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1.
Environ Microbiol ; 24(2): 626-642, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35102700

RESUMO

Thermococcales, a major order of archaea inhabiting the iron- and sulfur-rich anaerobic parts of hydrothermal deep-sea vents, have been shown to rapidly produce abundant quantities of pyrite FeS2 in iron-sulfur-rich fluids at 85°C, suggesting that they may contribute to the formation of 'low temperature' FeS2 in their ecosystem. We show that this process operates in Thermococcus kodakarensis only when zero-valent sulfur is directly available as intracellular sulfur vesicles. Whether in the presence or absence of zero-valent sulfur, significant amounts of Fe3 S4 greigite nanocrystals are formed extracellularly. We also show that mineralization of iron sulfides induces massive cell mortality but that concomitantly with the formation of greigite and/or pyrite, a new generation of cells can grow. This phenomenon is observed for Fe concentrations of 5 mM but not higher suggesting that above a threshold in the iron pulse all cells are lysed. We hypothesize that iron sulfides precipitation on former cell materials might induce the release of nutrients in the mineralization medium further used by a fraction of surviving non-mineralized cells allowing production of new alive cells. This suggests that biologically induced mineralization of iron-sulfides could be part of a survival strategy employed by Thermococcales to cope with mineralizing high-temperature hydrothermal environments.


Assuntos
Thermococcales , Thermococcus , Ecossistema , Ferro/química , Sulfetos/química
2.
Extremophiles ; 23(1): 141-149, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30467661

RESUMO

Diverse DNA repair mechanisms are essential to all living organisms. Some of the most widespread repair systems allow recovery of genome integrity in the face of UV radiation. Here, we show that the hyperthermophilic archaeon Thermococcus nautili possesses a remarkable ability to recovery from extreme chromosomal damage. Immediately following UV irradiation, chromosomal DNA of T. nautili is fragmented beyond recognition. However, the extensive UV-induced double-stranded breaks (DSB) are repaired over the course of several hours, allowing restoration of growth. DSBs also disrupted plasmid DNA in this species. Similar to the chromosome, plasmid integrity was restored during an outgrowth period. Intriguingly, the topology of recovered pTN1 plasmids differed from control strain by being more positively supercoiled. As reverse gyrase (RG) is the only enzyme capable of inducing positive supercoiling, our results suggest the activation of RG activity by UV-induced stress. We suggest simple UV stress could be used to study archaeal DNA repair and responses to DSB.


Assuntos
DNA Arqueal/efeitos da radiação , DNA Super-Helicoidal/efeitos da radiação , Thermococcus/efeitos da radiação , Raios Ultravioleta , Proteínas Arqueais/metabolismo , Quebras de DNA de Cadeia Dupla , Fragmentação do DNA , DNA Girase/metabolismo , Reparo do DNA , Plasmídeos/efeitos da radiação , Thermococcus/genética
3.
Biochimie ; 118: 356-64, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26234734

RESUMO

The euryarchaeon Thermococcus prieurii inhabits deep-sea hydrothermal vents, one of the most extreme environments on Earth, which is reduced and enriched with heavy metals. Transmission electron microscopy and cryo-electron microscopy imaging of T. prieurii revealed the production of a plethora of diverse membrane vesicles (MVs) (from 50 nm to 400 nm), as is the case for other Thermococcales. T. prieurii also produces particularly long nanopods/nanotubes, some of them containing more than 35 vesicles encased in a S-layer coat. Notably, cryo-electron microscopy of T. prieurii cells revealed the presence of numerous intracellular dark vesicles that bud from the host cells via interaction with the cytoplasmic membrane. These dark vesicles are exclusively found in conjunction with T. prieurii cells and never observed in the purified membrane vesicles preparations. Energy-Dispersive-X-Ray analyses revealed that these dark vesicles are filled with sulfur. Furthermore, the presence of these sulfur vesicles (SVs) is exclusively observed when elemental sulfur was added into the growth medium. In this report, we suggest that these atypical vesicles sequester the excess sulfur not used for growth, thus preventing the accumulation of toxic levels of sulfur in the host's cytoplasm. These SVs transport elemental sulfur out of the cell where they are rapidly degraded. Intriguingly, closely related archaeal species, Thermococcus nautili and Thermococcus kodakaraensis, show some differences about the production of sulfur vesicles. Whereas T. kodakaraensis produces less sulfur vesicles than T. prieurii, T. nautili does not produce such sulfur vesicles, suggesting that Thermococcales species exhibit significant differences in their sulfur metabolic pathways.


