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1.
Cell ; 178(4): 820-834.e14, 2019 08 08.
Artículo en Inglés | MEDLINE | ID: mdl-31398339

RESUMEN

Delineating ecologically meaningful populations among microbes is important for identifying their roles in environmental and host-associated microbiomes. Here, we introduce a metric of recent gene flow, which when applied to co-existing microbes, identifies congruent genetic and ecological units separated by strong gene flow discontinuities from their next of kin. We then develop a pipeline to identify genome regions within these units that show differential adaptation and allow mapping of populations onto environmental variables or host associations. Using this reverse ecology approach, we show that the human commensal bacterium Ruminococcus gnavus breaks up into sharply delineated populations that show different associations with health and disease. Defining populations by recent gene flow in this way will facilitate the analysis of bacterial and archaeal genomes using ecological and evolutionary theory developed for plants and animals, thus allowing for testing unifying principles across all biology.


Asunto(s)
Clostridiales/genética , Flujo Génico , Microbiota/genética , Adaptación Fisiológica/genética , Alelos , Colitis Ulcerosa/microbiología , Enfermedad de Crohn/microbiología , Transferencia de Gen Horizontal , Genoma Bacteriano , Humanos , Modelos Genéticos , Tasa de Mutación , Filogenia , Polimorfismo de Nucleótido Simple , Prochlorococcus/genética , Sulfolobus/genética , Vibrio/genética
2.
Cell ; 179(1): 165-179.e18, 2019 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-31539494

RESUMEN

The three-dimensional organization of chromosomes can have a profound impact on their replication and expression. The chromosomes of higher eukaryotes possess discrete compartments that are characterized by differing transcriptional activities. Contrastingly, most bacterial chromosomes have simpler organization with local domains, the boundaries of which are influenced by gene expression. Numerous studies have revealed that the higher-order architectures of bacterial and eukaryotic chromosomes are dependent on the actions of structural maintenance of chromosomes (SMC) superfamily protein complexes, in particular, the near-universal condensin complex. Intriguingly, however, many archaea, including members of the genus Sulfolobus do not encode canonical condensin. We describe chromosome conformation capture experiments on Sulfolobus species. These reveal the presence of distinct domains along Sulfolobus chromosomes that undergo discrete and specific higher-order interactions, thus defining two compartment types. We observe causal linkages between compartment identity, gene expression, and binding of a hitherto uncharacterized SMC superfamily protein that we term "coalescin."


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas de Archaea/metabolismo , Sulfolobus/citología , Sulfolobus/genética , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Cromosomas de Archaea/genética , Replicación del ADN/genética , ADN de Archaea/metabolismo , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Sitios Genéticos/genética , Modelos Genéticos , Complejos Multiproteicos/metabolismo , Plásmidos/genética , Unión Proteica/genética , Transcripción Genética
3.
Mol Cell ; 79(5): 741-757.e7, 2020 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-32730741

RESUMEN

Cmr-ß is a type III-B CRISPR-Cas complex that, upon target RNA recognition, unleashes a multifaceted immune response against invading genetic elements, including single-stranded DNA (ssDNA) cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific single-stranded RNA (ssRNA) hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-ß, unveiling how binding of the target RNA regulates the Cmr2 activities. Cryoelectron microscopy (cryo-EM) analysis revealed the unique subunit architecture of Cmr-ß and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA-bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5' tag in the CRISPR RNAs (crRNA) and the 3' antitag of the target RNA activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self- versus non-self-recognition. These findings highlight the diverse defense strategies of type III complexes.


