Microcins are gene-encoded antimicrobial peptides with low-molecular masses (< 10 kDa) secreted by Enteriobacteriaceae. They play a role in microbial competitions within the intestinal microflora by exerting potent antibacterial activity against phylogenetically-related bacteria, with minimal inhibitory concentrations in the nanomolar range. We have previously characterized the first siderophore-microcin, MccE492m, an 84-residue peptide that carries a linear trimer of dihydroxybenzoyl serine (DHBS), a catechol-type siderophore. MccE492m is the modified form of microcin E492 (MccE492). Escherichia coli Nissle 1917 (O6:K5:H1) is a non-pathogenic commensal faecal isolate forming the basis of the probiotic preparation Mutaflor® used for treatment of various gastrointestinal disorders and diseases. E. coli Nissle 1917 has been shown to produce two bactericidal activities identified as microcins H47 and M (MccM). The genetic system responsible for production and secretion of MccM and for immunity of the producing strain to the microcin has been sequenced previously. However, MccM had never been isolated until now. Here we describe the expression and purification of MccM and show that it carries such a posttranslational modification. We also characterize the recognition mechanisms and biosynthetic pathways of this novel class of potent antibacterial peptides.