MOLECULAR GENETICS AND MECHANISM OF PROTEIN ANTIBIOTICS

University Of Maryland College Pk Campus, College Park MD

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Abstract

Certain strains of Bacillus subtilis produce subtilin, which is a broad- spectrum ribosomally-synthesized antibiotic peptide that contains many unusual amino acids (dehydroalanine, dehydrobutyrine, lanthionine, beta- methyllanthionine) as a consequence of posttranslational modifications of serine, threonine and cysteine residues present in the precursor peptide. Recent studies in this laboratory have used site-directed mutagenesis of the subtilin prepeptide gene to construct and express a structural analog of subtilin, in which the change of a single amino-acid residue resulted in dramatic enhancement of its chemical and antimicrobial properties. This proves the potential of using a genetic engineering approach to the design and construction of improved and novel antibiotics that may have expanded therapeutic potential with respect their natural forms. Because of the malleability of protein structures, it is realistic to hope that these analogs could be targeted to a variety of infectious agents, perhaps even including viruses. The purpose of this project is to increase our understanding of how to use mutagenesis for structure-function studies by acquiring fundamental information about the mechanism by which the cellular machinery recognizes the subtilin precursor peptide, the steps in the biosynthetic pathway, and the involvement of the prepeptide leader sequence in maturation and secretion. Because the natural producer of subtilin is an uncharacterized strain, the capability to produce subtilin was transformed into B. subtilis 168, for which an enormous amount of genetic information is available. We have exploited the well-established genetic tools available for strain 168 to construct and express mutant genes in which the subtilin leader region (has no normal secretion signal) is fused to PhoA as a reporter. Expression of fusion showed that the leader region directs secretion of the PhoA reporter through a secretion pathway that exists only in the subtilin-producing mutant of 168; but not in wild-type 168. This implies that subtilin-producing cells have a novel and uncharacterized secretion system that specifically recognizes the subtilin leader sequence. The secretion apparatus may also contain the enzymes that catalyze the post-translational modifications. This will be explored by examining the fusion proteins for the presence of the unusual amino acid residues, using a combination of N-terminal sequence, total amino acid composition, and NMR-spectroscopy. A variety of fusion proteins will be constructed for the purpose of localizing the recognition signals in the precursor peptide as being in the leader region, the mature region, or distributed throughout the peptide. Site-directed mutagenesis of the unusual amino acids will be used to explore their roles in the chemical properties and the antibiotic mechanism of subtilin. Finally, we will determine whether the five ORFs that have been identified in the spa operon are the only genes required to support subtilin biosynthesis, and if not, locate and identify any additional genes. The roles of the ORFs will be explored by mutating them one at a time using in-frame deletions, and inferring the role of each ORF in the biosythetic pathway by determining which step in the pathway is interrupted by each mutation.