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Daniel Kockelkorn - One of the best experts on this subject based on the ideXlab platform.
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Identification of missing genes and enzymes for autotrophic carbon fixation in crenarchaeota.
Journal of bacteriology, 2010Co-Authors: W. H. Ramos-vera, M. Weiss, Eric Strittmatter, Daniel KockelkornAbstract:Two autotrophic carbon fixation cycles have been identified in Crenarchaeota. The dicarboxylate/4-hydroxybutyrate cycle functions in anaerobic or microaerobic autotrophic members of the Thermoproteales and Desulfurococcales. The 3-hydroxypropionate/4-hydroxybutyrate cycle occurs in aerobic autotrophic Sulfolobales; a similar cycle may operate in autotrophic aerobic marine Crenarchaeota. Both cycles form succinyl-coenzyme A (CoA) from acetyl-CoA and two molecules of inorganic carbon, but they use different means. Both cycles have in common the (re)generation of acetyl-CoA from succinyl-CoA via identical intermediates. Here, we identified several missing enzymes/genes involved in the seven-step conversion of succinyl-CoA to two molecules of acetyl-CoA in Thermoproteus neutrophilus (Thermoproteales), Ignicoccus hospitalis (Desulfurococcales), and Metallosphaera sedula (Sulfolobales). The identified enzymes/genes include succinyl-CoA reductase, succinic semialdehyde reductase, 4-hydroxybutyrate-CoA ligase, bifunctional crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase, and beta-ketothiolase. 4-Hydroxybutyryl-CoA dehydratase, which catalyzes a mechanistically intriguing elimination of water, is well conserved and rightly can be considered the key enzyme of these two cycles. In contrast, several of the other enzymes evolved from quite different sources, making functional predictions based solely on genome interpretation difficult, if not questionable.
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malonic semialdehyde reductase succinic semialdehyde reductase and succinyl coenzyme a reductase from metallosphaera sedula enzymes of the autotrophic 3 hydroxypropionate 4 hydroxybutyrate cycle in Sulfolobales
Journal of Bacteriology, 2009Co-Authors: Daniel KockelkornAbstract:A 3-hydroxypropionate/4-hydroxybutyrate cycle operates during autotrophic CO2 fixation in various members of the Crenarchaea. In this cycle, as determined using Metallosphaera sedula, malonyl-coenzyme A (malonyl-CoA) and succinyl-CoA are reductively converted via their semialdehydes to the corresponding alcohols 3-hydroxypropionate and 4-hydroxybutyrate. Here three missing oxidoreductases of this cycle were purified from M. sedula and studied. Malonic semialdehyde reductase, a member of the 3-hydroxyacyl-CoA dehydrogenase family, reduces malonic semialdehyde with NADPH to 3-hydroxypropionate. The latter compound is converted via propionyl-CoA to succinyl-CoA. Succinyl-CoA reduction to succinic semialdehyde is catalyzed by malonyl-CoA/succinyl-CoA reductase, a promiscuous NADPH-dependent enzyme that is a paralogue of aspartate semialdehyde dehydrogenase. Succinic semialdehyde is then reduced with NADPH to 4-hydroxybutyrate by succinic semialdehyde reductase, an enzyme belonging to the Zn-dependent alcohol dehydrogenase family. Genes highly similar to the Metallosphaera genes were found in other members of the Sulfolobales. Only distantly related genes were found in the genomes of autotrophic marine Crenarchaeota that may use a similar cycle in autotrophic carbon fixation.
