Microcin E492 (MccE492) is an antimicrobial peptide secreted by Klebsiella pneumoniae. Recombinant Escherichia coli VCS257 carrying the genetic determinants of MccE492 production and immunity (plasmid pJAM229) were engineered to secrete higher amounts of active microcin in the culture medium. A channel-forming mechanism was proposed for MccE492 based on studies performed with a partially purified microcin. In an attempt to better characterize MccE492 structure and mode of action, we optimized a protocol of production and purification of MccE492 secreted by the recombinant E. coli strain cultured in minimum medium (M63-glucose). MccE492 was purified to homogeneity as controlled by silver-stained SDS-PAGE and MALDI-TOF-MS, with an extraction yield of 1.9 mg per liter of culture. The mature form purified from the culture supernatant corresponds to an anionic and hydrophobic (poly)peptide with a molecular mass at m/z 7887.5 (MALDI-TOF-MS). The first five N-terminal amino acids (Gly-Glu-Thr-Asp-Pro-) together with aminoacid composition showed that it is generated from the precursor molecule by elimination of a 19-residue leader sequence. Preliminary CD conformational studies showed that MccE492 adopts either a random coil structure in aqueous solution or a partly alpha-helical structure in the presence of 0.3% SDS, that suggests it could gain its active form in contact with membranes. The spectrum of activity was determined against Gram-positive, Gram-negative bacteria as well as yeast and filamentous fungi. MccE492 was found to be specifically active against Enterobacteriaceae with MIC values ranging from 20-80 nM on E. coli to 2-8 µM on K. pneumoniae. This activity was bactericidal against all the tested strains. At high peptide-concentrations (5-10 µM), MccE492 permeabilized E. coli ML35 outer and inner membranes, as indicated by a beta-galactosidase assay. Nonetheless, no leakage of encapsulated carboxyfluoresceine could be observed from neutral or negatively-charged liposomes when treated with MccE492. Comparison of the MccE492 concentrations needed for growth inhibition and permeabilizing activity on E. coli ML35 indicates that the bactericidal activity does not result exclusively from membrane disruption, but might involve a more complex mechanism.