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  • 1. Bosley, Katrine S
    et al.
    Botchan, Michael
    Bredenoord, Annelien L
    Carroll, Dana
    Charo, R Alta
    Charpentier, Emmanuelle
    Cohen, Ron
    Corn, Jacob
    Doudna, Jennifer
    Feng, Guoping
    Greely, Henry T
    Isasi, Rosario
    Ji, Weihzi
    Kim, Jin-Soo
    Knoppers, Bartha
    Lanphier, Edward
    Li, Jinsong
    Lovell-Badge, Robin
    Martin, G Steven
    Moreno, Jonathan
    Naldini, Luigi
    Pera, Martin
    Perry, Anthony C F
    Venter, J Craig
    Zhang, Feng
    Zhou, Qi
    CRISPR germline engineering: the community speaks2015In: Nature Biotechnology, ISSN 1087-0156, E-ISSN 1546-1696, Vol. 33, no 5, p. 478-486Article in journal (Refereed)
  • 2. Broglia, Laura
    et al.
    Materne, Solange
    Lecrivain, Anne-Laure
    Hahnke, Karin
    Le Rhun, Anaïs
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Institute for Biology, Humboldt University, Berlin, Germany; Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
    RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B2018In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 15, no 10, p. 1336-1347Article in journal (Refereed)
    Abstract [en]

    Endoribonuclease Y (RNase Y) is a crucial regulator of virulence in Gram-positive bacteria. In the human pathogen Streptococcus pyogenes, RNase Y is required for the expression of the major secreted virulence factor streptococcal pyrogenic exotoxin B (SpeB), but the mechanism involved in this regulation remains elusive. Here, we demonstrate that the 5′ untranslated region of speB mRNA is processed by several RNases including RNase Y. In particular, we identify two RNase Y cleavage sites located downstream of a guanosine (G) residue. To assess whether this nucleotide is required for RNase Y activity in vivo, we mutated it and demonstrate that the presence of this G residue is essential for the processing of the speB mRNA 5′ UTR by RNase Y. Although RNase Y directly targets and processes speB, we show that RNase Y-mediated regulation of speB expression occurs primarily at the transcriptional level and independently of the processing in the speB mRNA 5′ UTR. To conclude, we demonstrate for the first time that RNase Y processing of an mRNA target requires the presence of a G. We also provide new insights on the speB 5′ UTR and on the role of RNase Y in speB regulation.

  • 3. Charpentier, E
    et al.
    Courvalin, P
    Antibiotic resistance in Listeria spp.1999In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 43, no 9, p. 2103-2108Article in journal (Refereed)
  • 4. Charpentier, E
    et al.
    Courvalin, P
    Emergence of the trimethoprim resistance gene dfrD in Listeria monocytogenes BM4293.1997In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 41, no 5, p. 1134-1136Article in journal (Refereed)
    Abstract [en]

    The sequence of the trimethoprim resistance gene of the 3.7-kb plasmid (pIP823) that confers high-level resistance (MIC, 1,024 microg/ml) to Listeria monocytogenes BM4293 was determined. The gene was identical to dfrD recently detected in Staphylococcus haemolyticus MUR313. The corresponding protein, S2DHFR, represents the second class of high-level trimethoprim-resistant dihydrofolate reductase identified in gram-positive bacteria. We propose that trimethoprim resistance in L. monocytogenes BM4293 could originate in staphylococci.

  • 5. Charpentier, E
    et al.
    Gerbaud, G
    Courvalin, P
    Characterization of a new class of tetracycline-resistance gene tet(S) in Listeria monocytogenes BM4210.1993In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 131, no 1, p. 27-34Article in journal (Refereed)
    Abstract [en]

    The nucleotide sequence of the tetracycline (Tc)-minocycline (Mc)-resistance determinant of plasmid pIP811 from Listeria monocytogenes BM4210 has been determined. The gene, designated tet(S), was identified by analysis of the start and stop codons as a coding sequence of 1923 bp, corresponding to a protein with a calculated M(r) of 72,912. The apparent 68-kDa size estimated by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of the protein characterized in a cell-free coupled transcription-translation system was in good agreement with the calculated value. The tet(S) gene product exhibits 79 and 72% amino acid identity with Tet(M) from Streptococcus pneumoniae and Tet(O) from Campylobacter coli, respectively. The distribution of tet(S) in strains of Gram+ and Gram- genera resistant to Tc (TcR) and Mc (McR) was studied by hybridization under high stringency using a 590-bp intragenic probe. Homology with tet(S) was detected in two clinical isolates of L. monocytogenes isolated in different geographical areas.

  • 6. Charpentier, E
    et al.
    Gerbaud, G
    Courvalin, P
    Presence of the Listeria tetracycline resistance gene tet(S) in Enterococcus faecalis.1994In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 38, no 10, p. 2330-2335Article in journal (Refereed)
    Abstract [en]

    Two hundred thirty-eight tetracycline- and minocycline-resistant clinical isolates of Enterococcus and Streptococcus spp. were investigated by dot blot hybridization for the presence of nucleotide sequences related to tet(S) (first detected in Listeria monocytogenes BM4210), tet(K), tet(L), tet(M), tet(O), tet(P), and tet(Q) genes. The tet(S) determinant was found in 22 strains of Enterococcus faecalis, associated with tet(M) in 9 of these isolates and further associated with tet(L) in 3 of these strains. tet(M) was detected in all strains of Streptococcus spp. and in all but 10 isolates of Enterococcus spp.; tet(L) was found in 93 enterococci and tet(O) was found in single isolates of E. faecalis and Streptococcus milleri. No hybridization with the tet(K), tet(P), and tet(Q) probes was observed. Transfer of tet(S) by conjugation to E. faecalis or to E. faecalis and L. monocytogenes was obtained from 8 of the 10 E. faecalis strains harboring only this tet gene. Hybridization experiments with DNAs of four donors and of the corresponding transconjugants suggested that tet(S) was located in the chromosome. These results indicate that the genetic support of tet(S) in E. faecalis is different from that in L. monocytogenes, where it is carried by self-transferable plasmids, and confirm the notion of exchange of genetic information between Enterococcus and Listeria spp. in nature.

