Abstract
Decreased biomass growth in iron (Fe)-limited Pseudomonas is generally attributed to downregulated expression of Fe-requiring proteins accompanied by an increase in siderophore biosynthesis. Here, we applied a stable isotope-assisted metabolomics approach to explore the underlying carbon metabolism in glucose-grown Pseudomonas putida KT2440. Compared to Fe-replete cells, Fe-limited cells exhibited a sixfold reduction in growth rate but the glucose uptake rate was only halved, implying an imbalance between glucose uptake and biomass growth. This imbalance could not be explained by carbon loss via siderophore production, which accounted for only 10% of the carbon-equivalent glucose uptake. In lieu of the classic glycolytic pathway, the Entner-Doudoroff (ED) pathway in Pseudomonas is the principal route for glucose catabolism following glucose oxidation to gluconate. Remarkably, gluconate secretion represented 44% of the glucose uptake in Fe-limited cells but only 2% in Fe-replete cells. Metabolic (13) C flux analysis and intracellular metabolite levels under Fe limitation indicated a decrease in carbon fluxes through the ED pathway and through Fe-containing metabolic enzymes. The secreted siderophore was found to promote dissolution of Fe-bearing minerals to a greater extent than the high extracellular gluconate. In sum, bypasses in the Fe-limited glucose metabolism were achieved to promote Fe availability via siderophore secretion and to reroute excess carbon influx via enhanced gluconate secretion.