Introduction (ciliates as microbial eukaryotic models)

Ciliated protozoa emerged about 109 years ago (Early Cambrian) before fungi and other eukaryotic models. Ciliates are one of three major evolutionary lineages that make up the alveolates, the other two lineages being dinoflagellates and parasitic apicomplexa. Ciliated protozoa are free-living eukaryotic microorganisms with a cosmopolitan distribution, adapted for living in both terrestrial and aquatic ecosystems. They are believed to be important grazers of bacteria and other microorganisms and this activity appears to stimulate the rates of carbon and nitrogen cycling in soils [1]. The absence of a cell wall in the vegetative stage of these eukaryotic microorganisms offers a higher sensitivity to environmental pollutants than other microorganisms, such as yeasts and bacteria, and therefore a faster cellular response [2–4]. A general feature of ciliates is nuclear dimorphism: two types of nuclei (micronucleus and macronucleus) are present in the same cell [5]. The ciliatology has given rise to two main ciliate models, Tetrahymena thermophila and Paramecium tetraurelia, which owe their importance to their “domestication” or adaptation to microbial laboratory work: they grow rapidly, giving a high cell density (approximately 106 cells/ml) in a variety of media and culture conditions, they are relatively large microorganisms (40–50 μm), which makes their manipulation easier, and they have a typical eukaryotic life cycle and sexual interaction (conjugation), which makes genetic analysis possible. Several good genetic analysis tools (e.g., nullisomic and knockout strains) and molecular genetics tools have been developed in both ciliate models, and the macronuclear genomes have already been sequenced in each model [6, 7]. Ciliates are eukaryotic cells with some metabolic traits that are more similar to those of human cells than yeasts or other eukaryotic microorganisms. After completion of the genome sequencing, both ciliate models show that they share a higher degree of functional conservation with human genes than do other microbial models. This can be seen in the better matching of relevant ciliate coding sequences to those in humans compared with nonciliate microbial models, e.g., humans and T. thermophila share more orthologs with each other (2,280) than are shared between humans and the yeast Saccharomyces cerevisiae (2,097) [6]. This is a good reason to use ciliates in ecotoxicology studies as models for humans or as eukaryotic models to analyze cell–metal interactions, representing an alternative to animal tests [2, 4]. Ciliates have contributed to fundamental biological discoveries such as lysosomes and peroxisomes, cilia components, the discovery of the first eukaryotic retrotranscriptase (telomerase), autocatalytic RNA capacity (ribozyme), and the function of histone acetylation in gene expression regulation. Finally, over the last 10 years, ciliates have increased our general knowledge of both metallothioneins (MTs) [8] and their different responses to metal stress [9–11].

Ciliate heavy metal bioaccumulation and metallothioneins

Among the six well-known metal resistance mechanisms present in prokaryotic and eukaryotic organisms, ciliates have at least two main mechanisms [3]: intracellular sequestration (bioaccumulation) of metal ions by binding to proteins (MTs) to prevent metal-sensitive cellular target interactions [12], and extracellular sequestration or biosorption, in which metal cations are passively adsorbed by extracellular polymers, cell walls, or capsules [13]. The latter resistance mechanism is present exclusively in ciliate resting cysts [3]. A rapid biotransformation of arsenate by biomethylation and biovolatilization has very recently been reported in T. thermophila [14]. Therefore, a third metal resistance mechanism (biotransformation) is also possible in ciliated protozoa. A fourth potential metal resistance mechanism in ciliates might be an active export, which is supported by (1) the large number (485) of putative genes encoding membrane transporters for inorganic cations in T. thermophila [6] and very probably in other ciliates, and (2) the analysis of several expression gene libraries from T. thermophila after metal treatments (unpublished data) revealed the presence of about 4.6% complementary DNAs (cDNAs) encoding membrane transporters, indicating the relative importance of these genes in metal detoxification processes. Among ciliates, metal bioaccumulation is the best known resistance mechanism. It involves intracellular sequestration by MTs, forming metal–protein complexes which are accumulated into vacuoles and finally released from the cell as nontoxic metallic complexes. From ciliate toxicological data, metal bioaccumulation seems to be a specific feature of each ciliate strain [9]. In general, the metal bioaccumulation ranking in ciliates is Zn ≫ Cd > Cu. During ciliate metal bioaccumulation, Cd may displace Zn when both are present in the medium, so ciliates respond in a similar way to pluricellular organisms and mammalian tissues, in which the presence of Cd may cause a cellular Zn deficiency [15]. This competition between Cd and Zn has been detected in several ciliates, such as Tetrahymena sp. [16], Uronema marinum [17], Drepanomonas revoluta, Euplotes sp., and Uronema marinum [9]. Ciliate metal bioaccumulation has been detected by both transmission electron microscopy [18–20] and fluorescence microscopy [9, 11].