Asunto(s)
Inmunidad Adaptativa/fisiología , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteínas Arqueales/química , Proteínas Arqueales/fisiología , Proteínas Arqueales/ultraestructura , Proteínas Asociadas a CRISPR/ultraestructura , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , Microscopía por Crioelectrón , ADN de Cadena Simple/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , ARN Mensajero/metabolismo , Relación Estructura-Actividad , Sulfolobus/genética , Sulfolobus/fisiología
4.
Nature ; 577(7791): 572-575, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31942067

RESUMEN

The CRISPR system in bacteria and archaea provides adaptive immunity against mobile genetic elements. Type III CRISPR systems detect viral RNA, resulting in the activation of two regions of the Cas10 protein: an HD nuclease domain (which degrades viral DNA)1,2 and a cyclase domain (which synthesizes cyclic oligoadenylates from ATP)3-5. Cyclic oligoadenylates in turn activate defence enzymes with a CRISPR-associated Rossmann fold domain6, sculpting a powerful antiviral response7-10 that can drive viruses to extinction7,8. Cyclic nucleotides are increasingly implicated in host-pathogen interactions11-13. Here we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease AcrIII-1 is widely distributed in archaeal and bacterial viruses and in proviruses. The enzyme uses a previously unknown fold to bind cA4 specifically, and a conserved active site to rapidly cleave this signalling molecule, allowing viruses to neutralize the type III CRISPR defence system. The AcrIII-1 family has a broad host range, as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. Our findings highlight the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.


Asunto(s)
Sistemas CRISPR-Cas/inmunología , Endonucleasas/metabolismo , Interacciones Microbiota-Huesped/inmunología , Sulfolobus/virología , Proteínas Virales/metabolismo , Virus/enzimología , Nucleótidos de Adenina/química , Nucleótidos de Adenina/metabolismo , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/metabolismo , ADN Viral/metabolismo , Endonucleasas/química , Modelos Moleculares , Nucleótidos Cíclicos/química , Nucleótidos Cíclicos/metabolismo , Oligorribonucleótidos/química , Oligorribonucleótidos/metabolismo , Filogenia , Transducción de Señal , Sulfolobus/genética , Sulfolobus/inmunología , Sulfolobus/metabolismo , Proteínas Virales/química , Proteínas Virales/clasificación , Virus/inmunología
5.
Nucleic Acids Res ; 52(8): 4644-4658, 2024 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-38375885

RESUMEN

Pseudouridine, one of the most abundant RNA modifications, is synthesized by stand-alone or RNA-guided pseudouridine synthases. Here, we comprehensively mapped pseudouridines in rRNAs, tRNAs and small RNAs in the archaeon Sulfolobus islandicus and identified Cbf5-associated H/ACA RNAs. Through genetic deletion and in vitro modification assays, we determined the responsible enzymes for these modifications. The pseudouridylation machinery in S. islandicus consists of the stand-alone enzymes aPus7 and aPus10, and six H/ACA RNA-guided enzymes that account for all identified pseudouridines. These H/ACA RNAs guide the modification of all eleven sites in rRNAs, two sites in tRNAs, and two sites in CRISPR RNAs. One H/ACA RNA shows exceptional versatility by targeting eight different sites. aPus7 and aPus10 are responsible for modifying positions 13, 54 and 55 in tRNAs. We identified four atypical H/ACA RNAs that lack the lower stem and the ACA motif and confirmed their function both in vivo and in vitro. Intriguingly, atypical H/ACA RNAs can be modified by Cbf5 in a guide-independent manner. Our data provide the first global view of pseudouridylation in archaea and reveal unexpected structures, substrates, and activities of archaeal H/ACA RNPs.


