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: Staphylococcus aureus CzrA is a zinc-dependent transcriptional repressor from the ubiquitous ArsR family of metal sensor proteins. Zn(II) binds to a pair of intersubunit C-terminal alpha5-sensing sites, some 15 A distant from the DNA-binding interface, and allosterically inhibits DNA binding. This regulation is characterized by a large allosteric coupling free energy (DeltaGc) of approximately +6 kcal mol(-1), the molecular origin of which is poorly understood. Here, we report the solution quaternary structure of homodimeric CzrA bound to a palindromic 28-bp czr operator, a structure that provides an opportunity to compare the two allosteric "end" states of an ArsR family sensor. Zn(II) binding drives a quaternary structural switch from a "closed" DNA-binding state to a low affinity "open" conformation as a result of a dramatic change in the relative orientations of the winged helical DNA binding domains within the dimer. Zn(II) binding also effectively quenches both rapid and intermediate timescale internal motions of apo-CzrA while stabilizing the native state ensemble. In contrast, DNA binding significantly enhances protein motions in the allosteric sites and reduces the stability of the alpha5 helices as measured by H-D solvent exchange. This study reveals how changes in the global structure and dynamics drive a long-range allosteric response in a large subfamily of bacterial metal sensor proteins, and provides insights on how other structural classes of ArsR sensor proteins may be regulated by metal binding.Proceedings of the National Academy of Sciences 10/2009; 106(43):18177-82. · 9.68 Impact Factor
Article: Oxidation of a single active site suffices for the functional inactivation of the dimeric Bacillus subtilis OhrR repressor in vitro.[show abstract] [hide abstract]
ABSTRACT: Bacillus subtilis OhrR is a dimeric repressor that senses organic peroxides and regulates the expression of the OhrA peroxiredoxin. Derepression results from oxidation of an active site cysteine which ultimately results in formation of a mixed disulfide with a low molecular weight thiol, a cyclic sulfenamide, or overoxidation to the sulfinic or sulfonic acids. We expressed a single-chain OhrR (scOhrR) in which the two monomers were connected by a short amino-acid linker. scOhrR variants containing only one active site cysteine were fully functional as repressors and still responded, albeit with reduced efficacy, to organic peroxides in vivo. Biochemical analyses indicate that oxidation at a single active site is sufficient for derepression regardless of the fate of the active site cysteine. scOhrR with only one active site cysteine in the amino-terminal domain is inactivated at rates comparable to wild-type whereas when the active site is in the carboxyl-terminal domain the protein is inactivated much more slowly. The incomplete derepression noted for single active site variants of scOhrR in vivo is consistent with the hypothesis that protein reduction regenerates active repressor and that, in the cell, oxidation of the second active site may also contribute to derepression.Nucleic Acids Research 02/2009; 37(4):1174-81. · 8.03 Impact Factor
Article: Genome-wide transcriptional response of the archaeon Thermococcus gammatolerans to cadmium.[show abstract] [hide abstract]
ABSTRACT: Thermococcus gammatolerans, the most radioresistant archaeon known to date, is an anaerobic and hyperthermophilic sulfur-reducing organism living in deep-sea hydrothermal vents. Knowledge of mechanisms underlying archaeal metal tolerance in such metal-rich ecosystem is still poorly documented. We showed that T. gammatolerans exhibits high resistance to cadmium (Cd), cobalt (Co) and zinc (Zn), a weaker tolerance to nickel (Ni), copper (Cu) and arsenate (AsO(4)) and that cells exposed to 1 mM Cd exhibit a cellular Cd concentration of 67 µM. A time-dependent transcriptomic analysis using microarrays was performed at a non-toxic (100 µM) and a toxic (1 mM) Cd dose. The reliability of microarray data was strengthened by real time RT-PCR validations. Altogether, 114 Cd responsive genes were revealed and a substantial subset of genes is related to metal homeostasis, drug detoxification, re-oxidization of cofactors and ATP production. This first genome-wide expression profiling study of archaeal cells challenged with Cd showed that T. gammatolerans withstands induced stress through pathways observed in both prokaryotes and eukaryotes but also through new and original strategies. T. gammatolerans cells challenged with 1 mM Cd basically promote: 1) the induction of several transporter/permease encoding genes, probably to detoxify the cell; 2) the upregulation of Fe transporters encoding genes to likely compensate Cd damages in iron-containing proteins; 3) the induction of membrane-bound hydrogenase (Mbh) and membrane-bound hydrogenlyase (Mhy2) subunits encoding genes involved in recycling reduced cofactors and/or in proton translocation for energy production. By contrast to other organisms, redox homeostasis genes appear constitutively expressed and only a few genes encoding DNA repair proteins are regulated. We compared the expression of 27 Cd responsive genes in other stress conditions (Zn, Ni, heat shock, γ-rays), and showed that the Cd transcriptional pattern is comparable to other metal stress transcriptional responses (Cd, Zn, Ni) but not to a general stress response.PLoS ONE 01/2012; 7(7):e41935. · 4.09 Impact Factor