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3 hydroxypropionyl coenzyme a dehydratase and acryloyl coenzyme a reductase enzymes of the autotrophic 3 hydroxypropionate 4 hydroxybutyrate cycle in the Sulfolobales
Journal of Bacteriology, 2009Co-Authors: Robin Teufel, Daniel Kockelkorn, Johannes W Kung, Birgit E Alber, Georg FuchsAbstract:A 3-hydroxypropionate/4-hydroxybutyrate cycle operates in autotrophic CO2 fixation in various Crenarchaea, as studied in some detail in Metallosphaera sedula. This cycle and the autotrophic 3-hydroxypropionate cycle in Chloroflexus aurantiacus have in common the conversion of acetyl-coenzyme A (CoA) and two bicarbonates via 3-hydroxypropionate to succinyl-CoA. Both cycles require the reductive conversion of 3-hydroxypropionate to propionyl-CoA. In M. sedula the reaction sequence is catalyzed by three enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the CoA- and MgATP-dependent formation of 3-hydroxypropionyl-CoA. The next two enzymes were purified from M. sedula or Sulfolobus tokodaii and studied. 3-Hydroxypropionyl-CoA dehydratase, a member of the enoyl-CoA hydratase family, eliminates water from 3-hydroxypropionyl-CoA to form acryloyl-CoA. Acryloyl-CoA reductase, a member of the zinc-containing alcohol dehydrogenase family, reduces acryloyl-CoA with NADPH to propionyl-CoA. Genes highly similar to the Metallosphaera CoA synthetase, dehydratase, and reductase genes were found in autotrophic members of the Sulfolobales. The encoded enzymes are only distantly related to the respective three enzyme domains of propionyl-CoA synthase from C. aurantiacus, where this trifunctional enzyme catalyzes all three reactions. This indicates that the autotrophic carbon fixation cycles in Chloroflexus and in the Sulfolobales evolved independently and that different genes/enzymes have been recruited in the two lineages that catalyze the same kinds of reactions.
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a 3 hydroxypropionate 4 hydroxybutyrate autotrophic carbon dioxide assimilation pathway in archaea
Science, 2007Co-Authors: Ivan A. Berg, Daniel Kockelkorn, Wolfgang Buckel, Georg FuchsAbstract:The assimilation of carbon dioxide (CO2) into organic material is quantitatively the most important biosynthetic process. We discovered that an autotrophic member of the archaeal order Sulfolobales, Metallosphaera sedula, fixed CO2 with acetyl–coenzyme A (acetyl-CoA)/propionyl-CoA carboxylase as the key carboxylating enzyme. In this system, one acetyl-CoA and two bicarbonate molecules were reductively converted via 3-hydroxypropionate to succinyl-CoA. This intermediate was reduced to 4-hydroxybutyrate and converted into two acetyl-CoA molecules via 4-hydroxybutyryl-CoA dehydratase. The key genes of this pathway were found not only in Metallosphaera but also in Sulfolobus, Archaeoglobus, and Cenarchaeum species. Moreover, the Global Ocean Sampling database contains half as many 4-hydroxybutyryl-CoA dehydratase sequences as compared with those found for another key photosynthetic CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase-oxygenase. This indicates the importance of this enzyme in global carbon cycling.
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A 3-hydroxypropionate/4-hydroxybutyrate autotrophic carbon dioxide assimilation pathway in archaea. Science 318:1782–1786
2007Co-Authors: Ivan A. Berg, Daniel Kockelkorn, Wolfgang Buckel, Georg FuchsAbstract:We proposed that the 3-hydroxypropionate/4-hydroxybutyrate cycle might be important in global carbon cycling based on the abundance of related autotrophic Crenarchaea in the ocean and the high number of gene sequences for a key enzyme of the cycle. Here, we counter the specific criticisms raised by Ettema and Andersson. We recently elucidated a previously un-known autotrophic CO2 assimilation path-way termed the 3-hydroxypropionate/ 4-hydroxybutyrate cycle, which operates in the extreme thermophile Metallosphaera sedula (Crenarchaea) (1). This pathway is likely to exist in other autotrophic members of the order Sulfolobales and possibly in some mesophilic Crenarchaea. We proposed that the pathway might be important in global carbon cycling based on the abundance of related autotrophic Crenarchaea in the ocean (2, 3) and the high number of gene sequences for a key enzyme o
Roger A Garrett - One of the best experts on this subject based on the ideXlab platform.
