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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    • "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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    • "During long-term virus-free culturing, the host DNA of laboratory strains has served as the only DNA sources, in this case, naïve adaptation could be detrimental because it does not discriminate between non-self versus self DNA well (12–14), and host-derived spacers will lead to toxic effects or even cell death in the presence of an active interference pathway. Thus, we speculate that, for some, if not all, laboratory strains, naïve adaptation may have been inactivated or the whole system been silenced by, for example, H-NS (30,31), which may underlie the vacancy of adaptation reports for these systems. "
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    ABSTRACT: The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system mediates adaptive immunity against foreign nucleic acids in prokaryotes. However, efficient adaptation of a native CRISPR to purified viruses has only been observed for the type II-A system from a Streptococcus thermophilus industry strain, and rarely reported for laboratory strains. Here, we provide a second native system showing efficient adaptation. Infected by a newly isolated virus HHPV-2, Haloarcula hispanica type I-B CRISPR system acquired spacers discriminatively from viral sequences. Unexpectedly, in addition to Cas1, Cas2 and Cas4, this process also requires Cas3 and at least partial Cascade proteins, which are involved in interference and/or CRISPR RNA maturation. Intriguingly, a preexisting spacer partially matching a viral sequence is also required, and spacer acquisition from upstream and downstream sequences of its target sequence (i.e. priming protospacer) shows different strand bias. These evidences strongly indicate that adaptation in this system strictly requires a priming process. This requirement, if validated also true for other CRISPR systems as implied by our bioinformatic analysis, may help to explain failures to observe efficient adaptation to purified viruses in many laboratory strains, and the discrimination mechanism at the adaptation level that has confused scientists for years.
    Nucleic Acids Research 11/2013; 42(4). DOI:10.1093/nar/gkt1154 · 9.11 Impact Factor
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