| Pseudomonas fluorescens detoxifies aluminum (Al) in association with oxalic acid and phosphatidylethanolamine (PE). This study was aimed at elucidating the role of the intermediary metabolism of the cell in generating these moieties. Results presented here show a major reconfiguration of the metabolic pathways in response to Al-stress. This metabolic shift allowed the cell to divert precursors towards the production of key metabolites required to synthesize oxalate and PE that are pivotal in Al detoxification. The activity of isocitrate lyase (ICL), an enzyme that participates in the cleavage of isocitrate to glyoxylate and succinate incurred a 4-fold increase in the Al-stressed cells while glyoxylate dehydrogenase (GDH), the enzyme mediating the oxidation of glyoxylate to yield oxalate, underwent an 8-fold increase in activity in cells harvested from Al-rich media. There was also a marked increase in activity of such enzymes as pyruvate dehydrogenase (PDH) (4-fold), glucose-6-phosphate dehydrogenase (G6PDH) (1.5-fold), phosphogluconate dehydrogenase (PGDH) (1.5-fold) and a soluble NADP-dependent isocitrate dehydrogenase (IDH) (1.5-fold). However, the activity of alpha-ketoglutarate dehydrogenase (KDH) and a membrane-bound NAD-dependent IDH appeared to diminish by 3- and 1.5-fold respectively. Interestingly, even though the cells were grown in a citrate and/or Al-citrate media, phosphoenolpyruvate carboxykinase (PEPCK) activity appeared to be reduced (1.5-fold) in Al medium. In addition there was no change in activity of malate synthase (MS), an enzyme often expressed in conjunction with isocitrate lyase. The data presented also demonstrate the tendency of these enzymatic activities to return to near control levels once Al has been extruded and immobilized as a gelatinous residue consisting of oxalate and PE. For instance, when transferred to a fresh control medium, Al-stressed cells experienced a sharp decline in ICL activity. Blue native gel electrophoresis and western blot analyses revealed that this fluctuation in ICL activity was modulated by a change in the concentration of the enzyme. 13C NMR analyses revealed the de novo synthesis of PE and oxalate when the cells were exposed to Al. Although the activity of acetyl-CoA carboxylase (ACC) did not show any variation in activity when the cells were subjected to the Al media, the three NADPH generating enzymes, namely G6PDH, PGDH, NADP-dependent IDH did exhibit elevated activities in Al-stressed media. This suggests a deliberate modification of the metabolic pathways that enables the organism to circumvent the stress of Al. The shift in the TCA cycle, glycolysis, the glyoxylate cycle, the pentose phosphate pathway, and gluconeogenesis provides the necessary metabolites to generate the moieties required to immobilize and neutralize the toxic influence of the trivalent metal. |
