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  • 1.
    Amlinger, Lina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hoekzema, Mirthe
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 10392Article in journal (Refereed)
    Abstract [en]

    CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

  • 2. Andersson, Anders F.
    et al.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Eriksson, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Rosenlund, Magnus
    Bernander, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Nilsson, Peter
    Global analysis of mRNA stability in the archaeon Sulfolobus2006In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 7, no 10, p. R99-Article in journal (Refereed)
    Abstract [en]

    Background: Transcript half-lives differ between organisms, and between groups of genes within the same organism. The mechanisms underlying these differences are not clear, nor are the biochemical properties that determine the stability of a transcript. To address these issues, genome-wide mRNA decay studies have been conducted in eukaryotes and bacteria. In contrast, relatively little is known about RNA stability in the third domain of life, Archaea. Here, we present a microarray-based analysis of mRNA half-lives in the hyperthermophilic crenarchaea Sulfolobus solfatoricus and Sulfolobus acidocaldarius, constituting the first genome-wide study of RNA decay in archaea. Results: The two transcriptomes displayed similar half-life distributions, with medians of about five minutes. Growth-related genes, such as those involved in transcription, translation and energy production, were over-represented among unstable transcripts, whereas uncharacterized genes were over-represented among the most stable. Half-life was negatively correlated with transcript abundance and, unlike the situation in other organisms, also negatively correlated with transcript length. Conclusion: The mRNA half-life distribution of Sulfolobus species is similar to those of much faster growing bacteria, contrasting with the earlier observation that median mRNA half-life is proportional to the minimal length of the cell cycle. Instead, short half-lives may be a general feature of prokaryotic transcriptomes, possibly related to the absence of a nucleus and/or more limited post-transcriptional regulatory mechanisms. The pattern of growth-related transcripts being among the least stable in Sulfolobus may also indicate that the short half-lives reflect a necessity to rapidly reprogram gene expression upon sudden changes in environmental conditions.

  • 3.
    Andersson, Anders F.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Evolution. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Pelve, Erik A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Lindeberg, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Nilsson, Peter
    Bernander, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Replication-biased genome organisation in the crenarchaeon Sulfolobus2010In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 11, p. 454-Article in journal (Refereed)
    Abstract [en]

    Background: Species of the crenarchaeon Sulfolobus harbour three replication origins in their single circular chromosome that are synchronously initiated during replication. Results: We demonstrate that global gene expression in two Sulfolobus species is highly biased, such that early replicating genome regions are more highly expressed at all three origins. The bias by far exceeds what would be anticipated by gene dosage effects alone. In addition, early replicating regions are denser in archaeal core genes (enriched in essential functions), display lower intergenic distances, and are devoid of mobile genetic elements. Conclusion: The strong replication-biased structuring of the Sulfolobus chromosome implies that the multiple replication origins serve purposes other than simply shortening the time required for replication. The higher-level chromosomal organisation could be of importance for minimizing the impact of DNA damage, and may also be linked to transcriptional regulation.

  • 4.
    Bernander, Rolf
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Ettema, Thijs J. G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Comparative and functional analysis of the archaeal cell cycle2010In: Cell Cycle, ISSN 1538-4101, E-ISSN 1551-4005, Vol. 9, no 4, p. 795-806Article in journal (Refereed)
    Abstract [en]

    The temporal and spatial organization of the chromosome replication, genome segregation and cell division processes is less well understood in species belonging to the Archaea, than in those from the Bacteria and Eukarya domains. Novel insights into the regulation and key components of the Sulfolobus acidocaldarius cell cycle have been obtained through genome-wide analysis of cell cycle-specific gene expression, followed by cloning and characterization of gene products expressed at different cell cycle stages. Here, the results of the transcript profiling are further explored, and potential key players in archaeal cell cycle progression are highlighted in an evolutionary context, by comparing gene expression patterns and gene conservation between three selected microbial species from different domains of life. We draw attention to novel putative nucleases and helicases implicated in DNA replication, recombination and repair, as well as to potential genome segregation factors. Focus is also placed upon regulatory features, including transcription factors and protein kinases inferred to be involved in the execution of specific cell cycle stages, and regulation through metabolic coupling is discussed.

