From heavy metal‐binders to biosensors: Ciliate metallothioneins discussed

Metallothioneins (MTs) are ubiquitous proteins with the capacity to bind heavy metal ions (mainly Cd, Zn or Cu), and they have been found in animals, plants, eukaryotic and prokaryotic micro‐organisms. We have carried out a comparative analysis of ciliate MTs (Tetrahymena species) to well‐known MTs from other organisms, discussing their exclusive features, such as the presence of aromatic amino acid residues and almost exclusive cysteine clusters (CCC) present in cadmium‐binding metallothioneins (CdMTs), higher heavy metal‐MT stoichiometry values, and a strictly conserved modular–submodular structure. Based on this last feature and an extensive gene duplication, we propose a possible model for the evolutionary history of T. thermophila MTs. We also suggest possible functions for these MTs from consideration of their differential gene expressions and discuss the potential use of these proteins and/or their gene promoters for designing molecular or whole‐cell biosensors for a fast detection of heavy metals in diverse polluted ecosystems.     

Ciliate metallothioneins: unique microbial eukaryotic heavy-metal-binder molecules

This article represents an updated review of ciliate metallothioneins (Tetrahymena species) including a comparative analysis with regard to well-known metallothioneins (MTs) from other organisms and discussion of their exclusive features. It opens with an introduction to ciliates, summarizing the main characteristics of these eukaryotic microorganisms and their use as cellular models to study metallothioneins and metal–eukaryotic cell interactions. It has been experimentally proved that at least three different metal resistance mechanisms exist in ciliates, of which bioaccumulation is the most studied. Structural comparative analysis reveals that Tetrahymena MTs have unique characteristics, such as longer length, a considerably higher cysteine content, different metal–MT stoichiometry values, the presence of new cysteine clusters, and a strictly conserved modular–submodular structure. Gene expression analysis reveals a multistress and differential response to diverse metals and other environmental stressors, which corroborates the classification of these MTs. An in silico analysis of the promoter sequences of some MT genes reveals the presence of conserved motifs that are probably involved in gene expression regulation. We also discuss the great advantages of the first ciliate whole-cell biosensors based on MT promoters from Tetrahymena thermophila to detect heavy metal ions in environmental samples.     

Nematode and snail metallothioneins

Metallobiologists have, at large, neglected soil dwelling invertebrates; exceptions are the nematode (Caenorhabditis elegans) and snails (Helix pomatia and Cantareus aspersus). This review aims to compare and contrast the molecular, protein and cellular mechanisms of the multifunctional nematode and snail metallothioneins (MTs). The C. elegans genome contains two MT genes, mtl-1, which is constitutively expressed in the pharynx and likely to act as an essential and/or toxic metal sensor, and mtl-2, which plays a negligible role under normal physiological conditions but is strongly induced (as mtl-1) in intestinal cells upon metal exposure. It has been possible to follow the intricate phenotypic responses upon the knockdown/knockout of single and multiple MT isoforms and we have started to decipher the multifunctional role of C. elegans MTs. The snails have contributed to our understanding regarding MT evolution and diversity, structure and metal-specific functionality. The H. pomatia and C. aspersus genomes contain at least three MT isoform genes. CdMT is responsible for cadmium detoxification, CuMT is involved in copper homeostasis and Cd/CuMT is a putative ancestral MT possibly only of minor importance in metal metabolism. Further investigations of nematode, snail and other invertebrate MTs will allow the development of alternative biomarker approaches and lead to an improved understanding of metallobiology, protein evolution and toxicogenomics.     

Two new members of the Tetrahymena multi-stress-inducible metallothionein family: Characterization and expression analysis of T. rostrata Cd/Cu metallothionein genes.