Asunto(s)
Seudouridina , ARN de Archaea , ARN de Transferencia , Sulfolobus , Seudouridina/metabolismo , Sulfolobus/genética , Sulfolobus/metabolismo , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , ARN de Archaea/genética , ARN de Archaea/metabolismo , ARN de Archaea/química , ARN Ribosómico/metabolismo , ARN Ribosómico/genética , Proteínas Arqueales/metabolismo , Proteínas Arqueales/genética , Procesamiento Postranscripcional del ARN , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo
6.
RNA ; 29(5): 551-556, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36759127

RESUMEN

Analysis of the profile of the tRNA modifications in several Archaea allowed us to observe a novel modified uridine in the V-loop of several tRNAs from two species: Pyrococcus furiosus and Sulfolobus acidocaldarius Recently, Ohira and colleagues characterized 2'-phosphouridine (Up) at position 47 in tRNAs of thermophilic Sulfurisphaera tokodaii, as well as in several other archaea and thermophilic bacteria. From the presence of the gene arkI corresponding to the RNA kinase responsible for Up47 formation, they also concluded that Up47 should be present in tRNAs of other thermophilic Archaea Reanalysis of our earlier data confirms that the unidentified residue in tRNAs of both P. furiosus and S. acidocaldarius is indeed 2'-phosphouridine followed by m5C48. Moreover, we find this modification in several tRNAs of other Archaea and of the hyperthermophilic bacterium Aquifex aeolicus.


Asunto(s)
Archaea , Sulfolobus , Archaea/genética , Bacterias/genética , Sulfolobus/genética
7.
Nucleic Acids Res ; 51(4): 1707-1723, 2023 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-36715325

RESUMEN

Cell cycle regulation is of paramount importance for all forms of life. Here, we report that a conserved and essential cell cycle-specific transcription factor (designated as aCcr1) and its viral homologs control cell division in Sulfolobales. We show that the transcription level of accr1 reaches peak during active cell division (D-phase) subsequent to the expression of CdvA, an archaea-specific cell division protein. Cells over-expressing the 58-aa-long RHH (ribbon-helix-helix) family cellular transcription factor as well as the homologs encoded by large spindle-shaped viruses Acidianus two-tailed virus (ATV) and Sulfolobus monocaudavirus 3 (SMV3) display significant growth retardation and cell division failure, manifesting as enlarged cells with multiple chromosomes. aCcr1 over-expression results in downregulation of 17 genes (>4-fold), including cdvA. A conserved motif, aCcr1-box, located between the TATA-binding box and the translation initiation site of 13 out of the 17 highly repressed genes, is critical for aCcr1 binding. The aCcr1-box is present in the promoters and 5' UTRs of cdvA genes across Sulfolobales, suggesting that aCcr1-mediated cdvA repression is an evolutionarily conserved mechanism by which archaeal cells dictate cytokinesis progression, whereas their viruses take advantage of this mechanism to manipulate the host cell cycle.


Asunto(s)
Sulfolobus , Factores de Transcripción , Factores de Transcripción/genética , Archaea , División Celular , Sulfolobus/genética , Regulación de la Expresión Génica
8.
J Gen Virol ; 105(7)2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38959058

RESUMEN

The family Turriviridae includes viruses with a dsDNA genome of 16-17 kbp. Virions are spherical with a diameter of approximately 75 nm and comprise a host-derived internal lipid membrane surrounded by a proteinaceous capsid shell. Members of the family Turriviridae infect extremophilic archaea of the genera Sulfolobus and Saccharolobus. Viral infection results in cell lysis for Sulfolobus turreted icosahedral virus 1 infection but other members of the family can be temperate. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Turriviridae, which is available at ictv.global/report/turriviridae.


Asunto(s)
Virus ADN , Genoma Viral , Virión , Virus ADN/clasificación , Virus ADN/genética , Virus ADN/ultraestructura , Virión/ultraestructura , Virus de Archaea/clasificación , Virus de Archaea/genética , Virus de Archaea/ultraestructura , Virus de Archaea/fisiología , Sulfolobus/virología , Sulfolobus/genética , ADN Viral/genética
9.
J Virol ; 96(24): e0143822, 2022 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-36448807

