Microcins are gene-encoded potent antimicrobial peptides secreted by enterobacteria. Microcin E492 is secreted by the wild type Klebsiella pneumoniae and recombinant E. coli strains under both an unmodified (u- MccE492) and a post-translationally modified form (MccE492), endowed with higher activity. The modification consists of a C-glucosylated linear trimer of N-(2,3-dihydroxybenzoyl)-L-serine, a catecholate siderophore related to salmochelins and enterobactin. MccE492 was thus the first siderophore-peptide to be isolated. MccE492 was produced under conditions inhibiting various biosynthetic pathways. HPLC and mass spectrometry were used to monitor MccE492/u-MccE492 production. Free aromatic amino acids and high iron concentrations inhibited the acquisition of the post-translational modification. Exogenous enterobactin restored this biosynthesis in a bacterial strain deficient in enterobactin synthesis. Gene disruption experiments allowed identifying the mceABCDEFGHIJ-encoded enzymes responsible for the posttranslational modification of the precursor MceA. mceC and mceD are required for the synthesis of MccE492 post-translational modification. Moreover, those genes are efficiently complemented by genes homologous to iroB and iroD, which are involved in salmochelin biosynthesis. Thus, two precursors, MceA and enterobactin, are the substrates of the MccE492 enzyme machinery and u-MccE492 is an incompletely processed form of the microcin. Modification of MccE492 would involve this sequence of events: enterobactin C-glycosylation by MceC, hydrolyzis of C-glucosylated enterobactin by MceD, transfer of the linearized enterobactin onto MceA by MceI and J, and finally concomitant cleavage and export of the mature siderophore-peptide.