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1.
Artigo em Inglês | MEDLINE | ID: mdl-37074136

RESUMO

In the present study, we attempt to clarify the taxonomic positions of Picrophilus oshimae and Picrophilus torridus. The 16S rRNA gene sequence similarity between P. oshimae DSM 9789T and P. torridus DSM9790T (99.4 %) was above the threshold value (98.6 %) for bacterial species delineation. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between P. oshimae DSM 9789T and P. torridus DSM9790T were higher than the threshold values (95-96 % for ANI and 70 % for dDDH) for bacterial species delineation. The present results indicate that Picrophilus torridus Zillig et al. 1996 is a later heterotypic synonym of Picrophilus oshimae Schleper et al. 1996.


Assuntos
Ácidos Graxos , Thermoplasmales , Análise de Sequência de DNA , Técnicas de Tipagem Bacteriana , RNA Ribossômico 16S/genética , Filogenia , DNA Bacteriano/genética , Composição de Bases , Ácidos Graxos/química , Thermoplasmales/genética , Hibridização de Ácido Nucleico
2.
Biophys Chem ; 296: 106991, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36905840

RESUMO

Heliorhodopsin (HeR) is a seven-helical transmembrane protein with a retinal chromophore that corresponds to a new rhodopsin family. HeR from the archaebacterium Thermoplasmatales archaeon (TaHeR) exhibits unique features, such as the inverted protein orientation in the membrane compared to other rhodopsins and a long photocycle. Here, we used solid-state nuclear magnetic resonance (NMR) spectroscopy to investigate the 13C and 15N NMR signals of the retinal chromophore and protonated Schiff base (RPSB) in TaHeR embedded in POPE/POPG membrane. Although the 14- and 20-13C retinal signals indicated 13-trans/15-anti (all-trans) configurations, the 20-13C chemical shift value was different from that of other microbial rhodopsins, indicating weakly steric hinderance between Phe203 and the C20 methyl group. 15N RPSB/λmax plot deviated from the linear correlation based on retinylidene-halide model compounds. Furthermore, 15N chemical shift anisotropy (CSA) suggested that Ser112 and Ser234 polar residues distinguish the electronic environment tendencies of RPSB from those of other microbial rhodopsins. Our NMR results revealed that the retinal chromophore and the RPSB in TaHeR exhibit unique electronic environments.


Assuntos
Retinaldeído , Thermoplasmales , Retinaldeído/química , Retinaldeído/metabolismo , Bases de Schiff/química , Rodopsina/química , Rodopsina/metabolismo , Rodopsinas Microbianas/química , Espectroscopia de Ressonância Magnética/métodos , Thermoplasmales/metabolismo , Archaea/metabolismo
3.
J Biol Chem ; 298(7): 102111, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35690147

RESUMO

Mevalonate 3,5-bisphosphate decarboxylase is involved in the recently discovered Thermoplasma-type mevalonate pathway. The enzyme catalyzes the elimination of the 3-phosphate group from mevalonate 3,5-bisphosphate as well as concomitant decarboxylation of the substrate. This entire reaction of the enzyme resembles the latter half-reactions of its homologs, diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, which also catalyze ATP-dependent phosphorylation of the 3-hydroxyl group of their substrates. However, the crystal structure of mevalonate 3,5-bisphosphate decarboxylase and the structural reasons of the difference between reactions catalyzed by the enzyme and its homologs are unknown. In this study, we determined the X-ray crystal structure of mevalonate 3,5-bisphosphate decarboxylase from Picrophilus torridus, a thermoacidophilic archaeon of the order Thermoplasmatales. Structural and mutational analysis demonstrated the importance of a conserved aspartate residue for enzyme activity. In addition, although crystallization was performed in the absence of substrate or ligands, residual electron density having the shape of a fatty acid was observed at a position overlapping the ATP-binding site of the homologous enzyme, diphosphomevalonate decarboxylase. This finding is in agreement with the expected evolutionary route from phosphomevalonate decarboxylase (ATP-dependent) to mevalonate 3,5-bisphosphate decarboxylase (ATP-independent) through the loss of kinase activity. We found that the binding of geranylgeranyl diphosphate, an intermediate of the archeal isoprenoid biosynthesis pathway, evoked significant activation of mevalonate 3,5-bisphosphate decarboxylase, and several mutations at the putative geranylgeranyl diphosphate-binding site impaired this activation, suggesting the physiological importance of ligand binding as well as a possible novel regulatory system employed by the Thermoplasma-type mevalonate pathway.


