Characterization of the temperate actinophage ϕA7 DNA and its deletion derivatives

Abstract

SummaryA restriction map of ø A7 DNA (46·7 kb) was established for nine endonucleases (Bc/I,ClaI,EcoRI,EcoRV,HpaI,PvuI,SacII,SphI andXbaI) which cut the phage genome up to 11 times. There were no sites forBamHI,Bg/II,HindIII,PstI,PvuII,SacI orSa/I. ø A7 DNA, circularized through its cohesive ends, could integrate into the genome of severalStreptomyceshosts, to form stable lysogens. Integration occurred by recombination between unique attachment sites on the phage (attP) and the host (attB) genomes. TheattPsite has been located on the ø A7 restriction map. Deletion mutants of ø A7 DNA were obtained by selecting for pyrophosphate- or EDTA-resistant clones. The deletions occurred either near the left-hand end of the conventional restriction map, or about 18 kb from the right-hand end, close to, but not affecting the uniqueSacII site. Together, the deletions defined at least 7·9 of DNA (16·9% of the phage genome) non-essential for plaque formation. ø A7 DNA was introduced intoS. lividansprotoplasts by liposome-assisted transfection. Since the phage does not adsorb to intact cells of this strain, and therefore does not form plaques, an overlay ofS. antibioticusspores was used to detect the infectious progeny released by the protoplasts. Using this technique, ø A7 could be introduced intoS. antibioticuswith an efficiency of about 6 × 106p.f.u. per μg DNA (equivalent to 3 × 10-4p.f.u. per DNA molecule).