  • 5. Brouns, Stan J. J.
    et al.
    Walther, Jasper
    Snijders, Ambrosius P. L.
    van de Werken, Harmen J. G.
    Willemen, Hanneke L. D. M.
    Worm, Petra
    de Vos, Marjon G. J.
    Andersson, Anders
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Mazon, Hortense F. M.
    van den Heuvel, Robert H. H.
    Nilsson, Peter
    Salmon, Laurent
    de Vos, Willem M.
    Wright, Phillip C.
    Bernander, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    van der Oost, John
    Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 37, p. 27378-27388Article in journal (Refereed)
    Abstract [en]

    The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for D-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to be differentially expressed compared with growth on D-glucose. These genes were heterologously overexpressed in Escherichia coli, and the recombinant proteins were purified and biochemically studied. This showed that D-arabinose is oxidized to 2-oxoglutarate by the consecutive action of a number of previously uncharacterized enzymes, including a D-arabinose dehydrogenase, a D-arabinonate dehydratase, a novel 2-keto-3-deoxy-D-arabinonate dehydratase, and a 2,5-dioxopentanoate dehydrogenase. Promoter analysis of these genes revealed a palindromic sequence upstream of the TATA box, which is likely to be involved in their concerted transcriptional control. Integration of the obtained biochemical data with genomic context analysis strongly suggests the occurrence of pentose oxidation pathways in both Archaea and Bacteria, and predicts the involvement of additional enzyme components. Moreover, it revealed striking genetic similarities between the catabolic pathways for pentoses, hexaric acids, and hydroxyproline degradation, which support the theory of metabolic pathway genesis by enzyme recruitment.

  • 6. Brouns, Stan J
    et al.
    Jore, Matthijs M
    Lundgren, Magnus
    Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University.
    Westra, Edze R
    Slijkhuis, Rik J
    Snijders, Ambrosius P
    Dickman, Mark J
    Makarova, Kira S
    Koonin, Eugene V
    Van Der Oost, John
    Small CRISPR RNAs guide antiviral defense in prokaryotes2008In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 321, no 5891, p. 960-964Article in journal (Refereed)
    Abstract [en]

    Prokaryotes acquire virus resistance by integrating short fragments of viral nucleic acid into clusters of regularly interspaced short palindromic repeats (CRISPRs). Here we show how virus-derived sequences contained in CRISPRs are used by CRISPR-associated (Cas) proteins from the host to mediate an antiviral response that counteracts infection. After transcription of the CRISPR, a complex of Cas proteins termed Cascade cleaves a CRISPR RNA precursor in each repeat and retains the cleavage products containing the virus-derived sequence. Assisted by the helicase Cas3, these mature CRISPR RNAs then serve as small guide RNAs that enable Cascade to interfere with virus proliferation. Our results demonstrate that the formation of mature guide RNAs by the CRISPR RNA endonuclease subunit of Cascade is a mechanistic requirement for antiviral defense.

  • 7.
    Ettema, Thijs J. G.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Lindas, Ann-Christin
    Hjort, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Poplawski, Andrzej B.
    Kaessmann, Henrik
    Grogan, Dennis W.
    Kelman, Zvi
    Andersson, Anders F.
    Pelve, Erik A.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Rolf Bernander (1956-2014): pioneer of the archaeal cell cycle Obituary2014In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 92, no 5, p. 903-909Article in journal (Refereed)
    Abstract [en]

    On 19 January 2014 Rolf (Roffe') Bernander passed away unexpectedly. Rolf was a dedicated scientist; his research aimed at unravelling the cell biology of the archaeal domain of life, especially cell cycle-related questions, but he also made important contributions in other areas of microbiology. Rolf had a professor position in the Molecular Evolution programme at Uppsala University, Sweden for about 8 years, and in January 2013 he became chair professor at the Department of Molecular Biosciences, The Wenner-Gren Institute at Stockholm University in Sweden. Rolf was an exceptional colleague and will be deeply missed by his family and friends, and the colleagues and co-workers that he leaves behind in the scientific community. He will be remembered for his endless enthusiasm for science, his analytical mind, and his quirky sense of humour.