We report the cloning and characterization of two new metallothionein (MT) genes (TrosMTT1 and TrosMTT2), isolated as cDNAs, from the ciliated protozoa Tetrahymena rostrata. The TrosMTT1 inferred protein has been identified as a CdMT and included into the 7a subfamily of Tetrahymena MTs, while TrosMTT2 has been identified as a CuMT (including it into 7b subfamily), due to its similarity to TpigMT-2 and its significant induction by copper. TrosMTT1 protein sequence reveals a remarkably regular and hierarchical modular organization, as it is known for other Tetrahymena CdMTs, showing a bi-modular structure. TrosMTT2 presents a structural organization based on CKCX(2-5)CKC repeats, like it occurs in other Tetrahymena CuMTs, indicating that an evolutionary history based on intra-gene duplications might be also possible. Both are also multi-stress-inducible genes because they are induced by other heavy metals and stressors, as it has been shown by quantitative real-time RT-PCR. It is the first time that the gene expression of a putative Tetrahymena CuMT is analyzed by quantitative PCR, confirming it as a CuMT. These two new Tetrahymena MTs complete, at present, the actual view of this protein superfamily, and corroborate the unique features of ciliate MTs. Furthermore, both, a comparative analysis of relative gene expression values obtained by quantitative RT-PCR on other Tetrahymena MT genes and an analysis of the different Tetrahymena MTs based on the different Cys clusters of these proteins are carried out, which show an update view of Tetrahymena MT gene family.     

Functional comparision between truncated MTT1 and truncated MTT2 from Tetrahyemna thermophila

Metallothioneins (MTs) are low-molecular-weight proteins with high Cys content and high metal-chelating ability. CdMT and CuMT subfamilies present different characteristics in Tetrahymena. To explore the effect of the cysteine arrangement and sequence length of MTs for binding different metal ions, MTT1, truncated MTT1 (TM1), MTT2, and truncated MTT2 (TM2) were expressed in E. coli. The half-maximal inhibiting concentrations (IC₅₀) of Cd²⁺ and Cu⁺ for the recombinant strains were different. Furthermore, E. coli cells expressing MTT1 and TM1 exhibited higher accumulating ability for Cd²⁺ than cells expressing MTT2 and TM2. However, the opposite is true for Cu⁺. The binding ability of the different recombinant proteins to Cd²⁺ and Cu⁺ were also different. MTT1 and truncated mutant TM1 were the preference for Cd²⁺, whereas MTT2 and truncated mutant TM2 were the preference for Cu⁺ coordination. These results showed that metal ion tolerance and accumulation ability not only depended on cysteine arrangement pattern but also on sequence length of MT in Tetrahymena.     

Spectroscopic characterization of a fruit-specific metallothionein: M. acuminata MT3

Metallothioneins (MTs) are ubiquitous low molecular mass, cysteine-rich proteins with the ability to bind d¹⁰ metal ions in the form of metal-thiolate clusters. In contrast to the vertebrate forms, knowledge about the properties of members of the plant metallothionein family is still scarce. The amino acid sequences of plant MTs are distinctively different to the sequences of other MT species. The protein under investigation, Musa acuminata (banana) MT3, belongs to the plant MT fruit-specific p3 subfamily. With a total of 10 cysteine residues, MT3 features a cysteine content and percentage that is more comparable to fungal and prokaryotic MTs than to the well characterized mammalian iso-forms. The gene sequence encoding MT3 was cloned into a suitable vector and the protein was recombinantly overexpressed in Escherichia coli. MT3 is able to coordinate a maximum of four divalent d¹⁰ metal ions under the formation of metal-thiolate clusters. The hitherto unknown spectroscopic behavior of MT3 in combination with the metal ions Zn²⁺, Cd²⁺, Pb²⁺, and Hg²⁺ will be presented and gives rise to the existence of a weaker metal ion coordination site. The pH stability of the investigated zinc and cadmium clusters is comparable to the values found for other plant metallothioneins though significantly lower than for the mammalian iso-forms. Possible metal-thiolate cluster structures will additionally be discussed.     

Variations in plant metallothioneins: the heavy metal hyperaccumulator <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis> as a study case

Plant metallothioneins (MTs) are extremely diverse and are thought to be involved in metal homeostasis or detoxification. Thlaspi caerulescens is a model Zn/Cd hyperaccumulator and thus constitutes an ideal system to study the variability of these MTs. Two T. caerulescens cDNAs (accession: 665511; accession: 665515), that are highly homologous to type 1 and type 2 Arabidopsis thaliana MTs, have been isolated using a functional screen for plant cDNAs that confer Cd tolerance to yeast. However, TcMT1 has a much shorter N-terminal domain than that of A. thaliana and so lacks Cys motifs conserved through all the plant MTs classified as type 1. A systematic search in plant databases allowed the detection of MT-related sequences. Sixty-four percent fulfil the criteria for MT classification described in Cobbett and Goldsbrough (2002) and further extend our knowledge about other conserved residues that might play an important role in plant MT structure. In addition, 34% of the total MT-related sequences cannot be classified strictly as they display modifications in the conserved residues according to the current plant MTs’ classification. The significance of this variability in plant MT sequences is discussed. Functional complementation in yeast was used to assess whether these variations may alter the MTs’ function in T. caerulescens. Regulation of the expression of MTs in T. caerulescens was also investigated. TcMT1 and TcMT2 display higher expression in T. caerulescens than in A. thaliana. Moreover, their differential expression patterns in organs and in response to metal exposure, suggest that the two types of MTs may have diverse roles and functions in T. caerulescens.     

