ANALYSIS OF DIPHTHERIA-TOXIN REPRESSOR-OPERATOR INTERACTIONS AND CHARACTERIZATION OF A MUTANT REPRESSOR WITH DECREASED BINDING-ACTIVITY FORDIVALENT METALS

The diphtheria toxin repressor (DtxR) is an Fe2+-activated protein wit h sequence-specific DNA-binding activity for the diphtheria toxin (tox ) operator. Under high-iron conditions in Corynebacterium diphtheriae, DtxR represses toxin and siderophore biosynthesis as well as iron upt ake. DtxR and a mutant repressor with His-47 substituted for Arg-47, d esignated DtxR-R47H, were purified and compared. Six different divalen t cations (Cd2+, Co2+, Fe2+, Mn2+, Ni2+, and Zn2+) activated the seque nce-specific DNA-binding activity of DtxR and enabled it to protect th e tox operator from DNase I digestion, but Cu2+ failed to activate Dtx R. Hydroxyl radical footprinting experiments indicated that DtxR binds symmetrically about the dyad axis of the tox operator. Methylation pr otection experiments demonstrated that DtxR binding alters the suscept ibility to methylation of three G residues within the AT-rich tox oper ator. These findings suggest that two or more monomers of DtxR are inv olved in binding to the tox operator, with symmetrical DNA-protein int eractions occurring at each end of the palindromic operator. In this r egard, DtxR resembles several other well-characterized prokaryotic rep ressor proteins but differs dramatically from the Fe2+-activated ferri c uptake repressor protein (Fur) of Escherichia coli. The concentratio n of Co2+ required to activate DtxR-R47H was at least 10-fold greater than that needed to activate DtxR, but the sequence-specific DNA bindi ng of activated DtxR-R47H was indistinguishable from that of wild-type DtxR. The markedly deficient repressor activity of DtxR-R47H is consi stent with a significant decrease in its binding activity for divalent cations.