Microcin E492 (MccE492), a hydrophobic and anionic 84-residue antimicrobial peptide from Klebsiella pneumoniae, was first isolated as an unmodified peptide (7886 u). We have isolated a posttranslationally modified form of MccE492 (MccE492m, 8717 u) with more potent antimicrobial properties than MccE492. MccE492m is the first example of a novel type of antibacterial peptides that we call siderophore-peptides. MccE492 and MccE492m were submitted to chymotrypsin digestion. A 1771.6 u fragment purified by RP-HPLC from the MccE492m digest contained the entire 831.2 Da posttranslational modification. This fragment was submitted to MS analyses, using hybrid ESI-QqTOF and nanoESI-ion trap instruments, and to an NMR study at 800 MHz. CID experiments on the [M+2H]2+ ion gave two independent series of b- and y-ions. The y-ions were shifted to upper m/z ratios consistent with an 831.2 Da mass increase, while the b-ions were unshifted, indicating the modification is localized on the MccE492 C-terminal serine. The NMR data together with further MSn experiments identified the complete structure of the modification. It carries N-(2,3-dihydroxybenzoyl)-L-serine, which belongs to a group of catechol-type siderophores involved in iron uptake by enterobacteria. Moreover, MSn experiments in presence of FeCl3 showed that the modification was able to capture Fe3+. We show that recognition of both MccE492 and MccE492m is mediated by iron/siderophore receptors at the surface of sensitive bacteria. Therefore, we propose that improvement of MccE492 antimicrobial activity upon modification results from an increase of the microcin/receptor affinity.