Assuntos
Carboxiliases/química , Thermoplasmales/enzimologia , Trifosfato de Adenosina/metabolismo , Carboxiliases/metabolismo , Redes e Vias Metabólicas , Ácido Mevalônico/metabolismo
4.
Nat Commun ; 13(1): 1735, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35365607

RESUMO

Micrarchaeota is a distinctive lineage assigned to the DPANN archaea, which includes poorly characterised microorganisms with reduced genomes that likely depend on interactions with hosts for growth and survival. Here, we report the enrichment of a stable co-culture of a member of the Micrarchaeota (Ca. Micrarchaeum harzensis) together with its Thermoplasmatales host (Ca. Scheffleriplasma hospitalis), as well as the isolation of the latter. We show that symbiont-host interactions depend on biofilm formation as evidenced by growth experiments, comparative transcriptomic analyses and electron microscopy. In addition, genomic, metabolomic, extracellular polymeric substances and lipid content analyses indicate that the Micrarchaeon symbiont relies on the acquisition of metabolites from its host. Our study of the cell biology and physiology of a Micrarchaeon and its host adds to our limited knowledge of archaeal symbioses.


Assuntos
Thermoplasmales , Archaea/genética , Biofilmes , Genoma Arqueal , Filogenia , Thermoplasmales/genética , Thermoplasmales/metabolismo
5.
Elife ; 102021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33739282

RESUMO

Helicases utilize nucleotide triphosphate (NTP) hydrolysis to translocate along single-stranded nucleic acids (NA) and unwind the duplex. In the cell, helicases function in the context of other NA-associated proteins such as single-stranded DNA binding proteins. Such encounters regulate helicase function, although the underlying mechanisms remain largely unknown. Ferroplasma acidarmanus xeroderma pigmentosum group D (XPD) helicase serves as a model for understanding the molecular mechanisms of superfamily 2B helicases, and its activity is enhanced by the cognate single-stranded DNA binding protein replication protein A 2 (RPA2). Here, optical trap measurements of the unwinding activity of a single XPD helicase in the presence of RPA2 reveal a mechanism in which XPD interconverts between two states with different processivities and transient RPA2 interactions stabilize the more processive state, activating a latent 'processivity switch' in XPD. A point mutation at a regulatory DNA binding site on XPD similarly activates this switch. These findings provide new insights on mechanisms of helicase regulation by accessory proteins.


Assuntos
Proteínas de Bactérias/metabolismo , Proteína de Replicação A/metabolismo , Thermoplasmales/enzimologia , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo , Sítios de Ligação , Pinças Ópticas
6.
Protein Pept Lett ; 28(6): 675-679, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33243110

RESUMO

BACKGROUND: CsaA is among the few chaperones which are present in both bacteria and archaea, but absent in eukaryotes. There are no reports on interactome analysis of CsaA from archaea, till date. Identification of binding partners of CsaA might be helpful in understanding CsaA-associated processes in Picrophilus torridus an extreme thermoacidophilic euryarchaeon. OBJECTIVES: The present study was conducted to identify the binding partners of CsaA of P. torridus (PtCsaA). METHODS: The binding partners of PtCsaA were isolated and identified using a pull down assay and liquid chromatography-mass spectrometry (LC-MS). RESULTS: The results revealed twelve potential binding partners of CsaA. These were thermosome subunits (Q6KZS2 and Q6L132), nascent polypeptide-associated complex protein (Q6L1N3), elongation factor 1-alpha (Q6L202), uncharacterized protein (Q6L0Y6), citrate synthase (Q6L0M8), asparaginyl- tRNA synthetase (Q6L0M5), succinyl-CoA synthetase beta chain (Q6L0B4), pyruvate ferredoxin oxidoreductase alpha and beta chain proteins (Q6KZA7 and Q6KZA6, respectively), malate dehydrogenase (Q6L0C3) and reversed fumarylacetoacetase (Q6KZ97). Functional categorization revealed that of these, six proteins were involved in energy metabolic pathways, three were archaeal chaperones, two were involved in translation and one might be a transcription regulator. STRING-based analysis of the protein-protein interactions of the experimental interactome revealed strong interactions among them. CONCLUSION: PtCsaA might be a multifaceted protein which besides translation might also play important role in metabolic processes of P. torridus. However, further experiments investigating the binding partners of CsaA in other archaea are required for a better understanding of CsaA-associated processes in archaea.


