Open AccessCharacterization of the temperate actinophage ϕA7 DNA and its deletion derivatives
Author(s) -
Luis A. Díaz,
Carlos Hardisson,
M. Rosario Rodicio
Publication year - 1991
Publication title -
microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.352
H-Index - 35
eISSN - 1465-2080
pISSN - 1350-0872
DOI - 10.1099/00221287-137-2-293
Subject(s) - restriction enzyme , lysogen , biology , microbiology and biotechnology , dna , temperateness , genome , restriction map , ecori , restriction site , cosmid , bacteriophage , plasmid , restriction fragment , genetics , escherichia coli , gene
Summary A restriction map of ø A7 DNA (46·7 kb) was established for nine endonucleases ( Bc /I, Cla I, Eco RI, Eco RV, Hpa I, Pvu I, Sac II, Sph I and Xba I) which cut the phage genome up to 11 times. There were no sites for Bam HI, Bg /II, Hind III, Pst I, Pvu II, Sac I or Sa /I. ø 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 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 unique Sac II 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 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, ø A7 could be introduced into S. antibioticus with an efficiency of about 6 × 10 6 p.f.u. per μg DNA (equivalent to 3 × 10 -4 p.f.u. per DNA molecule).