Spectroscopic properties of the metalloregulatory Cd(II) and Pb(II) sites of S. aureus pI258 CadC

Staphylococcus aureus pI258 CadC is an extrachromosomally encoded metalloregulatory repressor protein from the ArsR superfamily which negatively regulates the expression of the cad operon in a metal-dependent fashion. The metalloregulatory hypothesis holds that direct binding of thiophilic divalent cations including Cd(II), Pb(II), and Zn(II) by CadC allosterically regulates the DNA binding activity of CadC to the cad operator/promoter (O/P). This report presents a detailed characterization of the metal binding and DNA binding properties of wild-type CadC. The results of analytical ultracentrifugation experiments suggest that both apo- and Cd(1)-CadC are stable or weakly dissociable homodimers characterized by a K(dimer) = 3.0 x 10(6) M(-1) (pH 7.0, 0.20 M NaCl, 25.0 degrees C) with little detectable effect of Cd(II) on the dimerization equilibrium. As determined by optical spectroscopy, the stoichiometry of Cd(II) and Pb(II) binding is approximately 0.7-0.8 mol/mol of wild-type CadC monomer. Chelator (EDTA) competition binding isotherms reveal that Cd(II) binds very tightly, with K(Cd) = 4.3 (+/-1.8) x 10(12) M(-1). The results of UV-Vis and X-ray absorption spectroscopy of the Cd(1) complex are consistent with a tetrathiolate (S(4)) complex formed by four cysteine ligands. The (113)Cd NMR spectrum reveals a single resonance of delta = 622 ppm, consistent with an S(3)(N,O) or unusual upfield-shifted S(4) complex. The Pb(II) complex reveals two prominent absorption bands at 350 nm (epsilon = 4000 M(-1) cm(-1)) and 250 nm (epsilon = 41 000 M(-1) cm(-1)), spectral properties consistent with three or four thiolate ligands to the Pb(II) ion. The change in the anisotropy of a fluorescein-labeled oligonucleotide containing the cad O/P upon binding CadC and analyzed using a dissociable CadC dimer binding model reveals that apo-CadC forms a high-affinity complex [K(a) = (1.1 +/- 0.3) x 10(9) M(-1); pH 7.0, 0.40 M NaCl, 25 degrees C], the affinity of which is reduced approximately 300-fold upon the binding of a single molar equivalent of Cd(II) or Pb(II). The implications of these findings on the mechanism of metalloregulation are discussed.     

The soft metal ion binding sites in the Staphylococcus aureus pI258 CadC Cd(II)/Pb(II)/Zn(II)-responsive repressor are formed between subunits of the homodimer

The Staphylococcus aureus plasmid pI258 CadC is a homodimeric repressor that binds Cd(II), Pb(II), and Zn(II) and regulates expression of the cadAC operon. CadC binds two Cd(II) ions per dimer, with a tetrathiolate binding site composed of residues Cys(7), Cys(11), Cys(58), and Cys(60). It is not known whether each site consists of residues from a single monomer or from residues contributed by both subunits. To examine whether Cys(7) and Cys(11) are spatially proximate to Cys(58) and Cys(60) of the same subunit or of the other subunit, homodimers with the same cysteine mutation in each subunit and heterodimers containing different cysteine mutations in the two subunits were reacted with 4,6-bis(bromomethyl)-3,7-dimethyl-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione, which cross-links thiol groups that are within 3-6 A of each other. Cys(7) or Cys(11) cross-linked only with Cys(58) or Cys(60) on the other subunit. The data demonstrate that Cys(7) and Cys(11) from one monomer are within 3-6 A of either Cys(58) or Cys(60) in the other monomer. The results of this study strongly indicate that each of the two Cd(II) binding sites in the CadC homodimer is composed of Cys(7) and Cys(11) from one monomer and Cys(58) and Cys(60) from the other monomer.     

