State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
J Bacteriol. 2013 Feb;195(4):867-75. doi: 10.1128/JB.01688-12. Epub 2012 Dec 14.
The adaptive immune system comprising CRISPR (clustered regularly interspaced short palindromic repeats) arrays and cas (CRISPR-associated) genes has been discovered in a wide range of bacteria and archaea and has recently attracted comprehensive investigations. However, the subtype I-B CRISPR-Cas system in haloarchaea has been less characterized. Here, we investigated Cas6-mediated RNA processing in Haloferax mediterranei. The Cas6 cleavage site, as well as the CRISPR transcription start site, was experimentally determined, and processing of CRISPR transcripts was detected with a progressively increasing pattern from early log to stationary phase. With genetic approaches, we discovered that the lack of Cas1, Cas3, or Cas4 unexpectedly resulted in a decrease of CRISPR transcripts, while Cas5, Cas6, and Cas7 were found to be essential in stabilizing mature CRISPR RNA (crRNA). Intriguingly, we observed a CRISPR- and Cas3-independent inhibition of a defective provirus, in which the putative Cascade (CRISPR-associated complex for antiviral defense) proteins (Cas5, Cas6, Cas7, and Cas8b) were indispensably required. A sequence carried by a proviral transcript was found to be homologous to the CRISPR repeat RNA and vulnerable to Cas6-mediated cleavage, implying a distinct interference mechanism that may account for this unusual inhibition. These results provide fundamental information for the subtype I-B CRISPR-Cas system in halophilic archaea and suggest diversified mechanisms and multiple physiological functions for the CRISPR-Cas system.
CRISPR（成簇规律间隔短回文重复）阵列和 cas（CRISPR 相关）基因组成的适应性免疫系统已在广泛的细菌和古菌中被发现，并最近引起了广泛的研究。然而，嗜盐古菌中的 I-B 型 CRISPR-Cas 系统的特征较少。在这里，我们研究了 Haloferax mediterranei 中的 Cas6 介导的 RNA 加工。通过实验确定了 Cas6 的切割位点以及 CRISPR 的转录起始位点，并从早期对数期到静止期检测到 CRISPR 转录物的加工呈逐渐增加的模式。通过遗传方法，我们发现 Cas1、Cas3 或 Cas4 的缺失出乎意料地导致 CRISPR 转录物减少，而 Cas5、Cas6 和 Cas7 被发现对稳定成熟的 CRISPR RNA（crRNA）是必不可少的。有趣的是，我们观察到一种 CRISPR 和 Cas3 独立的缺陷前病毒的抑制作用，其中推定的 Cascade（抗病毒防御的 CRISPR 相关复合物）蛋白（Cas5、Cas6、Cas7 和 Cas8b）是必不可少的。一种由前病毒转录本携带的序列与 CRISPR 重复 RNA 同源，容易受到 Cas6 介导的切割，暗示存在一种独特的干扰机制，可能解释这种不寻常的抑制作用。这些结果为嗜盐古菌中的 I-B 型 CRISPR-Cas 系统提供了基本信息，并表明 CRISPR-Cas 系统具有多样化的机制和多种生理功能。
