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)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23550 |
| Date | 10 1900 |
| Creators | Marshall, Christopher G |
| Contributors | Wright, G.D., Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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