Morphogenesis of bacteriophage T4 in extracts of mutant-infected cells.

The complex structure of bacteriophage T4 includes a variety of proteins' which become assembled into mature particles during intracellular development of the virus. Some insight into the genetic control of this process has been provided by physiological studies with conditional lethal mutants, which show that over 40 phage genes are involved in T4 morphogenesis2 (Fig. 1). However, the mechanisms by which components are assembled have remained obscure, due in part to the lack of a suitable system for their study. In the experiments reported below, conditional lethal mutants of strain T4D have been exploited to develop an in vitro system in which several of the steps in phage morphogenesis can be demonstrated. Methods and Materials.-Incubations and growth of liquid cultures were carried out at 30'C unless otherwise indicated. Previously described procedures were employed for preparation and assay of phage stocks, and for complementation tests between amber (am) mutants.2 Phage strains: All strains are derivatives of T4D unless otherwise indicated. Wild type, the rI mutant r48, and most of the am mutants employed have been described previously.2 Am mutants of phage T2L were isolated and characterized by R. L. Russell. Mutations carried by the T2 am strains were assigned to homologous T4 genes on the basis of T2 am X T4 am complementation tests. Additional information on the phenotypic defects of the various mutants is given in Figure 1 and the tables. Escherichia coli host strains were employed as follows: CR 63 (permissive for am mutants) for all phage assays and preparation of stocks; B/5 (nonpermissive) for preparation of infected cell extracts; S/6 (nonpermissive) as a selective plating indicator for am+ phage; B/2, S/4 (resistant to adsorption of T2 and T4, respectively) for adsorption experiments (see Table 1). Media and reagents: H broth, used for growth of bacteria, and EHA top and bottom agar, used for plating assays, were prepared as described previously.' Buffer contained Na2HPO4 (0.039 M), KH2PO4 (0.022 M), NaCl (0.07 M), and MgSO4 (0.01 M) at pH 7.4. Crystalline bovine pancreatic DNase was obtained from Sigma Chemical Co. Tail-fiberless particles were prepared using a multiple am mutant (X4E) defective in the tailfiber genes 34, 35, 37, and 38 (am mutations: B25, A455, B252, N52, B280, and B262). A culture of coli B/5 was grown to 4 X 108 cells/ml, infected with X4E phage at a multiplicity (m.o.i.) of 4, aerated for 3 hr, and then treated with CHCl3 to lyse the infected cells. The defective particles were purified by two cycles of lowand high-speed centrifugation and suspended in buffer. The particle concentration was estimated from the optical density of the suspension at 265 miu, assuming OD265 = 1.0 for a suspension of 1.2 X 1011 particles/ml.4 Fewer than 0.01% of the particles in such preparations formed plaques when plated on CR 63 indicator bacteria. Infected-cell extracts: A culture of B/5 was grown in H broth at 37°C to 4 X 108 cells/ml, cooled to 30°C, infected (0 min) at m.o.i -4 with phage of the desired genotype, and aerated vigorously at 30°C. Assays of surviving bacteria were generally made to verify that most of the cells were infected. At 30 min the culture was rapidly chilled by pouring into large iced Erlenmeyer flasks. (Following infection with wild-type T4D at 30'C, intracellular phage first appear at 23 min and spontaneous cell lysis does not begin until about 40 min.) The chilled culture was concentrated about 200 times by centrifugation at 5000 X g for 8 min and resuspension of the viscous pellet in buffer containing DNase (10 pg/ml). Microscope counts indicated 10-20% recovery of intact cells. (The low recovery is probably due to the fragility of the cells at this stage of the latent period.) The resuspended pellet was frozen at -70°C in a dry ice-ethanol