Analysis of the acquired microbial defence mechanism against foreign DNA by the CRISPR system
Final Report Abstract
The main goal of this study was the characterization of the CRISPR-Cas defense system in E. coli K12 with focus on: 1) transcriptional regulation of the CRISPR-Cas activity; 2) the analysis of Cascade components and the Cascade-mediated targeting of double-stranded DNA and 3) the mechanism of spacer selection and incorporation into the CRISPR locus. (1) We could identify an extended -10 promoter within the leader DNA, which directs the transcription of the CRISPR locus. A second promoter, Pcas, was identified upstream of the casA gene, from which the transcription of the polycistronic Cascade-cas1-cas2 mRNA is initiated. We discovered that the DNA-binding protein H-NS inhibits the Pcas promoter through cooperative occupation of the promoter rendering the promoter inaccessible to RNA polymerase. The formation of mature crRNAs only occurred in the absence of H-NS or in the presence of elevated levels of the LeuO protein, which binds at two sites within the Pcas promoter region and competes with the cooperative spreading of H-NS along the DNA. The LeuO-dependent activation of the Pcas promoter could also be achieved by constitutive expression of BglJ. However, the processing of the precursor crRNA still remained inactive in BglJ-expressing cells, indicating additional regulation of Cascade expression/activity at a post-transcriptional or later level by unknown factors. (2) We analysed the binding of crRNA-loaded Cascade complexes to doublestranded target DNA by enzymatic and chemical footprint studies. The results showed that the targeting of double-stranded DNA occurs by the formation of an R-loop through a specific base-pairing of the crRNA with the complementary DNA strand and displacement of the non-complementary strand. (3) In order to unravel the mechanism of spacer DNA acquisition, we initiated experiments to detect Cas1-mediated cleavage sites in the course of spacer integration. We were able to visualize a putative intermediate of spacer integration in vivo, whose composition led to formulation of a novel model for the acquisition of new spacer DNA by a coupled cleavage-ligation reaction. Mutational studies of CRISPR repeat sequence or the Cas1 protein allowed us to correlate the formation of these integration intermediates with the ability to acquire new spacer DNA. The concerted cleavage-ligation (integrase) activity of Cas1 protein could be verified by preliminary in vitro assays. Furthermore, functional and structural studies on the Csn2 protein from Streptococcus agalactiae were conducted, which is essential for uptake of new spacer DNA in type II-A CRISPR-Cas systems. We could show that four monomers of Csn2 form a ring-shaped tetramer, which binds to the ends of a doublestranded DNA through its central hole and slides inward along the DNA, suggesting a pivotal role of Csn2 during the integration of exogenous DNA by end-joing.
Publications
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H-NS mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO. Mol. Microbiol. 77: 1380–1393 (2010)
Westra ER, Pul Ü, Heidrich N, Jore MM, Lundgren M, Stratmann T, Wurm R, Raine A, Mescher M, van Heereveld L, Mastop M, Wagner EGH, Schnetz K, van der Oost J, Wagner R, Brouns SJJ
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Identification and Characterization of E. coli CRISPR-cas Promoters and their Silencing by H-NS. Mol. Microbiol., 75:1495-1512 (2010)
Pul Ü, Wurm R, Arslan Z, Geißen R, Hofmann N, Wagner R
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Mikrobielles "Immunsystem": Abwehr gegen Fremd-DNA durch das bakterielle CRISPR/Cas-System. BIOspektrum 4, 393-395 (2011)
Wagner R, Pul Ü
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Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat. Struc. Mol. Biol. 18: 529–536 (2011)
Jore MM, Lundgren M, van Duin E, Bultema JB, Westra ER, Waghmare SP, Wiedenheft B, Pul Ü, Wurm R, Wagner R, Beijer M, Barendregt RA, Zhou K, Snijders APL, Dickman MJ, Doudna JA, Boekema EJ, Heck AJR, van der Oost J, Brouns SJJ
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CRISPR - a bacterial immunity system based on small RNAs. In: From Nucleic Acids Sequences to Molecular Medicine, Erdmann, Volker A.; Barciszewski, Jan (Eds.), Springer-Verlag IX, 121-143 (2012)
Wagner R, Pul Ü
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The crystal structure of the CRISPR-associated protein Csn2 from Streptococcus agalactiae. J. Struct. Biol. 178 (3), 350-62 (2012)
Ellinger P, Arslan Z, Wurm R, Ries B, MacKenzie C, Pfeffer K, Panjikar S, Wagner R, Schmitt L, Gohlke H, Pul Ü, Smits SHJ
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Das Immunsystem der Prokaryoten: CRISPR-Cas. BiuZ 43(3), 158-165 (2013)
Marchfelder A, Maier L-K, Heidrich N, Pul Ü
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Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2. Nucl Acids Res, 41 (12), 6347-6359 (2013)
Arslan Z, Wurm R, Brener O, Ellinger P, Nagel-Steger L, Oesterhelt F, Schmitt L, Willbold D, Wagner R, Gohlke H, Smits SHJ, Pul Ü
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RcsB-BglJ mediated activation of Cascade operon does not induce the maturation of CRISPR RNAs in E. coli K12. RNA Biol. 10 (5), 708-715 (2013)
Arslan Z, Stratmann T, Wurm R, Wagner R, Schnetz K, Pul Ü
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Regulation of CRISPR-based immune responses. In: CRISPR-Cas systems: RNA-mediated Adaptive Immunity in Bacteria and Archaea, Barrangou, Rodolphe; van der Oost, John (Eds.) , Springer-Verlag X, 93-113 (2013)
Arslan Z, Westra ER, Wagner R, Pul Ü
