Genetic and biochemical studies were conducted on S. toyocaensis NRRL 15009, a gram-positive spomlating filamentous bacterium, and producer of the glycopeptide antibiotic A47934. This compound is structurally similar to the clinically important antibiotic vancomycin, and the recent spread of vancomycin-resistant enterococci (VRE) in North American hospitals has driven the need for new glycopeptides with enhanced activities. Studies were aimed at developing an understanding of the mechanism of A47934 biosynthesis inS. toyocaensis NRRL 15009, as well as the mechanism of resistance employed by this organism. Two cosmid clones, containing a partial A47934 biosynthesis gene cluster on a total of65 kilobases of S. toyocaensis NRRL 15009 chromosomal DNA, were isolated for study. Preliminary sequencing indicates the presence of several genes predicted in glycopeptide assembly, such as peptide synthetases and glycosyltransfe~ases. Furthermore, using a oligonucleotide probe designed to identify D-alanine-D-alanine ligases, an 8.1 kilobase chromosomal fragment was isolated from S. toyocaensis NRRL 15009 and found to contain genes very similar to VRE vanH, vanA and vanX. Phylogenetic analysis of the predicted products of these genes showed them to be more similar to the VRE enzymes than any other in each enzyme class. These genes were also found in the vancomycin producer A. orienta/is C329.2 and several other glycopeptide antibiotic producing organisms. Not only does this imply that these organisms employ a mechanism of resistance similar to clinical VRE, it also suggests that these organisms may have been the source of the VRE genes. The enzymes VanHst and DdlN were studied in some detail and found to have biochemical properties similar to their corresponding VRE enzymes VanH and VanA, respectively. Given that the latter group of enzymes has physical properties that have impeded detailed analysis of enzyme mechanism, these new enzymes could find use as model systems in drug development programs. / Thesis / Doctor of Philosophy (PhD)