Programmed Deviations of Ribosomes From Standard Decoding in Archaea.

Genetic code decoding, initially considered to be universal and immutable, is now known to be flexible. In fact, in specific genes, ribosomes deviate from the standard translational rules in a programmed way, a phenomenon globally termed recoding. Translational recoding, which has been found in all domains of life, includes a group of events occurring during gene translation, namely stop codon readthrough, programmed ± 1 frameshifting, and ribosome bypassing. These events regulate protein expression at translational level and their mechanisms are well known and characterized in viruses, bacteria and eukaryotes. In this review we summarize the current state-of-the-art of recoding in the third domain of life. In Archaea, it was demonstrated and extensively studied that translational recoding regulates the decoding of the 21st and the 22nd amino acids selenocysteine and pyrrolysine, respectively, and only one case of programmed -1 frameshifting has been reported so far in Saccharolobus solfataricus P2. However, further putative events of translational recoding have been hypothesized in other archaeal species, but not extensively studied and confirmed yet. Although this phenomenon could have some implication for the physiology and adaptation of life in extreme environments, this field is still underexplored and genes whose expression could be regulated by recoding are still poorly characterized. The study of these recoding episodes in Archaea is urgently needed.

Xyloglucan Oligosaccharides Hydrolysis by Exo-Acting Glycoside Hydrolases from Hyperthermophilic Microorganism Saccharolobus solfataricus.

In the field of biocatalysis and the development of a bio-based economy, hemicellulases have attracted great interest for various applications in industrial processes. However, the study of the catalytic activity of the lignocellulose-degrading enzymes needs to be improved to achieve the efficient hydrolysis of plant biomasses. In this framework, hemicellulases from hyperthermophilic archaea show interesting features as biocatalysts and provide many advantages in industrial applications thanks to their stability in the harsh conditions encountered during the pretreatment process. However, the hemicellulases from archaea are less studied compared to their bacterial counterpart, and the activity of most of them has been barely tested on natural substrates. Here, we investigated the hydrolysis of xyloglucan oligosaccharides from two different plants by using, both synergistically and individually, three glycoside hydrolases from Saccharolobus solfataricus: a GH1 ß-gluco-/ß-galactosidase, a α-fucosidase belonging to GH29, and a α-xylosidase from GH31. The results showed that the three enzymes were able to release monosaccharides from xyloglucan oligosaccharides after incubation at 65 °C. The concerted actions of ß-gluco-/ß-galactosidase and the α-xylosidase on both xyloglucan oligosaccharides have been observed, while the α-fucosidase was capable of releasing all α-linked fucose units from xyloglucan from apple pomace, representing the first GH29 enzyme belonging to subfamily A that is active on xyloglucan.

Transcript Regulation of the Recoded Archaeal α-l-Fucosidase In Vivo.

Genetic decoding is flexible, due to programmed deviation of the ribosomes from standard translational rules, globally termed "recoding". In Archaea, recoding has been unequivocally determined only for termination codon readthrough events that regulate the incorporation of the unusual amino acids selenocysteine and pyrrolysine, and for -1 programmed frameshifting that allow the expression of a fully functional α-l-fucosidase in the crenarchaeon Saccharolobus solfataricus, in which several functional interrupted genes have been identified. Increasing evidence suggests that the flexibility of the genetic code decoding could provide an evolutionary advantage in extreme conditions, therefore, the identification and study of interrupted genes in extremophilic Archaea could be important from an astrobiological point of view, providing new information on the origin and evolution of the genetic code and on the limits of life on Earth. In order to shed some light on the mechanism of programmed -1 frameshifting in Archaea, here we report, for the first time, on the analysis of the transcription of this recoded archaeal α-l-fucosidase and of its full-length mutant in different growth conditions in vivo. We found that only the wild type mRNA significantly increased in S. solfataricus after cold shock and in cells grown in minimal medium containing hydrolyzed xyloglucan as carbon source. Our results indicated that the increased level of fucA mRNA cannot be explained by transcript up-regulation alone. A different mechanism related to translation efficiency is discussed.

Spatial Metagenomics of Three Geothermal Sites in Pisciarelli Hot Spring Focusing on the Biochemical Resources of the Microbial Consortia.

