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CONSTRUCTION OF A Pseudomonas aeruginosa DIHYDROOROTASE MUTANT AND THE DISCOVERY OF A NOVEL LINK BETWEEN PYRIMIDINE BIOSYNTHETIC INTERMEDIATES AND THE ABILITY TO PRODUCE VIRULENCE FACTORS

By Dayna Michelle Brichta

Abstract

The ability to synthesize pyrimidine nucleotides is essential for most organisms. Pyrimidines are required for RNA and DNA synthesis, as well as cell wall synthesis and the metabolism of certain carbohydrates. Recent findings, however, indicate that the pyrimidine biosynthetic pathway and its intermediates maybe more important for bacterial metabolism than originally thought. Maksimova et al., 1994, reported that a P. putida M, pyrimidine auxotroph in the third step of the pathway, dihydroorotase (DHOase), failed to produce the siderophore pyoverdin. We created a PAO1 DHOase pyrimidine auxotroph to determine if this was also true for P. aeruginosa. Creation of this mutant was a two-step process, as P. aeruginosa has two pyrC genes (pyrC and pyrC2), both of which encode active DHOase enzymes. The pyrC gene was inactivated by gene replacement with a truncated form of the gene. Next, the pyrC2 gene was insertionally inactivated with the aacC1 gentamicin resistance gene, isolated from pCGMΩ. The resulting pyrimidine auxotroph produced significantly less pyoverdin than did the wild type. In addition, the mutant produced 40 % less of the phenazine antibiotic, pyocyanin, than did the wild type. As both of these compounds have been reported to be vital to the virulence response of P. aeruginosa, we decided to test the ability of the DHOase mutant strain to produce othe

Year: 2003
OAI identifier: oai:CiteSeerX.psu:10.1.1.961.2901
Provided by: CiteSeerX
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