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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.

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.

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 p