Isolation and Characterization of Covalently Closed Circular Dna Associated with Chromosomal and Membrane Fraction from Streptomyces Ambofaciens (original) (raw)

1982, The Journal of Antibiotics

Covalently closed circular (ccc) DNAs were isolated by a technique involving alkaline denaturation from the spiramycin producer Streptonryces annbofaciens KA-1028 and also from spiramycin non-producing strains AF-1 I and QN-25; plasmids could not be detected in these strains by a cleared lysate method. It was found that most of the ccc DNA in these strains was present in the chromosomal and membrane fractions. These ccc DNAs had identical mobilities in agarose gel electrophoresis. The size was calculated to be 53.1 x 106 daltons from the contour length measurements. The ccc DNA gave one fragment on digestion with Hind 111, three fragments with Eco Rl, and twenty-eight fragments with Bain HI. Various ccc DNAs have been isolated from streptomycetes1~13) Their involvement in various biological functions, such as antibiotic production14) and resistance4.11), fertility2,15) , differentiation16~18) and release of extracellular enzyme19,20), has been studied; the involvement of plasmids in antibiotic production in streptomycetes has been suggested by "curing experiments"16). Isolation of ccc DNAs from streptomycetes has generally been performed by cleared lysate methods although YAGISAWA et al.11 and OMURA et al.12) employed alkaline denaturation. THE JOURNAL OF ANTIBIOTICS APR. 1982 This paper deals with the isolation and distribution of plasmid DNA from spiramycin-producing and non-producing strains of S. ambofaciens and with the characterization of pSAI DNA. Materials and Methods Organisms Streptomyces ambofaciens KA-1028 (ISP-5053), a spiramycin-producer, and strains AF-11 and QN-25, spiramycin non-producing strains, were used. Strains AF-11 and QN-25 were non-producing strains obtained by treatment of S. ambofaciens KA-1028 with acriflavine and quinacrine, respectively. Chemicals CsCl was purchased from Nakarai Chemicals Ltd., Kyoto, Japan, lysozyme and ethidium bromide from Sigma Chemicals Co., RNase A from Boehringer Mannheim Gm13H, restriction endonucleases, Eco R1, Hlnd 111, and Bam HI, from Takara Shuzo Co., Ltd., Kyoto, Japan, and [methyl-1H]thymidine (41 Ci/mmole) and [2-14C]thymidine (42 mCi/mmole) from New England Nuclear. Media and Growth Strains were incubated in Tryptic Soy Broth (BBL) at 27°C for 48 hours, and 0.2 ml of the culture was transferred into 10 ml of a medium containing 0.25 % glucose, 0.5 low phosphate content Casamino Acids (when low phosphate content Casamino Acids was not used, mycelial pellets were often formed that interfered with lysozyme digestion), 0.3 % L-asparagine • H2O, 0.3 % NaCl, 0.05 % MgSO4 • 7H2O, 0.1% NaNO2, 0.01 % CaCl2.2H2O, 0.009 % KH2PO4, 0.4 % trace elements solution, 0.02 % 2'-deoxyadenosine, 0.1 M tris-(hydroxymethyl)aminomethane hydrochloride (tris-HCl), pH 7.2, and 1-10 t1Ci of [methyl-3H]thymidine (41 Ci/mmole) or 1 1LCi of [2-11C]thymidine (42 mCi/mmole) per ml and then cultivated at 27°C for 18 hours. Mycelia were harvested by centrifugation at 12,000 xg for 10 minutes. Low phosphate content Casamino Acids was prepared as follows. Twenty grams of Casamino Acids (Difco) was dissolved in 100 ml of distilled water and then 2.54 g of MgCl2.6H2O and 11 ml of concentrated NH4OH were added. The stirred mixture was chilled at 0°C for at least 2 hours, and centrifuged at 2,000 xg for 5 minutes at 0°C to remove the precipitate. The excess ammonia was evaporated from the supernatant fluid under reduced pressure. After the pH was adjusted to 7.0 with I N HCl, the solution was lyophilized to give a powder of low phosphate content Casamino Acids.