  • 8. Götz, Dorothee
    et al.
    Paytubi, Sonia
    Munro, Stacey
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Bernander, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    White, Malcolm F.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Responses of hyperthermophilic crenarchaea to UV irradiation2007In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 8, no 10, p. R220-Article in journal (Refereed)
    Abstract [en]

    Background:

    DNA damage leads to cellular responses that include the increased expression of DNA repair genes, repression of DNA replication and alterations in cellular metabolism. Archaeal information processing pathways resemble those in eukaryotes, but archaeal damage response pathways remain poorly understood.

    Results:

    We analyzed the transcriptional response to UV irradiation in two related crenarchaea, Sulfolobus solfataricus and Sulfolobus acidocaldarius. Sulfolobus species encounter high levels of DNA damage in nature, as they inhabit high temperature, aerobic environments and are exposed to sunlight. No increase in expression of DNA repair genes following UV irradiation was observed. There was, however, a clear transcriptional response, including repression of DNA replication and chromatin proteins. Differential effects on the expression of the three transcription factor B ( tfb) genes hint at a mechanism for the modulation of transcriptional patterns in response to DNA damage. TFB3, which is strongly induced following UV irradiation, competes with TFB1 for binding to RNA polymerase in vitro, and may act as a repressor of transcription or an alternative transcription factor for certain promoters.

    Conclusion:

    A clear response to DNA damage was observed, with down-regulation of the DNA replication machinery, changes in transcriptional regulatory proteins, and up-regulation of the biosynthetic enzymes for beta-carotene, which has UV protective properties, and proteins that detoxify reactive oxygen species. However, unlike eukaryotes and bacteria, there was no induction of DNA repair proteins in response to DNA damage, probably because these are expressed constitutively to deal with increased damage arising due to high growth temperatures.

  • 9. Jore, Matthijs
    et al.
    Lundgren, Magnus
    Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Wageningen, The Netherlands.
    van Duijn, Esther
    Bultema, Jelle
    Westra, Edze
    Waghmare, Sakharam
    Wiedenheft, Blake
    Pul, Ümit
    Wurm, Reinhild
    Wagner, Rolf
    Beijer, Marieke
    Barendregt, Arjan
    Zhou, Kaihong
    Snijders, Ambrosius
    Dickman, Mark
    Doudna, Jennifer
    Boekema, Egbert
    Heck, Albert
    van der Oost, John
    Brouns, Stan
    Structural basis for CRISPR RNA-guided DNA recognition by Cascade2011In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 18, no 5, p. 529-536Article in journal (Refereed)
    Abstract [en]

    The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

  • 10.
    Lundgren, Magnus
    Wageningen University, Wageningen, The Netherlands.
    Archaeal Chromosome2008In: Encyclopedia of Life Sciences, Chichester, England: John Wiley & Sons , 2008Chapter in book (Refereed)
    Abstract [en]

    The Archaea is one of the three domains of life, and the least known. Sequencing ofmany archaeal chromosomes has shown that archaea display many eukaryotic features,despite sharing their appearance and range of genome size with bacteria. Chromatinorganization, replication and transcription are similar to eukaryotes while operonorganization and transcription regulation are similar to bacteria.

