The long-term objective of this research proposal is to understand the biosynthesis and maturation of microcin E492m. Microcins are antibiotic peptides that bacteria produce and release to inhibit or kill neighboring bacteria of different species. MccE492m is post-translationally modified with an iron-chelating siderophore/salmochelin moiety. This modification allows the microcin to bind to catecholate siderophore receptor proteins on nearby bacterial cells, which affords more efficient uptake. Additionally, siderophore- producing bacteria are among the most virulent, and MccE492m production therefore provides a strategy for certain bacterial strains to defeat their most competitive neighbors. The logic of MccE492m assembly is currently unknown and unveiling the details of this process will help provide strategies for the development of new antibiotic drugs. Toward this goal, proteins expressed by the MccE492 gene cluster will be cloned, over-expressed and their activities and substrate specificities characterized in vitro. Particular emphasis will be placed on understanding the reactivity of MceC, a putative C-glycosyltransferase, and MceD, a putative esterase, that show amino acid homology with proteins of the iroA gene cluster responsible for salmochelin production. A detailed mechanistic investigation of the MccE492m post-translational modification will also be undertaken. This modification is remarkable since it involves formation of an ester linkage between a glycosylated siderophore and a C-terminal serine residue of the MccE492 peptide. Proteins of the MccE492 gene cluster, including Mcel, a putative acyltransferase, and several other proteins of unknown function will be considered in these studies and their role(s) established. Once the mechanistic details of this post- translational modification are elucidated, substrate recognition and specificity will be probed. Of particular interest is to determine if the entire microcin peptide or only the C-terminus or serine residue is required for recognition. If the catalytic system can form ester linkages between the glycosylated siderophore and substrates other than the full-length MccE492 peptide, the machinery will offer a new approach for preparing siderophore-toxin conjugates, which may be useful antibiotics. Lastly, a combination of fluorescence labeling and time-lapse confocal microscopy will be employed to visualize MccE492m uptake into prokaryotic and eukaryotic cells and to ascertain if cytoplasmic entry occurs. These biochemical studies are of particular relevance to public health because antibiotic resistance is a serious problem worldwide. Unveiling the fundamental biochemistry behind MccE492m biosynthesis will provide new insights for the development of novel antibiotics for virulent siderophore-producing and gram-negative bacterial strains.
