A zinc(II)/lead(II)/cadmium(II)-inducible operon from the Cyanobacterium anabaena is regulated by AztR, an alpha3N ArsR/SmtB metalloregulator.
ABSTRACT A novel Zn(II)/Pb(II)/Cd(II)-responsive operon that consists of genes encoding a Zn(II)/Pb(II) CPx-ATPase efflux pump (aztA) and a Zn(II)/Cd(II)/Pb(II)-specific SmtB/ArsR family repressor (aztR) has been identified and characterized from the cyanobacterium Anabaena PCC 7120. In vivo real time quantitative RT-PCR assays reveal that both aztR and aztA expression are induced by divalent metal ions Zn(II), Cd(II), and Pb(II) but not by other divalent [Co(II), Ni(II)] or monovalent metal ions [Cu(I) and Ag(I)]. The introduction of a plasmid containing the azt operon into a Zn(II)/Cd(II)-hypersensitive Escherichia coli strain GG48 functionally restores Zn(II) and Pb(II) resistance with a limited effect on Cd(II) resistance. Gel mobility shift assays and aztR O/P-lacZ induction experiments confirm that AztR is the metal-regulated repressor of this operon. In vitro biochemical and mutagenesis studies indicate that AztR contains a sole metal-binding site, designated the alpha3N site, that binds Zn(II), Cd(II), and Pb(II) with a high affinity. Optical absorption spectra of Co(II)- and Cd(II)-substituted AztR and (113)Cd NMR spectroscopy of (113)Cd(II)-substituted AztR reveal that the sole alpha3N site in AztR is a CadC-like distorted tetrahedral S(3)(N,O) metal site. The first metal-coordination shell in the AztR alpha3N site differs from other alpha3N family members that sense Cd(II)/Pb(II) and those alpha5 repressors that sense Zn(II)/Co(II). Our results reveal that the alpha3N site in AztR mediates derepression of the azt operon in the presence of Zn(II), as well as Cd(II) and Pb(II); this might have provided Anabaena with an evolutionary advantage to adapt to heavy-metal-rich environments, while maintaining homeostasis of an essential metal ion, Zn(II).
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ABSTRACT: Cyanobacteria represent the largest and most diverse group of prokaryotes capable of performing oxygenic photosynthesis and are frequently found in environments contaminated with heavy metals. Several studies have been performed in these organisms in order to better understand the effects of metals such as Zn, Cd, Cu, Ni and Co. In Synechocystis sp. PCC 6803, genes involved in Ni, Co, Cu and Zn resistance have been reported. However, proteomic studies for the identification of proteins modulated by heavy metals have not been carried out. In the present work, we have analyzed the proteomic pattern alterations of the cyanobacterium Synechocystis sp. PCC 6803 in response to Ni, Co and Cd in order to identify the metabolic processes affected by these metals. We show that some proteins are commonly regulated in response to the different metal ions, including ribulose1,5-bisphosphate carboxylase and the periplasmic iron-binding protein FutA2, while others, such as chaperones, were specifically induced by each metal. We also show that the main processes affected by the metals are carbon metabolism and photosynthesis, since heavy metals affect proteins required for the correct functioning of these activities. Biological Significance This is the first report on the proteomic profile of Synechocystis sp. PCC 6803 wild type and mutant strains for the identification of proteins affected by the heavy metals Ni, Co and Cd. We have identified proteins commonly responsive to all three metals and also chaperones specifically modulated by each metal. Our data also supports previous studies that suggest the existence of additional sensor systems for Co.Journal of proteomics 03/2014; · 5.07 Impact Factor
