Unusual site-specific DNA integration into the highly active pseudo-attB of the Streptomyces albus J1074 genome

The phi C31-encoded recombination system has become a widely used tool for genetic analysis of streptomycetes, gene therapy and generation of transgenic animals. However, the application of this system, even in the context of its natural host genus, Streptomyces, may require a specific approach for each species. In this study, we have identified a novel pseudo-attB site, called pseB4, for integration of vectors using the phi C31 system. More than 90 % of clones contained two copies of pSET152- or pOJ436-based cosmids, after their introduction into S. albus. The efficiency of the integration of phi C31-based vectors into pseB4 is therefore comparable to that of the integration into attB. Moreover, in contrast with integration into the native attB, integration into pseB4 is not polar and does not require a complementary sequence in the TT-core region. Furthermore, an analysis of conjugation frequency revealed mutual inhibition of plasmid integration into either site when both the attB and pseB4 sites were present in the genome.