Unraveling the metabolic networks involved in the utilization of L-glutamine in Pseudomonas fluorescens exposed to nutritional stress

Abstract

Sulfur plays an essential role in oxidative homeostasis due to its participation in sulfhydryl groups (SH). A disruption of this vital nutrient is known to promote oxidative stress and activates a plethora of anti-oxidative strategies. Phosphate, a micronutrient that is part of adenosine triphosphate (ATP), the main molecule used as energy and other macromolecules in living cells. The stress response to sulfur and phosphate deficiency in Pseudomonas fluorescens was investigated with emphasis on ROS detoxification, and energy production. Metabolite profiling was performed by High Performance Liquid Chromatography (HPLC), enzymatic analysis was done using Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) and gene expression assessment of targeted genes was performed with SYBR-Green real-time PCR (qPCR). When cultured in a sulfur-deficient medium with glutamine as the sole carbon and nitrogen source, the microbe reconfigures its metabolism aimed at the enhanced synthesis of NADPH, an antioxidant and the limited production of NADH, a pro-oxidant. The up-regulation of isocitrate dehydrogenase (ICDH)-NADP+ dependent in the soluble fraction of the cells obtained from the S-deficient media results in enhanced NADPH synthesis. This reaction is aided by the concomitant increase in NAD kinase (NADK) activity. The latter converts NAD+ into NADP+ in the presence of ATP. Additionally, the microbe reprograms its metabolic pathways to produce KG and regenerate this keto-acid from succinate, a by-product of ROS detoxification. Succinate semialdehyde dehydrogenase (SSADH) and KG decarboxylase (KDC) work in partnership to synthesize KG. This process is further aided by the increased activity of the enzymes glutamate decarboxylase (GDC) and γ-amino-butyrate transaminases (GABA-T). Taken together, the data point to a metabolic network involving isocitrate, KG, and ICDH that converts NADH into NADPH in P. fluorescens subjected to a S-deprived environment. Finally, when cultured in low phosphate environments, the microbe can produce ATP via substrate level phosphorylation (SLP), in a mechanism involving the reductive isocitrate dehydrogenase (ICDH-NADH), isocitrate lyase (ICL), malate synthase (MS) as well as phosphoenol pyruvate carboxylase (PEPC), phosphoenol pyruvate synthase (PEPS) and pyruvate phosphate dikinase (PPDK). This metabolic reprogramming ensures the survival of the microbe and reveals the central role metabolism plays in cellular adaptation to abiotic stress.