Characterization of a metalloregulatory bismuth(III) site in Staphylococcus aureus pI258 CadC repressor

Staphylococcus aureus pI258 CadC is a metal sensor protein that regulates the expression of the cad operon which encodes metal ion resistance proteins involved in the efficient efflux of Cd(II), Pb(II), Zn(II) and, according to one report, Bi(III) ions. In this paper, direct evidence is presented that Bi(III) binds to CadC and negatively regulates cad operator/promoter (O/P) binding. Optical absorption spectroscopy reveals that dimeric CadC binds approximately 0.8 mol equivalents of Bi(III) per CadC monomer to form a coordination complex characterized by three S(-)-->Bi(III) ligand-to-metal charge transfer transitions, with the longest wavelength absorption band centered at 415 nm (epsilon(415)=4000 M(Bi)(-1) cm(-1)). UV-Vis absorption spectra of wild-type and mutant Cys-->Gly (Ser) substitution CadC mutants compared to [Bi(DTT)(2)], [Bi(GSH)(3)] and [Bi(NAC)](3) model complexes reveal that Cys7, Cys11, Cys60 and Cys58 directly coordinate Bi(III) in a tetrathiolate coordination complex. The apparent affinity derived from a Bi(III)-displacement optical titration with Cd(II) is estimated to be K(Bi)< or =10(12) M(-1). Apo-CadC binds with high affinity [ K(a)=1.1(+/-0.3)x10(9) M(-1); 0.40 M NaCl, pH 7.0, 25 degrees C] to a 5'-fluorescein-labeled cad O/P oligonucleotide,while the binding of one molar equivalent of Bi(III) per CadC monomer (Bi(1)-CadC) reduces the affinity by approximately 170-fold. Strikingly, Bi(III)-responsive negative regulation of cad O/P binding is abrogated for Bi(1)-C60G CadC and severely disrupted in Bi(1)-C7G CadC, whose relative affinity is reduced only 10-fold. The mechanism of Bi(III)-responsive metalloregulation is discussed, based on the findings presented here. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0336-9.     

Cd(II), Pb(II) and Zn(II) ions regulate expression of the metal-transporting P-type ATPase ZntA in Escherichia coli

Feeding bioassay results established that the soybean cysteine proteinase inhibitor N (soyacystatin N, scN) substantially inhibits growth and development of western corn rootworm (WCR), by attenuating digestive proteolysis [Zhao, Y. et al. (1996) Plant Physiol. 111, 1299-1306]. Recombinant scN was more inhibitory than the potent and broad specificity cysteine proteinase inhibitor E-64. WCR digestive proteolytic activity was separated by mildly denaturing SDS-PAGE into two fractions and in-gel assays confirmed that the proteinase activities of each were largely scN-sensitive. Since binding affinity to the target proteinase [Koiwa, H. et al. (1998) Plant J. 14, 371-380] governs the effectiveness of scN as a proteinase inhibitor and an insecticide, five peptides (28-33 kDa) were isolated from WCR gut extracts by scN affinity chromatographic separation. Analysis of the N-terminal sequence of these peptides revealed similarity to a cathepsin L-like cysteine proteinase (DvCAL1, Diabrotica virgifera virgifera cathepsin L) encoded by a WCR cDNA. Our results indicate that cathepsin L orthologs are pivotal digestive proteinases of WCR larvae, and are targets of plant defensive cystatins (phytocystatins), like scN.     

A family of polyamino phenolic macrocyclic ligands. Acid-base and coordination properties towards Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II) ions