Terrestrial hot springs are of great interest to the general public and to scientists alike due to their unique and extreme conditions. These have been sought out by geochemists, astrobiologists, and microbiologists around the globe who are interested in their chemical properties, which provide a strong selective pressure on local microorganisms. Drivers of microbial community composition in these springs include temperature, pH, in-situ chemistry, and biogeography. Microbes in these communities have evolved strategies to thrive in these conditions by converting hot spring chemicals and organic matter into cellular energy. Following our previous metagenomic analysis of Pisciarelli hot springs (Naples, Italy), we report here the comparative metagenomic study of three novel sites, formed in Pisciarelli as result of recent geothermal activity. This study adds comprehensive information about phylogenetic diversity within Pisciarelli hot springs by peeking into possible mechanisms of adaptation to biogeochemical cycles, and high applicative potential of the entire set of genes involved in the carbohydrate metabolism in this environment (CAZome). This site is an excellent model for the study of biodiversity on Earth and biosignature identification, and for the study of the origin and limits of life.

Discovery of hyperstable carbohydrate-active enzymes through metagenomics of extreme environments.

The enzymes from hyperthermophilic microorganisms populating volcanic sites represent interesting cases of protein adaptation and biotransformations under conditions where conventional enzymes quickly denature. The difficulties in cultivating extremophiles severely limit access to this class of biocatalysts. To circumvent this problem, we embarked on the exploration of the biodiversity of the solfatara Pisciarelli, Agnano (Naples, Italy), to discover hyperthermophilic carbohydrate-active enzymes (CAZymes) and to characterize the entire set of such enzymes in this environment (CAZome). Here, we report the results of the metagenomic analysis of two mud/water pools that greatly differ in both temperature and pH (T = 85 °C and pH 5.5; T = 92 °C and pH 1.5, for Pool1 and Pool2, respectively). DNA deep sequencing and following in silico analysis led to 14 934 and 17 652 complete ORFs in Pool1 and Pool2, respectively. They exclusively belonged to archaeal cells and viruses with great genera variance within the phylum Crenarchaeota, which reflected the difference in temperature and pH of the two Pools. Surprisingly, 30% and 62% of all of the reads obtained from Pool1 and 2, respectively, had no match in nucleotide databanks. Genes associated with carbohydrate metabolism were 15% and 16% of the total in the two Pools, with 278 and 308 putative CAZymes in Pool1 and 2, corresponding to ~ 2.0% of all ORFs. Biochemical characterization of two CAZymes of a previously unknown archaeon revealed a novel subfamily GH5_19 ß-mannanase/ß-1,3-glucanase whose hemicellulose specificity correlates with the vegetation surrounding the sampling site, and a novel NAD+ -dependent GH109 with a previously unreported ß-N-acetylglucosaminide/ß-glucoside specificity. DATABASES: The sequencing reads are available in the NCBI Sequence Read Archive (SRA) database under the accession numbers SRR7545549 (Pool1) and SRR7545550 (Pool2). The sequences of GH5_Pool2 and GH109_Pool2 are available in GenBank database under the accession numbers MK869723 and MK86972, respectively. The environmental data relative to Pool1 and Pool2 (NCBI BioProject PRJNA481947) are available in the Biosamples database under the accession numbers SAMN09692669 (Pool1) and SAMN09692670 (Pool2).

Identification of a novel esterase from the thermophilic bacterium Geobacillus thermodenitrificans NG80-2.

In the framework of the discovery of new thermophilic enzymes of potential biotechnological interest, we embarked in the characterization of a new thermophilic esterase from the thermophilic bacterium Geobacillus thermodenitrificans. The phylogenetic analysis of the GTNG_0744 esterase indicated that the sequence belongs to the enterochelin/enterobactin esterase group, which have never been recognized as a family in the lipases/esterase classification. These enzymes catalyze the last step in the acquisition of environmental Fe3+ through siderophore hydrolysis. In silico analysis revealed, for the first time, that the machinery for the uptake of siderophores is present in G. thermodenitrificans. The purified recombinant enzyme, EstGtA3, showed different substrate specificity from known enterochelin/enterobactin esterases, recognizing short chain esters with a higher specificity constant for 4-NP caprylate. The enzyme does not require cofactors for its activity, is active in the pH range 7.0-8.5, has highest activity at 60 °C and is 100% stable when incubated for 16 h at 55 °C. DTT, ß-mercaptoethanol and Triton X-100 have an activating effect on the enzymatic activity. Organic solvents have in general a negative effect on the enzyme, but n-hexane is a strong activator up to 150, making EstGtA3 a good candidate for applications in biotechnology.

