Identification of iron and zinc responsive TonB-dependent transporters in Pseudomonas aeruginosa.

Pseudomonas aeruginosa employs a repertoire of TonB-dependent transporters (TBDTs) to acquire essential nutrients, such as iron-siderophore complexes, enabling its adaptation to diverse and often hostile environments. While the roles of some TBDTs in virulence and metal acquisition are well-established, many remain poorly characterized, despite their potential contributions to pathogenicity. Using a domain-based detection approach, we show that TBDTs are broadly conserved across diverse P. aeruginosa isolates and remain highly prevalent in multidrug-resistant (MDR) strains, further highlighting their clinical relevance. Notably, these transporters can be exploited as gateways for antibiotic delivery, as exemplified by the siderophore-antibiotic conjugate cefiderocol. To identify metal specificity TBDTs, we combined bioinformatic promoter analysis with fluorescent transcriptional reporters to validate their response to iron and zinc limitation. This approach identified 19 iron-responsive (bringing it to 21 iron-responsive TBDTs with previously published data) and 4 zinc-responsive TBDTs, including transporters with previously unknown substrates, and revealed that promoter-associated regulatory motifs, rather than protein homology, predict metal-specific regulation. Beyond this initial classification, the reporter library serves as a versatile tool for exogenous siderophore detection by monitoring transporter activity in response to their presence. Furthermore, single-cell analysis of TBDT promoter activity during co-culture with an enterobactin-producing Klebsiella pneumoniae strain revealed homogeneous expression of key TBDTs in P. aeruginosa populations, suggesting coordinated metal acquisition strategies under competitive conditions.