Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 8 de 8
Filter
Add more filters










Database
Language
Publication year range
1.
Nat Commun ; 10(1): 4797, 2019 10 22.
Article in English | MEDLINE | ID: mdl-31641111

ABSTRACT

The S-layer is a proteinaceous surface lattice found in the cell envelope of bacteria and archaea. In most archaea, a glycosylated S-layer constitutes the sole cell wall and there is evidence that it contributes to cell shape maintenance and stress resilience. Here we use a gene-knockdown technology based on an endogenous CRISPR type III complex to gradually silence slaB, which encodes the S-layer membrane anchor in the hyperthermophilic archaeon Sulfolobus solfataricus. Silenced cells exhibit a reduced or peeled-off S-layer lattice, cell shape alterations and decreased surface glycosylation. These cells barely propagate but increase in diameter and DNA content, indicating impaired cell division; their phenotypes can be rescued through genetic complementation. Furthermore, S-layer depleted cells are less susceptible to infection with the virus SSV1. Our study highlights the usefulness of the CRISPR type III system for gene silencing in archaea, and supports that an intact S-layer is important for cell division and virus susceptibility.


Subject(s)
Archaeal Proteins/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , Sulfolobus solfataricus/cytology , Sulfolobus solfataricus/virology , Archaeal Proteins/genetics , Cell Wall/genetics , Cell Wall/metabolism , Chromosomes, Archaeal , Fuselloviridae , Gene Knockdown Techniques , Gene Silencing , Genetic Complementation Test , Glycosylation , Host-Pathogen Interactions/genetics , Sulfolobus solfataricus/genetics
2.
Nucleic Acids Res ; 44(9): 4233-42, 2016 05 19.
Article in English | MEDLINE | ID: mdl-27098036

ABSTRACT

The Sulfolobales host a unique family of crenarchaeal conjugative plasmids some of which undergo complex rearrangements intracellularly. Here we examined the conjugation cycle of pKEF9 in the recipient strain Sulfolobus islandicus REY15A. The plasmid conjugated and replicated rapidly generating high average copy numbers which led to strong growth retardation that was coincident with activation of CRISPR-Cas adaptation. Simultaneously, intracellular DNA was extensively degraded and this also occurred in a conjugated Δcas6 mutant lacking a CRISPR-Cas immune response. Furthermore, the integrated forms of pKEF9 in the donor Sulfolobus solfataricus P1 and recipient host were specifically corrupted by transposable orfB elements, indicative of a dual mechanism for inactivating free and integrated forms of the plasmid. In addition, the CRISPR locus of pKEF9 was progressively deleted when conjugated into the recipient strain. Factors influencing activation of CRISPR-Cas adaptation in the recipient strain are considered, including the first evidence for a possible priming effect in Sulfolobus The 3-Mbp genome sequence of the donor P1 strain is presented.


Subject(s)
DNA, Archaeal/genetics , Sulfolobus solfataricus/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Evolution, Molecular , Plasmids/genetics , Sulfolobus solfataricus/cytology , Sulfolobus solfataricus/growth & development
3.
BMC Mol Biol ; 15: 18, 2014 Sep 09.
Article in English | MEDLINE | ID: mdl-25200003

ABSTRACT

BACKGROUND: Reverse gyrases are DNA topoisomerases characterized by their unique DNA positive-supercoiling activity. Sulfolobus solfataricus, like most Crenarchaeota, contains two genes each encoding a reverse gyrase. We showed previously that the two genes are differently regulated according to temperature and that the corresponding purified recombinant reverse gyrases have different enzymatic characteristics. These observations suggest a specialization of functions of the two reverse gyrases. As no mutants of the TopR genes could be obtained in Sulfolobales, we used immunodetection techniques to study the function(s) of these proteins in S. solfataricus in vivo. In particular, we investigated whether one or both reverse gyrases are required for the hyperthermophilic lifestyle. RESULTS: For the first time the two reverse gyrases of S. solfataricus have been discriminated at the protein level and their respective amounts have been determined in vivo. Actively dividing S. solfataricus cells contain only small amounts of both reverse gyrases, approximately 50 TopR1 and 125 TopR2 molecules per cell at 80°C. S. solfataricus cells are resistant at 45°C for several weeks, but there is neither cell division nor replication initiation; these processes are fully restored upon a return to 80°C. TopR1 is not found after three weeks at 45°C whereas the amount of TopR2 remains constant. Enzymatic assays in vitro indicate that TopR1 is not active at 45°C but that TopR2 exhibits highly positive DNA supercoiling activity at 45°C. CONCLUSIONS: The two reverse gyrases of S. solfataricus are differently regulated, in terms of protein abundance, in vivo at 80°C and 45°C. TopR2 is present both at high and low temperatures and is therefore presumably required whether cells are dividing or not. By contrast, TopR1 is present only at high temperature where the cell division occurs, suggesting that TopR1 is required for controlling DNA topology associated with cell division activity and/or life at high temperature. Our findings in vitro that TopR1 is able to positively supercoil DNA only at high temperature, and TopR2 is active at both temperatures are consistent with them having different functions within the cells.


