New Bacillus subtilis vector, pSSβ, as genetic tool for site-specific integration and excision of cloned DNA, and prophage elimination

The Journal of General and Applied Microbiology

article

Shota Suzuki¹  Sachie Osada³  Daisuke Imamura¹  Tsutomu Sato¹ 

[1] Research Center of Micro-Nano Technology, Hosei University;Department of Life Science, College of Science, Rikkyo University;Department of Frontier Bioscience, Hosei University

关键词: site-specific integration vector;    site-specific recombination;    SPβ;    SSR;    Bacillus subtilis;    integrase;    RDF;   

DOI  :  10.2323/jgam.2021.10.004

学科分类：微生物学和免疫学

来源: Applied Microbiology, Molecular and Cellulrar Biosciences Research Foundation

【 摘 要 】

Site-specific recombination (SSR) systems areemployed in many genetic mobile elements, including temperate phages, for their integration andexcision. Recently, they have also been used as toolsfor applications in fields ranging from basic to synthetic biology. SPβ is a temperate phage of theSiphoviridae family found in the laboratory standard Bacillus subtilis strain 168. SPβ encodes a serine-type recombinase, SprA, and recombinationdirectionality factor (RDF), SprB. SprA catalyzesrecombination between the attachment site of thephage, attP, and that of the host, attB, to integratephage genome into the attB site of the host genomeand generate attL and attR at both ends of theprophage genome. SprB works in conjunction withSprA and switches from attB/attP to attL/R recombination, which leads to excision of the prophage.In the present study, we took advantage of thishighly efficient recombination system to develop asite-specific integration and excision plasmid vector,named pSSβ. It was constructed using pUC plasmidand the SSR system components, attP, sprA and sprBof SPβ. pSSβ was integrated into the attB site witha significantly high efficiency, and the resultingpSSβ-integrated strain also easily eliminated pSSβitself from the host genome by the induction of SprBexpression with xylose. This report presents twoapplications using pSSβ that are particularly suitable for gene complementation experiments and fora curing system of SPβ prophage, that may serve asa model system for the removal of prophages inother bacteria.

【 授权许可】

Unknown