Enterobacteria efficiently regulate their respiratory metabolism in response to availability of alternate electron acceptors such as oxygen, nitrate and fumarate. This regulation is coordinated to ensure use of the most efficient respiratory pathway. In the absence of oxygen, nitrate, the preferred anaerobic electron acceptor, induces synthesis of enzymes for nitrate respiration (formate dehydrogenase-N and nitrate reductase) while repressing the synthesis of other anaerobic respiratory enzymes such as fumarate reductase. The long-term goals of this project are to understand the physiological and genetic mechanisms by which nitrate coordinately regulates the synthesis of different anaerobic pathways in different ways. Previous work has identified two genes, narL and narX, which are required for nitrate induction of nitrate reductase synthesis and nitrate repression of fumarate reductase synthesis. The narL gene product, NARL, is hypothesized to be a DNA-binding protein that regulates transcription in response to nitrate. The narX gene product, NARX, is hypothesized to convert NARL to its repressor form. NARL and NARX show sequence similarity to the group of """"""""two-component regulatory systems"""""""" involved in signal transduction. This project will define the roles of narL and narX in regulation by isolating and characterizing altered function mutations in these genes, by expressing narL and narX independently of each other under various growth conditions, and by analyzing binding of NARL to its target DNA sites. The structure and expression of the narL complex operon will be analyzed by operon and gene fusions and by transcript mapping, in order to understand the regulation of narX and narL expression. A search will be made for a hypothetical gene, """"""""narQ"""""""", that may be required for conversion of NARL to its activator form. Finally, the structural genes for formate dehydrogenase-N will be identified, and their regulation by nitrate and NARL will be analyzed by genetic and molecular biological methods. Together, these studies will provide a more detailed view of how nitrate regulates and coordinates anaerobic metabolism.
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