Fu Qiang, Li Shiyu, Wang Zhaofei, Shan Wenya, Ma Jingjiao, Cheng Yuqiang, Wang Hengan, Yan Yaxian, Sun Jianhe
Shanghai Key Laboratory of Veterinary Biotechnology, Key Laboratory of Urban Agriculture (South), Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong UniversityShanghai, China.
Front Microbiol. 2017 Apr 18;8:652. doi: 10.3389/fmicb.2017.00652. eCollection 2017.
Shiga toxin-converting bacteriophages (Stx phages) carry the gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. There is limited understanding of the effect that an () 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 Stx2 phage lysogen. The Δ mutant (MG1655Δ) of the K-12 strain MG1655 was obtained. The Δ 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 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 Δ 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 protects bacteria against Stx2 phage lysogeny by inhibiting the phage release and toxin production of the lysogen.
志贺毒素转换噬菌体（Stx噬菌体）携带stx基因，可将非致病性细菌菌株转化为产志贺毒素的细菌。人们对I型（I-E）成簇规律间隔短回文重复序列（CRISPR）-Cas适应性免疫系统对Stx噬菌体溶原菌的影响了解有限。我们研究了热稳定类核结构蛋白（H-NS）突变介导的CRISPR-Cas激活及其对Stx2噬菌体溶原菌的影响。获得了K-12菌株MG1655的Δhns突变体（MG1655Δhns）。与野生型菌株相比，Stx噬菌体溶源感染后产生的Δhns突变体溶原菌生长状态受到抑制，群体行为减弱，包括生物膜形成和群集运动。随后验证了H-NS突变对CRISPR-Cas活性的去抑制作用。结果表明，stx基因表达上调，野生型CRISPR质粒的转化效率降低，这可能表明CRISPR-Cas系统被激活。此外，通过激活包含Stx2噬菌体Min27原间隔区插入片段的CRISPR-Cas系统，研究了CRISPR-Cas对Stx2噬菌体溶原菌的功能。研究了四种携带工程化CRISPR的溶原菌的噬菌体释放和毒素产生情况。值得注意的是，在携带Min27间隔区的Δhns突变体溶原菌的上清液中，丝裂霉素C诱导后子代噬菌体释放和毒素产生均受到抑制。这些观察结果表明，H-NS突变激活的CRISPR-Cas系统在改变Stx2噬菌体溶原菌的影响方面发挥作用。我们的研究结果表明，H-NS突变介导的大肠杆菌CRISPR-Cas激活通过抑制溶原菌的噬菌体释放和毒素产生来保护细菌免受Stx2噬菌体溶原化。
