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Journal of Bacteriology, February 2008, p. 1401-1412, Vol. 190, No. 4
0021-9193/08/$08.00+0     doi:10.1128/JB.01415-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Diversity, Activity, and Evolution of CRISPR Loci in Streptococcus thermophilus{triangledown} ,{dagger}

Philippe Horvath,1 Dennis A. Romero,2 Anne-Claire Coûté-Monvoisin,1 Melissa Richards,2 Hélène Deveau,3 Sylvain Moineau,3 Patrick Boyaval,1 Christophe Fremaux,1 and Rodolphe Barrangou2*

Danisco France SAS, BP10, F-86220 Dangé-Saint-Romain, France,1 Danisco USA, Inc., 3329 Agriculture Drive, Madison, Wisconsin 53716,2 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, Canada3

Received 31 August 2007/ Accepted 21 November 2007

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 after 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 the 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 coevolution of host and viral genomes.

* Corresponding author. Mailing address: Danisco USA, Inc., 3329 Agriculture Drive, Madison, WI 53716. Phone: (608) 395-2648. Fax: (608) 395-2721. E-mail: rodolphe.barrangou{at}danisco.com

{triangledown} Published ahead of print on 7 December 2007.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, February 2008, p. 1401-1412, Vol. 190, No. 4
0021-9193/08/$08.00+0     doi:10.1128/JB.01415-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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