The long term goal of this research proposal is to elucidate the molecular mechanism(s) of diphtheria tox regulation. It is widely known that the expression of diphtheria toxin by Corynebacterium diphtheriae is dependent upon a) lysogenic conversion of non-toxigenic strains to toxigenicity, and b) the physiologic state of the host bacterium. In particular, the depletion of exogenous iron from the culture medium such that iron becomes the growth rate limiting substrate has been directly linked to the expression of the diphtheria tox gene. Based upon both biochemical and genetic analyses, Dr. Murphy has proposed that the regulation of tox expression was mediated through an iron-binding repressor that is encoded by the bacterial host. Since conjugation, transfection, and transformation have not been well developed in C. diphtheriae, the investigators have examined the expression of tox gene products in recombinant strains of Escherichia coli. These studies have shown that the expression of the tox gene is not responsive to exogenous iron and is constitutive. Moreover, the expression of tox gene products has been shown to initiate from the same transcriptional start points in E. coli and C. diphtheriae. The investigator has constructed a diphtheria tox regulatory region / lacZ transcriptional fusion, and has introduced that fusion into the chromosome of E. coli in single copy. This strain was used to screen genomic libraries of C. diphtheriae for determinants that suppress lacZ expression. The investigator has cloned and sequenced a gene, dtxR, from the non-toxogenic C7(-) strain of C. diphtheriae that regulates the expression of beta-galactosidase from the tox: lacZ fusion. Importantly, the regulatory activity of the cloned factor is dependent upon the concentration of exogenous iron. This proposal requests funds to express, purify, and investigate the interaction between the diphtheria toxin regulatory element, dtxR, and the tox operator.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI021628-08
Application #
3131833
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1984-05-01
Project End
1994-02-28
Budget Start
1992-03-01
Budget End
1993-02-28
Support Year
8
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Boston University
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02118
Stapleton, Brian; Walker, Lawrence R; Logan, Timothy M (2013) Zn(II) stimulation of Fe(II)-activated repression in the iron-dependent repressor from Mycobacterium tuberculosis. Biochemistry 52:1927-38
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Murphy, John R (2011) Mechanism of diphtheria toxin catalytic domain delivery to the eukaryotic cell cytosol and the cellular factors that directly participate in the process. Toxins (Basel) 3:294-308
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