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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

During the long history of co-evolution with protists, bacteria have evolved defense strategies to avoid grazing and survive phagocytosis. These mechanisms allow bacteria to exploit phagocytic cells as a protective niche in which to escape from environmental stress and even replicate. Importantly, these anti-grazing mechanisms can function as virulence factors when bacteria infect humans. Here, we discuss how protozoan predation exerts a selective pressure driving bacterial virulence and shaping their genomes, and how bacteria-protist interactions might contribute to the spread of antibiotic resistance as well. We provide examples to demonstrate that besides being voracious bacterial predators, protozoa can serve as melting pots where intracellular organisms exchange genetic information, or even “training grounds” where some pathogens become hypervirulent after passing through. In this special issue, we would like to emphasize the tremendous impact of bacteriaprotist interactions on human health and the potential of amoebae as model systems to study biology and evolution of a variety of pathogens. Besides, a better understanding of bacteria-protist relationships will help us expand our current understanding of bacterial virulence and, likely, how pathogens emerge., Ministerio de Economía y Competitividad (MINECO), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

El proyecto de innovación tiene como principal objetivo implementar la metodología de enseñanza-aprendizaje blended learning en las clases prácticas de la asignatura “Biología de la contaminación” (Grado en Biología UCM, cuarto curso) creando una colección de laboratorios virtuales en sinergia con las sesiones de docencia práctica presencial. De esta forma la colección de recursos digitales que reune este proyecto complementará la formación que reciben los alumnos en las clases prácticas de laboratorio de la asignatura, y permitirá adaptar las prácticas a un formato de educación online o semipresencial en el contexto de situaciones similares a la actual crisis sanitaria derivada de la COVID-19.

In this mini-review, the potential of using microorganisms to design biosensors for detecting environmental pollutants is analyzed and discussed. A distinction is made between a classical biosensor (CB) and a whole-cell biosensor (WCB), emphasizing their structural components and the possibility of using whole microorganisms as their bioreceptor elements. The advantages and disadvantages of using prokaryotic microorganisms as opposed to eukaryotic microorganisms are described. Likewise, the advantages of using protozoa ciliates) over other eukaryotic microorganisms are also shown. We analyze the current bibliography on biosensors built on microorganisms as bioreceptors of pollutant molecules, such as inorganic (metal (loid)s) or organic (xenobiotics). New trends, such as the prokaryotic riboswitches, microbial two-component systems where the pollutant can be simultaneously detected and bioremediated, along with advances in synthetic biology, are shown as promising tools in the design of environmental biosensors.

Metallothioneins (MT) are multi-stress proteins mainly involved in metal detoxification. MT gene expression is normally induced by a broad variety of stimulus and its gene expression regulation mainly occurs at a transcriptional level. Conserved motifs in the Tetrahymena thermophila MT promoters have been described. These motifs show a consensus sequence very similar to AP-1 sites, and bZIP type transcription factors might participate in the MT gene expression regulation. In this research work, we characterize four AP-1 transcription factors in each of four different analyzed Tetrahymena species, detecting a high conservation among them. Each AP-1 molecule has its counterpart in the other three Tetrahymena species. A comparative qRT-PCR analysis of these AP-1 genes have been carried out in different T. thermophila strains (including metal-adapted, knockout and/or knockdown strains among others), and under different metal-stress conditions (1 or 24 h Cd2+, Cu2+, or Pb2+ treatments). The possible interaction of these transcription factors with the conserved AP-1 motifs present in MT promoters has been corroborated by protein-DNA interaction experiments. Certain connection between the expression patterns of the bZIP and MT genes seems to exist. For the first time, and based on our findings, a possible gene expression regulation model including both AP-1 transcription factors and MT genes from the ciliate T. thermophila has been elaborated., Ministerio de Economía y Competitividad (MINECO), Ministerio de Educación, Cultura y Deporte (MECD), 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.

