Site-specific integration of corynephage phi16: construction of an integration vector

Phi16, a temperate phage induced from Corynebacterium glutamicum ATCC 21792, lysogenizes its host via site-specific recombination. The phage attachment site, attP, was located to a 6.5 kb BamHI fragment of the phi16 genome. This fragment also contained phi16 integrative functions. The minimal phage DNA fragment required for integration was defined. This 1630 bp region contained a large open reading frame, int, encoding a protein of 416 amino acids with similarity in its carboxyl-terminal domain to tyrosine recombinases and particularly to the Xer recombinases. The comparison of the nucleotide sequences of attB, attL, attR, and attP identified a common 29 bp sequence, the core sequence. It lies 11 bp downstream of the 3' end of the integrase gene. phi16 integrase was shown to catalyse site-specific integration in trans to attP with an efficiency of 5x10(3) integrants per microg DNA. The integrating fragment catalysed integration in several Corynebacterium strains that are not infected by phi16, thus enlarging the host spectrum of integrating vectors derived from phi16. In these strains, the phi16 attB site was located in a conserved intergenic region and lies downstream of a clp gene.