The acid-base and coordination properties towards Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II) of four polyamino-phenol macrocycles 15-hydroxy-3,6,9-triazabicyclo[9.3.1]pentadeca-11,13,1¹⁵-triene L1, 18-hydroxy-3,6,9,12-tetraazabicyclo[12.3.1]octadeca-14,16,1¹⁸-triene L2, 21-hydroxy-3,6,9,12,15-pentaazabicyclo[15.3.1]enaicosa-17,19,1²¹-triene L3 and 24-hydroxy-3,6,9,12,15,18-hexaazabicyclo[18.3.1]tetraicosa-20,22,1²⁴-triene L4 are reported. The protonation and stability constants were determined by means of potentiometric measurements in 0.15 mol dm⁻³ NMe₄Cl aqueous solution at 298.1 K. L1 forms highly unsaturated Co(II), Cu(II), Zn(II) and Cd(II) mononuclear complexes that are prone to give dimeric dinuclear species with [(MH₋₁L1)₂]²⁺ stoichiometry, in solution. L2 forms stable Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II) mononuclear complexes that can coordinate external species as OH⁻ anion, giving hydroxylated complexes at alkaline pH. L3 forms stable Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Pb(II) mononuclear complexes and Co(II), Ni(II), Cu(II) and Zn(II) dinuclear [M₂H₋₁L3]³⁺ species. L4 forms stable mono- and dinuclear Co(II), Cu(II), Zn(II) and Cd(II) complexes, but only mononuclear species with Pb(II). The effect of macrocyclic size is considered in the discussion of results.     

Impact of Humin on Soil Adsorption and Remediation of Cd(II), Pb(II), and Cu(II)

In situ immobilization constitutes a promising technology for the mitigation of contaminants, through the reduction of metal bioavailability and mobility. This study investigated the adsorption isotherms and kinetic characteristics of humin extracted from peat soils. We also studied the influences of the pH, ionic strengths, and soluble organic matter concentrations of soil solutions on the adsorptive properties of humin, and compared its ability to detoxify potentially toxic metals in both actual and simulated soil solutions. The study results indicated that humin contains a massive population of oxygen-containing functional groups. Its adsorption capacity for Pb(II) was greater than that for Cu(II), which exceeded that for Cd(II). The adsorption of humin for Pb(II) conformed to the Freundlich model, while the adsorption of humin for Cd(II) and Cu(II) followed the Langmuir model. The adsorption kinetics of humin with respect to potentially toxic metals aligned well with second-order kinetics equations. As the pH was elevated, the potentially toxic metal adsorption by humin increased rapidly. Electrolyte ions and tartaric acids in solution both inhibited the adsorption of potentially toxic metals by humin, and its ability to inactivate potentially toxic metals. This was shown to be improved in actual field soil solutions in contrast to simulated soil solutions.     

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.     

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.     

Membrane topology of the p1258 CadA Cd(II)/Pb(II)/Zn(II)-translocating P-type ATPase

Plasmid pl258 carries the cadA gene that confers resistance to cadmium, lead, and zinc. CadA catalyzes ATP-dependent cadmium efflux from cells of Staphylococcus aureus. It is a member of the superfamily of P-type ATPases and belongs to the subfamily of soft metal ion pumps. In this study the membrane topology of this P-type ATPase was determined by constructing fusions with the topological reporter genes phoA or lacZ. A series of 44 C-terminal truncated CadAs were fused with one or the other reporter gene, and the activity of each chimeric protein was determined. In addition, the location of the first transmembrane segment was determined by immunoblot analysis. The results are consistent with the pl258 CadA ATPase having eight transmembrane segments. The first 109 residues is a cytosolic domain that includes the Cys(X)₂Cys motif that distinguishes soft metal ion-translocating P-type ATPases from their hard metal ion-translocating homologues. Another feature of soft metal ion P-type ATPases is the CysProCys motif, which is found in the sixth transmembrane segment of CadA. The phosphorylation site and ATP binding domain conserved in all P-type ATPases are situated within the large cytoplasmic loop between the sixth and seventh transmembrane segments.     

Formation of binary complexes of Pb(II), Cd(II) and Hg(II) with maleic acid in CTAB-water mixtures

Abstract

Complexation of toxic metal ions with maleic acid in (0.0–2.5% w/v) cetyltrimethylammonium bromide (CTAB)–water mixtures has been studied pH-metrically at ambient conditions and an ionic strength of 0.16 mol L^-1. The existence of different binary species was established from modelling studies using the computer program MINIQUAD75. The best-fit chemical models were selected based on statistical parameters such as the crystallographic R factor and sum of the squares of residuals in mass-balance equations. The models for binary complex systems contain the chemical species ML₂, ML₂H and ML₃ for Pb(II), Cd(II) and Hg(II) in CTAB–water mixtures. The trend in the variation of stability constants with change in the mole fraction of the medium was explained based on electrostatic and non-electrostatic forces. Distribution of the species with pH at different compositions of CTAB–water mixtures was also presented.     

