Del Giudice, Immacolata (2013) Molecular characterization of aromatic compound and heavy metal detoxification systems in thermophilic microorganisms: impact on biomonitoring and bioremediation. [Tesi di dottorato]

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Abstract

Both arsenic and aromatic compounds are naturally present in the environment but human activities, such as the chemical and pharmaceutical industries, use of fossil fuels and pesticides, have contributed to their anomalous accumulation in the biosphere, determining severe damages to all living organisms. Many microorganisms possess tuned mechanisms for sensing the level of pollutants in their growth environment and controlling intracellular concentrations according to their biochemical needs. In this PhD thesis a mechanism for aromatic compound detoxification and a strategy for arsenic resistance have been identified and characterized in Sulfolobus solfataricus P2 and in Thermus thermophilus HB27, respectively. In S. solfataricus we characterized BldR2, as a new member of the MarR transcriptional factor family, and reported the physiological, biochemical, and biophysical investigation of its stability and DNA binding ability. Transcriptional analysis revealed the upregulation of bldR2 expression by aromatic compounds and allowed the identification of cis-acting sequences. BldR2 is a dimer in solution and possesses a high stability against temperature and chemical denaturing agents; the protein binds site-specifically to its own promoter and the alcohol dehydrogenase gene and the MarR-like operon promoters, as well as to the putative promoter of the operon encoding components of antimicrobial peptide transport system, located immediately upstream of its gene. Benzaldehyde and salicylate, the ligands of BldR2, are antagonists of DNA binding. Two single-point mutants of BldR2 have been produced and characterized; the results point to arginine 19 as a key amino acid involved in protein dimerization, while the introduction of a serine in position 65 increases the DNA affinity of the protein, making it comparable with those of other members of the MarR family. Regarding to the arsenic resistance mechanism, T. thermophilus exhibited a good tolerance to high concentrations of 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+ Staphylococcus aureus bacterium, and a putative chromosomal transcriptional regulator (TtsmtB) gene, encoding a protein homologous to the members of ArsR/SmtB family. Differently from the characterized arsenic resistance genes of many microorganisms, TtarsC and TtsmtB are part of two operons including genes not apparently related to arsenic resistance; qRT-PCR showed that TtarsC expression was four-fold increased when arsenate was added to the growth medium, whereas TtsmtB expression was two-fold increased in the presence of both arsenate and arsenite. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant proteins, proved that, like ArsC of S. aureus, TtArsC was indeed a thioredoxin-coupled arsenate reductase and exhibited also weak phosphatase activity; TtSmtB was able to bind putative promoter regions of its own gene, of the operon including TtsmtB and of the operon including TtarsC; arsenite was antagonist of DNA binding by TtSmtB. The catalytic role of the first cysteine (Cys7) of TtArsC was also ascertained by site directed mutagenesis. All the results identify TtArsC and TtSmtB as the main actors in the arsenic resistance in T. thermophilus giving the first structural-functional characterization of thermophilic components of the arsenic detoxification mechanism. Comprehensive knowledge on the molecular and genetic basis of detoxification, besides being stimulating from an evolutionary point of view, also represents an important starting point for developing efficient and selective environmental bioremediation approaches.

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