Ph.D. (Doctor of Philosophy)
Department of Biological Sciences
Escherichia coli synthesize an important component of the electron transport chain, Coenzyme Q (Ubiquinone; Q) from the shikimate pathway intermediate, chorismate. Two isofunctional enzymes UbiD and UbiX are responsible for the decarboxylation step in Q biosynthesis. It was shown by Gulmezian et al. (2007) that the loss of ubiX+ gene leads to a reduction in growth in both rich and minimal media as well as decrease in Q. They further reported that UbiX is responsible for controlling the activity of UbiG O-methyltransferase, involved in two O-methylation reactions in Q biosynthesis. Contrary to these results, in this study we show that the [Delta]ubiX had no effect on the growth of E. coli in succinate minimal medium and on Q biosynthesis. The growth and levels of Q, were however drastically decreased when the gene ubiD+ was deleted. The decreased levels of Q synthesized by [Delta]ubiD is due to the activity of UbiX. It was further shown that UbiX was not involved in controlling the activity of UbiG, since the expression of UbiG was not affected in the [Delta]ubiX deletion mutant. Phylogenetic analysis of the two methyltrasferases, UbiG and UbiE demonstrated the possibility of horizontal gene transfer between closely related bacteria. The first of the three hydroxylations in Q biosynthesis, contrary to previous studies was shown to be carried out by UbiI (VisC) by Chehade et al. (2013). In this study we demonstrate that a [Delta]visC deletion mutant grew to wild-type levels on succinate minimal medium and synthesized wild-type levels of Q, proving that VisC was not involved in the aerobic biosynthesis of Q. Our results show that, [Delta]ubiB deletion mutant failed to grow on succinate minimal medium and accumulated 2-octaprenylphenol, the substrate for this hydroxylation step. It was recently reported that, in Uropathogenic E. coli (UPEC), VisC is required for biofilm formation and expression of Type I pili resulting in increased virulence under aerobic conditions (Floyd et al., 2016). The prenyl sidechain in Q is synthesized in most bacteria using the non-mevalonate or methylerythritol-phosphate (MEP) pathway (Rodriguez-Concepcion and Boronat, 2002), whereas humans and a few genera of bacteria use the mevalonate pathway (Endo, 1992). This difference was utilized in designing novel antimicrobials targeted against the various enzymes of the MEP pathway. Our study involved the anti-bacterial testing of over 350 such compounds against nine potential bacteria including pathogens like Pseudomonas aeruginosa, Bacillus cereus, Klebsiella pneumoniae and Mycobacterium smegmatis. Over 200 compounds were found to have inhibitory effects on multiple bacteria and this will contribute to research for new antibiotics, which is the need of the hour.
Ghose, Debarati, "Studies on selected enzymes involved in ubiquinone biosynthesis in Escherichia coli" (2018). Graduate Research Theses & Dissertations. 5104.
Northern Illinois University
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