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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.
    Berghoff, Bork A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Justus Liebig Univ, Inst Mikrobiol & Mol Biol, D-35392 Giessen, Germany..
    Hoekzema, Mirthe
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Aulbach, Lena
    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.
    Two regulatory RNA elements affect TisB-dependent depolarization and persister formation2017In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 103, no 6, p. 1020-1033Article in journal (Refereed)
    Abstract [en]

    Bacterial survival strategies involve phenotypic diversity which is generated by regulatory factors and noisy expression of effector proteins. The question of how bacteria exploit regulatory RNAs to make decisions between phenotypes is central to a general understanding of these universal regulators. We investigated the TisB/IstR-1 toxin-antitoxin system of Escherichia coli to appreciate the role of the RNA antitoxin IstR-1 in TisB-dependent depolarization of the inner membrane and persister formation. Persisters are phenotypic variants that have become transiently drug-tolerant by arresting growth. The RNA antitoxin IstR-1 sets a threshold for TisB-dependent depolarization under DNA-damaging conditions, resulting in two sub-populations: polarized and depolarized cells. Furthermore, our data indicate that an inhibitory 5 UTR structure in the tisB mRNA serves as a regulatory RNA element that delays TisB translation to avoid inappropriate depolarization when DNA damage is low. Investigation of the persister sub-population further revealed that both regulatory RNA elements affect persister levels as well as persistence time. This work provides an intriguing example of how bacteria exploit regulatory RNAs to control phenotypic heterogeneity.

  • 3.
    Hoekzema, Mirthe
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Small RNAs, Big Consequences: Post-transcriptional Regulation and Adaptive Immunity in Bacteria2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    It is nowadays widely accepted that non-coding RNAs play important roles in post-transcriptional regulation of genes in all kingdoms of life. In bacteria, the largest group of RNA regulators are the small RNAs (sRNAs). Almost all sRNAs act through anti-sense base-pairing with target mRNAs, and by doing so regulate their translation and/or stability. As important modulators of gene expression, sRNAs are involved in all aspects of bacterial physiology. My studies aimed to deepen our understanding of the mechanisms behind sRNA-mediated gene regulation. We have shown that translation of the di-guanylate-cyclase YdaM, a major player in the biofilm regulatory cascade, is repressed by the sRNAs OmrA and OmrB. OmrAB require the RNA chaperone protein Hfq for efficient regulation. Interestingly, our results suggest a non-canonical mechanism for Hfq-mediated ydaM-OmrA/B base-pairing. Instead of serving as RNA interaction platform, Hfq restructures the ydaM mRNA to enable sRNA binding. We also addressed the question of how bacteria utilize regulatory RNAs to create phenotypic heterogeneity by studying the role of the tisB/istR-1 type 1 toxin-antitoxin system in SOS-induced persister cell formation in E. coli.

    In addition, I have investigated the prokaryotic CRISPR-Cas immune system, which has led to the development of two molecular tools. The CRISPR-Cas adaptive immune system consists of a CRISPR array, where palindromic repeats are interspaced by unique spacer sequences derived from foreign genetic elements, and the CRISPR-associated (Cas) proteins. In the adaptation stage, memory is created by insertion of spacer sequences into the CRISPR array. We developed a fluorescent reporter that accurately and sensitively detects spacer integration events (denoted: “acquisition”) in single cells and in real-time. In the effector stage of immunity, crRNAs, consisting of one spacer-repeat unit, associate with the Cas proteins to form a ribonucleoprotein complex that surveys the cell for invader DNA. Target identification occurs by base-pairing between the crRNA and the complementary sequence in the target nucleic acid, which triggers degradation. We repurposed the E. coli type I-E CRISPR-Cas effector complex Cascade for specific reprogrammable transcriptional gene silencing.

    The studies presented herein thus contributes to our understanding of RNA-based target identification for gene regulation and adaptive immunity.

    List of papers
    1. Hfq-dependent mRNA unfolding promotes sRNA-based inhibition of translation
    Open this publication in new window or tab >>Hfq-dependent mRNA unfolding promotes sRNA-based inhibition of translation
    2019 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, article id e101199Article in journal (Refereed) Published
    Abstract [en]

    Small RNAs post-transcriptionally regulate many processes inbacteria. Base-pairing of sRNAs near ribosome-binding sites inmRNAs inhibits translation, often requiring the RNA chaperoneHfq. In the canonical model, Hfq simultaneously binds sRNAs andmRNA targets to accelerate pairing. Here, we show that theEscher-ichia colisRNAs OmrA and OmrB inhibit translation of the diguany-late cyclase DgcM (previously: YdaM), a player in biofilmregulation. In OmrA/B repression ofdgcM, Hfq is not required as anRNA interaction platform, but rather unfolds an inhibitory RNAstructure that impedes OmrA/B binding. This restructuring involvesdistal face binding of Hfq and is supported by RNA structuremapping. A corresponding mutant protein cannot support inhibi-tionin vitroandin vivo; proximal and rim mutations have negligi-ble effects. Strikingly, OmrA/B-dependent translational inhibitionin vitrois restored, in complete absence of Hfq, by a deoxyoligori-bonucleotide that base-pairs to the biochemically mapped Hfq siteindgcMmRNA. We suggest that Hfq-dependent RNA structureremodeling can promote sRNA access, which represents a mecha-nism distinct from an interaction platform model.

