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Characterization of the biochemical activity of the open reading frame "YqiQ" of Bacillus subiltilis

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
William T. Booth II (Creator)
Institution
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/
Advisor
Jason Reddick

Abstract: The goal of this research is to provide a greater chemical understanding of the mechanisms by which Bacillus subtilis transforms itself into a spore during times of nutrient deprivation. The area of interest of this study lies in the mother cell metabolic gene (mmg) operon, which is activated by a sigma-E transcription factor, during the early stages of sporulation. This operon is proposed to play roles in fatty acid metabolism, the methyl citric acid cycle, and the citric acid cycle during the intermediate stages of sporulation. The genes within this operon: mmgA,mmgB, mmgC, mmgD, mmgE, and yqiQ encode for proteins that have proposed biochemical roles to promote the above activities. The goal of this thesis is to biochemically confirm the activity of the protein encoded by the yqiQ gene. Our hypothesis is that this gene encodes for a 2-methylisocitrate lyase, the last enzyme of the methylcitric acid cycle. Methylisocitrate lyases catabolize 2-methylisocitrate into pyruvate and succinate, interconnecting the activities of the methylcitric acid cycle and the citric acid cycle of the bacterium. This pathway ultimately allows for the entrance of propionate from the metabolism of odd-chain fatty acids into the bacteria's citric acid cycle to promote ATP production. From work done in this lab previously, yqiQ has been cloned, and its protein was isolated and purified. In this work we optimized the yield of this purification producing approximately 2.17 milligrams per liter of culture. We are studying the "reverse" reaction by utilizing an assay which uses equimolar concentrations of pyruvate and succinate to produce 2-methylisocitrate (1). The reverse reaction is being conducted due to the lack of commercial availability of 2-methylisocitrate. Results have been able to provide partial evidence of this conversion over time. In order to investigate the proposed "forward" reaction mechanism, we are also working on the chemical synthesis of 2-methylisocitrate.

Additional Information

Publication
Thesis
Language: English
Date: 2011
Keywords
Bacillus subtilis, Sporulation, yqiQ gene
Subjects
Bacillus subtilis $x Genetics