Adsorption of Cu(II), Cd(II), and Pb(II) from aqueous single metal solutions by cellulose and mercerized cellulose chemically modified with succinic anhydride

This work describes the preparation of new chelating material from mercerized cellulose. The first part treats the chemical modification of non-mercerized cellulose (cell 1) and mercerized cellulose (cell 2) with succinic anhydride. Mass percent gains (mpg) and degree of succinylation (DS) of cell 3 (from cell 1) and cell 4 (from cell 2) were calculated. Cell 4 in relation to cell 3 exhibited an increase in mpg and in the concentration of carboxylic functions of 68.9% and 2.8 mmol/g, respectively. Cells 5 and 6 were obtained by treatment of cells 3 and 4 with bicarbonate solution to release the carboxylate functions and characterized by FTIR. The second part compares the adsorption capacity of cells 5 and 6 for Cu2+, Cd2+, and Pb2+ ions in an aqueous single metal solution. Adsorption isotherms were developed using Langmuir model. Cell 6 in relation to cell 5 exhibited an increase in Qmax for Cu2+ (30.4 mg/g), Cd2+ (86.0 mg/g) and Pb2+ (205.9 mg/g).     

Identification of complexes containing glutathione with As(III), Sb(III), Cd(II), Hg(II), Tl(I), Pb(II) or Bi(III) by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) of mixtures containing glutathione (GSH) and nitrates, oxides or chlorides of the heavy metals, arsenic, antimony, cadmium, mercury, thallium, lead or bismuth allows for definitive identification of complexes in the gas phase. In the positive ion mode, spectra show prominent m/z peaks that are assigned to monocations of general formulae [E(GSH)-xH]+ (E = Cd, Hg, Tl, Pb, As, Sb or Bi; x = 0, 1 or 2), [E(GSH)2-xH]+ (E = Hg, As, Sb, or Bi; x = 1 or 2), [E(GSH)3-xH]+ (E = As, Sb or Bi; x = 2), [E2(GSH)-xH]+ (E = Tl or Pb; x = 1 or 3), [E2(GSH)2-xH]+ (E = Bi; x = 5), [E2(GSH)3-xH]+ (E = Bi; x = 5), and/or [E3(GSH)-xH]+ (E = Tl; x = 2). Spectra obtained in the negative ion mode give m/z peaks observed in assigned to monoanionic species that correspond to some of the monocationic species listed above with two protons removed. The results demonstrate the potential application of ESI-MS as a versatile and efficient approach to study toxic heavy metals in biological systems. In addition, the observations provide a foundation database to understand the chemistry of these heavy metals with bio-molecules.     

Binding of Cd(II), Pb(II), and Zn(II) to a type 1 metallothionein from maize (<Emphasis Type="Italic">Zea mays</Emphasis>)

Metallothioneins (MTs) are a family of ubiquitous, low-molecular-mass, cysteine-rich proteins that play a significant role in maintaining intracellular metal homeostasis, eliminating metal toxification, and protecting cells against oxidative damages. Research activity on plant MTs, although known for 30 years, has only moderately increased in the past few years. In this study, a type 1 MT from maize (Zea mays) (ZmMT1) was successfully expressed in Escherichia coli strain BL21 (DE3). The UV absorption spectra recorded after the reconstitution of apo-ZmMT1 with different metals demonstrated that ZmMT1 can coordinate up to six Zn(II) ions, six Cd(II) ions, and even higher amounts of Pb(II). In addition, the general metal ion coordination abilities of ZmMT1 characterized by pH-dependent zinc-, lead- and cadmium-binding stability and by the competitive reaction with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) were evaluated. Results showed that the affinity of metal ions for the recombinant form of ZmMT1 can be arranged as follows: Cd(II)?>?Pb(II)?>?Zn(II). The observation revealed that chelating agents, such as ethylene diamine tetraacetic acid (EDTA) and ATP, accelerate the oxidation of ZmMT1 in the following order: EDTA???l-histidine?>?ATP?≈?citrate. Meanwhile, commonly used buffers increase the reactivity of ZmMT1 with DTNB in the following order: PBS?>?Tris–HCl?>?HEPES.     

