Structure of the chromosomal insertion site for pSAM2: functional analysis in Escherichia coli

The element pSAM2 from Streptomyces ambofaciens integrates into the chromosome through site-specific recombination between the element ( att  P) and the chromosomal ( att  B) sites. These regions share an identity segment of 58 bp extending from the anti-codon loop through the 3' end of a tRNA^Pro gene. To facilitate the study of the att  B site, the int and xis genes, expressed from an inducible promoter, and att  P from pSAM2 were cloned on plasmids in Escherichia coli . Compatible plasmids carrying the different att  B regions to be tested were introduced in these E . coli strains. Under these conditions, Int alone could promote site-specific integration; Int and Xis were both required for site-specific excision. This experimental system was used to study the sequences required in att  B for efficient site-specific recombination. A 26 bp sequence, centred on the anti-codon loop region and not completely included in the identity segment, retained all the functionality of att  B; shorter sequences allowed integration with lower efficiencies. By comparing the 26-bp-long att  B with att  P, according to the Lambda model, we propose that B and B', C and C' core-type Int binding sites consist of 9 bp imperfect inverted repeats separated by a 5 bp overlap region.