Microcin J25 (MccJ25) is a naturally occurring cyclic antimicrobial peptide. This 21-residue peptide, produced by Escherichia coli, inhibits the RNA polymerase from phylogenetically related bacterial species. It adopts an original lasso structure in which the C-terminal tail of the peptide is threaded into the ring formed by the side chain-to-backbone peptide bond between Gly1 and Glu8. Upon folding, the C-terminal tail of MccJ25 adopts a beta-hairpin structure, the two bulky aromatic side chains from Phe19 and Tyr20 being positioned on each side of the ring, thus firmly blocking the tail into the ring. MccJ25 is amazingly resistant to extreme treatments, such as temperature, pH or chaotropic agents, compared to its linear variant. The Val11-Pro16 beta-hairpin region was found to be required for recognition by the FhuA receptor at the outer membrane of E. coli while the N-terminal ring-tail region was critical for inhibition of the RNA polymerase. The genetic determinants required for MccJ25 production, mcjABCD, were entirely sequenced. It was shown that MccJ25 is expressed as a 58-amino acid inactive precursor, McjA, which further undergoes post-translational modifications before secretion by the McjD export protein. The post-translational modifications of McjA are thought to involve McjB and McjC, which do not display homologies to other known proteins, but are necessary for the production of mature MccJ25. In order to identify the enzyme machinery that generates MccJ25 lasso structure and simultaneously provides it with antibacterial activity, we have cloned the structural gene mcjA, as well as the genes encoding the two putative modification enzymes, mcjB and mcjC. McjA, McjB and McjC were overexpressed in E. coli as fusion proteins bearing a His6 tag in N-terminal position, and subsequently purified by nickel affinity chromatography. We followed MccJ25 production upon McjA processing by McjB and McjC in vitro. We detected the acquisition of the three-dimensional lasso structure, concomitant with ring formation and precursor cleavage by HLPC coupled with mass spectrometry (LC-MS). The subsequent acquisition of antibacterial activity, specific for MccJ25, was assessed by antimicrobial assays. We were able to reconstitute the biosynthesis of MccJ25 in vitro and showed that McjB and McjC are the sole bacterial components responsible for the lasso structure of MccJ25.