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In Corynebacterium glutamicum formation of glc-1-P (α-glucose-1-phosphate) from glc-6-P (glucose-6-phosphate) by α-Pgm (phosphoglucomutase) is supposed to be crucial for synthesis of glycogen and the cell wall precursors trehalose and rhamnose. Furthermore, Pgm is probably necessary for glycogen degradation and maltose utilization as glucan phosphorylases of both pathways form glc-1-P. We here show that C. glutamicum possesses at least two Pgm isoenzymes, the cg2800 (pgm) encoded enzyme contributing most to total Pgm activity. By inactivation of pgm we created C. glutamicum IMpgm showing only about 12% Pgm activity when compared to the parental strain. We characterized both strains during cultivation with either glucose or maltose as substrate and observed that (i) the glc-1-P content in the WT (wild-type) and the mutant remained constant independent of the carbon source used, (ii) the glycogen levels in the pgm mutant were lower during growth on glucose and higher during growth on maltose, and (iii) the morphology of the mutant was altered with maltose as a substrate. We conclude that C. glutamicum employs glycogen as carbon capacitor to perform glc-1-P homeostasis in the exponential growth phase and is therefore able to counteract limited Pgm activity for both anabolic and catabolic metabolic pathways.  相似文献   

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Corynebacterium glutamicum strains NC-2 were able to grow on xylose as sole carbon sources in our previous work. Nevertheless, it exhibited the major shortcoming that the xylose consumption was repressed in the presence of glucose. So far, regarding C. glutamicum, there are a number of reports on ptsG gene, the glucose-specific transporter, involved in glucose metabolism. Recently, we found ptsG had influence on xylose utilization and investigated the ptsG gene in response to xylose utilization in C. glutamicum with the aim to improve xylose consumption and simultaneously utilized glucose and xylose. The ptsG-deficient mutant could grow on xylose, while exhibiting noticeably reduced growth on xylose as sole carbon source. A mutant deficient in ptsH, a general PTS gene, exhibited a similar phenomenon. When complementing ptsG gene, the mutant ΔptsG-ptsG restored the ability to grow on xylose similarly to NC-2. These indicate that ptsG gene is not only essential for metabolism on glucose but also important in xylose utilization. A ptsG-overexpressing recombinant strain could not accelerate glucose or xylose metabolism. When strains were aerobically cultured in a sugar mixture of glucose and xylose, glucose and xylose could not be utilized simultaneously. Interestingly, the ΔptsG strain could co-utilize glucose and xylose under oxygen-deprived conditions, though the consumption rate of glucose and xylose dramatically declined. It was the first report of ptsG gene in response to xylose utilization in C. glutamicum.  相似文献   

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Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP):maltose phosphotransferase system (PTS). The maltose‐specific PTS permease is encoded by the malT gene. However, E. faecalis lacks a malA gene encoding a 6‐phospho‐α‐glucosidase, which in B. subtilis hydrolyses maltose 6′‐P into glucose and glucose 6‐P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose 6‐P into glucose 1‐P and glucose 6‐P. However, purified MalP phosphorolyses maltose but not maltose 6′‐P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose 6′‐P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose 1‐P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose 6′‐P restored growth on maltose. MapP catalyses the dephosphorylation of intracellular maltose 6′‐P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose 1‐P. MapP therefore connects PTS‐mediated maltose uptake to maltose phosphorylase‐catalysed metabolism. Dephosphorylation assays with a wide variety of phospho‐substrates revealed that MapP preferably dephosphorylates disaccharides containing an O‐α‐glycosyl linkage.  相似文献   

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Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3Δ nor sam3Δ single mutant is defective in polyamine transport, while the dur3Δ sam3Δ double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2Δ mutant. Studies of Agp2 localization indicate that in the double mutant dur3Δ sam3Δ, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport.  相似文献   

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To characterize the mechanisms involved in glucose transport, in the filamentous fungus Aspergillus nidulans, we have identified four glucose transporter encoding genes hxtB-E. We evaluated the ability of hxtB-E to functionally complement the Saccharomyces cerevisiae EBY.VW4000 strain that is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae HxtB-E were targeted to the plasma membrane. The expression of HxtB, HxtC and HxtE was able to restore growth on glucose, fructose, mannose or galactose, indicating that these transporters accept multiple sugars as a substrate through an energy dependent process. A tenfold excess of unlabeled maltose, galactose, fructose, and mannose were able to inhibit glucose uptake to different levels (50 to 80 %) in these s. cerevisiae complemented strains. Moreover, experiments with cyanide-m-chlorophenylhydrazone (CCCP), strongly suggest that hxtB, -C, and –E mediate glucose transport via active proton symport. The A. nidulans ΔhxtB, ΔhxtC or ΔhxtE null mutants showed ~2.5-fold reduction in the affinity for glucose, while ΔhxtB and -C also showed a 2-fold reduction in the capacity for glucose uptake. The ΔhxtD mutant had a 7.8-fold reduction in affinity, but a 3-fold increase in the capacity for glucose uptake. However, only the ΔhxtB mutant strain showed a detectable decreased rate of glucose consumption at low concentrations and an increased resistance to 2-deoxyglucose.  相似文献   