  • 7. Charpentier, E
    et al.
    Gerbaud, G
    Jacquet, C
    Rocourt, J
    Courvalin, P
    Incidence of antibiotic resistance in Listeria species.1995In: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 172, no 1, p. 277-281Article in journal (Refereed)
    Abstract [en]

    To define the prevalence of antibiotic resistance in Listeria species pathogenic for humans and animals, 1100 isolates (60 from cases of listeriosis and 1040 from food and environment) collected worldwide were screened. Of the 61 tetracycline- and minocycline-resistant strains (37 Listeria monocytogenes), 57 harbored tet(M); 4 non-L. monocytogenes isolates contained tet(S). One Listeria innocua isolate was also resistant to streptomycin and contained the tet(M) and aad6 genes. An L. monocytogenes isolate was trimethoprim-resistant, a characteristic not reported previously in Listeria species, because of the presence of a yet-uncharacterized gene. Three clinical isolates of L. monocytogenes were resistant to low levels of streptomycin. Since the tet(M), tet(S), and aad6 genes are common in enterococci and streptococci, these data suggest transfer from the latter to Listeria species. Uniform susceptibility to tetracycline, minocycline, trimethoprim, and streptomycin cannot be assumed any longer for Listeria species.

  • 8. Charpentier, E
    et al.
    Lavker, R M
    Acquista, E
    Cowin, P
    Plakoglobin suppresses epithelial proliferation and hair growth in vivo.2000In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 149, no 2, p. 503-520Article in journal (Refereed)
    Abstract [en]

    Plakoglobin regulates cell adhesion by providing a modulatable connection between both classical and desmosomal cadherins and their respective cytoskeletal linker proteins. Both plakoglobin and the related protein beta-catenin are posttranscriptionally upregulated in response to Wnt-1 in cultured cells. Upregulation of beta-catenin has been implicated in potentiating hyperproliferation and tumor formation. To investigate the role of plakoglobin in these functions we expressed a full-length (PG) and an NH(2)-terminally truncated form of plakoglobin (DeltaN80PG) in mouse epidermis and hair follicles, tissues which undergo continuous and easily observed postnatal renewal and remodeling. Expression of these constructs results in stunted hair growth, a phenotype that has also been observed in transgenic mice expressing Wnt3 and Dvl2 (Millar et al. 1999). Hair follicles from PG and DeltaN80PG mice show premature termination of the growth phase (anagen) of the hair cycle, an event that is regulated in part by FGF5 (Hebert et al. 1994). The proliferative rate of the epidermal cells was reduced and apoptotic changes, which are associated with entry into the regressive phase of the hair follicle cycle (catagen), occurred earlier than usual.

  • 9. Charpentier, E
    et al.
    Novak, R
    Tuomanen, E
    Regulation of growth inhibition at high temperature, autolysis, transformation and adherence in Streptococcus pneumoniae by clpC.2000In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 37, no 4, p. 717-26Article in journal (Refereed)
    Abstract [en]

    The ClpC ATPase is a subfamily of HSP100/Clp molecular chaperones-regulators of proteolysis. By screening a library of loss of function mutants for the ability to survive treatment with penicillin, we identified the gene clpC. The corresponding protein was identified as a ClpC ATPase, sharing strong peptide sequence identity with ClpC of Bacillus subtilis, Listeria monocytogenes and Lactococcus lactis. Northern blot experiments showed that expression of clpC was induced in response to high temperature (40-42 degrees C) versus 37 degrees C, suggesting that ClpC is a heat shock protein. Insertional duplication mutagenesis of clpC resulted in increased tolerance to high temperature; a result in contrast to other bacterial Clp proteases. The clpC-deficient mutant formed long chains and failed to undergo lysis after treatment with penicillin or vancomycin. The effect of the clpC mutation extended to deficiency of adherence to the human type II alveolar cells. Finally, the clpC disruption resulted in decreased genetic transformation. Western blot analysis demonstrated that the mutant failed to express pneumolysin and the choline-binding proteins LytA, CbpA, CbpE, CbpF, CbpJ. These results suggest that the heat shock protein ClpC plays an essential complex pleiotropic role in pneumococcal physiology, including cell growth under heat stress, cell division, autolysis, adherence and transformation.

  • 10. Charpentier, E
    et al.
    Tuomanen, E
    Mechanisms of antibiotic resistance and tolerance in Streptococcus pneumoniae.2000In: Microbes and infection, ISSN 1286-4579, E-ISSN 1769-714X, Vol. 2, no 15, p. 1855-64Article in journal (Refereed)
    Abstract [en]

    Streptococcus pneumoniae is a major pathogen causing potentially life-threatening community-acquired diseases in both the developed and developing world. Since 1967, there has been a dramatic increase in the incidence of penicillin-resistant and multiply antibiotic-resistant pneumococci worldwide. Prevention of access of the antibiotic to the target, inactivation of the antibiotic and alteration of the target are mechanisms that S. pneumoniae has developed to resist antibiotics. Recent studies on antibiotic-tolerant pneumococcal mutants permitted development of a novel model for the control of bacterial cell death.

  • 11.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany; Hannover Medical School, Hannover, Germany.
    CRISPR-Cas9: how research on a bacterial RNA-guided mechanism opened new perspectives in biotechnology and biomedicine2015In: EMBO Molecular Medicine, ISSN 1757-4676, E-ISSN 1757-4684, Vol. 7, no 4, p. 363-365Article in journal (Refereed)
  • 12.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Programmable RNA-guided Cas9 endonuclease: a novel tool for genome engineering2014In: Transgenic research, ISSN 0962-8819, E-ISSN 1573-9368, Vol. 23, no 1, p. 188-189Article in journal (Other academic)
  • 13.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Institute for Biology, Humboldt University, Berlin, Germany.
    Spotlight on... Emmanuelle Charpentier2018In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 365, no 4, article id fnx271Article in journal (Other academic)
  • 14. Charpentier, Emmanuelle
    et al.
    Anton, Ana I
    Barry, Peter
    Alfonso, Berenice
    Fang, Yuan
    Novick, Richard P
    Novel cassette-based shuttle vector system for Gram-positive bacteria.2004In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 70, no 10, p. 6076-6085Article in journal (Refereed)
    Abstract [en]

