Microcin B17: posttranslational modifications and their biological implications.

In the invisible world of microorganisms, starvation and war is a routine part of life. Lack of nutrition in the environment and overpopulation by diffferent bacteria lead to various strategies to ensure the survival of one species and its dominance over others. The most effective strategy is the secretion of antibiotics to kill, or inhibit the growth of, other bacterial species. Elucidation of structures of antibiotics, their biogenesis, and their modes of action over the years has revealed many fascinating aspects of biochemical and genetic regulation of prokaryotic cells and has facilitated the generation of more effective antibiotics. Despite its long history, the field of antibiotics is so fertile and vast that discoveries are still being made at an unabated pace. Among the recently discovered antibiotics, the microcins are an illuminating example of the richness of chemistry and biology associated with antibiotics and nature's ingenuity in evolving a wide variety of unusual antibiotic structures that are imaginable only in the wildest dreams of synthetic chemists. This is elegantly underscored by the discovery of previously unknown posttranslational modifications of a member of the microcin family, microcin (Mcc) B17, as reported by Kolter and colleagues in this journal (1) and by Jung and colleagues elsewhere (2). Microcins belong to a growing family of peptide antibiotics -that are synthesized on the ribosome from RNA templates rather than by multienzyme complexes from secondary metabolites (3, 4). Unlike many high molecular mass polypeptide antibiotics, such as colicin, the microcins have relatively low molecular mass (<5 kDa) and are often posttranslationally modified. Based on their mechanism of action, microcins can be classified into three types: type A inhibits metabolic enzymes, type B prevents DNA replication, and type D impairs the cell energy-generating system. MccB17, a type B microcin, has been the focus of genetic and molecular biological studies by several groups due, in part, to its intriguing chemical and biological properties. As a result, much has been learned about the biosynthesis and mechanism of action of MccB17. The complete information for the structure of MccB17 and most of the proteins involved in its posttranslational modifications and function is encoded on a plasmid in a single operon consisting of seven genes mcbA to G (Fig. 1). Although there is a low level of transcription of the mcb operon during logarithmic growth, its high level of transcription is induced by the ompR gene product only when the host cell enters stationary phase. After transcription, the precursor McbA with an N-terminal leader sequence is translated at least 2 hr before the other gene products, ensuring a high substrate/ modifying enzyme ratio through an uncharacterized translational control mechanisms. Posttranslational modifications of McbA are catalyzed by three enzymes-McbB, McbC, and McbD-to give proMccB17. The N-terminal leader peptide is cleaved by a cellular protease PmbA to yield the mature MccB17, which is immediately and actively transported out of the cell by McbE and McbF. The entrance of MccB17 into a target cell is mediated by OmpF on the outer membrane and by SbmA on the inner membrane. Once inside the target cell, MccB17 binds to DNA gyrase, causing double-stranded DNA breaks and triggering the SOS response, which eventually leads to DNA degradation and cell death, reminiscent of apoptosis that occurs in mammalian cells. The extremely high potency of MccB17 is evident as a single molecule is sufficient to kill a bacterial cell. To avoid "friendly fire" from MccB17 either before export or after its reentry into the host cell, McbG, together with McbE and McbF, confers immunity of host cells to MccB17 by an unknown mechanism. Thus, the mcb operon provides host cells with a combination of both offensive and defensive systems. The use of bacterial genetics and molecular biology has led to the identification of DNA gyrase as the molecular target of MccB17 (5) and the cloning and sequencing of all seven genes in the mcb operon (6, 7). It was established that the structural gene for the precursor of MccB17, mcbA, encodes a 69-amino acid polypeptide with the first 26 residues as the leader sequence, which is absent in mature MccB17. It was also known that the posttranslational modifications of MccB17 involve three enzymes McbB to D. For several years, however, the nature of the modifications that occur in MccB17 remained a mystery. Now, by using several chemical techniques including UV, NMR, mass spectroscopy, and chemical degradations, two groups have independently solved this mystery (1, 2). In the process, they uncovered different types of posttranslational modifications involving both amino acid side chains and peptide backbones to form oxazoles and thiazoles. The mature MccB17 consists of 43 amino acids according to its DNA coding sequence. Of the 43 residues, it is predicted that 26 are glycines, 6 are serines, and 4 are cysteines. But amino acid analysis left 6 glycines and 4 of 6 serines unaccounted for. Additional analysis showed that all 4 cysteines are also missing. UV spectroscopic analysis revealed chromophores characteristic of conjugated heterocycles; mass spectrometry analysis indicated a loss of 160 mass units from the translated polypeptides. Proton NMR analysis gave eight aromatic protons uncharacteristic of any of the predicted amino acid side chains. With additional 13C and 15N NMR data and analysis of a seine mutant (S39N), the posttranslational modifications in MccB17 were determined by Kolter and colleagues (1) to be the conversion of 4 serines into oxazoles and 4 cysteines into thiazoles involving the condensation of either serine hydroxyl groups or cysteine thiol groups with carbonyl groups of the preceding residue (6 glycine residues and 2 from each of a preformed thiazole or oxazole) followed by dehydrogenation to form the aromatic heterocycles. The evidence was abundant and clear; the conclusions are unambiguous. The same conclusions were reached independently by Jung and coworkers through overlapping but not identical approaches (2). This finding throws new light on the biosynthesis of MccB17 and paves the way for further studying its mechanisms of biosynthesis and action at the molecular level. Of the three enzymes involved in the posttranslational modifications, no specific functions have been ascribed to them in spite of their sequence determi-