副溶血弧菌TF2中整合接合元件ICEVpaTF2的生物信息学特征和剪切、环化活性

Bioinformatics characteristics and excision/cyclization activities of an ICE element, ICEVpaTF2, in Vibrio parahaemolyticus TF2

摘要:

[目的] 从副溶血弧菌TF2基因组框架序列中拼接获得整合性接合元件（ICE）ICEVpaTF2的全序列，分析其基因组学特征；研究ICEVpaTF2是否具有从基因组中剪切、环化活性以及剪切、环化时attB和attP位点如何形成。[方法] 对副溶血弧菌TF2基因组框架序列进行RAST注释，发现其基因组中可能存在一个完整的ICE元件，命名为ICEVpaTF2，经过人工拼接和PCR扩增测序验证，得到完整的ICEVpaTF2序列，并对ICEVpaTF2再次进行RAST注释和ICE元件部分特征分析。通过PCR检测，探索ICEVpaTF2是否能够从基因组剪切并环化。通过attL、attR、attB、attP位点序列比较，探索attB和attP重组特征。[结果] ICEVpaTF2全长83588 bp，包含SXT/R391家族ICE元件的52个保守核心基因，它们与ICE切除、整合、自我转移和调节机制相关。ICEVpaTF2也包含5个外源基因插入的热点区（HS）、2个可变区（VR）以及3个非典型插入位点。HS和VR包含了大量可变基因，它们负责编码限制性修饰系统、DNA修复系统或毒素-抗毒素系统等，赋予宿主广泛适应性功能，ICEVpaTF2也包含独特未知功能基因。通过对int、xis二个核心保守基因种系发生分析，发现ICEVpaTF2的int、xis分别属于以R391和SXT为代表的亚群。ICEVpaTF2在attL和attR处发生剪切并完成环化，新形成杂合重组的attB和attP位点。[结论] ICEVpaTF2属于SXT/R391家族，是一个具备自我剪切和环化能力的完整ICE元件，剪切后新形成的attB和attP位点由attL、attR杂合重组形成。

Abstract:

[Objective] To obtain complete sequence of an integrating conjugative element (ICE), ICEVpaTF2, from Vibrio parahaemolyticus TF2, and analyze its genomic characteristics; to investigate if ICEVpaTF2 has excision/cyclization activities from host genome and how att sites recombine during excision/cyclization. [Methods] The genomic frame sequences of V. parahaemolyticus TF2 was first annotated by RAST, which led to the finding of a potential and complete ICE element, named ICEVpaTF2. After manual splicing, PCR amplification followed by sequencing verification, a complete sequence of ICEVpaTF2 was obtained, and then it was annotated and characterized again. PCR detection and subsequent sequencing were performed to explore whether ICEVpaTF2 could excise from its host genome and cyclize itself and to characterize the forming of attB and attP by comparing the sequences of attL, attR, attB and attP. [Results] ICEVpaTF2, with a total length of 83588 bp, contains 52 conserved core genes of SXT/R391 ICE elements, which are related to excision, integration, self-transfer, and regulatory mechanisms. ICEVpaTF2 also contains five hot spots (HS), two variable regions (VR), and three atypical insertion sites accommodating variable genes. HS and VR contain a large number of variable genes, encoding for restrictive modification system, DNA repair system, toxin-antitoxin system, and etc., which confer the host extensive adaptive profits to the bacterial host. ICEVpaTF2 also contains unique genes with unknown functions. Through phylogenetic analysis of the two core conserved genes, int and xis, it was found that int and xis of ICEVpaTF2 belong to subgroups represented by that of R391 and SXT, respectively. ICEVpaTF2 excises at chromosomal attL and attR sites and cyclizes resulting in the forming of new heterozygous and recombined attB and attP sites. [Conclusion] ICEVpaTF2 belongs to the SXT/R391 family and it is a complete ICE element harboring self-excision and cyclization activities. New attB and attP sites are generated by hybrid recombination between primary attL and attR sites.