Mechanism of action of Escherichia coli ribonuclease III. Stringent chemical requirement for the glutamic acid 117 side chain and Mn2+ rescue of the Glu117Asp mutant

Escherichia coli ribonuclease III (EC 3.1.24) is a double-strand- (ds-) specific endoribonuclease involved in the maturation and decay of cellular, phage, and plasmid RNAs. RNase III is a homodimer and requires Mg(2+) to hydrolyze phosphodiesters. The RNase III polypeptide contains an N-terminal catalytic (nuclease) domain which exhibits eight highly conserved acidic residues, at least one of which (Glu117) is important for phosphodiester hydrolysis but not for substrate binding [Li and Nicholson (1996) EMBO J. 15, 1421-1433]. To determine the side chain requirements for activity, Glu117 was changed to glutamine or aspartic acid. The mutant proteins were purified as (His)(6)-tagged species, and both exhibited normal homodimeric behavior as shown by chemical cross-linking. The Glu117Gln mutant is unable to cleave substrate in vitro under all tested conditions but can bind substrate. The Glu117Asp mutant also is defective in cleavage while able to bind substrate. However, low level activity is observed at extended reaction times and high enzyme concentrations, with an estimated catalytic efficiency approximately 15 000-fold lower than that of RNase III. The activity of the Glu117Asp mutant but not that of the Glu117Gln mutant can be greatly enhanced by substituting Mn(2+) for Mg(2+), with the catalytic efficiency of the Glu117Asp-Mn(2+) holoenzyme approximately 400-fold lower than that of the RNase III-Mn(2+) holoenzyme. For RNase III, a Mn(2+) concentration of 1 mM provides optimal activity, while concentrations >5 mM are inhibitory. In contrast, the Glu117Asp mutant is not inhibited by high concentrations of Mn(2+). Finally, high concentrations of Mg(2+) do not inhibit RNase III nor relieve the Mn(2+)-dependent inhibition. In summary, these experiments establish the stringent functional requirement for a precisely positioned carboxylic acid group at position 117 and reveal two classes of divalent metal ion binding sites on RNase III. One site binds either Mg(2+) or Mn(2+) and supports catalysis, while the other site is specific for Mn(2+) and confers inhibition. Glu117 is important for the function of both sites. The implications of these findings on the RNase III catalytic mechanism are discussed.     

Heterodimer-based analysis of subunit and domain contributions to double-stranded RNA processing by Escherichia coli RNase III in vitro

Members of the RNase III family are the primary cellular agents of dsRNA (double-stranded RNA) processing. Bacterial RNases III function as homodimers and contain two dsRBDs (dsRNA-binding domains) and two catalytic sites. The potential for functional cross-talk between the catalytic sites and the requirement for both dsRBDs for processing activity are not known. It is shown that an Escherichia coli RNase III heterodimer that contains a single functional wt (wild-type) catalytic site and an inactive catalytic site (RNase III[E117A/wt]) cleaves a substrate with a single scissile bond with a k(cat) value that is one-half that of wt RNase III, but exhibits an unaltered K(m). Moreover, RNase III[E117A/wt] cleavage of a substrate containing two scissile bonds generates singly cleaved intermediates that are only slowly cleaved at the remaining phosphodiester linkage, and in a manner that is sensitive to excess unlabelled substrate. These results demonstrate the equal probability, during a single binding event, of placement of a scissile bond in a functional or nonfunctional catalytic site of the heterodimer and reveal a requirement for substrate dissociation and rebinding for cleavage of both phosphodiester linkages by the mutant heterodimer. The rate of phosphodiester hydrolysis by RNase III[E117A/wt] has the same dependence on Mg(2+) ion concentration as that of the wt enzyme, and exhibits a Hill coefficient (h) of 2.0+/-0.1, indicating that the metal ion dependence essentially reflects a single catalytic site that employs a two-Mg(2+)-ion mechanism. Whereas an E. coli RNase III mutant that lacks both dsRBDs is inactive, a heterodimer that contains a single dsRBD exhibits significant catalytic activity. These findings support a reaction pathway involving the largely independent action of the dsRBDs and the catalytic sites in substrate recognition and cleavage respectively.     