  • 11.
    Lundgren, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Andersson, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Chen, Lanming
    Nilsson, Peter
    Bernander, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Three replication origins in Sulfolobus species: synchronous initiation of chromosome replication and asynchronous termination2004In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 101, no 18, p. 7046-7051Article in journal (Refereed)
    Abstract [en]

    Chromosome replication origins were mapped in vivo in the two hyperthermophilic archaea, Sulfolobus acidocaldarius and Sulfolobus solfataricus, by using microarray-based marker frequency analysis. Bidirectional replication was found to be initiated in near synchrony from three separate sites in both organisms. Two of the three replication origins in each species were located in the vicinity of a cdc6/orc1 replication initiation gene, whereas no known replication-associated gene could be identified near the third origin in either organism. In contrast to initiation, replication termination occurred asynchronously, such that certain replication forks continued to progress for >40 min after the others had terminated. In each species, all replication forks advanced at similar DNA polymerization rates; this was found to be an order of magnitude below that displayed by Escherichia coli and thus closer to eukaryotic elongation rates. In S. acidocaldarius, a region containing short regularly spaced repeats was found to hybridize aberrantly, as compared to the rest of the chromosome, raising the possibility of a centromere-like function.

  • 12.
    Lundgren, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Bernander, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Archaeal cell cycle progress2005In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 8, no 6, p. 662-668Article in journal (Refereed)
    Abstract [en]

    The discovery of multiple chromosome replication origins in Sulfolobus species has added yet another eukaryotic trait to the archaea, and brought new levels of complexity to the cell cycle in terms of initiation of chromosome replication, replication termination and chromosome decatenation. Conserved repeated DNA elements — origin recognition boxes — have been identified in the origins of replication, and shown to bind the Orc1/Cdc6 proteins involved in cell cycle control. The origin recognition boxes aid in the identification and characterization of new origins, and their conservation suggests that most archaea have a similar replication initiation mechanism. Cell-cycle-dependent variation in Orc1/Cdc6 levels has been demonstrated, reminiscent of variations in cyclin levels during the eukaryotic cell cycle. Information about archaeal chromosome segregation is also accumulating, including the identification of a protein that binds to short regularly spaced repeats that might constitute centromer-like elements. In addition, studies of cell-cycle-specific gene expression have potential to reveal, in the near future, missing components in crenarchaeal chromosome replication, genome segregation and cell division. Together with an increased number of physiological and cytological investigations of the overall organization of the cell cycle, rapid progress of the archaeal cell cycle field is evident, and archaea, in particular Sulfolobus species, are emerging as simple and powerful models for the eukaryotic cell cycle.

  • 13.
    Lundgren, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Bernander, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Genome-wide transcription map of an archaeal cell cycle2007In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 104, no 8, p. 2939-2944Article in journal (Refereed)
    Abstract [en]

    Relative RNA abundance was measured at different cell-cyclestages in synchronized cultures of the hyperthermophilic archaeonSulfolobus acidocaldarius. Cyclic induction was observed for >160genes, demonstrating central roles for transcriptional regulationand cell-cycle-specific gene expression in archaeal cell-cycle pro-gression. Many replication genes were induced in a cell-cycle-specific manner, and novel replisome components are likely to beamong the genes of unknown function with similar inductionpatterns. Candidate genes for the unknown genome segregationand cell division machineries were also identified, as well as seventranscription factors likely to be involved in cell-cycle control. Twoserine-threonine protein kinases showed distinct cell-cycle-specificinduction, suggesting regulation of the archaeal cell cycle alsothrough protein modification. Two candidate recognition ele-ments, CCR boxes, for transcription factors in control of cell-cycleregulons were identified among gene sets with similar inductionkinetics. The results allow detailed characterization of the genomesegregation, division, and replication processes and may, becauseof the extensive homologies between the archaeal and eukaryoticinformation machineries, also be applicable to core features of theeukaryotic cell cycle.

