Characterization of the temperate actinophage phi A7 DNA and its deletion derivatives.
A restriction map of phi A7 DNA (46.7 kb) was established for nine endonucleases (BclI, ClaI, EcoRI, EcoRV, HpaI, PvuI, SacII, SphI and XbaI) which cut the phage genome up to 11 times. There was no sites for BamHI, BglII, HindIII, PstI, PvuII, SacI or SalI. phi A7 DNA, circularized through its cohesive ends, could integrate into the genome of several Streptomyces hosts, to form stable lysogens. Integration occurred by recombination between unique attachment sites on the phage (attP) and the host (attB) genomes. The attP site has been located on the phi A7 restriction map. Deletion mutants of phi 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 unique SacII site. Together, the deletions defined at least 7.9 kb of DNA (16.9% of the phage genome) non-essential for plaque formation. phi A7 DNA was introduced into S. lividans protoplasts by liposome-assisted transfection. Since the phage does not adsorb to intact cells of this strain, and therefore does not form plaques, an overlay of S. antibioticus spores was used to detect the infectious progeny released by the protoplasts. Using this technique, phi A7 could be introduced into S. antibioticus with an efficiency of about 6 x 10(6) p.f.u. per micrograms DNA (equivalent to 3 x 10(-4) p.f.u. per DNA molecule).
Diaz
L A
LA
Departamento de Biología Funcional, Area de Microbiología, Universidad de Oviedo, Spain.
Hardisson
C
C
Rodicio
M R
MR
D001287:Attachment Sites, Microbiological; D001435:Bacteriophages / Q000235:genetics* / Q000502:physiology; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics*; D011756:Diphosphates / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D008242:Lysogeny; D009154:Mutation; D015183:Restriction Mapping; D013302:Streptomyces; D013303:Streptomyces antibioticus; D014162:Transfection; D010948:Viral Plaque Assay
D004279:DNA, Viral; D011756:Diphosphates; D004492:Edetic Acid; C003319:sodium pyrophosphate