A study of the metabolic adaptation provoked by decreased aconitase activity and increased ROS production as a consequence of aluminum stress in Pseudomonas fluorescens

The goal of this study was to evaluate the metabolic strategy invoked by Pseudomonas fluorescens to survive decreased aconitase (Acn; E.C. 4.2.1.3) activity elicited by aluminum (Al). Al is known to interfere with Fe homeostasis, by limiting the bioavailability of Fe, and creates an oxidative environment. As a result, Acn an Fe-dependent and oxygen-sensitive enzyme was severely impeded. 13C NMR studies revealed an olefinic peak attributable to aconitate upon incubation of the soluble cell-free extract (CFE) from the control cells with citrate while such a peak was not evident in the CFE from Al-stressed cells. BN-PAGE (blue native polyacrylamide electrophoresis) analysis indicated an inversed relationship between Al concentration and Acn activity. However, immunoblotting and two dimensional (2D) BN-PAGE activity staining techniques revealed no significant correlation between Al and Acn protein levels. UV/Vis spectroscopy demonstrated that the [4Fe--4S] cluster of Acn, essential for enzymatic activity was perturbed. The decrease in Acn activity and the increase in reactive oxygen species (ROS) production were countered by a major metabolic reconfiguration. The increased activity of two NADP+-dependent isocitrate dehydrogenases (IDH; E.C. 1.1.1.42) and isocitrate lyase (ICL; E.C. 4.1.3.1) enabled the organism to metabolize citrate by promoting isocitrate degradation even when Acn was markedly diminished. Further protection from the oxidative environment appeared to be mediated by the increased production of the antioxidant NADPH via G6PDH and 6PGDH in Al-challenged cells. Hence a major metabolic shift appears to be initiated by P. fluorescens in an effort to nullify the toxic influence of Al and ensure its survival.