A Cu(I)-sensing ArsR family metal sensor protein with a relaxed metal selectivity profile

ArsR (or ArsR/SmtB) family metalloregulatory homodimeric repressors collectively respond to a wide range of metal ion inducers in regulating homeostasis and resistance of essential and nonessential metal ions in bacteria. BxmR from the cyanobacterium Osciliatoria brevis is the first characterized ArsR protein that senses both Cu (I)/Ag (I) and divalent metals Zn (II)/Cd (II) in cells by regulating the expression of a P-type ATPase efflux pump (Bxa1) and an intracellular metallothionein (BmtA). We show here that both pairs of predicted alpha3N and alpha5 sites bind metal ions, but with distinct physicochemical and functional metal specificities. Inactivation of the thiophilic alpha3N site via mutation (C77S) abolishes regulation by both Cd (II) and Cu (I), while Zn (II) remains a potent allosteric negative effector of operator/promoter binding (Delta G c >or= +3.2 kcal mol (-1)). In contrast, alpha5 site mutant retains regulation by all four metal ions, albeit with a smaller coupling free energy (Delta G c approximately +1.7 (+/-0.1) kcal mol (-1)). Unlike the other metals ions, the BxmR dimer binds 4 mol equiv of Cu (I) to form an alpha3N binuclear Cu (I) 2S 4 cluster by X-ray absorption spectroscopy. BxmR is thus distinguishable from other closely related ArsR family sensors, in having evolved a metalloregulatory alpha3N site that can adopt an expanded range of coordination chemistries while maintaining redundancy in the response to Zn (II). The evolutionary implications of these findings for the ArsR metal sensor family are discussed.     

A zinc(II)/lead(II)/cadmium(II)-inducible operon from the Cyanobacterium anabaena is regulated by AztR, an alpha3N ArsR/SmtB metalloregulator

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).     

The chromosomally encoded cation diffusion facilitator proteins DmeF and FieF from Wautersia metallidurans CH34 are transporters of broad metal specificity

Genomic sequencing of the beta-proteobacterium Wautersia (previously Ralstonia) metallidurans CH34 revealed the presence of three genes encoding proteins of the cation diffusion facilitator (CDF) family. One, CzcD, was previously found to be part of the high-level metal resistance system Czc that mediates the efflux of Co(II), Zn(II), and Cd(II) ions catalyzed by the CzcCBA cation-proton antiporter. The second CDF protein, FieF, is probably mainly a ferrous iron detoxifying protein but also mediated some resistance against other divalent metal cations such as Zn(II), Co(II), Cd(II), and Ni(II) in W. metallidurans or Escherichia coli. The third CDF protein, DmeF, showed the same substrate spectrum as FieF, but with different preferences. DmeF plays the central role in cobalt homeostasis in W. metallidurans, and a disruption of dmeF rendered the high-level metal cation resistance systems Czc and Cnr ineffective against Co(II). This is evidence for the periplasmic detoxification of substrates by RND transporters of the heavy metal efflux family subgroup.     

A novel cyanobacterial SmtB/ArsR family repressor regulates the expression of a CPx-ATPase and a metallothionein in response to both Cu(I)/Ag(I) and Zn(II)/Cd(II)

A novel SmtB/ArsR family metalloregulator, denoted BxmR, has been identified and characterized from the cyanobacterium Oscillatoria brevis. Genetic and biochemical evidence reveals that BxmR represses the expression of both bxa1, encoding a CPx-ATPase metal transporter, as well as a divergently transcribed operon encoding bxmR and bmtA, a heavy metal sequestering metallothionein. Derepression of the expression of all three genes is mediated by both monovalent (Ag(I) and Cu(I)) and divalent (Zn(II) and Cd(II)) heavy metal ions, a novel property among SmtB/ArsR metal sensors. Electrophoretic gel mobility shift experiments reveal that apoBxmR forms multiple resolvable complexes with oligonucleotides containing a single 12-2-12 inverted repeat derived from one of the two operator/promoter regions with similar apparent affinities. Preincubation with either monovalent or divalent metal ions induces disassembly of both the BxmR-bxa1 and BxmR-bxmR/bmtA operator/promoter complexes. Interestingly, the temporal regulation of expression of bxa1 and bmtA mRNAs is different in O. brevis with bxa1 induced first upon heavy metal treatment, followed by bmtA/bxmR. A dynamic interplay among Bxa1, BmtA, and BxmR is proposed that maintains metal homeostasis in O. brevis by balancing the relative rates of metal storage and efflux of multiple heavy metal ions.     