Although metal bioaccumulation has been detected in diverse phylogenetically distant ciliates, the presence of MTs has been reported in only two ciliate genera (Tetrahymena and Paramecium). With respect to the latter, no experimental data on its gene expression under metal stress have been given, so it should not be considered until expression experiments have been reported. Therefore, we have only considered as ciliate MTs those that are exclusively from different Tetrahymena species.

Ciliate MTs have been included in the MT superfamily classification system as family 7, which has been divided into two main subfamilies [8, 21, 22]: subfamily 7a, or CdMTs, and subfamily 7b, or CuMTs. Both subfamilies differ in metal induction patterns (Cd/Zn or Cu) and the pattern of Cys residue clustering [8, 22–26].

Most Tetrahymena MT genes were isolated as cDNA molecules and their alignment with the corresponding genomic DNA sequences revealed the absence of introns in all these MT genes. Genes with expression levels that change rapidly in response to environmental stress contain significantly lower intron numbers. The presence of introns can delay regulatory responses and they are selected against genes with transcripts that require rapid adjustment to survive environmental changes [27]. In general, the gene expression of Tetrahymena MT genes is very fast, as reported in a CdMT gene (MT-1) from T. pigmentosa, in which an approximately tenfold increase in this transcript was detected after 30 min Cd treatment [28].

Structural comparative analysis of Tetrahymena metallothioneins

Twelve complete putative CdMT cDNA sequences from different Tetrahymena species have been reported and submitted to GenBank: two T. pyriformis sequences [abbreviated TpyrMT-1 (AJ005080) and TpyrMT-2 (AY765220)], three T. thermophila sequences [TtheMTT1 (DQ517937/EF195745/AY061892), TtheMTT3 (EF195744), and TtheMTT5 (DQ517936)], one T. tropicalis sequence (TtroMTT1; EF185997), one T. rostrata sequence (TrosMTT1; EU627174), one T. pigmentosa sequence (TpigMT-1; EU420056), one T. vorax sequence (TvorMT1; HQ166889), one T. mobilis sequence (TmobMT1; HQ166888), and two T. hegewischi sequences [ThegMT1 (HQ166890) and ThegMT2 (HQ166891)] [22–26, 28–30]. Four complete putative CuMT cDNA sequences from different Tetrahymena species have been already reported: two T. thermophila sequences [(TtheMTT2 (AY204351) and TtheMTT4 (AY660008)], one T. pigmentosa sequence (TpigMT-2; AF479586), and one T. rostrata sequence (TrosMTT2; EU627175) [22, 23, 28, 31].