Assuntos
Proteínas de Bactérias , Chaperonas Moleculares , Thermoplasmales/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cromatografia Líquida , Espectrometria de Massas , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas
7.
J Phys Chem Lett ; 11(20): 8604-8609, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32940480

RESUMO

Heliorhodopsin (HeR), a recently discovered new rhodopsin family, has an inverted membrane topology compared to animal and microbial rhodopsins, and no ion-transport activity. The slow photocycle of HeRs suggests a light-sensor function, although the function remains unknown. HeRs exhibit no specific binding of monovalent cations or anions. Despite this, ATR-FTIR spectroscopy in the present study demonstrates binding of Zn2+ to HeR from Thermoplasmatales archaeon (TaHeR). The biding of Zn2+ to 0.2 mM Kd is accompanied by helical structural perturbations without altering its color. Even though ion-specific FTIR spectra were observed for many divalent cations, only helical structural perturbations were observed for Zn2+-binding. Similar results were obtained for HeR 48C12. These findings suggest a possible modification of HeR function by Zn2+.


Assuntos
Rodopsina/química , Rodopsinas Microbianas/química , Zinco/química , Cátions Bivalentes/química , Cobalto/química , Cor , Cobre/química , Luz , Modelos Moleculares , Níquel/química , Ligação Proteica , Conformação Proteica , Thermoplasmales/metabolismo
8.
Biochem Biophys Res Commun ; 533(3): 262-267, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-32951839

RESUMO

Microbial rhodopsins comprise an opsin protein with seven transmembrane helices and a retinal as the chromophore. An all-trans retinal is covalently bonded to a lysine residue through the retinal Schiff base (RSB) and stabilized by a negatively charged counterion. The distance between the RSB and counterion is closely related to the light energy absorption. However, in heliorhodopsin-48C12 (HeR-48C12), while E107 acts as the counterion, E107D mutation exhibits an identical absorption spectrum to the wild-type, suggesting that the distance does not affect its absorption spectra. Here we present the 2.6 Å resolution crystal structure of the Thermoplasmatales archaeon HeR E108D mutant, which also has an identical absorption spectrum to the wild-type. The structure revealed that D108 does not form a hydrogen bond with the RSB, and its counterion interaction becomes weaker. Alternatively, the serine cluster, S78, S112, and S238 form a distinct interaction network around the RSB. The absorption spectra of the E to D and S to A double mutants suggested that S112 influences the spectral shift by compensating for the weaker counterion interaction. Our structural and spectral studies have revealed the unique spectral shift mechanism of HeR and clarified the physicochemical properties of HeRs.


Assuntos
Substituição de Aminoácidos , Proteínas Arqueais/química , Retinaldeído/química , Rodopsinas Microbianas/química , Thermoplasmales/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Clonagem Molecular , Cor , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retinaldeído/metabolismo , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/metabolismo , Bases de Schiff/química
9.
Extremophiles ; 23(6): 783-792, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31549249

RESUMO

Intracellular ß-galactosidase (E.C 3.2.1.23) produced by the thermoacidophilic archeon Picrophilus torridus DSM 9790 was purified to homogeneity using a combination of DEAE Sepharose, gel filtration, hydroxyapatite and chromatofocusing chromatographies. LC-MS/MS analysis was used to confirm the identity of the purified protein. The enzyme was found to be a homotrimer, with a molecular mass of 157.0 kDa and an isoelectric point of 5.7. To our knowledge, this enzyme has the lowest pH optimum of any intracellular ß-galactosidase characterized to date. Maximal activity was exhibited at acidic pH values of 5.0-5.5 and at 70 °C. The enzyme retained > 95% activity after heating to 70 °C for 1 h, or after incubation at pH 5.5 for 1 h. The enzyme may be of interest for high-temperature bioprocessing, such as in the production of lactulose. This investigation suggests that the ß-galactosidase activity produced by P. torridus is potentially more useful than several enzymes already characterized for such an application.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/isolamento & purificação , Temperatura Alta , Thermoplasmales/enzimologia , beta-Galactosidase/química , beta-Galactosidase/isolamento & purificação , Estabilidade Enzimática , Microbiologia Industrial
10.
Nature ; 574(7776): 132-136, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31554965

RESUMO

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the ß-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.


