Mechanism Of Escherichia Coli JM101 Central Carbon Metabolism In Response To Superoxide Stress

The cellular responses of bacteria to superoxide stress can be used to model adaptation to severe environmental changes. Superoxide stress promotes the excessive production of reactive oxygen species (ROS) that have detrimental effects on cell metabolic and other physiological activities. To antagonize such effects, the cell needs to regulate a range of metabolic reactions in a coordinated way, so that coherent metabolic responses are generated by the cellular metabolic reaction network as a whole. In the present study, we have used a quantitative metabolic flux analysis approach, together with measurement of gene expression and activity of key enzymes, to investigate changes in central carbon metabolism that occur in Escherichia coli in response to paraquat-induced superoxide stress. The cellular regulatory mechanisms involved in the observed global flux changes are discussed.Metabolic flux analysis (MFA) using 13C labeling has been frequently used to follow the intracellular fluxes in the central metabolism in bacteria, yeast, filamentous fungi, and animal cells. 13C-labeled metabolites can be monitored throughout the metabolic system and their distribution in certain metabolites can be measured either by two-dimensional nuclear magnetic resonance (2D NMR) or by gas chromatography/mass spectrometry (GC-MS). From these measurements, intracellular fluxes can then be estimated by parameter fitting procedures. The 13C-FLUX software was used for estimation of optimal flux values and 90% confidence intervals of flux values were calculated by Monte Carlo sampling method.In the present study, we used MFA to investigate the metabolic response of E. coli exposed to paraquat (PQ), a known inducer of oxidative stress. We have cultivated wild type E. coli cells in the normal minimum medium and in a PQ-containing one using chemostat cultivations. After comparing some general growth parameters and metabolite production parameters under the two conditions, we have used steady state 13C flux analysis to determine the metabolic flux distributions in the central carbon metabolism network. The network comprises the Embden-Meyerhof pathway (EMP), the pentose phosphate (PP) pathway, the Entner Dourodouf (ED) pathway, the tricarboxylic acid cycle (TCA cycle), the anaplerotic reaction, and the glyoxylate shunt. Flux analysis based on nuclear magnetic resonance (NMR) and mass spectroscopy (MS) measurements and computation provided quantitative results on the metabolic fluxes redistribution of the E. coli central carbon network under paraquat-induced oxidative stress. The metabolic fluxes of the glycolytic pathway were redirected to the pentose phosphate pathway (PP pathway). The production of acetate increased significantly, the fluxes associated with the TCA cycle decreased, and the fluxes in the glyoxylate shunt increased in response to oxidative stress. These global flux changes resulted in an increased ratio of NADPH:NADH and in the accumulation ofα-ketoglutarate.We have quantified a range of changes that occur in metabolic carbon flux during PQ stress in E. coli. These changes are, to a large extent, coherent and lead to systematic adjustments of cellular physiological states. One major adjustment is the increased NADPH generation and decreased NADH generation. This reflects a cellular strategy whereby efficiency is traded for survival under stressful conditions. Our results provide direct data of specific changes in the metabolic fluxes leading to such systematic changes, and suggest that global redistributions of metabolic fluxes upon superoxide exposure may have been achieved through the regulation of key enzyme expression/activities.More generally, our study provide an example in which metabolic flux analyses present direct measurements of the physiological states of cells, while gene expression and proteomics studies measure the molecular states. In complex systems such as the metabolic networks, the different molecular processes that eventually determine the physiological states are tightly coupled to each other; i.e., there may not always be simple, process-by-process correspondence between changes in the physiological states and in the molecular states of cells. For instance, we have seen that the reduced akd flux is associated with the inactivation, but not the reduced expression, of AKGDH. Occasionally, reduced fluxes are found with unchanged or even increased expression of the respective genes and/or activities of associated enzymes (for example, the pgi step and the associated PGI enzyme). These types of results highlight the important complementarity between different types of systems level approaches. Used together, these approaches can provide comprehensive and undistorted pictures of how microorganisms respond to oxidative stress or other drastic environmental challenges.Metabolic flux analysis provided a quantitative and global picture of responses of the E. coli central carbon metabolic network to oxidative stress. Systematic adjustments of cellular physiological state clearly occurred in response to changes in metabolic fluxes induced by oxidative stress. Quantitative flux analysis therefore could reveal the physiological state of the cell at the systems level and is a useful complement to molecular systems approaches, such as proteomics and transcription analyses.