27 results on '"Fiorentino, Gabriella"'
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2. Additional file 3 of Prebiotic properties of Bacillus coagulans MA-13: production of galactoside hydrolyzing enzymes and characterization of the transglycosylation properties of a GH42 β-galactosidase
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Aulitto, Martina, Strazzulli, Andrea, Sansone, Ferdinando, Cozzolino, Flora, Monti, Maria, Moracci, Marco, Fiorentino, Gabriella, Limauro, Danila, Bartolucci, Simonetta, and Contursi, Patrizia
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Additional file 3: Table S2. Purification table of BcGalB. Table S3. Relative activity of BcGalB in presence of chemicals (PPTX 43 KB)
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- 2021
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3. Food waste mining within circular agro-industry. The value chain of wine, olive oil, and dairy product
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Oliveira, Mariana, Santagata, Remo, Ncube, Amos, Colella, Maria, Panfilo, Carolina, Coccozza, Annalisa, Fiorentino, Gabriella, Zucaro, Amalia, and Ulgiati, Sergio
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- 2019
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4. MOESM1 of Characterization of a promiscuous cadmium and arsenic resistance mechanism in Thermus thermophilus HB27 and potential application of a novel bioreporter system
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Antonucci, Immacolata, Gallo, Giovanni, Limauro, Danila, Contursi, Patrizia, Ribeiro, Ana, Blesa, Alba, JosĂŠ Berenguer, Bartolucci, Simonetta, and Fiorentino, Gabriella
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Additional file 1: Table S1. Strains used in this work classified according to their genotype. Table S2. Oligonucleotides used in this work. Table S3. Plasmids used in this work classified according to their features. Figure S1. Growth curves of T. thermophilus HB27 transformed with the vector pMHTtarsXpbgaA in the absence (circle) and presence of 100 ÎźM Cd(II) (triangle).
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- 2018
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5. MOESM1 of Thermus thermophilus as source of thermozymes for biotechnological applications: homologous expression and biochemical characterization of an Îą-galactosidase
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Aulitto, Martina, Fusco, Salvatore, Fiorentino, Gabriella, Limauro, Danila, Pedone, Emilia, Bartolucci, Simonetta, and Contursi, Patrizia
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Additional file 1: Table S1. Purification table of EcGalA. Table S2. Purification table of TtGalA.
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- 2017
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6. L'attività delle dogane comunitarie: procedure e regimi
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Fiorentino, Gabriella
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Evoluzione del processo di armonizzazione comunitaria in materia doganale, con particalare riferimento alle procedure ed ai regimi doganali.
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- 2015
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7. Oxygen: friend or foe? Archaeal superoxide dismutases in the protection of intra- and extracellular oxidative stress
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CANNIO R., FIORENTINO, GABRIELLA, MORANA A., ROSSI M., BARTOLUCCI, SIMONETTA, Cannio, R., Fiorentino, Gabriella, Morana, A., Rossi, M., and Bartolucci, Simonetta
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chemistry.chemical_classification ,Reactive oxygen species ,Antioxidant ,biology ,Cellular respiration ,Chemistry ,Superoxide ,Superoxide Dismutase ,medicine.medical_treatment ,Oxidative phosphorylation ,medicine.disease_cause ,Archaea ,Superoxide dismutase ,Oxygen ,chemistry.chemical_compound ,Oxidative Stress ,Biochemistry ,medicine ,Extracellular ,biology.protein ,antioxidant defense system ,Oxidative stress - Abstract
Both "environmental chemistry" and metabolic biochemical reactions can constantly generate in vivo free radicals and other oxygen-derived species that can cause severe damage to almost all biomolecules, especially to DNA, proteins, and lipids. The superoxide anion has been shown to be the most readily generated and spread radical among organisms and it is a common intermediate of oxidative stress processes in the cells. The antioxidant defense system of superoxide dismutases (SOD) scavenges and minimizes the formation of this radical, and thus plays a major role in reducing cumulative oxidative damage in different cell compartments both in aerobic and anaerobic cells. In the cell, cytosol SODs are constitutively present and induced by many oxidative agents able to raise the superoxide concentrations. Presence of SODs, however, in extracellular cell-associated locations demonstrates how valuable they are in maintaining the integrity of cells against oxidative stress generated by the cell environment, particularly upon increased oxygenation. Because SODs have recently been found in Archaea, which are prokaryotes, sometimes living in extreme environments, even in anaerobic ones, these enzymes can be considered essential: they may have allowed the evolution of aerobic respiration starting from an ancient form of oxygen-insensitive life.
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- 2000
8. Circular economy paths in the olive oil industry: a Life Cycle Assessment look into environmental performance and benefits
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Amos Ncube, Gabriella Fiorentino, Carolina Panfilo, Maria De Falco, Sergio Ulgiati, Ncube, Amo, Fiorentino, Gabriella, Panfilo, Carolina, DE FALCO, Maria, and Ulgiati, Sergio
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Circular economy ,Valorization of organic residues ,Linear production ,Life Cycle Assessment ,Olive oil ,Biorefinery ,Life Cycle Assessment · Olive oil · Valorization of organic residues · Biorefinery · Linear production · Circular economy ,General Environmental Science - Abstract
Purpose The olive oil sector in Italy has a significant socio-economic, environmental, and cultural relevance. However, the environmental impacts of production and consumption models are considerable, mainly due to the demand for large quanti- ties of resources (fuels, chemicals) and to the environmental impacts of residues’ disposal. Due to the scarcity of resources and climate change concerns, circular economy principles based on industrial ecology concepts are emerging. In this paper, the principles of circular economy were specifically applied to the olive oil supply chain, to improve the environmental sustainability of the sector. Methods The production chain of extra virgin olive oil was analyzed using the Life Cycle Assessment method, based on primary data from an oil farm and mill in Southern Italy. The environmental impacts were evaluated through the SimaPro software and the ReCiPe 2016 Mid-point (H) Impact Assessment Method, with reference to the functional unit of 1-L bot- tle of extra virgin olive oil. Some circular improvement options were investigated, comparing the impacts generated by (i) extra virgin olive oil linear production without valorization of by-products, (ii) extra virgin olive oil linear production with allocation of total impacts to co-products, and (iii) two circular production systems, incorporating improvements such as replacement of diesel with biodiesel and of electricity from the national grid with energy recovered from residues. Results and discussion The environmental impacts of the business-as-usual production pattern were identified for possible improvements. In all phases of the production chain of organic extra virgin olive oil, the most affected impact categories were human carcinogenic toxicity, marine ecotoxicity, and terrestrial ecotoxicity. As expected, the major contributions to almost all the analyzed impact categories were determined by the agricultural phase (92.65%), followed by the bottling phase (7.13%) and the oil extraction phase (0.22%). The valorization of by-products was considered by widening the system boundaries to ensure the environmental sustainability by developing circular patterns that feedback waste materials to upstream steps of the same process. The environmental impacts resulted lower in almost all the impact categories, with the major benefits gained in the global warming and fossil depletion impact categories. Conclusions The analysis proved that the reuse of pomace, prunings, and exhausted cooking oil initially considered as waste can bring benefits from an environmental point of view to the larger scale of the economy, by replacing fossil fuels, as well as to the olive oil chain itself, by providing the needed energy for production.
