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1.
Nat Commun ; 12(1): 5281, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489402

RESUMO

The archaeal phylum Woesearchaeota, within the DPANN superphylum, includes phylogenetically diverse microorganisms that inhabit various environments. Their biology is poorly understood due to the lack of cultured isolates. Here, we analyze datasets of Woesearchaeota 16S rRNA gene sequences and metagenome-assembled genomes to infer global distribution patterns, ecological preferences and metabolic capabilities. Phylogenomic analyses indicate that the phylum can be classified into ten subgroups, termed A-J. While a symbiotic lifestyle is predicted for most, some members of subgroup J might be host-independent. The genomes of several Woesearchaeota, including subgroup J, encode putative [FeFe] hydrogenases (known to be important for fermentation in other organisms), suggesting that these archaea might be anaerobic fermentative heterotrophs.


Assuntos
Archaea/genética , Proteínas Arqueais/genética , Genoma Arqueal , Hidrogenase/genética , RNA Arqueal/genética , RNA Ribossômico 16S/genética , Sequência de Aminoácidos , Anaerobiose/genética , Archaea/classificação , Archaea/enzimologia , Proteínas Arqueais/metabolismo , Evolução Biológica , Fermentação , Processos Heterotróficos/genética , Hidrogenase/metabolismo , Metagenoma , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
2.
Nucleic Acids Res ; 49(16): 9444-9458, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34387688

RESUMO

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg2+-dependent cleavage of the 5' leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250-500 mM Mg2+ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg2+ requirement to ∼10-20 mM and improves the rate and fidelity of cleavage. To understand the Mg2+- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPRC domain and Cy5-RPRS domain), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg2+-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg2+ cooperativity. Our findings highlight how Mg2+ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.


Assuntos
Archaea/enzimologia , Magnésio/metabolismo , RNA Arqueal/genética , Ribonuclease P/genética , Conformação de Ácido Nucleico/efeitos dos fármacos , Pyrococcus furiosus/enzimologia , Pyrococcus furiosus/genética , Precursores de RNA/genética , RNA Arqueal/ultraestrutura , RNA Catalítico , Ribonuclease P/ultraestrutura
3.
Science ; 372(6541)2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33926924

RESUMO

CRISPR-Cas systems provide RNA-guided adaptive immunity in prokaryotes. We report that the multisubunit CRISPR effector Cascade transcriptionally regulates a toxin-antitoxin RNA pair, CreTA. CreT (Cascade-repressed toxin) is a bacteriostatic RNA that sequesters the rare arginine tRNAUCU (transfer RNA with anticodon UCU). CreA is a CRISPR RNA-resembling antitoxin RNA, which requires Cas6 for maturation. The partial complementarity between CreA and the creT promoter directs Cascade to repress toxin transcription. Thus, CreA becomes antitoxic only in the presence of Cascade. In CreTA-deleted cells, cascade genes become susceptible to disruption by transposable elements. We uncover several CreTA analogs associated with diverse archaeal and bacterial CRISPR-cas loci. Thus, toxin-antitoxin RNA pairs can safeguard CRISPR immunity by making cells addicted to CRISPR-Cas, which highlights the multifunctionality of Cas proteins and the intricate mechanisms of CRISPR-Cas regulation.


Assuntos
Proteínas Associadas a CRISPR/fisiologia , Sistemas CRISPR-Cas/fisiologia , Haloarcula/fisiologia , RNA Arqueal/fisiologia , Sistemas Toxina-Antitoxina/fisiologia , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Análise Mutacional de DNA , Regulação da Expressão Gênica em Archaea , Haloarcula/genética , Óperon , RNA de Transferência de Arginina/metabolismo , Sistemas Toxina-Antitoxina/genética
4.
Nucleic Acids Res ; 49(3): 1662-1687, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33434266

RESUMO

Ribosomes are intricate molecular machines ensuring proper protein synthesis in every cell. Ribosome biogenesis is a complex process which has been intensively analyzed in bacteria and eukaryotes. In contrast, our understanding of the in vivo archaeal ribosome biogenesis pathway remains less characterized. Here, we have analyzed the in vivo role of the almost universally conserved ribosomal RNA dimethyltransferase KsgA/Dim1 homolog in archaea. Our study reveals that KsgA/Dim1-dependent 16S rRNA dimethylation is dispensable for the cellular growth of phylogenetically distant archaea. However, proteomics and functional analyses suggest that archaeal KsgA/Dim1 and its rRNA modification activity (i) influence the expression of a subset of proteins and (ii) contribute to archaeal cellular fitness and adaptation. In addition, our study reveals an unexpected KsgA/Dim1-dependent variability of rRNA modifications within the archaeal phylum. Combining structure-based functional studies across evolutionary divergent organisms, we provide evidence on how rRNA structure sequence variability (re-)shapes the KsgA/Dim1-dependent rRNA modification status. Finally, our results suggest an uncoupling between the KsgA/Dim1-dependent rRNA modification completion and its release from the nascent small ribosomal subunit. Collectively, our study provides additional understandings into principles of molecular functional adaptation, and further evolutionary and mechanistic insights into an almost universally conserved step of ribosome synthesis.


Assuntos
Archaea/enzimologia , Metiltransferases/metabolismo , RNA Arqueal/metabolismo , RNA Ribossômico/metabolismo , Archaea/genética , Movimento Celular , Crenarchaeota/enzimologia , Euryarchaeota/enzimologia , Haloferax volcanii/enzimologia , Metiltransferases/fisiologia , Biossíntese de Proteínas , RNA Arqueal/química , RNA Ribossômico/química , Subunidades Ribossômicas Menores de Arqueas/enzimologia
5.
RNA ; 27(2): 133-150, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33184227

RESUMO

The large ribosomal RNAs of eukaryotes frequently contain expansion sequences that add to the size of the rRNAs but do not affect their overall structural layout and are compatible with major ribosomal function as an mRNA translation machine. The expansion of prokaryotic ribosomal RNAs is much less explored. In order to obtain more insight into the structural variability of these conserved molecules, we herein report the results of a comprehensive search for the expansion sequences in prokaryotic 5S rRNAs. Overall, 89 expanded 5S rRNAs of 15 structural types were identified in 15 archaeal and 36 bacterial genomes. Expansion segments ranging in length from 13 to 109 residues were found to be distributed among 17 insertion sites. The strains harboring the expanded 5S rRNAs belong to the bacterial orders Clostridiales, Halanaerobiales, Thermoanaerobacterales, and Alteromonadales as well as the archael order Halobacterales When several copies of a 5S rRNA gene are present in a genome, the expanded versions may coexist with normal 5S rRNA genes. The insertion sequences are typically capable of forming extended helices, which do not seemingly interfere with folding of the conserved core. The expanded 5S rRNAs have largely been overlooked in 5S rRNA databases.


Assuntos
Genoma Arqueal , Genoma Bacteriano , RNA Arqueal/genética , RNA Bacteriano/genética , RNA Ribossômico 5S/genética , Alteromonadaceae/classificação , Alteromonadaceae/genética , Alteromonadaceae/metabolismo , Pareamento de Bases , Sequência de Bases , Clostridiales/classificação , Clostridiales/genética , Clostridiales/metabolismo , Firmicutes/classificação , Firmicutes/genética , Firmicutes/metabolismo , Halobacteriales/classificação , Halobacteriales/genética , Halobacteriales/metabolismo , Conformação de Ácido Nucleico , Filogenia , RNA Arqueal/química , RNA Arqueal/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Ribossômico 5S/química , RNA Ribossômico 5S/metabolismo , Thermoanaerobacterium/classificação , Thermoanaerobacterium/genética , Thermoanaerobacterium/metabolismo
6.
Biomolecules ; 10(12)2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33302546

RESUMO

Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants.


Assuntos
Proteínas Arqueais/química , DNA Polimerase beta/química , DNA Arqueal/química , Hexosefosfatos/química , Nucleotídeos/química , RNA Arqueal/química , Thermococcus/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , DNA Arqueal/genética , DNA Arqueal/metabolismo , Evolução Molecular Direcionada/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Hexosefosfatos/metabolismo , Cinética , Simulação de Dinâmica Molecular , Mutação , Conformação de Ácido Nucleico , Nucleotídeos/genética , Nucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas , RNA Arqueal/genética , RNA Arqueal/metabolismo , Especificidade por Substrato , Thermococcus/enzimologia
7.
BMC Genomics ; 21(1): 797, 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33198623

RESUMO

BACKGROUND: The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3' to 5' direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3'-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging. RESULTS: To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon Sulfolobus solfataricus. A relatively high proportion (17-19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5' parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3'-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5'-ends of RNAs was detected. CONCLUSIONS: In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5'-ends of genes suggests simultaneous binding of both RNA ends by the S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3'-5' direction.


Assuntos
Proteínas Arqueais , Exossomos , Sulfolobus solfataricus , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/genética , Exossomos/metabolismo , RNA/genética , Estabilidade de RNA , RNA Arqueal/genética , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/metabolismo
8.
Nucleic Acids Res ; 48(19): 11068-11082, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33035335

RESUMO

tRNAs play a central role during the translation process and are heavily post-transcriptionally modified to ensure optimal and faithful mRNA decoding. These epitranscriptomics marks are added by largely conserved proteins and defects in the function of some of these enzymes are responsible for neurodevelopmental disorders and cancers. Here, we focus on the Trm11 enzyme, which forms N2-methylguanosine (m2G) at position 10 of several tRNAs in both archaea and eukaryotes. While eukaryotic Trm11 enzyme is only active as a complex with Trm112, an allosteric activator of methyltransferases modifying factors (RNAs and proteins) involved in mRNA translation, former studies have shown that some archaeal Trm11 proteins are active on their own. As these studies were performed on Trm11 enzymes originating from archaeal organisms lacking TRM112 gene, we have characterized Trm11 (AfTrm11) from the Archaeoglobus fulgidus archaeon, which genome encodes for a Trm112 protein (AfTrm112). We show that AfTrm11 interacts directly with AfTrm112 similarly to eukaryotic enzymes and that although AfTrm11 is active as a single protein, its enzymatic activity is strongly enhanced by AfTrm112. We finally describe the first crystal structures of the AfTrm11-Trm112 complex and of Trm11, alone or bound to the methyltransferase inhibitor sinefungin.


Assuntos
Proteínas Arqueais , Archaeoglobus fulgidus/enzimologia , RNA Arqueal/metabolismo , RNA de Transferência/metabolismo , tRNA Metiltransferases , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , tRNA Metiltransferases/química , tRNA Metiltransferases/metabolismo
9.
RNA ; 26(12): 1957-1975, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32994183

RESUMO

To improve and complete our knowledge of archaeal tRNA modification patterns, we have identified and compared the modification pattern (type and location) in tRNAs of three very different archaeal species, Methanococcus maripaludis (a mesophilic methanogen), Pyrococcus furiosus (a hyperthermophile thermococcale), and Sulfolobus acidocaldarius (an acidophilic thermophilic sulfolobale). Most abundant isoacceptor tRNAs (79 in total) for each of the 20 amino acids were isolated by two-dimensional gel electrophoresis followed by in-gel RNase digestions. The resulting oligonucleotide fragments were separated by nanoLC and their nucleotide content analyzed by mass spectrometry (MS/MS). Analysis of total modified nucleosides obtained from complete digestion of bulk tRNAs was also performed. Distinct base- and/or ribose-methylations, cytidine acetylations, and thiolated pyrimidines were identified, some at new positions in tRNAs. Novel, some tentatively identified, modifications were also found. The least diversified modification landscape is observed in the mesophilic Methanococcus maripaludis and the most complex one in Sulfolobus acidocaldarius Notable observations are the frequent occurrence of ac4C nucleotides in thermophilic archaeal tRNAs, the presence of m7G at positions 1 and 10 in Pyrococcus furiosus tRNAs, and the use of wyosine derivatives at position 37 of tRNAs, especially those decoding U1- and C1-starting codons. These results complete those already obtained by others with sets of archaeal tRNAs from Methanocaldococcus jannaschii and Haloferax volcanii.


Assuntos
Mathanococcus/genética , Nucleotídeos/química , Pyrococcus furiosus/genética , RNA de Transferência/química , RNA de Transferência/genética , Sulfolobus acidocaldarius/genética , Sequência de Bases , Conformação de Ácido Nucleico , RNA Arqueal/química , RNA Arqueal/genética
10.
Appl Environ Microbiol ; 86(23)2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-32978127

RESUMO

Long-term nitrogen field fertilization often results in significant changes in nitrifying communities that catalyze a key step in the global N cycle. However, whether microcosm studies are able to inform the dynamic changes in communities of ammonia-oxidizing bacteria (AOB) and archaea (AOA) under field conditions remains poorly understood. This study aimed to evaluate the transcriptional activities of nitrifying communities under in situ conditions, and we found that they were largely similar to those of 13C-labeled nitrifying communities in the urea-amended microcosms of soils that had received different N fertilization regimens for 22 years. High-throughput sequencing of 16S rRNA genes and transcripts suggested that Nitrosospira cluster 3-like AOB and Nitrososphaera viennensis-like AOA were significantly stimulated in N-fertilized fresh soils. Real-time quantitative PCR demonstrated that the significant increase of AOA and AOB in fresh soils upon nitrogen fertilization could be preserved in the air-dried soils. DNA-based stable-isotope probing (SIP) further revealed the greatest labeling of Nitrosospira cluster 3-like AOB and Nitrosospira viennensis-like AOA, despite the strong advantage of AOB over AOA in the N-fertilized soils. Nitrobacter-like nitrite-oxidizing bacteria (NOB) played more important roles than Nitrospira-like NOB in urea-amended SIP microcosms, while the situation was the opposite under field conditions. Our results suggest that long-term fertilization selected for physiologically versatile AOB and AOA that could have been adapted to a wide range of substrate ammonium concentrations. It also provides compelling evidence that the dominant communities of transcriptionally active nitrifiers under field conditions were largely similar to those revealed in 13C-labeled microcosms.IMPORTANCE The role of manipulated microcosms in microbial ecology has been much debated, because they cannot entirely represent the in situ situation. We collected soil samples from 20 field plots, including 5 different treatments with and without nitrogen fertilizers for 22 years, in order to assess active nitrifying communities by in situ transcriptomics and microcosm-based stable-isotope probing. The results showed that chronic N enrichment led to competitive advantages of Nitrosospira cluster 3-like AOB over N. viennensis-like AOA in soils under field conditions. Microcosm labeling revealed similar results for active AOA and AOB, although an apparent discrepancy was observed for nitrite-oxidizing bacteria. This study suggests that the soil microbiome represents a relatively stable community resulting from complex evolutionary processes over a large time scale, and microcosms can serve as powerful tools to test the theory of environmental filtering on the key functional microbial guilds.


Assuntos
Archaea/isolamento & purificação , Bactérias/isolamento & purificação , Nitrogênio/metabolismo , Microbiologia do Solo , Archaea/genética , Bactérias/genética , Fertilizantes/análise , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , RNA Arqueal/análise , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Genética
11.
Appl Environ Microbiol ; 86(19)2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32709727

RESUMO

Global marine sediments harbor a large and highly diverse microbial biosphere, but the mechanism by which this biosphere is established during sediment burial is largely unknown. During burial in marine sediments, concentrations of easily metabolized organic compounds and total microbial cell abundance decrease. However, it is unknown whether some microbial clades increase with depth. We show total population increases in 38 microbial families over 3 cm of sediment depth in the upper 7.5 cm of White Oak River (WOR) estuary sediments. Clades that increased with depth were more often associated with one or more of the following: anaerobes, uncultured, or common in deep marine sediments relative to those that decreased. Maximum doubling times (in situ steady-state growth rates could be faster to balance cell decay) were estimated as 2 to 25 years by combining sedimentation rate with either quantitative PCR (qPCR) or the product of the fraction read abundance of 16S rRNA genes and total cell counts (FRAxC). Doubling times were within an order of magnitude of each other in two adjacent cores, as well as in two laboratory enrichments of Cape Lookout Bight (CLB), NC, sediments (average difference of 28% ± 19%). qPCR and FRAxC in sediment cores and laboratory enrichments produced similar doubling times for key deep subsurface uncultured clades Bathyarchaeota (8.7 ± 1.9 years) and Thermoprofundales/MBG-D (4.1 ± 0.7 years). We conclude that common deep subsurface microbial clades experience a narrow zone of growth in shallow sediments, offering an opportunity for selection of long-term subsistence traits after resuspension events.IMPORTANCE Many studies show that the uncultured microbes that dominate global marine sediments do not actually increase in population size as they are buried in marine sediments; rather, they exist in a sort of prolonged torpor for thousands of years. This is because, although studies have shown biomass turnover in these clades, no evidence has ever been found that deeper sediments have larger populations for specific clades than shallower layers. We discovered that they actually do increase population sizes during burial, but only in the upper few centimeters. This suggests that marine sediments may be a vast repository of mostly nongrowing microbes with a thin and relatively rapid area of cell abundance increase in the upper 10 cm, offering a chance for subsurface organisms to undergo natural selection.


Assuntos
Archaea/crescimento & desenvolvimento , Bactérias/crescimento & desenvolvimento , Sedimentos Geológicos/microbiologia , Microbiota , Rios/microbiologia , Anaerobiose , North Carolina , RNA Arqueal/análise , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , Análise de Sequência de RNA
12.
Nature ; 583(7817): 638-643, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32555463

RESUMO

N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.


Assuntos
Acetilação , Citidina/análogos & derivados , Células Eucarióticas/metabolismo , Evolução Molecular , RNA/química , RNA/metabolismo , Archaea/química , Archaea/citologia , Archaea/genética , Archaea/crescimento & desenvolvimento , Sequência Conservada , Microscopia Crioeletrônica , Citidina/metabolismo , Células Eucarióticas/citologia , Células HeLa , Humanos , Modelos Moleculares , Acetiltransferases N-Terminal/metabolismo , RNA Arqueal/química , RNA Arqueal/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Temperatura
13.
Microb Ecol ; 80(4): 793-808, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32572534

RESUMO

We describe the geochemistry and microbial diversity of a pristine environment that resembles an acid rock drainage (ARD) but it is actually the result of hydrothermal and volcanic influences. We designate this environment, and other comparable sites, as volcanic influenced acid rock drainage (VARD) systems. The metal content and sulfuric acid in this ecosystem stem from the volcanic milieu and not from the product of pyrite oxidation. Based on the analysis of 16S rRNA gene amplicons, we report the microbial community structure in the pristine San Cayetano Costa Rican VARD environment (pH = 2.94-3.06, sulfate ~ 0.87-1.19 g L-1, iron ~ 35-61 mg L-1 (waters), and ~ 8-293 g kg-1 (sediments)). San Cayetano was found to be dominated by microorganisms involved in the geochemical cycling of iron, sulfur, and nitrogen; however, the identity and abundance of the species changed with the oxygen content (0.40-6.06 mg L-1) along the river course. The hypoxic source of San Cayetano is dominated by a putative anaerobic sulfate-reducing Deltaproteobacterium. Sulfur-oxidizing bacteria such as Acidithiobacillus or Sulfobacillus are found in smaller proportions with respect to typical ARD. In the oxic downstream, we identified aerobic iron-oxidizers (Leptospirillum, Acidithrix, Ferrovum) and heterotrophic bacteria (Burkholderiaceae bacterium, Trichococcus, Acidocella). Thermoplasmatales archaea closely related to environmental phylotypes found in other ARD niches were also observed throughout the entire ecosystem. Overall, our study shows the differences and similarities in the diversity and distribution of the microbial communities between an ARD and a VARD system at the source and along the oxygen gradient that establishes on the course of the river.


Assuntos
Archaea/isolamento & purificação , Bactérias/isolamento & purificação , Microbiota/fisiologia , Oxigênio/análise , Costa Rica , Concentração de Íons de Hidrogênio , RNA Arqueal/análise , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , Rios , Erupções Vulcânicas
14.
RNA Biol ; 17(10): 1480-1491, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32552320

RESUMO

RNase J is a prokaryotic 5'-3' exo/endoribonuclease that functions in mRNA decay and rRNA maturation. Here, we report a novel duplex unwinding activity of mpy-RNase J, an archaeal RNase J from Methanolobus psychrophilus, which enables it to degrade duplex RNAs with hairpins up to 40 bp when linking a 5' single-stranded overhangs of ≥ 7 nt, corresponding to the RNA channel length. A 6-nt RNA-mpy-RNase J-S247A structure reveals the RNA-interacting residues and a steric barrier at the RNA channel entrance comprising two archaeal loops and two helices. Mutagenesis of the residues key to either exoribonucleolysis or RNA translocation diminished the duplex unwinding activity. Substitution of the residues in the steric barrier yielded stalled degradation intermediates at the duplex RNA regions. Thus, an exoribonucleolysis-driven and steric occlusion-based duplex unwinding mechanism was identified. The duplex unwinding activity confers mpy-RNase J the capability of degrading highly structured RNAs, including the bacterial REP RNA, and archaeal mRNAs, rRNAs, tRNAs, SRPs, RNase P and CD-box RNAs, providing an indicative of the potential key roles of mpy-RNase J in pleiotropic RNA metabolisms. Hydrolysis-coupled duplex unwinding activity was also detected in a bacterial RNase J, which may use a shared but slightly different unwinding mechanism from archaeal RNase Js, indicating that duplex unwinding is a common property of the prokaryotic RNase Js.


Assuntos
Archaea/enzimologia , Archaea/genética , Conformação de Ácido Nucleico , RNA Arqueal/química , RNA Arqueal/genética , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , Ribonucleases/metabolismo , Hidrólise , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Clivagem do RNA , RNA Arqueal/metabolismo , RNA de Cadeia Dupla/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
15.
BMC Microbiol ; 20(1): 130, 2020 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-32448158

RESUMO

BACKGROUND: RNAs perform many functions in addition to supplying coding templates, such as binding proteins. RNA-protein interactions are important in multiple processes in all domains of life, and the discovery of additional protein-binding RNAs expands the scope for studying such interactions. To find such RNAs, we exploited a form of ribosomal regulation. Ribosome biosynthesis must be tightly regulated to ensure that concentrations of rRNAs and ribosomal proteins (r-proteins) match. One regulatory mechanism is a ribosomal leader (r-leader), which is a domain in the 5' UTR of an mRNA whose genes encode r-proteins. When the concentration of one of these r-proteins is high, the protein binds the r-leader in its own mRNA, reducing gene expression and thus protein concentrations. To date, 35 types of r-leaders have been validated or predicted. RESULTS: By analyzing additional conserved RNA structures on a multi-genome scale, we identified 20 novel r-leader structures. Surprisingly, these included new r-leaders in the highly studied organisms Escherichia coli and Bacillus subtilis. Our results reveal several cases where multiple unrelated RNA structures likely bind the same r-protein ligand, and uncover previously unknown r-protein ligands. Each r-leader consistently occurs upstream of r-protein genes, suggesting a regulatory function. That the predicted r-leaders function as RNAs is supported by evolutionary correlations in the nucleotide sequences that are characteristic of a conserved RNA secondary structure. The r-leader predictions are also consistent with the locations of experimentally determined transcription start sites. CONCLUSIONS: This work increases the number of known or predicted r-leader structures by more than 50%, providing additional opportunities to study structural and evolutionary aspects of RNA-protein interactions. These results provide a starting point for detailed experimental studies.


Assuntos
Regiões 5' não Traduzidas , Archaea/genética , Bactérias/genética , RNA Ribossômico/química , Archaea/metabolismo , Bacillus subtilis/genética , Bactérias/metabolismo , Escherichia coli/genética , Regulação da Expressão Gênica em Archaea , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Arqueal/genética , RNA Bacteriano/genética , RNA Ribossômico/genética , Proteínas Ribossômicas/metabolismo
16.
Appl Environ Microbiol ; 86(11)2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32245755

RESUMO

Anthropogenic activity impacts stream ecosystems, resulting in a loss of diversity and ecosystem function; however, little is known about the response of aquatic microbial communities to changes in land use. Here, microbial communities were characterized in 82 headwater streams across a gradient of urban and agricultural land uses using 16S rRNA gene amplicon sequencing and compared to a rich data set of physicochemical variables and traditional benthic invertebrate indicators. Microbial diversity and community structures differed among watersheds with high agricultural, urban, and forested land uses, and community structure differed in streams classified as being in good, fair, poor, and very poor condition using benthic invertebrate indicators. Microbial community similarity decayed with geodesic distance across the study region but not with environmental distance. Stream community respiration rates ranged from 21.7 to 1,570 mg O2 m-2 day-1 and 31.9 to 3,670 mg O2 m-2 day-1 for water column and sediments, respectively, and correlated with nutrients associated with anthropogenic influence and microbial community structure. Nitrous oxide (N2O) concentrations ranged from 0.22 to 4.41 µg N2O liter-1; N2O concentration was negatively correlated with forested land use and was positively correlated with dissolved inorganic nitrogen concentrations. Our findings suggest that stream microbial communities are impacted by watershed land use and can potentially be used to assess ecosystem health.IMPORTANCE Stream ecosystems are frequently impacted by changes in watershed land use, resulting in altered hydrology, increased pollutant and nutrient loads, and habitat degradation. Macroinvertebrates and fish are strongly affected by changes in stream conditions and are commonly used in biotic indices to assess ecosystem health. Similarly, microbes respond to environmental stressors, and changes in community composition alter key ecosystem processes. The response of microbes to habitat degradation and their role in global biogeochemical cycles provide an opportunity to use microbes as a monitoring tool. Here, we identify stream microbes that respond to watershed urbanization and agricultural development and demonstrate that microbial diversity and community structure can be used to assess stream conditions and ecosystem functioning.


Assuntos
Archaea/isolamento & purificação , Bactérias/isolamento & purificação , Microbiota , Rios/microbiologia , Agricultura , Archaea/classificação , Bactérias/classificação , Cidades , Maryland , RNA Arqueal/análise , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , Estações do Ano
17.
Mol Genet Genomics ; 295(3): 775-785, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32170429

RESUMO

The regulatory networks involved in the uptake and metabolism of different nitrogen sources in response to their availability are crucial in all organisms. Nitrogen metabolism pathways have been studied in detail in archaea such as the extreme halophilic archaeon Haloferax mediterranei. However, knowledge about nitrogen metabolism regulation in haloarchaea is very scarce, and no transcriptional regulators involved in nitrogen metabolism have been identified to date. Advances in the molecular biology field have revealed that many small RNAs (sRNAs) are involved in the regulation of a diverse metabolic pathways. Surprisingly, no studies on regulation mediated by sRNAs have focused on the response to environmental fluctuations in nitrogen in haloarchaea. To identify sRNAs involved in the transcriptional regulation of nitrogen assimilation genes in Haloferax mediterranei and, thus, propose a novel regulatory mechanism, RNA-Seq was performed using cells grown in the presence of two different nitrogen sources. The differential transcriptional expression analysis of the RNA-Seq data revealed differences in the transcription patterns of 102 sRNAs according to the nitrogen source, and the molecular functions, cellular locations and biological processes with which the target genes were associated were predicted. These results enabled the identification of four sRNAs that could be directly related to the regulation of genes involved in nitrogen metabolism. This work provides the first proposed regulatory mechanism of nitrogen assimilation-related gene expression by sRNAs in haloarchaea as an alternative to transcriptional regulation mediated by proteins.


Assuntos
Proteínas Arqueais/genética , Regulação da Expressão Gênica em Archaea , Haloferax mediterranei/genética , Haloferax mediterranei/metabolismo , Nitrogênio/metabolismo , RNA Arqueal/genética , Pequeno RNA não Traduzido/genética , Perfilação da Expressão Gênica , Haloferax mediterranei/crescimento & desenvolvimento
18.
Appl Environ Microbiol ; 86(11)2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32220837

RESUMO

Most of the microbial degradation in oil reservoirs is believed to take place at the oil-water transition zone (OWTZ). However, a recent study indicates that there is microbial life enclosed in microliter-sized water droplets dispersed in heavy oil of Pitch Lake in Trinidad and Tobago. This life in oil suggests that microbial degradation of oil also takes place in water pockets in the oil-bearing rock of an oil leg independent of the OWTZ. However, it is unknown whether microbial life in water droplets dispersed in oil is a generic property of oil reservoirs rather than an exotic exception. Hence, we took samples from three heavy-oil seeps, Pitch Lake (Trinidad and Tobago), the La Brea Tar Pits (California, USA), and an oil seep on the McKittrick oil field (California, USA). All three tested oil seeps contained dispersed water droplets. Larger droplets between 1 and 10 µl revealed high cell densities of up to 109 cells ml-1 Testing for ATP content and LIVE/DEAD staining showed that these populations consist of active and viable microbial cells with an average of 60% membrane-intact cells and ATP concentrations comparable to those of other subsurface ecosystems. Microbial community analyses based on 16S rRNA gene amplicon sequencing revealed the presence of known anaerobic oil-degrading microorganisms. Surprisingly, the community analyses showed similarities between all three oil seeps, revealing common OTUs, although the sampling sites were thousands of kilometers apart. Our results indicate that small water inclusions are densely populated microhabitats in heavy oil and possibly a generic trait of degraded-oil reservoirs.IMPORTANCE Our results confirmed that small water droplets in oil are densely populated microhabitats containing active microbial communities. Since these microhabitats occurred in three tested oil seeps which are located thousands of kilometers away from each other, such populated water droplets might be a generic trait of biodegraded oil reservoirs and might be involved in the overall oil degradation process. Microbial degradation might thus also take place in water pockets in the oil-bearing oil legs of the reservoir rock rather than only at the oil-water transition zone.


Assuntos
Archaea/isolamento & purificação , Bactérias/isolamento & purificação , Microbiota , Campos de Petróleo e Gás/microbiologia , Microbiologia da Água , Archaea/classificação , Bactérias/classificação , California , Lagos , Los Angeles , RNA Arqueal/análise , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , Trinidad e Tobago , Água/química
19.
J Bacteriol ; 202(8)2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32041795

RESUMO

Archaeosine (G+) is a structurally complex modified nucleoside found quasi-universally in the tRNA of Archaea and located at position 15 in the dihydrouridine loop, a site not modified in any tRNA outside the Archaea G+ is characterized by an unusual 7-deazaguanosine core structure with a formamidine group at the 7-position. The location of G+ at position 15, coupled with its novel molecular structure, led to a hypothesis that G+ stabilizes tRNA tertiary structure through several distinct mechanisms. To test whether G+ contributes to tRNA stability and define the biological role of G+, we investigated the consequences of introducing targeted mutations that disrupt the biosynthesis of G+ into the genome of the hyperthermophilic archaeon Thermococcus kodakarensis and the mesophilic archaeon Methanosarcina mazei, resulting in modification of the tRNA with the G+ precursor 7-cyano-7-deazaguansine (preQ0) (deletion of arcS) or no modification at position 15 (deletion of tgtA). Assays of tRNA stability from in vitro-prepared and enzymatically modified tRNA transcripts, as well as tRNA isolated from the T. kodakarensis mutant strains, demonstrate that G+ at position 15 imparts stability to tRNAs that varies depending on the overall modification state of the tRNA and the concentration of magnesium chloride and that when absent results in profound deficiencies in the thermophily of T. kodakarensis IMPORTANCE Archaeosine is ubiquitous in archaeal tRNA, where it is located at position 15. Based on its molecular structure, it was proposed to stabilize tRNA, and we show that loss of archaeosine in Thermococcus kodakarensis results in a strong temperature-sensitive phenotype, while there is no detectable phenotype when it is lost in Methanosarcina mazei Measurements of tRNA stability show that archaeosine stabilizes the tRNA structure but that this effect is much greater when it is present in otherwise unmodified tRNA transcripts than in the context of fully modified tRNA, suggesting that it may be especially important during the early stages of tRNA processing and maturation in thermophiles. Our results demonstrate how small changes in the stability of structural RNAs can be manifested in significant biological-fitness changes.


Assuntos
Guanosina/análogos & derivados , Methanosarcina/metabolismo , RNA Arqueal/genética , RNA de Transferência/genética , Thermococcus/metabolismo , Guanosina/metabolismo , Methanosarcina/química , Methanosarcina/genética , Estabilidade de RNA , RNA Arqueal/química , RNA Arqueal/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Thermococcus/química , Thermococcus/genética
20.
Int J Biol Macromol ; 150: 705-713, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32057853

RESUMO

Aminoacyl tRNA synthetase (AARS) plays an important role in transferring each amino acid to its cognate tRNA. Specifically, tyrosyl tRNA synthetase (TyrRS) is involved in various functions including protection from DNA damage due to oxidative stress, protein synthesis and cell signaling and can be an attractive target for controlling the pathogens by early inhibition of translation. TyrRS has two disordered regions, which lack a stable 3D structure in solution, and are involved in tRNA synthetase catalysis and stability. One of the disordered regions undergoes disorder-to-order transition (DOT) upon complex formation with tRNA whereas the other remains disordered (DR). In this work, we have explored the importance of these disordered regions using molecular dynamics simulations of both free and RNA-complexed states. We observed that the DOT and DR regions of the first subunit acts as a flap and interact with the acceptor arm of the tRNA. The DOT-DR flap closes when tyrosine (TyrRSTyr) is present at the active site of the complex and opens in the presence of tyrosine monophosphate (TyrRSYMP). The DOT and DR regions of the second subunit interact with the anticodon stem as well as D-loop of the tRNA, which might be involved in stabilizing the complex. The anticodon loop of the tRNA binds to the structured region present in the C-terminal of the protein, which is observed to be flexible during simulations. Detailed energy calculations also show that TyrRSTyr complex has stronger binding energy between tRNA and protein compared to TyrRSYMP; on the contrary, the anticodon is strongly bound in TyrRSYMP. The results obtained in the present study provide additional insights for understanding catalysis and the involvement of disordered regions in Tyr transfer to cognate tRNA.


Assuntos
Proteínas Arqueais/química , Methanocaldococcus/química , RNA Arqueal/química , RNA de Transferência de Tirosina/química , Tirosina-tRNA Ligase/química , Tirosina/química , Proteínas Arqueais/metabolismo , Methanocaldococcus/metabolismo , RNA Arqueal/metabolismo , RNA de Transferência de Tirosina/metabolismo , Tirosina/metabolismo , Tirosina-tRNA Ligase/metabolismo
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