CHARACTERIZATION OF AN IRON-DEPENDENT REGULATORY PROTEIN (IDER) OF MYCOBACTERIUM-TUBERCULOSIS AS A FUNCTIONAL HOMOLOG OF THE DIPHTHERIA-TOXIN REPRESSOR (DTXR) FROM CORYNEBACTERIUM-DIPHTHERIAE

The DtxR protein from Corynebacterium diphtheriae is an iron-dependent repressor that regulates transcription from the tax, IRP1, and IRP2 p romoters, A gene from virulent Mycobacterium tuberculosis H37Rv was re cently shown to encode a protein, here designated iron-dependent regul ator (IdeR), that is almost 60% homologous to DtxR from C. diphtheriae . A 750-bp PCR-derived DNA fragment carrying the M. tuberculosis ideR allele was subcloned to both high- and low-copy-number vectors, In Esc herichia coli, transcription from the C. diphtheriae tor, IRP1, and IR P2 promoters was strongly repressed by ideR under high-iron conditions , and ideR restored normal iron-dependent expression of the corynebact erial siderophore in the C. diphtheriae dtxR mutant C7(beta)hm723. The M. tuberculosis IdeR protein was overexpressed in E. coil and purifie d to near homogeneity by nickel affinity chromatography, Gel mobility shift experiments revealed that IdeR bound to a DNA fragment that carr ied the C. diphtheriae tox promoter/operator sequence, DNase I footpri nt analysis demonstrated that IdeR, in the presence of Cd2+, Co2+, Fe2 +, Mn2+, Ni2+, or Zn2+, protected an approximately 30-bp region on DNA fragments carrying the tox, IRP1, or IRP2 promoter/operator sequences , IdeR reacted very weakly in Western blots (immunoblots) with antiser um against the C. diphtheriae DtxR protein, suggesting that the immuno dominant epitopes of DtxR may be located in its poorly conserved carbo xyl-terminal domain.