Transfection in Streptomyces virginiae

Until now, transformation with linear DNA has not been demonstrated in Streptomycetes, although it has been established in a thermophilic Actinomyces species, Thermoactinomyces vulgaris (Hopwood & Wright, 1972). The main tool used in Streptomycete genetics is conjugation (Hopwood, 1967). In the wake of a genetic map for Streptomyces coelicolor, maps for other strains were constructed by means of conjugation, and they exhibited strong similarities to that for S. coelicolor (Hopwood, 1973). To have a complementary tool to study Streptomyces genetics on a fine scale, we screened strains including S. coelicolor for uptake of [3H]DNA during the growth cycle, starting from germinating spores (Roelants et al., 1976). Streptomyces virginiae A.T.C.C. 13161 and, toalesserextent Streptomyceskasugaensis, were found to take up DNA [with a maximum incorporation of 2% of the exogenous DNA (lOpg/ml) added]. The uptake curve suggested strongly a competence phase that seemed to occur during the early stationary phase. In the other strains the DNA was either very poorly taken up or was degraded. S. virginiae was further investigated for biological expression of the DNA taken up. Phage DNA was chosen for this purpose, and the present communication describes an assay of transfection by means of the phage S1 DNA. (Transfection is the process by which bacteriophage DNA is expressed after uptake by the production of bacteriophages that conform to the original in both phenotype and genotype.) Description and growth of the phage. Phage S1 is a virulent phage first used by Roelants et al. (1968). It was found to grow only on S. virginiae and did not lyse 15 other Streptomycetes or six different species of the Nocardiae. Morphologically, phage S1 has an oval head and a long flexible tail terminated by a fixation structure. Phage infection requires Ca2+. Phage growth can be initiated at any stage in the cell cycle from the germination of spores to the late stationary phase. One-step growth experiments (Dowding, 1973) showed a lag phase of 90-120min and an exponential phase of 210min. The total growth cycle of the phage lasted about 5 h. Phage were grown either on liquid medium (AC broth, Difco) or on agar plates (yeast-extract/malt agar, Difco) by the double-layer technique (Eisenstark, 1967). After confluent lysis, the phage was harvested and purified on CsCl gradients. The density of the phage in a CsCl gradient was 1.520g/cm3. Phage DNA extraction. A purified and concentrated phage suspension containing about loi2 plaque-forming units (p.f.u.)/ml was exposed to sodium dodecyl sulphate ( 1 %, final concn.) during 4min, then twice treated with an equal volume of phenol saturated with 0.01 M-NaCI (Thomas & Abelson, 1967). The suspension required very gentle manipulation. After centrifugation and decantation, the treatment was completed with extensive dialysis in salinelcitrate buffer at 4°C. Phage DNA was found to be uncontaminated with p.f.u. by spreading on plates inoculated with S. virginiae spores and incubated from 2 to 10h at 29°C before layering the phage. Preparations treated with phenol that were not treated with sodium dodecyl sulphate still contained some p.f.u. The molecular weight of the final DNA preparation was determined by viscosimetry with the low-shear Beckman viscosimeter as described by Eigner & Doty (1965). Maximum values for mol.wt. were around 30000000. The density of DNA as determined by centrifugation in CsCl gradients was 1.725g/cm3, slightly lower than that for host-cell DNA. Transfection assay. Cells of the S. virginiae mutant SV 664 (methionine-requiring) were grown in minimal medium supplemented with 10% (v/v) glycerol, casamino acids (200pg/ml) and methionine (20pg/ml) to produce the expected com-