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- 2022
9. Insight into CAZymes of Alicyclobacillus mali FL18: Characterization of a New Multifunctional GH9 Enzyme
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Miriam Carbonaro, Martina Aulitto, Giovanni Gallo, Patrizia Contursi, Danila Limauro, Gabriella Fiorentino, Carbonaro, Miriam, Aulitto, Martina, Gallo, Giovanni, Contursi, Patrizia, Limauro, Danila, and Fiorentino, Gabriella
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Alicyclobacillu ,multifunctional glycosyl hydrolase ,lignocellulose waste ,Organic Chemistry ,General Medicine ,cellulose ,Catalysis ,Computer Science Applications ,Inorganic Chemistry ,thermozymes ,CAZyme ,renewable sources ,Alicyclobacillus ,CAZymes ,multifunctional glycosyl hydrolases ,Physical and Theoretical Chemistry ,renewable source ,Molecular Biology ,Spectroscopy - Abstract
In the bio-based era, cellulolytic and hemicellulolytic enzymes are biocatalysts used in many industrial processes, playing a key role in the conversion of recalcitrant lignocellulosic waste biomasses. In this context, many thermophilic microorganisms are considered as convenient sources of carbohydrate-active enzymes (CAZymes). In this work, a functional genomic annotation of Alicyclobacillus mali FL18, a recently discovered thermo-acidophilic microorganism, showed a wide reservoir of putative CAZymes. Among them, a novel enzyme belonging to the family 9 of glycosyl hydrolases (GHs), named AmCel9, was identified; in-depth in silico analyses highlighted that AmCel9 shares general features with other GH9 members. The synthetic gene was expressed in Escherichia coli and the recombinant protein was purified and characterized. The monomeric enzyme has an optimal catalytic activity at pH 6.0 and has comparable activity at temperatures ranging from 40 °C to 70 °C. It also has a broad substrate specificity, a typical behavior of multifunctional cellulases; the best activity is displayed on β-1,4 linked glucans. Very interestingly, AmCel9 also hydrolyses filter paper and microcrystalline cellulose. This work gives new insights into the properties of a new thermophilic multifunctional GH9 enzyme, that looks a promising biocatalyst for the deconstruction of lignocellulose.
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- 2022
10. Alicyclobacillus mali FL18 as a Novel Source of Glycosyl Hydrolases: Characterization of a New Thermophilic β-Xylosidase Tolerant to Monosaccharides
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Flora Salzano, Martina Aulitto, Gabriella Fiorentino, Emilia Pedone, Patrizia Contursi, Danila Limauro, Salzano, Flora, Aulitto, Martina, Fiorentino, Gabriella, Pedone, Emilia, Contursi, Patrizia, and Limauro, Danila
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Inorganic Chemistry ,thermophile ,Organic Chemistry ,β-xylosidase ,General Medicine ,Physical and Theoretical Chemistry ,Alicyclobacillus mali ,glycosyl hydrolase ,Molecular Biology ,thermophiles ,glycosyl hydrolases ,Spectroscopy ,Catalysis ,Computer Science Applications - Abstract
A thermo-acidophilic bacterium, Alicyclobacillus mali FL18, was isolated from a hot spring of Pisciarelli, near Naples, Italy; following genome analysis, a novel putative β-xylosidase, AmβXyl, belonging to the glycosyl hydrolase (GH) family 3 was identified. A synthetic gene was produced, cloned in pET-30a(+), and expressed in Escherichia coli BL21 (DE3) RIL. The purified recombinant protein, which showed a dimeric structure, had optimal catalytic activity at 80 °C and pH 5.6, exhibiting 60% of its activity after 2 h at 50 °C and displaying high stability (more than 80%) at pH 5.0–8.0 after 16 h. AmβXyl is mainly active on both para-nitrophenyl-β-D-xylopyranoside (KM 0.52 mM, kcat 1606 s−1, and kcat/KM 3088.46 mM−1·s−1) and para-nitrophenyl-α-L-arabinofuranoside (KM 10.56 mM, kcat 2395.8 s−1, and kcat/KM 226.87 mM−1·s−1). Thin-layer chromatography showed its ability to convert xylooligomers (xylobiose and xylotriose) into xylose, confirming that AmβXyl is a true β-xylosidase. Furthermore, no inhibitory effect on enzymatic activity by metal ions, detergents, or EDTA was observed except for 5 mM Cu2+. AmβXyl showed an excellent tolerance to organic solvents; in particular, the enzyme increased its activity at high concentrations (30%) of organic solvents such as ethanol, methanol, and DMSO. Lastly, the enzyme showed not only a good tolerance to inhibition by xylose, arabinose, and glucose, but was activated by 0.75 M xylose and up to 1.5 M by both arabinose and glucose. The high tolerance to organic solvents and monosaccharides together with other characteristics reported above suggests that AmβXyl may have several applications in many industrial fields.
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- 2022
11. Self-assembling thermostable chimeras as new platform for arsenic biosensing
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Alessandra Piscitelli, Giovanni Gallo, Gabriella Fiorentino, Ilaria Sorrentino, Paola Giardina, Rosanna Puopolo, Alan Le Goff, Département de Chimie Moléculaire (DCM), Université Grenoble Alpes (UGA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Puopolo, Rosanna, Sorrentino, Ilaria, Gallo, Giovanni, Piscitelli, Alessandra, Giardina, Paola, Le Goff, Alan, and Fiorentino, Gabriella
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0301 basic medicine ,Immobilized enzyme ,Arsenate Reductases ,Hydrophobin ,Molecular biology ,Science ,[SDV]Life Sciences [q-bio] ,Recombinant Fusion Proteins ,02 engineering and technology ,Biosensing Techniques ,medicine.disease_cause ,Pleurotus ,Article ,Arsenic ,Fungal Proteins ,03 medical and health sciences ,medicine ,Escherichia coli ,Electrochemistry ,[CHIM]Chemical Sciences ,Humans ,10. No inequality ,Multidisciplinary ,biology ,Chemistry ,Thermophile ,Thermus thermophilus ,021001 nanoscience & nanotechnology ,biology.organism_classification ,Enzymes, Immobilized ,Combinatorial chemistry ,Fusion protein ,030104 developmental biology ,Arsenate reductase ,enzyme immobilization, chimeric proteins, arsenic biosensor, hydrofobin, thermostable arsenate reductase, gold electrode ,Medicine ,0210 nano-technology ,Biosensor - Abstract
The correct immobilization and orientation of enzymes on nanosurfaces is a crucial step either for the realization of biosensors, as well as to guarantee the efficacy of the developed biomaterials. In this work we produced two versions of a chimeric protein, namely ArsC-Vmh2 and Vmh2-ArsC, which combined the self-assembling properties of Vmh2, a hydrophobin from Pleurotus ostreatus, with that of TtArsC, a thermophilic arsenate reductase from Thermus thermophilus; both chimeras were heterologously expressed in Escherichia coli and purified from inclusion bodies. They were characterized for their enzymatic capability to reduce As(V) into As(III), as well as for their immobilization properties on polystyrene and gold in comparison to the native TtArsC. The chimeric proteins immobilized on polystyrene can be reused up to three times and stored for 15 days with 50% of activity loss. Immobilization on gold electrodes showed that both chimeras follow a classic Langmuir isotherm model towards As(III) recognition, with an association constant (KAsIII) between As(III) and the immobilized enzyme, equal to 650 (± 100) L mol−1 for ArsC-Vmh2 and to 1200 (± 300) L mol−1 for Vmh2-ArsC. The results demonstrate that gold-immobilized ArsC-Vmh2 and Vmh2-ArsC can be exploited as electrochemical biosensors to detect As(III).
