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.
    Full-text · Article · Dec 2014 · Microbiology
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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.
    Full-text · Article · Jun 2014 · Nucleic Acids Research
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    • "The cas genes expression in E. coli K12 is activated by LeuO but repressed by heat-stable nucleoid-structuring protein (H-NS) (Pul et al., 2010; Westra et al., 2010). LeuO, a LysR-type transcription factor, belongs to the helix–turn–helix-type transcriptional regulator (Henikoff et al., 1988; Ueguchi et al., 1998), whereas H-NS, a dominant histone-like protein, is expressed during exponential growth (Ali Azam et al., 1999). "
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    ABSTRACT: The CRISPR/Cas system is an important aspect in bacterial immunology. The anti-phage activity of the CRISPR system has been established using synthetic CRISPR spacers, but in vivo studies of endogenous CRISPR spacers are relatively scarce. Here, we showed that bacteriophage P1 titer in Escherichia coli decreased in the glucose-containing medium compared with that in the absence of glucose. This glucose effect of E. coli against phage P1 infection disappeared in cse3 deletion mutants. The effect on the susceptibility to phage P1 was associated with cAMP receptor protein (CRP)-mediated repression of cas genes transcription and crRNA maturation. Analysis of the regulatory element in the cse1 promoter region revealed a novel CRP-binding site, which overlapped with a LeuO-binding site. Furthermore, the limited sequence identity between endogenous spacers and the phage P1 genome was necessary and sufficient for CRISPR-mediated repression of phage P1 replication. Trans-expression of the third and seventh spacers in the CRISPR I region or third and sixth spacers in the CRISPR II region effectively reduced phage P1 titers in the CRISPR deletion mutants. These results demonstrate a novel regulatory mechanism for cas repression by CRP and provide evidence that endogenous spacers can repress phage P1 replication in E. coli.
    Full-text · Article · Apr 2014 · Molecular Microbiology
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