In Table 1, we compare several features of Tetraymena MTs with regard to MTs present in most organisms. They are considerably longer than vertebrate MTs. Their lengths fall in the top 1% of the more than 500 nonredundant Cys-rich (more than 25% Cys residues) proteins in GenBank annotated as MT-like, so, likewise, they have molecular masses greater than those of vertebrate MTs (Table 1). They are classified as stable proteins with an estimated half-life of about 30 h. Their average hydropathicity values are −0.54 (CdMTs) and −0.44 (CuMTs) (indicating their hydrophilic character and a higher protein flexibility), which are lower than those for vertebrate MTs (−0.15 as an average value) [32]. A general feature of MTs is the relative scarcity of aromatic amino acids. All Tetrahymena CuMTs and some CdMTs (TpyrMT-1, TpyrMT-2, TrosMTT1, TmobMT1, and TvorMT1) lack aromatic amino acids, but other Tetrahymena CdMTs are less typical because they contain aromatic residues (Phe > Tyr > Trp). Another infrequent residue in the amino acid sequences of MTs is the His residue. It is generally absent in MTs, as in most Tetrahymena MTs. However, as recently reported, His residues, the most frequent Zn ligands in enzymatic sites, are also present (one to four residues) in MTs from a variety of species (bacteria, fungi, plants, and animals), thereby enhancing the relative affinity for Zn in comparison with Cd [33]. Only one Tetrahymena CdMT (TtheMTT3) and two CuMTs (TtheMTT2 and TtheMTT4) contain His residues.

Table 1 Comparison of the main features of Tetrahymena metallothionein (MTs) with regard to other (mainly vertebrate) MTs
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Another general feature of MTs (including those from Tetrahymena) (Table 1) is the extreme asymmetry in the ratio of positively charged amino acids Lys and Arg. Other peculiar amino acid asymmetry ratios of Tetrahymena MTs with regard to vertebrate MTs are the Ile to Leu and the Ser to Thr ratios (Table 1). We do not know the meaning (if any) of these differences.

Cys residues are the main elements in MT composition because they are the chelating points for heavy metals, forming metal–thiolate clusters [34, 35]. Although the average Cys percentage in Tetrahymena MTs is within the average Cys percentage of vertebrate MTs, the total amount of Tetrahymena MT Cys residues is considerably higher (up to 54 residues) (Table 2) than that in vertebrate MTs (seven to 21 residues). These Cys residues are arranged in clusters, such as XCCX, CXC, and XXCXX (unclustered Cys residues), where X is any amino acid. These are typical clusters in MTs, but possible additional clusters (CCC, CXCC, or CXCXC) have been found in Tetrahymena CdMTs (Table 2). CuMTs from this ciliate only have typical MT clusters (Table 2). In Tetrahymena CdMTs, about 32% of the total Cys residues are clustered in the CCC motif, which is occasionally found in MTs from other organisms, such as a CdMT from the annelid Eisenia foetida, a putative MT from the yeast Yarrowia (Candida) lipolytica (P41928), a CuMT from the arthropod Callinectes sapidus, and an MT isoform from the mollusk Crassostrea virginica [36–38]. XCCX motifs are also abundant in Tetrahymena CdMTs (about 33.8% of the total Cys residues), whereas in CuMTs, Cys residues are mostly clustered in the CXC motif (89.4% of the total Cys residues) (Table 2).

Table 2 Distribution of Cys cluster types among Tetrahymena MTs
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Considering that in vertebrate MTs all Cys residues are involved in heavy metal binding and the stoichiometry is therefore Cd7(Cys)20 for CdMTs or Cu12(Cys)20 for CuMTs, and assuming that this is also applicable to Tetrahymena MTs, we can calculate the theoretical binding capacity of these MTs (Table 1). For instance, TtheMTT1, TvorMT1, and ThegMT2 might bind approximately 17 Cd atoms, TtheMTT3 15 Cd atoms, TtheMTT5 eight Cd atoms, TrosMTT1, TpigMT-1, and ThegMT1 approximately 12 Cd atoms, TtroMTT1 16 Cd atoms, TmobMT1 18 Cd atoms, TpyrMT-2 19 Cd atoms, and TpyrMT-1 11 Cd atoms. Some of these data have been experimentally corroborated: 12 Cd2+ per mole of protein or 11 Cd2+ per polypeptide for TpyrMT-1 [39, 40]. A stable Cd16(Cys)48 complex in vitro has recently been suggested [41] for TtheMTT1 (a value similar to the theoretical value assigned to this CdMT) and Cd11(Cys)32 has been suggested for TtheMTT2, with the metal-to-Cys ratio being about 1:3 for both MTs. It was found that Cu2+ cannot replace Cd from the Cd16–TtheMTT1 complex, but Cu2+ can replace Cd2+ from the Cd11–TtheMTT2 complex. These data confirm the classification of TtheMTT1 and TtheMTT2 as CdMT and CuMT, respectively. With regard to Tetrahymena CuMTs, TtheMTT2 and TtheMTT4 (which are identical proteins, approximately 99% identity) [23] might bind approximately 19 Cu+, TpigMT-2 16 Cu+, and TrosMTT2 13 Cu+. Although a more intensive analysis of the real metal binding capacity of these ciliate MTs is necessary, we can conclude that the number of metal ions per molecule that these MTs might bind is considerably higher than that of vertebrate MTs (Table 1).