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CRISPR-based immune systems of the Sulfolobales: complexity and diversity
2020Co-Authors: Roger A Garrett, Chandra S. Kenchappa, Susanne Erdmann, Shiraz A Shah, Gisle Vestergaard, Ling Deng, Soley Gudbergsdottir, Qunxin SheAbstract:Abstract CRISPR (cluster of regularly interspaced palindromic repeats)/Cas and CRISPR/Cmr systems of Sulfolobus, targeting DNA and RNA respectively of invading viruses or plasmids are complex and diverse. We address their classification and functional diversity, and the wide sequence diversity of RAMP (repeat-associated mysterious protein)-motif containing proteins encoded in Cmr modules. Factors influencing maintenance of partially impaired CRISPR-based systems are discussed. The capacity for whole CRISPR transcripts to be generated despite the uptake of transcription signals within spacer sequences is considered. Targeting of protospacer regions of invading elements by Cas protein-crRNA (CRISPR RNA) complexes exhibit relatively low sequence stringency, but the integrity of protospacer-associated motifs appears to be important. Different mechanisms for circumventing or inactivating the immune systems are presented
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CRISPR: Evolution, Mechanisms and Infection 1449 SMV1 virus-induced CRISPR spacer acquisition from the conjugative plasmid pMGB1 in
2016Co-Authors: Sulfolobus Solfataricus P, Shiraz A Shah, Susanne Erdmann, Roger A GarrettAbstract:Organisms of the crenarchaeal order Sulfolobales carry complex CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune systems. These systems are modular and show extensive structural and functional diversity, especially in their interference complexes. The primary targets are an exceptional range of diverse viruses, many of which propagate stably within cells and follow lytic life cycles without producing cell lysis. These properties are consistent with the difficulty of activating CRISPR spacer uptake in the laboratory, but appear to conflict with the high complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2 that was co-infected with the virus SMV1 (Sulfolobus monocaudavirus 1). Although we reaffirm that protospacer selection is essentially a random process with respect to the pMGB1 genome, we identified single spacer sequences specific for each of CRISPR loci C, D and E that, exceptionally, occurred in many sequenced clones. Moreover, the same sequence was reproducibly acquired for a given locus in independent experiments, consistent with it being the first protospacer to be selected. There was also a small protospacer bias (1.6:1) to the antisense strand of protein genes. In addition, new experiments demonstrated that spacer acquisition in the previously inactive CRISPR locus A could be induced on freeze– thawing of the infected cells, suggesting that environmental stress can facilitate activation. Coincidentally with spacer acquisition, a mobile OrfB element was deleted from pMGB1, suggesting that interplay can occur between spacer acquisition and transposition. Viruses and conjugative plasmids of the Sulfolobales All known viruses infecting members of the Sulfolobales exhibit dsDNA genomes, either linear for the Lipothrixvi-ridae, Rudiviridae and Ampullaviridae or circular for th
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Genome sequence of a novel archaeal fusellovirus assembled from the metagenome of a mexican hot spring.
Genome announcements, 2013Co-Authors: Luis E. Servín-garcidueñas, Roger A Garrett, Xu Peng, Esperanza Martínez-romeroAbstract:The consensus genome sequence of a new member of the family Fuselloviridae designated as SMF1 (Sulfolobales Mexican fusellovirus 1) is presented. The complete circular genome was recovered from a metagenomic study of a Mexican hot spring. SMF1 exhibits an exceptional coding strand bias and a reduced set of fuselloviral core genes.
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genome sequence of a novel archaeal rudivirus recovered from a mexican hot spring
Genome Announcements, 2013Co-Authors: Luis E Servingarciduenas, Roger A Garrett, Xu Peng, Esperanza MartinezromeroAbstract:ABSTRACT We report the consensus genome sequence of a novel GC-rich rudivirus, designated SMR1 ( Sulfolobales Mexican rudivirus 1), assembled from a high-throughput sequenced environmental sample from a hot spring in Los Azufres National Park in western Mexico.
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Solution properties of the archaeal CRISPR DNA repeat-binding homeodomain protein Cbp2
Nucleic acids research, 2013Co-Authors: Chandra S. Kenchappa, Roger A Garrett, Pétur O. Heidarsson, Birthe B. Kragelund, Flemming M. PoulsenAbstract:Clustered regularly interspaced short palindromic repeats (CRISPR) form the basis of diverse adaptive immune systems directed primarily against invading genetic elements of archaea and bacteria. Cbp1 of the crenarchaeal thermoacidophilic order Sulfolobales, carrying three imperfect repeats, binds specifically to CRISPR DNA repeats and has been implicated in facilitating production of long transcripts from CRISPR loci. Here, a second related class of CRISPR DNA repeat-binding protein, denoted Cbp2, is characterized that contains two imperfect repeats and is found amongst members of the crenarchaeal thermoneutrophilic order Desulfurococcales. DNA repeat-binding properties of the Hyperthermus butylicus protein Cbp2Hb were characterized and its three-dimensional structure was determined by NMR spectroscopy. The two repeats generate helix-turn-helix structures separated by a basic linker that is implicated in facilitating high affinity DNA binding of Cbp2 by tethering the two domains. Structural studies on mutant proteins provide support for Cys7 and Cys28 enhancing high thermal stability of Cbp2Hb through disulphide bridge formation. Consistent with their proposed CRISPR transcriptional regulatory role, Cbp2Hb and, by inference, other Cbp1 and Cbp2 proteins are closely related in structure to homeodomain proteins with linked helix-turn-helix (HTH) domains, in particular the paired domain Pax and Myb family proteins that are involved in eukaryal transcriptional regulation.
