Antonucci, Immacolata (2016) DEVELOPMENT OF MOLECULAR SYSTEMS FOR THE DETECTION OF ENVIRONMENTAL ARSENIC. [Tesi di dottorato]

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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.

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