Title

Control of Pyrimidine Biosynthesis in Pseudomonas Aurantiaca by Pyrimidines

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

Date of Award

Summer 2018

Abstract

The control of de novo pyrimidine biosynthesis in the bacterium Pseudomonas aurantiaca ATCC 33663 was investigated. This pseudomonad may be useful as a biocontrol agent, and it is important to learn more about its nucleic metabolism including pyrimidine biosynthesis. Moreover, by determining the regulation of pyrimidine biosynthesis in P. aurantiaca, its regulation can be compared to the regulation of other close taxonomically related species of Pseudomonas. This research examined now active the pyrimidine biosynthetic pathway is in this pseudomonad. It was observed that all the pathway enzyme activities were higher in succinate-grown P. aurantiaca ATCC 33663 cells than in glucose-grown cells. The influence of supplementing exogenous pyrimidine bases on the pyrimidine biosynthetic pathway enzyme activities in cells grown in minimal medium containing 0.4% glucose or succinate as a carbon source was also analyzed to learn if metabolites of orotic acid or uracil affected the pyrimidine biosynthetic enzyme activities in P. aurantiaca ATCC 33633 regulated by metabolites of orotic acid or uracil. Pyrimidine supplementation could increase or decrease pyrimidine biosynthetic enzyme activities depending upon the enzyme assayed. To determine if pyrimidine limitation initiates pathway enzyme overproduction due to repressor scarcity, two pyrimidine auxotrophic mutants from P. aurantiaca were isolated by conventional mutagenesis and resistance to 5-fluoroorotic acid. One mutant strain DW-1 was deficient for orotate phosphoribosyltransferase activity while the second mutant strain DW-2 lacked orotidine 5’-monophosphate decarboxylase activity (strain DW-2). After pyrimidine limitation of both auxotrophic mutant strains, derepression of pathway enzyme synthesis was seen with strain DW-1 exhibiting the greatest derepression of three pyrimidine biosynthetic enzyme activities independent of carbon source. A lesser degree of derepression of enzyme synthesis was observed for mutant strain DW-2. Aspartate transcarbamoylase enzyme in P. aurantiaca was highly inhibited by PPi, AMP, UMP, and GMP at the level of enzyme activity. It was possible to conclude that the results provide evidence of transcriptional control of pyrimidine biosynthesis by pyrimidine metabolites and in vitro regulation of transcarbamoylase activity in P. aurantiaca.

Advisor

Thomas P West

Subject Categories

Biochemistry, Biophysics, and Structural Biology | Chemistry | Life Sciences | Physical Sciences and Mathematics

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