uu.seUppsala University Publications
Change search
Link to record
Permanent link

Direct link
BETA
Lundgren, Magnus
Publications (10 of 24) Show all publications
Amlinger, L., Hoekzema, M., Wagner, G. E. H., Koskiniemi, S. & Lundgren, M. (2017). Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition. Scientific Reports, 7, Article ID 10392.
Open this publication in new window or tab >>Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition
Show others...
2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 10392Article in journal (Refereed) Published
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.

Keywords
quorum sensing controls, escherichia-coli, cas adaptation, immune-system, host factor, dna, resistance, elements, repeats, bacteriophage
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-334399 (URN)10.1038/s41598-017-10876-z (DOI)000408997700091 ()
Available from: 2017-12-08 Created: 2017-12-08 Last updated: 2017-12-08Bibliographically approved
Amlinger, L., Hoekzema, M., Wagner, G. E. H., Koskiniemi, S. & Lundgren, M. (2017). Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition. Scientific Reports, 7, Article ID 10392.
Open this publication in new window or tab >>Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition
Show others...
2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 10392Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2017
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-335401 (URN)10.1038/s41598-017-10876-z (DOI)000408997700091 ()28871175 (PubMedID)
Funder
Swedish Research CouncilWenner-Gren FoundationsThe Royal Swedish Academy of SciencesScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2017-12-05 Created: 2017-12-05 Last updated: 2018-02-28Bibliographically approved
Rath, D., Amlinger, L., Hoekzema, M., Devulapally, P. R. & Lundgren, M. (2015). Efficient programmable gene silencing by Cascade. Nucleic Acids Research, 43(1), 237-246
Open this publication in new window or tab >>Efficient programmable gene silencing by Cascade
Show others...
2015 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 1, p. 237-246Article in journal (Refereed) Published
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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-249042 (URN)10.1093/nar/gku1257 (DOI)000350207100026 ()25435544 (PubMedID)
Available from: 2015-04-23 Created: 2015-04-10 Last updated: 2018-02-28Bibliographically approved
Rath, D., Amlinger, L., Rath, A. & Lundgren, M. (2015). The CRISPR-Cas immune system: Biology, mechanisms and applications. Biochimie, 117, 119-128
Open this publication in new window or tab >>The CRISPR-Cas immune system: Biology, mechanisms and applications
2015 (English)In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 117, p. 119-128Article, review/survey (Refereed) Published
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.

Keywords
CRISPR, Cascade, Cas9, Cas3, Cmr/Csm
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-266137 (URN)10.1016/j.biochi.2015.03.025 (DOI)000361864900013 ()25868999 (PubMedID)
Funder
Swedish Research CouncilWenner-Gren Foundations
Available from: 2015-11-17 Created: 2015-11-05 Last updated: 2017-12-01Bibliographically approved
Ettema, T. J. G., Lindas, A.-C., Hjort, K., Poplawski, A. B., Kaessmann, H., Grogan, D. W., . . . Svärd, S. G. (2014). Rolf Bernander (1956-2014): pioneer of the archaeal cell cycle Obituary. Molecular Microbiology, 92(5), 903-909
Open this publication in new window or tab >>Rolf Bernander (1956-2014): pioneer of the archaeal cell cycle Obituary
Show others...
2014 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 92, no 5, p. 903-909Article in journal (Refereed) Published
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.

National Category
Microbiology Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-228890 (URN)10.1111/mmi.12608 (DOI)000337560500001 ()
Note

Erratum in Molecular Microbiology, 2014:93, issue 3, p 582, doi: 10.1111/mmi.12691

Available from: 2014-07-23 Created: 2014-07-22 Last updated: 2018-01-11Bibliographically approved
Raedts, J., Lundgren, M., Kengen, S. W. M., Li, J.-P. & van der Oost, J. (2013). A Novel Bacterial Enzyme with D-Glucuronyl C5-epimerase Activity. Journal of Biological Chemistry, 288(34), 24332-24339
Open this publication in new window or tab >>A Novel Bacterial Enzyme with D-Glucuronyl C5-epimerase Activity
Show others...
2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 34, p. 24332-24339Article in journal (Refereed) Published
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-219878 (URN)10.1074/jbc.M113.476440 (DOI)000330612300005 ()
Available from: 2014-03-12 Created: 2014-03-06 Last updated: 2017-12-05Bibliographically approved
van Duijn, E., Barbu, I. M., Barendreg, A., Jore, M. M., Wiedenheft, B., Lundgren, M., . . . Heck, A. J. R. (2012). 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 aeruginosa. Molecular & Cellular Proteomics, 11(11), 1430-1441
Open this publication in new window or tab >>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 aeruginosa
Show others...
2012 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, no 11, p. 1430-1441Article in journal (Refereed) Published
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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-194374 (URN)10.1074/mcp.M112.020263 (DOI)000313277100025 ()
Available from: 2013-02-13 Created: 2013-02-13 Last updated: 2017-12-06Bibliographically approved
Jore, M., Lundgren, M., van Duijn, E., Bultema, J., Westra, E., Waghmare, S., . . . Brouns, S. (2011). Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nature Structural & Molecular Biology, 18(5), 529-536
Open this publication in new window or tab >>Structural basis for CRISPR RNA-guided DNA recognition by Cascade
Show others...
2011 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 18, no 5, p. 529-536Article in journal (Refereed) Published
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.

National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-166885 (URN)10.1038/nsmb.2019 (DOI)21460843 (PubMedID)
Available from: 2012-01-16 Created: 2012-01-16 Last updated: 2017-12-08Bibliographically approved
Bernander, R., Lundgren, M. & Ettema, T. J. G. (2010). Comparative and functional analysis of the archaeal cell cycle. Cell Cycle, 9(4), 795-806
Open this publication in new window or tab >>Comparative and functional analysis of the archaeal cell cycle
2010 (English)In: Cell Cycle, ISSN 1538-4101, E-ISSN 1551-4005, Vol. 9, no 4, p. 795-806Article in journal (Refereed) Published
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.

Keywords
archaea, caulobacter, cell cycle, cell division, chromosome replication, crenarchaeota, cytokinesis, genome segregation, mitosis, saccharomyces, sulfolobus
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-136815 (URN)10.4161/cc.9.4.10674 (DOI)000275811600038 ()20139715 (PubMedID)
Available from: 2010-12-14 Created: 2010-12-14 Last updated: 2017-12-11Bibliographically approved
Westra, E. R., Pul, Ü., Heidrich, N., Jore, M. M., Lundgren, M., Stratmann, T., . . . Brouns, S. J. J. (2010). H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO. Molecular Microbiology, 77(6), 1380-1393
Open this publication in new window or tab >>H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO
Show others...
2010 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 77, no 6, p. 1380-1393Article in journal (Refereed) Published
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.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-134681 (URN)10.1111/j.1365-2958.2010.07315.x (DOI)000281831400006 ()20659289 (PubMedID)
Available from: 2010-12-01 Created: 2010-11-30 Last updated: 2017-12-12Bibliographically approved
Organisations

Search in DiVA

Show all publications