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A mesophilic, hyperacidophilic archaeon, strain M1T, was isolated from a rock sample from Vulcano Island, Italy. Cells of this organism were cocci with an average diameter of 1 µm. Some cells possessed filaments. The strain grew in the range of temperatures between 15 and 52 °C and pH 0.5-4.0 with growth optima at 40 °C and pH 1.0. Strain M1T was aerobic and chemoorganotrophic, growing on complex substrates, such as casamino acids, trypticase, tryptone, yeast and beef extracts. No growth at expenses of oxidation of elemental sulphur or reduced sulphur compounds, pyrite, or ferrous sulphate was observed. The core lipids were glycerol dibiphytanyl glycerol tetraether lipids (membrane spanning) with 0 to 4 cyclopentane moieties and archaeol, with trace amounts of hydroxy archaeol. The dominant quinone was MK-7â:â7. The genome size of M1T was 1.67 Mbp with a G+C content of 39.76 mol%, and both characteristics were well within the common range for Thermoplasmatales. The phylogenetic analysis based on 16S rRNA gene sequence placed the strain M1T within the order Thermoplasmatales with sequence identities of 90.9, 90.3 and 90.5% to the closest SSU rRNA gene sequences from organisms with validly published names, Thermoplasma acidophilum, Thermoplasma volcanium and Thermogymnomonas acidicola, respectively. Based on the results of our genomic, phylogenetic, physiological and chemotaxonomic studies, we propose that strain M1T (=DSM 116605T=JCM 36570T) represents a new genus and species, Oxyplasma meridianum gen. nov., sp. nov., within the order Thermoplasmatales.
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Composição de Bases , DNA Arqueal , Filogenia , RNA Ribossômico 16S , Análise de Sequência de DNA , RNA Ribossômico 16S/genética , DNA Arqueal/genética , Itália , Thermoplasmales/genética , Thermoplasmales/classificação , Thermoplasmales/isolamento & purificação , Sedimentos Geológicos/microbiologia , Genoma ArquealRESUMO
Hydrothermal vents are geographically widespread and host microorganisms with robust enzymes useful in various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of Firmicutes, Proteobacteria, and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus species and to some extent Fontimonas and Schleiferia species. The screening at 37 to 70°C of metagenomic fosmid libraries from above enrichment cultures identified three hydrolases (IS10, IS11, and IS12), all derived from yet-uncultured Chloroflexota and showing low sequence identity (33 to 56%) to characterized enzymes. Enzymes expressed in Escherichia coli exhibited maximal esterase activity at 70 to 90°C, with IS11 showing the highest thermostability (90% activity after 20-min incubation at 80°C). IS10 and IS12 were highly substrate promiscuous and hydrolyzed all 51 monoester substrates tested. Enzymes were active with PLA, polyethylene terephthalate model substrate, and mycotoxin T-2 (IS12). IS10 and IS12 had a classical α/ß-hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in their hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 Å revealed the presence of a N-terminal ß-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 included catalytic Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain enclosed the catalytic cleft like a lid and contributed to substrate binding. Our study identified novel thermotolerant carboxylesterases with a broad substrate range, including polyesters and mycotoxins, for potential applications in biotechnology. IMPORTANCE High-temperature-active microbial enzymes are important biocatalysts for many industrial applications, including recycling of synthetic and biobased polyesters increasingly used in textiles, fibers, coatings and adhesives. Here, we identified three novel thermotolerant carboxylesterases (IS10, IS11, and IS12) from high-temperature enrichment cultures from Ischia hydrothermal vents and incubated with biobased polymers. The identified metagenomic enzymes originated from uncultured Chloroflexota and showed low sequence similarity to known carboxylesterases. Active sites of IS10 and IS12 had the largest effective volumes among the characterized prokaryotic carboxylesterases and exhibited high substrate promiscuity, including hydrolysis of polyesters and mycotoxin T-2 (IS12). Though less promiscuous than IS10 and IS12, IS11 had a higher thermostability with a high temperature optimum (80 to 90°C) for activity and hydrolyzed polyesters, and its crystal structure revealed an unusual lipocalin domain likely involved in substrate binding. The polyesterase activity of these enzymes makes them attractive candidates for further optimization and potential application in plastics recycling.