Decimal numbers of Cys residue percentages from all Tetrahymena MTs are pure or semipure periodic numbers, indicating the existence of a periodic pattern of these amino acid residues in the protein sequence [8], as has also been detected in most MTs from other organisms. A remarkably regular and hierarchical modular organization has been reported in Tetrahymena CdMTs (Fig. 1) [8, 22, 23]. All members of Tetrahymena subfamily 7a (CdMTs) are readily and unambiguously divided into segments initially defined by the criterion that every segment carries a CXCCK motif at its C-terminus (with several exceptions) (Fig. 1), denominated as “modules.” These range in length from 27 to 56 amino acids and are separated by two to nine amino acid “linkers.” From this point of view, TpyrMT-1, TtheMTT5, TrosMTT1, TpigMT-1, and ThegMT1 present a bimodular structure, TtheMTT1, TtheMTT3, TtroMTT1, TvorMT1, and TmobMT1 have three modules, ThegMT2 has four modules, and TpyrMT-2 (the longest MT) has five modules (Fig. 1). These modules appear to consist of two types of submodule: type 1 submodules have the consensus sequence C2–3X6C2X6 (with few exceptions), whereas the complete type 2 submodules might be represented as C2X6–8 + CXCXXCXC1–2X1–2 (approximately half of a type 1 submodule plus the C-terminus) (where the last “X” is K in approximately 48%, Q in 45%, or E/N in 3%). In most cases, these modules are formed by two type 1 submodules and one type 2 submodule (Fig. 1), although several exceptions are observed, such as TpyrMT-2 (which has four modules formed by only one type 1 submodule and half of a type 2 submodule). The second or third module of several CdMTs has two type 1 submodules and half of a type 2 submodule (Fig. 1).

Fig. 1
figure 1

Modular structure of Tetrahymena subfamily 7a (Cd metallothioneins, CdMT). Multiple alignments were made using the T-coffee program, followed by visual inspection and manual adjustment. See the text for further explanation. Areas shaded in gray show conserved Cys residues in submodules. Areas shaded in blue and yellow show Lys (K) or Gln (Q) residues, respectively. His (H) residues are shown by red shading. sm1 type 1 submodule, C-ter C-terminus

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Tetrahymena CuMTs (subfamily 7b) do not have such clear modular structures as CdMTs, but from their alignment a structural organization based on CKCX2–5CXC repeats (where in a few cases Lys may be substituted by other amino acids) might be considered [23].

Another independent structural feature that also differentiates both Tetrahymena MT subfamilies is the physical relationship of Lys to Cys residues along the polypeptide backbone. The Lys residues of CuMTs have a strong tendency to be juxtaposed with Cys (CKC), as occurs in mammalian MTs. On the other hand, in subfamily 7a (CdMTs), Cys-juxtaposed Lys residues are more scarce and are concentrated in type 2 submodules. It has been reported that having an adjacent basic residue considerably decreases the pK value and consequently the reactivity of Cys residues [42]. Therefore, it is possible that Tetrahymena CuMTs may have a metal buffering capacity that differs from the one theoretically assigned to them.

All these differential structural features of Tetrahymena MTs have led to them being divided into two well-characterized subfamilies (7a, or CdMTs, and 7b, or CuMTs) [22]. The proposed phylogenetic tree obtained from the alignments of amino acid sequences [23] corroborates this classification.

