The morphology of the intracellular development of bacteriophage 425 in Bacillus subtilis 168M has been correlated with nucleic acid synthesis in infected cells. Host deoxyribonucleic acid (DNA) synthesis was shut off by a phage-induced enzyme within 5 min after infection, and another phage-mediated function extensively degraded host DNA at the time of cell lysis. Synthesis of phage DNA in infected cells began within 5 min and continued until late in the rise period. After phage DNA synthesis

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... d coinciding with lysis, much of the unpackaged, newly synthesized phage DNA was degraded. Studies of thin sections of 4)25 infected cells suggested that unfilled capsids may be precursors to filled capsids in the packaging process. To assess dependence of capsid formation on phage DNA replication, cells were either treated with mitomycin C and infected with normal phage or infected with ultraviolet-irradiated (99% killed) 425. Only empty capsids were found in these cells, indicating that capsid production may be independent of the presence of newly synthesized viral DNA. Our previous morphological studies on the intracellular development of bacteriophage )25 in Bacillus subtilis 168M suggested that 425 head capsids were formed and then filled with deoxyribonucleic acid (DNA) (Liljemark and Anderson, Bacteriol. Proc., p. 164, 1968). These results prompted us to correlate intracellular morphological changes with the kinetics of host and phage DNA synthesis to understand better the 4)25 infection process in B. subtilis and to compare this infection with other B. subtilis phage systems. The present report provides morphological evidence that empty capsids are formed when DNA replication is inhibited. MATERIALS AND METHODS Phage and host. Phage 025 was originally obtained from B. E. Reilly. B. subtilis 168M (ind-) was the host strain in these studies, and all experiments we e conducted in Difco Antibiotic Medium No. 3. Phage 425 has a latent period of 20 to 25 min and a rise period of 40 to 50 min under the conditions used in this study. The generation time of B. subtilis 168M was 30 min. Electron microscopy. Infected and uninfected cells were fixed and dehydrated for electron microscopy by the method of Kellenberger et al. (4). The cells were embedded in Epon 812 by a modification of the procedure of Luft (6) . Thin sections were cut on an LKB Ultratome, mounted on carbon-coated Formvarcovered grids, and doubly stained either with uranyl acetate and Millonig's lead hydroxide (9) or uranyl acetate and Reynold's lead citrate (11). All electron micrographs were taken with an RCA EMU-3G electron microscope. Cell fractionation and radioisotope assay procedures. 3H-thymidine incorporation was followed by placing 0.2 ml of culture containing the radioisotope in 3 ml of iced 10% trichloroacetic acid. After 60 min, the cold mixture was filtered through 25-mm membrane filters (Millipore Corp., Bedford, Mass.; pore size, 0.45 ,m) and washed with 10 ml of iced 10% trichloroacetic acid. The filters were allowed to dry at room temperature and assayed for radioactivity as described below. Cells grown in the presence of 32P-phosphate were fractionated to isolate the ribonucleic acid (RNA) and DNA components by the method of Schmidt and Thannhauser (13), as modified by Schachtele and Rogers (12). 32PO4-labeled RNA and DNA were assayed by placing 0.1-ml samples on Whatman no. 3 paper discs (22 mm) mounted on stick pins. After drying, the discs were placed in counting vials containing 5 ml of scintillation fluid, which consisted of 100 mg of 1 ,4-bis-2-(5-phenyloxazolyl)-benzene and 4 g of 2, 5-diphenyloxazole per liter of toluene. Samples were counted with a Packard Tri-Carb liquid scintillation spectrometer. RESULTS Intracellular development of 025. Samples of a culture of B. subtilis 168M infected with 425 at a multiplicity of four particles per bacterium were taken at 10-min intervals from the time of phage 114 on May 4, 2020 by guest