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Hidrolases de Éster Carboxílico , Fontes Hidrotermais , Hidrolases de Éster Carboxílico/metabolismo , Polímeros , Hidrolases/metabolismo , Poliésteres , Plásticos , Especificidade por SubstratoRESUMO
The Calvin-Benson-Bassham (CBB) cycle assimilates CO2 for the primary production of organic matter in all plants and algae, as well as in some autotrophic bacteria. The key enzyme of the CBB cycle, ribulose-bisphosphate carboxylase/oxygenase (RubisCO), is a main determinant of de novo organic matter production on Earth. Of the three carboxylating forms of RubisCO, forms I and II participate in autotrophy, and form III so far has been associated only with nucleotide and nucleoside metabolism. Here, we report that form III RubisCO functions in the CBB cycle in the thermophilic chemolithoautotrophic bacterium Thermodesulfobium acidiphilum, a phylum-level lineage representative. We further show that autotrophic CO2 fixation in T. acidiphilum is accomplished via the transaldolase variant of the CBB cycle, which has not been previously demonstrated experimentally and has been considered unlikely to occur. Thus, this work reveals a distinct form of the key pathway of CO2 fixation.
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Processos Autotróficos , Proteínas de Bactérias/metabolismo , Firmicutes/enzimologia , Fotossíntese , Ribulose-Bifosfato Carboxilase/metabolismo , Dióxido de Carbono/metabolismo , Sequestro de Carbono , Redes e Vias MetabólicasRESUMO
RNA interference (RNAi)-based therapeutics hold the potential for dominant genetic disorders, enabling sequence-specific inhibition of pathogenic gene products. We aimed to direct RNAi for the selective suppression of the heterozygous GNAO1 c.607 G > A variant causing GNAO1 encephalopathy. By screening short interfering RNA (siRNA), we showed that GNAO1 c.607G>A is a druggable target for RNAi. The si1488 candidate achieved at least twofold allelic discrimination and downregulated mutant protein to 35%. We created vectorized RNAi by incorporating the si1488 sequence into the short hairpin RNA (shRNA) in the adeno-associated virus (AAV) vector. The shRNA stem and loop were modified to improve the transcription, processing, and guide strand selection. All tested shRNA constructs demonstrated selectivity toward mutant GNAO1, while tweaking hairpin structure only marginally affected the silencing efficiency. The selectivity of shRNA-mediated silencing was confirmed in the context of AAV vector transduction. To conclude, RNAi effectors ranging from siRNA to AAV-RNAi achieve suppression of the pathogenic GNAO1 c.607G>A and discriminate alleles by the single-nucleotide substitution. For gene therapy development, it is crucial to demonstrate the benefit of these RNAi effectors in patient-specific neurons and animal models of the GNAO1 encephalopathy.
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Encefalopatias , Terapia Genética , Animais , Humanos , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Alelos , Encefalopatias/genética , Vetores Genéticos/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genéticaRESUMO
The development of personalized medicine for genetic diseases requires preclinical testing in the appropriate animal models. GNAO1 encephalopathy is a severe neurodevelopmental disorder caused by heterozygous de novo mutations in the GNAO1 gene. GNAO1 c.607 G>A is one of the most common pathogenic variants, and the mutant protein Gαo-G203R likely adversely affects neuronal signaling. As an innovative approach, sequence-specific RNA-based therapeutics such as antisense oligonucleotides or effectors of RNA interference are potentially applicable for selective suppression of the mutant GNAO1 transcript. While in vitro validation can be performed in patient-derived cells, a humanized mouse model to rule out the safety of RNA therapeutics is currently lacking. In the present work, we employed CRISPR/Cas9 technology to introduce a single-base substitution into exon 6 of the Gnao1 to replace the murine Gly203-coding triplet (GGG) with the codon used in the human gene (GGA). We verified that genome-editing did not interfere with the Gnao1 mRNA or Gαo protein synthesis and did not alter localization of the protein in the brain structures. The analysis of blastocysts revealed the off-target activity of the CRISPR/Cas9 complexes; however, no modifications of the predicted off-target sites were detected in the founder mouse. Histological staining confirmed the absence of abnormal changes in the brain of genome-edited mice. The created mouse model with the "humanized" fragment of the endogenous Gnao1 is suitable to rule out unintended targeting of the wild-type allele by RNA therapeutics directed at lowering GNAO1 c.607 G>A transcripts.