Gene duplication is one of the main steps in the generation and evolution of new genes and seems to be the main mechanism involved in MT evolution, as noted by several authors [43–45]. This assumption is easily deduced from the existence of conserved Cys pattern repeats throughout the MT polypeptide molecule. A similar hypothesis has been reported to explain Tetrahymena MT evolution [22, 23, 30, 46], and an updated model to explain the possible evolutionary history of Tetrahymena CdMTs has recently been reported [8].

Gene expression and multistress response

MTs can be induced by an extensive range of different stimuli or environmental stressors, such as metals, oxidative stress, heat or cold shocks, hormones, cytokines, pH changes, starvation, and a large variety of chemicals or drugs. For this reason, these proteins are considered as multistress proteins. MTs do not seem to be essential proteins, but rather provide survival advantages during times of stress [47, 48].

Likewise, the Tetrahymena MT family presents a multistress response because its gene expression can be induced by very different stressors [22–24, 30, 49]. Comparisons of quantitative real-time PCR values obtained for Tetrahymena MT genes by various authors is not easy, owing to differences in experimental conditions and/or quantitative reverse transcription PCR (qRT-PCR) protocols. However, general qualitative considerations may be inferred from already published qRT-PCR data (Table 3). In general, the induction values of Tetrahymena CdMT genes caused by Cd are higher than those caused by Cu (Cd > Cu), whereas in the only CuMT gene analyzed by qRT-PCR (TrosMTT2) to date the average induction value caused by Cu is higher than that caused by Cd (Cu > Cd) (Table 3). Secondly, the ranking of relative fold-induction values can change depending on the duration of metal treatment. When arsenate (As5+) is used, it occupies the second or third place of the fold-induction ranking for Tetrahymena CdMTs (Table 3); however, the CuMT gene (TrosMTT2) is downregulated after 1 or 24 h treatment. Thirdly, Pb induces the expression of Tetrahymena MT genes and occupies first or third place in the relative fold-induction ranking. Several Tetrahymena CdMT genes achieve their highest expression levels under Pb treatment, such as TtheMTT5 and TrosMTT1 [22, 23]. MTs are induced by Pb in rats, humans, and fishes [50–52]. Pb is second to Cd in its ability to displace Zn from hepatic ZnMT and can displace Cd from the CdMT complex [53, 54]. In plants, transcriptome analysis reveals that many genes respond similarly to Pb and Cd [55]. It has recently [56] been reported that La3+ induces the expression of both TtheMTT1 and TtheMTT2 genes, whereas fluorescence analysis indicates that La3+ binds to both T. thermophila MTs.

Table 3 Comparison of relative fold-induction rank of Tetrahymena MT genes under different heavy metal stress, obtained by quantitative reverse transcription PCR analysis
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Oxidative stress caused by H2O2 or paraquat induces MT gene expression, including both Tetrahymena CdMT and CuMT genes [22–24, 30, 49]. It has also been suggested that TtheMTT1 and TtheMTT2 have antioxidant ability, after the appearance of disulfide bonds from the CdMT complexes was detected when they reacted with NO in vitro [40]. However, other Tetrahymena CdMT genes (TpyrMT-2 and TrosMTT1) are not significantly induced by H2O2 or paraquat [23, 24]. Starvation (the most common environmental stress), mainly after 24 h exposure, induces the gene expression of several Tetrahymena MT genes [22, 23]. The TtheMTT5 gene is expressed during ciliate conjugation (a sexual process induced by starvation), as revealed by the existence of several expressed sequence tags encoding this gene, isolated from conjugating cells [22].

