Dataset Information

Specific metal recognition in nickel trafficking.

ABSTRACT: Nickel is an essential metal for a number of bacterial species that have developed systems for acquiring, delivering, and incorporating the metal into target enzymes and controlling the levels of nickel in cells to prevent toxic effects. As with other transition metals, these trafficking systems must be able to distinguish between the desired metal and other transition metal ions with similar physical and chemical properties. Because there are few enzymes (targets) that require nickel for activity (e.g., Escherichia coli transports nickel for hydrogenases made under anaerobic conditions, and Helicobacter pylori requires nickel for hydrogenase and urease that are essential for acid viability), the "traffic pattern" for nickel is relatively simple, and nickel trafficking therefore presents an opportunity to examine a system for the mechanisms that are used to distinguish nickel from other metals. In this review, we describe the details known for examples of uptake permeases, metallochaperones and proteins involved in metallocenter assembly, and nickel metalloregulators. We also illustrate a variety of mechanisms, including molecular recognition in the case of NikA protein and examples of allosteric regulation for HypA, NikR, and RcnR, employed to generate specific biological responses to nickel ions.

SUBMITTER: Higgins KA

PROVIDER: S-EPMC3502001 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Specific metal recognition in nickel trafficking.

Higgins Khadine A KA Carr Carolyn E CE Maroney Michael J MJ

Biochemistry 20120928 40

Nickel is an essential metal for a number of bacterial species that have developed systems for acquiring, delivering, and incorporating the metal into target enzymes and controlling the levels of nickel in cells to prevent toxic effects. As with other transition metals, these trafficking systems must be able to distinguish between the desired metal and other transition metal ions with similar physical and chemical properties. Because there are few enzymes (targets) that require nickel for activi ...[more]

PMID: 22970729

Similar Datasets

Project description:Helicobacter pylori , a pathogen that colonizes the human stomach, requires the nickel-containing metalloenzymes urease and NiFe-hydrogenase to survive this low pH environment. The maturation of both enzymes depends on the metallochaperone, HypA. HypA contains two metal sites, an intrinsic zinc site and a low-affinity nickel binding site. X-ray absorption spectroscopy (XAS) shows that the structure of the intrinsic zinc site of HypA is dynamic and able to sense both nickel loading and pH changes. At pH 6.3, an internal pH that occurs during acid shock, the zinc site undergoes unprecedented ligand substitutions to convert from a Zn(Cys)(4) site to a Zn(His)(2)(Cys)(2) site. NMR spectroscopy shows that binding of Ni(II) to HypA results in paramagnetic broadening of resonances near the N-terminus. NOEs between the beta-CH(2) protons of Zn cysteinyl ligands are consistent with a strand-swapped HypA dimer. Addition of nickel causes resonances from the zinc binding motif and other regions to double, indicating more than one conformation can exist in solution. Although the structure of the high-spin, 5-6 coordinate Ni(II) site is relatively unaffected by pH, the nickel binding stoichiometry is decreased from one per monomer to one per dimer at pH = 6.3. Mutation of any cysteine residue in the zinc binding motif results in a zinc site structure similar to that found for holo-WT-HypA at low pH and is unperturbed by the addition of nickel. Mutation of the histidines that flank the CXXC motifs results in a zinc site structure that is similar to holo-WT-HypA at neutral pH (Zn(Cys)(4)) and is no longer responsive to nickel binding or pH changes. Using an in vitro urease activity assay, it is shown that the recombinant protein is sufficient for recovery of urease activity in cell lysate from a HypA deletion mutant, and that mutations in the zinc-binding motif result in a decrease in recovered urease activity. The results are interpreted in terms of a model wherein HypA controls the flow of nickel traffic in the cell in response to nickel availability and pH.

Project description:The recent development of a custom cDNA microarray platform for one of thé standard organisms in aquatic toxicology, Daphnia magna, opened up new ways to mechanistic insights of toxicological responses. In this study, gene expression and (sub)organismal responses (Cellular Energy Allocation, growth) were assayed after short-term waterborne metal exposure. Microarray analysis of Ni-exposed daphnids revealed several affected functional gene classes, of which the largest ones were involved in different metabolic processes (mainly protein and chitin related processes), cuticula turnover, transport and signal transduction. Furthermore, genes involved in oxygen transport and heme metabolism (hemoglobin, δ-aminolevilunate synthase) were down-regulated. Applying a Partial Least Squares regression on nickel fingerprints and biochemical (sub)organismal parameters revealed a set of co-varying genes (hemoglobin, RNA terminal phosphate cyclase, a ribosomal protein and an “unknown” gene fragment). An inverse relationship was seen between the mRNA expression levels of different cuticula proteins and available energy reserves. In addition to the nickel exposure, daphnids were exposed to binary mixtures of nickel and cadmium or nickel and lead. Using multivariate analysis techniques, the mixture gene expression fingerprints (Ni2++Cd2+, Ni2++Pb2+) were compared to those of the single metal treatments (Ni2+, Cd2+, Pb2+). It was hypothesized that the molecular fingerprints of the mixtures would be additive combinations of the gene expression profiles of the individual compounds present in the mixture. However, our results clearly showed additionally affected pathways after mixture treatment (e.g. additional affected genes involved in carbohydrate catabolic processes and proteolysis), indicating interactive molecular responses which are not merely the additive sum of the individual metals. These findings, although indicative of the complex nature of mixture toxicity evaluation, underline the potential of a toxicogenomics approach in gaining more mechanistic information on the effects of single compounds and mixtures.