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- 2020
12. Biochemical characterization of a thermostable endomannanase/endoglucanase from Dictyoglomus turgidum
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Francesca Anna Fusco, Emilia Pedone, Simonetta Bartolucci, Danila Limauro, Gabriella Fiorentino, Patrizia Contursi, Raffaele Ronca, Fusco, FRANCESCA ANNA, Ronca, Raffaele, Fiorentino, Gabriella, Pedone, EMILIA MARIA, Contursi, Patrizia, Bartolucci, Simonetta, and Limauro, Danila
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Dictyoglomus turgidum ,Thermotolerance ,0301 basic medicine ,Glucomannan ,Cellulase ,medicine.disease_cause ,Microbiology ,Endomannanase, Endoglucanase, Thermophilic enzyme, Dictyoglomus turgidum ,Substrate Specificity ,Mannans ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Enzyme Stability ,Gram-Negative Bacteria ,Mannosidases ,medicine ,Glycoside hydrolase ,Thermal stability ,Endoglucanase ,Escherichia coli ,chemistry.chemical_classification ,Endomannanase ,biology ,Extreme Heat ,General Medicine ,Glucanase ,biology.organism_classification ,Thermophilic enzyme ,030104 developmental biology ,Enzyme ,Biochemistry ,chemistry ,Carboxymethylcellulose Sodium ,biology.protein ,Molecular Medicine ,Bacteria - Abstract
Dictyoglomus turgidum is a hyperthermophilic, anaerobic, gram-negative bacterium that shows an array of putative glycoside hydrolases (GHs) encoded by its genome, a feature that makes this microorganism very interesting for biotechnological applications. The aim of this work is the characterization of a hyperthermophilic GH5, Dtur_0671, of D. turgidum, annotated as endoglucanase and herein named DturCelB in agreement to DturCelA, which was previously characterized. The synthetic gene was expressed in Escherichia coli. The purified recombinant enzyme is active as a monomer (40 kDa) and CD structural studies showed a conserved α/β structure at different temperatures (25 and 70 °C) and high thermoresistance (Tm of 88 °C). Interestingly, the enzyme showed high endo-β-1,4-mannanase activity vs various mannans, but low endo-β-1,4 glucanase activity towards carboxymethylcellulose. The K M and V max of DturCelB were determined for both glucomannan and CMC: they were 4.70 mg/ml and 473.1 μmol/min mg and 1.83 mg/ml and 1.349 μmol/min mg, respectively. Its optimal activity towards temperature and pH resulted to be 70 °C and pH 5.4, respectively. Further characterization highlighted good thermal stability (~ 50% of enzymatic activity after 2 h at 70 °C) and pH stability over a broad range (> 90% of activity after 1 h in buffer, ranging pH 5–9); resistance to chemicals was also observed.
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- 2017
13. Enzymatic Antioxidant Signatures in Hyperthermophilic Archaea
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Danila Limauro, Gabriella Fiorentino, Simonetta Bartolucci, Emilia Pedone, Pedone, Emilia, Fiorentino, Gabriella, Bartolucci, Simonetta, and Limauro, Danila
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0301 basic medicine ,Antioxidant ,archaea ,Physiology ,medicine.medical_treatment ,Thioredoxin reductase ,030106 microbiology ,Clinical Biochemistry ,antioxidant enzymes, archaea, hyperthermophiles, oxidative stress, reactive oxygen species ,Review ,Oxidative phosphorylation ,medicine.disease_cause ,Biochemistry ,Superoxide dismutase ,03 medical and health sciences ,antioxidant enzymes ,medicine ,oxidative stress ,hyperthermophiles ,Molecular Biology ,reactive oxygen species ,chemistry.chemical_classification ,Reactive oxygen species ,biology ,Chemistry ,lcsh:RM1-950 ,Cell Biology ,lcsh:Therapeutics. Pharmacology ,030104 developmental biology ,Catalase ,biology.protein ,ROS scavengers ,Thioredoxin ,Oxidative stress - Abstract
To fight reactive oxygen species (ROS) produced by both the metabolism and strongly oxidative habitats, hyperthermophilic archaea are equipped with an array of antioxidant enzymes whose role is to protect the biological macromolecules from oxidative damage. The most common ROS, such as superoxide radical (O2−.) and hydrogen peroxide (H2O2), are scavenged by superoxide dismutase, peroxiredoxins, and catalase. These enzymes, together with thioredoxin, protein disulfide oxidoreductase, and thioredoxin reductase, which are involved in redox homeostasis, represent the core of the antioxidant system. In this review, we offer a panorama of progression of knowledge on the antioxidative system in aerobic or microaerobic (hyper)thermophilic archaea and possible industrial applications of these enzymes.