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The advancement of precision medicine critically depends on the robustness and specificity of the carriers used for the targeted delivery of effector molecules in the human body. Numerous nanocarriers have been explored in vivo, to ensure the precise delivery of molecular cargos via tissue-specific targeting, including the endocrine part of the pancreas, thyroid, and adrenal glands. However, even after reaching the target organ, the cargo-carrying vehicle needs to enter the cell and then escape lysosomal destruction. Most artificial nanocarriers suffer from intrinsic limitations that prevent them from completing the specific delivery of the cargo. In this respect, extracellular vesicles (EVs) seem to be the natural tool for payload delivery due to their versatility and low toxicity. However, EV-mediated delivery is not selective and is usually short-ranged. By inserting the viral membrane fusion proteins into exosomes, it is possible to increase the efficiency of membrane recognition and also ease the process of membrane fusion. This review describes the molecular details of the viral-assisted interaction between the target cell and EVs. We also discuss the question of the usability of viral fusion proteins in developing extracellular vesicle-based nanocarriers with a higher efficacy of payload delivery. Finally, this review specifically highlights the role of Gag and RNA binding proteins in RNA sorting into EVs.
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Exossomos/metabolismo , Transporte de RNA , Proteínas Virais de Fusão/metabolismo , Proteínas da Matriz Viral/metabolismo , Animais , Interações Hospedeiro-Patógeno , Humanos , Fusão de MembranaRESUMO
[This corrects the article DOI: 10.3389/fmicb.2020.576520.].
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The virus infection, which visually looks like typical monoinfection, in fact may hide a great complex of different species. Without detailed analysis, we may miss the important interaction between pathogens, including new species. In the current study, we found the new species inside the mix of cubic and polyhedral occlusion bodies (OBs) isolated from the gypsy moth, Lymantria dispar L. (Ld). Transmission electron microscopy (TEM) revealed that into the one cadaver were OBs which belonged to baculovirus and cypoviruses. The baculovirus produced polyhedral OBs, while cypoviruses produced polyhedral and cubic OBs. Genomic analysis detected the multiple Ld nucleopolyhedroviruses, and cypoviruses were Hubei lepidoptera virus 3 and Dendrolimus punctatus cypovirus 1. This represents the first isolation of the Hubei lepidoptera virus 3 from the gypsy moth, proposed as "Lymantria dispar cypovirus 3". The RNAseq analysis also revealed the presence of Lymantria dispar iflavirus 1. The insecticidal activity of the mixed infection was comparable to that of typical baculovirus monoinfection. Thus, we demonstrate that i) the shape of OBs identified by light microscopy cannot be a robust indicator of viral species infecting the host; ii) only specific analysis may reveal the true composition of viral infection.
