The site-specific recombination system of actinophage TG1

Abstract

Actinophage TG1 forms stable lysogens by integrating at a unique site on chromosomes of Streptomyces strains. The phage (attP(TG1)) and bacterial (attB(TG1)) attachment sites for TG1 were deduced from comparative genomic studies on the TG1-lysogen and nonlysogen of Streptomyces avermitilis. The attB(TG1) was located within the 46-bp region in the dapC gene (SAV4517) encoding the putative N-succinyldiaminopimelate aminotransferase. TG1-lysogens of S. avermitilis, however, did not demand either lysine or diaminopimelate for growth, indicating that the dapC annotation of S. avermitilis requires reconsideration. A bioinformatic survey of DNA databases using the fasta program for the attB(TG1) sequence extracted possible integration sites from varied streptomycete genomes, including Streptomyces coelicolor A3(2) and Streptomyces griseus. The gene encoding the putative TG1 integrase (int(TG1)) was located adjacent to the attP(TG1) site. TG1 integrase deduced from the int(TG1) gene was a protein of 619 amino acids having a high sequence similarity to phiC31 integrase, especially at the N-terminal catalytic region. By contrast, sequence similarities at the C-terminal regions crucial for the recognition of attachment sites were moderate or low. The site-specific recombination systems based on TG1 integrase were shown to work efficiently not only in Streptomyces strains but also in heterologous Escherichia coli.

In vitro characterization of the site-specific recombination system based on actinophage TG1 integrase

“…We have previously demonstrated that the integrase system of actinophage TG1 works eYciently not only in homologous Streptomyces cells, but also in heterologous E. coli cells (Morita et al 2009). This indicates that integrase expressed from the int TG1 gene in E. coli cells catalyzes a site-speciWc recombination between attB and attP sites of TG1 phage without detrimental eVects on the host bacteria.…”

“…In a previous paper we have established an in vivo integration system based on actinohage TG1 and relevant attachment sites (Morita et al 2009). Although integrases of TG1 and C31 share a high sequence similarity, especially at the N-terminal catalytic domain, attachment sites (attP, attB) of two integrases were distinct and not interconvertible (Ikeda et al 2003;Morita et al 2009).…”

“…Although integrases of TG1 and C31 share a high sequence similarity, especially at the N-terminal catalytic domain, attachment sites (attP, attB) of two integrases were distinct and not interconvertible (Ikeda et al 2003;Morita et al 2009). The TG1-based integration system was demonstrated to function in heterologous Escherichia coli cells, suggesting that the TG1 integrase does not require host-speciWc factor(s).…”

Genetic approaches to generate hyper-producing strains of goadsporin: the relationships between productivity and gene duplication in secondary metabolite biosynthesis

“…pGSBC1 and pTYM1-GSBC contain the entire goadsporin biosynthetic gene cluster and actinomycete genome integration modules that consist of integration genes isolated from actinophages phiC31 14) and TG1. 15) Each integrase recognizes a different attB site in a genome, enabling the two plasmids to integrate into a single genome. After transconjugation, site-specific integrases phiC31 and TG1 catalyze unidirectional recombination between the vector attP and the chromosomal attB, and single integrated recombination occurs at the attB site.…”

New ΦBT1 site-specific integrative vectors with neutral phenotype in Streptomyces

“…The alternatives to the ΦC31 integrative site in Streptomyces are the ΦBT1 (Gregory et al 2003), TG1 (Morita et al 2009) and SV1 (Fayed et al 2014) integrative sites. The ΦBT1 derived vectors integrate into the attP site localized into the SCO4848 gene (S. coelicolor genome) (Gregory et al 2003), while the TG1 plasmids integrate at SCO3658 (Fayed et al 2014) and SV1 plasmids at SCO4383 (Fayed et al 2014).…”