Specific binding of the diphtheria tox regulatory element DtxR to the tox operator requires divalent heavy metal ions and a 9-base-pair interrupted palindromic sequence.
The structural gene for diphtheria toxin, tox, is carried by a family of closely related corynebacteriophages; however, the regulation of tox expression is controlled by a Corynebacterium diphtheriae-encoded regulatory element, dtxR. The molecular cloning and sequence analysis of dtxR was recently described. Previous studies have suggested that DtxR-mediated regulation of the diphtheria tox operator involves the formation of an iron-repressor complex, which specifically binds to the tox operator. We have expressed and purified DtxR from recombinant Escherichia coli. Immunoblot analysis shows DtxR to be a single M(r) 28,000 protein band in both recombinant E. coli and the C7(-) and C7hm723(-) strains of C. diphtheriae. In addition, we demonstrate that the binding of DtxR to a diphtheria tox promoter/operator probe requires the addition of Mn2+ to the reaction mixture; however, binding may be blocked by addition of the chelator 2,2'-dipyridyl, anti-DtxR antiserum, and excess unlabeled probe to the reaction mixture. Deletion of one of the 9-base-pair inverted repeat sequences from the tox operator results in a loss of DtxR binding. The results presented here demonstrate that regulation of diphtheria toxin expression by DtxR requires direct interaction between this regulatory factor and the tox operator in the presence of a divalent heavy metal ion.
Tao
X
X
Evans Department of Clinical Research, University Hospital, Boston, MA 02118.
Boyd
J
J
Murphy
J R
JR
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003353:Corynebacterium diphtheriae / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004167:Diphtheria Toxin / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008670:Metals; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins; D012045:Regulatory Sequences, Nucleic Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D004167:Diphtheria Toxin; C075586:DtxR protein, Corynebacterium diphtheriae; D008670:Metals; D011994:Recombinant Proteins