Jansen_MolMicro_2002

•clustered regularly interspaced short palindromic repeats (CRISPR)

•A characeristic of the CRISPRs, not seen in any otherclass of repetitive DNA, is that the repeats of theCRISPRs are interspaced by similarly sized nonrepetitiveDNA. The direct repeats varies in size from21 bp in Salmonella typhimurium to 37 bp in Streptococcuspyogenes, and they are clustered in one or severalloci on the chromosome.

•The CRISPR loci in clinical isolates of M. tuberculosisand S. pyogenes are extremely polymorphic, and thisstrain-dependent polymorphism has been exploited forepidemiological and taxonomic purposes (Kamerbeeket al., 1997; Hoe et al., 1999). The short repeat sequenceitself is remarkably well conserved in clinical isolates;however, the number of repeats and spacers differs fromstrain to strain (van Embden et al., 2000).

The number of CRISPR loci varied from 1 in somespecies, e.g. M. tuberculosis and Neisseria meningitidis,to 20 in M. jannaschii. The number of repeats within theCRISPR loci varied greatly, from two repeats to as manyas 124 repeats in M. thermoautotrophicum (see Table 1).

Despite the absence of sequence similarity betweenthe repeat sequences of the species, the repeat sequencesshare some common features. In most of therepeat sequences a lose dyad symmetry can be recognized(Mojica et al., 2000). The nucleotides involved in thedyad symmetry are mainly located at the termini of therepeats and often include the complementary sequencesGTT and AAC.

The repeat sequences within a given CRISPR locuswere generally identical with rare single-basepair substitutions.Although the majority of the repeats in differentCRISPR loci of the same organism were highly similaror identical, some were very dissimilar, such as those inAquifex aeolicus, Archaeoglobus fulgidus, Pasteurellamultocida, Pyrococcus horikoshii and Streptococcus pyogenes.Each of these species carried several CRISPR lociand the repeat sequences of the various CRISPR lociwithin the species may differ completely from each other.

The DNA stretches between the repeats (the spacers)were present as a single copy in a CRISPR locus withvery few exceptions. Exceptions were found in a CRISPRlocus of M. thermoautotrophicum, in which small clustersof repeats and spacers had duplicates in the CRISPRlocus and in M. tuberculosis that carried two identicalspacers in the CRISPR region (Smith et al., 1997; vanEmbden et al., 2000). None of the spacers of a givenorganism shared sequence similarity with spacers ofother organisms. This also holds true when the spacer

sequences of closely related species were compared, e.g.E. coli and S. enterica or the Streptococcus and thePyrococcus species (Table 1).

Comparison of the genes that flank the CRISPR loci inthe genomes of different prokaryotic species showed aclear homology among four genes. We designated thesegenes the CRISPR-associated genes, cas1 to cas4

Barrangou_Science_2007.pdf

Streptococcus thermophilus is a low G+C Gram-positive bacterium and a key species ex- ploited in the formulation of dairy culture sys- tems for the production of yogurt and cheese.

These results reveal that, on becoming resistant to bacteriophages, the CRISPR1 locus was modified by the integration of novel spacers, apparently derived from phage DNA

The phage-resistance profile seemed correlated to the spacer content, such that strains with spacers showing 100% identity to sequences conserved in both phages were resistant to both phages, such as spacers S3, S6, and S7. In contrast, when nucleotide polymor- phisms were observed between the spacer and the phage sequence [from 1 to 15 single-nucleotide

polymorphisms (SNPs) over 29 or 30 nucleo- tides], the spacer did not seem to provide re- sistance, such as spacers S1, S2, S4, S5, and S8

These findings suggest that the presence of a CRISPR spacer identical to a phage sequence provides resistance against phages containing this particular sequence.

In the process of generating strain WTФ 8 5 8 +S1S2∆CRISPR1, we created WTФ858

+S1S2::pR, a variant that contains the inte- gration vector with a single repeat inserted be- tween the cas genes and the native CRISPR1 locus (Fig. 3). Unexpectedly, strain WTФ858

+S1S2::pR was sensitive to phage 858, although spacers S1 and S2 remained on the chromosome (Fig. 3). Similarly, the WTФ2972

+S4::pS1S2 construct lost the resistance to phage 2972, although spacer S4 is present in the chromosome (Fig. 3). These results indicated that spacers alone did not provide resistance, and perhaps, that they have to be in a particular genetic context to be effective.

The cas5 inactivation re- sulted in loss of the phage resistance (Fig. 3), and perhaps Cas5 acts as a nuclease, because it contains an HNH-type nuclease motif. In con- trast, inactivating cas7 did not alter the resist- ance to phage 858 (Fig. 3). Interestingly, we were repeatedly unable to generate CRISPR1 phage-resistant mutants from the cas7 knock- out, perhaps because Cas7 is involved in the synthesis and/or insertion of new spacers and additional repeats.

When we tested the sensitivity of the phage- resistant mutants, we found that plaque formation was dramatically reduced, but that a relatively small population of bacteriophage retained the ability to infect the mutants. We further analyzed phage variants derived from phage 858 that retained the ability to infect WTФ858

+S1S2. In par- ticular, we investigated the sequence of the ge- nome region corresponding to additional spacers S1 and S2 in two virulent phage variants. In both cases, the genome sequence of the phage var- iant had mutated, and two distinct SNPs were identified in the sequence corresponding to spacer S1 (fig. S3).

Bhaya_AnnRevGenet_2011

Finally, the ability to dynamically acquire foreign DNA and subsequently use it to fight off invading genetic material has elements of an acquired and heritable immunity system, reflecting a Lamarckian mode of evolution (64).

Westra_NatMicroRev_2014

Poster