Title

Regulation of Pyrimidine Biosynthesis in Pseudomonas chlororaphis

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

Date of Award

Spring 2020

Abstract

The pyrimidine biosynthetic pathway includes five enzymes unique to pyrimidine formation. The enzymes in the pathway are known to be aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and orotidine 5' -monophosphate decarboxylase. Initially, the effect of pyrimidine supplementation on the levels of the pyrimidine biosynthetic enzyme activities in the bacterium Pseudomonas chlororaphis ATCC 17414 was examined. In ATCC 17414 cells, transcarbamoylase and decarboxylase activities were repressed by orotic acid or uracil addition independent of carbon source. The isolation oftwo pyrimidine auxotrophic mutants of P. chlororaphis using chemical mutagenesis and 5-fluororotic acid resistance was reported. The mutants required uracil or cytosine as a pyrimidine source to support their growth. One mutant strain, designated BW-1, was found to be deficient for OMP decarboxylase activity while the other mutant strain, designated BW-2, had reduced phosphoribosyltransferase activity. Having isolated pyrimidine biosynthetic pathway mutants, the mutant cells were subjected to pyrimidine limitation to learn whether nucleotide depletion affects the synthesis of the pyrimidine biosynthetic enzyme activities since it should be possible to observe derepression of the synthesis of the pyrimidine pathway enzymes. Transcarbamoylase, dihydroorotase, dehydrogenase and phosphoribosyltransferase activities were all derepressed following one hour of pyrimidine limitation of strain BW-1 cells. When strain BW-2 was subjected to pyrimidine limitation for 2 hours, all five pyrimidine biosynthetic enzyme activities were derepressed independent of carbon source. Lastly, a determination was made to explore the regulation of the enzyme aspartate transcarbamoylase by pyrophosphate and ribonucleotides in P. chlororaphis ATCC 17414 cells. The enzyme was found to be highly inhibited by uridine 5' -monophosphate, uridine 5' -diphosphate, cytidine 5' -monophosphate and guanosine 5' -monophospahte in the glucose-grown ATCC 17414 cells. In conclusion, the five pyrimidine biosynthetic pathway enzymes were noted to be regulated at the level of enzyme synthesis plus transcarbamoylase activity was controlled at the level of enzyme activity.

Advisor

Thomas P. West

Subject Categories

Chemistry | Physical Sciences and Mathematics

Link to Full Text
COinS