| The aim of this study was to elucidate and characterize the metabolic circuits evoked in response to oxidative stress in the soil bacterium Pseudomonas fluorescens. When the microbe was grown in menadione (100muM) the activity and expression of catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) were sharply increased. There was also a marked increment in such NADPH-generating enzymes as isocitrate dehydrogenase NADP+-dependent (ICDH), glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme (ME), and glutamate dehydrogenase NADP+-dependent (GDH). However, the NADH producing enzymes alpha-ketoglutarate dehydrogenase (alpha-KGDH) and isocitrate dehydrogenase (ICDH) NAD+-dependent that are linked to the TCA cycle were drastically reduced, as were the electron carriers Complex I and II. 13C NMR, spectroscopic, and electrophoretic evidence implicate alpha-ketoglutarate as a potent scavenger of ROS. Metabolic pathways aimed at enhanced production of NADPH and alpha-ketoglutarate and at a reduction of NADH formation were evident. A metabolic network mediated by pyruvate carboxylase (PC), isocitrate lyase (ICL), malate synthase (MS), malate dehydrogenase (MDH), phosphoenol pyruvate carboxykinase (PEPCK), and pyruvate kinase (PK) was operative in an effort to convert NADH to NADPH. Furthermore, NAD+ and NADH kinases were up-regulated in menadione-stressed cells. Hence, a metabolic shift in favour of NADPH and alpha-ketoglutarate production, coupled with a decrease in NADH formation is pivotal for the survival of this organism challenged by an oxidative stress. |
