The aim of this research project is to understand the general mechanisms responsible for controlling anaerobic gene expression in the enteric bacterium, Escherichia coli. We are using the fumarate reductase (frdABCD) genes of this organism as a model to examine how the presence of alternative cellular electron acceptors are able to affect gene expression. The frdABCD genes encode the fumarate reductase enzyme complex that functions in the final step of anaerobic respiration to convert fumarate, as the terminal electron acceptor, to succinate. It is membrane bound and contains both flavin and non-heme iron centers. Expression of theses genes occurs optimally under anaerobic growth conditions in the presence of fumarate and in the absence of nitrate. As yet, little is known about the mechanisms responsible for this phenomenon. We wish to elucidate the molecular and biochemical basis of the anaerobic induction and the nitrate repression processes. We will study the role of the fnr, narR, and narL gene products in these processes and determine the mechanisms by which they act. An analysis of cis-acting sites for oxygen/redox state control of frdABCD gene expression will be performed to identify their locations(s) in the frd regulatory region. Chemical and enzymatic methods will be used to establish the domains for the FNR binding. The FNR protein will also be characterized to understand its role in sensing the anaerobic state. Mutagenesis of the fnr gene will be done to identify essential regions in FNR for activating transcription during anaerobiosis. An analysis of cis-acting sites for nitrate control of frdABCD gene expression will also be performed to understand where the narR and narL gene productions act to control also be performed to understand where the narR and narL gene products act to control frdABCD expression. The narR and NarL genes will be characterized with respect to sequence, transcription, and function via mutational analysis to obtain mutations that are altered for nitrate sensing and signaling. The gene products will be purified and characterized to understand the interactions necessary for the repression process. We believe that fumarate reductase is an excellent model system to examine the induction and regulation of genes whose products are required under anaerobic growth conditions. Little is known about how these genes are induced or repressed in response to oxygen and other electron acceptors. This oxygen phenomenon is involved in regulation of the electron transport process, photosynthesis and nitrogen fixation in a variety of bacterial species.
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