Date of Award

2002

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

Supervisor

Professor G.D. Wright

Abstract

With the recent crisis in hospital-acquired infections, a renewed sense of urgency for studying bacterial species carrying antibiotic resistance determinants has developed. Much of the focus has been centered on the glycopeptide antibiotics including vancomycin, which represent the last main line of defense against many hospital-acquired infections. These clinically pivotal antibiotics are produced by filamentous bacteria in the order Actinomycetes including the genus Streptomyces. The investigations presented in this thesis were undertaken to address fundamental questions of glycopeptide production and resistance in the glycopeptide-producing Streptomyces toyocaensis NRRL 15009 and non-producing Streptomyces coeficolor A3(2). Specifically, questions with respect to the regulation of antibiotic production and resistance have been addressed in light of the relatively recent isolation of protein kinase genes with high similarity to those of eukaryotic origin. A link between glycopeptide production, resistance and protein phosphorylation in S. toyocaensis NRRL 15009 is presented as a basis for a subsequent degenerate PCR strategy to clone putative protein kinase genes from this organism. Using the PCR strategy four putative protein kinase gene fragments were cloned and one of these, stoPK-1 was isolated in its entirety and characterized by biochemical and genetic methods. Analysis of StoPK-1 revealed a bona fide serine/threonine protein kinase with localization to membrane fractions. Disruption of the genomic stoPK-1 gene in S. toyocaensis NRRL 15009 brought about an increased sensitivity to oxidative stress which could be reversed by supplying stoPK-l in trans, but not with a catalytically dead mutant, suggesting a role for the active kinase in responding to oxidative stress in S. toyocaensis NRRL 15009. Additionally, studies examining glycopeptide resistance were undertaken through genomic gene disruption and complementation on the previously isolated vancomycin resistance gene cluster (vanHAX), supporting a role for this cluster in Streptomyces similar to its function in yancomycin resistant gnterococcus (VRE). Furthermore, analysis of Streptomyces DNA sequences revealed putative two component regulatory systems composed of histidine kinase and response regulator pairs associated with the vanHAX genes in S. coelicolor A3(2) and the glycopeptide biosynthesis genes in S. toyocaensis NRRL 15009. These putative VanRS systems share homology to proteins shown to be critical for inducible resistance in VRE, and these observations encouraged an assessment of the response regulator (VanR) homologue in regulating resistance in S. coelicolor A3(2). The results of these studies support the conclusion that the regulation of vancomycin resistance in Streptomyces is similar to Enterococcus and provide further evidence that these soil-born bacteria may be the original source of the clinically important resistance genes.

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