We propose to extend our studies on the regulation of the expression of the nar operon and the biogenesis of the nitrate reductase complex in Escherichia coli with the following general aims: (a) To define the molecular mechanisms involved in induction of specific gene expression by anaerobic conditions and the modulation of that expression by specific electron acceptors and (b) to define the temporal sequence and molecular mechanisms involved int he biogenesis of the functional, membrane-bound complex. Nitrate reductase is a membrane-bound enzyme in Escherichia coli which permits this facultative anaerobe to grow using nitrate as an electron acceptor under anaerobic conditions. This enzyme is induced under anaerobic conditions along with a network of enzymes which permits E. coli to derive energy by fermentation or by anaerobic respiration using exogenous electron acceptors. The expression of the nar operon, which encodes nitrate reductase, is regulated at the level of transcription by a positive regulator of a number of anaerobically expressed enzymes, Fnr, as well as a specific positive regulatory factor, NarL, which is activated by the presence of nitrate. We propose to study the molecular mechanisms involved in the anaerobic activation of Fnr and the activation of transcription of the nar operon by these two trans-acting systems. Membrane-bound nitrate reductase is composed of three subunits (alpha, beta, gamma), a molybdenum-pterium cofactor, nonheme ion and a cytochrome b component. this complex is organized in the cell membrane so that reduction of nitrate to nitrite coupled to oxidation of fermentation pathway intermediates or products drives the formation of a proton gradient. The alpha and beta subunits form a complex which can reduce nitrate only with artificial electron donors while the gamma subunit is a hydrophobic cytochrome b component which apparently anchors the complex to the membrane in a physiologically functional form. We propose to utilize the cloned nar operon along with mutants unable to produce specific subunits to determine the temporal events and molecular mechanisms involved in subunit synthesis, cofactor addition and assembly of the functional complex in the cell membrane.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM019511-18
Application #
3269664
Study Section
Bacteriology and Mycology Subcommittee 1 (BM)
Project Start
1976-06-01
Project End
1994-07-31
Budget Start
1989-08-01
Budget End
1990-07-31
Support Year
18
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225
Walker, M S; DeMoss, J A (1994) NarL-phosphate must bind to multiple upstream sites to activate transcription from the narG promoter of Escherichia coli. Mol Microbiol 14:633-41
Walker, M S; DeMoss, J A (1993) Phosphorylation and dephosphorylation catalyzed in vitro by purified components of the nitrate sensing system, NarX and NarL. J Biol Chem 268:8391-3
Walker, M S; DeMoss, J A (1992) Role of alternative promoter elements in transcription from the nar promoter of Escherichia coli. J Bacteriol 174:1119-23
Bonnefoy, V; DeMoss, J A (1992) Identification of functional cis-acting sequences involved in regulation of narK gene expression in Escherichia coli. Mol Microbiol 6:3595-602
Dubourdieu, M; DeMoss, J A (1992) The narJ gene product is required for biogenesis of respiratory nitrate reductase in Escherichia coli. J Bacteriol 174:867-72
Dong, X R; Li, S F; DeMoss, J A (1992) Upstream sequence elements required for NarL-mediated activation of transcription from the narGHJI promoter of Escherichia coli. J Biol Chem 267:14122-8
Walker, M S; DeMoss, J A (1991) Promoter sequence requirements for Fnr-dependent activation of transcription of the narGHJI operon. Mol Microbiol 5:353-60
DeMoss, J A; Hsu, P Y (1991) NarK enhances nitrate uptake and nitrite excretion in Escherichia coli. J Bacteriol 173:3303-10
Sodergren, E J; DeMoss, J A (1988) narI region of the Escherichia coli nitrate reductase (nar) operon contains two genes. J Bacteriol 170:1721-9
Sodergren, E J; Hsu, P Y; DeMoss, J A (1988) Roles of the narJ and narI gene products in the expression of nitrate reductase in Escherichia coli. J Biol Chem 263:16156-62

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