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Deletion of TRK1 and TRK2 abolishes high-affinity K+ uptake in Saccharomyces cerevisiae, resulting in the inability to grow on typical synthetic growth medium unless it is supplemented with very high concentrations of potassium. Selection for spontaneous suppressors that restored growth of trk1Δ trk2Δ cells on K+-limiting medium led to the isolation of cells with unusual gain-of-function mutations in the glucose transporter genes HXT1 and HXT3 and the glucose/galactose transporter gene GAL2. 86Rb uptake assays demonstrated that the suppressor mutations conferred increased uptake of the ion. In addition to K+, the mutant hexose transporters also conferred permeation of other cations, including Na+. Because the selection strategy required such gain of function, mutations that disrupted transporter maturation or localization to the plasma membrane were avoided. Thus, the importance of specific sites in glucose transport could be independently assessed by testing for the ability of the mutant transporter to restore glucose-dependent growth to cells containing null alleles of all of the known functional glucose transporter genes. Twelve sites, most of which are conserved among eukaryotic hexose transporters, were revealed to be essential for glucose transport. Four of these have previously been shown to be essential for glucose transport by animal or plant transporters. Eight represented sites not previously known to be crucial for glucose uptake. Each suppressor mutant harbored a single mutation that altered an amino acid(s) within or immediately adjacent to a putative transmembrane domain of the transporter. Seven of 38 independent suppressor mutations consisted of in-frame insertions or deletions. The nature of the insertions and deletions revealed a striking DNA template dependency: each insertion generated a trinucleotide repeat, and each deletion involved the removal of a repeated nucleotide sequence.  相似文献   

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Little is known about the molecular basis of biofilm formation in Listeria monocytogenes. The superoxide dismutase (SOD) of the deletion mutant of lm.G_1771 gene, which encodes for a putative ABC transporter permease, is highly expressed in biofilm. In this study, the sod gene deletion mutant Δsod, and double deletion mutant of the sod and lm. G_1771 genes Δ1771Δsod were used to investigate the role of SOD and its relationship to the expression of the putative ABC transporter permease in biofilm formation. Our results showed that the ability to form a biofilm was significantly reduced in the Δsod mutant and the Δ1771Δsod double mutant. Both Δsod and Δ1771Δsod mutants exhibited slow growth phenotypes and produced more reactive oxygen species (ROS). The growth was inhibited in the mutants by methyl viologen (MV, internal oxygen radical generator) treatment. In addition, the expression of one oxidation resistance gene (kat), two stress regulators encoding genes (perR and sigB), and one DNA repair gene (recA) were analyzed in both the wild-type L. monocytogenes 4b G and the deletion mutants by RT-qPCR. The expression levels of the four genes were increased in the deletion mutants when biofilms were formed. Taken together, our data indicated that SOD played an important role in biofilm formation through coping with the oxidant burden in deficient antioxidant defenses.  相似文献   

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Btn2p, a novel coiled-coil protein, is up-regulated in btn1Δ yeast strains, and this up-regulation is thought to contribute to maintaining a stable vacuolar pH in btn1Δ strains (D. A. Pearce, T. Ferea, S. A. Nosel, B. Das, and F. Sherman, Nat. Genet. 22:55-58, 1999). We now report that Btn2p interacts biochemically and functionally with Rsg1p, a down-regulator of the Can1p arginine and lysine permease. Rsg1p localizes to a distinct structure toward the cell periphery, and strains lacking Btn2p (btn2Δ strains) fail to correctly localize Rsg1p. btn2Δ strains, like rsg1Δ strains, are sensitive for growth in the presence of the arginine analog canavanine. Furthermore, btn2Δ strains, like rsg1Δ strains, demonstrate an elevated rate of uptake of [14C]arginine, which leads to increased intracellular levels of arginine. Overexpression of BTN2 results in a decreased rate of arginine uptake. Collectively, these results indicate that altered levels of Btn2p can modulate arginine uptake through localization of the Can1p-arginine permease regulatory protein, Rsg1p. Our original identification of Btn2p was that it is up-regulated in the btn1Δ strain which serves as a model for the lysosomal storage disorder Batten disease. Btn1p is a vacuolar/lysosomal membrane protein, and btn1Δ suppresses both the canavanine sensitivity and the elevated rate of uptake of arginine displayed by btn2Δ rsg1Δ strains. We conclude that Btn2p interacts with Rsg1p and modulates arginine uptake. Up-regulation of BTN2 expression in btn1Δ strains may facilitate either a direct or indirect effect on intracellular arginine levels.  相似文献   