The metal binding preference of metallothioneins (MTs) groups them in two extreme subsets, the Zn/Cd- and the Cu-thioneins. Ciliates harbor the largest MT gene/protein family reported so far, in-cluding 5 paralogs that exhibit relatively low sequence similarity, excepting MTT2 and MTT4. In Tet-rahymena thermophila, three MTs (MTT1, MTT3 and MTT5) were considered Cd-thioneins and two (MTT2 and MTT4) Cu-thioneins, according to gene expression inducibility and phylogenetic analysis. In this study, the metal-binding abilities of the five MTT proteins were characterized, to obtain information about the folding and stability of their cognate- and non-cognate metal complexes, and to characterize the T. thermophila MT system at protein level. Hence, the five MTTs were recombinantly synthesized as Zn2+-, Cd2+- or Cu+-complexes, which were analyzed by electrospray mass spectrometry (ESI-MS), circular dichroism (CD), and UV-vis spectrophotometry. Among the Cd-thioneins, MTT1 and MTT5 were optimal for Cd2+ coordination, yielding unique Cd17- and Cd8- complexes, respectively. When binding Zn2+, they rendered a mixture of Zn-species. Only MTT5 was capable to coordinate Cu+, although yielding heteronuclear Zn-, Cu-species or highly unstable Cu-homometallic species. MTT3 exhibited poor binding abilities both for Cd2+ and for Cu+, and although not optimally, it yielded the best result when coordinating Zn2+. The two Cu-thioneins, MTT2 and MTT4 isoforms formed homometallic Cu-complexes (major Cu20-MTT) upon synthesis in Cu-supplemented hosts. Contrarily, they were unable to fold into stable Cd-complexes, while Zn-MTT species were only recovered for MTT4 (major Zn10-MTT4). Thus, the metal binding preferences of the five T. thermophila MTs correlate well with their previous classification as Cd- and Cu-thioneins, and globally, they can be classified from Zn/Cd- to Cu-thioneins according to the gradation: MTT1>MTT5>MTT3>MTT4>MTT2. The main mechanisms underlying the evolution and special, Ministerio de Economía y Competitividad (MINECO), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

This review analyzes the advantages and disadvantages of using eukaryotic microorganisms to design whole-cell biosensors (WCBs) for monitoring environmental heavy metal pollution in soil or aquatic habitats. Basic considerations for designing a eukaryotic WCB are also shown. A comparative analysis of the promoter genes used to design WCBs is carried out, and the sensitivity and reproducibility of the main reporter genes used is also reviewed. Three main eukaryotic taxonomic groups are considered: yeasts, microalgae, and ciliated protozoa. Models that have been widely analyzed as potential WCBs are the Saccharomyces cerevisiae model among yeasts, the Tetrahymena thermophila model for ciliates and Chlamydomonas model for microalgae. The advantages and disadvantages of each microbial group are discussed, and a ranking of sensitivity to the same type of metal pollutant from reported eukaryotic WCBs is also shown. General conclusions and possible future developments of eukaryotic WCBs are reported., Ministerio de Economía y Competitividad (MINECO), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

La evaluación de la contaminación por metales pesados se debe realizar en base a parámetros como la biodisponibilidad de metales, su toxicidad y genotoxicidad. Para ello se utilizan los llamado biosensores celulares, y los ciliados, como microorganismos eucariotas móviles y sin pared celular, constituyen una excelente herramienta para ello. Aunque T. thermophila presenta un gen codificante (TtPCS) para una proteína homóloga a fitoquelatín sintasas, la ausencia de fitoquelatinas en este ciliado, demostrada en este trabajo, hace pensar que el principal mecanismo de resistencia a metales pesados (bioacumulación) en T. thermophila, consiste en la inmovilización de los cationes metálicos en el citoplasma por metalotioneínas (MTs), cuya síntesis se induce rápidamente cuando la célula es expuesta a metales y otras condiciones de estrés celular. Actualmente se conocen once MTs en distintas especies del género Tetrahymena, y de acuerdo a sus características estructurales, filogenéticas y el patrón de expresión de los genes que las codifican, las hemos agrupado en dos subfamilias distintas: 7a (Cd-MTs) y 7b (Cu-MTs). Las Cd-MTs de Tetrahymena comparten una marcada estructura modular, que podría haber contribuido al inusual tamaño de estas MTs, superior respecto a las MTs del resto de organismos. Aprovechando la gran capacidad de respuesta a metales de los promotores de MTT1 y MTT5 de T. thermophila hemos construido dos biosensores celulares, que bajo control de estos promotores sobreexpresan el gen de la luciferasa eucariota (lucFF) cuando la célula es expuesta a distintos metales pesados. Las cepas MTT1Luc y MTT5Luc representan los biosensores eucariotas capaces de detectar las menores concentraciones de metales pesados no esenciales (Cd, Pb, As, Hg), con una sensibilidad de detección de 5-50 nM dependiendo del metal. Además, los biosensores construidos son capaces de detectar la presencia de metales pesados en forma biodisponible en distintas muestras ambientales de origen