The major aims of this research project are to further elucidate the regulatory mechanisms required for nitrate- and molybdate-dependent gene expression in Escherichia coli. This enteric bacterium is a model pathogenic and commensal enteric microorganism: it can detect nitrate and a variety of other respiratory substrates in the environment and respond accordingly by synthesizing different respiratory complexes in response to changing environmental signals. This strategy ensures optimal production of energy to support of cell maintenance reactions, biosynthesis, and cell growth. In the studies proposed herein, we will use several model nitrate/nitrite-controlled genes in this organism (e.g., fumarate reductase (frdABCD), NarG and NapF nitrate reductase (narGHJI, and napA) nitrite reductase (nrf and nirB), and DMSO/TMAO reductase (dmsABC) genes to examine how the alternative environmental signals are detected and how the resulting information is then utilized to modulate gene expression. Since these processes are common to many facultative animal and plant pathogens, the proposed molecular and biochemical studies should provide insight concerning how less well understood microbes manage the anaerobic transitions with respect to nitrate. We will examine the basis for nitrate, nitrite, and molybdate-dependent control of gene expression by the Nar and ModE regulatory circuits. We will extend our studies to further examine how the NarX, NarQ, NarP, and NarL proteins detect and transduce signals to modulate gene control. We will examine how the ModE protein provides for the molybdate-dependent control molybdate-containing enzymes to fine-tune expression of the anaerobic respiratory pathway genes. The Nar and Mode gene families will be identified by microarray analysis. Finally, the physiology of nitrate control will be examined by use of continuous culture methods. ? ?
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