The aim of this research proposal is to gain a clearer understanding of the mechanisms for the induction, synthesis and turnover of the fumarate reductase complex in Escherichia coli. This oxido-reductase enzyme complex is membrane bound and contains both flavoprotein and non-heme iron centers. Synthesis of the four individual polypeptides of the fumarate reductase complex occurs only under anaerobic growth conditions when fumarate is available for use as a terminal electron acceptor. Synthesis of the complex is repressed when oxygen or nitrate is present. Little is known about this induction and/or repression phenomenon at either the genetic or the molecular level. We plan to examine the mechanisms for the transcriptional regulation of the four genes encoding the individual polypeptides of the membrane bound fumarate reductase complex using recombinant DNA techniques. Mutations resulting in altered fumarate reductase expression as well as mutations in the structural genes for fumarate reductase will be generated to increase our understanding of both the regulatory phenomenon and biochemistry of the complex. The in vivo expression and turnover of the fumarate reductase complex under inducing and non inducing conditions will also be examined using gene fusion and immunological techniques. The fumarate, nitrate and FNR regulator proteins affecting frd expression will be studied using genetic and molecular approaches. Experiments directed towards understanding the activation of fumarate reductase expression by the positive regulatory protein, FNR, will be undertaken. The transcriptional regulation of the fnr gene encoding FNR will be examined to locate the promoter and associated regulatory control regions. 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 fumarate, nitrate and oxygen. Although the study of fumarate reductase in E. coli is interesting in its own right, this system may also serve as a useful model for understanding facultative metabolism in other enteric bacteria.
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