Prolylpeptide Binding by the Prokaryotic SH3-like Domain of the Diphtheria Toxin Repressor:  A Regulatory Switch†,‡

Biochemistry ◽  
2005 ◽  
Vol 44 (1) ◽  
pp. 40-51 ◽  
Author(s):  
Gregory P. Wylie ◽  
Vijayaraghavan Rangachari ◽  
Ewa A. Bienkiewicz ◽  
Vedrana Marin ◽  
Nilakshee Bhattacharya ◽  
...  
Keyword(s):  
Diphtheria Toxin ◽  
Diphtheria Toxin Repressor

Structure of the metal-ion-activated diphtheria toxin repressor/ tox operator complex

Nature ◽  
10.1038/28893 ◽  
1998 ◽  
Vol 394 (6692) ◽  
pp. 502-506 ◽  
Author(s):  
André White ◽  
Xiaochun Ding ◽  
Johanna C. vanderSpek ◽  
John R. Murphy ◽  
Dagmar Ringe
Keyword(s):  
Diphtheria Toxin ◽  
Metal Ion ◽  
Diphtheria Toxin Repressor

scholarly journals Construction and Characterization of Transposon Insertion Mutations in Corynebacterium diphtheriae That Affect Expression of the Diphtheria Toxin Repressor (DtxR)

2002 ◽  
Vol 184 (20) ◽  
pp. 5723-5732 ◽  
Author(s):  
Diana Marra Oram ◽  
Ana Avdalovic ◽  
Randall K. Holmes
Keyword(s):  
Oxidative Stress ◽  
Diphtheria Toxin ◽  
Corynebacterium Diphtheriae ◽  
Insertion Mutant ◽  
Toxin Gene ◽  
Wild Type ◽  
Transposon Insertion ◽  
Diphtheria Toxin Repressor ◽  
Mutant Strains

ABSTRACT Transcription of the bacteriophage-borne diphtheria toxin gene tox is negatively regulated, in response to intracellular Fe2+ concentration, by the chromosomally encoded diphtheria toxin repressor (DtxR). Due to a scarcity of tools, genetic analysis of Corynebacterium diphtheriae has primarily relied on analysis of chemically induced and spontaneously occurring mutants and on the results of experiments with C. diphtheriae genes cloned in Escherichia coli or analyzed in vitro. We modified a Tn5-based mutagenesis technique for use with C. diphtheriae, and we used it to construct the first transposon insertion libraries in the chromosome of this gram-positive pathogen. We isolated two insertions that affected expression of DtxR, one 121 bp upstream of dtxR and the other within an essential region of the dtxR coding sequence, indicating for the first time that dtxR is a dispensable gene in C. diphtheriae. Both mutant strains secrete diphtheria toxin when grown in medium containing sufficient iron to repress secretion of diphtheria toxin by wild-type C. diphtheriae. The upstream insertion mutant still produces DtxR in decreased amounts and regulates siderophore secretion in response to iron in a manner similar to its wild-type parent. The mutant containing the transposon insertion within dtxR does not produce DtxR and overproduces siderophore in the presence of iron. Differences in the ability of the two mutant strains to survive oxidative stress also indicated that the upstream insertion retained slight DtxR activity, whereas the insertion within dtxR abolished DtxR activity. This is the first evidence that DtxR plays a role in protecting the cell from oxidative stress.

scholarly journals Metal stoichiometry and functional studies of the diphtheria toxin repressor

2003 ◽  
Vol 100 (7) ◽  
pp. 3808-3813 ◽  
Author(s):  
M. M. Spiering ◽  
D. Ringe ◽  
J. R. Murphy ◽  
M. A. Marletta
Keyword(s):  
Diphtheria Toxin ◽  
Functional Studies ◽  
Diphtheria Toxin Repressor

Anion-Coordinating Residues at Binding Site 1 Are Essential for the Biological Activity of the Diphtheria Toxin Repressor

1999 ◽  
Vol 67 (4) ◽  
pp. 1806-1811
Author(s):  
Joanne Goranson-Siekierke ◽  
Ehmke Pohl ◽  
Wim G. J. Hol ◽  
Randall K. Holmes
Keyword(s):  
Biological Activity ◽  
Binding Site ◽  
Diphtheria Toxin ◽  
Diphtheria Toxin Repressor

scholarly journals Anion-Coordinating Residues at Binding Site 1 Are Essential for the Biological Activity of the Diphtheria Toxin Repressor

1999 ◽  
Vol 67 (4) ◽  
pp. 1806-1811 ◽  
Author(s):  
Joanne Goranson-Siekierke ◽  
Ehmke Pohl ◽  
Wim G. J. Hol ◽  
Randall K. Holmes
Keyword(s):  
Binding Site ◽  
Metal Binding ◽  
Diphtheria Toxin ◽  
Cation Binding ◽  
Anion Binding ◽  
Divalent Metal Ions ◽  
X Ray Crystallography ◽  
Diphtheria Toxin Repressor ◽  
Cation Binding Site ◽  
Binding Residues

ABSTRACT The homodimeric diphtheria toxin repressor (DtxR) uses Fe2+ as a corepressor, binds to iron-regulated promoters, and negatively regulates the syntheses of diphtheria toxin, corynebacterial siderophore, and several other Corynebacterium diphtheriae products. The crystal structure of DtxR shows that the second domain of each monomer has two binding sites for Fe2+ or certain other divalent metal ions. In addition, site 1 binds a sulfate or phosphate anion, suggesting that phosphate may function intracellularly as a co-corepressor. The effects of alanine substitutions for selected residues in sites 1 and 2 were determined by measuring the β-galactosidase activities of a tox operator/promoter-lacZ reporter construct in Escherichia coli strains expressing each DtxR variant. Our studies demonstrated that single alanine substitutions for the anion-binding residues in site 1 (R80A, S126A, or N130A) caused severely decreased DtxR activity, similar to the effects of alanine substitutions for metal-binding residues in site 2 (C102A, E105A, or H106A) and greater than the effects of alanine substitutions for metal-binding residues in site 1 (H79A, E83A, or H98A) reported previously by other investigators. Various combinations of alanine substitutions for site 1 and site 2 residues were also analyzed to further elucidate the roles of these cation- and anion-binding ligands in DtxR activity. Furthermore, the interaction between residue E20 in the DNA binding domain and R80 in anion/cation binding site 1 was analyzed, and the E20A variant of DtxR was shown to have a phenotype indistinguishable from that of the R80A variant. Our data demonstrated for the first time that the anion-binding residues R80, S126, and N130 at site 1 are essential for DtxR activity. The data also showed that the interaction of E20 in domain 1 with R80 in domain 2, first revealed by X-ray crystallography in apo-DtxR and holo-DtxR, is a structural feature of DtxR that is important for its repressor activity.

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