Nisin and subtilin are peptide antibiotics that are synthesized ribosomally and converted to mature active forms by post-transcriptional modifications. Because their structures are gene-encoded, they are amenable to controlled structural modification by mutagenesis. The powerful tools of genetic engineering can therefore by used to construct new forms of these antibiotics with novel and useful properties, that may help alleviate the growing problem of antibiotic resistance in bacterial populations; which is steadily reducing our arsenal of useful antibacterial agents. The structural gene for subtilin has recently been cloned in Bacillus subtilis by workers in this laboratory. With the gene available for the first time, it will be possible to study the structure and organization of the subtilin structural genes, and the structure and organization of the subtilin structural genes, and the peptide precursor from which subtilin is made. The precursor can be used to test hypotheses about the biosynthetic pathway for subtilin. Our results from restriction mapping of cloned subtilin genes suggest they may be in a transposon, that may also encode the post-transcriptional processing enzymes. This hypothesis will be tested by transferring the genetic element to a non-subtilin producing strain, and looking for subtilin production. If this is successful, the nucleic acid sequence will be studied in detail. Also, we will attempt to construct an expression system that will produce the antibiotic more efficiently than is now possible. We have evidence that the dehydro residues in nisin are bifunctional; one reacting with membrane sulfhydryl groups and another acting to "anchor" the antibiotic to the cell surface. This will be studied further by incorporation studies of radioactive nisin that has been synthesized in vivo from labeled precursors.