The Streptomyces Genome Contains Multiple Pseudo-attB Sites for the φC31-Encoded Site-Specific Recombination System

The integrase from the Streptomyces phage φC31 is a member of the serine recombinase family of site-specific recombinases and is fundamentally different from that of λ or its relatives. Moreover, φC31 int/attP is used widely as an essential component of integration vectors (such as pSET152) employed in the genetic analysis of Streptomyces species. φC31 or integrating plasmids containing int/attP have been shown previously to integrate at a locus, attB, in the chromosome. The DNA sequences of the attB sites of various Streptomyces species revealed nonconserved positions. In particular, the crossover site was narrowed to the sequence 5′TT present in both attP and attB. Strains of Streptomyces coelicolor and S. lividans were constructed with a deletion of the attB site (ΔattB), and pSET152 was introduced into these strains by conjugation. Thus, secondary or pseudo-attB sites were identified by Southern blotting and after rescue of plasmids containing DNA flanking the insertion sites from the chromosome. The sequences of the integration sites had similarity to those of attB. Analysis of the insertions of pSET152 into both attB(+) and ΔattB strains indicated that this plasmid can integrate at several loci via independent recombination events within a transconjugant.