Acta Microbiologica 11. (1964)

4. szám - Balassa, R.–Gábor, M.: Transformation of Streptomycin Markers in Rough Strains of Rhizobium lupini. Transformation of Streptomycin-Dependence

330 R. BALASSA and M. GÁBOR Materials and methods Bacterial strains. Rough strains of Rhizobium lupini were used. The strains H-13-1 and H-13-3 were streptomycin-sensitive [4], the others trains were streptomycin-dependent mutants of strains H-13-1 and H-13-3. The dependent strains were obtained by single-step selec­tion of spontaneous mutants from sensitive strains or by selection of dependent transformants in experiments of transformation of streptomycin-dependence. During the selection of spon­taneous mutants, we obtained one dependent colony, selected with lOOO/^g/ml of streptomycin from lXlO11 cells (strain H-13-1) and 5ХЮ9 cells (strain H-13-3), respectively. The selected strains grew slowly on media containing 100 or 300 ^g/ml streptomycin. The streptomycin requirement of the dependent strains obtained by transformation of streptomycin-dependence was only 300 jug/ml. The response to streptomycin of the strains used is shown in Table I. Media. The nutrient medium contained (per liter) K2HP04, 3.6 g; KH2P04, 0.4 g; MgS04 • 7H20, 0.05 g; NaCl, 0.5 g; ferric ammonium citrate, 4.0 mg; agar, 20 g. The pH was adjusted, to 7.0 — 7.2. To the synthetic medium were added glucose to a final concen­tration of 5 g, casein hydrolysate 1 g, and yeast extract 1 g, for 1000 ml [2]. Preparation of DNA. The bacteria were centrifuged at the end exponential growth, resuspended in 0AM NaCl sodium citrate, and lysed by the addition of 0.2 volume of 5 per cent deoxycholate for 10 —15 min at 55°C. The material was deproteinized 5 — 7 times by the Sevag method with chloroform and purified by repeated precipitation in 3 volumes or 1 volume of alcohol, respectively. The DNA thus obtained was stored by the method of Zamenhof in 0.14 M NaCl and 0.015 M sodium citrate at pH 7.4, at 4°C. The DNA content of the prepa­rations was estimated according to DlSCHE [2]. The transforming extract contained no viable bacteria. Deoxyribonuclease (DN-ase). The DN-ase used was prepared in the Institute of Medical Chemistry, University Medical School, Budapest. Transformation technique. A 20 hrs old culture (108 bacteria per ml) was diluted to a final concentration of 108 bacteria/ml into 10 — 15 ml fresh supplemented medium, containing 5 —19 /ig/ml of the transforming DNA. The transformation reaction was stopped, if necessary, by the addition of DN-ase at a final concentration of 2.5 /ig/ml. In other cases the transforma­tion reaction lasted 8, 24 or 48 hours without addition of streptomycin to the medium. Thereaf­ter the transformed population was plated on the selective medium, containing 30, 100, 300 or 1000 /ig/ml streptomycin, and incubated for 48 to 72 hours at 30°C. As controls, cultures of cells grown under identical conditions but without transforming DNA were plated on the selective media to verify that the number of spontaneous mutations bearing the marker under study was insignificant. The frequency of transformation was calculated in per cent of the total number of cells, estimated on medium lacking streptomycin. The colonies grown on the selective medium were tested for character by suspending in 0.5 ml of saline some colonies picked up at random, and streaking a loopful on media contain­ing 0, 30, 50, 100, 300 or 1000 ^g/ml streptomycin. After 48 to 72 hours incubation at 30°C growth was scored from — to Н--Т-Ы--К or the colonies were counted. Results Genetic analysis of streptomycin-dependent donors (spontaneous mutants or transformants) have revealed the following. Table II gives the data of ex­periments performed to study whether streptomycin-dependence could he transferred by transformation, and to determine the most suitable conditions for detecting the streptomycin-dependent transformants. The recipient bac­teria were sensitive to streptomycin, transformation was carried out without streptomycin (Tables I, II). In the experiment with the DNA of the streptomycin-dependent strain 1-str-d (1000) (Table II, exp. 1/74) seven normal-sized colonies and one small colony among ten, which appeared on selective medium, were streptomycin­­independent and had a high level of resistance. Testing of two very small

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