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Agents that form methylation adducts in DNA are highly mutagenic and carcinogenic, and organisms have evolved specialized cellular pathways devoted to their repair, including DNA alkyltransferases. These are proteins conserved in eucarya, bacteria and archaea, acting by a unique reaction mechanism, which leads to direct repair of DNA alkylation damage and irreversible protein alkylation. The alkylated form of DNA alkyltransferases is inactive, and in eukaryotes, it is rapidly directed to degradation. We report here in vitro and in vivo studies on the DNA alkyltransferase from the thermophilic archaeon Sulfolobus solfataricus (SsOGT). The development of a novel, simple, and sensitive fluorescence-based assay allowed a careful characterization of the SsOGT biochemical and DNA binding activities. In addition, transcriptional and post-translational regulation of SsOGT by DNA damage was studied. We show that although the gene transcription is induced by alkylating agent treatment, the protein is degraded in vivo by an alkylation-dependent mechanism. These experiments suggest a striking conservation, from archaea to humans, of this important pathway safeguarding genome stability.

Metagenomic analysis applied to specialized environments offers the possibility to understand the evolution of complex biotypes. In particular, metagenomics of extreme environments, which might resemble those of primitive Earth, is of interest for the study of the origin, evolution and distribution of extremophiles (organisms thriving at these conditions) as primitive forms of life populating our planet and, in an astrobiological perspective, other bodies in the universe. The Pisciarelli solfatara, located in the Phelgrean Fields (Naples, Italy), is characterized by a remarkable diversity in terms of temperature and pHs of the different mud pools and solfataric vents [1]. Here several thermoacidophilic species, such as Acidianus, Archaeoglobus spp, Pyrobaculum, Sulfolobus, and several viruses have been isolated [1]. In order to map the composition of this biotope in terms of number and abundance of the different species, we decided to carry out a metagenomic deep sequencing project on soil and water/mud pools of Pisciarelly solfatara. We will show our preliminary results derived by the sampling in the main water pool. Total DNA was extracted from the pellets obtained by centrifuging water samples and analysed by using Illumina paired-end sequencing technology. In a single run, we produced~3.0 Gb total nucleotides. These reads were assembled into 1,543 contigs with an N50 of 15863 bp，and a total of 17,652 Open Reading Frames (ORFs) were identified. These ORFs were subjected to BLAST similarity searches and annotated with KEGG, eggNOG and NR database respectively. The clean reads rate was 99.7% and a total of 17,652 Open Reading Frames (ORFs) were identified. The majority (77%) of the species found belonged to the Crenarcheaota phylum, which branches deeply in the phylogenetic tree of life, followed by Proteobacteria (22%), and, in much lesser amounts, Euryarchaeota, Actinobacteria, and others. The number of species identified is much higher than those found by a conventional PCR-based approach [2]. Details on the species composition and on the realtive abundance of each ORF will be described together with the astrobiological implications.

The second generation bioethanol represents a main challenge in global efforts to utilize renewable resources rather than fossil fuels. However, the close association of cellulose and hemicelluloses to lignin in the plant cell wall makes it difficult to degrade lignocellulose into fermentable sugars. Consequently, pretreatments are necessary to make the polysaccharides more accessible to the enzymes, but the high temperature and extreme pH conditions required give rise to problems when using conventional enzymes in the saccharification step (Galbe and Zacchi 2002). Microorganisms thriving in habitats characterized by harsh conditions, and the enzymes derived therein, represent a helpful tool in the development of bioethanol production processes. In fact, they allow bioconversions at non-conventional conditions under which common biocatalysts are denatured. The use of high operational temperatures allows energy savings by reducing the cooling cost after high temperature pretreatments, and, in ethanol production, thermophilic conditions permit ethanol evaporation allowing harvest during fermentation.

Pompe disease (PD) is a metabolic myopathy due to the deficiency of the lysosomal enzyme alpha-glucosidase (GAA). The only approved treatment for this disorder, enzyme replacement with recombinant human GAA (rhGAA), has shown limited therapeutic efficacy in some PD patients. Pharmacological chaperone therapy (PCT), either alone or in combination with enzyme replacement, has been proposed as an alternative therapeutic strategy. However, the chaperones identified so far also are active site-directed molecules and potential inhibitors of target enzymes. We demonstrated that N-acetylcysteine (NAC) is a novel allosteric chaperone for GAA. NAC improved the stability of rhGAA as a function of pH and temperature without disrupting its catalytic activity. A computational analysis of NAC-GAA interactions confirmed that NAC does not interact with GAA catalytic domain. NAC enhanced the residual activity of mutated GAA in cultured PD fibroblasts and in COS7 cells overexpressing mutated GAA. NAC also enhanced rhGAA efficacy in PD fibroblasts. In cells incubated with NAC and rhGAA, GAA activities were 3.7-8.7-fold higher than those obtained in cells treated with rhGAA alone. In a PD mouse model the combination of NAC and rhGAA resulted in better correction of enzyme activity in liver, heart, diaphragm and gastrocnemia, compared to rhGAA alone. Received 12 May 2012; accepted 3 July 2012; advance online publication 18 September 2012. doi:10.1038/mt.2012.152

Agents that form methylation adducts in DNA are highly mutagenic and carcinogenic, and organisms have evolved specialized cellular pathways devoted to their repair, including DNA alkyltransferases. These are proteins conserved in eucarya, bacteria and archaea, acting by a unique reaction mechanism, which leads to direct repair of DNA alkylation damage and irreversible protein alkylation. The alkylated form of DNA alkyltransferases is inactive, and in eukaryotes, it is rapidly directed to degradation. We report here in vitro and in vivo studies on the DNA alkyltransferase from the thermophilic archaeon Sulfolobus solfataricus (SsOGT). The development of a novel, simple, and sensitive fluorescence-based assay allowed a careful characterization of the SsOGT biochemical and DNA binding activities. In addition, transcriptional and post-translational regulation of SsOGT by DNA damage was studied. We show that although the gene transcription is induced by alkylating agent treatment, the protein is degraded in vivo by an alkylation-dependent mechanism. These experiments suggest a striking conservation, from archaea to humans, of this important pathway safeguarding genome stability.

Reverse gyrase is a peculiar DNA topoisomerase, specific of hyperthermophilic Archaea and Bacteria, which has the unique ability of introducing positive supercoiling into DNA molecules. Although the function of the enzyme has not been established directly, it has been suggested to be involved in DNA protection and repair. We show here that the enzyme is degraded after treatment of Sulfolobus solfataricus cells with the alkylating agent MMS. MMS-induced reverse gyrase degradation is highly specific, since (i) neither hydroxyurea (HU) nor puromycin have a similar effect, and (ii) topoisomerase VI and two chromatin components are not degraded. Reverse gyrase degradation does not depend on protein synthesis. Experiments in vitro show that direct exposure of cell extracts to MMS does not induce reverse gyrase degradation; instead, extracts from MMS-treated cells contain some factor(s) able to degrade the enzyme in extracts from control cells. In vitro, degradation is blocked by incubation with divalent metal chelators, suggesting that reverse gyrase is selectively degraded by a metal-dependent protease in MMS-treated cells. In addition, we find a striking concurrence of extensive genomic DNA degradation and reverse gyrase loss in MMS-treated cells. These results support the hypothesis that reverse gyrase plays an essential role in DNA thermoprotection and repair in hyperthermophilic organisms.