Shiraz A Shah - One of the best experts on this subject based on the ideXlab platform.
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CRISPR-based immune systems of the Sulfolobales: complexity and diversity
2020Co-Authors: Roger A Garrett, Chandra S. Kenchappa, Susanne Erdmann, Shiraz A Shah, Gisle Vestergaard, Ling Deng, Soley Gudbergsdottir, Qunxin SheAbstract:Abstract CRISPR (cluster of regularly interspaced palindromic repeats)/Cas and CRISPR/Cmr systems of Sulfolobus, targeting DNA and RNA respectively of invading viruses or plasmids are complex and diverse. We address their classification and functional diversity, and the wide sequence diversity of RAMP (repeat-associated mysterious protein)-motif containing proteins encoded in Cmr modules. Factors influencing maintenance of partially impaired CRISPR-based systems are discussed. The capacity for whole CRISPR transcripts to be generated despite the uptake of transcription signals within spacer sequences is considered. Targeting of protospacer regions of invading elements by Cas protein-crRNA (CRISPR RNA) complexes exhibit relatively low sequence stringency, but the integrity of protospacer-associated motifs appears to be important. Different mechanisms for circumventing or inactivating the immune systems are presented
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CRISPR: Evolution, Mechanisms and Infection 1449 SMV1 virus-induced CRISPR spacer acquisition from the conjugative plasmid pMGB1 in
2016Co-Authors: Sulfolobus Solfataricus P, Shiraz A Shah, Susanne Erdmann, Roger A GarrettAbstract:Organisms of the crenarchaeal order Sulfolobales carry complex CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune systems. These systems are modular and show extensive structural and functional diversity, especially in their interference complexes. The primary targets are an exceptional range of diverse viruses, many of which propagate stably within cells and follow lytic life cycles without producing cell lysis. These properties are consistent with the difficulty of activating CRISPR spacer uptake in the laboratory, but appear to conflict with the high complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2 that was co-infected with the virus SMV1 (Sulfolobus monocaudavirus 1). Although we reaffirm that protospacer selection is essentially a random process with respect to the pMGB1 genome, we identified single spacer sequences specific for each of CRISPR loci C, D and E that, exceptionally, occurred in many sequenced clones. Moreover, the same sequence was reproducibly acquired for a given locus in independent experiments, consistent with it being the first protospacer to be selected. There was also a small protospacer bias (1.6:1) to the antisense strand of protein genes. In addition, new experiments demonstrated that spacer acquisition in the previously inactive CRISPR locus A could be induced on freeze– thawing of the infected cells, suggesting that environmental stress can facilitate activation. Coincidentally with spacer acquisition, a mobile OrfB element was deleted from pMGB1, suggesting that interplay can occur between spacer acquisition and transposition. Viruses and conjugative plasmids of the Sulfolobales All known viruses infecting members of the Sulfolobales exhibit dsDNA genomes, either linear for the Lipothrixvi-ridae, Rudiviridae and Ampullaviridae or circular for th
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smv1 virus induced crispr spacer acquisition from the conjugative plasmid pmgb1 in sulfolobus solfataricus p2
Biochemical Society Transactions, 2013Co-Authors: Susanne Erdmann, Shiraz A ShahAbstract:Organisms of the crenarchaeal order Sulfolobales carry complex CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune systems. These systems are modular and show extensive structural and functional diversity, especially in their interference complexes. The primary targets are an exceptional range of diverse viruses, many of which propagate stably within cells and follow lytic life cycles without producing cell lysis. These properties are consistent with the difficulty of activating CRISPR spacer uptake in the laboratory, but appear to conflict with the high complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2 that was co-infected with the virus SMV1 (Sulfolobus monocaudavirus 1). Although we reaffirm that protospacer selection is essentially a random process with respect to the pMGB1 genome, we identified single spacer sequences specific for each of CRISPR loci C, D and E that, exceptionally, occurred in many sequenced clones. Moreover, the same sequence was reproducibly acquired for a given locus in independent experiments, consistent with it being the first protospacer to be selected. There was also a small protospacer bias (1.6:1) to the antisense strand of protein genes. In addition, new experiments demonstrated that spacer acquisition in the previously inactive CRISPR locus A could be induced on freeze–thawing of the infected cells, suggesting that environmental stress can facilitate activation. Coincidentally with spacer acquisition, a mobile OrfB element was deleted from pMGB1, suggesting that interplay can occur between spacer acquisition and transposition.
