Bacteriophage T4 head morphogenesis. On the nature of gene 49-defective heads and their role as intermediates.

Abstract Following infection under non-permissive conditions, T4 mutants defective in gene 49 accumulate structures which appear in the electron microscope to be empty phage heads. These structures are seen in extracts prepared under a variety of conditions, as well as in sections of the mutant-infected cells. The 49-defective heads (300 s) can be separated from phage particles (1000 s) by sedimentation through a sucrose gradient. A temperature-sensitive gene 49 mutant, ts C9, accumulates 300 s heads following infection at 41.5 °C, but can be “rescued” by a shift-down to 25 °C during the latter half of the latent period. Evidence from pulse-chase isotopic labeling experiments suggests that the 49-defective heads are intermediates in head formation. 14 C-Labeled lysine, incorporated into the 300 s fraction at 41.5 °C, is rapidly and almost quantitatively transferred into the 1000 s phage particle fraction following a chase with an excess of unlabeled lysine and a shift to low temperature. The same result is observed when puromycin (200 μg/ml.) or chloramphenicol (200 μg/ml.) is added to the culture before temperature shift, suggesting that the inactive gene 49 product produced at high temperature becomes active at low temperature. In pulse-chase experiments carried out with wild-type T4-infected cells during the latter half of the latent period, the labeling kinetics of the 300 s and phage particle fractions support a precursor-product relationship. Conservation of the 300 s head structures during conversion to phage is demonstrated by 13 C- 15 N density labeling of ts C9-infected cells at 41.5 °C followed by transfer to 12 C- 14 N medium, shift to low temperature, isolation and lysis of the phage particles formed and centrifugation of the phage ghosts to equilibrium in CsCl solution. The 300 s heads made before temperature shift in ts C9 infection incorporate some DNA labeled with [ 3 H]thymidine after temperature shift, suggesting that gene 49-defective heads are not completely filled with DNA in the infected cell. They are probably partially filled, however, since [ 3 H]thymidine labeling and density analysis show that the isolated 300 s component contains 12 to 32% of the normal complement of DNA in a DNase-resistant form.