Cloning of the two pyruvate kinase isoenzyme structural genes from Escherichia coli: the relative roles of these enzymes in pyruvate biosynthesis

Ponce, Elizabeth; Flores, Noemí; Martı́nez, Alfredo; Valle, Fernando; Bolívar, Francisco

doi:10.1128/jb.177.19.5719-5722.1995

Cited by 103 publications

(101 citation statements)

References 9 publications

(4 reference statements)

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“…and its pyruvate kinase-deficient but otherwise isogenic derivative PB25 (pykA::kan pykF::cat) were used (35). Inocula were grown overnight from a picked colony in baffled shake flasks at 37°C in M9 minimal medium containing 5 g of glucose per liter, 42 mM Na 2 HPO 4 , 22 mM KH 2 PO 4 , 19 mM NH 4 Cl, and 9 mM NaCl.…”

Section: Methodsmentioning

confidence: 99%

“…To investigate quantitative physiological effects of pyruvate kinase knockout, we grew E. coli JM101 and its pyruvate kinase-deficient derivative PB25 (35) in aerobic chemostats under glucose or ammonia limitation (Table 1), which represent bioenergetically very different regimens with profound effects on cellular physiology (31,42).…”

Section: Physiology Of E Coli Jm101 and Pb25 In Chemostat Culturementioning

confidence: 99%

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Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli

et al. 2002

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The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13 C labeling experiments with [U-13 C 6 ]glucose in glucoseor ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model. The general response to disruption of both pyruvate kinase isoenzymes in E. coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme. Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions. In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E. coli, with a turnover that is comparable to the specific glucose uptake rate. Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E. coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate. Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.The central carbon pathways fulfill anabolic and catabolic functions by providing cofactors and building blocks for macromolecular synthesis as well as energy. While some singlegene knockout mutations in central metabolism preclude growth on glucose, a majority of such variations can potentially be compensated for either by isoenzymes (39) or by a rerouting of carbon fluxes through alternative pathways (7,15,20,42). Usually, the phenotypes of knockout mutants is characterized by quantitative physiological analysis, and conclusions on intracellular metabolism are then based on qualitative interpretation of these results. To reveal cause and effect relationships, however, it is important to gain deeper insight into the complex metabolic responses at the level of intracellular metabolite concentrations and fluxes. These intracellular carbon fluxes, or in vivo reaction rates, are per se nonmeasurable quantities that cannot usually be inferred directly from in vitro enzyme activities because not all in vivo effector concentrations are known. Hence, the intracellular reaction rates are commonly estimated by methods of metabolic flux analysis, which provide a holistic perspective on metabolism (52, 54).The most common approach is based on flux balancing analysis within a stoichiometric model of cellular metabolism (26,41, 50, 52). In this approach, uptake and secretion rates and biosynthetic requirements are balanced in a stoichiometric model, assuming quasi-steady-state material balances on the intermediates. In many cases, however, limited extracellular data and stoichiometric constraints lead to underdetermined systems, so that addition...

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Section: Methodsmentioning

confidence: 99%

Section: Physiology Of E Coli Jm101 and Pb25 In Chemostat Culturementioning

confidence: 99%

Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli

et al. 2002

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“…1C). The pyk gene encodes a pyruvate kinase enzyme involved in pyruvate biosynthesis (38), and ptsN encodes a phosphotransferase system in E. coli (22). Plasmid pVS166, containing pyk::lacZ, expressed the same levels of ␤-galactosidase activity in the wild-type, VS94 (luxS mutant), and VS95 (VS94 complemented) strains' backgrounds (Fig.…”

Section: Construction Of Ehec Luxs Mutantmentioning

confidence: 99%

Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

et al. 2001

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Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenic luxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in the luxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, the luxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 m/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with -like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

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“…While PykF 58 crystal structures are available and show the tetrameric organisation typical of PK enzymes 59 [8,9], structural data on PykA homologues are missing. In activity assays, PykF significantly 60 surpasses PykA activity under all conditions tested [6, 10,11] leaving open the question of 61 why these bacteria need two isoenzymes. In E. coli, the deletion of both pyk genes increases 62 expression and activity of phosphoenolpyruvate carboxylase (PEPC), a fact that indicates the 63 rerouting of carbon fluxes via PEPC [12].…”

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confidence: 99%