The metal specificity and selectivity of ZntA from Escherichia coli using the acylphosphate intermediate

ZntA from Escherichia coli is a P-type ATPase that confers resistance to Pb(II), Zn(II), and Cd(II) in vivo. We had previously shown that purified ZntA shows ATP hydrolysis activity with the metal ions Pb(II), Zn(II), and Cd(II). In this study, we utilized the acylphosphate formation activity of ZntA to further investigate the substrate specificity of ZntA. The site of phosphorylation was Asp-436, as expected from sequence alignments. We show that in addition to Pb(II), Zn(II), and Cd(II), ZntA is active with Ni(II), Co(II), and Cu(II), but not with Cu(I) and Ag(I). Thus, ZntA is specific for a broad range of divalent soft metal ions. The activities with Ni(II), Co(II), and Cu(II) are extremely low; the activities with these non-physiological substrates are 10-20-fold lower compared with the values obtained with Pb(II), Zn(II), and Cd(II). Similar results were obtained with DeltaN-ZntA, a ZntA derivative lacking the amino-terminal metal binding domain. By characterizing the acylphosphate formation reaction in ZntA in detail, we show that a step prior to enzyme phosphorylation, most likely the metal ion binding step, is the slow step in the reaction mechanism in ZntA. The low activities with Ni(II), Co(II), and Cu(II) are because of a further decrease in the rate of binding of these metal ions. Thus, metal ion selectivity in ZntA and possibly other P1-type ATPases is based on the charge and the ligand preference of particular metal ions but not on their size.     

Synthesis, spectroscopic, structural and complexation studies of a new tetra-naphthylmethylene pendant-armed macrocyclic ligand

A novel emissive tetra-naphthylmethylene pendant-armed macrocyclic ligand and a series of complexes with monovalent and divalent metal ions have been synthesized. Solid compounds have been isolated as mononuclear (Co(II), Cu(II) and Zn(II)) or dinuclear (Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Ag(I)), complexes, depending on the counterions used. The chemical and photophysical properties of the free ligand, the protonation behavior and its metal complexes have been investigated in solution. UV-Vis spectroscopy has revealed a 1:1 binding stoichiometry for Cu(II), Zn(II), Cd(II), Ni(II) and Co(II), and 2:1 molar ratio for Ag(I). In chloroform, the free ligand presents two emission bands related to the monomer naphthalene emission and a red-shifted band attibutable to an exciplex due to a charge transfer from the nitrogen lone electron pair to the excited chromophore. Upon protonation of the free amines or due to metal complexation, the exciplex band disappears. The crystal structure of [Ag₂L(NO₃)₂] is also reported. The structure reveals that both metal ions are into the macrocyclic cavity in a distorted square plane {AgN₃O} environment. Each Ag(I) atom interacts with two neighbouring amine nitrogen atoms, one pyridine nitrogen and one oxygen atom from a monodentate nitrate ion.     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.     

Resistance to cadmium, cobalt, zinc, and nickel in microbes.

The divalent cations of cobalt, zinc, and nickel are essential nutrients for bacteria, required as trace elements at nanomolar concentrations. However, at micro- or millimolar concentrations, Co2+, Zn2+, and Ni2+ (and "bad ions" without nutritional roles such as Cd2+) are toxic. These cations are transported into the cell by constitutively expressed divalent cation uptake systems of broad specificity, i.e., basically Mg2+ transport systems. Therefore, in case of a heavy metal stress, uptake of the toxic ions cannot be reduced by a simple down-regulation of the transport activity. As a response to the resulting metal toxicity, metal resistance determinants evolved which are mostly plasmid-encoded in bacteria. In contrast to that of the cation Hg2+, chemical reduction of Co2+, Zn2+, Ni2+, and Cd2+ by the cell is not possible or sensible. Therefore, other than mutations limiting the ion range of the uptake system, only two basic mechanisms of resistance to these ions are possible (and were developed by evolution): intracellular complexation of the toxic metal ion is mainly used in eucaryotes; the cadmium-binding components are phytochelatins in plant and yeast cells and metallothioneins in animals, plants, and yeasts. In contrast, reduced accumulation based on an active efflux of the cation is the primary mechanism developed in procaryotes and perhaps in Saccharomyces cerevisiae. All bacterial cation efflux systems characterized to date are plasmid-encoded and inducible but differ in energy-coupling and in the number and types of proteins involved in metal transport and in regulation. In the gram-positive multiple-metal-resistant bacterium Staphylococcus aureus, Cd2+ (and probably Zn2+) efflux is catalyzed by the membrane-bound CadA protein, a P-type ATPase. However, a second protein (CadC) is required for full resistance and a third one (CadR) is hypothesized for regulation of the resistance determinant. The czc determinant from the gram-negative multiple-metal-resistant bacterium Alcaligenes eutrophus encodes proteins required for Co2+, Zn2+, and Cd2+ efflux (CzcA, CzcB, and CzcC) and regulation of the czc determinant (CzcD). In the current working model CzcA works as a cation-proton antiporter, CzcB as a cation-binding subunit, and CzcC as a modifier protein required to change the substrate specificity of the system from Zn2+ only to Co2+, Zn2+, and Cd2+.     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.     