Assuntos
Vesículas Secretórias/metabolismo , Enxofre/metabolismo , Thermococcus/metabolismo , Inativação Metabólica/fisiologia , Microscopia Eletrônica , Espectrometria por Raios X
4.
J Virol ; 87(1): 124-36, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23055559

RESUMO

The structural and functional analysis of the protein AvtR encoded by Acidianus filamentous virus 6 (AFV6), which infects the archaeal genus Acidianus, revealed its unusual structure and involvement in transcriptional regulation of several viral genes. The crystal structure of AvtR (100 amino acids) at 2.6-Å resolution shows that it is constituted of a repeated ribbon-helix-helix (RHH) motif, which is found in a large family of bacterial transcriptional regulators. The known RHH proteins form dimers that interact with DNA using their ribbon to create a central ß-sheet. The repeated RHH motifs of AvtR superpose well on such dimers, but its central sheet contains an extra strand, suggesting either conformational changes or a different mode of DNA binding. Systematic evolution of ligands by exponential enrichment (SELEX) experiments combined with systematic mutational and computational analysis of the predicted site revealed 8 potential AvtR targets in the AFV6 genome. Two of these targets were studied in detail, and the complex role of AvtR in the transcriptional regulation of viral genes was established. Repressing transcription from its own gene, gp29, AvtR can also act as an activator of another gene, gp30. Its binding sites are distant from both genes' TATA boxes, and the mechanism of AvtR-dependent regulation appears to include protein oligomerization starting from the protein's initial binding sites. Many RHH transcriptional regulators of archaeal viruses could share this regulatory mechanism.


Assuntos
Acidianus/virologia , Proteínas de Ligação a DNA/química , Lipothrixviridae/química , Proteínas Virais/química , Acidianus/genética , Sequência de Aminoácidos , Cristalografia por Raios X , Análise Mutacional de DNA , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Lipothrixviridae/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas Virais/genética
6.
Protein Sci ; 18(4): 850-5, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19319959

RESUMO

We present here the 2.6A resolution crystal structure of the pT26-6p protein, which is encoded by an ORF of the plasmid pT26-2, recently isolated from the hyperthermophilic archaeon, Thermococcus sp. 26,2. This large protein is present in all members of a new family of mobile elements that, beside pT26-2 include several virus-like elements integrated in the genomes of several Thermococcales and Methanococcales (phylum Euryarchaeota). Phylogenetic analysis suggested that this protein, together with its nearest neighbor (organized as an operon) have coevolved for a long time with the cellular hosts of the encoding mobile element. As the sequences of the N and C-terminal regions suggested a possible membrane association, a deletion construct (739 amino acids) was used for structural analysis. The structure consists of two very similar beta-sheet domains with a new topology and a five helical bundle C-terminal domain. Each of these domains corresponds to a unique fold that has presently not been found in cellular proteins. This result supports the idea that proteins encoded by plasmid and viruses that have no cellular homologues could be a reservoir of new folds for structural genomic studies.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/genética , Sequências Repetitivas Dispersas , Thermococcus/química , Thermococcus/genética , Cristalografia por Raios X , Filogenia , Plasmídeos , Conformação Proteica , Multimerização Proteica , Homologia Estrutural de Proteína
8.
Nucleic Acids Res ; 33(7): 2310-7, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15849317

RESUMO

Type II DNA topoisomerases have been classified into two families, Topo IIA and Topo IIB, based on structural and mechanistic dissimilarities. Topo IIA is the target of many important antibiotics and antitumoural drugs, most of them being inactive on Topo IIB. The effects and mode of action of Topo IIA inhibitors in vitro and in vivo have been extensively studied for the last twenty-five years. In contrast, studies of Topo IIB inhibitors were lacking. To document this field, we have studied two Hsp90 inhibitors (radicicol and geldanamycin), known to interact with the ATP-binding site of Hsp90 (the Bergerat fold), which is also present in Topo IIB. Here, we report that radicicol inhibits the decatenation and relaxation activities of Sulfolobus shibatae DNA topoisomerase VI (a Topo IIB) while geldanamycin does not. In addition, radicicol has no effect on the Topo IIA Escherichia coli DNA gyrase. In agreement with their different effects on DNA topoisomerase VI, we found that radicicol can theoretically fit in the ATP-binding pocket of the DNA topoisomerase VI 'Bergerat fold', whereas geldanamycin cannot. Radicicol inhibited growths of Sulfolobus acidocaldarius (a crenarchaeon) and of Haloferax volcanii (a euryarchaeon) at the same doses that inhibited DNA topoisomerase VI in vitro. In contrast, the bacteria E.coli was resistant to this drug. Radicicol thus appears to be a very promising compound to study the mechanism of Topo IIB in vitro, as well as the biological roles of these enzymes in vivo.


Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactonas/farmacologia , Inibidores da Topoisomerase II , Proteínas Arqueais , Benzoquinonas , Proliferação de Células/efeitos dos fármacos , DNA Topoisomerases Tipo II , Inibidores Enzimáticos/química , Haloferax volcanii/citologia , Haloferax volcanii/efeitos dos fármacos , Haloferax volcanii/enzimologia , Lactamas Macrocíclicas , Lactonas/química , Macrolídeos , Quinonas/farmacologia , Sulfolobus acidocaldarius/citologia , Sulfolobus acidocaldarius/efeitos dos fármacos , Sulfolobus acidocaldarius/enzimologia
9.
Biochem Soc Trans ; 32(Pt 2): 184-7, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15046568

RESUMO

This minireview summarizes our current knowledge about archaeal genetic elements in the hyperthermophilic order Thermococcales in the phylum Euryarchaeota. This includes recent work on the first virus of Pyrococcus, PAV1, the discovery of novel unique virus morphotypes in hot deep-sea environments, and preliminary observations on novel cryptic plasmids. We also review the work accomplished over the last 5 years in the development of genetic tools for members of the Pyrococcus and Thermococcus genera, mainly in our laboratories.


Assuntos
Genoma Arqueal , Pyrococcus/genética , Thermococcales/genética , Thermococcus/genética , Antígenos Arqueais/química , DNA Arqueal/ultraestrutura , Vetores Genéticos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Transmissão , Compostos Organometálicos/farmacologia , Plasmídeos/metabolismo , Transgenes , Vírus/genética
10.
Nucleic Acids Res ; 32(4): 1439-47, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-14990749

RESUMO

We showed previously that rad50 and mre11 genes of thermophilic archaea are organized in an operon-like structure with a third gene (nurA) encoding a 5' to 3' exonuclease. Here, we show that the rad50, mre11 and nurA genes from the hyperthermophilic archaeon Sulfolobus acidocaldarius are co-transcribed with a fourth gene encoding a DNA helicase. This enzyme (HerA) is the prototype of a new class of DNA helicases able to utilize either 3' or 5' single-stranded DNA extensions for loading and subsequent DNA duplex unwinding. To our knowledge, DNA helicases capable of translocating along the DNA in both directions have not been identified previously. Sequence analysis of HerA shows that it is a member of the TrwB, FtsK and VirB4/VirD4 families of the PilT class NTPases. HerA homologs are found in all thermophilic archaeal species and, in all cases except one, the rad50, mre11, nurA and herA genes are grouped together. These results suggest that the archaeal Rad50-Mre11 complex might act in association with a 5' to 3' exonuclease (NurA) and a bipolar DNA helicase (HerA) indicating a probable involvement in the initiation step of homologous recombination.


Assuntos
Proteínas Arqueais/genética , DNA Helicases/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Genes Arqueais , Sequência de Aminoácidos , DNA Helicases/classificação , DNA Helicases/metabolismo , Temperatura Alta , Dados de Sequência Molecular , Óperon , Alinhamento de Sequência , Sulfolobus acidocaldarius/genética , Transcrição Gênica
11.
Mol Genet Genomics ; 266(1): 72-8, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11589580

RESUMO

Hyperthermophilic archaea of the genus Pyrococcus are resistant to gamma radiation, suggesting that efficient mechanisms for DNA repair exist in these organisms. To determine whether protective mechanisms might also be implicated in this radioresistance, we have estimated the linear density of DNA double-stranded breaks caused by gamma irradiation in the genomic DNA of two Pyrococcus species, using Escherichia coli and the radioresistant bacterium Deinococcus radiodurans as controls. The linear density of double-stranded breaks was essentially the same in all four microorganisms when irradiation was carried under similar anaerobic conditions, indicating that no specific DNA protection mechanisms exist in Pyrococcus species. Using one- and two-dimensional gel electrophoresis we compared the protein patterns from Pyrococcus abyssi and P. furiosus cells that had or had not been exposed to gamma rays. We did not detect any significant protein induction following DNA damage in either species.


