Siderophore systems at play: system interactions as drivers of diversification in pathogenic .

Unlabelled: The -HPI genomic island encodes the salicylate-type siderophore piscibactin, a key virulence factor in several pathogenic species. To advance our understanding of the siderophore metabolome (siderome) in , we combined high-resolution metabolomics with genetic and phenotypic analyses of biosynthetic mutants. LC-MS/MS and Feature-Based Molecular Networking (FBMN) revealed the presence of known siderophores, vanchrobactin and piscibactin/photoxenobactin-like siderophores, alongside two non-reported catecholate analogs of the formed siderophores, 2-hydroxypiscibactin (2-OH-Pcb) and 2-hydroxyphotoxenobactin E (2-OH-PxbE). Functional characterization of mutants defective in piscibactin or vanchrobactin production demonstrated biosynthetic and uptake-level interactions between these systems. We show that the salicylate-activating enzyme Irp5 is a promiscuous aryl acid adenylation enzyme capable of activating 2,3-dihydroxybenzoate (DHBA) and driving the synthesis of catecholate-piscibactin/photoxenobactin and fluorinated analogs through precursor-directed biosynthesis. Our data support photoxenobactin E as the terminal product of the piscibactin/photoxenobactin pathway in . Comparative genomics revealed frequent co-occurrence of -HPI with other siderophore systems, especially catecholate-type systems such as vanchrobactin. These findings provide new insight into the molecular basis of virulence in and highlight the ecological and evolutionary significance of siderophore system interplay, which may enhance metabolic diversity, promote environmental adaptability, and preserve virulence under mutational pressure.

Importance: Iron acquisition is critical for bacterial survival and virulence, especially under the iron-limited conditions encountered in host environments. This study uncovers unexpected siderophore diversification within the piscibactin pathway, including two novel catecholate analogs and fluorinated derivatives produced via precursor-directed biosynthesis. We identify Irp5 as a versatile salicylate-activating enzyme that drives this metabolic flexibility. Moreover, the piscibactin and vanchrobactin systems interact at both biosynthetic and uptake levels, forming a resilient and adaptable iron acquisition network that supports virulence. These findings advance our understanding of siderophore-mediated iron acquisition and illustrate how enzymatic promiscuity and system interplay can be leveraged for synthetic biology and antimicrobial development. Given the widespread distribution of -HPI in , including human pathogens, our study provides a foundation for the rational design of antimicrobial strategies.