RESUMEN

All living organisms have evolved DNA damage response (DDR) strategies in coping with threats to the integrity of their genome. In response to DNA damage, Sulfolobus islandicus activates its DDR network in which Orc1-2, an ortholog of the archaeal Orc1/Cdc6 superfamily proteins, plays a central regulatory role. Here, we show that pretreatment with UV irradiation reduced virus genome replication in S. islandicus infected with the fusellovirus SSV2. Like treatment with UV or the DNA-damaging agent 4-nitroquinoline-1-oxide (NQO), infection with SSV2 facilitated the expression of orc1-2 and significantly raised the cellular level of Orc1-2. The inhibitory effect of UV irradiation on the virus DNA level was no longer apparent in the infected culture of an S. islandicus orc1-2 deletion mutant strain. On the other hand, the overexpression of orc1-2 decreased virus genomic DNA by ~102-fold compared to that in the parent strain. Furthermore, as part of the Orc1-2-mediated DDR response genes for homologous recombination repair (HRR), cell aggregation and intercellular DNA transfer were upregulated, whereas genes for cell division were downregulated. However, the HRR pathway remained functional in host inhibition of SSV2 genome replication in the absence of UpsA, a subunit of pili essential for intercellular DNA transfer. In agreement with this finding, lack of the general transcriptional activator TFB3, which controls the expression of the ups genes, only moderately affected SSV2 genome replication. Our results demonstrate that infection of S. islandicus by SSV2 triggers the host DDR pathway that, in return, suppresses virus genome replication. IMPORTANCE Extremophiles thrive in harsh habitats and thus often face a daunting challenge to the integrity of their genome. How these organisms respond to virus infection when their genome is damaged remains unclear. We found that the thermophilic archaeon Sulfolobus islandicus became more inhibitory to genome replication of the virus SSV2 after preinfection UV irradiation than without the pretreatment. On the other hand, like treatment with UV or other DNA-damaging agents, infection of S. islandicus by SSV2 triggers the activation of Orc1-2-mediated DNA damage response, including the activation of homologous recombination repair, cell aggregation and DNA import, and the repression of cell division. The inhibitory effect of pretreatment with UV irradiation on SSV2 genome replication was no longer observed in an S. islandicus mutant lacking Orc1-2. Our results suggest that DNA damage response is employed by S. islandicus as a strategy to defend against virus infection.


Asunto(s)
Fuselloviridae , Sulfolobus , Daño del ADN/genética , Reparación del ADN/genética , Fuselloviridae/genética , Sulfolobus/genética , Sulfolobus/efectos de la radiación , Sulfolobus/virología , Replicación Viral , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Rayos Ultravioleta , 4-Nitroquinolina-1-Óxido/farmacología , Complejo de Reconocimiento del Origen/genética , Complejo de Reconocimiento del Origen/metabolismo
10.
World J Microbiol Biotechnol ; 39(4): 90, 2023 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-36752840

RESUMEN

Endonuclease V (EndoV), which is widespread in bacteria, eukarya and Archaea, can cleave hypoxanthine (Hx)-containing DNA or RNA strand, and play an essential role in Hx repair. However, our understanding on archaeal EndoV's function remains incomplete. The model archaeon Sulfolobus islandicus REY15A encodes a putative EndoV protein (Sis-EndoV). Herein, we probed the biochemical characteristics of Sis-EndoV and dissected the roles of its seven conserved residues. Our biochemical data demonstrate that Sis-EndoV displays maximum cleavage efficiency at above 60 °C and at pH 7.0-9.0, and the enzyme activity is dependent on a divalent metal ion, among which Mg2+ is optimal. Importantly, we first measured the activation energy for cleaving Hx-containing ssDNA by Sis-EndoV to be 9.6 ± 0.8 kcal/mol by kinetic analyses, suggesting that chemical catalysis might be a rate-limiting step for catalysis. Mutational analyses show that residue D38 in Sis-EndoV is essential for catalysis, but has no role in DNA binding. Furthermore, we first revealed that residues Y41 and D189 in Sis-EndoV are involved in both DNA cleavage and DNA binding, but residues F77, H103, K156 and F161 are only responsible for DNA binding.