Assuntos
Rodopsinas Microbianas/química , Thermoplasmales/química , Bacteriorodopsinas/química , Sítios de Ligação , Cristalografia por Raios X , Microscopia de Força Atômica , Modelos Moleculares , Dobramento de Proteína , Multimerização Proteica , Retinaldeído/química , Rodopsinas Microbianas/ultraestrutura
11.
Genes (Basel) ; 10(6)2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31208064

RESUMO

"Candidatus Micrarchaeota" are widely distributed in acidic environments; however, their cultivability and our understanding of their interactions with potential hosts are very limited. Their habitats were so far attributed with acidic sites, soils, peats, freshwater systems, and hypersaline mats. Using cultivation and culture-independent approaches (16S rRNA gene clonal libraries, high-throughput amplicon sequencing of V3-V4 region of 16S rRNA genes), we surveyed the occurrence of these archaea in geothermal areas on Kamchatka Peninsula and Kunashir Island and assessed their taxonomic diversity in relation with another type of low-pH environment, acid mine drainage stream (Wales, UK). We detected "Ca. Micrarchaeota" in thermophilic heterotrophic enrichment cultures of Kunashir and Kamchatka that appeared as two different phylotypes, namely "Ca. Mancarchaeum acidiphilum"-, and ARMAN-2-related, alongside their potential hosts, Cuniculiplasma spp. and other Thermoplasmatales archaea without defined taxonomic position. These clusters of "Ca. Micrarchaeota" together with three other groups were also present in mesophilic acid mine drainage community. Present work expands our knowledge on the diversity of "Ca. Micrarchaeota" in thermophilic and mesophilic acidic environments, suggests cultivability patterns of acidophilic archaea and establishes potential links between low-abundance species of thermophilic "Ca. Micrarchaeota" and certain Thermoplasmatales, such as Cuniculiplasma spp. in situ.


Assuntos
Ácidos/química , Archaea/genética , Microbiologia do Solo , Thermoplasmales/genética , Archaea/química , Archaea/classificação , Ecossistema , Água Doce/microbiologia , Genoma Arqueal/genética , Fontes Termais , Filogenia , RNA Ribossômico 16S/genética , Rios/microbiologia , Solo/química , Thermoplasmales/química , País de Gales
12.
J Phys Chem B ; 123(11): 2507-2512, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30742768

RESUMO

Heliorhodopsins (HeR) constitute a new rhodopsin family and show only <15% sequence identities with type-1 and type-2 rhodopsins. The large difference in amino acid sequence between HeRs and other rhodopsins raises a question whether their biological function is triggered by efficient and rapid photoisomerization of the retinal chromophore as in the case of other rhodopsins. We performed femtosecond time-resolved absorption measurements of two HeRs, HeR 48C12 and HeR from Thermoplasmatales archaeon SG8-52-1. Both HeRs exhibit excited-state absorption around 480 nm and stimulated emission in the >650 nm region, and these transient signals decay concomitantly with appearance of photoproduct absorption on a subpicosecond time scale. The observed spectral change indicates that ultrafast retinal photoisomerization proceeds in the femtosecond time region. The transient spectra and dynamics of HeRs are surprisingly similar to those of type-1 rhodopsins, despite remarkable differences in amino acid arrangement in the hydrophobic region of the retinal binding site.


Assuntos
Proteínas Arqueais/química , Rodopsinas Microbianas/química , Thermoplasmales/química , Isomerismo , Cinética , Espectrofotometria
13.
Extremophiles ; 23(2): 177-187, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30600357

