Iron-dependent metabolic rewiring drives tryptophan-associated melanin production in a clinical Pseudomonas aeruginosa strain

ABSTRACT Pseudomonas aeruginosa is a human opportunistic pathogen, capable of producing a wide range of metabolites, including pyomelanin, which results from alterations in tyrosine catabolism. In contrast, melanin synthesis from tryptophan has never been reported in Pseudomonas . In this study, we applied an integrated multi-omics approach to characterize physiological adaptations associated with the emergence of a tryptophan-associated melanin-like pigment in P. aeruginosa PAH, a persistently re-isolated strain from a cystic fibrosis patient. Structural analysis revealed the production of a previously unreported melanin-like polymer produced under tryptophan supplementation, coexisting with a pyomelanin-like soluble fraction. This melanin exhibited iron-reduction capacity, supporting a potential physiological role in iron acquisition within the infective niche. Genome analysis identified mutations affecting iron acquisition, including genes involved in pyoverdine biosynthesis. Transcriptomic profiling revealed a coordinated reprogramming of iron homeostasis, characterized not only by the downregulation of pyoverdine-associated genes but also by altered expression of alternative siderophore systems and a broader set of genes, such as redox response pathways. Non-targeting metabolomic analyses uncovered the accumulation of indole-derived compounds. Inhibitors of tyrosine and tryptophan catabolism demonstrated a metabolic shift between these pathways. Notably, iron availability emerged as a key regulator of this system, with iron limitation promoting pigment production and associated metabolic states. Integrating these data sets, we developed an integrative model linking iron homeostasis, genomic variation, transcriptional and metabolic reprogramming, and melanin production in P. aeruginosa PAH. Overall, tryptophan arises as a key compound for melanin production in a Pseudomonas strain, expanding the diversity of melanins synthesized by this genus. IMPORTANCE This study reveals a previously unrecognized way by which Pseudomonas aeruginosa can produce melanin, a protective pigment linked to survival and persistence in hostile environments. Here, we report how a novel melanin pigment, produced in tryptophan-supplemented media, can reduce iron and is linked to iron homeostasis in a clinical P. aeruginosa strain. Employing a multi-omics approach, we show that this pathway operates primarily through anthranilate and indolic intermediates and is favored under iron-limited conditions. This finding deepens current understanding about pigment diversity in bacteria and highlights the remarkable metabolic flexibility of P. aeruginosa during infection. Since the same phenotype was observed again in year-later isolates from the same patient, our results suggest a potential role of this trait in long-term infection and adaptation.