CorA affects tolerance of Escherichia coli and Salmonella enterica serovar Typhimurium to the lactoperoxidase enzyme system but not to other forms of oxidative stress

The enzyme lactoperoxidase is part of the innate immune system in vertebrates and owes its antimicrobial activity to the formation of oxidative reaction products from various substrates. In a previous study, we have reported that, with thiocyanate as a substrate, the lactoperoxidase system elicits a distinct stress response in Escherichia coli MG1655. This response is different from but partly overlapping with the stress responses to hydrogen peroxide and to superoxide. In the current work, we constructed knockouts in 10 lactoperoxidase system-inducible genes to investigate their role in the tolerance of E. coli MG1655 to this antimicrobial system. Five mutations resulted in a slightly increased sensitivity, but one mutation (corA) caused hypersensitivity to the lactoperoxidase system. This hypersensitive phenotype was specific to the lactoperoxidase system, since neither the sensitivity to hydrogen peroxide nor to the superoxide generator plumbagin was affected in the corA mutant. Salmonella enterica serovar Typhimurium corA had a similar phenotype. Although corA encodes an Mg2+ transporter and at least three other inducible open reading frames belonged to the Mg2+ regulon, repression of the Mg stimulon by Mg2+ did not change the lactoperoxidase sensitivity of either the wild-type or corA mutant. Prior exposure to 0.3 mM Ni2+, which is also transported by CorA, strongly sensitized MG1655 but not the corA mutant to the lactoperoxidase system. Furthermore, this Ni2+-dependent sensitization was suppressed by the CorA-specific inhibitor Co(III) hexaammine. These results indicate that CorA affects the lactoperoxidase sensitivity of E. coli by modulating the cytoplasmic concentrations of transition metals that enhance the toxicity of the lactoperoxidase system.     

Monitoring the structure of Escherichia coli RNase P RNA in the presence of various divalent metal ions

Lead(II)-induced cleavage can be used as a tool to probe conformational changes in RNA. In this report, we have investigated the conformation of M1 RNA, the catalytic subunit of Escherichia coli RNase P, by studying the lead(II)-induced cleavage pattern in the presence of various divalent metal ions. Our data suggest that the overall conformation of M1 RNA is very similar in the presence of Mg(2+), Mn(2+), Ca(2+), Sr(2+) and Ba(2+), while it is changed compared to the Mg(2+)-induced conformation in the presence of other divalent metal ions, Cd(2+) for example. We also observed that correct folding of some M1 RNA domains is promoted by Pb(2+), while folding of other domain(s) requires the additional presence of other divalent metal ions, cobalt(III) hexamine or spermidine. Based on the suppression of Pb(2+) cleavage at increasing concentrations of various divalent metal ions, our findings suggest that different divalent metal ions bind with different affinities to M1 RNA as well as to an RNase P hairpin-loop substrate and yeast tRNA(Phe). We suggest that this approach can be used to obtain information about the relative binding strength for different divalent metal ions to RNA in general, as well as to specific RNA divalent metal ion binding sites. Of those studied in this report, Mn(2+) is generally among the strongest RNA binders.     

Characterization of structure and metal ions specificity of Co2+-binding RNA aptamers

Studies on RNA motifs capable of binding metal ions have largely focused on Mg(2+)-specific motifs, therefore information concerning interactions of other metal ions with RNA is still very limited. Application of the in vitro selection approach allowed us to isolate two RNA aptamers that bind Co(2+) ions. Structural analysis of their secondary structures revealed the presence of two motifs, loop E and "kissing" loop complex, commonly occurring in RNA molecules. The Co(2+)-induced cleavage method was used for identification of Co(2+)-binding sites after the determination of the optimal cleavage conditions. In the aptamers, Co(2+) ions seem to bind to N7 atoms of purines, inducing cleavage of the adjacent phosphodiester bonds, similarly as is the case with yeast tRNA(Phe). Although the in vitro selection experiment was carried out in the presence of Co(2+) ions only, the aptamers displayed broader metal ions specificity. This was shown by inhibition of Co(2+)-induced cleavages in the presence of the following transition metal ions: Zn(2+), Cd(2+), Ni(2+), and Co(NH(3))(6)(3+) complex. On the other hand, alkaline metal ions such as Mg(2+), Ca(2+), Sr(2+), and Ba(2+) affected Co(2+)-induced cleavages only slightly. Multiple metal ions specificity of Co(2+)-binding sites has also been reported for other in vitro selected or natural RNAs. Among many factors that influence metal specificity of the Co(2+)-binding pocket, chemical properties of metal ions, such as their hardness as well as the structure of the coordination site, seem to be particularly important.     

