H-NS Mutation-Mediated CRISPR-Cas Activation Inhibits Phage Release and Toxin Production of Escherichia coli Stx2 Phage Lysogen.
H-NS Mutation-Mediated CRISPR-Cas Activation Inhibits Phage Release and Toxin Production of Escherichia coli Stx2 Phage Lysogen.
复制标题
H-NS 突变介导的 CRISPR-Cas 激活抑制大肠杆菌 Stx2 噬菌体溶原菌的噬菌体释放和毒素产生
DOI：
10.3389/fmicb.2017.00652
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发表时间：
2017
影响因子：
5.2
通讯作者：
Sun J
中科院分区：
文献类型：
--
作者：
Fu Q;Li S;Wang Z;Shan W;Ma J;Cheng Y;Wang H;Yan Y;Sun J
志贺毒素转化噬菌体（Stx噬菌体）携带Stx基因并将非致病性菌株转化为产生志贺毒素的细菌。目前对大肠杆菌（E. coli）聚集规律间隔短回文重复序列(CRISPR)-Cas适应性免疫系统对Stx噬菌体溶原的影响了解有限。我们研究了热稳定核结构（H-NS）突变介导的CRISPR-Cas激活及其对大肠杆菌Stx2噬菌体溶原的影响。获得大肠杆菌K-12菌株MG1655的Δhns突变体（MG1655Δhns）。与野生型菌株相比，Stx噬菌体溶原感染后产生的Δhns突变体溶原具有抑制生长状态，表现出较低的群体行为，包括生物膜形成和群体运动。然后验证了H-NS突变对CRISPR-Cas活性的去抑制作用。结果显示，cas2基因表达上调，野生型CRISPR质粒转化效率降低，这可能表明CRISPR- cas系统被激活。此外，通过激活含有Stx2噬菌体Min27原间隔区插入的CRISPR-Cas系统，研究了CRISPR-Cas对Stx2噬菌体溶原的功能。研究了四种携带工程crispr的溶原的噬菌体释放和毒素产生。值得注意的是，在含有Min27间隔物的Δhns突变体溶原的上清中，丝裂霉素C诱导后，子代噬菌体的释放和毒素的产生均受到抑制。这些观察结果表明，H-NS突变激活的CRISPR-Cas系统在修饰Stx2噬菌体溶原的作用中起作用。我们的研究结果表明，H-NS突变介导的CRISPR-Cas在大肠杆菌中的激活通过抑制噬菌体释放和溶原的毒素产生来保护细菌免受Stx2噬菌体的溶原作用。
Shiga toxin-converting bacteriophages (Stx phages) carry the stx gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. There is limited understanding of the effect that an Escherichia coli (E. coli) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas adaptive immune system has on Stx phage lysogen. We investigated heat-stable nucleoid-structuring (H-NS) mutation-mediated CRISPR-Cas activation and its effect on E. coli Stx2 phage lysogen. The Δhns mutant (MG1655Δhns) of the E. coli K-12 strain MG1655 was obtained. The Δhns mutant lysogen that was generated after Stx phage lysogenic infection had a repressed growth status and showed subdued group behavior, including biofilm formation and swarming motility, in comparison to the wild-type strain. The de-repression effect of the H-NS mutation on CRISPR-Cas activity was then verified. The results showed that cas gene expression was upregulated and the transformation efficiency of the wild-type CRISPR plasmids was decreased, which may indicate activation of the CRISPR-Cas system. Furthermore, the function of CRISPR-Cas on Stx2 phage lysogen was investigated by activating the CRISPR-Cas system, which contains an insertion of the protospacer regions of the Stx2 phage Min27. The phage release and toxin production of four lysogens harboring the engineered CRISPRs were investigated. Notably, in the supernatant of the Δhns mutant lysogen harboring the Min27 spacer, both the progeny phage release and the toxin production were inhibited after mitomycin C induction. These observations demonstrate that the H-NS mutation-activated CRISPR-Cas system plays a role in modifying the effects of the Stx2 phage lysogen. Our findings indicated that H-NS mutation-mediated CRISPR-Cas activation in E. coli protects bacteria against Stx2 phage lysogeny by inhibiting the phage release and toxin production of the lysogen.