Chen Fuguang, Wang Di, Lu Tongyan, Li Shaowu
Department of Aquatic Animal Health, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin, China.
Key Laboratory of Aquatic Animal Diseases and Immune Technology of Heilongjiang Province, Harbin, China.
Front Microbiol. 2023 Jun 15;14:1181303. doi: 10.3389/fmicb.2023.1181303. eCollection 2023.
is the causative agent of rainbow trout fry syndrome and bacterial cold-water disease in salmonid fish worldwide. As an important fish pathogen, is frequently exposed to multiple invading genetic elements in natural environments. Endonuclease Cas9 provides bacteria with adaptive interference against invading genetic elements. Previous studies revealed that several strains harbored a type II-C Cas9 called Fp1Cas9, but little is known about the potential role of this endonuclease against invading genetic elements. In this work, we identified a gene encoding a novel type II-C Cas9 called Fp2Cas9 from strain CN46. Through bacterial RNA sequencing, we demonstrated active transcription of both Fp2Cas9 and pre-crRNAs in strain CN46. Bioinformatics analysis further revealed that the transcription of Fp2Cas9 and pre-crRNAs was driven by a newly integrated promoter sequence and a promoter element embedded within each CRISPR repeat, respectively. To formally demonstrate that Fp2Cas9 and associated crRNAs yielded functional interference in strain CN46, a plasmid interference assay was performed, resulting in adaptive immunity to target DNA sequences in bacteriophages. Phylogenetic analysis demonstrated that Fp2Cas9 was present only in several isolates. Phylogenetic analysis revealed that this novel endonuclease was probably acquired through horizontal gene transfer from the CRISPR-Cas9 system in an unidentified species. Comparative genomics analysis further showed that the Fp2Cas9 was integrated into the type II-C CRISPR-Cas locus in strain CN38 instead of the original Fp1Cas9. Taken together, our results shed light on the origin and evolution of Fp2Cas9 gene and demonstrated that this novel endonuclease provided adaptive interference against bacteriophage infections.
是全球鲑科鱼类虹鳟鱼苗综合征和细菌性冷水病的病原体。作为一种重要的鱼类病原体，在自然环境中经常暴露于多种入侵的遗传元件。核酸内切酶Cas9为细菌提供了对入侵遗传元件的适应性干扰。先前的研究表明，几种菌株含有一种名为Fp1Cas9的II-C型Cas9，但关于这种核酸内切酶对入侵遗传元件的潜在作用知之甚少。在这项工作中，我们从菌株CN46中鉴定出一个编码新型II-C型Cas9（称为Fp2Cas9）的基因。通过细菌RNA测序，我们证明了菌株CN46中Fp2Cas9和前体crRNA的活性转录。生物信息学分析进一步表明，Fp2Cas9和前体crRNA的转录分别由新整合的启动子序列和每个CRISPR重复序列中嵌入的启动子元件驱动。为了正式证明Fp2Cas9和相关的crRNA在菌株CN46中产生功能性干扰，进行了质粒干扰试验，从而对噬菌体中的靶DNA序列产生适应性免疫。系统发育分析表明，Fp2Cas9仅存在于几种分离株中。系统发育分析表明，这种新型核酸内切酶可能是通过水平基因转移从一个未鉴定物种的CRISPR-Cas9系统中获得的。比较基因组学分析进一步表明，Fp2Cas9在菌株CN38中整合到II-C型CRISPR-Cas位点，而不是原来的Fp1Cas9。综上所述，我们的结果揭示了Fp2Cas9基因的起源和进化，并证明了这种新型核酸内切酶对噬菌体感染提供了适应性干扰。
