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.

A novel arsenate reductase from the bacterium Thermus thermophilus HB27: its role in arsenic detoxification

LIMAURO, DANILA;ANTONUCCI, IMMACOLATA;BARTOLUCCI, SIMONETTA;FIORENTINO, GABRIELLA
2013

Abstract

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.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/594026
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