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Under specific environmental conditions, the yeast Saccharomyces cerevisiae can undergo a morphological switch to a pseudohyphal growth pattern. Pseudohyphal differentiation is generally studied upon induction by nitrogen limitation in the presence of glucose. It is known to be controlled by several signaling pathways, including mitogen-activated protein kinase, cyclic AMP-protein kinase A (cAMP-PKA), and Snf1 kinase pathways. We show that the alpha-glucoside sugars maltose and maltotriose, and especially sucrose, are more potent inducers of filamentation than glucose. Sucrose even induces filamentation in nitrogen-rich media and in the mep2Δ/mep2Δ ammonium permease mutant on ammonium-limiting medium. We demonstrate that glucose also inhibits filamentation by means of a pathway parallel to the cAMP-PKA pathway. Deletion of HXK2 shifted the pseudohyphal growth pattern on glucose to that of sucrose, while deletion of SNF4 abrogated filamentation on both sugars, indicating a negative role of glucose repression and a positive role for Snf1 activity in the control of filamentation. In all strains and in all media, sucrose induction of filamentation is greatly diminished by deletion of the sucrose/glucose-sensing G-protein-coupled receptor Gpr1, whereas it has no effect on induction by maltose and maltotriose. The competence of alpha-glucoside sugars to induce filamentation is reflected in the increased expression of the cell surface flocculin gene FLO11. In addition, sucrose is the only alpha-glucoside sugar capable of rapidly inducing FLO11 expression in a Gpr1-dependent manner, reflecting the sensitivity of Gpr1 for this sugar and its involvement in rapid sucrose signaling. Our study identifies sucrose as the most potent nutrient inducer of pseudohyphal growth and shows that glucose inactivation of Snf1 kinase signaling is responsible for the lower potency of glucose.  相似文献   

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Thermoanaerobacter ethanolicus is a gram-positive thermophile that produces considerable amounts of ethanol from soluble sugars and polymeric substrates, including starch. Growth on maltose, a product of starch hydrolysis, was associated with the production of a prominent membrane-associated protein that had an apparent molecular weight of 43,800 and was not detected in cells grown on xylose or glucose. Filter-binding assays revealed that cell membranes bound maltose with high affinity. Metabolic labeling of T. ethanolicus maltose-grown cells with [14C]palmitic acid showed that this protein was posttranslationally acylated. A maltose-binding protein was purified by using an amylose resin affinity column, and the binding constant was 270 nM. Since maltase activity was found only in the cytosol of fractionated cells and unlabeled glucose did not compete with radiolabeled maltose for uptake in whole cells, it appeared that maltose was transported intact. In whole-cell transport assays, the affinity for maltose was approximately 40 nM. Maltotriose and α-trehalose competitively inhibited maltose uptake in transport assays, whereas glucose, cellobiose, and a range of disaccharides had little effect. Based on these results, it appears that T. ethanolicus possesses a high-affinity, ABC type transport system that is specific for maltose, maltotriose, and α-trehalose.  相似文献   

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The transport of glucose across the bacterial cell membrane of Thermoanaerobacter thermosulfuricus (Clostridium thermohydrosulfuricum) Rt8.B1 was governed by a permease which did not catalyze concomitant substrate transport and phosphorylation and thus was not a phosphoenolpyruvate-dependent phosphotransferase. Glucose uptake was carrier mediated, could not be driven by an artificial membrane potential (Δψ) in the presence or absence of sodium, and was not sensitive to inhibitors which dissipate the proton motive force (Δp; tetrachlorosalicylanilide, N,N-dicyclohexylcarboiimide, and 2,4-dinitrophenol), and no uptake of the nonmetabolizable analog 2-deoxyglucose could be demonstrated. The glucokinase apparent Km for glucose (0.21 mM) was similar to the Kt (affinity constant) for glucose uptake (0.15 mM), suggesting that glucokinase controls the rate of glucose uptake. Inhibitors of ATP synthesis (iodoacetate and sodium fluoride) also inhibited glucose uptake, and this effect was due to a reduction in the level of ATP available to glucokinase for glucose phosphorylation. These results indicated that T. thermosulfuricus Rt8.B1 lacks a concentrative uptake system for glucose and that uptake is via facilitated diffusion, followed by ATP-dependent phosphorylation by glucokinase. In T. thermosulfuricus Rt8.B1, glucose is metabolized by the Embden-Meyerhof-Parnas pathway, which yields 2 mol of ATP (G. M. Cook, unpublished data). Since only 1 mol of ATP is used to transport 1 mol of glucose, the energetics of this system are therefore similar to those found in bacteria which possess a phosphotransferase.  相似文献   

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