Peptidic models for the binding of Pb(II), Bi(III) and Cd(II) to mononuclear thiolate binding sites

Herein, we evaluate the binding of Pb(II) and Bi(III) to cysteine-substituted versions of the TRI peptides [AcG-(LKALEEK)₄G-NH₂] which have previously been shown to bind Hg(II) and Cd(II) in unusual geometries as compared with small-molecule thiol ligands in aqueous solutions. Studies of Pb(II) and Bi(III) with the peptides give rise to complexes consistent with the metal ions bound to three sulfur atoms with M–S distances of 2.63 and 2.54 Å, respectively. Competition experiments between the metal ions Pb(II), Cd(II), Hg(II) and Bi(III) for the peptides show that Hg(II) has the highest affinity, owing to the initial formation of the extremely strong HgS₂ bond. Cd(II) and Pb(II) have comparable binding affinities at pH > 8, while Bi(III) displays the weakest affinity, following the model, M(II) + (TRI LXC)₃ ^3? → M(II)(TRI LXC)₃ ^?. While the relevant equilibria for Hg(II) binding to the TRI peptides corresponds to a strong first step forming Hg(TRI LXC)₂(HTRI LXC), followed by a single deprotonation to give Hg(TRI LXC)₃ ^?, the binding of Cd(II) and Pb(II) is consistent with initial formation of M(II)(TRI LXC)(HTRI LXC)₂ ⁺ at pH < 5 followed by a two-proton dissociation step (pK _a2) yielding M(II)(TRI LXC)₃ ^?. Pb(II)(TRI LXC)(HTRI LXC)₂ ⁺ converts to Pb(II)(TRI LXC)₃ ^? at slightly lower pH values than the corresponding Cd(II)–peptide complexes. In addition, Pb(II) displays a lower pK _a of binding to the “d”-substituted peptide, (TRI L12C, pK _a2 = 12.0) compared with the “a”-substituted peptide, (TRI L16C, pK _a2 = 12.6), the reverse of the order seen for Hg(II) and Cd(II). Pb(II) also showed a stronger binding affinity for TRI L12C (K _bind = 3.2 × 10⁷ M^?1) compared with that with TRI L16C (K _bind = 1.2 × 10⁷ M^?1) at pH > 8.     

Synergistic Removal of Pb(II), Cd(II) and Humic Acid by Fe3O4@Mesoporous Silica-Graphene Oxide Composites

The synergistic adsorption of heavy metal ions and humic acid can be very challenging. This is largely because of their competitive adsorption onto most adsorbent materials. Hierarchically structured composites containing polyethylenimine-modified magnetic mesoporous silica and graphene oxide (MMSP-GO) were here prepared to address this. Magnetic mesoporous silica microspheres were synthesized and functionalized with PEI molecules, providing many amine groups for chemical conjugation with the carboxyl groups on GO sheets and enhanced the affinity between the pollutants and the mesoporous silica. The features of the composites were characterized using TEM, SEM, TGA, DLS, and VSM measurements. Series adsorption results proved that this system was suitable for simultaneous and efficient removal of heavy metal ions and humic acid using MMSP-GO composites as adsorbents. The maximum adsorption capacities of MMSP-GO for Pb(II) and Cd (II) were 333 and 167 mg g⁻¹ caculated by Langmuir model, respectively. HA enhances adsorption of heavy metals by MMSP-GO composites due to their interactions in aqueous solutions. The underlying mechanism of synergistic adsorption of heavy metal ions and humic acid were discussed. MMSP-GO composites have shown promise for use as adsorbents in the simultaneous removal of heavy metals and humic acid in wastewater treatment processes.     

