Inverse stable isotope labeling (InverSIL) links predicted catecholate siderophore gene clusters to their products in diverse bacteria.

Bacteria produce high-affinity, iron-chelating secondary metabolites called siderophores to access insoluble Fe(III) in their environments. Genome mining has revealed many predicted siderophore biosynthetic gene clusters (BGCs) in bacterial genomes; however, the structures of their siderophore products remain mostly undetermined. This limits our molecular-level understanding of how bacteria acquire iron. Here, we apply inverse stable isotope labeling (InverSIL) to rapidly connect predicted siderophore BGCs to their products. With InverSIL, bacteria are grown on IMPORTANCE: Iron acquisition is important for microbial survival, and bacteria produce secondary metabolites called siderophores to scavenge iron from the environment. While bacterial genome sequences show many predicted genes for making siderophores, most remain unlinked to their metabolic products. Understanding which siderophores bacteria produce is critical for elucidating microbial iron acquisition strategies, ecological interactions, and potential roles in host-microbe interactions. Here, we demonstrate how inverse stable isotope labeling (InverSIL) can rapidly link predicted siderophore gene clusters to their corresponding metabolites. By applying InverSIL to diverse bacterial strains, we validate known siderophore products and uncover unexpected products, highlighting the limitations of current