Transport and detoxification systems for transition metals,heavy metals and metalloids in eukaryotic and prokaryotic microbes

Transition metals, heavy metals and metalloids are usually toxic in excess, but a number of transition metals are essential trace elements. In all cells there are mechanisms for metal ion homeostasis that frequently involve a balance between uptake and efflux systems. This review will briefly describe ATP-coupled resistance pumps. ZntA and CadA are bacterial P-type ATPases that confers resistance to Zn(II), Cd(II) and Pb(II). Homologous copper pumps include the Menkes and Wilson disease proteins and CopA, an Escherichia coli pump that confers resistance to Cu(I). For resistance to arsenicals and antimonials there are several different families of transporters. In E. coli the ArsAB ATPase is a novel system that confers resistance to As(III) and Sb(III). Eukaryotic arsenic resistance transporters include Acr3p and Ycf1p of Saccharomyces cerevisiae. These systems provide resistance to arsenite [As(III)]. Arsenate [As(V)] detoxification involves reduction of As(V) to As(III), a process catalyzed by arsenate reductase enzymes. There are three families of arsenate reductases, two found in bacterial systems and a third identified in S. cerevisiae.     

Interplay of different transporters in the mediation of divalent heavy metal resistance in Pseudomonas putida KT2440

According to in silico analysis, the genome of Pseudomonas putida KT2440 encodes at least four Zn/Cd/Pb efflux transporters-two P-type ATPases (CadA1 and CadA2) and two czc chemiosmotic transporters (CzcCBA1 and CzcCBA2). In this study we showed that all these transporters are functional, but under laboratory conditions only two of them were involved in the mediation of heavy metal resistance in P. putida KT2440. CadA2 conferred Cd(2+) and Pb(2+) resistance, whereas CzcCBA1 was involved in export of Zn(2+), Cd(2+), and possibly Pb(2+). CadA1, although nonfunctional in P. putida, improved Zn(2+) resistance and slightly improved Cd(2+) resistance when it was expressed in Escherichia coli. CzcCBA2 contributed to Zn resistance of a czcA1-defective P. putida strain or when the CzcA2 subunit was overexpressed in a transporter-deficient strain. It seemed that CzcA2 could complex with CzcC1 and CzcB1 subunits and therefore complement the loss of CzcA1. The CzcCBA2 transporter itself, however, did not function. Expression of cadA1, cadA2, and czcCBA1 was induced by heavy metals, and the expression levels were dependent on the growth medium and growth phase. Expression of cadA2 and czcCBA1 was nonspecific; both genes were induced by Zn(2+), Cd(2+), Pb(2+), Ni(2+), Co(2+), and Hg(2+). On the other hand, remarkably, expression of cadA1 was induced only by Zn(2+). Possible roles of distinct but simultaneously functioning transporters are discussed.     

A bacterial view of the periodic table: genes and proteins for toxic inorganic ions

Essentially all bacteria have genes for toxic metal ion resistances and these include those for Ag+, AsO2-, AsO4(3-), Cd2+ Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, TeO3(2-), Tl+ and Zn2+. The largest group of resistance systems functions by energy-dependent efflux of toxic ions. Fewer involve enzymatic transformations (oxidation, reduction, methylation, and demethylation) or metal-binding proteins (for example, metallothionein SmtA, chaperone CopZ and periplasmic silver binding protein SilE). Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. For example, Cd2+-efflux pumps of bacteria are either inner membrane P-type ATPases or three polypeptide RND chemiosmotic complexes consisting of an inner membrane pump, a periplasmic-bridging protein and an outer membrane channel. In addition to the best studied three-polypeptide chemiosmotic system, Czc (Cd2+, Zn2+, and Co2), others are known that efflux Ag+, Cu+, Ni2+, and Zn2+. Resistance to inorganic mercury, Hg2+ (and to organomercurials, such as CH3Hg+ and phenylmercury) involve a series of metal-binding and membrane transport proteins as well as the enzymes mercuric reductase and organomercurial lyase, which overall convert more toxic to less toxic forms. Arsenic resistance and metabolizing systems occur in three patterns, the widely-found ars operon that is present in most bacterial genomes and many plasmids, the more recently recognized arr genes for the periplasmic arsenate reductase that functions in anaerobic respiration as a terminal electron acceptor, and the aso genes for the periplasmic arsenite oxidase that functions as an initial electron donor in aerobic resistance to arsenite.