Understanding the 7‐Cys module amplification of C. neoformans metallothioneins: how high capacity Cu‐binding polypeptides are built to neutralize host nutritional immunity

Cryptococcus neoformans metallothioneins (MTs), CnMT1 and CnMT2, have been identified as essential infectivity and virulence factors of this pathogen. Both MTs are unusually long Cu‐thioneins, exhibiting protein architecture and metal‐binding abilities compatible with the hypothesis of resulting from three and five tandem repetitions of 7‐Cys motives, respectively, each of them folding into Cu₅‐clusters. Through the study of the Zn(II)‐ and Cu(I)‐binding capabilities of several CnMT1 truncated mutants, we show that a 7‐Cys segment of CnMT1 folds into Cu₅‐species, of additive capacity when joined in tandem. All the obtained Cu‐complexes share practically similar architectural features, if judging by their almost equivalent CD fingerprints, and they also share their capacity to restore copper tolerance in MT‐devoid yeast cells. Besides the analysis of the modular composition of these long fungal MTs, we evaluate the features of the Cys‐rich stretch spacer and flanking sequences that allow the construction of stable metal clusters by adjacent union of binding modules. Overall, our data support a mechanism by which some microbial MTs may have evolved to enlarge their original metal co‐ordination capacity under the specific selective pressure of counteracting the Cu‐based immunity mechanisms evolved by the infected hosts.     

Isolation of Zn-responsive genes from two accessions of the hyperaccumulator plant <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis>

Several populations with different metal tolerance, uptake and root-to-shoot transport are known for the metal hyperaccumulator plant Thlaspi caerulescens. In this study, genes differentially expressed under various Zn exposures were identified from the shoots of two T. caerulescens accessions (calaminous and non-calaminous) using fluorescent differential display RT-PCR. cDNA fragments from 16 Zn-responsive genes, including those encoding metallothionein (MT) type 2 and type 3, MRP-like transporter, pectin methylesterase (PME) and Ole e 1-like gene as well as several unknown genes, were eventually isolated. The full-length MT2 and MT3 sequences differ from those previously isolated from other Thlaspi accessions, possibly representing new alleles or isoforms. Besides the differential expression in Zn exposures, the gene expression was dependent on the accession. Thlaspi homologues of ClpP protease and MRP transporter were induced at high Zn concentrations. MT2 and PME were expressed at higher levels in the calaminous accession. The MTs and MRP transporter expressed in transgenic yeasts were capable of conferring Cu and Cd tolerance, whereas the Ole e 1-like gene enhanced toxicity to these metals. The MTs increased yeast intracellular Cd content. As no significant differences were found between Arabidopsis and Thlaspi MTs, they apparently do not differ in their capacity to bind metals. However, the higher levels of MT2 in the calaminous accession may contribute to the Zn-adapted phenotype.     

Barcodes Reveal 48 New Species of Tetrahymena,Dexiostoma, and Glaucoma: Phylogeny,Ecology, and Biogeography of New and Established Species

Tetrahymena mitochondrial cox1 barcodes and nuclear SSUrRNA sequences are particularly effective at distinguishing among its many cryptic species. In a project to learn more about Tetrahymena natural history, the majority of >1,000 Tetrahymena‐like fresh water isolates were assigned to established Tetrahymena species with the remaining assigned to 37 new species of Tetrahymena, nine new species of Dexiostoma and 12 new species of Glaucoma. Phylogenetically, all but three Tetrahymena species belong to the well‐established “australis” or “borealis” clades; the minority forms a divergent “paravorax” clade. Most Tetrahymena species are micronucleate, but others are exclusively amicronucleate. The self‐splicing intron of the LSUrRNA precursor is absent in Dexiostoma and Glaucoma and was likely acquired subsequent to the “australis/borealis” split; in some instances, its sequence is diagnostic of species. Tetrahymena americanis, T. elliotti, T. gruchyi n. sp., and T. borealis, together accounted for >50% of isolates, consistent with previous findings for established species. The biogeographic range of species found previously in Austria, China, and Pakistan was extended to the Nearctic; some species show evidence of population structure consistent with endemism. Most species were most frequently collected from ponds or lakes, while others, particularly Dexiostoma species, were collected most often from streams or rivers. The results suggest that perhaps hundreds of species remain to be discovered, particularly if collecting is global and includes hosts of parasitic forms.     

