Polymorphism of CRISPR shows separated natural groupings of <i>Shigella</i> subtypes and evidence of horizontal transfer of CRISPR

<p>Clustered, regularly interspaced, short palindromic repeats (CRISPR) act as an adaptive RNA-mediated immune mechanism in bacteria. They can also be used for identification and evolutionary studies based on polymorphisms within the CRISPR locus. We amplified and analyzed 6 CRISPR loci from 237 <i>Shigella</i> strains belonging to the 4 species groups, as well as 13 <i>Escherichia coli</i> strains. The CRISPR-associated (cas) gene sequence arrays of these strains were screened and compared. The CRISPR sequences from <i>Shigella</i> were conserved among subtypes, suggesting that CRISPR may represent a new identification tool for the detection and discrimination of <i>Shigella</i> species. Secondary structure analysis showed a different stem-loop structure at the terminal repeat, suggesting a distinct recognition mechanism in the formation of crRNA. In addition, the presence of “self-target” spacers and polymorphisms within CRISPR in <i>Shigella</i> indicated a selective pressure for inhibition of this system, which has the potential to damage “self DNA.” Homology analysis of spacers showed that CRISPR might be involved in the regulation of virulence transmission. Phylogenetic analysis based on CRISPR sequences from <i>Shigella</i> and <i>E. coli</i> indicated that although phenotypic properties maintain convergent evolution, the 4 <i>Shigella</i> species do not represent natural groupings. Surprisingly, comparative analysis of <i>Shigella</i> repeats with other species provided new evidence for CRISPR horizontal transfer. Our results suggested that CRISPR analysis is applicable for the detection of <i>Shigella</i> species and for investigation of evolutionary relationships.</p>