Determination of the structure of Escherichia coli glyoxalase I suggests a structural basis for differential metal activation

The metalloenzyme glyoxalase I (GlxI) converts the nonenzymatically produced hemimercaptal of cytotoxic methylglyoxal and glutathione to nontoxic S-D-lactoylglutathione. Human GlxI, for which the structure is known, is active in the presence of Zn(2+). Unexpectedly, the Escherichia coli enzyme is inactive in the presence of Zn(2+) and is maximally active with Ni(2+). To understand this difference in metal activation and also to obtain a representative of the bacterial enzymes, the structure of E. coli Ni(2+)-GlxI has been determined. Structures have also been determined for the apo enzyme as well as complexes with Co(2+), Cd(2+), and Zn(2+). It is found that each of the protein-metal complexes that is catalytically active has octahedral geometry. This includes the complexes of the E. coli enzyme with Ni(2+), Co(2+), and Cd(2+), as well as the structures reported for the human Zn(2+) enzyme. Conversely, the complex of the E. coli enzyme with Zn(2+) has trigonal bipyramidal coordination and is inactive. This mode of coordination includes four protein ligands plus a single water molecule. In contrast, the coordination in the active forms of the enzyme includes two water molecules bound to the metal ion, suggesting that this may be a key feature of the catalytic mechanism. A comparison of the human and E. coli enzymes suggests that there are differences between the active sites that might be exploited for therapeutic use.     

Magnesium transport in Salmonella typhimurium: the influence of new mutations conferring Co2+ resistance on the CorA Mg2+ transport system

The CorA Mg2+ transport system of Salmonella typhimurium mediates both influx and efflux of Mg2+. Mutations at the corA locus (83.5 min) confer resistance to Co2+. Using transposon mutagenesis, three additional Co2+ resistance loci (corB, corC, and corD) were found and mapped to 55, 15, and 3min, respectively, on the S. typhimurium chromosome. No mutations corresponding to the reported corB locus at 95 min in Escherichia coli were obtained. The corB, corC, and corD mutations confer levels of Co2+ resistance intermediate between those of the wild-type and corA mutations. Isogenic strains were constructed containing combinations of transposon insertion mutations in each of the three Co(2+)-resistance loci to assess their influence on the CorA Mg2+ transport system. The Vmax and Km values for 28Mg2+ or for 57Co2+ and 63Ni2+ influx, analogues of Mg2+ transported by the CorA system, were changed less than twofold compared with the wild-type values, regardless of the mutation(s) present. However, while efflux of 28Mg2+ through the CorA system was decreased threefold in strains carrying one or two mutant alleles among corB, corC, or corD, efflux was completely abolished in either a corA or a corBCD strain. Thus, although the corA gene product is necessary and sufficient to mediate Mg2+ influx, Mg2+ efflux requires the presence of a wild-type allele of at least one of the corB, corC or corD loci.     

Cobalt- and nickel-binding property of cullin-2.

Treatment with divalent metal ions such as cobalt (Co(2+)) or nickel (Ni(2+)) result in the stabilization of hypoxia-inducible factor-1alpha (HIF1alpha). Recently, HIF1alpha was shown to be ubiquitinated by an E3-ligase complex and be subsequently targeted for proteasomal degradation. In this study, we demonstrated that Co(2+) and Ni(2+) specifically bind to cullin-2. Mutant analysis revealed that cullin-2 possesses at least three sites for the binding. Furthermore, fluorescence spectroscopy revealed that only Co(2+) and Ni(2+) have the binding activity to cullin-2, but other metal ions, including Cu(2+), Ca(2+), Mg(2+), Mn(2+), and Zn(2+), did not. Finally, we found that Co(2+) and Ni(2+) do not bind to any components of the E3-ligase other than cullin-2, suggesting that cullin-2 is a key target of Co(2+) and Ni(2+). Interestingly, Co(2+) did not affect the complex formation of the ligase, suggesting that the metal binding to cullin-2 affects the function, but not the assembly of the E3-ligase.     

