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A strain of the protozoan ciliate Tetrahymena thermophila adapted to increasing Pb(II) concentrations over two years has shown that one of the resistance mechanisms to this extreme metal stress is the lead biomineralization to chloropyromorphite, one of the most stable minerals in the earth's crust. Several techniques such as microanalysis coupled to transmission and scanning electron microscopy (X-Ray Energy Disperse Spectroscopy), fluorescence microscopy and X-ray power diffraction analysis have revealed the presence of chloropyromorphite as crystalline aggregates of nano-globular structure, together with the presence of other secondary lead minerals. This is the first time that the existence of this type of biomineralization in a ciliate protozoan is described. The Pb(II) bioremediation capacity of this strain has shown that it can remove >90 % of the toxic soluble lead from the medium. A quantitative proteomic analysis of this strain has revealed the main molecular-physiological elements involved in adaptation to Pb(II) stress: increased activity of proteolytic systems against lead proteotoxicity, occurrence of metallothioneins to immobilize Pb(II) ions, antioxidant enzymes to mitigate oxidative stress, and an intense vesicular trafficking presumably involved in the formation of vacuoles where pyromorphite accumulates and is subsequently excreted, together with an enhanced energy metabolism. As a conclusion, all these results have been compiled into an integrated model that could explain the eukaryotic cellular response to extreme lead stress., Ministerio de Ciencia y Competitividad, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Among the epigenetic mechanisms based on non-coding RNA are microRNAs (miRNAs) that are involved in the post-transcriptional regulation of mRNAs. In many organisms, the expression of genes involved in the cellular response to biotic or abiotic stress depends on the regulation, generally inhibitory, performed by miRNAs. For the first time in the eukaryotic microorganism (ciliate-model) Tetrahymena thermophila, miRNAs involved in the posttranscriptional regulation of transcripts linked to the response to cadmium have been isolated and analyzed. Forty de novo miRNAs (we named tte-miRNAs) have been isolated from control and Cd-treated populations (1 or 24 h exposures). An exhaustive comparative analysis of the features of these mature tte-miRNAs and their precursor sequences (pre-tte-miRNAs) confirms that they are true miRNAs. In addition to the three types of miRNA isoforms previously described in other organisms, two new types are also described among the ttemiRNAs studied. A certain percentage of the pre-tte-miRNA sequences are in introns from genes with many introns, and have been defined as 5′, 3′-tailed mirtrons. A qRT-PCR analysis of selected tte-miRNAs together with some of their targets has validated them. Cd is one of the most toxic metals for the cell, which must defend itself against its toxicity by various mechanisms, such as expulsion by membrane pumps, chelation by metallothioneins, among others. Like other toxic metals, Cd also causes a well-known series of cellular effects such as intense proteotoxicity. Many of the targets that are regulated by the tte-miRNAs are transcripts encoding proteins that fit into these defense mechanisms and toxic metal effects., Ministerio de Economía y Competitividad (España), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

CRUE-CSIC (Acuerdos Transformativos 2022), A comparative analysis of toxicities of both arsenic forms (arsenite and arsenate) in the model eukaryotic microorganism Tetrahymena thermophila (ciliate protozoa) has shown the presence of various detoxification mechanisms and cellular effects comparable to those of animal cells under arsenic stress. In the wild type strain SB1969 arsenate is almost 2.5 times more toxic than arsenite. According to the concentration addition model used in binary metallic mixtures their toxicities show an additive effect. Using fluorescent assays and flow cytometry, it has been detected that As(V) generates elevated levels of ROS/RNS compared to As(III). Both produce the same levels of superoxide anion, but As(V) also causes greater increases in hydrogen peroxide and peroxynitrite. The mitochondrial membrane potential is affected by both As(V) and As(III), and electron microscopy has also revealed that mitochondria are the main target of both arsenic ionic forms. Fusion/fission and swelling mitochondrial and mitophagy, together with macroautophagy, vacuolization and mucocyst extruction are mainly associated to As(V) toxicity, while As(III) induces an extensive lipid metabolism dysfunction (adipotropic effect). Quantitative RT-PCR analysis of some genes encoding antioxidant proteins or enzymes has shown that glutathione and thioredoxin metabolisms are involved in the response to arsenic stress. Likewise, the function of metallothioneins seems to be crucial in arsenic detoxification processes, after using both metallothionein knockout and knockdown strains and cells overexpressing metallothionein genes from this ciliate. The analysis of the differential toxicity of As(III) and As(V) shown in this study provides cytological and molecular tools to be used as biomarkers for each of the two arsenic ionic forms., Ministerio de Economía y Competitividad (España), Depto. de Biodiversidad, Ecología y Evolución, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

In the present review, we discussed the concept of metallothionein (MT) and analyzed the structural and functional diversity of microbial MTs, grouping them into three main groups; bacteria, fungi, and protists. Structural diversity is analyzed based on the primary, secondary, and/or tertiary structure of the proteins. Their ability to bind different metals is also analyzed in each microbial group. An in silico and phylogenetic analysis of MT sequences from the main microbial groups has been carried out. The wide functional diversity of these proteins and the regulation of the MT gene expression are also discussed. The presence of several paralog genes in many microorganisms provides a coordinated and multiple response against different types of environmental stressors. Likewise, the main possible biotechnological applications of these proteins are explored, such as molecular tools to design biosensors for evaluating metal contamination and in metal bioremediation.