    Our understanding of staphylococcal pathogenesis depends on reliable genetic tools for gene expression analysis and tracing of bacteria. Here, we have developed and evaluated a series of novel versatile Escherichia coli-staphylococcal shuttle vectors based on PCR-generated interchangeable cassettes. Advantages of our module system include the use of (i) staphylococcal low-copy-number, high-copy-number, thermosensitive and theta replicons and selectable markers (choice of erythromycin, tetracycline, chloramphenicol, kanamycin, or spectinomycin); (ii) an E. coli replicon and selectable marker (ampicillin); and (iii) a staphylococcal phage fragment that allows high-frequency transduction and an SaPI fragment that allows site-specific integration into the Staphylococcus aureus chromosome. The staphylococcal cadmium-inducible P(cad)-cadC and constitutive P(blaZ) promoters were designed and analyzed in transcriptional fusions to the staphylococcal beta-lactamase blaZ, the Vibrio fischeri luxAB, and the Aequorea victoria green fluorescent protein reporter genes. The modular design of the vector system provides great flexibility and variety. Questions about gene dosage, complementation, and cis-trans effects can now be conveniently addressed, so that this system constitutes an effective tool for studying gene regulation of staphylococci in various ecosystems.

  • 15. Charpentier, Emmanuelle
    et al.
    Courvalin, P
    Antibiotic resistance in Listeria spp1995In: Med. Maladies. Infect., Vol. 25, p. 225-232Article in journal (Refereed)
  • 16.
    Charpentier, Emmanuelle
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Doudna, Jennifer A.
    Biotechnology: rewriting a genome2013In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 495, no 7439, p. 50-51Article in journal (Other academic)
  • 17. Charpentier, Emmanuelle
    et al.
    Gerbaud, G
    Courvalin, P
    Conjugative mobilization of the rolling-circle plasmid pIP823 from Listeria monocytogenes BM4293 among gram-positive and gram-negative bacteria.1999In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 181, no 11, p. 3368-3374Article in journal (Refereed)
    Abstract [en]

    We determined the sequence and genetic organization of plasmid pIP823, which contains the dfrD gene; dfrD confers high-level trimethoprim resistance to Listeria monocytogenes BM4293 by synthesis of dihydrofolate reductase type S2. pIP823 possessed all the features of the pUB110/pC194 plasmid family, whose members replicate by the rolling-circle mechanism. The rep gene encoded a protein identical to RepU, the protein required for initiation of the replication of plasmids pTB913 from a thermophilic Bacillus sp. and pUB110 from Staphylococcus aureus. The mob gene encoded a protein with a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmidic recombination of pTB913 and pUB110. The host range of pIP823 was broad and included L. monocytogenes, Enterococcus faecalis, S. aureus, Bacillus subtilis, and Escherichia coli. In all these species, pIP823 replicated by generating single-stranded DNA and was stable. Conjugative mobilization of pIP823 was obtained by self-transferable plasmids between L. monocytogenes and E. faecalis, between L. monocytogenes and E. coli, and between strains of E. coli, and by the streptococcal conjugative transposon Tn1545 from L. monocytogenes to E. faecalis, and from L. monocytogenes and E. faecalis to E. coli. These data indicate that the gene flux observed in nature from gram-positive to gram-negative bacteria can occur by conjugative mobilization. Our results suggest that dissemination of trimethoprim resistance in Listeria spp. and acquisition of other antibiotic resistance determinants in this species can be anticipated.

  • 18.
    Charpentier, Emmanuelle
    et al.
    Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology.
    Hess, Wolfgang R
    RNA in bacteria: biogenesis, regulatory mechanisms and functions2015In: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 39, no 3, p. 277-279Article in journal (Refereed)
  • 19.
    Charpentier, Emmanuelle
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Marraffini, Luciano A.
    Editorial overview: Novel technologies in microbiology: Recent advances in techniques in microbiology2014In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 19, p. VIII-XArticle in journal (Refereed)
  • 20.
    Charpentier, Emmanuelle
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Marraffini, Luciano A.
    Harnessing CRISPR-Cas9 immunity for genetic engineering2014In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 19, p. 114-119Article in journal (Refereed)
    Abstract [en]

    CRISPR-Cas encodes an adaptive immune system that defends prokaryotes against infectious viruses and plasmids. Immunity is mediated by Cas nucleases, which use small RNA guides (the crRNAs) to specify a cleavage site within the genome of invading nucleic acids. In type II CRISPR-Cas systems, the DNA-cleaving activity is performed by a single enzyme Cas9 guided by an RNA duplex. Using synthetic single RNA guides, Cas9 can be reprogrammed to create specific double-stranded DNA breaks in the genomes of a variety of organisms, ranging from human cells to bacteria, and thus constitutes a powerful tool for genetic engineering. Here we describe recent advancements in our understanding of type II CRISPR-Cas immunity and how these studies led to revolutionary genome editing applications.

  • 21. Charpentier, Emmanuelle
    et al.
    Novak, R
    Bacterial death and antibiotics of the ß-lactam family2000In: Med Sciences, ISSN 0767-0974, Vol. 16, no 10, p. 1125-1127Article in journal (Refereed)
  • 22.
    Charpentier, Emmanuelle
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Richter, Hagen
    van der Oost, John
    White, Malcolm F.
    Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity2015In: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 39, no 3, p. 428-441Article, review/survey (Refereed)
    Abstract [en]

    CRISPR-Cas is an RNA-mediated adaptive immune system that defends bacteria and archaea against mobile genetic elements. Short mature CRISPR RNAs (crRNAs) are key elements in the interference step of the immune pathway. A CRISPR array composed of a series of repeats interspaced by spacer sequences acquired from invading mobile genomes is transcribed as a precursor crRNA (pre-crRNA) molecule. This pre-crRNA undergoes one or two maturation steps to generate the mature crRNAs that guide CRISPR-associated (Cas) protein(s) to cognate invading genomes for their destruction. Different types of CRISPR-Cas systems have evolved distinct crRNA biogenesis pathways that implicate highly sophisticated processing mechanisms. In Types I and III CRISPR-Cas systems, a specific endoribonuclease of the Cas6 family, either standalone or in a complex with other Cas proteins, cleaves the pre-crRNA within the repeat regions. In Type II systems, the trans-acting small RNA (tracrRNA) base pairs with each repeat of the pre-crRNA to form a dual-RNA that is cleaved by the housekeeping RNase III in the presence of the protein Cas9. In this review, we present a detailed comparative analysis of pre-crRNA recognition and cleavage mechanisms involved in the biogenesis of guide crRNAs in the three CRISPR-Cas types.

  • 23.
    Charpentier, Emmanuelle
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Schroeder, Renée
    RNA techniques for bacteria2007In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 10, no 3, p. 254-256Article in journal (Other academic)
  • 24.
    Chylinski, Krzysztof
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Le Rhun, Anaïs
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems2013In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 10, no 5, p. 726-737Article in journal (Refereed)
    Abstract [en]

    CRISPR-Cas is a rapidly evolving RNA-mediated adaptive immune system that protects bacteria and archaea against mobile genetic elements. The system relies on the activity of short mature CRISPR RNAs (crRNAs) that guide Cas protein(s) to silence invading nucleic acids. A set of CRISPR-Cas, type II, requires a trans-activating small RNA, tracrRNA, for maturation of precursor crRNA (pre-crRNA) and interference with invading sequences. Following co-processing of tracrRNA and pre-crRNA by RNase III, dual-tracrRNA:crRNA guides the CRISPR-associated endonuclease Cas9 (Csn1) to cleave site-specifically cognate target DNA. Here, we screened available genomes for type II CRISPR-Cas loci by searching for Cas9 orthologs. We analyzed 75 representative loci, and for 56 of them we predicted novel tracrRNA orthologs. Our analysis demonstrates a high diversity in cas operon architecture and position of the tracrRNA gene within CRISPR-Cas loci. We observed a correlation between locus heterogeneity and Cas9 sequence diversity, resulting in the identification of various type II CRISPR-Cas subgroups. We validated the expression and co-processing of predicted tracrRNAs and pre-crRNAs by RNA sequencing in five bacterial species. This study reveals tracrRNA family as an atypical, small RNA family with no obvious conservation of structure, sequence or localization within type II CRISPR-Cas loci. The tracrRNA family is however characterized by the conserved feature to base-pair to cognate pre-crRNA repeats, an essential function for crRNA maturation and DNA silencing by dual-RNA:Cas9. The large panel of tracrRNA and Cas9 ortholog sequences should constitute a useful database to improve the design of RNA-programmable Cas9 as genome editing tool.

  • 25.
    Chylinski, Krzysztof
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max F. Perutz Laboratories, University of Vienna, Austria .
    Makarova, Kira S.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany ; Hannover Medical School, Hannover, Germany .
    Koonin, Eugene V.
    Classification and evolution of type II CRISPR-Cas systems2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 10, p. 6091-6105Article in journal (Refereed)
    Abstract [en]

    The CRISPR-Cas systems of archaeal and bacterial adaptive immunity are classified into three types that differ by the repertoires of CRISPR-associated (cas) genes, the organization of cas operons and the structure of repeats in the CRISPR arrays. The simplest among the CRISPR-Cas systems is type II in which the endonuclease activities required for the interference with foreign deoxyribonucleic acid (DNA) are concentrated in a single multidomain protein, Cas9, and are guided by a co-processed dual-tracrRNA: crRNA molecule. This compact enzymatic machinery and readily programmable site-specific DNA targeting make type II systems top candidates for a new generation of powerful tools for genomic engineering. Here we report an updated census of CRISPR-Cas systems in bacterial and archaeal genomes. Type II systems are the rarest, missing in archaea, and represented in similar to 5% of bacterial genomes, with an over-representation among pathogens and commensals. Phylogenomic analysis suggests that at least three cas genes, cas1, cas2 and cas4, and the CRISPR repeats of the type II-B system were acquired via recombination with a type I CRISPR-Cas locus. Distant homologs of Cas9 were identified among proteins encoded by diverse transposons, suggesting that type II CRISPR-Cas evolved via recombination of mobile nuclease genes with type I loci.

  • 26.
    Deltcheva, Elitza
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Chylinski, Krzysztof
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sharma, Cynthia M
    Gonzales, Karine
    Chao, Yanjie
    Pirzada, Zaid A
    Eckert, Maria R
    Vogel, Jörg
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III2011In: Nature, Vol. 471, no 7340, p. 602-607Article in journal (Refereed)
    Abstract [en]

    CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence foreign nucleic acids in a sequence-specific manner. Although the maturation of crRNAs represents a key event in CRISPR activation, the responsible endoribonucleases (CasE, Cas6, Csy4) are missing in many CRISPR/Cas subtypes. Here, differential RNA sequencing of the human pathogen Streptococcus pyogenes uncovered tracrRNA, a trans-encoded small RNA with 24-nucleotide complementarity to the repeat regions of crRNA precursor transcripts. We show that tracrRNA directs the maturation of crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated Csn1 protein; all these components are essential to protect S. pyogenes against prophage-derived DNA. Our study reveals a novel pathway of small guide RNA maturation and the first example of a host factor (RNase III) required for bacterial RNA-mediated immunity against invaders.

  • 27.
    Doudna, Jennifer A.
    et al.
    Univ Calif Berkeley, Berkeley, CA 94720 USA.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Helmholtz Ctr Infect Res, Dept Regulat Infect Biol, D-38124 Braunschweig, Germany; Hannover Med Sch, D-30625 Hannover, Germany.
    The new frontier of genome engineering with CRISPR-Cas92014In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 346, no 6213, p. 1077-+Article, review/survey (Refereed)
    Abstract [en]

    The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

  • 28. Eggenschwiler, Reto
    et al.
    Moslem, Mohsen
    Fráguas, Mariane Serra
    Galla, Melanie
    Papp, Oliver
    Naujock, Maximilian
    Fonfara, Ines
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, Berlin, Germany.
    Gensch, Ingrid
    Wähner, Annabell
    Beh-Pajooh, Abbas
    Mussolino, Claudio
    Tauscher, Marcel
    Steinemann, Doris
    Wegner, Florian
    Petri, Susanne
    Schambach, Axel
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Cathomen, Toni
    Cantz, Tobias
    Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 38198Article in journal (Refereed)
    Abstract [en]

    Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puro Delta tk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells.

  • 29. Elsholz, Alexander K. W.
    et al.
    Birk, Marlene S.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Humboldt University, Berlin, Germany.
    Turgay, Kuersad
    Functional Diversity of AAA plus Protease Complexes in Bacillus subtilis2017In: Frontiers in Molecular Biosciences, ISSN 2296-889X, Vol. 4, article id 44Article, review/survey (Refereed)
    Abstract [en]

    Here, we review the diverse roles and functions of AAA+ protease complexes in protein homeostasis, control of stress response and cellular development pathways by regulatory and general proteolysis in the Gram-positive model organism Bacillus subtilis. We discuss in detail the intricate involvement of AAA+ protein complexes in controlling sporulation, the heat shock response and the role of adaptor proteins in these processes. The investigation of these protein complexes and their adaptor proteins has revealed their relevance for Gram-positive pathogens and their potential as targets for new antibiotics.

  • 30. Fineran, Peter C.
    et al.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Memory of viral infections by CRISPR-Cas adaptive immune systems: acquisition of new information2012In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 434, no 2, p. 202-209Article, review/survey (Refereed)
    Abstract [en]

    Multiple organisms face the threat of viral infections. To combat phage invasion, bacteria and archaea have evolved an adaptive mechanism of protection against exogenic mobile genetic elements, called CRISPR-Cas. In this defense strategy, phage infection is memorized via acquisition of a short invader sequence, called a spacer, into the CRISPR locus of the host genome. Upon repeated infection, the 'vaccinated' host expresses the spacer as a precursor RNA, which is processed into a mature CRISPR RNA (crRNA) that guides an endonuclease to the matching invader for its ultimate destruction. Recent efforts have uncovered molecular details underlying the crRNA biogenesis and interference steps. However, until recently the step of adaptation had remained largely uninvestigated. In this minireview, we focus on recent publications that have begun to reveal molecular insights into the adaptive step of CRISPR-Cas immunity, which is required for the development of the heritable memory of the host against viruses. 

  • 31.
    Fonfara, Ines
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany.
    Le Rhun, Anaïs
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany.
    Chylinski, Krzysztof
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Deptartment of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Austria.
    Makarova, Kira S.
    Lécrivain, Anne-Laure
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bzdrenga, Janek
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Koonin, Eugene V.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany ; Hannover Medical School, Hannover, Germany .
    Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 4, p. 2577-2590Article in journal (Refereed)
    Abstract [en]

    The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA: crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA: Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool.

  • 32.
    Fonfara, Ines
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig 38124, Germany; 3Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, Berlin 10117, Germany.
    Richter, Hagen
    Bratovic, Majda
    Le Rhun, Anaïs
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig 38124, Germany; 3Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, Berlin 10117, Germany.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig 38124, Germany; 3Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, Berlin 10117, Germany.
    The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA2016In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 532, no 7600, p. 517-520Article in journal (Refereed)
    Abstract [en]

    CRISPR-Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA(1). The well-studied types I, II and III utilize a set of distinct CRISPR-associated ( Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA)(2-5) and the mature crRNAs then guide a complex of Cas proteins ( Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA(6-12). In type II systems, RNase III cleaves pre-crRNA base-paired with trans-activating crRNA (tracrRNA) in the presence of Cas9 (refs 13, 14). The mature tracrRNA-crRNA duplex then guides Cas9 to cleave target DNA15. Here, we demonstrate a novel mechanism in CRISPR-Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5'-YTN- 3' protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5' overhang(16). The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V- A constitutes the most minimalistic of the CRISPR- Cas systems so far described.

  • 33. Fuhrmann, Jakob
    et al.
    Mierzwa, Beata
    Trentini, Debora B.
    Spiess, Silvia
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Lehner, Anita
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Clausen, Tim
    Structural Basis for Recognizing Phosphoarginine and Evolving Residue-Specific Protein Phosphatases in Gram-Positive Bacteria2013In: Cell Reports, ISSN 2211-1247, Vol. 3, no 6, p. 1832-1839Article in journal (Refereed)
    Abstract [en]

    Many cellular pathways are regulated by the competing activity of protein kinases and phosphatases. The recent identification of arginine phosphorylation as a protein modification in bacteria prompted us to analyze the molecular basis of targeting phosphoarginine. In this work, we characterize an annotated tyrosine phosphatase, YwlE, that counteracts the protein arginine kinase McsB. Strikingly, structural studies of YwlE reaction intermediates provide a direct view on a captured arginine residue. Together with biochemical data, the crystal structures depict the evolution of a highly specific phospho-arginine phosphatase, with the use of a size-and-polarity filter for distinguishing phosphorylated arginine from other phosphorylated side chains. To confirm the proposed mechanism, we performed bioinformatic searches for phosphatases, employing a similar selectivity filter, and identified a protein in Drosophila melanogaster exhibiting robust arginine phosphatase activity. In sum, our findings uncover the molecular framework for specific targeting of phospho-arginine and suggest that protein arginine (de) phosphorylation may be relevant in eukaryotes.

  • 34. Fuhrmann, Jakob
    et al.
    Schmidt, Andreas
    Spiess, Silvia
    Lehner, Anita
    Turgay, Kürsad
    Mechtler, Karl
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Clausen, Tim
    McsB is a protein arginine kinase that phosphorylates and inhibits the heat-shock regulator CtsR2009In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 324, no 5932, p. 1323-1327Article in journal (Refereed)
    Abstract [en]

    All living organisms face a variety of environmental stresses that cause the misfolding and aggregation of proteins. To eliminate damaged proteins, cells developed highly efficient stress response and protein quality control systems. We performed a biochemical and structural analysis of the bacterial CtsR/McsB stress response. The crystal structure of the CtsR repressor, in complex with DNA, pinpointed key residues important for high-affinity binding to the promoter regions of heat-shock genes. Moreover, biochemical characterization of McsB revealed that McsB specifically phosphorylates arginine residues in the DNA binding domain of CtsR, thereby impairing its function as a repressor of stress response genes. Identification of the CtsR/McsB arginine phospho-switch expands the repertoire of possible protein modifications involved in prokaryotic and eukaryotic transcriptional regulation.

  • 35. Garnier, Fabien
    et al.
    Janapatla, Rajendra Prasad
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Masson, Geoffrey
    Grélaud, Carole
    Stach, Jean François
    Denis, François
    Ploy, Marie-Cécile
    Insertion sequence 1515 in the ply gene of a type 1 clinical isolate of Streptococcus pneumoniae abolishes pneurnolysin expression2007In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 45, no 7, p. 2296-2297Article in journal (Refereed)
    Abstract [en]

    Abstract: A serotype 1 Streptococcus pneumoniae strain isolated by blood culture from a woman with pneumonia was found to harbor insertion sequence (IS) 1515 in the pneumolysin gene, abolishing pneumolysin expression. To our knowledge, this is the first report of an IS in the pneumolysin gene of S. pneumoniae.

  • 36. Gratz, Nina
    et al.
    Siller, Maria
    Schaljo, Barbara
    Pirzada, Zaid A
    Gattermeier, Irene
    Vojtek, Ivo
    Kirschning, Carsten J
    Wagner, Hermann
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kovarik, Pavel
    Akira, Shizuo
    Group A streptococcus activates type I interferon production and MyD88-dependent signaling without involvement of TLR2, TLR4, and TLR92008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 29, p. 19879-19887Article in journal (Refereed)
    Abstract [en]

    Bacterial pathogens are recognized by the innate immune system through pattern recognition receptors, such as Toll-like receptors (TLRs). Engagement of TLRs triggers signaling cascades that launch innate immune responses. Activation of MAPKs and NF-kappaB, elements of the major signaling pathways induced by TLRs, depends in most cases on the adaptor molecule MyD88. In addition, Gram-negative or intracellular bacteria elicit MyD88-independent signaling that results in production of type I interferon (IFN). Here we show that in mouse macrophages, the activation of MyD88-dependent signaling by the extracellular Gram-positive human pathogen group A streptococcus (GAS; Streptococcus pyogenes) does not require TLR2, a receptor implicated in sensing of Gram-positive bacteria, or TLR4 and TLR9. Redundant engagement of either of these TLR molecules was excluded by using TLR2/4/9 triple-deficient macrophages. We further demonstrate that infection of macrophages by GAS causes IRF3 (interferon-regulatory factor 3)-dependent, MyD88-independent production of IFN. Surprisingly, IFN is induced also by GAS lacking slo and sagA, the genes encoding cytolysins that were shown to be required for IFN production in response to other Gram-positive bacteria. Our data indicate that (i) GAS is recognized by a MyD88-dependent receptor other than any of those typically used by bacteria, and (ii) GAS as well as GAS mutants lacking cytolysin genes induce type I IFN production by similar mechanisms as bacteria requiring cytoplasmic escape and the function of cytolysins.

  • 37. Heckl, Dirk
    et al.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Germany ; Department of Regulation in Infection Biology, Hannover Medical School, Hannover, Germany.
    Toward whole-transcriptome editing with CRISPR-Cas92015In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 58, no 4, p. 560-562Article in journal (Other academic)
    Abstract [en]

    Targeted regulation of gene expression holds huge promise for biomedical research. In a series of recent publications (Gilbert et al., 2014; Konermann et al., 2015; Zalatan et al., 2015), sophisticated, multiplex-compatible transcriptional activator systems based on the CRISPR-Cas9 technology and genome-scale libraries advance the field toward whole-transcriptome control.

  • 38. Hille, Frank
    et al.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin 10117, Germany.
    CRISPR-Cas: biology, mechanisms and relevance2016In: Philosophical Transactions of the Royal Society of London. Biological Sciences, ISSN 0962-8436, E-ISSN 1471-2970, Vol. 371, no 1707, article id 20150496Article, review/survey (Refereed)
    Abstract [en]

    Prokaryotes have evolved several defence mechanisms to protect themselves from viral predators. Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) display a prokaryotic adaptive immune system that memorizes previous infections by integrating short sequences of invading genomes-termed spacers-into the CRISPR locus. The spacers interspaced with repeats are expressed as small guide CRISPR RNAs (crRNAs) that are employed by Cas proteins to target invaders sequence-specifically upon a reoccurring infection. The ability of the minimal CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new avenues in genome editing in a broad range of cells and organisms with high potential in therapeutical applications. While numerous scientific studies have shed light on the biochemical processes behind CRISPR-Cas systems, several aspects of the immunity steps, however, still lack sufficient understanding. This review summarizes major discoveries in the CRISPR-Cas field, discusses the role of CRISPR-Cas in prokaryotic immunity and other physiological properties, and describes applications of the system as a DNA editing technology and antimicrobial agent. This article is part of the themed issue 'The new bacteriology'.

  • 39. Hille, Frank
    et al.
    Richter, Hagen
    Wong, Shi Pey
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Institute for Biology, Humboldt University, Germany.
    Bratovic, Majda
    Ressel, Sarah
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Institute for Biology, Humboldt University, Germany.
    The Biology of CRISPR-Cas: Backward and Forward2018In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 172, no 6, p. 1239-1259Article, review/survey (Refereed)
    Abstract [en]

    In bacteria and archaea, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system against phages and other foreign genetic elements. Here, we review the biology of the diverse CRISPR-Cas systems and the major progress achieved in recent years in understanding the underlying mechanisms of the three stages of CRISPR-Cas immunity: adaptation, crRNA biogenesis, and interference. The ecology and regulation of CRISPR-Cas in the context of phage infection, the roles of these systems beyond immunity, and the open questions that propel the field forward are also discussed.

  • 40. Hurwitz, Julia L.
    et al.
    Jones, Bart G.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Institute for Infection Biology, Berlin, Germany; Humboldt University, Berlin, Germany.
    Woodland, David L.
    Hypothesis: RNA and DNA Viral Sequence Integration into the Mammalian Host Genome Supports Long-Term B Cell and T Cell Adaptive Immunity2017In: Viral immunology, ISSN 0882-8245, E-ISSN 1557-8976, Vol. 30, no 9, p. 628-632Article in journal (Other academic)
    Abstract [en]

    Viral sequence integration into the mammalian genome has long been perceived as a health risk. In some cases, integration translates to chronic viral infection, and in other instances, oncogenic gene mutations occur. However, research also shows that animal cells can benefit from integrated viral sequences (e.g., to support host cell development or to silence foreign invaders). Here we propose that, comparable with the clustered regularly interspaced short palindromic repeats that provide bacteria with adaptive immunity against invasive bacteriophages, animal cells may co-opt integrated viral sequences to support immune memory. We hypothesize that host cells express viral peptides from open reading frames in integrated sequences to boost adaptive B cell and T cell responses long after replicating viruses are cleared. In support of this hypothesis, we examine previous literature describing (1) viruses that infect acutely (e.g., vaccinia viruses and orthomyxoviruses) followed by unexplained, long-term persistence of viral nucleotide sequences, viral peptides, and virus-specific adaptive immunity, (2) the high frequency of endogenous viral genetic elements found in animal genomes, and (3) mechanisms with which animal host machinery supports foreign sequence integration.