Magnesium transport in Salmonella typhimurium: characterization of magnesium influx and cloning of a transport gene

The influx of Mg2+ in Salmonella typhimurium LT-2 was studied by both kinetic and genetic techniques. Wild-type cells grown in a high MgSO4 concentration (10 mM) exhibited a Km of 15 microM for Mg2+ influx, with a Vmax of 0.25 nmol of Mg2+ per min per 10(8) cells. The apparent Km decreased to 3 microM, and the Vmax increased 60% after growth in a low MgSO4 concentration (10 microM). Co2+ was a simple competitive inhibitor (Ki = 30 microM) of Mg2+ influx in cells grown in high Mg2+ concentrations but blocked only a portion of the Mg2+ influx in cells grown in low Mg2+ concentrations. Co2+ influx exhibited kinetics similar to those of Mg2+ influx (Km = 30 microM; Vmax = 0.5 nmol of Co2+ per min per 10(8) cells) but was not affected by growth conditions. Co2+ influx was competitively inhibited by both Mg2+ and Mn2+. Mutations affecting Mg2+ uptake were isolated by selection for spontaneous resistance to toxic levels of Co2+. One class of mutants designated corA mapped at 84 min near metE with the following gene order: corA, metE, zie-3161::Tn10, pepQ. A second class designated corB mapped at 98 min near pyrB. Mg2+ influx was decreased in a corA mutant strain (relative to that of the wild type) when grown in high Mg2+ concentrations but was restored when grown in low Mg2+ concentrations. Co2+ transport was completely abolished by the corA mutation under all growth conditions. Recombinant plasmids carrying the corA region from either Escherichia coli K-12 or S. typhimurium complemented the corA mutation in S. typhimurium, restoring uptake of both Co2+ and Mg2+ and conferring sensitivity to Co2+. The S. typhimurium corA gene was localized to a restriction fragment of approximately 1.5 kilobases.     

Mutational analysis of the nuclease domain of Escherichia coli ribonuclease III. Identification of conserved acidic residues that are important for catalytic function in vitro

The ribonuclease III superfamily represents a structurally distinct group of double-strand-specific endonucleases with essential roles in RNA maturation, RNA decay, and gene silencing. Bacterial RNase III orthologs exhibit the simplest structures, with an N-terminal nuclease domain and a C-terminal double-stranded RNA-binding domain (dsRBD), and are active as homodimers. The nuclease domain contains conserved acidic amino acids, which in Escherichia coli RNase III are E38, E41, D45, E65, E100, D114, and E117. On the basis of a previously reported crystal structure of the nuclease domain of Aquifex aeolicus RNase III, the E41, D114, and E117 side chains of E. coli RNase III are expected to be coordinated to a divalent metal ion (Mg(2+) or Mn(2+)). It is shown here that the RNase III[E41A] and RNase III[D114A] mutants exhibit catalytic activities in vitro in 10 mM Mg(2+) buffer that are comparable to that of the wild-type enzyme. However, at 1 mM Mg(2+), the activities are significantly lower, which suggests a weakened affinity for metal. While RNase III[E41A] and RNase III[D114A] have K(Mg) values that are approximately 2.8-fold larger than the K(Mg) of RNase III (0.46 mM), the RNase III[E41A/D114A] double mutant has a K(Mg) of 39 mM, suggesting a redundant function for the two side chains. RNase III[E38A], RNase III[E65A], and RNase III[E100A] also require higher Mg(2+) concentrations for optimal activity, with RNase III[E100A] exhibiting the largest K(Mg). RNase III[D45A], RNase III[D45E], and RNase III[D45N] exhibit negligible activities, regardless of the Mg(2+) concentration, indicating a stringent functional requirement for an aspartate side chain. RNase III[D45E] activity is partially rescued by Mn(2+). The potential functions of the conserved acidic residues are discussed in the context of the crystallographic data and proposed catalytic mechanisms.     

Intrinsic double-stranded-RNA processing activity of Escherichia coli ribonuclease III lacking the dsRNA-binding domain.

