Isolation and characterization of iron-independent positive dominant mutants of the diphtheria toxin repressor DtxR.
It is well known that the functional activity of the diphtheria toxin repressor DtxR is controlled by iron, which serves as an essential cofactor necessary for activation of target DNA binding by this regulatory element. In this communication, we describe the isolation and characterization of a unique series of DtxR mutants that are constitutively active and repress the expression of beta-galactosidase from a diphtheria tox promoter/operator-lacZ transcriptional fusion, even in the absence of iron. These self-activating mutants of DtxR (SAD) were isolated through the use of a positive selection system for the cloning of functional dtxR alleles and target DNA operator sites. Of the four independently isolated SAD mutants that were characterized, two (SAD2 and SAD11) were found to carry a single missense mutation (E175K) in their respective C-terminal SH3-like domains. In contrast, the mutant allele encoding SAD3 was found to carry a total of six missense mutations distributed throughout the N- and C-terminal domains of the repressor. Partial diploid analysis of strains carrying both native dtxR and alleles encoding either SAD2 or SAD3 demonstrate that these iron-independent mutants possess a positive dominant phenotype in the regulation of beta-galactosidase expression from a diphtheria tox promoter/operator-lacZ transcriptional fusion.
Sun
L
L
Evans Department of Clinical Research and Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA.
vanderSpek
J
J
Murphy
J R
JR
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D001665:Binding Sites; D003353:Corynebacterium diphtheriae / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification; D007501:Iron / Q000378:metabolism; D009154:Mutation
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C075586:DtxR protein, Corynebacterium diphtheriae; D007501:Iron