  • 41. Jinek, Martin
    et al.
    Chylinski, Krzysztof
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fonfara, Ines
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hauer, Michael
    Doudna, Jennifer A
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity2012In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 337, no 6096, p. 816-821Article in journal (Refereed)
    Abstract [en]

    Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

  • 42. Jinek, Martin
    et al.
    Jiang, Fuguo
    Taylor, David W.
    Sternberg, Samuel H.
    Kaya, Emine
    Ma, Enbo
    Anders, Carolin
    Hauer, Michael
    Zhou, Kaihong
    Lin, Steven
    Kaplan, Matias
    Iavarone, Anthony T.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Nogales, Eva
    Doudna, Jennifer A.
    Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation2014In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 343, no 6176, p. 1215-Article in journal (Refereed)
    Abstract [en]

    Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

  • 43. Labenski, Verena
    et al.
    Suerth, Julia D
    Barczak, Elke
    Heckl, Dirk
    Levy, Camille
    Bernadin, Ornellie
    Charpentier, Emmanuelle
    Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Williams, David A
    Fehse, Boris
    Verhoeyen, Els
    Schambach, Axel
    Alpharetroviral self-inactivating vectors produced by a superinfection-resistant stable packaging cell line allow genetic modification of primary human T lymphocytes.2016In: Biomaterials, ISSN 0142-9612, E-ISSN 1878-5905, Vol. 97, p. 97-109Article in journal (Refereed)
    Abstract [en]

    Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses. We developed an ARV platform for the transduction of primary human T lymphocytes. We demonstrated functional transgene transfer using the clinically relevant herpes-simplex-virus thymidine kinase variant TK.007. Proof-of-concept of alpharetroviral-mediated T-lymphocyte engineering was shown in vitro and in a humanized transplantation model in vivo. Furthermore, we established a stable, human alpharetroviral packaging cell line in which we deleted the entry receptor (SLC1A5) for RD114/TR-pseudotyped ARVs to prevent superinfection and enhance genomic integrity of the packaging cell line and viral particles. We showed that superinfection can be entirely prevented, while maintaining high recombinant virus titers. Taken together, this resulted in an improved production platform representing an economic strategy for translating the promising features of ARVs for therapeutic T-lymphocyte engineering.

  • 44. Labuhn, Maurice
    et al.
    Adams, Felix F.
    Ng, Michelle
    Knoess, Sabine
    Schambach, Axel
    Charpentier, Emmanuelle M.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Schwarzer, Adrian
    Mateo, Juan L.
    Klusmann, Jan-Henning
    Heckl, Dirk
    Refined sgRNA efficacy prediction improves large- and small-scale CRISPR-Cas9 applications2018In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 3, p. 1375-1385Article in journal (Refereed)
    Abstract [en]

    Genome editing with the CRISPR-Cas9 system has enabled unprecedented efficacy for reverse genetics and gene correction approaches. While off-target effects have been successfully tackled, the effort to eliminate variability in sgRNA efficacies-which affect experimental sensitivity-is in its infancy. To address this issue, studies have analyzed the molecular features of highly active sgRNAs, but independent cross-validation is lacking. Utilizing fluorescent reporter knock-out assays with verification at selected endogenous loci, we experimentally quantified the target efficacies of 430 sgRNAs. Based on this dataset we tested the predictive value of five recently-established prediction algorithms. Our analysis revealed a moderate correlation (r = 0.04 to r = 0.20) between the predicted and measured activity of the sgRNAs, and modest concordance between the different algorithms. We uncovered a strong PAM-distal GC-content-dependent activity, which enabled the exclusion of inactive sgRNAs. By deriving nine additional predictive features we generated a linear model-based discrete system for the efficient selection (r = 0.4) of effective sgRNAs (CRISPRater). We proved our algorithms' efficacy on small and large external datasets, and provide a versatile combined on-and off-target sgRNA scanning platform. Altogether, our study highlights current issues and efforts in sgRNA efficacy prediction, and provides an easily-applicable discrete system for selecting efficient sgRNAs.

  • 45.
    Latvala, S.
    et al.
    Natl Inst Hlth & Welf, Dept Infect Dis Surveillance & Control, Virol Unit, Helsinki, Finland.
    Makela, S. M.
    Natl Inst Hlth & Welf, Dept Infect Dis Surveillance & Control, Virol Unit, Helsinki, Finland.
    Miettinen, M.
    Valio Ltd R&D, Helsinki, Finland.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Julkunen, I.
    Natl Inst Hlth & Welf THL, Dept Infect Dis Surveillance & Control, Virol Unit, Helsinki 00271, Finland; Univ Turku, Dept Virol, Turku, Finland.
    Dynamin inhibition interferes with inflammasome activation and cytokine gene expression in Streptococcus pyogenes-infected human macrophages2014In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 178, no 2, p. 320-333Article in journal (Refereed)
    Abstract [en]

    In the present study, we have analysed the ability of Streptococcus pyogenes [Group A streptococcus (GAS)] to activate the NACHT-domain-, leucinerich repeat-and PYD-containing protein 3 (NALP3) inflammasome complex in human monocyte-derived macrophages and the molecules and signalling pathways involved in GAS-induced inflammatory responses. We focused upon analysing the impact of dynamin-dependent endocytosis and the role of major streptococcal virulence factors streptolysin O (SLO) and streptolysin S (SLS) in the immune responses induced by GAS. These virulence factors are involved in immune evasion by forming pores in host cell membranes, and aid the bacteria to escape from the endosome-lysosome pathway. We analysed cytokine gene expression in human primary macrophages after stimulation with live or inactivated wild-type GAS as well as with live SLO and SLS defective bacteria. Interleukin (IL)-1 beta, IL-10, tumour necrosis factor (TNF)-alpha and chemokine (C-X-C motif) ligand (CXCL)-10 cytokines were produced after bacterial stimulation in a dose-dependent manner and no differences in cytokine levels were seen between live, inactivated or mutant bacteria. These data suggest that streptolysins or other secreted bacterial products are not required for the inflammatory responses induced by GAS. Our data indicate that inhibition of dynamin-dependent endocytosis in macrophages attenuates the induction of IL-1 beta, TNF-alpha, interferon (IFN)-beta and CXCL-10 mRNAs. We also observed that pro-IL-1 beta protein was expressed and efficiently cleaved into mature-IL-1 beta via inflammasome activation after bacterial stimulation. Furthermore, we demonstrate that multiple signalling pathways are involved in GAS-stimulated inflammatory responses in human macrophages.

  • 46.
    Le Rhun, Anais
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, D-10117 Berlin, Germany; Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, D-38124 Braunschweig, Germany.
    Lecrivain, Anne-Laure
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, D-10117 Berlin, Germany.
    Reimegard, Johan
    Proux-Wera, Estelle
    Broglia, Laura
    Della Beffa, Cristina
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, D-10117 Berlin, Germany; Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, D-38124 Braunschweig, Germany; Humboldt University, D-10115 Berlin, Germany.
    Identification of endoribonuclease specific cleavage positions reveals novel targets of RNase III in Streptococcus pyogenes2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 5, p. 2329-2340Article in journal (Refereed)
    Abstract [en]

    A better understanding of transcriptional and post-transcriptional regulation of gene expression in bacteria relies on studying their transcriptome. RNA sequencing methods are used not only to assess RNA abundance but also the exact boundaries of primary and processed transcripts. Here, we developed a method, called identification of specific cleavage position (ISCP), which enables the identification of direct endoribonuclease targets in vivo by comparing the 5' and 3' ends of processed transcripts between wild type and RNase deficient strains. To demonstrate the ISCP method, we used as a model the double-stranded specific RNase III in the human pathogen Streptococcus pyogenes. We mapped 92 specific cleavage positions (SCPs) among which, 48 were previously described and 44 are new, with the characteristic 2 nucleotides 3' overhang of RNase III. Most SCPs were located in untranslated regions of RNAs. We screened for RNase III targets using transcriptomic differential expression analysis (DEA) and compared those with the RNase III targets identified using the ISCP method. Our study shows that in S. pyogenes, under standard growth conditions, RNase III has a limited impact both on antisense transcripts and on global gene expression with the expression of most of the affected genes being downregulated in an RNase III deletion mutant.

  • 47.
    Le Rhun, Anaïs
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Helmholtz Centre for Infection Research (HZI), Department of Regulation in Infection Biology, D-38124 Braunschweig, Germany.
    Beer, Yan Yan
    Helmholtz Centre for Infection Research (HZI), Department of Regulation in Infection Biology, D-38124 Braunschweig, Germany.
    Reimegård, Johan
    Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, S-75003 Uppsala, Sweden.
    Chylinski, Krzysztof
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Max F. Perutz Laboratories (MFPL), University of Vienna, A-1030 Vienna, Austria.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Helmholtz Centre for Infection Research (HZI), Department of Regulation in Infection Biology, Germany; Hannover Medical School (MHH), Hannover, Germany; Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, Berlin, Germany.
    RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes2016In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 13, no 2, p. 177-195Article in journal (Refereed)
    Abstract [en]

    Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.

  • 48.
    Le Rhun, Anaïs
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Small RNAs in streptococci2012In: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 9, no 4, p. 414-426Article in journal (Refereed)
    Abstract [en]

    The group of streptococci includes species responsible for severe diseases in humans. To adapt to their environment and infect their hosts, streptococci depend on precise regulation of gene expression. The last decade has witnessed increasing findings of small RNAs (sRNAs) having regulatory functions in bacteria. More recently, genome-wide screens revealed that streptococcal genomes also encode multiple sRNAs. Some sRNAs including the class of CRISPR RNAs (crRNAs) play critical roles in streptococcal adaptation and virulence. Analysis of sRNA mechanisms uncovered three sRNAs that target in trans mRNA (FasX), sRNA (tracrRNA) and DNA (crRNA). Overall, the current understanding of sRNA-mediated regulation in streptococci remains very limited. Given the complexity of regulatory networks and the number of recently predicted sRNAs, future research should reveal new functions and mechanisms for the streptococcal sRNAs. Here, we provide a comprehensive summary of the information available on the topic.

  • 49.
    Lécrivain, Anne-Laure
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Unit for the Science of Pathogens, D-10117 Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, D-10117 Berlin, Germany..
    Broglia, Laura
    Renault, Thibaud
    Hahnke, Karin
    Ahmed-Begrich, Rina
    Le Rhun, Anaïs
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Interplay between 3′-to-5′ exoRNases and RNase Y in Streptococcus pyogenes2018Manuscript (preprint) (Other academic)
  • 50.
    Lécrivain, Anne-Laure
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Le Rhun, Anaïs
    Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Renault, Thibaud T.
    Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; nstitute for Biology, Humboldt University, Berlin, Germany .
    Ahmed-Begrich, Rina
    Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Hahnke, Karin
    Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Charpentier, Emmanuelle
    Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; nstitute for Biology, Humboldt University, Berlin, Germany.
    In vivo 3′-to-5′ exoribonuclease targetomes of Streptococcus pyogenes2018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 46, p. 11814-11819Article in journal (Refereed)
    Abstract [en]

    mRNA decay plays an essential role in the control of gene expression in bacteria. Exoribonucleases (exoRNases), which trim transcripts starting from the 5′ or 3′ end, are particularly important to fully degrade unwanted transcripts and renew the pool of nucleotides available in the cell. While recent techniques have allowed genome-wide identification of ribonuclease (RNase) targets in bacteria in vivo, none of the 3′-to-5′ exoRNase targetomes (i.e., global processing sites) have been studied so far. Here, we report the targetomes of YhaM, polynucleotide phosphorylase (PNPase), and RNase R of the human pathogen Streptococcus pyogenes. We determined that YhaM is an unspecific enzyme that trims a few nucleotides and targets the majority of transcript ends, generated either by transcription termination or by endonucleolytic activity. The molecular determinants for YhaM-limited processivity are yet to be deciphered. We showed that PNPase clears the cell from mRNA decay fragments produced by endoribonucleases (endoRNases) and is the major 3′-to-5′ exoRNase for RNA turnover in S. pyogenes. In particular, PNPase is responsible for the degradation of regulatory elements from 5′ untranslated regions. However, we observed little RNase R activity in standard culture conditions. Overall, our study sheds light on the very distinct features of S. pyogenes 3′-to-5′ exoRNases.

12 1 - 50 of 71
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