Subject(s)
DNA Topoisomerases, Type I/metabolism , Sulfolobus solfataricus/cytology , Sulfolobus solfataricus/enzymology , Amino Acid Sequence , DNA Topoisomerases, Type I/analysis , DNA, Superhelical/metabolism , Hot Temperature , Molecular Sequence Data , Sulfolobus solfataricus/chemistry , Sulfolobus solfataricus/physiology
4.
Biochimie ; 94(7): 1578-87, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22503705

ABSTRACT

The archaeal exosome is a protein complex involved in the degradation and the posttranscriptional tailing of RNA. The proteins Rrp41, Rrp42, Rrp4, Csl4 and DnaG are major subunits of the exosome in Sulfolobus solfataricus. In vitro, Rrp41 and Rrp42 form a catalytically active hexamer, to which an RNA-binding cap of Rrp4 and/or Csl4 is attached. Rrp4 confers strong poly(A) specificity to the exosome. The majority of Rrp41 and DnaG is detectable in the insoluble fraction and is localized at the cell periphery. The aim of this study was to analyze whether there are differences in the composition of the soluble and the insoluble exosomes. We found that the soluble exosome contains less DnaG and less Csl4 than the insoluble exosome which co-sediments with ribosomal subunits in sucrose density gradients. EF1-alpha was co-precipitated with the soluble exosome from S100 fractions using DnaG-directed antibodies, and from density gradient fractions using Rrp41-specific antibodies, strongly suggesting that EF1-alpha is an interaction partner of the soluble exosome. Furthermore, Csl4 was co-immunoprecipitated with the exosome using Rrp4-specific antibodies and vice versa, demonstrating the presence of heteromeric RNA-binding caps in vivo. To address the mechanism for poly(A) recognition by Rrp4, an exosome with an RNA-binding cap composed of truncated Rrp4 lacking the KH domain was reconstituted and analyzed. Although the deletion of the KH domain negatively influenced the degradation activity of the exosome, the poly(A) specificity was retained, showing that the KH domain is dispensable for the strong poly(A) preference of Rrp4.


Subject(s)
Exosomes/metabolism , RNA, Bacterial/metabolism , Sulfolobus solfataricus/cytology , Sulfolobus solfataricus/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Exosomes/chemistry , Poly A/metabolism , Protein Structure, Tertiary , RNA Stability , RNA, Bacterial/chemistry , Solubility , Substrate Specificity , Sulfolobus solfataricus/genetics
5.
J Biol Inorg Chem ; 15(5): 729-36, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20221652

ABSTRACT

DNA-binding proteins from nutrient-starved cells (DPS) protect cells from oxidative stress by removing H(2)O(2) and iron. A new class of DPS-like proteins has recently been identified, with DPS-like protein from Sulfolobus solfataricus (SsDPS) being the best characterized to date. SsDPS protects cells from oxidative stress and is upregulated in response to H(2)O(2) but also in response to iron depletion. The ferroxidase active site of SsDPS is structurally similar to the active sites of manganese catalase and rat liver arginase. The present work shows that the ferroxidase center in SsDPS binds two Mn(2+) ions with K (D) = (1/K (1) K (2))(1/2) = 48(3) microM. The binding constant of the second Mn(2+) is significantly higher than that of the first, inducing dinuclear Mn(II) cluster formation for all but the lowest concentrations of added Mn(2+). In competition experiments, equimolar amounts of Fe(2+) were unable to displace the bound manganese. EPR spectroscopy of the Mn(2) (2+) cluster showed signals comparable to those of other characterized dimanganese clusters. The exchange coupling for the cluster was determined, J = -1.4(3) cm(-1) (H = -2JS (1) S (2)), and is within the range expected for a mu(1,1)-carboxylato bridge between the manganese ions. Manganese-bound SsDPS showed catalase activity at a rate 10-100 times slower than for manganese catalases. EPR spectra of SsDPS after addition of H(2)O(2) showed the appearance of an intermediate in the reaction with H(2)O(2).


Subject(s)
Catalase/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Manganese/metabolism , Stress, Physiological , Sulfolobus solfataricus/cytology , Sulfolobus solfataricus/metabolism , Catalase/chemistry , Catalytic Domain , Electron Spin Resonance Spectroscopy , Enzyme Activation , Hydrogen Peroxide/chemistry , Hydrogen Peroxide/metabolism , Iron/chemistry , Molecular Conformation , Organometallic Compounds/chemistry , Organometallic Compounds/metabolism , Oxidative Stress , Sulfolobus solfataricus/enzymology
6.
Biochem Soc Trans ; 37(Pt 1): 36-41, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19143598

