Highly Efficient In Vitro Site-Specific Recombination System Based on Streptomyces Phage φBT1 Integrase

Abstract

The Streptomyces phage BT1 encodes a site-specific integrase of the large serine recombinase subfamily. In this report, the enzymatic activity of the BT1 integrase was characterized in vitro. We showed that this integrase has efficient integration activity with substrate DNAs containing attB and attP sites, independent of DNA supercoiling or cofactors. Both intra-and intermolecular recombinations proceed with rapid kinetics. The recombination is highly specific, and no reactions are observed between pairs of sites including attB and attL, attB and attR, attP and attL, or attP and attR or between two identical att sequences; however, a low but significant frequency of excision recombination between attL and attR is observed in the presence of the BT1 integrase alone. In addition, for efficient integration, the minimal sizes of attB and attP are 36 bp and 48 bp, respectively. This site-specific recombination system is efficient and simple to use; thus, it could have applications for the manipulation of DNA in vitro.Site-specific recombinases have been widely used in genetic engineering: for example, in vitro cloning, plant and mammalian cell genome modification, and gene therapy (3,11,12,15,16). Nearly all site-specific recombinases can be classified as tyrosine recombinases, also known as the integrase family, or serine recombinases, also known as the resolvase/invertase family, based on comparisons of amino acid sequences and different mechanisms of catalysis; these two types use tyrosine or serine, respectively, to attack the DNA sugar-phosphate backbone (14,18,20). The process of strand exchange catalyzed by tyrosine recombinases involves a Holliday junction intermediate and cleavage and rejoining of the strands one by one. In contrast, serine-mediated recombination involves a process of double-strand breakage, followed by rotation and religation (9). The well-known tyrosine recombinase integrase recognizes two different attachment substrate sites, the 25-bp attB gene (in the bacterial chromosome) and the 240-bp attP gene (in the phage genome), which are inverted into attL and attR with the aid of the integration host factor (a chaperone). Furthermore, excision between attL and attR requires recombination directionality factor (RDF), and recombinations mediated by tyrosine recombinases require supercoiling of the substrate DNAs (10). In contrast, the extensively studied large serine recombinase from Streptomyces phage C31 is thought to catalyze unidirectional recombination between DNA substrates attB and attP, independent of DNA supercoiling and host cofactors (18). In addition, the attachment sites of the C31 integrase are smaller than those of most tyrosine recombinases, with minimal sizes of 34 bp for attB and 39 bp for attP (11,18).Streptomyces phage BT1, a homoimmune relative of C31, is inserted into the chromosomes of a wide range of Streptomyces spp. for lysogeny and encodes a large serine recombinase (5, 8). Similar to those of the C31 site-specific recombination system, the attB and attP sites of BT1 ...

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

“…The minimal sizes of substrate DNAs for the serine integrases are similar, but not identical. In general, the minimal sizes of attB sites for the serine integrases are less than 40 bp, but those of attP sites are larger than 40 bp (38 and 48 bp for Bxb1 integrase, Ghosh et al 2003; 36 and 48 bp for BT1 integrase, Zhang et al 2008).…”

“…The reversibility and substrate speciWcity of TG1 integrase Serine integrases reported so far are known to be irreversible for the integration reaction in the absence of accessory factors (directionality factor) except for BT1 integrase (Zhang et al 2008). Next, we examined whether TG1 integrase can reverse the integration reaction in the absence The recombined product in this reaction is a linear DNA molecule containing attL and attR.…”

Streptomyces temperate bacteriophage integration systems for stable genetic engineering of actinomycetes (and other organisms)

“…1). The mechanism of insertion was studied in vitro where it was shown that the minimal attB and attP sites comprise 36 and 48 bp, respectively [106]. The integration process was very eYcient with attB and attP substrates, but was also measurable with attL and attR sequences, implying that Int might excise BT1 in vivo at some frequency in the absence of other factors.…”

RifZ (AMED_0655) Is a Pathway-Specific Regulator for Rifamycin Biosynthesis in Amycolatopsis mediterranei

“…Briefly, primers 0655U-F/0655U-R and 0655D-F/0655D-R were separately used to amplify the upstream and downstream homologous arms of rifZ, and the PCR fragments were introduced into the XcmI site of pFDZ101 and pFDZ103 to obtain pDZL101 and pDZL103 (Table 2), respectively. pDZL101, pDZL103, and pTA0613 were linearized and tandemly assembled with pFDZ100 using BT1 integrase (38) to produce the knockout plasmid pDZL104, which was further electroporated into U32 to knock out rifZ. Mutants were selected on Bennet plates containing apramycin (50 g/ml) and verified by PCR employing primers 0655CH-UF/ 0655CH-UR and 0655CH-DF/0655CH-DR.…”