Cas3 is a limiting factor for CRISPR-Cas immunity in Escherichia coli cells lacking H-NS.
Cas3 is a limiting factor for CRISPR-Cas immunity in Escherichia coli cells lacking H-NS.
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DOI：
10.1186/s12866-016-0643-5
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发表时间：
2016-03-08
期刊：
影响因子：
4.2
通讯作者：
Ivančić-Baće I
中科院分区：
文献类型：
--
作者：
Majsec K;Bolt EL;Ivančić-Baće I
CRISPR-Cas系统对原核生物中可移动的遗传元件提供适应性免疫。在包括大肠杆菌在内的许多细菌中，一种名为Cascade的特殊核糖核蛋白复合体通过CRISPR编码的RNA(CrRNA)和入侵者DNA序列之间的干扰反应来产生免疫力。CASCADE通过短的“原间隔相邻基序”(PAM)序列和crRNA-DNA互补来识别入侵者DNA。这会引发Cas3蛋白对入侵者DNA的降解，并在某些情况下刺激捕获新的入侵者DNA Protspacers，由Cas1和Cas2蛋白作为“间隔区”并入CRISPR，从而增强免疫力。如果Cascade和CRISPR的转录抑制因子H-NS失活(ΔHNS)，则Cascade、Cas3和crRNA的共同表达有效地使大肠杆菌细胞对噬菌体裂解产生抵抗。我们进一步对CRISPR-Cas介导的噬菌体抗性在Δhns大肠杆菌细胞中的调节进行了遗传分析。我们观察到，大肠杆菌I-E型CRISPR-Cas介导的对噬菌体λ的抗性强烈地依赖于温度，当重复先前发表的实验程序时。进一步的遗传分析强调了培养条件对控制大肠杆菌中CRISPR免疫程度的重要性。这些数据表明，Cas3的表达水平是成功抗噬菌体的一个重要限制因素。值得注意的是，我们描述了新的鉴定，即Cas3也受到H-NS的转录控制，但这只在稳定期细胞中发挥作用。在这些实验系统中，对Cas3的调节是对生长阶段和生长温度的响应，影响了CRISPR-Cas免疫的效果。本文的在线版本(doi：10.1186/s12866-0160643-5)包含补充材料，授权用户可以使用。
CRISPR-Cas systems provide adaptive immunity to mobile genetic elements in prokaryotes. In many bacteria, including E. coli, a specialized ribonucleoprotein complex called Cascade enacts immunity by“ an interference reaction" between CRISPR encoded RNA (crRNA) and invader DNA sequences called “protospacers”. Cascade recognizes invader DNA via short “protospacer adjacent motif” (PAM) sequences and crRNA-DNA complementarity. This triggers degradation of invader DNA by Cas3 protein and in some circumstances stimulates capture of new invader DNA protospacers for incorporation into CRISPR as “spacers” by Cas1 and Cas2 proteins, thus enhancing immunity. Co-expression of Cascade, Cas3 and crRNA is effective at giving E. coli cells resistance to phage lysis, if a transcriptional repressor of Cascade and CRISPR, H-NS, is inactivated (Δhns). We present further genetic analyses of the regulation of CRISPR-Cas mediated phage resistance in Δhns E. coli cells. We observed that E. coli Type I-E CRISPR-Cas mediated resistance to phage λ was strongly temperature dependent, when repeating previously published experimental procedures. Further genetic analyses highlighted the importance of culture conditions for controlling the extent of CRISPR immunity in E. coli. These data identified that expression levels of cas3 is an important limiting factor for successful resistance to phage. Significantly, we describe the new identification that cas3 is also under transcriptional control by H-NS but that this is exerted only in stationary phase cells. Regulation of cas3 is responsive to phase of growth, and to growth temperature in E. coli, impacting on the efficacy of CRISPR-Cas immunity in these experimental systems. The online version of this article (doi:10.1186/s12866-016-0643-5) contains supplementary material, which is available to authorized users.