RESUMO

Here we report the chemical and microbial characterization of the surface water of a CO2-rich hydrothermal vent known in Costa Rica as Borbollones, located at Tenorio Volcano National Park. The Borbollones showed a temperature surrounding 60 °C, a pH of 2.4 and the gas released has a composition of ~ 97% CO2, ~ 0.07% H2S, ~ 2.3% N2 and ~ 0.12% CH4. Other chemical species such as sulfate and iron were found at high levels with respect to typical fresh water bodies. Analysis by 16S rRNA gene metabarcoding revealed that in Borbollones predominates an archaeon from the order Thermoplasmatales and one bacterium from the genus Sulfurimonas. Other sulfur- (genera Thiomonas, Acidithiobacillus, Sulfuriferula, and Sulfuricurvum) and iron-oxidizing bacteria (genera Sideroxydans, Gallionella, and Ferrovum) were identified. Our results show that CO2-influenced surface water of Borbollones contains microorganisms that are usually found in acid rock drainage environments or sulfur-rich hydrothermal vents. To our knowledge, this is the first microbiological characterization of a CO2-dominated hydrothermal spring from Central America and expands our understanding of those extreme ecosystems.


Assuntos
Bactérias/isolamento & purificação , Fontes Termais/microbiologia , Microbiota , Enxofre/metabolismo , Thermoplasmales/isolamento & purificação , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Filogenia , RNA Ribossômico 16S/genética , Thermoplasmales/classificação , Thermoplasmales/genética , Termotolerância
14.
Extremophiles ; 23(1): 1-7, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30499003

RESUMO

Recently, the order Thermoplasmatales was expanded through the cultivation and description of species Cuniculiplasma divulgatum and corresponding family Cuniculiplasmataceae. Initially isolated from acidic streamers, signatures of these archaea were ubiquitously found in various low-pH settings. Eight genomes with various levels of completeness are currently available, all of which exhibit very high sequence identities and genomic conservation. Co-existence of Cuniculiplasmataceae with archaeal Richmond Mine acidophilic nanoorganisms ('ARMAN')-related archaea representing an intriguing group within the "microbial dark matter" suggests their common fundamental environmental strategy and metabolic networking. The specific case of "Candidatus Mancarchaeum acidiphilum" Mia14 phylogenetically affiliated with "Ca. Micrarchaeota" from the superphylum "Ca. Diapherotrites" along with the presence of other representatives of 'DPANN' with significantly reduced genomes points at a high probability of close interactions between the latter and various Thermoplasmatales abundant in situ. This review critically assesses our knowledge on specific functional role and potential of the members of Cuniculiplasmataceae abundant in acidophilic microbiomes through the analysis of distribution, physiological and genomic patterns, and their interactions with 'ARMAN'-related archaea.


Assuntos
Genoma Arqueal , Filogenia , Thermoplasmales/genética , Metaboloma , Thermoplasmales/classificação , Thermoplasmales/metabolismo
15.
J Phys Chem Lett ; 9(22): 6431-6436, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30351947

RESUMO

Heliorhodopsins (HeRs) are a new category of retinal-bound proteins recently discovered through functional metagenomics analysis that exhibit obvious differences from type-1 microbial rhodopsins. We conducted the first detailed structural characterization of the retinal chromophore in HeRs using resonance Raman spectroscopy. The observed spectra clearly show that the Schiff base of the chromophore is protonated and forms a strong hydrogen bond to a species other than a water molecule, highly likely a counterion residue. The vibrational mode of the Schiff base of HeRs exhibits similarities with that of photosensory microbial rhodopsins, that is consistent with the previous proposal that HeRs function as photosensors. We also revealed unusual spectral features of the in-plane chain vibrations of the chromophore, suggesting an unprecedented geometry of the Schiff base caused by a difference in the retinal pocket structure of HeRs. These data demonstrate structural characteristics of the photoreceptive site in this novel type of rhodopsin family.


Assuntos
Proteínas Arqueais/química , Rodopsinas Microbianas/química , Bases de Schiff/química , Halobacterium salinarum/química , Ligação de Hidrogênio , Estrutura Molecular , Conformação Proteica , Análise Espectral Raman/métodos , Thermoplasmales/química , Vibração
16.
Cell Stress Chaperones ; 23(6): 1257-1274, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30178307

