The on–off switch of CRISPR immunity against phages in Escherichia coli

CRISPR/CAS is a novel prokaryotic defence system against cell invaders. A typical CRISPR/CAS system consists of one or more clusters of regularly interspaced short, often palindromic, repeats (CRISPR) followed at one side by an adenineand thymine-rich (AT-rich) leader sequence, and a variable set of CAS (CRISPR associated) genes. The recognition of this repeats family took place after the analysis of similarly arranged redundant sequences detected in 20 genomes of diverse lineages (Mojica et al., 2000). Its functionality was soon supported by the identification of the CAS genes (Jansen et al., 2002) and the transcription of CRISPR cassettes (Tang et al., 2002). A role in defence against invader genetic elements was proposed when the foreign origin of repeat intervening sequences (spacers) was established (Bolotin et al., 2005; Mojica et al., 2005; Pourcel et al., 2005). This activity was confirmed in 2007 (Barrangou et al., 2007), with the demonstration that Streptococcus thermophilus susceptible to a given phage becomes resistant by the acquisition of new spacers derived from the challenging virus. Likewise, plasmid transfer is prevented by the CRISPR/CAS system of Staphylococcus epidermidis (Marraffini and Sontheimer, 2008). Although the mechanism of CRISPR-based interference is not fully understood, a general working model can be drawn (Fig. 1A). Briefly, when exposed to foreign elements, cells harbouring a functional CRISPR/CAS system may become specifically immunized (this stage is termed adaptation or immunization) by the acquisition of new spacers derived from proto-spacers in the invader DNA. Whilst the mechanism of proto-spacer recognition is unknown, the conservation of proto-spacer adjacent motifs (PAMs) strongly suggests the involvement of these short signatures in immunization (Barrangou et al., 2007; Mojica et al., 2009). Transcription of the CRISPR array from the leader, and further processing of this pre-crRNA by CAS proteins of the Cascade complex (CasABCDE) provide small RNAs (crRNA) that contain one spacer flanked by two handles made of a few bases of the bordering repeats (Brouns et al., 2008; Marraffini and Sontheimer, 2008; Hale et al., 2008). These crRNAs act as nucleic acid ‘antibodies’, guiding the defence apparatus to the target: it is expected that base-pairing of crRNA with sequences fully complementary to the spacer, either in DNA (Marraffini and Sontheimer, 2008) or RNA (Hale et al., 2009) molecules, result in interference. Immunization and interference by CRISPR/CAS efficiently prevent phage infection in S. thermophilus (Barrangou et al., 2007). In contrast, neither insertion of new spacers nor interference with targeted invaders by nonmanipulated CRISPR loci has been documented in Escherichia coli so far. Nevertheless, overexpression of the CRISPR2/CAS-E system of E. coli (associated to Cse-subtype CAS genes) located in recombinant plasmids, provides the cell with high levels of protection against phage infection, suggesting that the apparent inactivity of the genomic loci could be due to constrained expression (Brouns et al., 2008). CRISPR2 repeats are arranged in 3 cassettes, CRISPR2.1 (also named CRISPR I), CRISPR2.3 (or CRISPR II) and CRISPR2.2 (or CRISPR III). CAS-E genes are located next to the CRISPR2.1 leader (Díez-Villaseñor et al., 2010). Regulation of CRISPR2/CAS-E has been recently studied in E. coli K12 (Pul et al., 2010). This work brought to the arena an eminent player, the histone-like nucleoid structuring protein (H-NS), a well-known dual regulator (more often repressor) of gene expression in Gram-negative bacteria. This protein binds and represses promoters of both CRISPR2 arrays and casABCD12 operon, indicating that CRISPR-mediated resistance may only be conferred when silencing by H-NS is circumvented. In this issue of Molecular Microbiology, Pougach et al. (2010) and Westra et al. (2010) independently show that a high protection against l is conferred by phage-matching spacers in hns-disrupted E. coli strains, demonstrating that derepression of CRISPR/CAS elements leads to efficient Accepted 24 July, 2010. *For correspondence. E-mail fmojica@ua.es; Tel. (+34) 965 90 9761; Fax (+34) 965 90 9569. Molecular Microbiology (2010) 77(6), 1341–1345 doi:10.1111/j.1365-2958.2010. 07326.x First published online 18 August 2010