Assuntos
DNA Arqueal/genética , Pyrococcus/efeitos da radiação , Tolerância a Radiação/genética , Dano ao DNA , Eletroforese/métodos , Escherichia coli/efeitos da radiação , Raios gama , Pyrococcus/genética , Especificidade da Espécie
12.
Proc Natl Acad Sci U S A ; 98(20): 11152-7, 2001 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-11562464

RESUMO

Although genome analyses have suggested parallels between archaeal and eukaryotic replication systems, little is known about the DNA replication mechanism in Archaea. By two-dimensional gel electrophoreses we positioned a replication origin (oriC) within 1 kb in the chromosomal DNA of Pyrococcus abyssi, an anaerobic hyperthermophile, and demonstrated that the oriC is physically linked to the cdc6 gene. Our chromatin immunoprecipitation assays indicated that P. abyssi Cdc6 and minichromosome maintenance (MCM) proteins bind preferentially to the oriC region in the exponentially growing cells. Whereas the oriC association of MCM was specifically inhibited by stopping DNA replication with puromycin treatment, Cdc6 protein stayed bound to the replication origin after de novo protein synthesis was inhibited. Our data suggest that archaeal and eukaryotic Cdc6 and MCM proteins function similarly in replication initiation and imply that an oriC association of MCM could be regulated by an unknown mechanism in Archaea.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromossomos de Archaea/genética , Proteínas de Ligação a DNA/metabolismo , Pyrococcus/genética , Pyrococcus/metabolismo , Origem de Replicação , Proteínas de Saccharomyces cerevisiae , Sequência de Bases , Cromatina/genética , Cromatina/metabolismo , Mapeamento Cromossômico , Sequência Consenso , Replicação do DNA , DNA Arqueal/química , DNA Arqueal/genética , Temperatura Alta , Dados de Sequência Molecular , Complexo de Reconhecimento de Origem , Sequências Repetitivas de Ácido Nucleico
13.
J Biol Chem ; 276(40): 37215-22, 2001 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-11485995

RESUMO

A key step in the DNA transport by type II DNA topoisomerase is the formation of a double-strand break with the enzyme being covalently linked to the broken DNA ends (referred to as the cleavage complex). In the present study, we have analyzed the formation and structure of the cleavage complex catalyzed by Sufolobus shibatae DNA topoisomerase VI (topoVI), a member of the recently described type IIB DNA topoisomerase family. A purification procedure of a fully soluble recombinant topoVI was developed by expressing both subunits simultaneously in Escherichia coli. Using this recombinant enzyme, we observed that the formation of the double-strand breaks on supercoiled or linear DNA is strictly dependent on the presence of ATP or AMP-PNP. This result suggests that ATP binding is required to stabilize an enzyme conformation able to cleave the DNA backbone. The structure of cleavage complexes on a linear DNA fragment have been analyzed at the nucleotide level. Similarly to other type II DNA topoisomerases, topoVI is covalently attached to the 5'-ends of the broken DNA. However, sequence analysis of the double-strand breaks revealed that they are all characterized by staggered two-nucleotide long 5' overhangs, contrasting with the four-base staggered double-strand breaks catalyzed by type IIA DNA topoisomerases. While no clear consensus sequences surrounding the cleavage sites could be described, interestingly A and T nucleotides are highly represented on the 5' extensions, giving a first insight on the preferred sequences recognized by this type II DNA topoisomerase.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA Topoisomerases Tipo II/metabolismo , DNA/metabolismo , Proteínas Arqueais , Sequência de Bases , Escherichia coli/enzimologia , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Análise de Sequência de DNA
19.
C R Acad Sci III ; 324(12): 1067-76, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11803805

RESUMO

The sequencing of several genomes from each of the three domains of life (Archaea, Bacteria and Eukarya) has provided a huge amount of data that can be used to gain insight about early cellular evolution. Some features of the universal tree of life based on rRNA polygenies have been confirmed, such as the division of the cellular living world into three domains. The monophyly of each domain is supported by comparative genomics. However, the hyperthermophilic nature of the 'last universal common ancestor' (LUCA) is not confirmed. Comparative genomics has revealed that gene transfers have been (and still are) very frequent in genome evolution. Nevertheless, a core of informational genes appears more resistant to transfer, testifying for a close relationship between archaeal and eukaryal informational processes. This observation can be explained either by a common unique history between Archaea and Eukarya or by an atypical evolution of these systems in Bacteria. At the moment, comparative genomics still does not allow to choose between a simple LUCA, possibly with an RNA genome, or a complex LUCA, with a DNA genome and informational mechanisms similar to those of Archaea and Eukarya. Further comparative studies on informational mechanisms in the three domains should help to resolve this critical question. The role of viruses in the origin and evolution of DNA genomes also appears an area worth of active investigations. I suggest here that DNA and DNA replication mechanisms appeared first in the virus world before being transferred into cellular organisms.


Assuntos
Evolução Biológica , DNA/genética , Genômica , Animais , Archaea/genética , Bactérias/genética , Células Eucarióticas
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