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- 2020
14. An ArsR/SmtB family member regulates arsenic resistance genes unusually arranged in Thermus thermophilus HB27
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Patrizia Contursi, Danila Limauro, Gabriella Fiorentino, Simonetta Bartolucci, Immacolata Antonucci, Ana Luisa Ribeiro, José Berenguer, Alba Blesa, Giovanni Gallo, Ministerio de Economía y Competitividad (España), Antonucci, Immacolata, Gallo, G, Limauro, Danila, Contursi, Patrizia, Ribeiro, Al, Blesa, A, Berenguer, J, Bartolucci, Simonetta, and Fiorentino, Gabriella
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0301 basic medicine ,Operon ,lcsh:Biotechnology ,030106 microbiology ,DNA binding proteins ,Bioengineering ,Applied Microbiology and Biotechnology ,Biochemistry ,Arsenic ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,lcsh:TP248.13-248.65 ,transcriptional regulation ,Promoter Regions, Genetic ,Gene ,Research Articles ,Arsenite ,biology ,Chemistry ,Thermus thermophilus ,Arsenate ,Promoter ,Gene Expression Regulation, Bacterial ,biology.organism_classification ,Arsenate reductase ,Multigene Family ,Ars operon ,metal resistance ,Research Article ,Biotechnology - Abstract
Arsenic resistance is commonly clustered in ars operons in bacteria; main ars operon components encode an arsenate reductase, a membrane extrusion protein, and an As-sensitive transcription factor. In the As-resistant thermophile Thermus thermophilus HB27, genes encoding homologues of these proteins are interspersed in the chromosome. In this article, we show that two adjacent genes, TtsmtB, encoding an ArsR/SmtB transcriptional repressor and, TTC0354, encoding a Zn/Cd-dependent membrane ATPase are involved in As resistance; differently from characterized ars operons, the two genes are transcribed from dedicated promoters upstream of their respective genes, whose expression is differentially regulated at transcriptional level. Mutants defective in TtsmtB or TTC0354 are more sensitive to As than the wild type, proving their role in arsenic resistance. Recombinant dimeric TtSmtB binds in vitro to both promoters, but its binding capability decreases upon interaction with arsenate and, less efficiently, with arsenite. In vivo and in vitro experiments also demonstrate that the arsenate reductase (TtArsC) is subjected to regulation by TtSmtB. We propose a model for the regulation of As resistance in T. thermophilus in which TtSmtB is the arsenate sensor responsible for the induction of TtArsC which generates arsenite exported by TTC0354 efflux protein to detoxify cells., Spanish Ministry of Economy and Competitiveness
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- 2017
15. Thermus thermophilus as source of thermozymes for biotechnological applications: homologous expression and biochemical characterization of an α-galactosidase
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Danila Limauro, Simonetta Bartolucci, Gabriella Fiorentino, Patrizia Contursi, Martina Aulitto, Salvatore Fusco, Emilia Pedone, Aulitto, Martina, Fusco, Salvatore, Fiorentino, Gabriella, Limauro, Danila, Pedone, EMILIA MARIA, Bartolucci, Simonetta, and Contursi, Patrizia
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0301 basic medicine ,Hot Temperature ,Themostability ,Bioengineering ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Microbiology ,law.invention ,Industrial Biotechnology ,03 medical and health sciences ,Recombinant expression ,Thermozymes ,Thermus thermophilus ,α-Galactosidase ,Biocatalysis ,Biotechnology ,Cloning, Molecular ,Enzyme Stability ,Escherichia coli ,Hydrogen-Ion Concentration ,Recombinant Proteins ,alpha-Galactosidase ,α-Galactosidase, Thermus thermophilus, Thermozymes, Recombinant expression, Themostability ,law ,medicine ,Thermolabile ,Gene ,chemistry.chemical_classification ,Alpha-galactosidase ,biology ,Research ,Thermophile ,Molecular ,biology.organism_classification ,030104 developmental biology ,Enzyme ,chemistry ,biology.protein ,Recombinant DNA ,Cloning - Abstract
Background The genus Thermus, which has been considered for a long time as a fruitful source of biotechnological relevant enzymes, has emerged more recently as suitable host to overproduce thermozymes. Among these, α-galactosidases are widely used in several industrial bioprocesses that require high working temperatures and for which thermostable variants offer considerable advantages over their thermolabile counterparts. Results Thermus thermophilus HB27 strain was used for the homologous expression of the TTP0072 gene encoding for an α-galactosidase (TtGalA). Interestingly, a soluble and active histidine-tagged enzyme was produced in larger amounts (5 mg/L) in this thermophilic host than in Escherichia coli (0.5 mg/L). The purified recombinant enzyme showed an optimal activity at 90 °C and retained more than 40% of activity over a broad range of pH (from 5 to 8). Conclusions TtGalA is among the most thermoactive and thermostable α-galactosidases discovered so far, thus pointing to T. thermophilus as cell factory for the recombinant production of biocatalysts active at temperature values over 90 °C. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0638-4) contains supplementary material, which is available to authorized users.
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- 2017
16. Conformational stability and ligand binding properties of BldR, a member of the MarR family, from Sulfolobus solfataricus
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Pompea Del Vecchio, Luigi Petraccone, Gabriella Fiorentino, Immacolata Del Giudice, Simonetta Bartolucci, Fiorentino, Gabriella, Del Giudice, I, Petraccone, Luigi, Bartolucci, Simonetta, and DEL VECCHIO, POMPEA GIUSEPPINA GRAZIA
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Protein Denaturation ,Conformational change ,Circular dichroism ,Stereochemistry ,Archaeal Proteins ,Dimer ,ved/biology.organism_classification_rank.species ,Biophysics ,Thermodynamic stability ,Biochemistry ,DSC ,Analytical Chemistry ,chemistry.chemical_compound ,Benzaldehyde binding ,Molecular Biology ,Guanidine ,Protein Unfolding ,Calorimetry, Differential Scanning ,Protein Stability ,Chemistry ,Effector ,ved/biology ,Circular Dichroism ,Sulfolobus solfataricus ,ITC ,Archaea ,Binding constant ,Recombinant Proteins ,Hyperthermophile ,DNA-Binding Proteins ,Kinetics ,Benzaldehydes ,Thermodynamics ,Chemical stability ,Winged-helix transcriptional regulator ,Protein Binding - Abstract
The multiple antibiotic resistance regulators (MarR) constitute a family of ligand-responsive transcriptional regulators ubiquitous among the bacterial and archaeal domains. BldR, an archaeal MarR member characterized from the hyperthermophilic crenarchaeon Sulfolobus solfataricus regulates its own expression and that of an alcohol dehydrogenase gene by binding to sequences in their promoters and responding to benzaldehyde as the effector molecule. In this study we assessed the thermodynamic stability of the protein BldR and its binding with benzaldehyde through biophysical measurements. The temperature- and denaturant-induced unfolding experiments, performed by means of circular dichroism (CD) and differential scanning calorimetry (DSC), showed that BldR has an extremely high thermal stability (Td = 108.9 °C) and a remarkable resistance against GuHCl (Cm = 5.3 M at 25 °C). The unfolding Gibbs energy, ΔdG (H2O), calculated by the linear extrapolation model from GuHCl-induced unfolding equilibrium curve, is 72.2 kJ mol− 1. ITC binding experiments showed that four benzaldehyde molecules bind to one BldR dimer with a binding constant Kb of 7.5·105 M− 1, being the binding entropically driven. ITC, CD and fluorescence results are consistent with a conformational change induced by benzaldehyde binding, further proving that this molecule is a specific effector for BldR modulating its DNA binding activity.