  • 14.
    Lundgren, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organism Biology, Molecular Evolution.
    Malandrin, Laurence
    Eriksson, Stefan
    Huber, Harald
    Bernander, Rolf
    Cell Cycle Characteristics of Crenarchaea: Unity among DiversityManuscript (Other academic)
  • 15.
    Lundgren, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Malandrin, Laurence
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Eriksson, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Huber, Harald
    Bernander, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Cell cycle characteristics of Crenarchaeota: unity among diversity2008In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 190, no 15, p. 5362-5367Article in journal (Refereed)
    Abstract [en]

    The hyperthermophilic archaea Acidianus hospitalis, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrobaculum calidifontis, and Sulfolobus tokodaii representing three different orders in the phylum Crenarchaeota were analyzed by flow cytometry and combined phase-contrast and epifluorescence microscopy. The overall organization of the cell cycle was found to be similar in all species, with a short prereplicative period and a dominant postreplicative period that accounted for 64 to 77% of the generation time. Thus, in all Crenarchaeota analyzed to date, cell division and initiation of chromosome replication occur in close succession, and a long time interval separates termination of replication from cell division. In Pyrobaculum, chromosome segregation overlapped with or closely followed DNA replication, and further genome separation appeared to occur concomitant with cellular growth. Cell division in P. aerophilum took place without visible constriction.

  • 16. Majernik, Alan I.
    et al.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    McDermott, Paul
    Bernander, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Chong, James P. J.
    DNA content and nucleoid distribution in Methanothermobacter thermautotrophicus2005In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 187, no 5, p. 1856-1858Article in journal (Refereed)
    Abstract [en]

    Flow cytometry and epifluorescence microscopy results for the euryarchaeon Methanothermobacter thermautotrophicus were consistent with filaments containing multiple cells. Filaments of one to four cells contained two to eight nucleoids. Single chromosome-containing cells were not observed. Filaments containing multiple genome copies displayed synchronous DNA replication initiation. Chromosome segregation occurred during replication or rapidly after replication termination.

  • 17. Raedts, John
    et al.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Kengen, Serve W. M.
    Li, Jin-Ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    van der Oost, John
    A Novel Bacterial Enzyme with D-Glucuronyl C5-epimerase Activity2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 34, p. 24332-24339Article in journal (Refereed)
    Abstract [en]

    Glycosaminoglycans are biologically active polysaccharides that are found ubiquitously in the animal kingdom. The biosynthesis of these complex polysaccharides involves complicated reactions that turn the simple glycosaminoglycan backbone into highly heterogeneous structures. One of the modification reactions is the epimerization of D-glucuronic acid to its C5-epimer L-iduronic acid, which is essential for the function of heparan sulfate. Although L-iduronic acid residues have been shown to exist in polysaccharides of some prokaryotes, there has been no experimental evidence for the existence of a prokaryotic D-glucuronyl C5-epimerase. This work for the first time reports on the identification of a bacterial enzyme with D-glucuronyl C5-epimerase activity. A gene of the marine bacterium Bermanella marisrubri sp. RED65 encodes a protein (RED65_08024) of 448 amino acids that has an overall 37% homology to the human D-glucuronic acid C5-epimerase. Alignment of this peptide with the human and mouse sequences revealed a 60% similarity at the carboxyl terminus. The recombinant protein expressed in Escherichia coli showed epimerization activity toward substrates generated from heparin and the E. coli K5 capsular polysaccharide, thereby providing the first evidence for bacterial D-glucuronyl C5-epimerase activity. These findings may eventually be used for modification of mammalian glycosaminoglycans.

