IRON-DEPENDENT CELLULAR REPROGRAMMING OF MIXED SUBSTRATE METABOLISM IN PSEUDOMONAS PUTIDA KT2440

Abstract

We investigated the consequences of Fe-limitation on the metabolism of mixed organic substrates. The following study presents a stable isotope-assisted metabolomics approach using 13C-labeled substrates and liquid chromatography-mass spectrometry (LC-MS) to evaluate the effects of Fe availability on co-utilization of glucose and benzoate in P. putida KT2440 cells. We provide new metabolic insights regarding nutrient dependent carbon metabolism of the biotechnologically important Pseudomonas species across three conditions: (1) Fe-replete condition, wherein the Fe concentration was within the optimal range for bacterial growth; (2) Fe-limited medium, in which no exogenous supply of Fe was provided; and (3), Fe-starved condition with ethylene-diamine-tetra-acetic-acid, a strong Fe chelator, in the growth a medium to evaluate the effects of very low Fe conditions. Our data revealed that Fe-limitation did not have any effect on the biomass growth of the cells but there was an overall increase in substrate consumption accompanied by secretion of the pyoverdine siderophore. Under the Fe starvation condition, there was a diauxic growth in which the cells first consumed benzoate in the growth phase and, upon benzoate depletion, the cells started consuming glucose in the second phase. Interestingly, pyoverdine secretion was enhanced during the benzoate consumption phase. These results thus demonstrate that the preferential intake of the aromatic substrate as a function of decreasing Fe availability was favorable to siderophore production. The present findings provide new insights into the metabolic underpinnings of nutrient-dependent carbon metabolism.