Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae RYC492. The genetic determinants for microcin synthesis and immunity were cloned in Escherichia coli VCS257 into the cosmid vector pHC79, starting from total DNA of K. pneumoniae RYC492. The microcin E492 expressed in E. coli had the same properties as that of K. pneumoniae, i.e., the same molecular weight, the ability to form ionic channels in planar phospholipid bilayers, and essentially identical biological properties. Microcin E492 expression in E. coli, like that in K. pneumoniae, was mainly in the exponential phase of growth, declining in the stationary phase. The immunity determinant was subcloned into the same vector, and its expression was found to disappear in the stationary phase. This phenomenon is not dependent on rpoS, the stationary-phase sigma factor.Microcins are a family of low-molecular-weight antibiotics produced by members of the family Enterobacteriaceae. Microcins, unlike colicins, are not induced by DNA-damaging agents, but like for colicins, bacterial strains producing a specific microcin are immune to the same microcin (3).Microcin E492 is a polypeptide with an M r of 6,000 (14) that is produced by Klebsiella pneumoniae RYC492 and is active on strains of Escherichia coli, Klebsiella, Salmonella, Citrobacter, Enterobacter, and Erwinia (6). The mechanism of action is through membrane depolarization (9) induced by the formation of pores in the bacterial membrane (14). Studies on channel-forming bacteriocins produced by gram-negative bacteria have been reported only for colicins, all of them of high molecular weight (reviewed in reference 5). The other described bacteriocin which has the cytoplasmic membrane as target is colicin V. This bacteriocin has been reclassified as a microcin because it has a molecular weight of 6,000 and does not have most of the properties which are associated with colicins (10). Despite the fact that microcin E492 has many feature in common with colicin V, they are not closely related, because ColV ϩ strains that have high-level immunity to colicin V are fully susceptible to microcin E492 (9). Those colicins which act by depolarizing the bacterial energy-transducing membrane in vivo are able to form aqueous channels in both closed liposomes and planar membranes. However, despite the fact that colicin V inhibits the capacity of E. coli to carry out active transport of proline and to generate a membrane potential (26), it has not been possible to find an effect of colicin V on asolectin liposomes, and it remains to be established whether this difference reflects a difference in the in vivo mode of action. Thus, so far the only microcin described to be a channel-forming bacteriocin is microcin E492 (14). Nevertheless, low-molecular-weight pore-forming bacteriocins of gram-positive bacteria have been described, among them the lantibiotic nisin and lactococcins A and B, which are nonlantibiotic heatstable bacteriocins (reviewed in references 1, 13, and 24). The former has...
The gene coding for the immunity protein (mceB) and the structural gene of microcin E492 (mceA), a low-molecular-weight channel-forming bacteriocin produced by a strain of Klebsiella pneumoniae, have been characterized. The microcin gene codes for a precursor protein of either 99 or 103 amino acids. Protein sequencing of the N-terminal region of microcin E492 unequivocally identified this gene as the microcin structural gene and indicated that this microcin is synthesized as a precursor protein that is cleaved at either amino acid 15 or 19, at a site resembling the double-glycine motif. The gene encoding the 95-amino-acid immunity protein (mceB) was identified by cloning the DNA segment that encodes only this polypeptide into an expression vector and demonstrating the acquisition of immunity to microcin E492. As expected, the immunity protein was found to be associated with the inner membrane. Analysis of the DNA sequence indicates that these genes belong to the same family as microcin 24, and they do not share structural motifs with any other known channel-forming bacteriocin. The organization of the microcin- and immunity protein-encoding genes suggests that they are coordinately expressed.
Bone development and remodeling are associated with changes in the pattern of vascularization. Here we show that endothelial cells isolated from rat liver or bovine aorta can greatly enhance bone formation when implanted in diffusion chambers with rat fetal calvarial cells. The latter cells are unable to form bone when implanted alone at low initial cell density. The amount of mineralization measured by calcium deposition was 70 times higher in chambers containing calvarial cells mixed with endothelial cells from isologous liver or bovine aorta than in chambers containing endothelial or calvarial cells alone. Alkaline phosphatase activity was increased 20-fold. Calvarial cells in the presence of demineralized bone matrix powder did not form bone when implanted under similar conditions. Endothelial cells implanted alone seemed to enhance neovascularization around the Millipore diffusion chambers.
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