title: A PNPase dependent CRISPR System in Listeria.
abstract: The human bacterial pathogen Listeria monocytogenes is emerging as a model organism to study RNA-mediated regulation in pathogenic bacteria. A class of non-coding RNAs called CRISPRs (clustered regularly interspaced short palindromic repeats) has been described to confer bacterial resistance against invading bacteriophages and conjugative plasmids. CRISPR function relies on the activity of CRISPR associated (cas) genes that encode a large family of proteins with nuclease or helicase activities and DNA and RNA binding domains. Here, we characterized a CRISPR element (RliB) that is expressed and processed in the L. monocytogenes strain EGD-e, which is completely devoid of cas genes. Structural probing revealed that RliB has an unexpected secondary structure comprising basepair interactions between the repeats and the adjacent spacers in place of canonical hairpins formed by the palindromic repeats. Moreover, in contrast to other CRISPR-Cas systems identified in Listeria, RliB-CRISPR is ubiquitously present among Listeria genomes at the same genomic locus and is never associated with the cas genes. We showed that RliB-CRISPR is a substrate for the endogenously encoded polynucleotide phosphorylase (PNPase) enzyme. The spacers of the different Listeria RliB-CRISPRs share many sequences with temperate and virulent phages. Furthermore, we show that a cas-less RliB-CRISPR lowers the acquisition frequency of a plasmid carrying the matching protospacer, provided that trans encoded cas genes of a second CRISPR-Cas system are present in the genome. Importantly, we show that PNPase is required for RliB-CRISPR mediated DNA interference. Altogether, our data reveal a yet undescribed CRISPR system whose both processing and activity depend on PNPase, highlighting a new and unexpected function for PNPase in "CRISPRology".
authors.affiliation: Unité des Interactions Bactéries-Cellules, Institut Pasteur, Paris, France ; INSERM, U604, Paris, France ; INRA, USC2020, Paris, France.
authors.forename: Nina
authors.initials: N
authors.lastname: Sesto
authors.affiliation: Unité de Génomique Evolutive des Microbes, Institut Pasteur, Paris, France ; CNRS, UMR3525, Paris, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Touchon
authors.affiliation: Control of Gene Expression, Instituto de Tecnologia Química e Biológica, Oeiras, Portugal.
authors.forename: José Marques
authors.initials: JM
authors.lastname: Andrade
authors.affiliation: Architecture et Réactivité de l'ARN Université de Strasbourg, CNRS, Strasbourg, France.
authors.forename: Jiro
authors.initials: J
authors.lastname: Kondo
authors.affiliation: Unité de Génomique Evolutive des Microbes, Institut Pasteur, Paris, France ; CNRS, UMR3525, Paris, France.
authors.forename: Eduardo P C
authors.initials: EP
authors.lastname: Rocha
authors.affiliation: Control of Gene Expression, Instituto de Tecnologia Química e Biológica, Oeiras, Portugal.
authors.forename: Cecilia Maria
authors.initials: CM
authors.lastname: Arraiano
authors.affiliation: Unité des Interactions Bactéries-Cellules, Institut Pasteur, Paris, France ; INSERM, U604, Paris, France ; INRA, USC2020, Paris, France.
authors.forename: Cristel
authors.initials: C
authors.lastname: Archambaud
authors.affiliation: Architecture et Réactivité de l'ARN Université de Strasbourg, CNRS, Strasbourg, France.
authors.forename: Éric
authors.initials: É
authors.lastname: Westhof
authors.affiliation: Architecture et Réactivité de l'ARN Université de Strasbourg, CNRS, Strasbourg, France.
authors.forename: Pascale
authors.initials: P
authors.lastname: Romby
authors.affiliation: Unité des Interactions Bactéries-Cellules, Institut Pasteur, Paris, France ; INSERM, U604, Paris, France ; INRA, USC2020, Paris, France.
authors.forename: Pascale
authors.initials: P
authors.lastname: Cossart
mesh_terms: D001426:Bacterial Proteins; D064113:CRISPR-Cas Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D016680:Genome, Bacterial; D006801:Humans; D008089:Listeria monocytogenes / Q000235:genetics*; D011117:Polyribonucleotide Nucleotidyltransferase / Q000235:genetics* / Q000378:metabolism; D022661:RNA, Untranslated / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D022661:RNA, Untranslated; D016601:RNA-Binding Proteins; D011117:Polyribonucleotide Nucleotidyltransferase