Updated project metadata. The development of new antibiotics against Gram-negative bacteria has to deal with the low permeability of the outer membrane. This obstacle can be overcome by utilizing siderophore-dependent iron uptake pathways as gates for antibiotic uptake. Iron-chelating siderophores are actively imported by bacteria, and their conjugation to antibiotics allows smuggling the latter into bacterial cells. Synthetic siderophore mimetics based on MECAM and DOTAM cores, both chelating iron via catechol groups, have been recently applied as versatile carriers of functional cargo. In the present study, we show that MECAM and the MECAM-ampicillin conjugate 3 transport iron into Pseudomonas aeruginosa cells via the catechol-type outer membrane transporters PfeA and PirA, and DOTAM solely via PirA. Differential proteomics and RTqPCR showed that MECAM import induced the expression of pfeA, whereas 3 led to an increase in the expression of pfeA and ampc, a gene conferring ampicillin resistance. The presence of DOTAM did not induce the expression of pirA, but upregulated the expression two zinc transporters (cntO and PA0781), pointing out that bacteria become zinc starved in the presence of this compound. Kinetic experiments with radioactive 55Fe demonstrated that iron uptake was as efficient as with the natural prototype siderophore enterobactin. The study provides a mechanistic and functional validation for DOTAM- and MECAM-based artificial siderophore mimetics as vehicles for the delivery of cargo into Gram-negative bacteria.