Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS. Mol Microbiol

Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany.
Molecular Microbiology (Impact Factor: 4.42). 03/2010; 75(6):1495-512. DOI: 10.1111/j.1365-2958.2010.07073.x
Source: PubMed


Inheritable bacterial defence systems against phage infection and foreign DNA, termed CRISPR (clustered regularly interspaced short palindromic repeats), consist of cas protein genes and repeat arrays interspaced with sequences originating from invaders. The Cas proteins together with processed small spacer-repeat transcripts (crRNAs) cause degradation of penetrated foreign DNA by unknown mechanisms. Here, we have characterized previously unidentified promoters of the Escherichia coli CRISPR arrays and cas protein genes. Transcription of precursor crRNA is directed by a promoter located within the CRISPR leader. A second promoter, directing cas gene transcription, is located upstream of the genes encoding proteins of the Cascade complex. Furthermore, we demonstrate that the DNA-binding protein H-NS is involved in silencing the CRISPR-cas promoters, resulting in cryptic Cas protein expression. Our results demonstrate an active involvement of H-NS in the induction of the CRISPR-cas system and suggest a potential link between two prokaryotic defence systems against foreign DNA.

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    • "The CRISPR array, or spacer array, is composed of direct repeat sequences that are interspaced with unique spacer sequences that are typically derived from mobile genetic elements such as bacteriophages and plasmids (Barrangou et al., 2007; Bolotin et al., 2005; Mojica et al., 2005; Pourcel et al., 2005). The AT-rich leader sequence lies directly upstream of each array and is thought to function as a promoter (Jansen et al., 2002; Pul et al., 2010). The spacer array is transcribed and processed into small CRISPR RNAs (crRNAs) each of which consists of the spacer flanked by portions of the direct repeat (Brouns et al., 2008; Hale et al., 2008, 2009; Lillestøl et al., 2006). "
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    ABSTRACT: Prokaryotic CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated genes) systems provide adaptive immunity from invasive genetic elements and encompass three essential features: i) cas genes, ii) a CRISPR array composed of spacers and direct repeats and iii) an AT-rich leader sequence upstream of the array. We performed in-depth sequence analysis of the CRISPR-Cas systems in >600 Salmonella, representing four clinically prevalent serovars. Each CRISPR-Cas feature is extremely conserved in the Salmonella, and the CRISPR1 locus is more highly conserved than CRISPR2. Array composition is serovar-specific, though no convincing evidence of recent spacer acquisition against exogenous nucleic acids exists. Only 12% of spacers match phage and plasmid sequences and self-targeting spacers are associated with direct repeat variants. High nucleotide identity (>99.9%) exists across the cas operon among isolates of a single serovar and in some cases this conservation extends across divergent serovars. These observations reflect historical CRISPR-Cas immune activity, showing that this locus has ceased undergoing adaptive events. Intriguingly, the high level of conservation across divergent serovars shows that the genetic integrity of these inactive loci is maintained over time, contrasting with the canonical view that inactive CRISPR loci degenerate over time. This thorough characterization of Salmonella CRISPR-Cas systems presents new insights into Salmonella CRISPR evolution, particularly with respect to cas gene conservation, leader sequences, organization of direct repeats and protospacer matches. Collectively, our data suggests that Salmonella CRISPR-Cas systems are no longer immunogenic; rather their impressive conservation indicates they may have an alternative function in Salmonella.
    Microbiology 12/2014; 161(Pt_2). DOI:10.1099/mic.0.000005 · 2.56 Impact Factor
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    • "Recent studies have shed light into the process of the adaptation, which is still the least understood stage of the CRISPR–Cas pathway (16–20). Usually, the repeat-spacer clusters are preceded by an AT-rich leader region that harbors the promoter for transcription of the array (21,22). It was consistently reported that the incorporation of new spacer occurs immediately next to the leader, pointing to a direct involvement of leader sequences in spacer uptake (18,23–25). "
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    ABSTRACT: The adaptation against foreign nucleic acids by the CRISPR–Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) depends on the insertion of foreign nucleic acid-derived sequences into the CRISPR array as novel spacers by still unknown mechanism. We identified and characterized in Escherichia coli intermediate states of spacer integration and mapped the integration site at the chromosomal CRISPR array in vivo. The results show that the insertion of new spacers occurs by site-specific nicking at both strands of the leader proximal repeat in a staggered way and is accompanied by joining of the resulting 5′-ends of the repeat strands with the 3′-ends of the incoming spacer. This concerted cleavage-ligation reaction depends on the metal-binding center of Cas1 protein and requires the presence of Cas2. By acquisition assays using plasmid-located CRISPR array with mutated repeat sequences, we demonstrate that the primary sequence of the first repeat is crucial for cleavage of the CRISPR array and the ligation of new spacer DNA.
    Nucleic Acids Research 06/2014; 42(12). DOI:10.1093/nar/gku510 · 9.11 Impact Factor
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    • "From a mechanistic point of view, this arrangement guarantees a streamlined processing of the pre-crRNA transcripts, immediate crRNA protection and the scanning for the complementary DNA target, localized within one protein complex. Additionally, the transcription of pre-crRNAs and cascade genes are strictly repressed by the global regulator H-NS and activated with the help of the transcription factor LeuO, ensuring a fast response to a viral attack (68–70). "
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    ABSTRACT: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems of type I use a Cas ribonucleoprotein complex for antiviral defense (Cascade) to mediate the targeting and degradation of foreign DNA. To address molecular features of the archaeal type I-A Cascade interference mechanism, we established the in vitro assembly of the Thermoproteus tenax Cascade from six recombinant Cas proteins, synthetic CRISPR RNAs (crRNAs) and target DNA fragments. RNA-Seq analyses revealed the processing pattern of crRNAs from seven T. tenax CRISPR arrays. Synthetic crRNA transcripts were matured by hammerhead ribozyme cleavage. The assembly of type I-A Cascade indicates that Cas3' and Cas3'' are an integral part of the complex, and the interference activity was shown to be dependent on the crRNA and the matching target DNA. The reconstituted Cascade was used to identify sequence motifs that are required for efficient DNA degradation and to investigate the role of the subunits Cas7 and Cas3'' in the interplay with other Cascade subunits.
    Nucleic Acids Research 02/2014; 42(8). DOI:10.1093/nar/gku120 · 9.11 Impact Factor
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