The Zinc-Responsive Regulator Zur Controls a Zinc Uptake System and Some Ribosomal Proteins inStreptomyces coelicolorA3(2)

Abstract

In various bacteria, Zur, a zinc-specific regulator of the Fur family, regulates genes for zinc transport systems to maintain zinc homeostasis. It has also been suggested that Zur controls zinc mobilization by regulating some ribosomal proteins. The antibiotic-producing soil bacterium Streptomyces coelicolor contains four genes for Fur family regulators, and one (named zur) is located downstream of the znuACB operon encoding a putative zinc uptake transporter. We found that zinc specifically repressed the level of znuA transcripts and that this level was derepressed in a Δzur mutant. Purified Zur existing as homodimers bound to the znuA promoter region in the presence of zinc, confirming the role of Zur as a zinc-responsive repressor. We analyzed transcripts for paralogous forms of ribosomal proteins L31 (RpmE1 and RpmE2) and L33 (RpmG2 and RpmG3) for their dependence on Zur and found that RpmE2 and RpmG2 with no zinc-binding motif of conserved cysteines (C's) were negatively regulated by Zur. C-negative RpmG3 and C-positive RpmE1 were not regulated by Zur. Instead, they were regulated by the sigma factor σR as predicted from their promoter sequences. The rpmE1 and rpmG3 genes were partially induced by EDTA in a manner dependent on σR, suggesting that zinc depletion may stimulate the σR regulatory system. This finding reflects a link between thiol-oxidizing stress and zinc depletion. We determined the Zur-binding sites within znuA and rpmG2 promoter regions by footprinting analyses and identified a consensus inverted repeat sequence (TGaaAatgatTttCA, where uppercase letters represent the nucleotides common to all sites analyzed). This sequence closely matches that for mycobacterial Zur and allows the prediction of more genes in the Zur regulon.

Graded expression of zinc-responsive genes through two regulatory zinc-binding sites in Zur

“…The binding reaction was carried out by incubating ∼3.07 fmol of labeled DNA with varying amounts (6.8 fmol ∼27.84 pmol) of purified Zur proteins as isolated in 20 μL of binding reaction buffer [20 mM Tris-HCl (pH 7.8), 50 mM KCl, 1 mM DTT, 0.1 mg of BSA/mL, 5% glycerol, 0.1 μg of poly (dI-dC)] for 1 h at room temperature. For zinc-depletion experiments, further incubation with TPEN (up to 25 μM, 30 min) was carried out, followed by gel electrophoresis (12). For zinc-addition experiments, DNA probes were preincubated in binding reaction buffer with added ZnSO 4 (up to 5 μM) and 5 μM TPEN for 30 min before adding EDTA-treated apo-Zur (170 nM), followed by further incubation (1 h) and electrophoresis.…”

“…Wild-type, C79S, C90S, and H84A mutant Zur proteins were purified from E. coli BL21 (DE3) cells containing pET3a-based recombinant plasmid, as previously described, with some modifications (12). EDTA was omitted in the buffer, except for preparing apo-Zur (SI Appendix).…”

“…RNA samples were prepared from wild-type and Δzur cells harboring chromosomally integrated copies of wild-type and mutated zur genes. Preparation of RNA, PCR amplification of gene-specific probes, radiolabeling of 5′ ends by 32 P, and S1-mapping analysis were done as described previously (12) and in the SI Appendix. Quantification of S1-protected bands was done through phosphor screen and image analyzer (FLA-2000; Fuji).…”

“…In many bacteria, zinc depletion causes inactivation of zinc-specific Zur to derepress zinc transporter genes (6). In Gram-positive bacteria, genes for zincless ribosomal proteins are derepressed to replace zinc-bound paralogs as a way of mobilizing zinc (7)(8)(9)(10)(11)(12). Coelibactin, a putative zincophore, has been recently reported to be regulated by Zur in Streptomyces coelicolor (13,14).…”

The plant pathogen Streptomyces scabies 87-22 has a functional pyochelin biosynthetic pathway that is regulated by TetR- and AfsR-family proteins

“…Iron metabolism in Gram-negative organisms and firmicutes is controlled by Fur (Hantke, 2001). Streptomycetes possess four Fur-family proteins, all of which have been characterized in the model streptomycete S. coelicolor, including CatR (SCO5206), Nur (SCO4180), Zur (SCO2508) and FurA (SCO0561); FurA has also been characterized in Streptomyces reticuli and is called FurS (Ahn et al, 2006;Hahn et al, 2000a, b;Ortiz de Orué Lucana & Schrempf, 2000;Shin et al, 2007;Zou et al, 1999). S. scabies contains four genes (SCAB30481, SCAB49681, SCAB61951 and SCAB9521) encoding Fur proteins orthologous to those of S. coelicolor and S. reticuli.…”