  • 18.
    Rath, Devashish
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Amlinger, Lina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hoekzema, Mirthe
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Devulapally, Praneeth Reddy
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Efficient programmable gene silencing by Cascade2015In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 1, p. 237-246Article in journal (Refereed)
    Abstract [en]

    Methods that permit controlled changes in the expression of genes are important tools for biological and medical research, and for biotechnological applications. Conventional methods are directed at individually changing each gene, its regulatory elements or its mRNA's translation rate. We demonstrate that the CRISPR-associated DNA-binding Cascade complex can be used for efficient, long-lasting and programmable gene silencing. When Cascade is targeted to a promoter sequence the transcription of the downstream gene is inhibited, resulting in dramatically reduced expression. The specificity of Cascade binding is provided by the integral crRNA component, which is easily designed to target virtually any stretch of DNA. Cascade targeted to the ORF sequence of the gene can also silence expression, albeit at lower efficiency. The system can be used to silence plasmid and chromosome targets, simultaneously target several genes and is active in different bacterial species and strains. The findings described here are an addition to the expanding range of CRISPR-based technologies and may be adapted to additional organisms and cell systems.

  • 19.
    Rath, Devashish
    et al.
    Bhabha Atom Res Ctr, Div Mol Biol, Mumbai 400085, Maharashtra, India..
    Amlinger, Lina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala Univ, Dept Cell & Mol Biol, SE-75124 Uppsala, Sweden..
    Rath, Archana
    Univ Mumbai, Dept Biotechnol, Mumbai 400098, Maharashtra, India..
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    The CRISPR-Cas immune system: Biology, mechanisms and applications2015In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 117, p. 119-128Article, review/survey (Refereed)
    Abstract [en]

    Viruses are a common threat to cellular life, not the least to bacteria and archaea who constitute the majority of life on Earth. Consequently, a variety of mechanisms to resist virus infection has evolved. A recent discovery is the adaptive immune system in prokaryotes, a type of system previously thought to be present only in vertebrates. The system, called CRISPR-Cas, provide sequence-specific adaptive immunity and fundamentally affect our understanding of virus host interaction. CRISPR-based immunity acts by integrating short virus sequences in the cell's CRISPR locus, allowing the cell to remember, recognize and clear infections. There has been rapid advancement in our understanding of this immune system and its applications, but there are many aspects that await elucidation making the field an exciting area of research. This review provides an overview of the field and highlights unresolved issues.

  • 20. Robinson, Nicholas P.
    et al.
    Dionne, Isabelle
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Marsh, Victoria L.
    Bernander, Rolf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Bell, Stephen D.
    Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus2004In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 116, no 1, p. 25-38Article in journal (Refereed)
    Abstract [en]

    Eukaryotic chromosomes possess multiple origins of replication, whereas bacterial chromosomes are replicated from a single origin. The archaeon Pyrococcus abyssi also appears to have a single origin, suggesting a common rule for prokaryotes. However, in the current work, we describe the identification of two active origins of replication in the single chromosome of the hyperthermophilic archaeon Sulfolobus solfataricus. Further, we identify conserved sequence motifs within the origins that are recognized by a family of three Sulfolobus proteins that are homologous to the eukaryotic initiator proteins Orc1 and Cdc6. We demonstrate that the two origins are recognized by distinct subsets of these Orc1/Cdc6 homologs. These data, in conjunction with an analysis of the levels of the three Orc1/Cdc6 proteins in different growth phases and cell cycle stages, lead us to propose a model for the roles for these proteins in modulating origin activity.

  • 21. van der Oost, John
    et al.
    Jore, Matthijs M
    Westra, Edze R
    Lundgren, Magnus
    Laboratory of Microbiology, Wageningen University.
    Brouns, Stan J J
    CRISPR-based adaptive and heritable immunity in prokaryotes2009In: TIBS -Trends in Biochemical Sciences. Regular ed., ISSN 0968-0004, E-ISSN 1362-4326, Vol. 34, no 8, p. 401-407Article, review/survey (Refereed)
    Abstract [en]

    The recently discovered CRISPR (clustered regularly interspaced short palindromic repeat) defense system protects bacteria and archaea against mobile genetic elements. This immunity system has the potential to continuously adjust its reach at the genomic level, implying that both gain and loss of information is inheritable. The CRISPR system consists of typical stretches of interspaced repetitive DNA (CRISPRs) and associated cas genes. Three distinct stages are recognized in the CRISPR defense mechanism: (i) adaptation of the CRISPR via the integration of short sequences of the invaders as spacers; (ii) expression of CRISPRs and subsequent processing to small guide RNAs; and (iii) interference of target DNA by the crRNA guides. Recent analyses of key Cas proteins indicate that, despite some functional analogies, this fascinating prokaryotic system shares no phylogenetic relation with the eukaryotic RNA interference system.

