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The ArsR/SmtB family of transcriptional regulators consists of metal responsive DNA binding proteins sharing a dimeric structure, a helix-turn-helix (HTH) or winged HTH DNA binding domain, and a metal binding box ELCV(C/G)D located within the HTH region. In microbial cells binding of these proteins to different heavy metals determines protein dissociation from target promoters and transcriptional activation. Metal binding mode and specificity vary very much among family members [1]. In a previous study we characterized TtSmtB from the thermophilic microorganism Thermus thermophilus HB27 through in vivo and in vitro approaches, showing that the protein is an arsenic responsive transcriptional repressor which regulates the downstream gene TTC0354 encoding a Zn2+/Cd2+ dependent membrane ATPase involved in arsenic transport outside the cell [2]. In the present study, we further characterized TtSmtB by an array of biochemical and biophysical approaches to deeply analyse its interaction with metal ions (different from arsenic) and target DNA; we show that the protein possesses significant thermal stability and binds other metal ions, such as cadmium and antimony with different affinity. To study the contribution of TtSmtB and TTC0354 in cadmium resistance in vivo, we generated transcriptional fusions of TtSmtB regulatory regions to LacZ in T. thermophilus HB27 wild type and a mutant strain in which TTC0354 was inactivated. These findings revealed the contribution of the system in cadmium resistance opening up to the realization of robust whole-cell biosensors for cadmium detection.

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).

Arsenic is an ubiquitous toxic metalloid naturally present in the soil, water and air that adversely affects human health. The abundance of arsenic in the environment has guided the evolution of multiple defence strategies in almost all microorganisms which must therefore sense the metalloid and regulate the transcription of genes coding for resistance proteins. In this sense microorganisms participate to the geochemical cycling of arsenic in their living environments, promoting or inhibiting arsenic release from sediment material (1). The thermophilic gram negative bacterium Thermus thermophilus HB27 is able to grow in the presence of both arsenate and arsenite in a range of concentrations which are lethal for other microorganisms. The putative resistance genes have not been found in a single resistance operon but associated to chromosomal genes apparently not functionally related. One of them codes for a thioredoxin-coupled arsenate reductase (TtArsC) which catalyzes the reduction of pentavalent arsenate to trivalent arsenite (2); the second codes for a transcriptional repressor (TtSmtB), sensitive to arsenic, belonging to the ArsR/SmtB family of transcriptional regulators. Here we present studies addressed to the elucidation of the role of TtarsC and TtsmtB in the arsenic resistance mechanism, among which the characterization of the recombinant TtArsC and TtSmtB proteins. The results obtained represent the starting point for the development of stable whole-cell or protein- based arsenic biosensors (3). THIS WORK IS SUPPORTED BY GRANTS FROM THE REGIONE CAMPANIA, LEGGE 5 (ITALY, CUP NUMBER E69D15000210002), AND FROM THE PROJECT BIO2013-4496R. REFERENCES: 1 ROSEN BP . “BIOCHEMISTRY OF ARSENIC DETOXIFICATION”. FEBS LETTERS 2002; 529:86-92. 2. DEL GIUDICE I, ET AL. “A NOVEL ARSENATE REDUCTASE FROM THE BACTERIUM THERMUS THERMOPHILUS HB27: ITS ROLE IN ARSENIC DETOXIFICATION”. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2013; 1834:2071-2079. 3. POLITI J ET AL. “INTERACTION OF THERMUS THERMOPHILUS ARSC ENZYME AND GOLD NANOPARTICLES NAKED-EYE ASSAYS SPECIATION BETWEEN AS(III) AND AS(V). NANOTECHNOLOGY. 2015;26(43):435703.