Adaptation to Cd(II) and Zn(II), and the caffeine-potentiated override of the G2 block induced by the checkpoint activated by DNA damage

ABSTRACT

Moderate and low concentrations of Zn(II) and Cd(II) were defined as those which depressed the rate of root elongation in Allium cepa L. to about 40 and 70% respectively of the control (17.3 ± 4.9 mm/day) at 25°C. At moderate concentrations, cells were detoxified from Cd(II), but not from Zn(II), by inducing the heavy metal chelators phytochelatins. Thus, root elongation further decreased (from 41 to 19% of the control) at moderate (0.05 mM) Cd(II) concentration upon addition of 0.25 mM L-buthionine-[S,R]-sulfoximine (BSO), a specific inhibitor of phytochelatin synthesis. On the other hand, cells were also detoxified from Zn(II) by an alternative mechanism, as the 42% inhibition displayed at 0.5 mM Zn(II) concentration was partially reversed (up to 79%) in the presence of BSO. Zn(II) activated the checkpoint pathway induced by DNA damage, as a transient G₂ block was produced; this block was partially cancelled by caffeine, so that chromosomal bridges (but no breaks) were observed in ana-telophase. On the other hand, Cd(II) did not activate the DNA damage checkpoint, as cells entered into anaphase with chromosomal breaks and bridges without any delay. Cd(II) may preclude the recognition of DNA damage by altering protein-DNA interactions, since 30% of the metaphases displayed clumped chromosomes. A minimum threshold was required to induce the adaptive responses described here, as BSO did not modify the reduction in root elongation rate recorded at low concentrations of both heavy metals.     

A second gene in the Staphylococcus aureus cadA cadmium resistance determinant of plasmid pI258.

Two open reading frames on a 3.7-kb BglII-XbaI fragment which encodes the Staphylococcus aureus cadA cadmium (and zinc) resistance determinant of plasmid pI258 were identified (G. Nucifora, L. Chu, T. K. Misra, and S. Silver, Proc. Natl. Acad. Sci. USA 86:3544-3548, 1989). The [35S]methionine-labelled protein products of the 727-amino-acid CadA ATPase and of the 122-amino-acid CadC polypeptide in Escherichia coli were identified by using the T7 RNA polymerase-promoter expression system. A truncated CadA polypeptide (402 amino acids) did not confer resistance in S. aureus but was expressed in E. coli under control of the T7 RNA polymerase-promoter. Removal of 678 nucleotides from the 5' end of the published sequence (which includes the cadA promoter) abolished resistance to cadmium, whereas a 146-nucleotide-shorter deletion was without effect. The cadC gene is needed in addition to cadA for full resistance to cadmium in S. aureus and Bacillus subtilis. cadC functions both in cis and in trans.     

A versatile and highly sensitive probe for Hg(II), Pb(II) and Cd(II) detection individually and totally in water samples

The detection of heavy metal ions using enzyme-linked immunosorbent assays (ELISA) has been reported by several research groups. However, highly sensitive and selective detection of total heavy metal ions using ELISA is a major technical limitation. Here we describe the development of a versatile and highly sensitive probe combining goat anti-mice IgG, colloidal gold nanoparticles (AuNPs) and horseradish peroxidase (HRP). We demonstrate the utility of this probe using three kinds of heavy metal complete antigens and three monoclonal antibodies (McAbs) in one ELISA system to establish a high-throughput screening protocol. The procedure was successfully applied to analysis of Hg(II), Pb(II) and Cd(II) individually and totally from different water samples. The sensitivities for the detection of Hg(II), Pb(II) and Cd(II) individually and totally are 27.4, 3.9, 15.8 and 18.2 nM, respectively. And all limit of detection (LODs) are lower than 1.2 nM. The recovery results obtained from the developed technique showed a good correlation (R² = 0.983) with those from ICP-MS. The major advantage of the probe is the versatility and high sensibility. The probe could be potentially used, upon demand, as a sensitive and versatile detector for a broad range of applications.