  • 22. van Duijn, Esther
    et al.
    Barbu, Ioana M.
    Barendreg, Arjan
    Jore, Matthijs M.
    Wiedenheft, Blake
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Westra, Edze R.
    Brouns, Stan J. J.
    Doudna, Jennifer A.
    van der Oost, John
    Heck, Albert J. R.
    Native Tandem and Ion Mobility Mass Spectrometry Highlight Structural and Modular Similarities in Clustered-Regularly-Interspaced Shot-Palindromic-Repeats (CRISPR)-associated Protein Complexes From Escherichia coli and Pseudomonas aeruginosa2012In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, no 11, p. 1430-1441Article in journal (Refereed)
    Abstract [en]

    The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to RNA-interference. Key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different CRISPR/Cas subtypes. Previous studies combined mass spectrometry, electron microscopy, and small angle x-ray scattering to demonstrate that the E. coli Cascade complex (405 kDa) and the P. aeruginosa Csy-complex (350 kDa) are similar in that they share a central spiral-shaped hexameric structure, flanked by associating proteins and one CRISPR RNA. Recently, a cryo-electron microscopy structure of Cascade revealed that the CRISPR RNA molecule resides in a groove of the hexameric backbone. For both complexes we here describe the use of native mass spectrometry in combination with ion mobility mass spectrometry to assign a stable core surrounded by more loosely associated modules. Via computational modeling subcomplex structures were proposed that relate to the experimental IMMS data. Despite the absence of obvious sequence homology between several subunits, detailed analysis of sub-complexes strongly suggests analogy between subunits of the two complexes. Probing the specific association of E. coli Cascade/crRNA to its complementary DNA target reveals a conformational change. All together these findings provide relevant new information about the potential assembly process of the two CRISPR-associated complexes.

  • 23. Westra, Edze R
    et al.
    Pul, Ümit
    Heidrich, Nadja
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Jore, Matthijs M
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Stratmann, Thomas
    Wurm, Reinhild
    Raine, Amanda
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mescher, Melina
    van Heereveld, Luc
    Mastop, Marieke
    Wagner, Gerhart E. H
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Schnetz, Karin
    van der Oost, John
    Wagner, Rolf
    Brouns, Stan J. J.
    H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO2010In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 77, no 6, p. 1380-1393Article in journal (Refereed)
    Abstract [en]

    The recently discovered prokaryotic CRISPR/Cas defence system provides immunity against viral infections and plasmid conjugation. It has been demonstrated that in Escherichia coli transcription of the Cascade genes (casABCDE) and to some extent the CRISPR array is repressed by heat-stable nucleoid-structuring (H-NS) protein, a global transcriptional repressor. Here we elaborate on the control of the E. coli CRISPR/Cas system, and study the effect on CRISPR-based anti-viral immunity. Transformation of wild-type E. coli K12 with CRISPR spacers that are complementary to phage Lambda does not lead to detectable protection against Lambda infection. However, when an H-NS mutant of E. coli K12 is transformed with the same anti-Lambda CRISPR, this does result in reduced sensitivity to phage infection. In addition, it is demonstrated that LeuO, a LysR-type transcription factor, binds to two sites flanking the casA promoter and the H-NS nucleation site, resulting in derepression of casABCDE12 transcription. Overexpression of LeuO in E. coli K12 containing an anti-Lambda CRISPR leads to an enhanced protection against phage infection. This study demonstrates that in E. coli H-NS and LeuO are antagonistic regulators of CRISPR-based immunity.

1 - 23 of 23
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