GlcNAc De-N-Acetylase from the Hyperthermophilic Archaeon Sulfolobus solfataricus.

Sulfolobus solfataricus is an aerobic crenarchaeal hyperthermophile with optimum growth at temperatures greater than 80°C and pH 2 to 4. Within the crenarchaeal group of Sulfolobales, N-acetylglucosamine (GlcNAc) has been shown to be a component of exopolysaccharides, forming their biofilms, and of the N-glycan decorating some proteins. The metabolism of GlcNAc is still poorly understood in Archaea, and one approach to gaining additional information is through the identification and functional characterization of carbohydrate active enzymes (CAZymes) involved in the modification of GlcNAc. The screening of S. solfataricus extracts allowed the detection of a novel α-N-acetylglucosaminidase (α-GlcNAcase) activity, which has never been identified in Archaea Mass spectrometry analysis of the purified activity showed a protein encoded by the sso2901 gene. Interestingly, the purified recombinant enzyme, which was characterized in detail, revealed a novel de-N-acetylase activity specific for GlcNAc and derivatives. Thus, assays to identify an α-GlcNAcase found a GlcNAc de-N-acetylase instead. The α-GlcNAcase activity observed in S. solfataricus extracts did occur when SSO2901 was used in combination with an α-glucosidase. Furthermore, the inspection of the genomic context and the preliminary characterization of a putative glycosyltransferase immediately upstream of sso2901 (sso2900) suggest the involvement of these enzymes in the GlcNAc metabolism in S. solfataricusIMPORTANCE In this study, a preliminary screening of cellular extracts of S. solfataricus allowed the identification of an α-N-acetylglucosaminidase activity. However, the characterization of the corresponding recombinant enzyme revealed a novel GlcNAc de-N-acetylase, which, in cooperation with the α-glucosidase, catalyzed the hydrolysis of O-α-GlcNAc glycosides. In addition, we show that the product of a gene flanking the one encoding the de-N-acetylase is a putative glycosyltransferase, suggesting the involvement of the two enzymes in the metabolism of GlcNAc. The discovery and functional analysis of novel enzymatic activities involved in the modification of this essential sugar represent a powerful strategy to shed light on the physiology and metabolism of Archaea.

Isolation and characterisation of a novel alpha-amylase from the extreme haloarchaeon Haloterrigena turkmenica.

An extracellular halophilic alpha-amylase (AmyA) was produced by the haloarchaeon Haloterrigena turkmenica grown in medium enriched with 0.2% (w/v) starch. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) analyses showed a major band at 66.0kDa and a peak of 54.0kDa, respectively. Analysis of tryptic fragments of the protein present in the major SDS-PAGE band by nano-LC-ESI-MS/MS led to identification of the alpha-amylase catalytic region, encoded by the htur2110 gene, as the protein possessing the described activity. Optimal values for activity were 55°C, pH 8.5 and 2M NaCl, and high thermostability was showed at 55°C and 3M NaCl. AmyA activity was enhanced by Triton X-100 and was not influenced by n-hexane and chloroform. Starch hydrolysis produced different oligomers with maltose as the smallest end-product. The efficiency of AmyA in degrading starch contained in agronomic residues was tested in grape cane chosen as model substrate. Preliminary results showed that starch was degraded making the enzyme a potential candidate for utilization of agro-industrial waste in fuel and chemicals production. AmyA is one of the few investigated amylases produced by haloarchaea, and the first alpha-amylase described among microorganisms belonging to the genus Haloterrigena.

Characterization of extra virgin olive oils produced with typical Italian varieties by their phenolic profile.

Evaluation of phenolic profile, antioxidant power, and protective capacity against oxidation of red blood cells (RBCs) of olive oil phenolic extracts (OOPEs) from several Italian varieties were studied. Phenolic profiles, and quantification of seven selected bioactive compounds were performed by RP-HPLC. OOPEs exhibited high antioxidant activity, and this capacity was positively related to their phenolic amount. In particular, OOPE5 (cv Gentile di Larino, Molise region) displayed the highest phenolic and ortho-diphenolic content as well as the strongest scavenging activity determined using 2,2'-diphenyl-1-picrylhydrazyl (DPPH) (87% DPPH inhibition). Protective capacity against stressed RBCs was investigated through the evaluation of methemoglobin (MetHb) and malondialdehyde (MDA) levels. OOPE5 was the most active against methemoglobin production (53.7% reduction), whereas OOPE1 (cv Lavagnina, Liguria region) showed the highest protection toward malondialdehyde (83.3% reduction). Overall the selected oils showed qualitative and quantitative differences in phenol composition, and this variability influenced their protective effect against oxidative damages.

