Deveau Hélène, Barrangou Rodolphe, Garneau Josiane E, Labonté Jessica, Fremaux Christophe, Boyaval Patrick, Romero Dennis A, Horvath Philippe, Moineau Sylvain
Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Groupe de Recherche en Ecologie Buccale, Félix d'Hérelle Reference Center for Bacterial Viruses, Université Laval, Quebec City, Quebec G1V 0A6, Canada.
J Bacteriol. 2008 Feb;190(4):1390-400. doi: 10.1128/JB.01412-07. Epub 2007 Dec 7.
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.
成簇规律间隔短回文重复序列（CRISPR）及其相关基因与细菌获得性噬菌体抗性机制有关。细菌可将短片段噬菌体衍生序列（间隔序列）整合到CRISPR基因座中，从而获得噬菌体抗性。在本研究中，我们进一步表征了嗜热链球菌中CRISPR1作为噬菌体抗性机制的效率。首先，我们表明CRISPR1与先前已知的噬菌体防御系统不同，并且对嗜热链球菌的两大类噬菌体均有效。对30个嗜热链球菌噬菌体不敏感突变体的分析表明，在CRISPR1中添加一个新的间隔序列是噬菌体攻击后最常见的结果，并且间隔序列的迭代添加会增加宿主的总体噬菌体抗性。添加的新间隔序列大小在29至31个核苷酸之间，其中30个核苷酸最为常见。将39个新获得的间隔序列与野生型噬菌体2972、858和DT1的完整基因组序列进行比较分析表明，新添加的间隔序列必须与噬菌体基因组中的一个区域（称为原间隔序列）相同，才能赋予噬菌体抗性表型。此外，我们在原间隔序列区域下游发现了一个对噬菌体抗性表型很重要的CRISPR1特异性序列（NNAGAAW）。最后，通过对20个突变噬菌体的分析，我们表明烈性噬菌体正在通过单核苷酸突变以及缺失快速进化，以应对CRISPR1。
