Site-specific integration of corynephage ε 16: construction of an integration vector

ε16, 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 ε16 genome. This fragment also contained ε16 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 particuliarly 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. ε16 integrase was shown to catalyse site-specific integration in trans to attP with an efficiency of 5 x 103integrants per μg DNA. The integrating fragment catalysed integration in several Corynebacterium strains that are not infected by ε16, thus enlarging the host spectrum of integrating vectors derived from ε16. In these strains, the ε16 attB site was located in a conserved intergenic region and lies downstream of a clp gene.