Person: Gutiérrez Fernández, Juan Carlos
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First Name
Juan Carlos
Last Name
Gutiérrez Fernández
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Biológicas
Department
Genética, Fisiología y Microbiología
Area
Microbiología
Identifiers
9 results
Search Results
Now showing 1 - 9 of 9
Item Structural and Functional Diversity of Microbial Metallothionein Genes(Microbial Diversity in the Genomic Era, 2018) Gutiérrez Fernández, Juan Carlos; Francisco Martínez, Patricia de; Amaro Torres, Francisco; Díaz, Silvia; Martín González, Ana María; Das, Surajit; Dash, Hirak RanjanIn 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.Item MicroRNAs in Tetrahymena thermophila: an epigenetic regulatory mechanism in the response to cadmium stress(Microbiological Research, 2023) Gonzalez David; Amaro Torres, Francisco; Gutiérrez Fernández, Juan CarlosAmong 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.Item Quantitative proteomic analyses of a Pb-adapted Tetrahymena thermophila strain reveal the cellular strategy to Pb(II) stress including lead biomineralization to chloropyromorphite(Science of the Total Environment, 2023) De Francisco Martínez, Patricia; Amaro Torres, Francisco; Martín González, Ana María; Aurelio Serrano; Gutiérrez Fernández, Juan CarlosA 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.Item AP-1 (bZIP) Transcription Factors as Potential Regulators of Metallothionein Gene Expression in Tetrahymena thermophila(Frontiers in Genetics, 2018) Francisco Martínez, Patricia de; Amaro Torres, Francisco; Martín González, Ana María; Gutiérrez Fernández, Juan CarlosMetallothioneins (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.Item Hints for Metal-Preference Protein Sequence Determinants: Different Metal Binding Features of the Five Tetrahymena thermophila Metallothioneins(International Journal of Biological Sciences, 2015) Espart, Anna; Marín, Maribel; Gil Moreno, Selene; Palacios, Óscar; Amaro Torres, Francisco; Martín González, Ana María; Gutiérrez Fernández, Juan Carlos; Capdevilla, Mercé; Atrian, SílviaThe 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 specialization of the MTT metal binding preferences may have been in-ternal tandem duplications, presence of doublet and triplet Cys patterns in Zn/Cd-thioneins, and op-timization of site specific amino acid determinants (Lys for Zn/Cd- and Asn for Cu-coordination).Item Heavy metal whole-cell biosensors using eukaryotic microorganisms: An updated critical review(Frontiers in Microbiology, 2015) Gutiérrez Fernández, Juan Carlos; Amaro Torres, Francisco; Martín González, Ana MaríaThis 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.Item Extreme metal adapted, knockout and knockdown strains reveal a coordinated gene expression among different Tetrahymena thermophila metallothionein isoforms(PLoS ONE, 2017) Francisco Martínez, Patricia de; Martín González, Ana María; Turkewitz, Aaron P.; Gutiérrez Fernández, Juan CarlosMetallothioneins (MT) constitute a superfamily of small cytosolic proteins that are able to bind metal cations through numerous cysteine (Cys) residues. Like other organisms the ciliate Tetrahymena thermophila presents several MT isoforms, which have been classified into two subfamilies (Cd- and Cu-metallothioneins). The main aim of this study was to examine the specific functions and transcriptional regulation of the five MT isoforms present in T. thermophila, by using several strains of this ciliate. After a laboratory evolution experiment over more than two years, three different T. thermophila strains adapted to extreme metal stress (Cd2+, Cu2+ or Pb2+) were obtained. In addition, three knockout and/or knockdown strains for different metallothionein (MT) genes were generated. These strains were then analyzed for expression of the individual MT isoforms. Our results provide a strong basis for assigning differential roles to the set of MT isoforms. MTT1 appears to have a key role in adaptation to Cd. In contrast, MTT2/4 are crucial for Cu-adaptation and MTT5 appears to be important for Pb-adaptation and might be considered as an “alarm” MT gene for responding to metal stress. Moreover, results indicate that likely a coordinated transcriptional regulation exists between the MT genes, particularly among MTT1, MTT5 and MTT2/4. MTT5 appears to be an essential gene, a first such report in any organism of an essential MT gene.Item Environmental Biosensors: A Microbiological View(Handbook of Cell Biosensors, 2020) Gutiérrez Fernández, Juan Carlos; Amaro Torres, Francisco; Díaz del Toro, Silvia; Martín González, Ana María; Thouand, G.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.Item Cellular response of adapted and non-adapted Tetrahymena thermophila strains to europium Eu(III) compounds(Biology, 2024) Alonso, Patricia; Blas, Javier; Amaro Torres, Francisco; De Francisco Martínez, Patricia; Martín González, Ana María; Gutiérrez Fernández, Juan CarlosEuropium is one of the most reactive lanthanides and humans use it in many different applications, but we still know little about its potential toxicity and cellular response to its exposure. Two strains of the eukaryotic microorganism model Tetrahymena thermophila were adapted to high concentrations of two Eu(III) compounds (EuCl3 or Eu2O3) and compared to a control strain and cultures treated with both compounds. In this ciliate, EuCl3 is more toxic than Eu2O3. LC50 values show that this microorganism is more resistant to these Eu(III) compounds than other microorganisms. Oxidative stress originated mainly by Eu2O3 is minimized by overexpression of genes encoding important antioxidant enzymes. The overexpression of metallothionein genes under treatment with Eu(III) compounds supports the possibility that this lanthanide may interact with the -SH groups of the cysteine residues from metallothioneins and/or displace essential cations of these proteins during their homeostatic function. Both lipid metabolism (lipid droplets fusing with europium-containing vacuoles) and autophagy are involved in the cellular response to europium stress. Bioaccumulation, together with a possible biomineralization to europium phosphate, seems to be the main mechanism of Eu(III) detoxification in these cells.