 388 (K391C), we observed behavior consistent with an increased preference for monovalent rather than divalent sugar 6-phosphate. Because positions 388 and 391 lie deep within the UhpT hydrophobic sector, these findings suggested that an extended length of TM11 may be accessible to external substrates and probes. To explore this issue, we used a panel of TM11 single cysteine variants to examine the transport of glucose 6-phosphate in the presence and absence of the membrane-impermeant, thiol-reactive agent p-chloromercuribenzosulfonate (PCMBS). Accessibility to PCMBS, together with the pattern of substrate protection against PCMBS inhibition, leads us to conclude that TM11 spans the membrane as an alpha-helix, with approximately two-thirds of its surface lining a substrate translocation pathway. We suggest that this feature is a general property of carrier proteins in the major facilitator superfamily and that for this reason residues in TM11 will serve to carry determinants of substrate selectivity.     

Purification and properties of DAHP synthase from Nocardia mediterranei

3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, the first enzyme of the shikimate pathway was isolated from Nocardia mediterranei. It has a molecular weight of approx. 135,000, and four identical subunits, each with a molecular weight of 35,000. The Km values for phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E-4-P) were 0.4 and 0.25 mM, respectively, and kinetic study showed that LTrp inhibited DAHP synthase activity, but was not competitive with respect to PEP or E-4-P. The enzyme activity was inhibited by excess of E-4-P added in the incubation system. D-ribose 5-phosphate (R-5-P), D-glucose 6-phosphate (G-6-P) or D-sedoheptulose 7-phosphate (Su-7-P) etc. inhibited DAHP synthase in cell-free extract, but on partially purified enzyme no inhibitory effect was detected. The indirect inhibition of R-5-P and other sugar phosphates was considered to be due to the formation of E-4-P catalyzed by the related enzymes present in cell-free extract.