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”.
Highlights
Listeria monocytogenes is a gram-positive foodborne pathogenic bacterium that has evolved two distinct lifestyles: a saprophytic one, primarily in decaying vegetation and a parasitic one in the tissues of mammals and birds, causing a disease known as listeriosis
CRISPR-Cas systems confer to bacteria and archaea an adaptive immunity that protects them against invading bacteriophages and plasmids
It is an unusual CRISPR that, as we demonstrate, has a secondary structure consisting of basepair interactions between the repeat sequence and the adjacent spacer
Summary
Listeria monocytogenes is a gram-positive foodborne pathogenic bacterium that has evolved two distinct lifestyles: a saprophytic one, primarily in decaying vegetation and a parasitic one in the tissues of mammals and birds, causing a disease known as listeriosis. In addition to protein determinants contributing to infection, Listeria possesses a virulence gene repertoire that expands to non-coding RNA (ncRNAs) molecules [2,3,4]. Bacterial ncRNAs are key regulatory molecules of metabolic, physiological and pathogenic processes and can be generally classified in four groups: a) the RNA regulatory elements located in the 59 untranslated regions (59UTRs) which regulate the expression of the corresponding mRNAs through the binding of various factors, like proteins (e.g. CsrA) and small metabolites (riboswitches) or by sensing environmental cues like temperature (thermosensors); b) the trans-acting small RNAs (sRNAs) regulating one or several target mRNAs located elsewhere on the chromosome; c) the sRNAs that sequester RNA-binding proteins; and d) the antisense transcripts (asRNAs), which overlap and are complementary to their target genes in the same genomic locus [5]. A CRISPR is defined by the alternating array of identical 20–40 nucleotides (nt) long repeat sequences, interspaced by non-repetitive spacer
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