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archaeal type ii toxin antitoxins
2013Co-Authors: Shiraz A Shah, Roger A GarrettAbstract:A few of the bacterial type II TA systems, primarily those involved in translational inhibition, occur widely throughout the archaeal domain. Using a bioinformatic approach, the frequency and distribution of these diverse TA loci were examined within completed genomes of 124 archaea that are distributed fairly evenly throughout the major archaeal phyla. Results for the frequency and diversity of TA loci are summarised for archaea isolated from environmental niches generally characterised by extreme conditions including high temperature, high salt concentrations, high pressures, extremes of pH or strictly anaerobic conditions. No clear correlations were found between the number of TA loci present and either the genome size or particular environmental conditions. Multiple TA loci tend to be concentrated in variable genomic regions where the occurrence of intra- or inter-genomic gene transfer are most prevalent. For members of the Sulfolobales which are uniformly rich in TA loci, a case is made for some TA systems facilitating maintenance of important genomic regions.
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crispr families of the crenarchaeal genus sulfolobus bidirectional transcription and dynamic properties
Molecular Microbiology, 2009Co-Authors: Reidun Lillestol, Hien Phan, Kim Brügger, Shiraz A Shah, Peter Redder, Jan Christiansen, Roger A GarrettAbstract:Summary Clusters of regularly interspaced short palindromic repeats (CRISPRs) of Sulfolobus fall into three main families based on their repeats, leader regions, associated cas genes and putative recognition sequences on viruses and plasmids. Spacer sequence matches to different viruses and plasmids of the Sulfolobales revealed some bias particularly for family III CRISPRs. Transcription occurs on both strands of the five repeat-clusters of Sulfolobus acidocaldarius and a repeat-cluster of the conjugative plasmid pKEF9. Leader strand transcripts cover whole repeat-clusters and are processed mainly from the 3′-end, within repeats, yielding heterogeneous 40–45 nt spacer RNAs. Processing of the pKEF9 leader transcript occurred partially in spacers, and was incomplete, probably reflecting defective repeat recognition by host enzymes. A similar level of transcripts was generated from complementary strands of each chromosomal repeat-cluster and they were processed to yield discrete ∼55 nt spacer RNAs. Analysis of the partially identical repeat-clusters of Sulfolobus solfataricus strains P1 and P2 revealed that spacer-repeat units are added upstream only when a leader and certain cas genes are linked. Downstream ends of the repeat-clusters are conserved such that deletions and recombination events occur internally.
Elise B Wilkes - One of the best experts on this subject based on the ideXlab platform.
