Shariat Nikki, Timme Ruth E, Pettengill James B, Barrangou Rodolphe, Dudley Edward G
1Department of Food Science, Center for Molecular Immunology and Infectious Disease and Center for Infectious Disease Dynamics, Pennsylvania State University, University Park, PA 16802, USA.
2Center for Food Safety and Applied Nutrition, US Food and Drug Administration, College Park, MD, USA.
Microbiology (Reading). 2015 Feb;161(2):374-386. doi: 10.1099/mic.0.000005.
Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide adaptive immunity from invasive genetic elements and encompass three essential features: (i) cas genes, (ii) a CRISPR array composed of spacers and direct repeats and (iii) an AT-rich leader sequence upstream of the array. We performed in-depth sequence analysis of the CRISPR-Cas systems in >600 Salmonella, representing four clinically prevalent serovars. Each CRISPR-Cas feature is extremely conserved in the Salmonella, and the CRISPR1 locus is more highly conserved than CRISPR2. Array composition is serovar-specific, although no convincing evidence of recent spacer acquisition against exogenous nucleic acids exists. Only 12 % of spacers match phage and plasmid sequences and self-targeting spacers are associated with direct repeat variants. High nucleotide identity (>99.9 %) exists across the cas operon among isolates of a single serovar and in some cases this conservation extends across divergent serovars. These observations reflect historical CRISPR-Cas immune activity, showing that this locus has ceased undergoing adaptive events. Intriguingly, the high level of conservation across divergent serovars shows that the genetic integrity of these inactive loci is maintained over time, contrasting with the canonical view that inactive CRISPR loci degenerate over time. This thorough characterization of Salmonella CRISPR-Cas systems presents new insights into Salmonella CRISPR evolution, particularly with respect to cas gene conservation, leader sequences, organization of direct repeats and protospacer matches. Collectively, our data suggest that Salmonella CRISPR-Cas systems are no longer immunogenic; rather, their impressive conservation indicates they may have an alternative function in Salmonella.
原核生物的CRISPR-Cas（成簇规律间隔短回文重复序列及其相关基因）系统提供针对入侵遗传元件的适应性免疫，包含三个基本特征：（i）cas基因，（ii）由间隔序列和同向重复序列组成的CRISPR阵列，以及（iii）阵列上游富含AT的前导序列。我们对代表四种临床流行血清型的600多种沙门氏菌中的CRISPR-Cas系统进行了深入的序列分析。每个CRISPR-Cas特征在沙门氏菌中都极为保守，并且CRISPR1位点比CRISPR2位点的保守性更高。尽管没有令人信服的证据表明最近有针对外源核酸获得间隔序列，但阵列组成具有血清型特异性。只有12%的间隔序列与噬菌体和质粒序列匹配，并且自我靶向间隔序列与同向重复序列变体相关。在单个血清型的分离株中，cas操纵子的核苷酸同一性很高（>99.9%），在某些情况下，这种保守性延伸到不同的血清型。这些观察结果反映了CRISPR-Cas的历史免疫活性，表明该位点已停止发生适应性事件。有趣的是，不同血清型之间的高度保守性表明这些无活性位点的遗传完整性随时间得以维持，这与无活性CRISPR位点随时间退化的传统观点形成对比。对沙门氏菌CRISPR-Cas系统的全面表征为沙门氏菌CRISPR的进化提供了新的见解，特别是在cas基因保守性、前导序列、同向重复序列的组织以及原间隔序列匹配方面。总体而言，我们的数据表明沙门氏菌的CRISPR-Cas系统不再具有免疫原性；相反，它们令人印象深刻的保守性表明它们在沙门氏菌中可能具有替代功能。
