Article Figures & Data
Figures
- FIG. 1.
Iron-dependent expression of the blr7895 and bll6680 genes in parent strain LO (Wt) or irr mutant strain LODTM5 (irr). mRNAs from cells grown in medium supplemented with no added iron (L) or with 12 μM FeCl3 (H) were analyzed by quantitative real-time PCR. The data are expressed as the relative starting quantity (SQ) of the respective mRNAs normalized to the housekeeping gene gapA. The data are expressed as the average of the results of three replicates ± the standard deviation. Error bars indicate the standard errors of the mean.
- FIG. 2.
Identification of Irr as a component in extracts of wild-type cells grown under iron limitation that binds to ICE DNA. (A) Extracts prepared from cells of parent strain LO (wt) or irr strain LODTM5 (irr) grown in medium supplemented with no iron (lanes L) or with 12 μM FeCl3 (lanes H) were incubated with 0.1 nM radiolabeled ICE DNA from blr7895 or bll6680. EMSA was carried out with free DNA probe (F) or DNA with 0.1 μg extract protein. Complexes were resolved on 6% nondenaturing gels and visualized by autoradiography. (B) Electrophoretic mobility supershift assays were carried out using free DNA probe (lanes F), 5 μg extract protein from wild-type cells grown in low-iron medium (lanes Ex), and extract plus anti-Irr antibodies at 1:100 [lanes Ab(H)] or 1:1,000 [lanes Ab(L)] dilutions in the binding reaction mixtures. The complexes were resolved and visualized as described for panel A. SS denotes the supershifted species in the presence of antibody.
- FIG. 3.
Binding of purified recombinant Irr to ICE DNA in vitro. (A) EMSA was carried out using ICE DNA corresponding to the blr7895 or bll6680 promoters. Binding reactions were carried out using 0.2 nM radiolabeled ICE DNA either without protein (lanes F) or with 200 nM Irr (lanes I). In addition, the reaction, running, and gel buffers were supplemented with 100 μM MnCl2, denoted as +Mn in the right panel. The complexes were resolved and visualized as described in the legend to Fig. 2. (B) Determination of Irr affinity for ICE DNA. EMSA was carried out using 0.1 nM radiolabeled ICE7895 or ICE6680 DNA with various concentrations of purified Irr. The signal intensities of bound and unbound DNA on the autoradiograms were quantified and are expressed as percent bound DNA. (C) Comparison of the mobilities of ICE complexes in purified Irr and in extracts of wild-type cells grown under iron limitation. EMSA was carried out as described in the legend to Fig. 2. Lanes: F, free DNA probe; Ex, probe with 5 μg protein extract; Ab, probe with extract plus 1:1,000 dilution of anti-Irr antibody; Irr, purified Irr.
- FIG. 4.
Inhibition of in vitro transcription by Irr. (A) A linear 241-bp DNA fragment that includes the blr7895 promoter region and is 167 bp downstream of the transcription start site was used as a template. The bent arrow denotes the transcription start site, and the white rectangle represents the ICE. (B) Transcription was carried out using a 4 nM DNA template and 0.5 μg purified B. japonicum RNA polymerase holoenzyme in the absence (−) or presence (+) of 200 nM Irr. The radiolabeled RNA products were separated by electrophoresis and visualized by autoradiography.
- FIG. 5.
Detection of Irr at the blr7895 and bll6680 gene promoters in vivo by cross-linking IP. Cross-linking of parent strain LO (Wt) (A) or irr strain LODTM5 (irr) (B) cells grown in low- or high-iron medium followed by cell breakage and co-IP was carried out as described in Materials and Methods. The primers used amplify the promoter region of blr7895, bll6680, and gapA. Lanes: in, input DNA; m, mock IP in the absence of antibody; ab, IP using anti-Irr antibodies. gapA is a control for a gene not regulated by Irr.