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co2 dependent carbon isotope fractionation in archaea part i modeling the 3hp 4hb pathway
Geochimica et Cosmochimica Acta, 2019Co-Authors: Ann Pearson, Sarah J Hurley, Felix J Elling, Elise B WilkesAbstract:The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) pathway of carbon fixation is found in thermophilic Crenarchaeota of the order Sulfolobales and in aerobic, ammonia-oxidizing Thaumarchaeota. Unlike all other known autotrophic carbon metabolisms, this pathway exclusively uses HCO_3- rather than CO_2 as the substrate for carbon fixation. Biomass produced by the 3HB/4HP pathway is relatively ^(13)C-enriched compared to biomass fixed by other autotrophic pathways, with total biosynthetic isotope effects (e_(Ar)) of ca. 3‰ in the Sulfolobales and ca. 20‰ in the Thaumarchaeota. Explanations for the difference between these values usually invoke the dual effects of thermophily and growth at low pH (low [HCO_3-]) for the former group vs. mesophily and growth at pH > 7 (high [HCO_3-]) for the latter group. Here we examine the model taxa Metallosphaera sedula and Nitrosopumilus maritimususing an isotope flux-balance model to argue that the primary cause of different e_(Ar) values more likely is the presence of carbonic anhydrase in M. sedula and its corresponding absence in N. maritimus. The results suggest that the pool of HCO_3-inside N. maritimus is out of isotopic equilibrium with CO_2 and that the organism imports < 10% HCO_3- from the extracellular environment. If correct and generalizable, the aerobic, ammonia-oxidizing marine Thaumarchaeota are dependent on passive CO_2 uptake and a slow rate of intracellular conversion to HCO_3-. Values of e_(Ar) should therefore vary in response to growth rate (μ) and CO_2 availability, analogous to eukaryotic algae, but in the opposite direction: e_(Ar) becomes smaller as [CO_(2(aq))] increases and/or μ decreases. Such an idea represents a testable hypothesis, both in the laboratory and in natural systems. Sensitivity to μ and CO_2 implies that measurements of e_(Ar) may hold promise as a pCO_2 paleobarometer.
Christa Schleper - One of the best experts on this subject based on the ideXlab platform.
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comparative crispr type iii based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing
RNA Biology, 2021Co-Authors: Isabelle Anna Zink, Thomas Fouqueau, Gabriel Tarrason Risa, Finn Werner, Buzz Baum, Udo Blasi, Christa SchleperAbstract:CRISPR type III systems, which are abundantly found in archaea, recognize and degrade RNA in their specific response to invading nucleic acids. Therefore, these systems can be harnessed for gene knockdown technologies even in hyperthermophilic archaea to study essential genes. We show here the broader usability of this posttranscriptional silencing technology by expanding the application to further essential genes and systematically analysing and comparing silencing thresholds and escape mutants. Synthetic guide RNAs expressed from miniCRISPR cassettes were used to silence genes involved in cell division (cdvA), transcription (rpo8), and RNA metabolism (smAP2) of the two crenarchaeal model organisms Saccharolobus solfataricus and Sulfolobus acidocaldarius. Results were systematically analysed together with those obtained from earlier experiments of cell wall biogenesis (slaB) and translation (aif5A). Comparison of over 100 individual transformants revealed gene-specific silencing maxima ranging between 40 and 75%, which induced specific knockdown phenotypes leading to growth retardation. Exceedance of this threshold by strong miniCRISPR constructs was not tolerated and led to specific mutation of the silencing miniCRISPR array and phenotypical reversion of cultures. In two thirds of sequenced reverted cultures, the targeting spacers were found to be precisely excised from the miniCRISPR array, indicating a still hypothetical, but highly active recombination system acting on the dynamics of CRISPR spacer arrays. Our results indicate that CRISPR type III - based silencing is a broadly applicable tool to study in vivo functions of essential genes in Sulfolobales which underlies a specific mechanism to avoid malignant silencing overdose.
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high spontaneous mutation rate in the hyperthermophilic archaeon sulfolobus solfataricus is mediated by transposable elements
Journal of Bacteriology, 2000Co-Authors: Erika Martusewitsch, Christoph Wilhelm Sensen, Christa SchleperAbstract:We have isolated uracil-auxotrophic mutants of the hyperthermophilic archaeon Sulfolobus solfataricus in order to explore the genomic stability and mutational frequencies of this organism and to identify complementable recipients for a selectable genetic transformation system. Positive selection of spontaneous mutants resistant to 5-fluoroorotate yielded uracil auxotrophs with frequencies of between 10−4 and 10−5 per sensitive, viable cell. Four different, nonhomologous insertion sequences (ISs) were identified at different positions within the chromosomal pyrEF locus of these mutants. They ranged in size from 1,058 to 1,439 bp and possessed properties typical of known transposable elements, i.e., terminal inverted repeats, flanking duplicated target sequences, and putative transposase genes encoding motifs that are indicative of the IS4-IS5 IS element families. Between 12 and 25 copies of each IS element were found in chromosomal DNAs by Southern analyses. While characteristic fingerprint patterns created by IS element-specific probes were observed with genomic DNA of different S. solfataricus strains, no homologous sequences were identified in DNA of other well-characterized strains of the order Sulfolobales.