The ribonuclease III superfamily represents a structurally related group of double-strand (ds) specific endoribonucleases which play key roles in diverse prokaryotic and eukaryotic RNA maturation and degradation pathways. A dsRNA-binding domain (dsRBD) is a conserved feature of the superfamily and is important for substrate recognition. RNase III family members also exhibit a "catalytic" domain, in part defined by a set of highly conserved amino acids, of which at least one (a glutamic acid) is important for cleavage but not for substrate binding. However, it is not known whether the catalytic domain requires the dsRBD for activity. This report shows that a truncated form of Escherichia coli RNase III lacking the dsRBD (RNase III[DeltadsRBD]) can accurately cleave small processing substrates in vitro. Optimal activity of RNase III[DeltadsRBD] is observed at low salt concentrations (<60 mM Na(+)), either in the presence of Mg(2+) (>25 mM) or Mn(2+) ( approximately 5 mM). At 60 mM Na(+) and 5 mM Mn(2+) the catalytic efficiency of RNase III[DeltadsRBD] is similar to that of RNase III at physiological salt concentrations and Mg(2+). In the presence of Mg(2+) RNase III[DeltadsRBD] is less efficient than the wild-type enzyme, due to a higher K(m). Similar to RNase III, RNase III[DeltadsRBD] is inhibited by high concentrations of Mn(2+), which is due to metal ion occupancy of an inhibitory site on the enzyme. RNase III[DeltadsRBD] retains strict specificity for dsRNA, as indicated by its inability to cleave (rA)(25), (rU)(25), or (rC)(25). Moreover, dsDNA, ssDNA, or an RNA-DNA hybrid are not cleaved. Low (micromolar) concentrations of ethidium bromide block RNase III[DeltadsRBD] cleavage of substrate, which is similar to the inhibition seen with RNase III and is indicative of an intercalative mode of inhibition. Finally, RNase III[DeltadsRBD] is sensitive to specific Watson-Crick base-pair substitutions which also inhibit RNase III. These findings support an RNase III mechanism of action in which the catalytic domain (i) can function independently of the dsRBD, (ii) is dsRNA-specific, and (iii) participates in cleavage site selection.     

Biochemical characterization of RNase HII from Aeropyrum pernix

Aeropyrum pernix contains one homolog of ribonuclease H (RNase H), A. pernix RNase HII (Ape-RNase HII). Activity characterization showed that Ape-RNase HII exhibited the highest activity in the presence of 5 mM Mn(2+), 1 mM Co(2+), or 10 mM Mg(2+), respectively; however, its cleavage efficiencies at different cleavage sites for Mn(2+) and Mg(2+) were different. Ape-RNase HII cleaved 12-bp RNA/DNA substrates at multiple sites and the optimum pH value was 11.0. Moreover, 16-bp DNA-r4-DNA/DNA and 13-bp DNA-r1-DNA/DNA chimeric substrates were cleaved at DNA-RNA junction. Ape-RNase HII was thermostable and the stabilization was enhanced with increased salt concentration. This work is believed to be the first in vitro functional study of Ape-RNase HII and the results should contribute to the analysis of RNase H of other archaeal species.     

The metallomics approach: use of Fe(II) and Cu(II) footprinting to examine metal binding sites on serum albumins

Metal binding to serum albumins is examined by oxidative protein-cleavage chemistry, and relative affinities of multiple metal ions to particular sites on these proteins were identified using a fast and reliable chemical footprinting approach. Fe(ii) and Cu(ii), for example, mediate protein cleavage at their respective binding sites on serum albumins, in the presence of hydrogen peroxide and ascorbate. This metal-mediated protein-cleavge reaction is used to evaluate the binding of metal ions, Na(+), Mg(2+), Ca(2+), Al(3+), Cr(3+), Mn(2+), Co(2+), Ni(2+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), and Ce(3+) to albumins, and the relative affinities (selectivities) of the metal ions are rapidly evaluated by examining the extent of inhibition of protein cleavage. Four distinct systems Fe(II)/BSA, Cu(II)/BSA, Fe(II)/HSA and Cu(II)/HSA are examined using the above strategy. This metallomics approach is novel, even though the cleavage of serum albumins by Fe(II)/Cu(II) has been reported previously by this laboratory and many others. The protein cleavage products were analyzed by SDS PAGE, and the intensities of the product bands quantified to evaluate the extent of inhibition of the cleavage and thereby evaluate the relative binding affinities of specific metal ions to particular sites on albumins. The data show that Co(II) and Cr(III) showed the highest degree of inhibition, across the table, followed by Mn(II) and Ce(III). Alakali metal ions and alkaline earth metal ions showed very poor affinity for these metal sites on albumins. Thus, metal binding profiles for particular sites on proteins can be obtained quickly and accurately, using the metallomics approach.     

Lead-catalyzed cleavage of ribonuclease P RNA as a probe for integrity of tertiary structure.