Introduction Microorganisms living in arsenic-rich geothermal environments act on arsenic using different biochemical strategies. So far, only limited insights have been obtained on the molecular mechanisms responsible for the arsenic resistance [1]. Investigation on the thermophilic bacterium Thermus thermophilus HB27 highlighted its tolerance to millimolar concentrations of As(V) and As(III). In fact, its genome encodes for an arsenate reductase, which is an important contributor in the arsenic resistance system [2]. In this study we focused on the role of the putative transcriptional regulator TtSmtB (TTC0353), belonging to ArsR/SmtB family, by in vivo and in vitro analysis. Methods A RT-PCR analysis was performed to identify TtSmtB transcriptional unit where putative internal promoters were found. TtSmtB was cloned in pET28b, expressed and the recombinant protein purified and characterized by EMSA assays for its ability to bind putative target promoters. A T. thermophilus TtsmtB- knocked strain was generated and characterized by qRT-PCR on putative TtSmtB target genes. Results TtSmtB gene is part of an operon of five genes not metabolically related with three arsenic responsive promoters upstream of TTC0351, TTC0353 and TTC0354. The recombinant protein is a dimer interacting with the target promoters. qRT-PCR on TtSmtB, TTC0351 and TTC0354 in the wild type showed an increased expression in the presence of arsenic. Furthermore, their expression in the knocked strain was always greater than in the wild type. Conclusions Our result show that TtSmtB is able to sense arsenic and acts as a transcriptional repressor of the entire operon in which it is encoded. 1) D. Páez-Espino, et al. 2009 Biometals, 22:117–130 2) Del Giudice I et al. 2013 Biochim Biophys Acta, 1834:2071-9

Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the toxic metalloid have only partially been examined[1]. In a recent study, we started an investigation on the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to both arsenate (V) and arsenite (III) in a millimolar range and owns in its genome a chromosomal arsenate reductase (TtarsC) which is an important contributor in the arsenic resistance[2]. With the aim to study the arsenic resistance regulation we focused on a putative transcriptional regulator (TtSmtB) sharing high identity with the members of the ArsR/SmtB family. TtSmtB is part of an operon including five genes not related to arsenic resistance with the exception of a putative metal-efflux transporter. Since by bioinformatics we found in the operon some putative internal promoters, by means of an in vivo reporter system[3], we identified those responsive to arsenate and arsenite. TtSmtB was also cloned and expressed in Escherichia coli. The purification of the recombinant protein, proved that TtSmtB is a DNA binding protein of 123 amino acids interacting with the in vivo active promoters. Preliminary EMSA assays in the presence of arsenite suggested its role as a derepressor.