Chestnut shell as unexploited source of fermentable sugars: effect of different pretreatment methods on enzymatic saccharification.

Chestnut shell (CS) is an agronomic residue mainly used for extraction of antioxidants or as adsorbent of metal ions. It also contains some polysaccharide that has not been considered as potential source of fermentable sugars for biofuel production until now. In this study, the effect of different pretreatment methods on CS was evaluated in order to obtain the greatest conversion of cellulose and xylan into fermentable sugars. Hot acid impregnation, steam explosion (acid-catalysed or not), and aqueous ammonia soaking (AAS) were selected as pretreatments. The pretreated biomass was subjected to saccharification with two enzyme cocktails prepared from commercial preparations, and evaluation of the best pretreatment and enzyme cocktail was based on the yield of fermentable sugars produced. As AAS provided the best result after preliminary experiments, enhancement of sugar production was attempted by changing the concentrations of ammonium hydroxide, enzymes, and CS. The optimal pretreatment condition was 10 % ammonium hydroxide, 70 °C, 22 h with CS at 5 % solid loading. After saccharification of the pretreated CS for 72 h at 50 °C and pH 5.0 with a cocktail containing cellulase (Accellerase 1500), beta-glucosidase (Accellerase BG), and xylanase (Accellerase XY), glucose and xylose yields were 67.8 and 92.7 %, respectively.

Evidence that the xylanase activity from Sulfolobus solfataricus Oalpha is encoded by the endoglucanase precursor gene (sso1354) and characterization of the associated cellulase activity.

Sulfolobus solfataricus strain Oalpha was previously isolated for its ability to grow on minimal medium supplemented with xylan as a carbon source. The strain exhibited thermostable xylanase activity but several attempts to identify the gene encoding for the activity failed. Further studies showed that the xylanase displayed activity on carboxymethylcellulose (CMC) and the new activity was characterized. It exhibited an optimal temperature and pH of 95 degrees C and 3.5, respectively, and a half-life of 53 min at 95 degrees C. The enzyme, which was demonstrated to be glycosylated, hydrolyzed CMC in an endo-manner releasing cellobiose and other cello-oligomers. Analysis of the tryptic fragments by tandem mass spectrometry led to identification of the endoglucanase precursor, encoded by the sso1354 gene, as the protein possessing dual activity. The efficiency of the SSO1354 protein in degrading cellulosic and hemicellulosic fractions contained in agronomic residues was tested at low pH and high temperature. Cellulose and xylan were degraded to glucose and xylose at 90 degrees C, pH 4 by an enzyme mix consisting of SSO1354 and additional glycosyl hydrolases from S. solfataricus Oalpha. Given its role in saccharification processes requiring high temperatures and acidic environments, SSO1354 represents an interesting candidate for the utilization of agro-industrial waste for fuel production.

Gene cloning and expression in Escherichia coli of a bi-functional beta-D-xylosidase/alpha-L-arabinosidase from Sulfolobus solfataricus involved in xylan degradation.

An open reading frame encoding a putative bi-functional beta-D-xylosidase/alpha-L-arabinosidase (Sso3032) was identified on the genome sequence of Sulfolobus solfataricus P2, the predicted gene product showing high amino-acid sequence similarity to bacterial and eukaryal individual beta-D-xylosidases and alpha-L-arabinosidases as well as bi-functional enzymes such as the protein from Thermoanaerobacter ethanolicus and barley. The sequence was PCR amplified from genomic DNA of S. solfataricus P2 and heterologous gene expression obtained in Escherichia coli, under optimal conditions for overproduction. Specific assays performed at 75 degrees C revealed the presence in the transformed E. coli cell extracts of this archaeal activity involved in sugar hydrolysis and specific for both substrates. The recombinant protein was purified by thermal precipitation of the host proteins and ethanol fractionation and other properties, such as high thermal activity and thermostability could be determined. The protein showed a homo-tetrameric structure with a subunit of molecular mass of 82.0 kDa which was in perfect agreement with that deduced from the cloned gene. Northern blot analysis of the xarS gene indicates that it is specifically induced by xylan and repressed by monosaccharides like D-glucose and L-arabinose.