Asunto(s)
Desoxirribonucleasa (Dímero de Pirimidina) , Sulfolobus , Desoxirribonucleasa (Dímero de Pirimidina)/química , Desoxirribonucleasa (Dímero de Pirimidina)/genética , Desoxirribonucleasa (Dímero de Pirimidina)/metabolismo , Sulfolobus/genética , Sulfolobus/metabolismo , Reparación del ADN , Daño del ADN , ADN
11.
Nucleic Acids Res ; 48(17): 9681-9693, 2020 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-32833023

RESUMEN

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.


Asunto(s)
Proteínas Arqueales/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Reparación del ADN , Sulfolobus/genética , Regiones no Traducidas 5' , Proteínas Arqueales/metabolismo , Sitios de Unión , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Daño del ADN , Perfilación de la Expresión Génica , Regulación de la Expresión Génica Arqueal , Genoma Arqueal , Mutación , Regiones Promotoras Genéticas , Sulfolobus/crecimiento & desarrollo
12.
Nucleic Acids Res ; 48(16): 9273-9284, 2020 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-32761152

RESUMEN

Nucleic acid-binding proteins of the Sac10b family, also known as Alba, are widely distributed in Archaea. However, the physiological roles of these proteins have yet to be clarified. Here, we show that Sis10b, a member of the Sac10b family from the hyperthermophilic archaeon Sulfolobus islandicus, was active in RNA strand exchange, duplex RNA unwinding in vitro and RNA unfolding in a heterologous host cell. This protein exhibited temperature-dependent binding preference for ssRNA over dsRNA and was more efficient in RNA unwinding and RNA unfolding at elevated temperatures. Notably, alanine substitution of a highly conserved basic residue (K) at position 17 in Sis10b drastically reduced the ability of this protein to catalyse RNA strand exchange and RNA unwinding. Additionally, the preferential binding of Sis10b to ssRNA also depended on the presence of K17 or R17. Furthermore, normal growth was restored to a slow-growing Sis10b knockdown mutant by overproducing wild-type Sis10b but not by overproducing K17A in this mutant strain. Our results indicate that Sis10b is an RNA chaperone that likely functions most efficiently at temperatures optimal for the growth of S. islandicus, and K17 is essential for the chaperone activity of the protein.


Asunto(s)
Proteínas Arqueales/genética , Proteínas de Unión al ADN/genética , Chaperonas Moleculares/genética , ARN/genética , Archaea/genética , Proteínas Arqueales/química , Proteínas de Unión al ADN/química , Chaperonas Moleculares/química , Unión Proteica/genética , Pliegue del ARN/genética , ARN Bicatenario/genética , Sulfolobus/química , Sulfolobus/genética
13.
Proc Natl Acad Sci U S A ; 116(50): 25278-25286, 2019 12 10.
Artículo en Inglés | MEDLINE | ID: mdl-31767763

RESUMEN

Surface protein layers (S-layers) often form the only structural component of the archaeal cell wall and are therefore important for cell survival. S-layers have a plethora of cellular functions including maintenance of cell shape, osmotic, and mechanical stability, the formation of a semipermeable protective barrier around the cell, and cell-cell interaction, as well as surface adhesion. Despite the central importance of S-layers for archaeal life, their 3-dimensional (3D) architecture is still poorly understood. Here we present detailed 3D electron cryomicroscopy maps of archaeal S-layers from 3 different Sulfolobus strains. We were able to pinpoint the positions and determine the structure of the 2 subunits SlaA and SlaB. We also present a model describing the assembly of the mature S-layer.


