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 tox, IRP1, and IRP2 promoters. A gene from virulent Mycobacterium tuberculosis H37Rv was recently shown to encode a protein, here designated iron-dependent regulator (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 Escherichia coli, transcription from the C. diphtheriae tox, IRP1, and IRP2 promoters was strongly repressed by ideR under high-iron conditions, and ideR restored normal iron-dependent expression of the corynebacterial siderophore in the C. diphtheriae dtxR mutant C7(beta)hm723. The M. tuberculosis IdeR protein was overexpressed in E. coli and purified to near homogeneity by nickel affinity chromatography. Gel mobility shift experiments revealed that IdeR bound to a DNA fragment that carried the C. diphtheriae tox promoter/operator sequence. DNAse I footprint 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 antiserum against the C. diphtheriae DtxR protein, suggesting that the immunodominant epitopes of DtxR may be located in its poorly conserved carboxyl-terminal domain.
Schmitt
M P
MP
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.
Predich
M
M
Doukhan
L
L
Smith
I
I
Holmes
R K
RK
D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D003001:Cloning, Molecular; D003353:Corynebacterium diphtheriae / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007501:Iron / Q000378:metabolism; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000235:genetics; D009875:Operator Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012097:Repressor Proteins / Q000502:physiology*; D017262:Siderophores / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C075586:DtxR protein, Corynebacterium diphtheriae; C096348:IdeR protein, Mycobacterium tuberculosis; D012333:RNA, Messenger; D012097:Repressor Proteins; D017262:Siderophores; D007501:Iron