The aim of this research proposal is to elucidate the mechanisms responsible for regulation of the anaerobic respiratory pathway genes of includes the fumarate reductase (frdABCD), nitrate reductase (narGHJI) and DMSO/TMAO reductase (damsABC) genes which encode membrane associate enzyme complexes that participate in electron transport reaction during anaerobic conditions. These processes generate a chemosmotic potential for subsequent ATP generation via the proton translocating ATPase, and for proton driven solute uptake and for cell motility. The induction of respiratory gene expression in response to anaerobiosis is due primarily to the fnr gene product, Fnr. We recently succeeded in purifying Fnr in a biologically active form which binds DNA only under anaerobic conditions. We plan to improve the Fnr purification protocol, to characterize Fnr biochemically to understand how it detects the anaerobic state. We will also characterize the DNA binding properties required for Fnr activation gene expression. The control of the anaerobic respiratory genes in response to nitrate is provided by the narX/narQ/narL gene products that comprise a two-component regulatory system. NarX and NarQ are required to detect nitrate and they can independently transfer a signal via phosphorylation to NarL which binds DNA to both negatively and positively regulate gene expression. To better understand these processes at the mechanistic levels, mutations, will be introduced into narX and narQ and the resulting proteins will be analyzed using in vivo and in vitro approaches. The role of phosphorylation in NarL activation and in NarL-DNA binding will also be examined. The transcription of the narX, narQ, and narL genes will be characterized to determine how the levels of the products vary in the cell. Lastly, the basis for additional aerobic/anaerobic control of the frdABCD and narGHJI genes that is independent for fnr will be examined. We believe that the frdABCD, dmsABC, and narGHJI genes of E. coli offer an excellent model system to examine the processes for anaerobic control of bacterial gene expression. Little is known about these processes in other enteric bacteria that are human and animal pathogens. These processes are also common to a large number of soil microorganisms that can adapt and respire anaerobically when oxygen becomes limited in the environment.
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