Asunto(s)
Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/ultraestructura , Sulfolobus/metabolismo , Microscopía por Crioelectrón , Dimerización , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Sulfolobus/química , Sulfolobus/genética , Sulfolobus/ultraestructura
14.
Int J Mol Sci ; 23(15)2022 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-35955649

RESUMEN

Type III CRISPR-Cas systems show the target (tg)RNA-activated indiscriminate DNA cleavage and synthesis of oligoadenylates (cOA) and a secondary signal that activates downstream nuclease effectors to exert indiscriminate RNA/DNA cleavage, and both activities are regulated in a spatiotemporal fashion. In III-B Cmr systems, cognate tgRNAs activate the two Cmr2-based activities, which are then inactivated via tgRNA cleavage by Cmr4, but how Cmr4 nuclease regulates the Cmr immunization remains to be experimentally characterized. Here, we conducted mutagenesis of Cmr4 conserved amino acids in Saccharolobus islandicus, and this revealed that Cmr4α RNase-dead (dCmr4α) mutation yields cell dormancy/death. We also found that plasmid-borne expression of dCmr4α in the wild-type strain strongly reduced plasmid transformation efficiency, and deletion of CRISPR arrays in the host genome reversed the dCmr4α inhibition. Expression of dCmr4α also strongly inhibited plasmid transformation with Cmr2αHD and Cmr2αPalm mutants, but the inhibition was diminished in Cmr2αHD,Palm. Since dCmr4α-containing effectors lack spatiotemporal regulation, this allows an everlasting interaction between crRNA and cellular RNAs to occur. As a result, some cellular RNAs, which are not effective in mediating immunity due to the presence of spatiotemporal regulation, trigger autoimmunity of the Cmr-α system in the S. islandicus cells expressing dCmr4α. Together, these results pinpoint the crucial importance of tgRNA cleavage in autoimmunity avoidance and in the regulation of immunization of type III systems.


Asunto(s)
Proteínas Asociadas a CRISPR , Sulfolobus , Autoinmunidad/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , ARN/genética , División del ARN , Sulfolobus/genética
15.
Int J Mol Sci ; 23(7)2022 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-35408816

RESUMEN

The winged helix superfamily comprises a large number of structurally related nucleic acid-binding proteins. While these proteins are often shown to bind dsDNA, few are known to bind ssDNA. Here, we report the identification and characterization of Sul7s, a novel winged-helix single-stranded DNA binding protein family highly conserved in Sulfolobaceae. Sul7s from Sulfolobus islandicus binds ssDNA with an affinity approximately 15-fold higher than that for dsDNA in vitro. It prefers binding oligo(dT)30 over oligo(dC)30 or a dG-rich 30-nt oligonucleotide, and barely binds oligo(dA)30. Further, binding by Sul7s inhibits DNA strand annealing, but shows little effect on the melting temperature of DNA duplexes. The solution structure of Sul7s determined by NMR shows a winged helix-turn-helix fold, consisting of three α-helices, three ß-strands, and two short wings. It interacts with ssDNA via a large positively charged binding surface, presumably resulting in ssDNA deformation. Our results shed significant light on not only non-OB fold single-stranded DNA binding proteins in Archaea, but also the divergence of the winged-helix proteins in both function and structure during evolution.


Asunto(s)
Proteínas de Unión al ADN , Sulfolobus , Archaea/metabolismo , ADN/química , ADN de Cadena Simple , Proteínas de Unión al ADN/metabolismo , Sulfolobus/genética
16.
Int J Mol Sci ; 23(17)2022 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-36077578

RESUMEN

CRISPR-Cas systems empower prokaryotes with adaptive immunity against invasive mobile genetic elements. At the first step of CRISPR immunity adaptation, short DNA fragments from the invaders are integrated into CRISPR arrays at the leader-proximal end. To date, the mechanism of recognition of the leader-proximal end remains largely unknown. Here, in the Sulfolobus islandicus subtype I-A system, we show that mutations destroying the proximal region reduce CRISPR adaptation in vivo. We identify that a stem-loop structure is present on the leader-proximal end, and we demonstrate that Cas1 preferentially binds the stem-loop structure in vitro. Moreover, we demonstrate that the integrase activity of Cas1 is modulated by interacting with a CRISPR-associated factor Csa3a. When translocated to the CRISPR array, the Csa3a-Cas1 complex is separated by Csa3a binding to the leader-distal motif and Cas1 binding to the leader-proximal end. Mutation at the leader-distal motif reduces CRISPR adaptation efficiency, further confirming the in vivo function of leader-distal motif. Together, our results suggest a general model for binding of Cas1 protein to a leader motif and modulation of integrase activity by an accessory factor.


