Resolvase-like recombination performed by the TP901-1 integrase

The site-specific recombination system of temperate lactococcal bacteriopha ge TP901-1 is unusual in several respects. First, the integrase belongs to the family of extended resolvases rather than to the lambda integrase famil y and second, in the presence of this integrase, a 56 bp attP fragment is s ufficient for efficient recombination with the chromosomal attB site in the host Lactococcus lactis subsp. cremoris MG1363. In the present work, this attB site was analysed and a 43 bp attB region was found to be the smallest fragment able to participate fully in recombination. In vitro studies show ed that the TP901-1 integrase binds this 43 bp attB fragment, the 56 bp att P and a larger attP fragment with equal affinity. Mutational analysis of th e 5 bp common core region (TCAAT) showed that the TC dinucleotide is essent ial for recombination, but not for binding of the integrase, whereas none o f the last three bases are important for recombination. When a number of at tL sites, obtained by recombination between an attB site containing a mutat ion in this TC dinucleotide and a wildtype attP site, were sequenced, a mix of sites with the wild-type or the mutated sequence was obtained. These re sults are consistent with the hypothesis that the TC dinucleotide constitut es the TP901-1 overlap region. A 2 bp overlap region has been observed in r ecombination reactions catalysed by all other members of the resolvase/inve rtase family tested so far. By selecting for attB sites with a decreased ab ility to participate in recombination, two bases located outside the core r egion of attB were shown to be involved in the in vitro binding of the TP90 1-1 integrase.