Iron-dependent Repressor Research Articles

Attenuation of virulence in Mycobacterium tuberculosis expressing a constitutively active iron repressor.

Iron is an essential nutrient for the survival of most organisms and has played a central role in the virulence of many infectious disease pathogens. Mycobacterial IdeR is an iron-dependent repressor that shows 80% identity in the functional domains with its corynebacterial homologue, DtxR (diphtheria toxin repressor). We have transformed Mycobacterium tuberculosis with a vector expressing an iron-independent, positive dominant, corynebacterial dtxR hyperrepressor, DtxR(E175K). Western blots of whole-cell lysates of M. tuberculosis expressing the dtxR(E175K) gene revealed the stable expression of the mutant protein in mycobacteria. BALB/c mice were infected by tail vein injection with 2 x 10(5) organisms of wild type or M. tuberculosis transformed with the dtxR mutant. At 16 weeks, there was a 1.2 log reduction in bacterial survivors in both spleen (P = 0.0002) and lungs (P = 0.006) with M. tuberculosis DtxR(E175K). A phenotypic difference in colonial morphology between the two strains also was noted. A computerized search of the M. tuberculosis genome for the palindromic consensus sequence to which DtxR and IdeR bind revealed six putative "iron boxes" within 200 bp of an ORF. Using a gel-shift assay we showed that purified DtxR binds to the operator region of five of these boxes. Attenuation of M. tuberculosis can be achieved by the insertion of a plasmid containing a constitutively active, iron-insensitive repressor, DtxR(E175K), which is a homologue of IdeR. Our results strongly suggest that IdeR controls genes essential for virulence in M. tuberculosis.

The battle for iron – crystallographic studies of the iron-dependent repressor proteins fromC. diptheriaandM. tuberculosis

The battle for iron – crystallographic studies of the iron-dependent repressor proteins from<i>C. diptheria</i>and<i>M. tuberculosis</i>

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.

Analysis of the complexity of gene regulation by fur in Vibrio cholerae

Iron concentration influences the expression of a number of genes involved in iron uptake and virulence in bacteria. In Escherichia coli, coordinate regulation of these genes by iron depends on the product of the fur gene, which acts as an iron-responsive, DNA-binding repressor protein. Several genes in Vibrio cholerae are also repressed by iron; and a fur gene, homologous to E. coli fur, has been previously cloned from this organism. The present study was undertaken to define the roles of Fur and iron in regulating gene expression in V. cholerae. V. cholerae strains with a mutation in fur by virtue of suicide plasmid integration into this gene showed derepressed expression of two previously characterized, iron-regulated genes, irgA and viuA, in high concentrations of iron; even in the fur mutants, however, residual two- to threefold regulation by iron persisted. The fur mutant strains constructed by suicide plasmid integration required antibiotic selection to maintain the mutation. To analyze further the effect of Fur and iron on gene regulation in V. cholerae without the need for antibiotic selection, we used in vivo marker exchange to construct a nonrevertible V. cholerae fur mutant. This V. cholerae fur mutant grew significantly less well in Luria-Bertani medium than the wild-type parent but grew slightly better than the wild type under iron-restricted conditions. The V. cholerae fur mutant was unable to utilize a number of carbon sources including glycerol, acetate, succinate, lactate, and fumarate, that supported growth of the wild-type strain on minimal media. We utilized two-dimensional gel electrophoresis of whole-cell protein extracts from the fur mutant and wild-type strains following growth in conditions of either low or high concentrations of iron to identify proteins regulated by iron and/or Fur. Twenty-two proteins were negatively regulated by iron in the wild-type strain but constitutively expressed in the fur mutant, consistent with the model of Fur as an iron-dependent repressor. However, many other proteins were regulated in a different manner by iron and/or Fur. Seventeen proteins were negatively regulated by iron but independent of Fur, suggesting the presence of an additional iron-dependent repressor(s). Six proteins were strongly iron regulated in the fur mutant but hardly expressed at all in the wild-type strain regardless of the iron concentration, suggesting an interaction between Fur and another iron regulatory mechanism. There were 11 proteins that were induced rather than repressed by iron, in four different regulatory classes. Gene regulation in V. cholerae by Fur and iron is much more complex than previously thought and is reminiscent of the Lrp regulon in E.coli.

Iron regulation of the cloned diphtheria toxin promoter in Escherichia coli.

Regulation of the diphtheria toxin promoter by iron was studied in Escherichia coli by using a galK transcriptional fusion. A fragment of the toxin (tox) operon containing the regulatory region was cloned from corynephage beta into a galK transcription vector such that expression of galK activity was controlled by the tox promoter. When E. coli N100 (a galK mutant) harboring this tox-galK fusion plasmid was grown in Luria broth, the specific activity of galactokinase remained constant throughout the exponential phase of growth. When bacteria were shifted from such high-iron medium into low-iron Luria broth, the specific activity of galactokinase increased rapidly, but induction of galactokinase was prevented by the addition of iron to the medium. Measurement of tox-specific mRNA by dot blot hybridization showed that this regulation occurred at the level of transcription. When the plasmid containing the tox-galK fusion was introduced into a fur mutant of E. coli, expression of galK was maximal in both high-iron and low-iron media; but repressibility of galK by iron in this strain was restored by complementation with the fur+ allele. The tox promoter has significant homology with the consensus sequence for other iron-regulated promoters of E. coli that are controlled by fur. These data indicate that the product of the fur gene can function in E. coli as an iron-dependent repressor for the tox promoter from corynephage beta.