Figure 1: The abundance of Zur and its genome-wide binding in S. coelicolor.

(a) Analytical western blot analysis of Zur. Exponentially grown S. coelicolor M145 cells were either untreated or treated with varying concentrations of chelator TPEN (5.9, 5.7, 5.5 and 5.0 μM) or 100 μM ZnSO₄ for 1 h before cell harvest. Crude cell extracts were analysed by western analysis, in parallel with quantified amount of purified Zur (1, 2 and 4 ng), using polyclonal antibodies against Zur. The amount of Zur in each loaded sample was estimated in ng, taking the band intensity of 1 ng purified Zur as 1.0. Average values with s.d.’s from three independent experimental samples were presented. (b) Zur-binding peaks throughout the whole genome from ChIP-chip analysis. The peak intensity values (y axis) were calculated from the average of the log₂ ratios of 10 highest consecutive probe signals for each Zur-enriched site. Known promoter sites of Zur-repressed genes were indicated with red arrows. A new promoter site with Zur-binding consensus sequence was indicated with a blue arrow. (c) The Zur binding motif was extracted from the highly enriched 172 Zur-binding regions by multiple EM for motif elicitation (MEME), with E-value of 3.9e-233. (d) The zinc-specific and Zur-dependent induction of the zitB gene. Transcripts from SCO6751 (zitB) gene were analysed by S1 mapping. Exponentially grown wild type (WT) and Δzur mutant cells were treated with 6 μM TPEN or various metal salts (ZnSO₄, CdCl₂, CoSO₄, FeSO₄, MnCl₂, NiSO₄ and CuSO₄) at 100 μM for 30 min before cell harvest. The amount of zitB transcript was quantified and presented in relative value with that in non-treated sample as 1.0. Values from three independent experiments were presented as average with s.d.’s. The P values for all the measurements in TPEN and zinc treatment to WT and TPEN treatment to Δzur mutant were <0.001 by Student’s t-test.