Asunto(s)
Proteínas Asociadas a CRISPR , Sulfolobus , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Integrasas/metabolismo , Motivos de Nucleótidos , Sulfolobus/genética , Sulfolobus/metabolismo
17.
Curr Issues Mol Biol ; 40: 231-266, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687344

RESUMEN

Over the last couple of decades there has been considerable progress in the identification and understanding of the mobile genetic elements that are exchanged between microbes in extremely acidic environments, and of the genes piggybacking on them. Numerous plasmid families, unique viruses of bizarre morphologies and lyfe cycles, as well as plasmid-virus chimeras, have been isolated from acidophiles and characterized to varying degrees. Growing evidence provided by omic-studies have shown that the mobile elements repertoire is not restricted to plasmids and viruses, but that a plethora of integrative elements ranging from miniature inverted repeat transposable elements to large integrative conjugative elements populate the genomes of acidophilic bacteria and archaea. This article reviews the diversity of elements that have been found to constitute the flexible genome of acidophiles. Special emphasis is put on the knowledge generated for Sulfolobus (archaea) and species of the bacterial genera Acidithiobacillus and Leptospirillum. Also, recent knowledge on the strategies used by acidophiles to contain deletereous exchanges while allowing innovation, and the emerging details of the molecular biology of these systems, are discussed. Major lacunae in our understanding of the mobilome of acidophilic prokaryotes and topics for further investigations are identified.


Asunto(s)
Acidithiobacillus/genética , Genoma Arqueal , Genoma Bacteriano , Sulfolobus/genética , Adaptación Fisiológica/genética , Virus de Archaea/genética , Elementos Transponibles de ADN/genética , Flujo Génico , Transferencia de Gen Horizontal , Genómica/métodos , Concentración de Iones de Hidrógeno , Filogenia , Plásmidos/genética , Sulfolobus/virología
18.
Mol Microbiol ; 113(4): 718-727, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31774609

RESUMEN

Characterizing the molecular interactions of viruses in natural microbial populations offers insights into virus-host dynamics in complex ecosystems. We identify the resistance of Sulfolobus islandicus to Sulfolobus spindle-shaped virus (SSV9) conferred by chromosomal deletions of pilin genes, pilA1 and pilA2 that are individually able to complement resistance. Mutants with deletions of both pilA1 and pilA2 or the prepilin peptidase, PibD, show the reduction in the number of pilins observed in TEM and reduced surface adherence but still adsorb SSV9. The proteinaceous outer S-layer proteins, SlaA and SlaB, are not required for adsorption nor infection demonstrating that the S-layer is not the primary receptor for SSV9 surface binding. Strains lacking both pilins are resistant to a broad panel of SSVs as well as a panel of unrelated S. islandicus rod-shaped viruses (SIRVs). Unlike SSV9, we show that pilA1 or pilA2 is required for SIRV8 adsorption. In sequenced Sulfolobus strains from around the globe, one copy of each pilA1 and pilA2 is maintained and show codon-level diversification, demonstrating their importance in nature. By characterizing the molecular interactions at the initiation of infection between S. islandicus and two different types of viruses we hope to increase the understanding of virus-host interactions in the archaeal domain.


