Date: August 2003 Creator: Brichta, Dayna Michelle Description:
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 gentam