    Keywords
    sRNA, Hfq, OmrA, OmrB, YdaM, Biofilm
    National Category
    Microbiology
    Identifiers
    urn:nbn:se:uu:diva-343525 (URN)10.15252/embj.2018101199 (DOI)000462892600006 ()30833291 (PubMedID)
    Funder
    Swedish Research Council
    Available from: 2018-02-28 Created: 2018-02-28 Last updated: 2019-05-03Bibliographically approved
    2. Two regulatory RNA elements affect TisB-dependent depolarization and persister formation
    Open this publication in new window or tab >>Two regulatory RNA elements affect TisB-dependent depolarization and persister formation
    2017 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 103, no 6, p. 1020-1033Article in journal (Refereed) Published
    Abstract [en]

    Bacterial survival strategies involve phenotypic diversity which is generated by regulatory factors and noisy expression of effector proteins. The question of how bacteria exploit regulatory RNAs to make decisions between phenotypes is central to a general understanding of these universal regulators. We investigated the TisB/IstR-1 toxin-antitoxin system of Escherichia coli to appreciate the role of the RNA antitoxin IstR-1 in TisB-dependent depolarization of the inner membrane and persister formation. Persisters are phenotypic variants that have become transiently drug-tolerant by arresting growth. The RNA antitoxin IstR-1 sets a threshold for TisB-dependent depolarization under DNA-damaging conditions, resulting in two sub-populations: polarized and depolarized cells. Furthermore, our data indicate that an inhibitory 5 UTR structure in the tisB mRNA serves as a regulatory RNA element that delays TisB translation to avoid inappropriate depolarization when DNA damage is low. Investigation of the persister sub-population further revealed that both regulatory RNA elements affect persister levels as well as persistence time. This work provides an intriguing example of how bacteria exploit regulatory RNAs to control phenotypic heterogeneity.

    Place, publisher, year, edition, pages
    WILEY, 2017
    National Category
    Biochemistry and Molecular Biology Microbiology
    Identifiers
    urn:nbn:se:uu:diva-320870 (URN)10.1111/mmi.13607 (DOI)000395758900008 ()27997707 (PubMedID)
    Funder
    German Research Foundation (DFG), BE 5210/1-1 BE 5210/2-1Swedish Research Council, VR 621-2010-5233
    Available from: 2017-04-26 Created: 2017-04-26 Last updated: 2018-02-28Bibliographically approved
    3. Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition
    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
    Cell and Molecular Biology
    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: 2020-02-04Bibliographically approved
    4. Efficient programmable gene silencing by Cascade
    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
  • 4.
    Hoekzema, Mirthe
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Romilly, Cedric
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Holmqvist, Erik
    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.
    Hfq-dependent mRNA unfolding promotes sRNA-based inhibition of translation2019In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, article id e101199Article in journal (Refereed)
    Abstract [en]

    Small RNAs post-transcriptionally regulate many processes inbacteria. Base-pairing of sRNAs near ribosome-binding sites inmRNAs inhibits translation, often requiring the RNA chaperoneHfq. In the canonical model, Hfq simultaneously binds sRNAs andmRNA targets to accelerate pairing. Here, we show that theEscher-ichia colisRNAs OmrA and OmrB inhibit translation of the diguany-late cyclase DgcM (previously: YdaM), a player in biofilmregulation. In OmrA/B repression ofdgcM, Hfq is not required as anRNA interaction platform, but rather unfolds an inhibitory RNAstructure that impedes OmrA/B binding. This restructuring involvesdistal face binding of Hfq and is supported by RNA structuremapping. A corresponding mutant protein cannot support inhibi-tionin vitroandin vivo; proximal and rim mutations have negligi-ble effects. Strikingly, OmrA/B-dependent translational inhibitionin vitrois restored, in complete absence of Hfq, by a deoxyoligori-bonucleotide that base-pairs to the biochemically mapped Hfq siteindgcMmRNA. We suggest that Hfq-dependent RNA structureremodeling can promote sRNA access, which represents a mecha-nism distinct from an interaction platform model.

  • 5.
    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.

1 - 5 of 5
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