Physiology study of Escherichia coli harboring high intracellular ATP driven by artificial Pck expression
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ABSTRACT:
ORGANISM(S): Escherichia coli BL21(DE3)
PROVIDER: GSE44928 | GEO | 2016/03/06
SECONDARY ACCESSION(S): PRJNA193227
REPOSITORIES: GEO
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Project description:Up-regulation of motility genes and biosynthesis genes were found in the pck expressing high ATP cell by transcriptome analysis while catabolism genes were down-regulated. Keywords: response depends on intracellular ATP concentration derived by pck or ppc overexpression 1. pck or ppc overexpressing E. coli at early log phase using glucose-minimal medium 2. pck or ppc overexpressing E. coli at chemostat culture (D=0.1 h-1)using LB-glucose medium
2010-06-09 | E-GEOD-8532 | biostudies-arrayexpress
Project description:Up-regulation of motility genes and biosynthesis genes were found in the pck expressing high ATP cell by transcriptome analysis while catabolism genes were down-regulated. Keywords: response depends on intracellular ATP concentration derived by pck or ppc overexpression
2007-07-26 | GSE8532 | GEO
Project description:Adenylate kinase 4 (AK4) is localized in the mitochondrial matrix, and is believed to be involved in stress, drug resistance, the malignant transformation of cancer, and ATP regulation. We produced AK4 knockdown HeLa cells by using shRNA and used these to analyze resistance, and found that sensitivity to hypoxia and drugs increased. 3 samples of HeLa cells haboring AK4 shRNA plasmids and 4 samples of HeLa cells haboring control shRNA plasmid.
2016-03-31 | E-GEOD-61843 | biostudies-arrayexpress
Project description:DNA affinity chromatography with the promoter region of the Corynebacterium glutamicum pck gene, encoding phosphoenolpyruvate carboxykinase (PEPCk), led to the isolation of four transcriptional regulators, i.e., RamA, GntR1, GntR2 and IolR. Determination of the PEPCk activity of the deletion mutants ΔramA, ΔgntR1ΔgntR2, and ΔiolR indicated that RamA represses pck during growth on glucose about twofold, whereas GntR1, GntR2, and IolR activate pck expression about twofold, irrespective whether glucose or acetate served as carbon source. The DNA binding sites of the four regulators in the pck promoter region were identified and their positions correlated with the predicted functions as repressor or activators. The iolR gene is located upstream and in divergent orientation to a iol gene cluster, encoding proteins involved in myo-inositol uptake and degradation. Comparative DNA microarray analysis of the ΔiolR mutant and the parental wild-type revealed strongly elevated (>100-fold) mRNA levels of the iol genes in the mutant, indicating that the primary function of IolR is the repression of the iol genes. IolR binding sites were identified in the promoter regions of iolC, iolT and iolR itself, which presumably is subject to negative autoregulation. A consensus DNA-binding motif was identified (5’-KGWCHTRACA-3’) which corresponds well to those of other GntR-type regulators of the HutC family. Taken together, our results disclose a complex regulation of the pck gene in C. glutamicum and identify IolR as an efficient repressor of genes involved in myo-inositol catabolism of this organism.
2013-07-29 | GSE44812 | GEO
Project description:DNA affinity chromatography with the promoter region of the Corynebacterium glutamicum pck gene, encoding phosphoenolpyruvate carboxykinase (PEPCk), led to the isolation of four transcriptional regulators, i.e., RamA, GntR1, GntR2 and IolR. Determination of the PEPCk activity of the deletion mutants M-NM-^TramA, M-NM-^TgntR1M-NM-^TgntR2, and M-NM-^TiolR indicated that RamA represses pck during growth on glucose about twofold, whereas GntR1, GntR2, and IolR activate pck expression about twofold, irrespective whether glucose or acetate served as carbon source. The DNA binding sites of the four regulators in the pck promoter region were identified and their positions correlated with the predicted functions as repressor or activators. The iolR gene is located upstream and in divergent orientation to a iol gene cluster, encoding proteins involved in myo-inositol uptake and degradation. Comparative DNA microarray analysis of the M-NM-^TiolR mutant and the parental wild-type revealed strongly elevated (>100-fold) mRNA levels of the iol genes in the mutant, indicating that the primary function of IolR is the repression of the iol genes. IolR binding sites were identified in the promoter regions of iolC, iolT and iolR itself, which presumably is subject to negative autoregulation. A consensus DNA-binding motif was identified (5M-bM-^@M-^Y-KGWCHTRACA-3M-bM-^@M-^Y) which corresponds well to those of other GntR-type regulators of the HutC family. Taken together, our results disclose a complex regulation of the pck gene in C. glutamicum and identify IolR as an efficient repressor of genes involved in myo-inositol catabolism of this organism. To identify genes that are potentially regulated by IolR, the transcriptome profile of the iolR deletion strain was compared to that of the WT using DNA microarray analysis. The strains were grown in CGXII minimal medium with 4% (wt/vol) glucose as sole carbon source and RNA was isolated from cells harvested in the early exponential growth phase (OD600 of about 5). The transcriptome comparison was performed in triplicate starting from independent cultures. The second replicate included a dye-swap.