Project description:The recent development of a custom cDNA microarray platform for one of thÃ© standard organisms in aquatic toxicology, Daphnia magna, opened up new ways to mechanistic insights of toxicological responses. In this study, gene expression and (sub)organismal responses (Cellular Energy Allocation, growth) were assayed after short-term waterborne metal exposure. Microarray analysis of Ni-exposed daphnids revealed several affected functional gene classes, of which the largest ones were involved in different metabolic processes (mainly protein and chitin related processes), cuticula turnover, transport and signal transduction. Furthermore, genes involved in oxygen transport and heme metabolism (hemoglobin, Î´-aminolevilunate synthase) were down-regulated. Applying a Partial Least Squares regression on nickel fingerprints and biochemical (sub)organismal parameters revealed a set of co-varying genes (hemoglobin, RNA terminal phosphate cyclase, a ribosomal protein and an âunknownâ gene fragment). An inverse relationship was seen between the mRNA expression levels of different cuticula proteins and available energy reserves. In addition to the nickel exposure, daphnids were exposed to binary mixtures of nickel and cadmium or nickel and lead. Using multivariate analysis techniques, the mixture gene expression fingerprints (Ni2++Cd2+, Ni2++Pb2+) were compared to those of the single metal treatments (Ni2+, Cd2+, Pb2+). It was hypothesized that the molecular fingerprints of the mixtures would be additive combinations of the gene expression profiles of the individual compounds present in the mixture. However, our results clearly showed additionally affected pathways after mixture treatment (e.g. additional affected genes involved in carbohydrate catabolic processes and proteolysis), indicating interactive molecular responses which are not merely the additive sum of the individual metals. These findings, although indicative of the complex nature of mixture toxicity evaluation, underline the potential of a toxicogenomics approach in gaining more mechanistic information on the effects of single compounds and mixtures. In general, a universal reference design was used. The following analysis (normalization and statistical analysis) was performed. Spots were identified and ratioâs quantified by means of the Genepix software 5.0 (Axon Instruments). Subsequently, a MIAME compliant platform, the BioArray Software Environment Database (BASE 1.2.12, http://www.islab.ua.ac.be/base/) was used for storage, further evaluation and succeeding statistical analysis of the microarray datasets. After a local background correction, spots with a foreground larger than the mean local background +2 standard deviations for one or both colours were selected for further analysis. The Log2 transformed Cy3/Cy5 ratio data were normalized by implementation of a Locally Weighed Scatterplot Smoothing (Lowess) (Yang et al., 2002) step, which is an intensity-based method. Significant vertical cut-off values of log -0.85 and 0.85 were determined through multiple self-self hybridizations. These were performed by labelling the same biological material with both Cy3 and Cy5 dyes and hybridizing them simultaneously on a microarray slide. To statistically evaluate the microarray results, Significance Analysis of Microarrays (SAM) described by Tusher et al. (2001) was applied on the Lowess normalized datasets. With this SAM approach a false discovery rate (FDR) below 5% was applied. As a result genes with log2 ratios outside the confidence interval of -0.85 and 0.85 were considered as differentially expressed gene fragments when FDR was below 5%.

Project description:A novel ArsR-SmtB family transcriptional repressor, KmtR, has been characterised from mycobacteria. Mutants of M. tuberculosis lacking kmtR show elevated expression of Rv2025c encoding a deduced CDF-family metal-exporter. KmtR-dependent repression of the cdf and kmtR operator-promoters was alleviated by nickel and cobalt in minimal medium. Electrophoretic mobility shift assays (EMSA) and fluorescence anisotropy (FA) show binding of purified KmtR to nucleotide sequences containing a region of dyad symmetry from the cdf and kmtR operator-promoters. A relatively large deltar(obs) in FA implies formation of high order apo-KmtR(n)-DNA and multiple complexes were detected by EMSA. Incubation of KmtR with cobalt inhibits DNA-complex assembly and metal-protein binding was confirmed by competition against 4-(2-pyridylazo)-resorcinol. KmtR is the second, to NmtR, characterised ArsR-SmtB sensor of nickel and cobalt from M. tuberculosis suggesting special significance for these ions in this pathogen. KmtR-dependent expression is elevated in complete medium with no increase in response to metals, while NmtR retains a response to nickel and cobalt under these conditions. Mixing equimolar apo-KmtR and apo-NmtR with 0.8 equivalents of nickel or cobalt gave nickel- and cobalt-dependent difference emission spectra similar to nickel(0.8)-KmtR and cobalt(0.8)-KmtR, respectively. Thus, KmtR has tighter affinities for nickel and cobalt than NmtR consistent with basal levels of these metals being sensed by KmtR but not NmtR in complete medium. More than a thousand genes encoding ArsR-SmtB related proteins are listed in databases and a proportion can be predicted to detect metals through known allosteric sites. KmtR has none of the previously defined sites. Substitution of His(88), Glu(101), His(102), His(110) or His(111) with Gln generated KmtR-variants that repress the cdf and kmtR operator-promoters even in elevated nickel and cobalt, revealing a new sensory site. Importantly, ArsR-SmtB sequence groupings do not correspond with the different sensory-motifs revealing that only the latter should be used to predict metal-sensing. Data is also available from http://bugs.sgul.ac.uk/E-BUGS-49

Dataset Information

Specific metal recognition in nickel trafficking.

Publications

Specific metal recognition in nickel trafficking.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets