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1.
Nucleic Acids Res ; 49(2): 1075-1093, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33398350

RESUMO

Defects in the posttranscriptional modifications of mitochondrial tRNAs have been linked to human diseases, but their pathophysiology remains elusive. In this report, we investigated the molecular mechanism underlying a deafness-associated tRNAIle 4295A>G mutation affecting a highly conserved adenosine at position 37, 3' adjacent to the tRNA's anticodon. Primer extension and methylation activity assays revealed that the m.4295A>G mutation introduced a tRNA methyltransferase 5 (TRMT5)-catalyzed m1G37 modification of tRNAIle. Molecular dynamics simulations suggested that the m.4295A>G mutation affected tRNAIle structure and function, supported by increased melting temperature, conformational changes and instability of mutated tRNA. An in vitro processing experiment revealed that the m.4295A>G mutation reduced the 5' end processing efficiency of tRNAIle precursors, catalyzed by RNase P. We demonstrated that cybrid cell lines carrying the m.4295A>G mutation exhibited significant alterations in aminoacylation and steady-state levels of tRNAIle. The aberrant tRNA metabolism resulted in the impairment of mitochondrial translation, respiratory deficiency, decreasing membrane potentials and ATP production, increasing production of reactive oxygen species and promoting autophagy. These demonstrated the pleiotropic effects of m.4295A>G mutation on tRNAIle and mitochondrial functions. Our findings highlighted the essential role of deficient posttranscriptional modifications in the structure and function of tRNA and their pathogenic consequence of deafness.


Assuntos
Perda Auditiva Neurossensorial/genética , Mutação Puntual , RNA de Transferência de Isoleucina/genética , Trifosfato de Adenosina/biossíntese , Adulto , Proteínas Arqueais/metabolismo , Autofagia , Sequência de Bases , Linhagem Celular , DNA Mitocondrial/genética , Grupos Étnicos/genética , Feminino , Pleiotropia Genética , Perda Auditiva Neurossensorial/etnologia , Humanos , Isoleucina/metabolismo , Masculino , Herança Materna , Potencial da Membrana Mitocondrial , Methanocaldococcus/enzimologia , Metilação , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Fosforilação Oxidativa , Linhagem , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , Proteínas Recombinantes/metabolismo , Aminoacilação de RNA de Transferência , Adulto Jovem , tRNA Metiltransferases/metabolismo
2.
Nat Commun ; 12(1): 629, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504778

RESUMO

Many transmembrane receptors have a desensitized state, in which they are unable to respond to external stimuli. The family of microbial rhodopsin proteins includes one such group of receptors, whose inactive or dark-adapted (DA) state is established in the prolonged absence of light. Here, we present high-resolution crystal structures of the ground (light-adapted) and DA states of Archaerhodopsin-3 (AR3), solved to 1.1 Å and 1.3 Å resolution respectively. We observe significant differences between the two states in the dynamics of water molecules that are coupled via H-bonds to the retinal Schiff Base. Supporting QM/MM calculations reveal how the DA state permits a thermodynamic equilibrium between retinal isomers to be established, and how this same change is prevented in the ground state in the absence of light. We suggest that the different arrangement of internal water networks in AR3 is responsible for the faster photocycle kinetics compared to homologs.


Assuntos
Proteínas Arqueais/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Água/química , Cristalografia por Raios X , Elétrons , Ligação de Hidrogênio , Isomerismo , Lipídeos/química , Conformação Molecular , Processamento de Proteína Pós-Traducional , Prótons , Retinaldeído/química , Retinaldeído/metabolismo
3.
Appl Environ Microbiol ; 87(6)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33419739

RESUMO

Dissimilatory iron reduction by hyperthermophilic archaea occurs in many geothermal environments and generally relies on microbe-mineral interactions that transform various iron oxide minerals. In this study, the physiology of dissimilatory iron and nitrate reduction was examined in the hyperthermophilic crenarchaeon type strain Pyrodictium delaneyi Su06. Iron barrier experiments showed that P. delaneyi required direct contact with the Fe(III) oxide mineral ferrihydrite for reduction. The separate addition of an exogenous electron shuttle (anthraquinone-2,6-disulfonate), a metal chelator (nitrilotriacetic acid), and 75% spent cell-free supernatant did not stimulate growth with or without the barrier. Protein electrophoresis showed that the c-type cytochrome and general protein compositions of P. delaneyi changed when grown on ferrihydrite relative to nitrate. Differential proteomic analyses using tandem mass tagged protein fragments and mass spectrometry detected 660 proteins and differential production of 127 proteins. Among these, two putative membrane-bound molybdopterin-dependent oxidoreductase complexes increased in relative abundance 60- to 3,000-fold and 50- to 100-fold in cells grown on iron oxide. A putative 8-heme c-type cytochrome was 60-fold more abundant in iron-grown cells and was unique to the Pyrodictiaceae There was also a >14,700-fold increase in a membrane transport protein in iron-grown cells. For flagellin proteins and a putative nitrate reductase, there were no changes in abundance, but a membrane nitric oxide reductase was more abundant on nitrate. These data help to elucidate the mechanisms by which hyperthermophilic crenarchaea generate energy and transfer electrons across the membrane to iron oxide minerals.IMPORTANCE Understanding iron reduction in the hyperthermophilic crenarchaeon Pyrodictium delaneyi provides insight into the diversity of mechanisms used for this process and its potential impact in geothermal environments. The ability of P. delaneyi to reduce Fe(III) oxide minerals through direct contact potentially using a novel cytochrome respiratory complex and a membrane-bound molybdopterin respiratory complex sets iron reduction in this organism apart from previously described iron reduction processes. A model is presented where obligatory H2 oxidation on the membrane coupled with electron transport and either Fe(III) oxide or nitrate reduction leads to the generation of a proton motive force and energy generation by oxidative phosphorylation. However, P. delaneyi cannot fix CO2 and relies on organic compounds from its environment for biosynthesis.


Assuntos
Compostos Férricos/metabolismo , Minerais/metabolismo , Nitratos/metabolismo , Pyrodictiaceae/metabolismo , Proteínas Arqueais/metabolismo , Ferro/metabolismo , Proteômica , Pyrodictiaceae/crescimento & desenvolvimento
4.
Nat Struct Mol Biol ; 28(1): 92-102, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33398171

RESUMO

Spo11, which makes DNA double-strand breaks (DSBs) that are essential for meiotic recombination, has long been recalcitrant to biochemical study. We provide molecular analysis of Saccharomyces cerevisiae Spo11 purified with partners Rec102, Rec104 and Ski8. Rec102 and Rec104 jointly resemble the B subunit of archaeal topoisomerase VI, with Rec104 occupying a position similar to the Top6B GHKL-type ATPase domain. Unexpectedly, the Spo11 complex is monomeric (1:1:1:1 stoichiometry), consistent with dimerization controlling DSB formation. Reconstitution of DNA binding reveals topoisomerase-like preferences for duplex-duplex junctions and bent DNA. Spo11 also binds noncovalently but with high affinity to DNA ends mimicking cleavage products, suggesting a mechanism to cap DSB ends. Mutations that reduce DNA binding in vitro attenuate DSB formation, alter DSB processing and reshape the DSB landscape in vivo. Our data reveal structural and functional similarities between the Spo11 core complex and Topo VI, but also highlight differences reflecting their distinct biological roles.


Assuntos
Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/metabolismo , Meiose/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Arqueais/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Microscopia de Força Atômica , Mutação/genética , Conformação de Ácido Nucleico , Recombinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
5.
Chem Commun (Camb) ; 57(4): 476-479, 2021 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-33326521

RESUMO

Replacing coenzyme F430, an Ni(i) F430-like cofactor derived from vitamin B12 (F430-B12) is revealed by DFT calculations to be able to catalyze methane formation in methyl-coenzyme M reductase with a barrier of 13.3 kcal mol-1, demonstrating the correctness of the route starting from vitamin B12. The structure-activity relationships of F430 and F430-B12 (especially the roles of the F ring) are discovered and several sources of inspiration promoting the application of F430-B12 are also obtained, coming closer to using F430 chemistry in man-made catalysis.


Assuntos
Metaloporfirinas/química , Oxirredutases/química , Vitamina B 12/análogos & derivados , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Teoria da Densidade Funcional , Metaloporfirinas/metabolismo , Metano/biossíntese , Methanobacteriaceae/enzimologia , Modelos Químicos , Estrutura Molecular , Níquel/química , Oxirredutases/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Termodinâmica , Vitamina B 12/metabolismo
6.
Nucleic Acids Res ; 49(D1): D803-D808, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33313828

RESUMO

Protein subcellular localization (SCL) is important for understanding protein function, genome annotation, and aids identification of potential cell surface diagnostic markers, drug targets, or vaccine components. PSORTdb comprises ePSORTdb, a manually curated database of experimentally verified protein SCLs, and cPSORTdb, a pre-computed database of PSORTb-predicted SCLs for NCBI's RefSeq deduced bacterial and archaeal proteomes. We now report PSORTdb 4.0 (http://db.psort.org/). It features a website refresh, in particular a more user-friendly database search. It also addresses the need to uniquely identify proteins from NCBI genomes now that GI numbers have been retired. It further expands both ePSORTdb and cPSORTdb, including additional data about novel secondary localizations, such as proteins found in bacterial outer membrane vesicles. Protein predictions in cPSORTdb have increased along with the number of available microbial genomes, from approximately 13 million when PSORTdb 3.0 was released, to over 66 million currently. Now, analyses of both complete and draft genomes are included. This expanded database will be of wide use to researchers developing SCL predictors or studying diverse microbes, including medically, agriculturally and industrially important species that have both classic or atypical cell envelope structures or vesicles.


Assuntos
Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Bases de Dados de Proteínas , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas de Bactérias/química , Parede Celular/química , Transporte Proteico , Frações Subcelulares/metabolismo , Interface Usuário-Computador
7.
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
8.
Nat Commun ; 11(1): 5016, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024106

RESUMO

Excitatory amino acid transporters (EAATs) are important in many physiological processes and crucial for the removal of excitatory amino acids from the synaptic cleft. Here, we develop and apply high-speed atomic force microscopy line-scanning (HS-AFM-LS) combined with automated state assignment and transition analysis for the determination of transport dynamics of unlabeled membrane-reconstituted GltPh, a prokaryotic EAAT homologue, with millisecond temporal resolution. We find that GltPh transporters can operate much faster than previously reported, with state dwell-times in the 50 ms range, and report the kinetics of an intermediate transport state with height between the outward- and inward-facing states. Transport domains stochastically probe transmembrane motion, and reversible unsuccessful excursions to the intermediate state occur. The presented approach and analysis methodology are generally applicable to study transporter kinetics at system-relevant temporal resolution.


Assuntos
Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Força Atômica/métodos , Sistemas de Transporte de Aminoácidos/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Razão Sinal-Ruído
9.
Nat Commun ; 11(1): 5490, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33127909

RESUMO

Halobacteria (henceforth: Haloarchaea) are predominantly aerobic halophiles that are thought to have evolved from anaerobic methanogens. This remarkable transformation most likely involved an extensive influx of bacterial genes. Whether it entailed a single massive transfer event or a gradual stream of transfers remains a matter of debate. To address this, genomes that descend from methanogen-to-halophile intermediates are necessary. Here, we present five such near-complete genomes of Marine Group IV archaea (Hikarchaeia), the closest known relatives of Haloarchaea. Their inclusion in gene tree-aware ancestral reconstructions reveals an intermediate stage that had already lost a large number of genes, including nearly all of those involved in methanogenesis and the Wood-Ljungdahl pathway. In contrast, the last Haloarchaea common ancestor gained a large number of genes and expanded its aerobic respiration and salt/UV resistance gene repertoire. Our results suggest that complex and gradual patterns of gain and loss shaped the methanogen-to-halophile transition.


Assuntos
Archaea/classificação , Archaea/genética , Euryarchaeota/genética , Genoma Arqueal , Filogenia , Archaea/metabolismo , Proteínas Arqueais/metabolismo , Euryarchaeota/classificação , Euryarchaeota/metabolismo , Evolução Molecular , Transferência Genética Horizontal , Genes Bacterianos , Metagenômica , Metano/metabolismo , Família Multigênica , RNA Ribossômico 16S/genética
10.
Appl Environ Microbiol ; 86(23)2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-32978128

RESUMO

Lipoic acid is a sulfur-containing cofactor and a component of the glycine cleavage system (GCS) involved in C1 compound metabolism and the 2-oxoacid dehydrogenases that catalyze the oxidative decarboxylation of 2-oxoacids. Lipoic acid is found in all domains of life and is generally synthesized as a lipoyl group on the H-protein of the GCS or the E2 subunit of 2-oxoacid dehydrogenases. Lipoyl synthase catalyzes the insertion of two sulfur atoms to the C-6 and C-8 carbon atoms of the octanoyl moiety on the octanoyl-H-protein or octanoyl-E2 subunit. Although the hyperthermophilic archaeon Thermococcus kodakarensis seemed able to synthesize lipoic acid, a classical lipoyl synthase (LipA) gene homolog cannot be found on the genome. In this study, we aimed to identify the lipoyl synthase in this organism. Genome information analysis suggested that the TK2109 and TK2248 genes, which had been annotated as biotin synthase (BioB), are both involved in lipoic acid metabolism. Based on the chemical reaction catalyzed by BioB, we predicted that the genes encode proteins that catalyze the lipoyl synthase reaction. Genetic analysis of TK2109 and TK2248 provided evidence that these genes are involved in lipoic acid biosynthesis. The purified TK2109 and TK2248 recombinant proteins exhibited lipoyl synthase activity toward a chemically synthesized octanoyl-octapeptide. These in vivo and in vitro analyses indicated that the TK2109 and TK2248 genes encode a structurally novel lipoyl synthase. TK2109 and TK2248 homologs are widely distributed among the archaeal genomes, suggesting that in addition to the LipA homologs, the two proteins represent a new group of lipoyl synthases in archaea.IMPORTANCE Lipoic acid is an essential cofactor for GCS and 2-oxoacid dehydrogenases, and α-lipoic acid has been utilized as a medicine and attracted attention as a supplement due to its antioxidant activity. The biosynthesis pathways of lipoic acid have been established in Bacteria and Eucarya but not in Archaea Although some archaeal species, including Sulfolobus, possess a classical lipoyl synthase (LipA) gene homolog, many archaeal species, including T. kodakarensis, do not. In addition, the biosynthesis mechanism of the octanoyl moiety, a precursor for lipoyl group biosynthesis, is also unknown for many archaea. As the enzyme identified in T. kodakarensis most likely represents a new group of lipoyl synthases in Archaea, the results obtained in this study provide an important step in understanding how lipoic acid is synthesized in this domain and how the two structurally distinct lipoyl synthases evolved in nature.


Assuntos
Proteínas Arqueais/genética , Sulfurtransferases/genética , Thermococcus/genética , Ácido Tióctico/biossíntese , Aminoácido Oxirredutases , Proteínas Arqueais/metabolismo , Complexos Multienzimáticos , Proteínas Recombinantes , Sulfurtransferases/metabolismo , Thermococcus/enzimologia , Transferases
11.
Nucleic Acids Res ; 48(19): 10986-10997, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-32997110

RESUMO

During DNA replication, the presence of 8-oxoguanine (8-oxoG) lesions in the template strand cause the high-fidelity (HiFi) DNA polymerase (Pol) to stall. An early response to 8-oxoG lesions involves 'on-the-fly' translesion synthesis (TLS), in which a specialized TLS Pol is recruited and replaces the stalled HiFi Pol for lesion bypass. The length of TLS must be long enough for effective bypass, but it must also be regulated to minimize replication errors by the TLS Pol. The exact position where the TLS Pol ends and the HiFi Pol resumes (i.e. the length of the TLS patch) has not been described. We use steady-state and pre-steady-state kinetic assays to characterize lesion bypass intermediates formed by different archaeal polymerase holoenzyme complexes that include PCNA123 and RFC. After bypass of 8-oxoG by TLS PolY, products accumulate at the template position three base pairs beyond the lesion. PolY is catalytically poor for subsequent extension from this +3 position beyond 8-oxoG, but this inefficiency is overcome by rapid extension of HiFi PolB1. The reciprocation of Pol activities at this intermediate indicates a defined position where TLS Pol extension is limited and where the DNA substrate is handed back to the HiFi Pol after bypass of 8-oxoG.


Assuntos
Proteínas Arqueais/metabolismo , Reparo do DNA , Replicação do DNA , DNA Arqueal/química , DNA Polimerase Dirigida por DNA/metabolismo , Archaea/enzimologia , Archaea/genética , Dano ao DNA , Guanina/análogos & derivados , Guanina/metabolismo
12.
PLoS One ; 15(8): e0237135, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822422

RESUMO

DNA-binding Transcription Factors (TFs) play a central role in regulation of gene expression in prokaryotic organisms, and similarities at the sequence level have been reported. These proteins are predicted with different abundances as a consequence of genome size, where small organisms contain a low proportion of TFs and large genomes contain a high proportion of TFs. In this work, we analyzed a collection of 668 experimentally validated TFs across 30 different species from diverse taxonomical classes, including Escherichia coli K-12, Bacillus subtilis 168, Corynebacterium glutamicum, and Streptomyces coelicolor, among others. This collection of TFs, together with 111 hidden Markov model profiles associated with DNA-binding TFs collected from diverse databases such as PFAM and DBD, was used to identify the repertoire of proteins putatively devoted to gene regulation in 1321 representative genomes of Archaea and Bacteria. The predicted regulatory proteins were posteriorly analyzed in terms of their genomic context, allowing the prediction of functions for TFs and their neighbor genes, such as genes involved in virulence, enzymatic functions, phosphorylation mechanisms, and antibiotic resistance. The functional analysis associated with PFAM groups showed diverse functional categories were significantly enriched in the collection of TFs and the proteins encoded by the neighbor genes, in particular, small-molecule binding and amino acid transmembrane transporter activities associated with the LysR family and proteins devoted to cellular aromatic compound metabolic processes or responses to drugs, stress, or abiotic stimuli in the MarR family. We consider that with the increasing data derived from new technologies, novel TFs can be identified and help improve the predictions for this class of proteins in complete genomes. The complete collection of experimentally characterized and predicted TFs is available at http://web.pcyt.unam.mx/EntrafDB/.


Assuntos
Archaea/genética , Proteínas Arqueais/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Escherichia coli K12/genética , Fatores de Transcrição/genética , Archaea/patogenicidade , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA Arqueal/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli K12/patogenicidade , Regulação da Expressão Gênica em Archaea , Regulação Bacteriana da Expressão Gênica , Genoma Arqueal , Genoma Bacteriano , Ligação Proteica , Fatores de Transcrição/metabolismo , Virulência/genética
13.
Appl Environ Microbiol ; 86(21)2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32826220

RESUMO

CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N2) fixation were targeted for dCas9-mediated repression. First, the nif operon (nifHI 1 I 2 DKEN) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNAs), one targeting the promoter and the other targeting nifD Remarkably, growth of M. acetivorans with N2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N2 Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1, encoding a subunit of the repressor of the nif operon, was targeted. The nrpR1 repression strain grew normally with N2 but had increased nif operon transcript abundance, consistent with NrpR1 acting as a repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivorans IMPORTANCE Genetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas, and a biofuel. The CRISPRi-dCas9 system in the model methanogen Methanosarcina acetivorans is, to our knowledge, the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens.


Assuntos
Proteínas Arqueais/genética , Sistemas CRISPR-Cas , Genes Arqueais/genética , Methanosarcina/genética , Fixação de Nitrogênio/genética , Proteínas Arqueais/metabolismo , Expressão Gênica , Methanosarcina/metabolismo
14.
Proc Natl Acad Sci U S A ; 117(33): 19914-19925, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747548

RESUMO

Apocarotenoids are important signaling molecules generated from carotenoids through the action of carotenoid cleavage dioxygenases (CCDs). These enzymes have a remarkable ability to cleave carotenoids at specific alkene bonds while leaving chemically similar sites within the polyene intact. Although several bacterial and eukaryotic CCDs have been characterized, the long-standing goal of experimentally visualizing a CCD-carotenoid complex at high resolution to explain this exquisite regioselectivity remains unfulfilled. CCD genes are also present in some archaeal genomes, but the encoded enzymes remain uninvestigated. Here, we address this knowledge gap through analysis of a metazoan-like archaeal CCD from Candidatus Nitrosotalea devanaterra (NdCCD). NdCCD was active toward ß-apocarotenoids but did not cleave bicyclic carotenoids. It exhibited an unusual regiospecificity, cleaving apocarotenoids solely at the C14'-C13' alkene bond to produce ß-apo-14'-carotenals. The structure of NdCCD revealed a tapered active site cavity markedly different from the broad active site observed for the retinal-forming Synechocystis apocarotenoid oxygenase (SynACO) but similar to the vertebrate retinoid isomerase RPE65. The structure of NdCCD in complex with its apocarotenoid product demonstrated that the site of cleavage is defined by interactions along the substrate binding cleft as well as selective stabilization of reaction intermediates at the scissile alkene. These data on the molecular basis of CCD catalysis shed light on the origins of the varied catalytic activities found in metazoan CCDs, opening the possibility of modifying their activity through rational chemical or genetic approaches.


Assuntos
Archaea/enzimologia , Proteínas Arqueais/química , Carotenoides/metabolismo , Dioxigenases/química , Archaea/química , Archaea/classificação , Archaea/genética , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carotenoides/química , Catálise , Domínio Catalítico , Dioxigenases/genética , Dioxigenases/metabolismo , Especificidade por Substrato , Synechocystis/química , Synechocystis/enzimologia , Synechocystis/genética
15.
Proc Natl Acad Sci U S A ; 117(33): 19904-19913, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747565

RESUMO

Asgard archaea genomes contain potential eukaryotic-like genes that provide intriguing insight for the evolution of eukaryotes. The eukaryotic actin polymerization/depolymerization cycle is critical for providing force and structure in many processes, including membrane remodeling. In general, Asgard genomes encode two classes of actin-regulating proteins from sequence analysis, profilins and gelsolins. Asgard profilins were demonstrated to regulate actin filament nucleation. Here, we identify actin filament severing, capping, annealing and bundling, and monomer sequestration activities by gelsolin proteins from Thorarchaeota (Thor), which complete a eukaryotic-like actin depolymerization cycle, and indicate complex actin cytoskeleton regulation in Asgard organisms. Thor gelsolins have homologs in other Asgard archaea and comprise one or two copies of the prototypical gelsolin domain. This appears to be a record of an initial preeukaryotic gene duplication event, since eukaryotic gelsolins are generally comprise three to six domains. X-ray structures of these proteins in complex with mammalian actin revealed similar interactions to the first domain of human gelsolin or cofilin with actin. Asgard two-domain, but not one-domain, gelsolins contain calcium-binding sites, which is manifested in calcium-controlled activities. Expression of two-domain gelsolins in mammalian cells enhanced actin filament disassembly on ionomycin-triggered calcium release. This functional demonstration, at the cellular level, provides evidence for a calcium-controlled Asgard actin cytoskeleton, indicating that the calcium-regulated actin cytoskeleton predates eukaryotes. In eukaryotes, dynamic bundled actin filaments are responsible for shaping filopodia and microvilli. By correlation, we hypothesize that the formation of the protrusions observed from Lokiarchaeota cell bodies may involve the gelsolin-regulated actin structures.


Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Archaea/metabolismo , Proteínas Arqueais/metabolismo , Gelsolina/metabolismo , Fatores de Despolimerização de Actina/química , Fatores de Despolimerização de Actina/genética , Actinas/química , Actinas/genética , Sequência de Aminoácidos , Archaea/química , Archaea/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Citoesqueleto/química , Citoesqueleto/genética , Citoesqueleto/metabolismo , Evolução Molecular , Gelsolina/química , Gelsolina/genética , Genoma Arqueal , Polimerização , Conformação Proteica em alfa-Hélice , Alinhamento de Sequência
16.
Proc Natl Acad Sci U S A ; 117(33): 20223-20234, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32759215

RESUMO

Nano-sized archaeota, with their small genomes and limited metabolic capabilities, are known to associate with other microbes, thereby compensating for their own auxotrophies. These diminutive and yet ubiquitous organisms thrive in hypersaline habitats that they share with haloarchaea. Here, we reveal the genetic and physiological nature of a nanohaloarchaeon-haloarchaeon association, with both microbes obtained from a solar saltern and reproducibly cultivated together in vitro. The nanohaloarchaeon Candidatus Nanohalobium constans LC1Nh is an aerotolerant, sugar-fermenting anaerobe, lacking key anabolic machinery and respiratory complexes. The nanohaloarchaeon cells are found physically connected to the chitinolytic haloarchaeon Halomicrobium sp. LC1Hm. Our experiments revealed that this haloarchaeon can hydrolyze chitin outside the cell (to produce the monosaccharide N-acetylglucosamine), using this beta-glucan to obtain carbon and energy for growth. However, LC1Hm could not metabolize either glycogen or starch (both alpha-glucans) or other polysaccharides tested. Remarkably, the nanohaloarchaeon's ability to hydrolyze glycogen and starch to glucose enabled growth of Halomicrobium sp. LC1Hm in the absence of a chitin. These findings indicated that the nanohaloarchaeon-haloarchaeon association is both mutualistic and symbiotic; in this case, each microbe relies on its partner's ability to degrade different polysaccharides. This suggests, in turn, that other nano-sized archaeota may also be beneficial for their hosts. Given that availability of carbon substrates can vary both spatially and temporarily, the susceptibility of Halomicrobium to colonization by Ca Nanohalobium can be interpreted as a strategy to maximize the long-term fitness of the host.


Assuntos
Halobacteriaceae/fisiologia , Nanoarchaeota/fisiologia , Polissacarídeos/metabolismo , Simbiose/fisiologia , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Técnicas de Cocultura , Regulação da Expressão Gênica em Archaea , Genoma Arqueal , Genômica , Filogenia
17.
Nucleic Acids Res ; 48(17): 9681-9693, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32833023

RESUMO

CRISPR-Cas system provides acquired immunity against invasive genetic elements in prokaryotes. In both bacteria and archaea, transcriptional factors play important roles in regulation of CRISPR adaptation and interference. In the model Crenarchaeon Sulfolobus islandicus, a CRISPR-associated factor Csa3a triggers CRISPR adaptation and activates CRISPR RNA transcription for the immunity. However, regulation of DNA repair systems for repairing the genomic DNA damages caused by the CRISPR self-immunity is less understood. Here, according to the transcriptome and reporter gene data, we found that deletion of the csa3a gene down-regulated the DNA damage response (DDR) genes, including the ups and ced genes. Furthermore, in vitro analyses demonstrated that Csa3a specifically bound the DDR gene promoters. Microscopic analysis showed that deletion of csa3a significantly inhibited DNA damage-induced cell aggregation. Moreover, the flow cytometry study and survival rate analysis revealed that the csa3a deletion strain was more sensitive to the DNA-damaging reagent. Importantly, CRISPR self-targeting and DNA transfer experiments revealed that Csa3a was involved in regulating Ups- and Ced-mediated repair of CRISPR-damaged host genomic DNA. These results explain the interplay between Csa3a functions in activating CRISPR adaptation and DNA repair systems, and expands our understanding of the lost link between CRISPR self-immunity and genome stability.


Assuntos
Proteínas Arqueais/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Reparo do DNA , Sulfolobus/genética , Regiões 5' não Traduzidas , Proteínas Arqueais/metabolismo , Sítios de Ligação , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Dano ao DNA , Perfilação da Expressão Gênica , Regulação da Expressão Gênica em Archaea , Genoma Arqueal , Mutação , Regiões Promotoras Genéticas , Sulfolobus/crescimento & desenvolvimento
18.
Nucleic Acids Res ; 48(17): 9589-9605, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32857850

RESUMO

Transcription termination defines accurate transcript 3'-ends and ensures programmed transcriptomes, making it critical to life. However, transcription termination mechanisms remain largely unknown in Archaea. Here, we reported the physiological significance of the newly identified general transcription termination factor of Archaea, the ribonuclease aCPSF1, and elucidated its 3'-end cleavage triggered termination mechanism. The depletion of Mmp-aCPSF1 in Methanococcus maripaludis caused a genome-wide transcription termination defect and disordered transcriptome. Transcript-3'end-sequencing revealed that transcriptions primarily terminate downstream of a uridine-rich motif where Mmp-aCPSF1 performed an endoribonucleolytic cleavage, and the endoribonuclease activity was determined to be essential to the in vivo transcription termination. Co-immunoprecipitation and chromatin-immunoprecipitation detected interactions of Mmp-aCPSF1 with RNA polymerase and chromosome. Phylogenetic analysis revealed that the aCPSF1 orthologs are ubiquitously distributed among the archaeal phyla, and two aCPSF1 orthologs from Lokiarchaeota and Thaumarchaeota could replace Mmp-aCPSF1 to terminate transcription of M. maripaludis. Therefore, the aCPSF1 dependent termination mechanism could be widely employed in Archaea, including Lokiarchaeota belonging to Asgard Archaea, the postulated archaeal ancestor of Eukaryotes. Strikingly, aCPSF1-dependent archaeal transcription termination reported here exposes a similar 3'-cleavage mode as the eukaryotic RNA polymerase II termination, thus would shed lights on understanding the evolutionary linking between archaeal and eukaryotic termination machineries.


Assuntos
Proteínas Arqueais/genética , Mathanococcus/genética , Ribonucleases/genética , Proteínas Arqueais/metabolismo , DNA Complementar/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Genoma Arqueal , Mutação , Filogenia , Ribonucleases/metabolismo , Transcrição Genética , Uridina
19.
Appl Environ Microbiol ; 86(21)2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32859593

RESUMO

Arsenate is a notorious toxicant that is known to disrupt multiple biochemical pathways. Many microorganisms have developed mechanisms to detoxify arsenate using the ArsC-type arsenate reductase, and some even use arsenate as a terminal electron acceptor for respiration involving arsenate respiratory reductase (Arr). ArsC-type reductases have been studied extensively, but the phylogenetically unrelated Arr system is less investigated and has not been characterized from Archaea Here, we heterologously expressed the genes encoding Arr from the crenarchaeon Pyrobaculum aerophilum in the euryarchaeon Pyrococcus furiosus, both of which grow optimally near 100°C. Recombinant P. furiosus was grown on molybdenum (Mo)- or tungsten (W)-containing medium, and two types of recombinant Arr enzymes were purified, one containing Mo (Arr-Mo) and one containing W (Arr-W). Purified Arr-Mo had a 140-fold higher specific activity in arsenate [As(V)] reduction than Arr-W, and Arr-Mo also reduced arsenite [As(III)]. The P. furiosus strain expressing Arr-Mo (the Arr strain) was able to use arsenate as a terminal electron acceptor during growth on peptides. In addition, the Arr strain had increased tolerance compared to that of the parent strain to arsenate and also, surprisingly, to arsenite. Compared to the parent, the Arr strain accumulated intracellularly almost an order of magnitude more arsenic when cells were grown in the presence of arsenite. X-ray absorption spectroscopy (XAS) results suggest that the Arr strain of P. furiosus improves its tolerance to arsenite by increasing production of less-toxic arsenate and nontoxic methylated arsenicals compared to that by the parent.IMPORTANCE Arsenate respiratory reductases (Arr) are much less characterized than the detoxifying arsenate reductase system. The heterologous expression and characterization of an Arr from Pyrobaculum aerophilum in Pyrococcus furiosus provides new insights into the function of this enzyme. From in vivo studies, production of Arr not only enabled P. furiosus to use arsenate [As(V)] as a terminal electron acceptor, it also provided the organism with a higher resistance to arsenate and also, surprisingly, to arsenite [As(III)]. In contrast to the tungsten-containing oxidoreductase enzymes natively produced by P. furiosus, recombinant P. aerophilum Arr was much more active with molybdenum than with tungsten. It is also, to our knowledge, the only characterized Arr to be active with both molybdenum and tungsten in the active site.


Assuntos
Proteínas Arqueais/genética , Arseniato Redutases/genética , Regulação da Expressão Gênica em Archaea , Pyrococcus furiosus/genética , Thermoproteaceae/genética , Proteínas Arqueais/metabolismo , Arseniato Redutases/metabolismo , Arsênico/metabolismo , Microrganismos Geneticamente Modificados/enzimologia , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Pyrococcus furiosus/enzimologia , Pyrococcus furiosus/metabolismo
20.
Sci Rep ; 10(1): 10946, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32616801

RESUMO

Mononuclear molybdoenzymes of the dimethyl sulfoxide reductase (DMSOR) family catalyze a number of reactions essential to the carbon, nitrogen, sulfur, arsenic, and selenium biogeochemical cycles. These enzymes are also ancient, with many lineages likely predating the divergence of the last universal common ancestor into the Bacteria and Archaea domains. We have constructed rooted phylogenies for over 1,550 representatives of the DMSOR family using maximum likelihood methods to investigate the evolution of the arsenic biogeochemical cycle. The phylogenetic analysis provides compelling evidence that formylmethanofuran dehydrogenase B subunits, which catalyze the reduction of CO2 to formate during hydrogenotrophic methanogenesis, constitutes the most ancient lineage. Our analysis also provides robust support for selenocysteine as the ancestral ligand for the Mo/W atom. Finally, we demonstrate that anaerobic arsenite oxidase and respiratory arsenate reductase catalytic subunits represent a more ancient lineage of DMSORs compared to aerobic arsenite oxidase catalytic subunits, which evolved from the assimilatory nitrate reductase lineage. This provides substantial support for an active arsenic biogeochemical cycle on the anoxic Archean Earth. Our work emphasizes that the use of chalcophilic elements as substrates as well as the Mo/W ligand in DMSORs has indelibly shaped the diversification of these enzymes through deep time.


Assuntos
Archaea/enzimologia , Proteínas Arqueais/metabolismo , Arsênico/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas com Ferro-Enxofre/metabolismo , Metano/metabolismo , Oxirredutases/metabolismo , Selênio/metabolismo , Evolução Molecular , Filogenia
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