Ph.D. (Doctor of Philosophy)
Department of Biological Sciences
The Gram negative facultative anaerobic bacterium E.coli contains the isoprenoid quinones ubiquinone (Q) and menaquinone (MK) since it is able to grow under both aerobic and anaerobic conditions. The Q biosynthetic genes ubiG, ubiH, and ubiF produce key intermediates in the pathway. To study the regulation of these genes, the putative regulatory region of each orf were linked to a promoterless lacZ+ gene contained on the plasmid pRS415 to construct ubiG’-lacZ+, ubiH’-lacZf, and ubiF’-lacZ+ operon fusions. These were integrated in single copy into E.coli MC4100 and into strains carrying various respiratory and regulatory mutations. Gene expression was studied by β-galactosidase assays under aerobic and anaerobic conditions in media containing a variety of carbon sources (glucose, xylose, glycerol, succinate, acetate, lactate, and pyruvate) and electron acceptors (oxygen, nitrate, and fumarate). UbiG, UbiH, and UbiF appear to be constitutively expressed under different growth conditions and the activity was not significantly affected by respiratory and regulatory mutations. It was found that the ubiH gene is part of an operon consisting of 5’ yg/B-pepP-ubiH-visC 3’. This has been verified through quantitative real-time PCR (QRT-PCR). Similar studies initiated with an MK biosynthetic gene menh. demonstrated the existence of a dicistronic operon that includes men A and rraA. In the MK biosynthetic pathway, men A is involved in the conversion of the naphthalenoid aromatic compound 1,4-dihydroxy-2-naphthoic acid (DHNA) to demethylmenaquinone (DMK) while RraA (regulator of RNAse E activity A) binds to the essential E.coli endonuclease RNAse E, inhibiting its RNA processing activity. It is intriguing that these two genes are linked and organized in an operon although they perform functionally different roles in E.coli. The presence of this operon has also been confirmed through lac fusion studies and QRT-PCR analyses. In summary, the above studies have established the identity of novel operons and have shed new light on the regulation of Q and MK biosynthetic genes in E.coli.
Royan, Sandhya Venantia, "Regulatory studies on selected quinone biosynthetic genes in Escherichia coli" (2006). Graduate Research Theses & Dissertations. 4691.
xiii, 155, pages
Northern Illinois University
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