Site-specific recombination by Gin of bacteriophage Mu: Inversions and deletions

Abstract

A 3000-bp invertible segment in the DNA of bacteriophage Mu determines the host range of the phage. The inversion is catalyzed by the phage-coded protein Gin; the recombination sites are short inverted repeats. Gin protein is only made in low amounts by Mu. To further investigate the Gin-mediated recombination reaction a Gin overproducing strain was constructed. The gin gene was cloned on a plasmid behind the p L-promoter of phage λ. This results in a 100-fold higher inversion frequency of a Mu gin phage as compared to the situation when Gin is expressed from its own promoter. A test system was developed suitable for the detection of Gin action in vivo and in vitro. the lacZ gene of E. coli was cloned within the invertible region in such a way that it is only expressed when the region is in one specific orientation. Thus inversions can be detected or selected as a switch from Lac − to Lac +. This system was used to determine the inversion frequency under different experimental conditions. The ability of Gin to catalyze deletions was investigated by inverting in vitro one of the two recombination sites using restriction enzymes and genetically marking the DNA between those sites. Deletions do occur, although at a lower frequency than inversions.