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- 2014
17. Identification and Physicochemical Characterization of BldR2 from Sulfolobus solfataricus, a Novel Archaeal Member of the MarR Transcription Factor Family
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Simonetta Bartolucci, Pompea Del Vecchio, Immacolata Del Giudice, Luigi Martino, Gabriella Fiorentino, Lorenzo Durante, Fiorentino, Gabriella, DEL GIUDICE, Immacolata, Bartolucci, Simonetta, Durante, Lorenzo, Martino, L, and DEL VECCHIO, POMPEA GIUSEPPINA GRAZIA
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Archaeal Proteins ,Molecular Sequence Data ,ved/biology.organism_classification_rank.species ,Biology ,Arginine ,Crystallography, X-Ray ,Biochemistry ,Serine ,chemistry.chemical_compound ,Amino Acid Sequence ,Promoter Regions, Genetic ,Protein Dimerization ,Transcription factor ,Peptide sequence ,Genetics ,chemistry.chemical_classification ,Alanine ,Base Sequence ,Sequence Homology, Amino Acid ,Protein Stability ,ved/biology ,Sulfolobus solfataricus ,Drug Resistance, Microbial ,Amino acid ,DNA-Binding Proteins ,DNA binding site ,chemistry ,Archaea - Hyperthermophile – Winged-helix transcriptional regulator Thermodynamic stability – Dimeric proteins – DNA binding protein ,Multigene Family ,Protein Multimerization ,DNA ,Protein Binding ,Transcription Factors - Abstract
The multiple antibiotic resistance regulators (MarR) constitute a family of ligand-responsive transcriptional regulators abundantly distributed throughout the bacterial and archaeal domains. Here we describe the identification and characterization of BldR2, as a new member of this family, in the archaeon Sulfolobus solfataricus and report physiological, biochemical, and biophysical investigation of its stability and DNA binding ability. Transcriptional analysis revealed the upregulation of BldR2 expression by aromatic compounds in the late-logarithmic growth phase and allowed the identification of cis-acting sequences. Our results suggest that BldR2 possesses in solution a dimeric structure and a high stability against both temperature and chemical denaturing agents; the protein binds site specifically to its own promoter, Sso1082, with a micromolar binding affinity at two palindromic sites overlapping TATA-BRE and the transcription start site. Benzaldehyde and salicylate, ligands of MarR members, are antagonists of binding of DNA by BldR2. Moreover, two single-point mutants of BldR2, R19A and A65S, properly designed for obtaining information about the dimerization and the DNA binding sites of the protein, have been produced and characterized. The results point out an involvement of BldR2 in the regulation of the stress response to aromatic compounds and point to arginine 19 as a key amino acid involved in protein dimerization, while the introduction of serine 65 increases the DNA affinity of the protein, making it comparable with those of other members of the MarR family.
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- 2011
18. Transcriptional Regulation of the Gene Encoding an Alcohol Dehydrogenase in the Archaeon Sulfolobus solfataricus Involves Multiple Factors and Control Elements
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Simonetta Bartolucci, Mosè Rossi, Raffaele Cannio, Gabriella Fiorentino, Fiorentino, G, Cannio, R, Rossi, Mose', Bartolucci, Simonetta, Fiorentino, Gabriella, R., Cannio, and M., Rossi
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Transcriptional Activation ,Transcription, Genetic ,5' Flanking Region ,Archaeal Proteins ,TATA box ,Molecular Sequence Data ,ved/biology.organism_classification_rank.species ,5' flanking region ,DNA Footprinting ,Biology ,Microbiology ,Sulfolobus ,Transcriptional regulation ,Promoter Regions, Genetic ,Molecular Biology ,Transcription factor ,Gene ,Regulation of gene expression ,Base Sequence ,ved/biology ,Sulfolobus solfataricus ,Alcohol Dehydrogenase ,Meeting Presentations ,Promoter ,TATA Box ,DNA binding protein ,Culture Media ,DNA-Binding Proteins ,DNA, Archaeal ,Biochemistry ,Benzaldehydes ,Gene Expression Regulation, Archaeal ,Transcription Factors - Abstract
A transcriptionally active region has been identified in the 5′ flanking region of the alcohol dehydrogenase gene of the crenarchaeon Sulfolobus solfataricus through the evaluation of the activity of putative transcriptional regulators and the role of the region upstream of the gene under specific metabolic circumstances. Electrophoretic mobility shift assays with crude extracts revealed protein complexes that most likely contain TATA box-associated factors. When the TATA element was deleted from the region, binding sites for both DNA binding proteins, such as the small chromatin structure-modeling Sso7d and Sso10b (Alba), and transcription factors, such as the repressor Lrs14, were revealed. To understand the molecular mechanisms underlying the substrate-induced expression of the adh gene, the promoter was analyzed for the presence of cis -acting elements recognized by specific transcription factors upon exposure of the cell to benzaldehyde. Progressive dissection of the identified promoter region restricted the analysis to a minimal responsive element (PAL) located immediately upstream of the transcription factor B-responsive element-TATA element, resembling typical bacterial regulatory sequences. A benzaldehyde-activated transcription factor (Bald) that specifically binds to the PAL cis -acting element was also identified. This protein was purified from heparin-fractionated extracts of benzaldehyde-induced cells and was shown to have a molecular mass of ∼16 kDa. The correlation between S. solfataricus adh gene activation and benzaldehyde-inducible occupation of a specific DNA sequence in its promoter suggests that a molecular signaling mechanism is responsible for the switch of the aromatic aldehyde metabolism as a response to environmental changes.
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- 2003
19. Differential antibody response to the Anopheles gambiae gSG6 and cE5 salivary proteins in individuals naturally exposed to bites of malaria vectors
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David Modiano, Valentina D. Mangano, Sodiomon B. Sirima, Issa Nebie, Gabriella Fiorentino, Raffaele Ronca, Bruno Arcà, Cinzia Rizzo, Fabrizio Lombardo, Rizzo, C, Lombardo, F, Ronca, R, Mangano, V, Sirima, S, Nèbiè, I, Fiorentino, Gabriella, Modiano, D, and Arcà, B.
