Intermediates in the Biosynthesis of Double-Stranded Ribonucleic Acids of Bacteriophage σ6

Pseudomonas phaseolicola infected with bacteriophage φ6 synthesized all three viral double-stranded RNA segments, three singlestranded RNAs, and three replicative intermediate-like RNAs in the presence of rifampin. The single-stranded RNA intermediates sedimented and electrophoresed along with melted viral double-stranded RNA, annealed with melted viral double-stranded RNA, and were transient in nature. The relative amounts of the single-stranded RNA intermediates varied during the infection cycle and were altered in the presence of chloramphenicol. The replicative intermediate-like RNAs sedimented faster than double-stranded RNA, failed to enter 2.5% polyacrylamide gels, eluted with double-stranded RNA from a CF-11 cellulose column, were precipitated with single-stranded RNA in 2 M LiCl, and yielded three genomesize pieces of double-stranded RNA upon digestion with RNase. These results are consistent with the hypothesis that complementary strands of the φ6 double-stranded RNAs are synthesized asynchronously during the infection cycle. Abbreviations: dsRNA, double-stranded RNA; ssRNA, single-stranded RNA; RI, replicative intermediate; NBY, nutrient broth/yeast extract medium; MSC, minimal salts/casein medium; STE buffer, 0.1 M NaCl, 50 mM Tris, 5 mM EDTA, (pH 8.0). The genome of φ6, a lipid-containing Pseudomonas phaseolicola bacteriophage, consists of three unique double-stranded RNA (dsRNA) segments of approximately 2.2, 3.2, and 5.0 × 106 daltons (1, 2). Recent work (Coplin et al., in preparation) indicates that the three segments are synthesized continuously throughout the infection cycle. If infected cells were labeled with [3H]uracil and then “chased” with excess unlabeled uracil, radioactivity continued to accumulate in dsRNA, suggesting that complementary strands of the dsRNAs are synthesized asynchronously, possibly via single-stranded RNA (ssRNA) intermediates. The present paper describes the identification and properties of two classes of RNA intermediates: (i) ssRNA transcripts formed from each segment, and (ii) the corresponding partially double-stranded branched RNAs which are analogous to the replicative intermediates (RI) of ssRNA bacteriophages. MATERIALS AND METHODS Growth and Infection of Bacteria. Phage lysates were prepared by suspending confluent lysis overlays in 5 ml of nutrient broth/yeast extract medium (NBY) (3) and clarifying by centrifugation. Supernatants were stored in 6% sucrose at –80°. P. phaseolicola HB10Y, host for phage φ6, was maintained on NBY agar (3). All experiments were done in minimal salts/casein (MSC) liquid medium which contains per liter of distilled water: 5.90 g of K2HPO4; 2.20 g of KH2PO4; 1.32 g of (NH4)2SO4; 0.20 g of MgSO4·7H2O; 1.54 mg of MnSO4·H2O; 8.8 mg of ZnSO4; 2.42 mg of FeCl3·6H2O; 10.0 g of sucrose; and 1.0 g of Difco casamino acids. The sucrose and casamino acids were autoclaved separately. To obtain consistent and maximal infection, a culture of the host that had been grown for 12 hr with shaking was subcultured at 26° and allowed to increase from 8 × 107 to 4 × 108 cells per ml. The culture was then chilled to 0° and phage were added at a ratio of 10 plaque-forming units (PFU) per cell. After an adsorption period of 30 to 60 min, infection was initiated by warming the culture to 26°. Rifampin Treatment, Pulse Labeling, and Extraction of RNA. Rifampin was routinely added to bacterial cultures to give a final concentration of 200 μg/ml 10 min before labeling. RNA was labeled with 3 μCi/ml of [5-3H]uracil (20.4 Ci/mmol) or [5,63H]uracil (40.7 Ci/mmol). Labeling was stopped by pouring cells over crushed frozen MSC medium, and the cells were harvested by centrifugation at 8,000 × g for 5 min. For extraction of dsRNA and RI, the cells were resuspended in 1.0 ml of STE buffer [50 mM Tris, 0.1 M NaC1, 5 mM EDTA (pH 8.0)] containing 0.1 ml of 25% sodium dodecyl sarcosine and 400 μg of washed bentonite. The suspension was placed at 65° for 5 min and then deproteinized with an equal volume of phenol-cresol (100 g of phenol, 11 ml of STE buffer, 14 ml of m-cresol, 100 mg of 8-hydroxyquinoline) at 65° for 5 min. The aqueous phase was removed and the sodium concentration adjusted to 0.3 M with 5 M sodium acetate, pH 5.5. RNA was precipitated with 3 volumes of 95% ethanol at –20°. ssRNA was extracted in the same manner except that it was deproteinized with equal volumes of phenol-cresol and chloroform at room temperature to prevent aggregation. Nucleic acids were purified by chromatography on CF-11 cellulose fiber columns (4). ssRNA eluted in 15% ethanol, while dsRNA and RI were eluted in buffer without alcohol. RI was separated from dsRNA by overnight precipitation at 4° with 2 M LiCl in STE buffer. Centrifugation and Electrophoresis. RNA samples were dissolved in 0.5 ml of 0.10 M NaCl, 10 mM Tris (pH 8.9) and centrifuged for 10.5 hr at 37,000 rpm at 4° in an SW 41 rotor on linear-logarithmic sucrose density gradients (5) equilibrated with the same buffer. The gradients were scanned photometrically at A254 nm with an ISCO density gradient fractionator, 0.25 ml fractions were Published in Proceedings of the National Academy of Sciences USA Vol. 72, No. 3 (March 1975), pp. 849–853. Copyright © 1975 National Academy of Sciences USA. Used by permission. Communicated by Myron K. Brakke, December 23, 1974. Intermediates in the Biosynthesis of Double-Stranded Ribonucleic Acids of Bacteriophage φ6 (Replicative intermediate/replication of double-stranded RNA/Pseudomonas phaseolicola) D. L. Coplin*, J. L. Van Etten, R. K. Koski, and A. K. Vidaver Department of Plant Pathology, University of Nebraska–Lincoln, Lincoln, NE 68503 *Present address: Department of Plant Pathology, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691.