Iron is an essential nutrient for most all living cells. Since iron has an extraordinarily low solubility at neutral pH (Ca., 10(-17 M), a variety of complex systems have evolved to facilitate the uptake of iron. The favorable redox potential and coordination chemistry of iron allows this element to be used intracellularly both as a cofactor for a variety of structural and metabolic functions. In addition, the regulation of virulence determinants (e. g., toxin, colonization factors, etc.) in most pathogenic microorganisms is also controlled by iron-activated regulatory factors. For example, the diphtheria tox repressor, DtxR, is activated in the presence of iron and other heavy metal ions and functions as a negative regulatory element in the control of diphtheria toxin expression, as well as other iron-sensitive genes in lysogenic toxigenic strains of Corynebacterium diphtheriae. Upon depletion of exogenous iron from the culture medium, the Fe/DtxR/DNA complex rapidly disassociates resulting in derepression of all iron-sensitive genes. Since conjugation, transfection, and transformation are not well developed in C. diphtheriae, we have used molecular cloning of dtxR and its target sequences in recombinant Escherichia coli in order to study the molecular biology and genetics of this iron-activated regulatory factor. The long term goals of this research are to develop a detailed understanding of the role played DtxR in the regulation of gene expression in C. diphtheriae. Other than the recent demonstration that DtxR controls the expression of siderophore genes in C. diphtheriae, virtually nothing is known about the global nature of DtxR-mediated regulation of iron sensitive gene expression. This proposal seeks continued support for the study of the molecular genetic analysis of DtxR and the determination of the role that this regulatory factor plays in the molecular pathogenesis of C. diphtheriae. In addition, we propose to solve the X-ray crystallographic structure of apoDtxR, the metal ion/DtxR complex, and the metal ion/DtxR/DNA complex. To the best of our knowledge DtxR is the first iron-activated regulatory element to be crystallized. The solution of these structures will provide the paradigm for the mechanism of action of other iron-activated regulatory elements in pathogenic microorganisms.
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