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

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.