Asunto(s)
Resistencia a la Enfermedad/genética , Proteínas Fimbrias/metabolismo , Fuselloviridae/fisiología , Interacciones Microbiota-Huesped , Rudiviridae/fisiología , Sulfolobus , Proteínas Fimbrias/genética , Fimbrias Bacterianas/metabolismo , Sulfolobus/genética , Sulfolobus/virología , Acoplamiento Viral
19.
Environ Microbiol ; 23(8): 4612-4630, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34190379

RESUMEN

Saccharolobus (formerly Sulfolobus) shibatae B12, isolated from a hot spring in Beppu, Japan in 1982, is one of the first hyperthermophilic and acidophilic archaeal species to be discovered. It serves as a natural host to the extensively studied spindle-shaped virus SSV1, a prototype of the Fuselloviridae family. Two additional Sa. shibatae strains, BEU9 and S38A, sensitive to viruses of the families Lipothrixviridae and Portogloboviridae, respectively, have been isolated more recently. However, none of the strains has been fully sequenced, limiting their utility for studies on archaeal biology and virus-host interactions. Here, we present the complete genome sequences of all three Sa. shibatae strains and explore the rich diversity of their integrated mobile genetic elements (MGE), including transposable insertion sequences, integrative and conjugative elements, plasmids, and viruses, some of which were also detected in the extrachromosomal form. Analysis of related MGEs in other Sulfolobales species and patterns of CRISPR spacer targeting revealed a complex network of MGE distributions, involving horizontal spread and relatively frequent host switching by MGEs over large phylogenetic distances, involving species of the genera Saccharolobus, Sulfurisphaera and Acidianus. Furthermore, we characterize a remarkable case of a virus-to-plasmid transition, whereby a fusellovirus has lost the genes encoding for the capsid proteins, while retaining the replication module, effectively becoming a plasmid.


Asunto(s)
Fuselloviridae , Sulfolobus , Archaea , Fuselloviridae/genética , Humanos , Filogenia , Análisis de Secuencia de ADN , Sulfolobus/genética
20.
Biochem Biophys Res Commun ; 585: 8-14, 2021 12 31.
Artículo en Inglés | MEDLINE | ID: mdl-34781059

RESUMEN

Inorganic pyrophosphatase catalyzes the conversion of pyrophosphate to phosphate and is often critical for driving reactions forward in cellular processes such as nucleic acid and protein synthesis. Commonly used methods for quantifying pyrophosphatase enzyme activity employ reacting liberated phosphate with a second molecule to produce absorbance changes or employing a second enzyme in coupled reactions to produce a product with a detectable absorbance. In this investigation, a novel [31P]-NMR spectroscopy-based assay was used to quantitatively measure the formation of phosphate and evaluate the activity of inorganic pyrophosphatase from the thermoacidophilic Crenarchaeota Sulfolobus islandicus. The enzymatic activity was directly measured via integration of the [31P] resonance associated with the phosphate product (δ = 2.1 ppm). Sulfolobus islandicus inorganic pyrophosphatase preferentially utilized Mg2+ as divalent cation and had pH and temperature optimums of 6.0 of 50 °C, respectively. The Vmax value was 850 µmol/min/mg and the Km for pyrophosphate was 1.02 mM. Sequence analysis indicates the enzyme is a Family I pyrophosphatase. Sulfolobus islandicus inorganic pyrophosphatase was shown to be inhibited by sodium fluoride with a IC50 of 2.26 mM, compared to a IC50 of 0.066 mM for yeast inorganic pyrophosphatase. These studies reveal that a [31P]-NMR spectroscopy-based assay is an effective method for analyzing catalysis by phosphate-producing enzymes.


Asunto(s)
Proteínas Arqueales/metabolismo , Pruebas de Enzimas/métodos , Pirofosfatasa Inorgánica/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Sulfolobus/enzimología , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Biocatálisis , Difosfatos/metabolismo , Concentración de Iones de Hidrógeno , Pirofosfatasa Inorgánica/genética , Cinética , Isótopos de Fósforo , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Sulfolobus/genética , Temperatura
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