Pseudomonas species thrive in different nutritional environments and can catabolize divergent carbon substrates. These capabilities have important implications for the role of these species in natural and engineered carbon processing. However, the metabolic phenotypes enabling Pseudomonas to utilize mixed substrates remain poorly understood. Here, we employed a multi-omics approach involving stable isotope tracers, metabolomics, fluxomics, and proteomics in Pseudomonas putida KT2440 to investigate the constitutive metabolic network that achieves co-utilization of glucose and benzoate, respectively a monomer of carbohydrate polymers and a derivative of lignin monomers. Despite nearly equal consumption of both substrates, metabolite isotopologues revealed nonuniform assimilation throughout the metabolic network. Gluconeogenic flux of benzoate-derived carbons from the tricarboxylic acid cycle did not reach the upper Embden-Meyerhof-Parnas pathway nor the pentose-phosphate pathway. These latter two pathways were populated exclusively by glucose-derived carbons through a cyclic connection with the Entner-Doudoroff pathway. We integrated the 13 C-metabolomics data with physiological parameters for quantitative flux analysis, demonstrating that the metabolic segregation of the substrate carbons optimally sustained biosynthetic flux demands and redox balance. Changes in protein abundance partially predicted the metabolic flux changes in cells grown on the glucose:benzoate mixture versus on glucose alone. Notably, flux magnitude and directionality were also maintained by metabolite levels and regulation of phosphorylation of key metabolic enzymes. These findings provide new insights into the metabolic architecture that affords adaptability of P. putida to divergent carbon substrates and highlight regulatory points at different metabolic nodes that may underlie the high nutritional flexibility of Pseudomonas species. This work was supported by an Integrative Graduate Education and Research Traineeship Fellowship from the National Science Foundation (to M. A. K.), graduate fellowships from Cornell University (to M. S. and R. A. W.), and a start-up package from Cornell University. The authors declare that they have no conflicts of interest with the contents of this article. This article contains Tables S1-S4 and Figs. S1-S6.

High-affinity iron (Fe) scavenging compounds, or siderophores, are widely employed by soil bacteria to survive scarcity in bioavailable Fe. Siderophore biosynthesis relies on cellular carbon metabolism, despite reported decrease in both carbon uptake and Fe-containing metabolic proteins in Fe-deficient cells. Given this paradox, the metabolic network required to sustain the Fe-scavenging strategy is poorly understood. Here, through multiple13C-metabolomics experiments with Fe-replete and Fe-limited cells, we uncover how soilPseudomonasspecies reprogram their metabolic pathways to prioritize siderophore biosynthesis. Across the three species investigated (Pseudomonas putidaKT2440,Pseudomonas protegensPf-5, andPseudomonas putidaS12), siderophore secretion is higher during growth on gluconeogenic substrates than during growth on glycolytic substrates. In response to Fe limitation, we capture decreased flux toward the tricarboxylic acid (TCA) cycle during the metabolism of glycolytic substrates but, due to carbon recycling to the TCA cycle via enhanced anaplerosis, the metabolism of gluconeogenic substrates results in an increase in both siderophore secretion (up to threefold) and Fe extraction (up to sixfold) from soil minerals. During simultaneous feeding on the different substrate types, Fe deficiency triggers a hierarchy in substrate utilization, which is facilitated by changes in protein abundances for substrate uptake and initial catabolism. Rerouted metabolism further promotes favorable fluxes in the TCA cycle and the gluconeogenesis–anaplerosis nodes, despite decrease in several proteins in these pathways, to meet carbon and energy demands for siderophore precursors in accordance with increased proteins for siderophore biosynthesis. Hierarchical carbon metabolism thus serves as a critical survival strategy during the metal nutrient deficiency.