Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1995 Nov;63(11):4284–4289. doi: 10.1128/iai.63.11.4284-4289.1995

Characterization of an iron-dependent regulatory protein (IdeR) of Mycobacterium tuberculosis as a functional homolog of the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae.

M P Schmitt 1, M Predich 1, L Doukhan 1, I Smith 1, R K Holmes 1
PMCID: PMC173608  PMID: 7591059

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (417.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bagg A., Neilands J. B. Molecular mechanism of regulation of siderophore-mediated iron assimilation. Microbiol Rev. 1987 Dec;51(4):509–518. doi: 10.1128/mr.51.4.509-518.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boyd J. M., Hall K. C., Murphy J. R. DNA sequences and characterization of dtxR alleles from Corynebacterium diphtheriae PW8(-), 1030(-), and C7hm723(-). J Bacteriol. 1992 Feb;174(4):1268–1272. doi: 10.1128/jb.174.4.1268-1272.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boyd J., Oza M. N., Murphy J. R. Molecular cloning and DNA sequence analysis of a diphtheria tox iron-dependent regulatory element (dtxR) from Corynebacterium diphtheriae. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5968–5972. doi: 10.1073/pnas.87.15.5968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Calderwood S. B., Mekalanos J. J. Iron regulation of Shiga-like toxin expression in Escherichia coli is mediated by the fur locus. J Bacteriol. 1987 Oct;169(10):4759–4764. doi: 10.1128/jb.169.10.4759-4764.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Farinha M. A., Kropinski A. M. Construction of broad-host-range plasmid vectors for easy visible selection and analysis of promoters. J Bacteriol. 1990 Jun;172(6):3496–3499. doi: 10.1128/jb.172.6.3496-3499.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fiss E. H., Yu S., Jacobs W. R., Jr Identification of genes involved in the sequestration of iron in mycobacteria: the ferric exochelin biosynthetic and uptake pathways. Mol Microbiol. 1994 Nov;14(3):557–569. doi: 10.1111/j.1365-2958.1994.tb02189.x. [DOI] [PubMed] [Google Scholar]
  7. Günter K., Toupet C., Schupp T. Characterization of an iron-regulated promoter involved in desferrioxamine B synthesis in Streptomyces pilosus: repressor-binding site and homology to the diphtheria toxin gene promoter. J Bacteriol. 1993 Jun;175(11):3295–3302. doi: 10.1128/jb.175.11.3295-3302.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hantke K. Cloning of the repressor protein gene of iron-regulated systems in Escherichia coli K12. Mol Gen Genet. 1984;197(2):337–341. doi: 10.1007/BF00330982. [DOI] [PubMed] [Google Scholar]
  9. Hantke K. Regulation of ferric iron transport in Escherichia coli K12: isolation of a constitutive mutant. Mol Gen Genet. 1981;182(2):288–292. doi: 10.1007/BF00269672. [DOI] [PubMed] [Google Scholar]
  10. Holmes R. K., Twiddy E. M. Characterization of monoclonal antibodies that react with unique and cross-reacting determinants of cholera enterotoxin and its subunits. Infect Immun. 1983 Dec;42(3):914–923. doi: 10.1128/iai.42.3.914-923.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kanei C., Uchida T., Yoneda M. Isolation from corynebacterium diphtheriae C7(beta) of bacterial mutants that produce toxin in medium with excess iron. Infect Immun. 1977 Oct;18(1):203–209. doi: 10.1128/iai.18.1.203-209.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Litwin C. M., Calderwood S. B. Role of iron in regulation of virulence genes. Clin Microbiol Rev. 1993 Apr;6(2):137–149. doi: 10.1128/cmr.6.2.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Oguiza J. A., Tao X., Marcos A. T., Martín J. F., Murphy J. R. Molecular cloning, DNA sequence analysis, and characterization of the Corynebacterium diphtheriae dtxR homolog from Brevibacterium lactofermentum. J Bacteriol. 1995 Jan;177(2):465–467. doi: 10.1128/jb.177.2.465-467.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pappenheimer A. M., Jr Diphtheria toxin. Annu Rev Biochem. 1977;46:69–94. doi: 10.1146/annurev.bi.46.070177.000441. [DOI] [PubMed] [Google Scholar]
  15. Qiu X., Verlinde C. L., Zhang S., Schmitt M. P., Holmes R. K., Hol W. G. Three-dimensional structure of the diphtheria toxin repressor in complex with divalent cation co-repressors. Structure. 1995 Jan 15;3(1):87–100. doi: 10.1016/s0969-2126(01)00137-x. [DOI] [PubMed] [Google Scholar]
  16. Schmitt M. P., Holmes R. K. Analysis of diphtheria toxin repressor-operator interactions and characterization of a mutant repressor with decreased binding activity for divalent metals. Mol Microbiol. 1993 Jul;9(1):173–181. doi: 10.1111/j.1365-2958.1993.tb01679.x. [DOI] [PubMed] [Google Scholar]
  17. Schmitt M. P., Holmes R. K. Characterization of a defective diphtheria toxin repressor (dtxR) allele and analysis of dtxR transcription in wild-type and mutant strains of Corynebacterium diphtheriae. Infect Immun. 1991 Nov;59(11):3903–3908. doi: 10.1128/iai.59.11.3903-3908.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schmitt M. P., Holmes R. K. Cloning, sequence, and footprint analysis of two promoter/operators from Corynebacterium diphtheriae that are regulated by the diphtheria toxin repressor (DtxR) and iron. J Bacteriol. 1994 Feb;176(4):1141–1149. doi: 10.1128/jb.176.4.1141-1149.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Schmitt M. P., Holmes R. K. Iron-dependent regulation of diphtheria toxin and siderophore expression by the cloned Corynebacterium diphtheriae repressor gene dtxR in C. diphtheriae C7 strains. Infect Immun. 1991 Jun;59(6):1899–1904. doi: 10.1128/iai.59.6.1899-1904.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schmitt M. P., Twiddy E. M., Holmes R. K. Purification and characterization of the diphtheria toxin repressor. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7576–7580. doi: 10.1073/pnas.89.16.7576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schwyn B., Neilands J. B. Universal chemical assay for the detection and determination of siderophores. Anal Biochem. 1987 Jan;160(1):47–56. doi: 10.1016/0003-2697(87)90612-9. [DOI] [PubMed] [Google Scholar]
  22. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tai S. P., Krafft A. E., Nootheti P., Holmes R. K. Coordinate regulation of siderophore and diphtheria toxin production by iron in Corynebacterium diphtheriae. Microb Pathog. 1990 Oct;9(4):267–273. doi: 10.1016/0882-4010(90)90015-i. [DOI] [PubMed] [Google Scholar]
  24. Tao X., Murphy J. R. Binding of the metalloregulatory protein DtxR to the diphtheria tox operator requires a divalent heavy metal ion and protects the palindromic sequence from DNase I digestion. J Biol Chem. 1992 Oct 25;267(30):21761–21764. [PubMed] [Google Scholar]
  25. Tao X., Murphy J. R. Determination of the minimal essential nucleotide sequence for diphtheria tox repressor binding by in vitro affinity selection. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9646–9650. doi: 10.1073/pnas.91.20.9646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tao X., Schiering N., Zeng H. Y., Ringe D., Murphy J. R. Iron, DtxR, and the regulation of diphtheria toxin expression. Mol Microbiol. 1994 Oct;14(2):191–197. doi: 10.1111/j.1365-2958.1994.tb01280.x. [DOI] [PubMed] [Google Scholar]
  27. Wang R. F., Kushner S. R. Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene. 1991 Apr;100:195–199. [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES