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Journal of Bacteriology, February 2008, p. 1390-1400, Vol. 190, No. 4
0021-9193/08/$08.00+0     doi:10.1128/JB.01412-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus{triangledown}

Hélène Deveau,1 Rodolphe Barrangou,2 Josiane E. Garneau,1 Jessica Labonté,1 Christophe Fremaux,3 Patrick Boyaval,3 Dennis A. Romero,2 Philippe Horvath,3 and Sylvain Moineau1*

Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Félix d'Hérelle Reference Center for Bacterial Viruses, Université Laval, Quebec City, Quebec G1V 0A6, Canada,1 Danisco, USA, Inc., 3329 Agriculture Drive, Madison, Wisconsin 53716,2 Danisco France SAS, BP10, F-86220 Dangé-Saint-Romain, France3

Received 31 August 2007/ Accepted 21 November 2007

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated genes are linked to a mechanism of acquired resistance against bacteriophages. Bacteria can integrate short stretches of phage-derived sequences (spacers) within CRISPR loci to become phage resistant. In this study, we further characterized the efficiency of CRISPR1 as a phage resistance mechanism in Streptococcus thermophilus. First, we show that CRISPR1 is distinct from previously known phage defense systems and is effective against the two main groups of S. thermophilus phages. Analyses of 30 bacteriophage-insensitive mutants of S. thermophilus indicate that the addition of one new spacer in CRISPR1 is the most frequent outcome of a phage challenge and that the iterative addition of spacers increases the overall phage resistance of the host. The added new spacers have a size of between 29 to 31 nucleotides, with 30 being by far the most frequent. Comparative analysis of 39 newly acquired spacers with the complete genomic sequences of the wild-type phages 2972, 858, and DT1 demonstrated that the newly added spacer must be identical to a region (named proto-spacer) in the phage genome to confer a phage resistance phenotype. Moreover, we found a CRISPR1-specific sequence (NNAGAAW) located downstream of the proto-spacer region that is important for the phage resistance phenotype. Finally, we show through the analyses of 20 mutant phages that virulent phages are rapidly evolving through single nucleotide mutations as well as deletions, in response to CRISPR1.

* Corresponding author. Mailing address: Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval, Quebec City, Quebec G1V 0A6, Canada. Phone: (418) 656-3712. Fax: (418) 656-2861. E-mail: Sylvain.Moineau{at}bcm.ulaval.ca

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

Journal of Bacteriology, February 2008, p. 1390-1400, Vol. 190, No. 4
0021-9193/08/$08.00+0     doi:10.1128/JB.01412-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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