ABSTRACT

Mechanisms involved in DNA repair and genome maintenance are essential for all organisms on Earth and have been studied intensively in bacteria and eukaryotes. Their analysis in extremely thermophilic archaea offers the opportunity to discover strategies for maintaining genome integrity of the relatively little explored third domain of life, thereby shedding light on the diversity and evolution of these central and important systems. These studies might also reveal special adaptations that are essential for life at high temperature. A number of investigations of the hyperthermophilic and acidophilic crenarchaeote Sulfolobus solfataricus have been performed in recent years. Mostly, the reactions to DNA damage caused by UV light have been analysed. Whole-genome transcriptomics have demonstrated that a UV-specific response in S. solfataricus does not involve the transcriptional induction of DNA-repair genes and it is therefore different from the well-known SOS response in bacteria. Nevertheless, the UV response in S. solfataricus is impressively complex and involves many different levels of action, some of which have been elucidated and shed light on novel strategies for DNA repair, while others involve proteins of unknown function whose actions in the cell remain to be elucidated. The present review summarizes and discusses recent investigations on the UV response of S. solfataricus on both the molecular biological and the cellular levels.


Subject(s)
DNA Damage , Models, Biological , Sulfolobus solfataricus/genetics , Sulfolobus solfataricus/radiation effects , Ultraviolet Rays , DNA Repair/genetics , Gene Expression Profiling , Sulfolobus solfataricus/cytology
7.
J Bacteriol ; 189(23): 8708-18, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17905990

ABSTRACT

In order to characterize the genome-wide transcriptional response of the hyperthermophilic, aerobic crenarchaeote Sulfolobus solfataricus to UV damage, we used high-density DNA microarrays which covered 3,368 genetic features encoded on the host genome, as well as the genes of several extrachromosomal genetic elements. While no significant up-regulation of genes potentially involved in direct DNA damage reversal was observed, a specific transcriptional UV response involving 55 genes could be dissected. Although flow cytometry showed only modest perturbation of the cell cycle, strong modulation of the transcript levels of the Cdc6 replication initiator genes was observed. Up-regulation of an operon encoding Mre11 and Rad50 homologs pointed to induction of recombinational repair. Consistent with this, DNA double-strand breaks were observed between 2 and 8 h after UV treatment, possibly resulting from replication fork collapse at damaged DNA sites. The strong transcriptional induction of genes which potentially encode functions for pilus formation suggested that conjugational activity might lead to enhanced exchange of genetic material. In support of this, a statistical microscopic analysis demonstrated that large cell aggregates formed upon UV exposure. Together, this provided supporting evidence to a link between recombinational repair and conjugation events.


Subject(s)
DNA Damage/radiation effects , DNA Repair/radiation effects , Gene Expression Regulation, Bacterial/radiation effects , Sulfolobus solfataricus/genetics , Sulfolobus solfataricus/radiation effects , Ultraviolet Rays , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Cycle/radiation effects , DNA Damage/genetics , DNA Repair/genetics , DNA, Bacterial/radiation effects , Dose-Response Relationship, Radiation , Sulfolobus solfataricus/cytology , Time Factors , Transcription, Genetic/radiation effects
8.
Mol Microbiol ; 62(4): 1076-89, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17078816

ABSTRACT

The eukaryotic exosome is a protein complex with essential functions in processing and degradation of RNA. Exosome-like complexes were recently found in Archaea. Here we characterize the exosome of Sulfolobus solfataricus. Two exosome fractions can be discriminated by density gradient centrifugation. We show that the Cdc48 protein is associated with the exosome from the 30S-50S fraction but not with the exosome of the 11.3S fraction. While only some complexes contain Cdc48, the archaeal DnaG-like protein was found to be a core exosome subunit in addition to Rrp4, Rrp41, Rrp42 and Csl4. Assays with depleted extracts revealed that the exosome is responsible for major ribonucleolytic activity in S. solfataricus. Various complexes consisting of the Rrp41-Rrp42 hexameric ring and Rrp4, Csl4 and DnaG were reconstituted. Dependent on their composition, different complexes showed variations in RNase activity indicating functional interdependence of the subunits. The catalytic activity of these complexes and of the native exosome can be ascribed to the Rrp41-Rrp42 ring, which degrades RNA phosphorolytically. Rrp4 and Csl4 do not exhibit any hydrolytic RNase activity, either when assayed alone or in context of the complex, but influence the activity of the archaeal exosome.


Subject(s)
Archaeal Proteins/chemistry , Multiprotein Complexes/chemistry , Sulfolobus solfataricus/chemistry , Adenosine Triphosphatases , Archaeal Proteins/metabolism , Cell Cycle Proteins/chemistry , DNA Primase/chemistry , DNA Primase/metabolism , Exoribonucleases/chemistry , Exoribonucleases/metabolism , Multiprotein Complexes/metabolism , RNA/metabolism , RNA Stability , Sulfolobus solfataricus/cytology , Sulfolobus solfataricus/enzymology , Valosin Containing Protein
SELECTION OF CITATIONS
SEARCH DETAIL
...