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ABSTRACT: Photosynthetic cyanobacteria are sensitive to toxicity of metal cadmium (Cd(2+)). Although metabolic responses against Cd(2+) exposure have been described, the related regulatory mechanism is still unclear in cyanobacteria. In this study, we identified in Synechocystis sp. PCC 6803 a response regulator (RR)-encoding gene sll0649, whose mutant was more sensitive to Cd(2+) stress. Further phenotypic analysis revealed that ∆sll0649 become more sensitive to Cu(2+), Fe(2+), Mn(2+) and Zn(2+) stress as well. Using a quantitative iTRAQ-LC-MS/MS proteomics approach, we showed that a total of 156 and 151 unique proteins were down- and up-regulated for at least 2 folds in the ∆sll0649 mutant grown under Cd(2+) stress, respectively. In addition, electrophoretic mobility shift assays showed that Sll0649 was able to bind directly to the upstream regions of sll1598 and slr0798, which encode an Mn(2+) transporter MntC and a Zn(2+) transporting P-type ATPases ZiaA, respectively, suggesting that Sll0649 was involved in Cd(2+) tolerance by regulating and maintaining intracellular metal homeostasis. The involvement of sll1598 and slr0798 genes in Cd(2+) tolerance was also verified by comparative mutant analyses. The study provided a proteomic description of the Cd(2+) response network mediated by the response regulator Sll0649, and revealed novel insights on the metal-tolerance mechanism in Synechocystis. Biological significance As a major pollutant on earth, Cd(2+) is toxic to both prokaryotic and eukaryotic organisms. It is thus important to obtain a better understanding of cellular response to Cd(2+) and the related regulatory mechanism. In this study, by screening 44 gene knockout mutants of putative RR-encoding genes in Synechocystis for their sensitivity change to Cd(2+) stress, we identified the orphan RR, Slr0649, involved in Cd(2+) tolerance in Synechocystis. The ∆sll0649 mutant was also found to be more sensitive to high-concentration Cu(2+), Fe(2+), Mn(2+) and Zn(2+), when compared with the wild type. Using an iTRAQ-LC-MSMS based quantitative proteomic analysis coupled with EMSAs, we found that, in addition to its positive regulation on genes directly related to Cd(2+) utilization, Sll0649 can also functions as a key positive regulator either directly or indirectly on expression of multiple genes related to transporting and utilization of several other metal ions. The study provided a proteomic description of the Cd(2+) response network mediated by the response regulator Sll0649, and revealed novel insights on the metal-tolerance mechanism in Synechocystis.Journal of proteomics 04/2014; · 5.07 Impact Factor
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ABSTRACT: A structural investigation of complexes formed between the Pb(2+) ion and glutathione (GSH, denoted AH(3) in its triprotonated form), the most abundant nonprotein thiol in biological systems, was carried out for a series of aqueous solutions at pH 8.5 and C(Pb(2+)) = 10 mM and in the solid state. The Pb L(III)-edge extended X-ray absorption fine structure (EXAFS) oscillation for a solid compound with the empirical formula [Pb(AH(2))]ClO(4) was modeled with one Pb-S and two short Pb-O bond distances at 2.64 ± 0.04 and 2.28 ± 0.04 Å, respectively. In addition, Pb···Pb interactions at 4.15 ± 0.05 Å indicate dimeric species in a network where the thiolate group forms an asymmetrical bridge between two Pb(2+) ions. In aqueous solution at the mole ratio GSH/Pb(II) = 2.0 (C(Pb(2+)) = 10 mM, pH 8.5), lead(II) complexes with two thiolate ligands form, characterized by a ligand-to-metal charge-transfer band (LMCT) S(-) → Pb(2+) at 317 nm in the UV-vis spectrum and mean Pb-S and Pb-(N/O) bond distances of 2.65 ± 0.04 and 2.51 ± 0.04 Å, respectively, from a Pb L(III)-edge EXAFS spectrum. For solutions with higher mole ratios, GSH/Pb(II) ≥ 3.0, electrospray ionization mass spectroscopy spectra identified a triglutathionyllead(II) complex, for which Pb L(III)-edge EXAFS spectroscopy shows a mean Pb-S distance of 2.65 ± 0.04 Å in PbS(3) coordination, (207)Pb NMR spectroscopy displays a chemical shift of 2793 ppm, and in the UV-vis spectrum, an S(-) → Pb(2+) LMCT band appears at 335 nm. The complex persists at high excess of GSH and also at ∼25 K in frozen glycerol (33%)/water glasses for GSH/Pb(II) mole ratios from 4.0 to 10 (C(Pb(2+)) = 10 mM) measured by Pb L(III)-edge EXAFS spectroscopy.Inorganic Chemistry 05/2012; 51(11):6285-98. · 4.59 Impact Factor