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Saliva ,IgG ,Anopheles gambiae ,030231 tropical medicine ,Pilot Projects ,Colombia ,Immunoglobulin G ,Anopheles gambiae, Salivary proteins, Immune response, IgG, IgG1, IgG4, Marker of exposure, Plasmodium transmission, Malaria epidemiology ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Antigen ,parasitic diseases ,Anopheles ,medicine ,Salivary proteins ,Immune response ,IgG1 ,IgG4 ,Marker of exposure ,Plasmodium transmission ,Malaria epidemiology ,Animals ,Humans ,Chile ,Salivary Proteins and Peptides ,030304 developmental biology ,0303 health sciences ,biology ,Research ,medicine.disease ,biology.organism_classification ,United States ,3. Good health ,Insect Vectors ,Malaria ,Infectious Diseases ,Immunology ,biology.protein ,Insect Proteins ,Parasitology ,Antibody ,Animal Distribution - Abstract
Background Mosquito saliva plays crucial roles in blood feeding but also evokes in hosts an anti-saliva antibody response. The IgG response to the Anopheles gambiae salivary protein gSG6 was previously shown to be a reliable indicator of human exposure to Afrotropical malaria vectors. We analyzed here the humoral response to the salivary anti-thrombin cE5 in a group of individuals from a malaria hyperendemic area of Burkina Faso. Methods ELISA was used to measure the anti-cE5 IgG, IgG1 and IgG4 antibody levels in plasma samples collected in the village of Barkoumbilen (Burkina Faso) among individuals of the Rimaibé ethnic group. Anti-gSG6 IgG levels were also determined for comparison. Anopheles vector density in the study area was evaluated by indoor pyrethrum spray catches. Results The cE5 protein was highly immunogenic and triggered in exposed individuals a relatively long-lasting antibody response, as shown by its unchanged persistence after a few months of absent or very low exposure (dry season). In addition cE5 did not induce immune tolerance, as previously suggested for the gSG6 antigen. Finally, IgG subclass analysis suggested that exposed individuals may mount a Th1-type immune response against the cE5 protein. Conclusions The anti-cE5 IgG response is shown here to be a sensitive indicator of human exposure to anopheline vectors and to represent an additional tool for malaria epidemiological studies. It may be especially useful in conditions of low vector density, to monitor transiently exposed individuals (i.e. travellers/workers/soldiers spending a few months in tropical Africa) and to evaluate the impact of insecticide treated nets on vector control. Moreover, the gSG6 and cE5 salivary proteins were shown to trigger in exposed individuals a strikingly different immune response with (i) gSG6 evoking a short-lived IgG response, characterized by high IgG4 levels and most likely induction of immune tolerance, and (ii) cE5 eliciting a longer-living IgG response, dominated by anti-cE5 IgG1 antibodies and not inducing tolerance mechanisms. We believe that these two antigens may represent useful reagents to further investigate the so far overlooked role of Anopheles saliva and salivary proteins in host early immune response to Plasmodium parasites. Electronic supplementary material The online version of this article (doi:10.1186/s13071-014-0549-8) contains supplementary material, which is available to authorized users.
- Published
- 2014
20. IgG1 and IgG4 antibody responses to the Anopheles gambiae salivary protein gSG6 in the sympatric ethnic groups Mossi and Fulani in a malaria hyperhendemic area of Burkina Faso
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David Modiano, Gabriella Fiorentino, Bruno Arcà, Issa Nebie, Raffaele Ronca, Sodiomon B. Sirima, Valentina D. Mangano, Cinzia Rizzo, Marita Troye-Blomberg, Fabrizio Lombardo, Rizzo, C, Ronca, R, Lombardo, F, Mangano, V, Sirima, Sb, Nèbiè, I, Fiorentino, Gabriella, Troye Blomberg, M, Modiano, D, and Arcà, B.
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Epidemiology ,Anopheles gambiae ,Protozoan Proteins ,lcsh:Medicine ,Disease Vectors ,Biochemistry ,Mosquitoes ,Immunoglobulin G ,Immune tolerance ,ADULT FEMALE MOSQUITO ,PLASMODIUM-FALCIPARUM MALARIA ,AEDES-AEGYPTI ,GLAND TRANSCRIPTS ,HUMORAL RESPONSE ,IMMUNE-RESPONSE ,TROPICAL AFRICA ,ANTIGENS ,MARKER ,INFECTION ,Allergies ,Medicine and Health Sciences ,Malaria, Falciparum ,lcsh:Science ,Child ,Immune Response ,Multidisciplinary ,Immune System Proteins ,biology ,3. Good health ,Circumsporozoite protein ,Insects ,Infectious Diseases ,saliva protein ,Insect Proteins ,Antibody ,Research Article ,Adult ,Adolescent ,Arthropoda ,Immunology ,Black People ,Antibodies ,Young Adult ,Immune system ,Antigen ,parasitic diseases ,Anopheles ,Burkina Faso ,medicine ,Immune Tolerance ,Parasitic Diseases ,Animals ,Humans ,Salivary Proteins and Peptides ,lcsh:R ,Immunity ,Organisms ,Biology and Life Sciences ,Proteins ,medicine.disease ,biology.organism_classification ,Virology ,Invertebrates ,Malaria ,Biomarker Epidemiology ,biology.protein ,lcsh:Q ,Clinical Immunology ,immunoglobulin - Abstract
Human antibody response to the Anopheles gambiae salivary protein gSG6 has recently emerged as a potentially useful tool for malaria epidemiological studies and for the evaluation of vector control interventions. However, the current understanding of the host immune response to mosquito salivary proteins and of the possible crosstalk with early response to Plasmodium parasites is still very limited. We report here the analysis of IgG1 and IgG4 subclasses among anti-gSG6 IgG responders belonging to Mossi and Fulani from Burkina Faso, two ethnic groups which are known for their differential humoral response to parasite antigens and for their different susceptibility to malaria. The IgG1 antibody response against the gSG6 protein was comparable in the two groups. On the contrary, IgG4 titers were significantly higher in the Fulani where, in addition, anti-gSG6 IgG4 antibodies appeared in younger children and the ratio IgG4/IgG1 stayed relatively stable throughout adulthood. Both gSG6-specific IgG1 and IgG4 antibodies showed a tendency to decrease with age whereas, as expected, the IgG response to the Plasmodium circumsporozoite protein (CSP) exhibited an opposite trend in the same individuals. These observations are in line with the idea that the An. gambiae gSG6 salivary protein induces immune tolerance, especially after intense and prolonged exposure as is the case for the area under study, suggesting that gSG6 may trigger in exposed individuals a Th2-oriented immune response.
- Published
- 2014
21. Functional and structural characterization of protein disulfide oxidoreductase from Thermus thermophilus HB27
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Simonetta Bartolucci, Danila Limauro, Patrizia Contursi, Emilia Pedone, Gabriella Fiorentino, Luciano Pirone, Pedone, E., Fiorentino, Gabriella, Pirone, L., Contursi, Patrizia, Bartolucci, Simonetta, and Limauro, Danila
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chemistry.chemical_classification ,Base Sequence ,ved/biology ,Oxidative folding ,Thioredoxin reductase ,Thermophile ,Thermus thermophilus ,Sulfolobus solfataricus ,ved/biology.organism_classification_rank.species ,Molecular Sequence Data ,Protein Disulfide Reductase (Glutathione) ,General Medicine ,Biology ,biology.organism_classification ,Microbiology ,Primer extension ,Biochemistry ,chemistry ,Bacterial Proteins ,Oxidoreductase ,protein disulfide oxidoreductase ,Molecular Medicine ,Thermostability - Abstract
The paper reports the characterization of a protein disulfide oxidoreductase (PDO) from the thermophilic Gram negative bacterium Thermus thermophilus HB27, identified as TTC0486 by genome analysis and named TtPDO. PDO members are involved in the oxidative folding, redox balance and detoxification of peroxides in thermophilic prokaryotes. Ttpdo was cloned and expressed in E. coli and the recombinant purified protein was assayed for the dithiol-reductase activity using insulin as substrate and compared with other PDOs characterized so far. In the thermophilic archaeon Sulfolobus solfataricus PDOs work as thiol-reductases constituting a peculiar redox couple with Thioredoxin reductase (SsTr). To get insight into the role of TtPDO, a hybrid redox couple with SsTr, homologous to putative Trs of T. thermophilus, was assayed. The results showed that SsTr was able to reduce TtPDO in a concentration dependent manner with a calculated K M of 34.72 μM, suggesting the existence of a new redox system also in thermophilic bacteria. In addition, structural characterization of TtPDO by light scattering and circular dichroism revealed the monomeric structure and the high thermostability of the protein. The analysis of the genomic environment suggested a possible clustering of Ttpdo with TTC0487 and TTC0488 (tlpA). Accordingly, transcriptional analysis showed that Ttpdo is transcribed as polycistronic messenger. Primer extension analysis allowed the determination of its 5′end and the identification of the promoter region.