RESUMO

Molecular chaperones are a diverse group of proteins that ensure proteome integrity by helping the proteins fold correctly and maintain their native state, thus preventing their misfolding and subsequent aggregation. The chaperone machinery of archaeal organisms has been thought to closely resemble that found in humans, at least in terms of constituent players. Very few studies have been ventured into system-level analysis of chaperones and their functioning in archaeal cells. In this study, we attempted such an analysis of chaperone-assisted protein folding in archaeal organisms through network approach using Picrophilus torridus as model system. The study revealed that DnaK protein of Hsp70 system acts as hub in protein-protein interaction network. However, DnaK protein was present only in a subset of archaeal organisms and absent from many archaea, especially members of Crenarchaeota phylum. Therefore, a similar network was created for another archaeal organism, Sulfolobus solfataricus, a member of Crenarchaeota. The chaperone network of S. solfataricus suggested that thermosomes played an integral part of hub proteins in archaeal organisms, where DnaK was absent. We further compared the chaperone network of archaea with that found in eukaryotic systems, by creating a similar network for Homo sapiens. In the human chaperone network, the UBC protein, a part of ubiquitination system, was the most important module, and interestingly, this system is known to be absent in archaeal organisms. Comprehensive comparison of these networks leads to several interesting conclusions regarding similarities and differences within archaeal chaperone machinery in comparison to humans.


Assuntos
Proteínas Arqueais/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Mapas de Interação de Proteínas , Sulfolobus/metabolismo , Thermoplasmales/metabolismo , Bases de Dados de Proteínas , Humanos , Dobramento de Proteína
17.
Biochemistry ; 57(26): 3797-3806, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29812914

RESUMO

The thermoacidophilic archaea Picrophilus torridus and Sulfolobus solfataricus catabolize glucose via a nonphosphorylative Entner-Doudoroff pathway and a branched Entner-Doudoroff pathway, respectively. Key enzymes for these Entner-Doudoroff pathways are the aldolases, 2-keto-3-deoxygluconate aldolase (KDG-aldolase) and 2-keto-3-deoxy-6-phosphogluconate aldolase [KD(P)G-aldolase]. KDG-aldolase from P. torridus (Pt-KDG-aldolase) is highly specific for the nonphosphorylated substrate, 2-keto-3-deoxygluconate (KDG), whereas KD(P)G-aldolase from S. solfataricus [Ss-KD(P)G-aldolase] is an enzyme that catalyzes the cleavage of both KDG and 2-keto-3-deoxy-6-phosphogluconate (KDPG), with a preference for KDPG. The structural basis for the high specificity of Pt-KDG-aldolase for KDG as compared to the more promiscuous Ss-KD(P)G-aldolase has not been analyzed before. In this work, we report the elucidation of the structure of Ss-KD(P)G-aldolase in complex with KDPG at 2.35 Å and that of KDG-aldolase from P. torridus at 2.50 Å resolution. By superimposition of the active sites of the two enzymes, and subsequent site-directed mutagenesis studies, a network of four amino acids, namely, Arg106, Tyr132, Arg237, and Ser241, was identified in Ss-KD(P)G-aldolase that interact with the negatively charged phosphate group of KDPG, thereby increasing the affinity of the enzyme for KDPG. This KDPG-binding network is absent in Pt-KDG-aldolase, which explains the low catalytic efficiency of KDPG cleavage.


Assuntos
Aldeído Liases/química , Proteínas Arqueais/química , Gluconatos/química , Sulfolobus solfataricus/enzimologia , Thermoplasmales/enzimologia , Modelos Moleculares , Domínios Proteicos , Relação Estrutura-Atividade
18.
Sci Rep ; 7(1): 3289, 2017 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-28607432

RESUMO

This study describes the laboratory cultivation of ARMAN (Archaeal Richmond Mine Acidophilic Nanoorganisms). After 2.5 years of successive transfers in an anoxic medium containing ferric sulfate as an electron acceptor, a consortium was attained that is comprised of two members of the order Thermoplasmatales, a member of a proposed ARMAN group, as well as a fungus. The 16S rRNA identity of one archaeon is only 91.6% compared to the most closely related isolate Thermogymnomonas acidicola. Hence, this organism is the first member of a new genus. The enrichment culture is dominated by this microorganism and the ARMAN. The third archaeon in the community seems to be present in minor quantities and has a 100% 16S rRNA identity to the recently isolated Cuniculiplasma divulgatum. The enriched ARMAN species is most probably incapable of sugar metabolism because the key genes for sugar catabolism and anabolism could not be identified in the metagenome. Metatranscriptomic analysis suggests that the TCA cycle funneled with amino acids is the main metabolic pathway used by the archaea of the community. Microscopic analysis revealed that growth of the ARMAN is supported by the formation of cell aggregates. These might enable feeding of the ARMAN by or on other community members.