Although the multistress character of Tetrahymena MTs is evident, a differential gene expression level exists under the same stressor among different MT isoforms from the same subfamily and the same Tetrahymena species, and among different species, as seen in different mammalian MT isoforms [57, 58]. The three CdMT isoform genes from T. thermophila present differential expression patterns. TtheMTT5 and TtheMTT1 seem to be general stress and metal detoxification MTs. Transformed strains of T. thermophila (GFPMTT5) with the plasmid construct P MTT1 ::GFP::MTT5 (which includes the MTT1 promoter, green fluorescent protein as the reporter gene, and the MTT5 open reading frame) have shown themselves to be about 10 times more resistant to Cd with regard to the wild-type strain (unpublished data), indicating that an increase in the CdMT (TtheMTT5) gene dose affects the Cd lethal concentration killing 50% of the cell population (LC50) value of this ciliate. It also indicates that the main function of this CdMT (TtheMTT5) is probably heavy metal detoxification. The TtheMTT3 isoform (a His-containing CdMT that can enhance the relative affinity for Zn, in comparison with Cd) could be involved in the intracellular homeostasis of Zn.

An interspecific stress response diversity is shown among MT genes of T. thermophila and T. rostrata with regard to heat-shock stress. Both TrosMTT1 and TrosMTT2 genes are considerably overexpressed at 42 °C (more than 100-fold induction), whereas T. thermophila MTs either show a low expression or are not expressed at all at this temperature [22, 23, 49]. These differences might be explained by the consideration that T. rostrata is a facultative parasite in various vertebrates and is probably more sensitive to temperature changes than other Tetrahymena species [59].

The induction of transcription of several MT genes by a variety of stress conditions suggests that cell exposure to one type of stress may lead to the acquisition of tolerance against another stress type. This cross-protection, detected in some organisms, suggests that there is at least partial overlap between genome responses to different types of stress [60]. This overlapping genome expression comprises general stress-responsive genes, such as heat-shock or MT genes.

MT genes of many organisms are mainly regulated at the transcriptional level [58]. Very few cis-acting regulatory elements have been identified experimentally in Tetrahymena. An MT conserved motif (MTCM1) was identified by in silico analysis in the putative promoter region of the three T. thermophila CdMT isoforms and the promoter region of the TpyrMT-1 gene [22]. Most of the MTCM1 copies include a sequence (TGANTCA, where “N” means any nucleotide) that is similar to the sequence (TGA[G/C]TCA) binding the eukaryotic AP-1 transcription factor [61]. In S. cerevisiae an AP-1 transcription factor (YAP-1) is involved in response to oxidative stress and metal resistance [62]. Likewise, transcription factors similar to AP-1 have been detected in the MT promoters of insects (Drosophila melanogaster) and mollusks (C. virginica) [37]. AP-1, also known as c-jun, is a member of the bZIP superfamily of eukaryotic DNA binding transcription factors.

This MTCM1 motif is present six times in TtheMTT1, twice in TtheMTT3, and 13 times in TtheMTT5 [22]. The promoter region of TtheMTT5 has a 416-bp tandem duplication (96% identical to one another); five copies of MTCM1 are present within each duplication and another three copies are outside the duplications and nearer the start codon. The quantitative expression analysis of the three T. thermophila CdMT genes showed that TtheMTT5 is the most strongly induced, whereas TtheMTT3 is the least induced by diverse environmental stressors (TtheMTT5 ≫ TtheMTT1 > TtheMTT3) [22]. The presence of the duplicated promoter region in TtheMTT5 might be related to the high expression level of this CdMT gene when compared with TtheMTT1 and TtheMTT3 genes. Likewise, the number of real polyA sites, detected from different cDNA libraries (seven in TtheMTT5, two in TtheMTT1, and one in TtheMTT3), might show different messenger RNA formation capacity levels.