Relationship between metallothionein and metal contents in red-blooded and white-blooded Antarctic teleosts

Metal bioaccumulation and metallothionein were investigated in different organs of the red-blooded teleost, Trematomus bernacchii and the haemoglobinless Chionodraco hamatus. Specimens of the two Antarctic fish were sampled from Terra Nova Bay (Ross Sea), and their levels of Cd, Cu and Zn in homogenates and in soluble fractions of liver, muscle, gills, heart and plasma were determined. Dosages of metallothioneins (MTs) or MT-like proteins were assayed by the silver saturation method in soluble fractions of the same organs. In both T. bernacchii and C. hamatus the highest MT contents were found in liver. Hepatic MT and Cd, Cu and Zn concentrations correlated positively in T. bernacchii, whereas in C. hamatus hepatic MT showed a positive correlation only with Cd. Positive correlations were also found between Cd and MT in gills of the two species. A metal-binding protein containing a high percentage of cysteine from C. hamatus was purified and compared with the MT from T. bernacchii and mammals. Accepted: 27 November 1999     

Comparative expression analysis of genes encoding metallothioneins in response to heavy metals and abiotic stresses in rice (Oryza sativa) and Arabidopsis thaliana

To get insights into the functions of metallothionein (MT) in plant response to multiple stresses, expressions of 10 rice MT genes (OsMTs) and 7 Arabidopsis MT genes (AtMTs) were comprehensively analyzed under combined heavy metal and salt stress. OsMT1a, OsMT1b, OsMT1c, OsMT1g, and OsMT2a were increased by different heavy metals. Notably, ABA remarkably increased OsMT4 up to 80-fold. Combined salt and heavy metals (Cd, Pb, Cu) synergistically increased OsMT1a, OsMT1c, and OsMT1g, whereas combined salt and H₂O₂ or ABA synergistically increased OsMT1a and OsMT4. Heavy metals decreased AtMT1c, AtMT2b, and AtMT3 but cold or ABA increased AtMT1a, AtMT1c, and AtMT2a. AtMT4a was markedly increased by salt stress. Combined salt and other stresses (Pb, Cd, H₂O₂) synergistically increased AtMT4a. Taken together, these findings suggest that MTs in monocot and dicot respond differently to combined stresses, which provides a valuable basis to further determine the roles of MTs in broad stress tolerance.     

Heavy metal and abiotic stress inducible metallothionein isoforms from <Emphasis Type="Italic">Prosopis juliflora</Emphasis> (SW) D.C. show differences in binding to heavy metals in vitro

Prosopis juliflora is a tree species that grows well in heavy metal laden industrial sites and accumulates heavy metals. To understand the possible contribution of metallothioneins (MTs) in heavy metal accumulation in P. juliflora, we isolated and compared the metal binding ability of three different types of MTs (PjMT1-3). Glutathione S-transferase fusions of PjMTs (GSTMT1-3) were purified from Escherichia coli cells grown in the presence of 0.3 mM cadmium, copper or zinc. Analysis of metal bound fusion proteins using atomic absorption spectrometry showed that PjMT1 bound higher levels of all three heavy metals as compared to PjMT2 and PjMT3. A comparative analysis of the genomic regions (including promoter for all three PjMTs) is also presented. All three PjMTs are induced by H₂O₂ and ABA applications. PjMT1 and PjMT2 are induced by copper and zinc respectively while PjMT3 is induced by copper, zinc and cadmium. Variation in induction of PjMTs in response to metal exposure and their differential binding to metals suggests that each MT has a specific role in P. juliflora. Of the three MTs analyzed, PjMT1 shows maximum heavy metal sequestration and is thus a potential candidate for use in heavy metal phytoremediation.