- Published
- 2014
22. Identification and molecular characterization of an endoglucanase gene, celS , from the extremely thermophilic archaeon Sulfolobus solfataricus
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Danila Limauro, Simonetta Bartolucci, Mosè Rossi, Gabriella Fiorentino, Raffaele Cannio, Limauro, Danila, Cannio, R., Fiorentino, Gabriella, Rossi, Mose', and Bartolucci, Simonetta
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Molecular Sequence Data ,ved/biology.organism_classification_rank.species ,Biology ,Microbiology ,Genes, Archaeal ,Sulfolobus ,Gene product ,Cellulase ,Amino Acid Sequence ,Cloning, Molecular ,Gene ,ved/biology ,Sulfolobus solfataricus ,Temperature ,Glycosyl hydrolase ,General Medicine ,Sulfolobus solfataricu ,biology.organism_classification ,Molecular biology ,Open reading frame ,Biochemistry ,Thermotoga maritima ,Pyrococcus furiosus ,Molecular Medicine ,Sequence Alignment ,Sequence Analysis ,Thermotoga neapolitana - Abstract
A genomic region upstream of the alcohol dehydrogenase ( Ssadh ) gene was cloned and sequenced from a library of Sulfolobus solfataricus MT4 strain. The isolated 4,040-bp DNA fragment revealed an open reading frame ( celS ), lying in the opposite direction to Ssadh , which showed significant similarity to endo - β -1,4-glucanases from Pyrococcus furiosus , Thermotoga maritima , and Thermotoga neapolitana . celS was shown to be a functional gene in vivo: a specific celS mRNA was detected by primer extension analysis showing a unique initiation transcription site coinciding with the ATG translation initiation codon. The specific gene product was detected as an extracellular cellulase after enzyme staining by carboxymethyl cellulose (CMC) SDS-PAGE, showing a molecular weight in agreement with that deduced from the open reading frame. Depending on growth conditions, different levels of cellulase activity and specific celS transcript were detected, revealing an inductive effect of CMC and suggesting a repressive role of glucose.
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- 2001
23. Structural analysis of BldR from Sulfolobus solfataricus provides insights into the molecular basis of transcriptional activation in Archaea by MarR family proteins
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Simonetta Bartolucci, Carlo Pedone, Raffaele Ronca, Anna Di Fiore, Gabriella Fiorentino, Pietro Amodeo, Giuseppina De Simone, Rosa Maria Vitale, Di Fiore, A, Fiorentino, Gabriella, Vitale, Rm, Ronca, Raffaele, Amodeo, P, Pedone, Carlo, Bartolucci, Simonetta, and De Simone, G.
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Models, Molecular ,Transcriptional Activation ,crystal structure ,Operon ,Archaeal Proteins ,ved/biology.organism_classification_rank.species ,Molecular Sequence Data ,Repressor ,MarR family ,Biology ,Crystallography, X-Ray ,Structural Biology ,Transcription (biology) ,Transcriptional regulation ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Gene ,Genetics ,Activator (genetics) ,ved/biology ,Sulfolobus solfataricus ,targeted molecular dynamics simulation ,Archaea ,Protein Structure, Tertiary ,DNA, Archaeal ,Trans-Activators ,Sequence Alignment ,Protein Binding - Abstract
The multiple antibiotic resistance regulator (MarR) family constitutes a significant class of transcriptional regulators whose members control a variety of important biological functions such as regulation of response to environmental stress, control of virulence factor production, resistance to antimicrobial agents, and regulation of aromatic catabolic pathways. Although the majority of MarR family members have been characterized as transcriptional repressors, a few examples of transcriptional activators have also been reported. BldR is a newly identified member of this family that has been demonstrated to act as a transcriptional activator in stress response to aromatic compounds in the crenarchaeon Sulfolobus solfataricus. In this work, we report findings on the BldR X-ray crystal structure and present a molecular modeling study on the complex that this protein forms with its cognate DNA sequence, thus providing the first detailed description of the DNA-binding mechanism of an archaeal activator belonging to the MarR family. Two residues responsible for the high binding specificity of this transcriptional regulator were also identified. Our studies demonstrated that, in Archaea, the capability of MarR family members to act as activators or repressors is not related to a particular DNA-binding mechanism but rather could be due to the position of the binding site on the target DNA. Moreover, since genes encoding MarR proteins often control transcription of operons that encode for multisubstrate efflux pumps, our results also provided important insights for the identification of new tools to overcome the microorganism's multidrug resistance.
- Published
- 2009
24. A novel E. coli biosensor for detecting aromatic aldehydes based on a responsive inducible archaeal promoter fused to the green fluorescent protein
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Gabriella Fiorentino, Simonetta Bartolucci, Raffaele Ronca, Fiorentino, Gabriella, Ronca, Raffaele, and Bartolucci, Simonetta
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Archaeal Proteins ,ved/biology.organism_classification_rank.species ,Green Fluorescent Proteins ,Molecular Sequence Data ,Context (language use) ,Biosensing Techniques ,Applied Microbiology and Biotechnology ,Green fluorescent protein ,Transcriptional regulation ,Gene expression ,Escherichia coli ,Cloning, Molecular ,Promoter Regions, Genetic ,Gene ,Transcription factor ,Reporter system ,Alcohol dehydrogenase ,Aldehydes ,biology ,Base Sequence ,ved/biology ,Sulfolobus solfataricus ,Alcohol Dehydrogenase ,General Medicine ,Archaea ,Molecular biology ,Biochemistry ,biology.protein ,Heterologous expression ,Detoxification ,Genetic Engineering ,Biotechnology - Abstract
A whole-cell bacterial biosensor for measuring aqueous concentrations of aromatic aldehydes was developed. It is based on the E. coli BL21DE3(RIL) expressing the green fluorescent protein under the control of an alcohol dehydrogenase inducible promoter belonging to the archaeon Sulfolobus solfataricus (Sso2536adh promoter). Since it was previously reported that the BldR regulatory protein is the transcription factor required for aromatic aldehyde response in S. solfataricus, the gene encoding for the sensor protein BldR was co-expressed in the biosensor strain on a different compatible plasmid. Gel mobility shift assays showed that the purified recombinant protein can bind specifically to the Sso2536adh promoter. We demonstrated the ability of the archaeal promoter and the BldR transcription factor to operate in a bacterial context to drive active gene expression in a hybrid archaeal/eukaryal fusion. Furthermore, the E. coli BL21DE3(RIL) biosensor strain displayed a specific response and high sensitivity to the different aromatic aldehydes used, suggesting its potential low-cost application to environmentally relevant samples.