Biotechnological use of ciliate metallothioneins

Bioassays and biosensors are good tools for assessing metal pollution because they have the ability to react with or detect only the available fraction of metal ions, whereas common analytical methods are unable to distinguish whether fractions of metals are available or unavailable to biological systems [63]. Tetrahymena MTs might be good tools (biological sensing elements) for developing classic biosensors because these proteins are larger than other MTs and have a higher metal binding capacity. In environmental biomonitoring, global parameters such as bioavailability, toxicity, and genotoxicity cannot be tested using molecular recognition or chemical analysis, and can only be assayed using whole cells. Over the last 10 years or so, the concept of the whole-cell biosensor (WCB) has been introduced by several authors as a very useful alternative to classical biosensors [64]. A WCB uses the whole prokaryotic or eukaryotic cell as a single reporter, incorporating both bioreceptor and traducer elements. In general, living systems used as WCBs are cells that are experimentally modified to incorporate the transducer capacity. Two types of bioassays using WCBs may be considered: “turn off” and “turn on” assays. In “turn on” assays, a quantifiable molecular reporter is fused to a specific gene promoter known to be activated by the target environmental pollutant (such as heavy metals). Some of the best gene promoter candidates to make a WCB are Tetrahymena MT gene promoters (such as those from TtheMTT1 and TtheMTT5 genes), which respond very quickly (less than 1 h) and strongly to heavy metal exposure. Indeed, both TtheMTT1 and TtheMTT2 promoters have already been used for overexpression of homolog and heterolog genes in T. thermophila, and are therefore important biotechnological tools for expressing eukaryotic proteins, some of which are difficult to express in prokaryotic cells [26, 31, 65, 66]. Recently, we have reported the first two ciliate WCBs to detect heavy metals [67]. These WCBs (MTT1Luc and MTT5Luc) are based on MT promoters from TtheMTT1 or TtheMTT5 genes of T. thermophila linked with the eukaryotic luciferase gene as the reporter gene (MTT1::LucFF and MTT5::LucFF). These transformed strains were used to design “turn on” bioassays to detect bioavailable heavy metals in polluted soil and aquatic samples in about 2 h. Validation of these WCBs was conducted using both artificial and natural samples, including methods for detecting false positives and negatives. The main features of these WCBs can be summarized in the following points:

  1. 1.

    Luciferase activity in MTT1Luc and MTT5Luc can be measured as efficiently in intact viable cells as in permeabilized cells, and a similar induction is detected with these in vivo and in vitro approaches [67]. This indicates that the luciferin uptake in Tetrahymena is more efficient than in other eukaryotic model systems that have been used as WCBs (e.g., WCBs based on S. cerevisiae or Caenorhabditis elegans require that cells first be permeabilized) [68, 69].

  2. 2.

    In both WCBs, all metals tested induce luciferase expression at concentrations lower than their respective LC50 values. Although both strains are effective WCBs, important differences between MTT1Luc and MTT5Luc exist: MTT5Luc responds more strongly to diverse heavy metals than MTT1Luc and a differential response for each type of metal has likewise been reported [67]. In general, these results are consistent with qRT-PCR analysis of the T. thermophila CdMT genes after similar metal treatments [22].

  3. 3.

    Experiments with previous EDTA exposures show that these WCBs only respond to bioavailable metals.

  4. 4.

    A comparison of the Tetrahymena MTT1Luc and MTT5Luc sensitivity levels with the sensitivity levels of eukaryotic or prokaryotic WCBs already published [67] shows that both Tetrahymena WCBs are often the most sensitive microbial eukaryotic metal biosensors, sometimes exceeding available bacterial WCBs as well.

  5. 5.

    Although a large number of metal WCBs have been developed, most have not been evaluated using natural samples [70]. Both MTT1Luc and MTT5Luc can detect bioavailable heavy metals in natural soil or aquatic samples, including samples with metal concentrations lower than the maximum values established by a European Union directive [67].

This first ciliate WCB might be the starting point for a more extensive use of these eukaryotic microbial models to design classic biosensors or WCBs for detecting diverse organic or inorganic environmental pollutants or biotechnologically relevant substances.

Concluding remarks

Ciliate MTs have unique features compared with MTs from other organisms. The most important of these are (1) an unusually high molecular mass and length with regard to most known MTs; (2) the presence of new Cys clusters, mainly CCC clusters; (3) a considerable number of Cys residues, allowing a greater total number of metal ions with which these MTs can bind in comparison with other MTs; (4) a highly conserved Cys periodic pattern, which divides amino acid sequences into modules and submodules; (5) two well-characterized and separate subfamilies with well-differentiated features; and (6) a fast and strong induction level of the gene expression, which constitutes a good molecular tool for designing biosensors of interest for environmental or biotechnological applications.