Assuntos
Técnicas de Cocultura/métodos , Fungos/crescimento & desenvolvimento , Laboratórios , Thermoplasmales/crescimento & desenvolvimento , Genoma Arqueal , Metagenoma , Filogenia , RNA Ribossômico 16S/genética , Transcriptoma/genética
19.
Acta Crystallogr F Struct Biol Commun ; 73(Pt 4): 184-195, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28368276

RESUMO

Single-wavelength anomalous dispersion (SAD) utilizing anomalous signal from native S atoms, or other atoms with Z ≤ 20, generally requires highly redundant data collected using relatively long-wavelength X-rays. Here, the results from two proteins are presented where the anomalous signal from serendipitously acquired surface-bound Ca atoms with an anomalous data multiplicity of around 10 was utilized to drive de novo structure determination. In both cases, the Ca atoms were acquired from the crystallization solution, and the data-collection strategy was not optimized to exploit the anomalous signal from these scatterers. The X-ray data were collected at 0.98 Šwavelength in one case and at 1.74 Šin the other (the wavelength was optimized for sulfur, but the anomalous signal from calcium was exploited for structure solution). Similarly, using a test case, it is shown that data collected at ∼1.0 Šwavelength, where the f'' value for sulfur is 0.28 e, are sufficient for structure determination using intrinsic S atoms from a strongly diffracting crystal. Interestingly, it was also observed that SHELXD was capable of generating a substructure solution from high-exposure data with a completeness of 70% for low-resolution reflections extending to 3.5 Šresolution with relatively low anomalous multiplicity. Considering the fact that many crystallization conditions contain anomalous scatterers such as Cl, Ca, Mn etc., checking for the presence of fortuitous anomalous signal in data from well diffracting crystals could prove useful in either determining the structure de novo or in accurately assigning surface-bound atoms.


Assuntos
Proteínas Arqueais/química , Proteínas de Bactérias/química , Cálcio/química , Proteínas do Ovo/química , Muramidase/química , Enxofre/química , Animais , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cátions Bivalentes , Galinhas/metabolismo , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Proteínas do Ovo/genética , Proteínas do Ovo/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Muramidase/genética , Muramidase/metabolismo , Conformação Proteica , Pseudomonas syringae/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Thermoplasmales/química , Difração de Raios X , Raios X
20.
Sci Rep ; 6: 39034, 2016 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-27966672

RESUMO

The order Thermoplasmatales (Euryarchaeota) is represented by the most acidophilic organisms known so far that are poorly amenable to cultivation. Earlier culture-independent studies in Iron Mountain (California) pointed at an abundant archaeal group, dubbed 'G-plasma'. We examined the genomes and physiology of two cultured representatives of a Family Cuniculiplasmataceae, recently isolated from acidic (pH 1-1.5) sites in Spain and UK that are 16S rRNA gene sequence-identical with 'G-plasma'. Organisms had largest genomes among Thermoplasmatales (1.87-1.94 Mbp), that shared 98.7-98.8% average nucleotide identities between themselves and 'G-plasma' and exhibited a high genome conservation even within their genomic islands, despite their remote geographical localisations. Facultatively anaerobic heterotrophs, they possess an ancestral form of A-type terminal oxygen reductase from a distinct parental clade. The lack of complete pathways for biosynthesis of histidine, valine, leucine, isoleucine, lysine and proline pre-determines the reliance on external sources of amino acids and hence the lifestyle of these organisms as scavengers of proteinaceous compounds from surrounding microbial community members. In contrast to earlier metagenomics-based assumptions, isolates were S-layer-deficient, non-motile, non-methylotrophic and devoid of iron-oxidation despite the abundance of methylotrophy substrates and ferrous iron in situ, which underlines the essentiality of experimental validation of bioinformatic predictions.


Assuntos
Ácidos/química , Ecossistema , Euryarchaeota/genética , Genoma Arqueal/genética , Thermoplasmales/genética , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , California , Euryarchaeota/classificação , Euryarchaeota/metabolismo , Genômica/métodos , Geografia , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Espanha , Thermoplasmales/metabolismo , Thermoplasmales/ultraestrutura , Reino Unido
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