Studies in the antimicrobial mechanism of the lantibiotics nisin and sublancin, and, A Bacillus subtilis 168 expression system that displays the lantibiotic antimicrobial peptide sublancin on the cell surface

In this work, the ability of nisin to bind to a variety of biological target structures was explored. Nisin was demonstrated as having the ability to bind non-ionic cellulose under mildly alkaline conditions (pH 8.0).;The dependence of binding on pH is consistent with nisin's role in autoregulation of its own biosynthesis and also may contribute its ability to provide a defense against its ecological-niche competitors. This unique ability to bind cellulose served as a potential new means for purification of nisin, and it was demonstrated that non-ionic cellulose could be used to prepare highly purified nisin from a culture of Lactococcus lactis 11454.;The possibility that the antimicrobial activity of nisin involves its ability to recognize specific polypeptide structural motifs was explored. Nisin was allowed to select for specific binding motifs from random 7-mer and 12-mer peptide libraries displayed on the surface of M13 bacteriophage. Nisin selected strongly for a motif having the consensus sequence PPS(T/S)XKL. The motif shared strong homology to the active-site motifs found in penicillin-binding proteins, which are membrane-bound enzymes involved in cell wall biosynthesis. The identification of this motif is consistent with nisin's ability to inhibit cell wall biosynthesis and suggests that the mechanism of nisin action includes its specific binding to the active-sites of penicillin-binding proteins, thereby inhibiting their activity.;A system was developed to display the lantibiotic sublancin on the surface of its Bacillus subtilis 168 producer cell. The displayed sublancin was found to be accessible by antibodies which are exposed to the producer cell surface. It was further demonstrated that this interaction allowed for cells that displayed the sublancin peptide could be purified away from cells that did not. This provides the basis of isolation of ligand-specific sublancin analogues from a combinatorial display library.;The antimicrobial mechanism of the lantibiotic sublancin was explored by identifying a biological target from Bacillus subtilis 168. A C-terminal 6xHis-tag was fused to sublancin by mutagenesis of the sublancin structural gene. After analysis of the tandem MS/MS spectra by the SEQUEST algorithm, the protein was identified as elongation factor Tu, which is a component of the protein synthetic machinery. The ability of sublancin to specifically bind to this protein suggests that its mechanism of action involves the inhibition of protein biosynthesis. (Abstract shortened by UMI.)...