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Mariposas , Nucleopoliedrovírus , Vírus de RNA , Animais , Larva , Nucleopoliedrovírus/genética , Vírus de RNA/genéticaRESUMO
Filterable microorganisms participate in dissolved organic carbon (DOC) cycling in freshwater systems, however their exact functional role remains unknown. We determined the taxonomic identity and community dynamics of prokaryotic microbiomes in the 0.22 µm-filtered fraction and unfiltered freshwater from the Conwy River (North Wales, UK) in microcosms and, using targeted metabolomics and 14C-labelling, examined their role in the utilization of amino acids, organic acids and sugars spiked at environmentally-relevant (nanomolar) concentrations. To identify changes in community structure, we used 16S rRNA amplicon and shotgun sequencing. Unlike the unfiltered water samples where the consumption of DOC was rapid, the filtered fraction showed a 3-day lag phase before the consumption started. Analysis of functional categories of clusters of orthologous groups of proteins (COGs) showed that COGs associated with energy production increased in number in both fractions with substrate addition. The filtered fraction utilized low-molecular-weight (LMW) DOC at much slower rates than the whole community. Addition of nanomolar concentrations of LMW DOC did not measurably influence the composition of the microbial community nor the rate of consumption across all substrate types in either fraction. We conclude that due to their low activity, filterable microorganisms play a minor role in LMW DOC processing within a short residence time of lotic freshwater systems.
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Microbiota , Compostos Orgânicos , Carbono , Água Doce , RNA Ribossômico 16S/genética , RiosRESUMO
Parys Mountain or Mynydd Parys (Isle of Anglesey, United Kingdom) is a mine-impacted environment, which accommodates a variety of acidophilic organisms. Our previous research of water and sediments from one of the surface acidic streams showed a high proportion of archaea in the total microbial community. To understand the spatial distribution of archaea, we sampled cores (0-20 cm) of sediment and conducted chemical analyses and taxonomic profiling of microbiomes using 16S rRNA gene amplicon sequencing in different core layers. The taxonomic affiliation of sequencing reads indicated that archaea represented between 6.2 and 54% of the microbial community at all sediment depths. Majority of archaea were associated with the order Thermoplasmatales, with the most abundant group of sequences being clustered closely with the phylotype B_DKE, followed by "E-plasma," "A-plasma," other yet uncultured Thermoplasmatales with Ferroplasma and Cuniculiplasma spp. represented in minor proportions. Thermoplasmatales were found at all depths and in the whole range of chemical conditions with their abundance correlating with sediment Fe, As, Cr, and Mn contents. The bacterial microbiome component was largely composed in all layers of sediment by members of the phyla Proteobacteria, Actinobacteria, Nitrospirae, Firmicutes, uncultured Chloroflexi (AD3 group), and Acidobacteria. This study has revealed a high abundance of Thermoplasmatales in acid mine drainage-affected sediment layers and pointed at these organisms being the main contributors to carbon, and probably to iron and sulfur cycles in this ecosystem.
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Marine hydrocarbon-degrading bacteria play an important role in natural petroleum biodegradation processes and were initially associated with man-made oil spills or natural seeps. There is no full clarity though on what, in the absence of petroleum, their natural niches are. Few studies pointed at some marine microalgae that produce oleophilic compounds (alkanes, long-chain fatty acids, and alcohols) as potential natural hosts of these bacteria. We established Dansk crude oil-based enrichment cultures with photobioreactor-grown marine microalgae cultures Pavlova lutheri and Nannochloropsis oculata and analyzed the microbial succession using cultivation and SSU (16S) rRNA amplicon sequencing. We found that petroleum enforced a strong selection for members of Alpha- and Gamma-proteobacteria in both enrichment cultures with the prevalence of Alcanivorax and Marinobacter spp., well-known hydrocarbonoclastic bacteria. In total, 48 non-redundant bacterial strains were isolated and identified to represent genera Alcanivorax, Marinobacter, Thalassospira, Hyphomonas, Halomonas, Marinovum, Roseovarius, and Oleibacter, which were abundant in sequencing reads in both crude oil enrichments. Our assessment of public databases demonstrated some overlaps of geographical sites of isolation of Nannochloropsis and Pavlova with places of molecular detection and isolation of Alcanivorax and Marinobacter spp. Our study suggests that these globally important hydrocarbon-degrading bacteria are associated with P. lutheri and N. oculata.