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- 2008
25. Retracted: Interaction of Thermus thermophilus ArsC enzyme and gold nanoparticles naked-eye assays speciation between As(III) and As(V)
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Luca De Stefano, Jane Politi, Sandra Casale, Gabriella Fiorentino, Jolanda Spadavecchia, Immacolata Antonucci, Politi, J, Spadavecchia, J, Fiorentino, Gabriella, Antonucci, Immacolata, Casale, S, and De Stefano, L.
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Arsenate Reductases ,biorecognition ,Inorganic chemistry ,Metal Nanoparticles ,chemistry.chemical_element ,Nanoparticle ,arsenate reductase ,Bioengineering ,Arsenic ,Polyethylene Glycols ,Bacterial Proteins ,Microscopy, Electron, Transmission ,Dynamic light scattering ,Spectroscopy, Fourier Transform Infrared ,General Materials Science ,Particle Size ,Electrical and Electronic Engineering ,Surface plasmon resonance ,Ions ,biology ,Thermus thermophilus ,Mechanical Engineering ,technology, industry, and agriculture ,General Chemistry ,Surface Plasmon Resonance ,biology.organism_classification ,Recombinant Proteins ,naked eye assay ,Arsenate reductase ,chemistry ,Mechanics of Materials ,Colloidal gold ,Spectrophotometry, Ultraviolet ,Gold ,Naked eye ,gold nanoparticle ,Nuclear chemistry - Abstract
The thermophilic bacterium Thermus thermophilus HB27 encodes chromosomal arsenate reductase (TtArsC), the enzyme responsible for resistance to the harmful effects of arsenic. We report on adsorption of TtArsC onto gold nanoparticles for naked-eye monitoring of biomolecular interaction between the enzyme and arsenic species. Synthesis of hybrid biological–metallic nanoparticles has been characterized by transmission electron microscopy (TEM), ultraviolet-visible (UV–vis), dynamic light scattering (DLS) and phase modulated infrared reflection absorption (PM-IRRAS) spectroscopies. Molecular interactions have been monitored by UV–vis and Fourier transform-surface plasmon resonance (FT-SPR). Due to the nanoparticles’ aggregation on exposure to metal salts, pentavalent and trivalent arsenic solutions can be clearly distinguished by naked-eye assay, even at 85 μM concentration. Moreover, the assay shows partial selectivity against other heavy metals.
- Published
- 2015
26. Development of a genetic system for hyperthermophilic Archaea: expression of a moderate thermophilic bacterial alcohol dehydrogenase gene in Sulfolobus solfataricus
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Santina Prato, Raffaele Cannio, Simonetta Bartolucci, Patrizia Contursi, M. Rossi, Gabriella Fiorentino, Contursi, Patrizia, R., Cannio, S., Prato, Fiorentino, Gabriella, Rossi, Mose', Bartolucci, Simonetta, Contursi, P., Cannio, R., Prato, S., and Fiorentino, G.
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"heterologous gene expression" ,"Sulfolobus solfataricus" ,Transcription, Genetic ,ved/biology.organism_classification_rank.species ,Genetic Vectors ,"shuttle vector " ,Microbiology ,Gene Expression Regulation, Enzymologic ,Sulfolobus ,Geobacillus stearothermophilus ,Plasmid ,Transformation, Genetic ,Shuttle vector ,Gene expression ,Genetics ,"alcohol dehydrogenase" ,Cloning, Molecular ,Promoter Regions, Genetic ,Molecular Biology ,Gene ,Alcohol dehydrogenase ,biology ,ved/biology ,Sulfolobus solfataricus ,Alcohol Dehydrogenase ,biology.organism_classification ,Molecular biology ,Terminator (genetics) ,Mutagenesis ,biology.protein ,Gene Expression Regulation, Archaeal ,hormones, hormone substitutes, and hormone antagonists ,Plasmids - Abstract
The Escherichia coli/Sulfolobus solfataricus shuttle vector pEXSs was used as a cloning vehicle for the gene transfer and expression of two bacterial genes in Sulfolobus solfataricus. The alcohol dehydrogenase (adh) from the moderate thermophilic Bacillus stearothermophilus (strain LLDR) and a mutagenised version encoding a less thermostable ADH enzyme were the selected genes. S. solfataricus adh promoter and aspartate aminotransferase terminator were used to drive the heterologous gene expression and to guarantee the correct termination of the transcripts, respectively. The constructed vectors were found to be able to carry these ‘passenger’ genes without undergoing any rearrangements. The active transcription of bacillar mRNAs was ascertained in vivo by RT-PCR. Transformed S. solfataricus expressed functional exogenous ADHs that showed unaffected kinetic and chemical–physical features.
- Published
- 2003
27. Decreasing the stability and changing the substrate specificity of the Bacillus stearothermophilus alcohol dehydrogenase by single amino acid replacements
- Author
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Raffaele Cannio, Gabriella Fiorentino, Simonetta Bartolucci, M. Rossi, Fiorentino, Gabriella, Cannio, R., Rossi, M., and Bartolucci, Simonetta
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Protein Denaturation ,Proline ,Glutamic Acid ,Bioengineering ,Bacillus stearothermophilu ,medicine.disease_cause ,Biochemistry ,Substrate Specificity ,Geobacillus stearothermophilus ,Residue (chemistry) ,Catalytic Domain ,Enzyme Stability ,Escherichia coli ,medicine ,Coenzyme binding ,Molecular Biology ,Guanidine ,Alcohol dehydrogenase ,chemistry.chemical_classification ,Binding Sites ,biology ,Thermophile ,Temperature ,alcohol dehydrogenase ,Hydrogen-Ion Concentration ,NAD ,Recombinant Proteins ,Amino acid ,Kinetics ,Amino Acid Substitution ,chemistry ,Alcohols ,Mutagenesis, Site-Directed ,biology.protein ,Salt bridge ,site-directed mutagenesis ,hormones, hormone substitutes, and hormone antagonists ,Biotechnology - Abstract
The gene encoding the alcohol dehydrogenase (adh-hT) from the thermophilic bacterium Bacillus stearothermophilus LLD-R strain has been overexpressed in Escherichia coli and the corresponding recombinant protein purified to homogeneity. Two putative structural determinants contributing to the higher stability of ADH-hT had been identified by comparison with the less thermostable ADH (ADH-T) from the less thermophilic B. stearothermophilus NCA 1503. In order to ascertain their role, mutations were designed to eliminate in ADH-hT a salt bridge at the N-terminus and a proline residue in the coenzyme binding domain replacing the amino acids located at the same positions in ADH-T. Three mutants--Glu11Lys, Pro242Ala, and Glu11Lys/Pro242Ala--were expressed at high level and the proteins purified and characterized. In general, the mutations had little effect on the activity, indicating that they were not disruptive. The thermal resistance was changed displaying quite additive effects.
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