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  • 1.
    Amlinger, Lina
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Hoekzema, Mirthe
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Koskiniemi, Sanna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Lundgren, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition2017Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikel-id 10392Artikel i tidskrift (Refereegranskat)
    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.
    Amlinger, Lina
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Hoekzema, Mirthe
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Koskiniemi, Sanna
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Lundgren, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition2017Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikel-id 10392Artikel i tidskrift (Refereegranskat)
    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.

  • 3.
    Berghoff, Bork A.
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi. Justus Liebig Univ, Inst Mikrobiol & Mol Biol, D-35392 Giessen, Germany..
    Hoekzema, Mirthe
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Aulbach, Lena
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Two regulatory RNA elements affect TisB-dependent depolarization and persister formation2017Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 103, nr 6, s. 1020-1033Artikel i tidskrift (Refereegranskat)
    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.

  • 4.
    Hoekzema, Mirthe
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Small RNAs, Big Consequences: Post-transcriptional Regulation and Adaptive Immunity in Bacteria2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    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.

    Delarbeten
    1. Hfq-dependent mRNA unfolding promotes sRNA-based inhibition of translation
    Öppna denna publikation i ny flik eller fönster >>Hfq-dependent mRNA unfolding promotes sRNA-based inhibition of translation
    2019 (Engelska)Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, artikel-id e101199Artikel i tidskrift (Refereegranskat) 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.

    Nyckelord
    sRNA, Hfq, OmrA, OmrB, YdaM, Biofilm
    Nationell ämneskategori
    Mikrobiologi
    Identifikatorer
    urn:nbn:se:uu:diva-343525 (URN)10.15252/embj.2018101199 (DOI)000462892600006 ()30833291 (PubMedID)
    Forskningsfinansiär
    Vetenskapsrådet
    Tillgänglig från: 2018-02-28 Skapad: 2018-02-28 Senast uppdaterad: 2019-05-03Bibliografiskt granskad
    2. Two regulatory RNA elements affect TisB-dependent depolarization and persister formation
    Öppna denna publikation i ny flik eller fönster >>Two regulatory RNA elements affect TisB-dependent depolarization and persister formation
    2017 (Engelska)Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 103, nr 6, s. 1020-1033Artikel i tidskrift (Refereegranskat) 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.

    Ort, förlag, år, upplaga, sidor
    WILEY, 2017
    Nationell ämneskategori
    Biokemi och molekylärbiologi Mikrobiologi
    Identifikatorer
    urn:nbn:se:uu:diva-320870 (URN)10.1111/mmi.13607 (DOI)000395758900008 ()27997707 (PubMedID)
    Forskningsfinansiär
    Deutsche Forschungsgemeinschaft (DFG), BE 5210/1-1 BE 5210/2-1Vetenskapsrådet, VR 621-2010-5233
    Tillgänglig från: 2017-04-26 Skapad: 2017-04-26 Senast uppdaterad: 2018-02-28Bibliografiskt granskad
    3. Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition
    Öppna denna publikation i ny flik eller fönster >>Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition
    Visa övriga...
    2017 (Engelska)Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikel-id 10392Artikel i tidskrift (Refereegranskat) 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.

    Ort, förlag, år, upplaga, sidor
    NATURE PUBLISHING GROUP, 2017
    Nationell ämneskategori
    Biologiska vetenskaper
    Identifikatorer
    urn:nbn:se:uu:diva-335401 (URN)10.1038/s41598-017-10876-z (DOI)000408997700091 ()28871175 (PubMedID)
    Forskningsfinansiär
    VetenskapsrådetWenner-Gren StiftelsernaKungliga VetenskapsakademienScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
    Tillgänglig från: 2017-12-05 Skapad: 2017-12-05 Senast uppdaterad: 2018-02-28Bibliografiskt granskad
    4. Efficient programmable gene silencing by Cascade
    Öppna denna publikation i ny flik eller fönster >>Efficient programmable gene silencing by Cascade
    Visa övriga...
    2015 (Engelska)Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, nr 1, s. 237-246Artikel i tidskrift (Refereegranskat) 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.

    Nationell ämneskategori
    Biokemi och molekylärbiologi
    Identifikatorer
    urn:nbn:se:uu:diva-249042 (URN)10.1093/nar/gku1257 (DOI)000350207100026 ()25435544 (PubMedID)
    Tillgänglig från: 2015-04-23 Skapad: 2015-04-10 Senast uppdaterad: 2018-02-28Bibliografiskt granskad
  • 5.
    Hoekzema, Mirthe
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Romilly, Cedric
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Holmqvist, Erik
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Wagner, Gerhart E. H.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Hfq-dependent mRNA unfolding promotes sRNA-based inhibition of translation2019Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, artikel-id e101199Artikel i tidskrift (Refereegranskat)
    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.

  • 6.
    Rath, Devashish
    et al.
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Amlinger, Lina
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Hoekzema, Mirthe
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Devulapally, Praneeth Reddy
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi.
    Lundgren, Magnus
    Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Mikrobiologi.
    Efficient programmable gene silencing by Cascade2015Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, nr 1, s. 237-246Artikel i tidskrift (Refereegranskat)
    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 - 6 av 6
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