2013-07-29 | E-GEOD-44812 | biostudies-arrayexpress
Project description:Saccharopolyspora erythraea is used for industrial-scale production of erythromycin. To explore the physiological role of co-factors in regulation of primary and secondary metabolism of S. erythraea, we initially overexpressed the endogenous F1-ATPase in an erythromycin high-producing strain, E3. The engineered strain is named EA. The F1-ATPase expression resulted in a lower [ATP]/[ADP] ratio, which was accompanied by a dramatic increased production of a reddish pigment and a decreased erythromycin production. Transcriptional analysis revealed that the intracellular [ATP]/[ADP] ratio appeared to exert a global regulation on the metabolism of S.erythraea, and the lower [ATP]/[ADP] ratio induced physiological changes to restore the energy balance, mainly via pathways that tend to produce ATP or NADH. The results also indicated a state of redox stress in the engineered strain, which was correlated to the alteration of electron transport at the branch of the terminal oxidases.
2020-02-26 | GSE134767 | GEO
Project description:The transcriptional regulator GntR1 downregultates the genes for gluconate catabolism and pentose phosphate pathway in Corynebacterium glutamicum. Gluconate lowers the DNA binding affinity of GntR1, which is probably the mechanism of gluconate-dependent induction of these genes. In addition, GntR1 positively regulates the ptsG, a gene encoding for a major glucose transporter, and the pck, a gene encoding phosphoenolpyruvate carboxykinase. Here, we searched for the new target of GntR1 at genome-wide scale by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analysis. This analysis identified 56 in vivo GntR1 binding sites, of which 7 sites were previously reported. The newly identified GntR1 sites include the upstream regions of carbon metabolism genes such as pyk, maeB, gapB, and icd. Binding of GntR1 to the promoter region of these genes was confirmed by electrophoretic mobility shift assay. The activity of the icd, gapB, and maeB promoters were reduced by the mutation at GntR1 binding site in contrast to the pyk promoter activity increased, indicating that GntR1 is a transcriptional activator of icd, gapB, and maeB and is a repressor of pyk. Thus, it is likely that GntR1 stimulates glucose uptake by inducing the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) gene while repressing pyk to increase PEP availability in the absence of gluconate. Repression of zwf and gnd may reduce NADPH supply which may be compensated by the induction of maeB, and icd. Upregulation of icd, gapB, and maeB and downregulation of pyk by GntR1 probably supports gluconeogenesis. ChIP-chip analyses using strain carrying FLAG-tagged gntR1 in the background of the wild type. Biological replicates: 2 replicates.
2014-07-15 | E-GEOD-58632 | biostudies-arrayexpress
Project description:The transcriptional regulator GntR1 downregulates the genes for gluconate catabolism and pentose phosphate pathway in Corynebacterium glutamicum. Gluconate lowers the DNA binding affinity of GntR1, which is probably the mechanism of gluconate-dependent induction of these genes. In addition, GntR1 positively regulates the ptsG, a gene encoding for a major glucose transporter, and the pck, a gene encoding phosphoenolpyruvate carboxykinase. Here, we searched for the new target of GntR1 at genome-wide scale by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analysis. This analysis identified 56 in vivo GntR1 binding sites, of which 7 sites were previously reported. The newly identified GntR1 sites include the upstream regions of carbon metabolism genes such as pyk, maeB, gapB, and icd. Binding of GntR1 to the promoter region of these genes was confirmed by electrophoretic mobility shift assay. The activity of the icd, gapB, and maeB promoters were reduced by the mutation at GntR1 binding site in contrast to the pyk promoter activity increased, indicating that GntR1 is a transcriptional activator of icd, gapB, and maeB and is a repressor of pyk. Thus, it is likely that GntR1 stimulates glucose uptake by inducing the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) gene while repressing pyk to increase PEP availability in the absence of gluconate. Repression of zwf and gnd may reduce NADPH supply which may be compensated by the induction of maeB, and icd. Upregulation of icd, gapB, and maeB and downregulation of pyk by GntR1 probably supports gluconeogenesis. Gene expression profile of the wild type at the exponential phase was compared with that of the rshA deletion mutant. Three independent experiments were performed.
2014-07-15 | E-GEOD-58631 | biostudies-arrayexpress
Project description:Effect pck or ppc expression on intracellular ATP
| PRJNA101665 | ENA
Project description:Wild-type C. glutamicum ATCC 13032 is known to possess two enzymes with anaplerotic (C4-directed) carboxylation activity, namely phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PCx). On the other hand, C3-directed decarboxylation can be catalyzed by the three enzymes phosphoenolpyruvate carboxykinase (PEPCk), oxaloacetate decarboxylase (ODx), and malic enzyme (ME). The resulting high metabolic flexibility at the anaplerotic node compromises the unambigous determination of its carbon and energy flux in C. glutamicum wild type. To circumvent this problem we performed a comprehensive analysis of selected single or double deletion mutants in the anaplerosis of wild-type C. glutamicum under defined D-glucose conditions. By applying well-controlled lab-scale bioreactor experiments in combination with untargeted proteomics, quantitative metabolomics and whole-genome sequencing hitherto unknown, and sometimes counter-intuitive, genotype-phenotype relationships in these mutants could be unraveled. In comparison to the wild type the four mutants C. glutamiucm pyc, C. glutamiucmpyc odx, C. glutamiucm ppc pyc and C. glutamiucm pck showed lowered specific growth rates and D-glucose uptake rates, underlining the importance of PCx and PEPCk activity for a balanced carbon and energy flux at the anaplerotic node. Most interestingly, the strain C. glutamiucm ppc pyc could be evolved to grow on D-glucose as the only source of carbon and energy, whereas this combination was previously considered lethal. The prevented anaplerotic carboxylation activity of PEPCx and PCx was found in the evolved strain to be compensated by an up-regulation of the glyoxylate shunt, potentially in combination with the 2-methylcitrate cycle.
2021-09-09 | PXD022622 | Pride