Arsenic is an ubiquitous toxic metalloid naturally present in the soil, water and air that adversely affects human health. The abundance of arsenic in the environment has guided the evolution of multiple defence strategies in almost all microorganisms which must therefore sense the metalloid and regulate the transcription of genes coding for resistance proteins. In this sense microorganisms participate to the geochemical cycling of arsenic in their living environments, promoting or inhibiting arsenic release from sediment material. The aim of this thesis has been the characterization at molecular level of the mechanisms of arsenic resistance in T. thermophilus and the realization of “Cell-Based” and “Enzyme-Based” biosensors for the detection of arsenic species in soils and waters. The thermophilic gram negative bacterium Thermus thermophilus HB27 is able to grow in the presence of both arsenate and arsenite in a range of concentrations which are lethal for other microorganisms. The putative resistance genes have not been found in a single resistance operon but associated to chromosomal genes apparently not functionally related. In particular we found a gene coding for a thioredoxin-coupled arsenate reductase (TtArsC) which catalyzes the reduction of pentavalent arsenate to trivalent arsenite; two genes (TTC1447, TTC0354) coding putative ArsB-like transporters; and a gene coding for a transcriptional repressor (TtSmtB) sensitive to arsenic, belonging to the ArsR/SmtB family of transcriptional regulators. TtsmtB is part of an operon containing putative internal promoters upstream of genes with no obvious functional relationship. The purified recombinant protein is a dimeric DNA binding protein able to bind in-vitro to target sequences and to dissociate upon arsenate and arsenite binding. Inactivation of the TtsmtB gene, in a T. thermophilus TtsmtB- mutant strain, induces the expression of the ars genes among which TtarsC and a putative efflux protein. These results prove that TtSmtB has a functional role in the regulation of the arsenic resistance. Analysing the TTC0351, TTC0353 and TTC0354 promoter activities in-vivo, through β-galactosidase reporter systems, it has been developed the first whole-cell arsenic biosensor based on the use of the thermophilic microorganism T. thermophilus. The biosensor response could be measured with reliability within 30 minutes of arsenate or arsenite addition, and have a minimum detection limit of 0.1 mM for both arsenate and arsenite. An intriguingly feature of this biosensor rely on its thermophilic nature, hence, despite not having a higher arsenic detection limit it could be more versatile, stable and strong in case of highly contaminated waters. Moreover, it has been developed an enzyme-based biosensor to screen for the presence of arsenic using TtArsC as biomolecular probe. TtArsC has been adsorbed on gold nanoparticles (AuNPs) and nanobiocomplexes demonstrating stability and the capacity to strongly bind the toxic ions. Interestingly, TtArsC-AuNPs interaction with arsenic can be followed by naked eye, since solutions completely change their colors. Therefore, a straightforward application in fast and low-cost screening of water can be envisaged. Finally, Geobacillus stearothermophilus has been isolated from a geothermal area near Naples known as Pisciarelli, and has been identified as a new arsenic tolerant microorganism. Our results made G. stearothermophilus a novel model of study for the development of new arsenic biosensing and bioremediation techniques and confirm the possibility of using Thermus thermophilus as biological systems (cellular or enzymatic) for the traceability of pollutants after a thorough molecular, structural and functional characterization of the components involved and their interactions.

Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the toxic metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to both arsenate and arsenite; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterised from the plasmid pI258 of the Gram+ bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase. It also exhibited weak phosphatase activity. The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important contributor in the arsenic resistance in T. thermophilus and give a structural-functional characterization of a thermophilic arsenate reductase.

Background: The characterization of the molecular determinants of metal resistance has potential biotechnological application in biosensing and bioremediation. In this context, the bacterium Thermus thermophilus HB27 is a metal tolerant thermophile containing a set of genes involved in arsenic resistance which, differently from other microbes, are not organized into a single operon. They encode the proteins: arsenate reductase, TtArsC, arsenic efflux membrane transporter, TtArsX, and transcriptional repressor, TtSmtB. Results: In this work we show that the arsenic efflux protein TtArsX and the arsenic responsive transcriptional repressor TtSmtB are required to provide resistance to cadmium. We analyzed the sensitivity to Cd(II) of mutants lacking TtArsX, finding that they are more sensitive to this metal than the wild type strain. In addition, using promoter probe reporter plasmids, we show that the transcription of TtarsX is also stimulated by the presence of Cd(II) in a TtSmtB-dependent way. Actually, a regulatory circuit composed of TtSmtB and a reporter gene expressed from the TtarsX promoter responds to variation in Cd(II), As(III) and As(V) concentrations. Conclusions: Our results demonstrate that the system composed by TtSmtB and TtArsX is responsible for both the arsenic and cadmium resistance in T. thermophilus. The data also support the use of T. thermophilus as a suitable chassis for the design and development of As-Cd biosensors., This research was carried out in the frame of the Project “Immobilization of ENzymes on hydrophobin-functionalized NAnomaterials” funded by the University of Napoli Federico II.

Reactive species of oxygen (ROS), responsible for oxidative stress, accumulate in various tissues damaged by burns, decubitus ulcers, and vascular lesions. Antioxidants play an important and well-documented role in healing of chronic and acute wounds. Rigenase®, a specific extract of Triticum vulgare manufactured by Farmaceutici Damor, is employed in products used for the regeneration of tissue injuries. In this work, we show that Rigenase® exhibits a scavenging effect toward free radicals, thus pointing to its relevant antioxidant activity.

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

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.