Pb(2+)-catalyzed cleavage of RNA has been shown previously to be a useful probe for tertiary structure. In the present study, Pb2+ cleavage patterns were identified for ribonuclease P RNAs from three phylogenetically disparate organisms, Escherichia coli, Chromatium vinosum, Bacillus subtilis, and for E. coli RNase P RNAs that had been altered by deletions. Each of the native RNAs undergoes cleavage at several sites in the core structure that is common to all bacterial RNase P RNAs. All the cleavages occur in non-paired regions of the secondary structure models of the RNAs, in regions likely to be involved in tertiary interactions. Two cleavage sites occur at homologous positions in all the native RNAs, regardless of sequence variation, suggesting common tertiary structural features. The Pb2+ cleavage sites in four deletion mutants of E. coli RNase P RNA differed from the native pattern, indicating alterations in the tertiary structures of the mutant RNAs. This conclusion is consistent with previously characterized properties of the mutant RNAs. The Pb2+ cleavage assay is thus a useful probe to reveal alteration of tertiary structure in RNase P RNA.     

Mutational analysis of an RNA internal loop as a reactivity epitope for Escherichia coli ribonuclease III substrates

The enzymatic cleavage of double-stranded (ds) RNA is an obligatory step in the maturation and decay of many cellular and viral RNAs. The primary agents of dsRNA processing are members of the ribonuclease III (RNase III) superfamily, which are highly conserved in eukaryotic and bacterial cells. Escherichia coli RNase III participates in the maturation of the ribosomal RNAs and in the maturation and decay of cellular and phage mRNAs. E. coli RNase III-dependent cleavage events can regulate gene expression by controlling mRNA stability and translational activity. RNase III recognizes its substrates and selects the scissile phosphodiester(s) by recognizing specific RNA sequence and structural elements, termed reactivity epitopes. Some E. coli RNase III substrates contain an internal loop, in which is located the single scissile phosphodiester. The specific features of the internal loop that establish the pattern of single-strand cleavage are not known. A mutational analysis of the asymmetric [4 nt/5 nt] internal loop of the phage T7 R1.1 substrate reveals that cleavage reactivity is largely independent of internal loop sequence. Instead, the [4/5] asymmetry per se is the primary determinant of cleavage of a single bond within the 5 nt strand of the internal loop. The T7 R1.1 internal loop lacks elements of local tertiary structure, as revealed by sensitivity to cleavage by terbium ion and by the ability of the internal loop to destabilize a small model duplex. The internal loop functions as a discrete structural element in that the pattern of cleavage can be controlled by the specific type of asymmetry. The implications of these findings are discussed in light of RNase III substrate function as a gene regulatory element.     

Physiological metal uptake by Nostoc punctiforme

Trace metals are required for many cellular processes. The acquisition of trace elements from the environment includes a rapid adsorption of metals to the cell surface, followed by a slower internalization. We investigated the uptake of the trace elements Co(2+), Cu(2+), Mn(2+), Ni(2+), and Zn(2+) and the non-essential divalent cation Cd(2+) in the cyanobacterium Nostoc punctiforme. For each metal, a dose response study based on cell viability showed that the highest non-toxic concentrations were: 0.5?μM Cd(2+), 2?μM Co(2+), 0.5?μM Cu(2+), 500?μM Mn(2+), 1?μM Ni(2+), and 18?μM Zn(2+). Cells exposed to these non-toxic concentrations with combinations of Zn(2+) and Cd(2+), Zn(2+) and Co(2+), Zn(2+) and Cu(2+) or Zn(2+) and Ni(2+), had reduced growth in comparison to controls. Cells exposed to metal combinations with the addition of 500?μM Mn(2+) showed similar growth compared to the untreated controls. Metal levels were measured after one and 72?h for whole cells and absorbed (EDTA-resistant) fractions and used to calculate differential uptake rates for each metal. The differences in binding and internalisation between different metals indicate different uptake processes exist for each metal. For each metal, competitive uptake experiments using (65)Zn showed that after 72?h of exposure Zn(2+) uptake was reduced by most metals particularly 0.5?μM Cd(2+), while 2?μM Co(2+) increased Zn(2+) uptake. This study demonstrates that N. punctiforme discriminates between different metals and favourably substitutes their uptake to avoid the toxic effects of particular metals.