Analysis of diphtheria toxin repressor-operator interactions and characterization of a mutant repressor with decreased binding activity for divalent metals

The diphtheria toxin repressor (DtxR) is an Fe²⁺-activated protein with 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 uptake. DtxR and a mutant repressor with His–47 substituted for Arg–47, designated DtxR-R47H, were purified and compared. Six different divalent cations (Cd²⁺, Co²⁺, Fe²⁺, Mn²⁺, Ni²⁺, and Zn²⁺) activated the sequence-specific DNA-binding activity of DtxR and enabled it to protect the fox operator from DNase I digestion, but Cu²⁺ failed to activate DtxR. Hydroxyl radical footprinting experiments indicated that DtxR binds symmetrically about the dyad axis of the tox operator. Methylation protection experiments demonstrated that DtxR binding alters the susceptibility to methylation of three G residues within the AT-rich tox operator. These findings suggest that two or more monomers of DtxR are involved in binding to the tox operator, with symmetrical DNA-protein interactions occurring at each end of the palindromic operator. In this regard, DtxR resembles several other well-characterized prokaryotic repressor proteins but differs dramatically from the Fe²⁺-activated ferric uptake repressor protein (Fur) of Escherichia coli. The concentration of Co²⁺ required to activate DtxR-R47H was at least 10-foid greater than that needed to activate DtxR, but the sequence-specific DNA binding of activated DtxR-R47H was indistinguishable from that of wild-type DtxR. The markedly deficient repressor activity of DtxR-R47H is consistent with a significant decrease in its binding activity for divalent cations.     

Extracellular deoxyribonuclease production by a thermophile, Streptomyces thermonitrificans

A thermophilic bacterial strain, Streptomyces thermonitrificans, produced high levels of extracellular deoxyribonuclease (DNase) when grown on NBG medium (containing 1% peptone, 0.3% beef extract, 1% glucose and 0.5% NaCl). Maximum DNase activity (140 U ml⁻¹) was obtained, in 24 h, when the culture was grown on modified NBG medium (containing 1.3% beef extract, 1% glucose, 0.5% NaCl and 50 μM Mn²⁺ at 45°C. The crude enzyme showed higher activity on native DNA than on sonicated and heat denatured DNA. Moreover, addition of Mn²⁺ in the assay mixture resulted in a significant stimulation (10–15 fold) of the enzyme activity. Received 24 November 1998/ Accepted in revised form 25 April 1999