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J. Bacteriol. doi:10.1128/JB.01415-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Diversity, activity and evolution of CRISPR loci in Streptococcus thermophilus

Danisco France SAS, BP10, F-86220 Dangé-Saint-Romain, France; Danisco USA Inc., 3329 Agriculture Drive, Madison, WI 53716, USA; and Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Groupe de Recherche en Ecologie Buccale, Faculté de Médecine Dentaire, Félix d'Hérelle Reference Center for Bacterial Viruses, Université Laval, G1K 7P4 Québec, Canada

^* To whom correspondence should be addressed. Email: rodolphe.barrangou{at}danisco.com.

	Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) are hypervariable loci widely distributed in prokaryotes, that provide acquired immunity against foreign genetic elements. Here, we characterize a novel Streptococcus thermophilus locus, CRISPR3, and experimentally demonstrate its ability to integrate novel spacers in response to bacteriophage. Also, we analyze CRISPR diversity and activity across three distinct CRISPR loci in several S. thermophilus strains. We show that both CRISPR repeats and cas genes are locus-specific and functionally coupled. A total of 124 strains were studied and 109 unique spacer arrangements were observed across the three CRISPR loci. Overall, 3,626 spacers were analyzed, including 2,829 for CRISPR1 (782 unique), 173 for CRISPR2 (16 unique) and 624 for CRISPR3 (154 unique). Sequence analysis of the spacers revealed homology and identity to phage sequences (77%), plasmid sequences (16%) and S. thermophilus chromosomal sequences (7%). Polymorphisms were observed for the CRISPR repeats, CRISPR spacers, cas genes, CRISPR motif, locus architecture and specific sequence content. Interestingly, CRISPR loci evolved both via polarized addition of novel spacers following exposure to foreign genetic elements and via internal deletion of spacers. We hypothesize that the level of diversity is correlated with relative CRISPR activity and propose that activity is highest for CRISPR1, followed by CRISPR3, while CRISPR2 may be degenerate. Globally, the dynamic nature of CRISPR loci might prove valuable for typing and comparative analyses of strains and microbial populations. Also, CRISPRs provide critical insights into the relationships between prokaryotes and their environments, notably the co-evolution of host and viral genomes.

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