尿路致病性大肠杆菌中Ⅰ型限制-修饰系统C5423-5425的鉴定

Characterization of a type I restriction-modification system C5423-5425 in uropathogenic Escherichia coli

摘要:

[目的] 多方面鉴定尿路致病性大肠杆菌中的Ⅰ型限制-修饰（restriction-modification，RM）系统C5423-5425，并揭示其功能与生理意义。[方法] 利用Lambda Red重组系统构建CFT073 DNA甲基化转移酶基因缺失株Δc5424；利用单分子实时测序分析以获得C5424甲基化修饰的位点与基序；利用转化效率试验说明RM系统抵御外源DNA的转化；利用转录组测序和荧光定量RT-PCR鉴定C5424的调控基因；利用软琼脂平板运动试验等方法研究细菌生理功能的变化。[结果] 用生物信息学方法鉴定了一个Ⅰ型RM系统；获得了c5424的突变株；鉴定了C5424修饰的靶序列G^mAGNNNNNNNGTCA/TG^mAC NNNNNNNCTC，并揭示了靶序列在基因组中的分布；C5424系统可以抵御外源DNA的进入；c5424的缺失显著影响17个基因的表达，包括运动相关基因motB和抗活性氯基因rclR等；c5424的缺失显著影响CFT073的运动能力和抗次氯酸的能力。[结论] 本文从多方面鉴定了尿路致病性大肠杆菌CFT073中的I型RM系统C5423-5425，这个系统对细菌具有重要的生理意义。本文对研究细菌的表观遗传学具有参考价值。

关键词:尿路致病性大肠杆菌;DNA甲基化转移酶;单分子实时测序;限制-修饰系统

Abstract:

[Objective] To characterize a type I restriction-modification (RM) system C5423-5425 in uropathogenic Escherichia coli CFT073 and to determine its function and physiological significance. [Methods] Lambda Red recombination system was used to construct methyltransferase gene deletion mutant Δc5424 in CFT073. The modification sites and recognition motif of C5424 methyltransferase were obtained by single molecule real-time sequencing analysis. A transformation efficiency assay was used to test that the RM system C5423-5425 can block transformation of exogenous DNA. Genes regulated by C5424 were identified by transcriptome sequencing and real-time quantitative PCR. The physiological function of c5424 was studied by soft agar plate motility assay. [Results] A type I RM system was identified by bioinformatics methods. A c5424 deletion mutant was constructed. We found that the C5424 recognition motif was G^mAGNNNNNNNGTCA/TG^mACNNNNNNNCTC, and its distribution in the genome was presented. C5424 contributed to reducing transformation of foreign DNA. Deletion of c5424 significantly affected the expression of 17 genes, including motB and rclR. Lacking c5424 significantly affected bacterial motility and the resistance to hypochlorous acid in CFT073. [Conclusion] In this paper, the type I RM system C5423-5425 was characterized in detail, and it is physiologically important for uropathogenic E. coli CFT073. Thus, our data provided valuable information for bacterial epigenetics studies.

Key words:uropathogenic Escherichia coli;DNA methyltransferase;single molecule real-time sequencing;restriction-modification system