46 VIRAL PROTEIN AND NUCLEIC ACID IN BACTERIOPHAGE GROWTH 



1. The unadsorbed fraction contained only 5 per cent of the original phage 

 particles in infective form, and only 13 per cent of the total sulfur. (Much 

 of this sulfur must be the material that is not adsorbable to whole bacteria.) 



2. About 80 per cent of the phage was inactivated. Most of the sulfur of 

 this phage, as well as most of the surviving phage, was found in the sediment 

 fraction. 



3. The supernatant fraction contained 40 per cent of the total phage DNA 

 (in a form labile to DNase) in addition to the DNA of the unadsorbed surviving 

 phage. The labile DNA amounted to about half of the DNA of the inactivated 

 phage particles, whose sulfur sedimented with the bacterial debris. 



4. Most of the sedimentable DNA could be accounted for either as surviving 

 phage, or as DNA labile to DNase, the latter amounting to about half the DNA 

 of the inactivated particles. 



Experiments of this kind are unsatisfactory in one respect: one cannot tell 

 whether the liberated DNA represents all the DNA of some of the inactivated 

 particles, or only part of it. 



Similar results were obtained when bacteria (strain B) were lysed by large 

 amounts of UV-killed phage T2 or T4 and then tested with P^Mabeled T2 

 and T4. The chief point of interest in this experiment is that bacterial debris 

 saturated with UV-killed T2 adsorbs T4 better than T2, and debris saturated 

 with T4 adsorbs T2 better than T4. As in the preceding experiment, some of 

 the adsorbed phage was not inactivated and some of the DNA of the inacti- 

 vated phage was not released from the debris. 



These experiments show that some of the cell receptors for T2 are different 

 from some of the cell receptors for T4, and that phage attaching to these spe- 

 cific receptors is inactivated by the same mechanism as phage attaching to 

 unselected receptors. This mechanism is evidently an active one, and not 

 merely the blocking of sites of attachment to bacteria. 



Removal of Phage Coats from Infected Bacteria. — Anderson (1951) has ob- 

 tained electron micrographs indicating that phage T2 attaches to bacteria 

 by its tail. If this precarious attachment is preserved during the progress of 

 the infection, and if the conclusions reached above are correct, it ought to 

 be a simple matter to break the empty phage membranes off the infected 

 bacteria, leaving the phage DNA inside the cells. 



The following experiments show that this is readily accomplished by strong 

 shearing forces applied to suspensions of infected cells, and further that in- 

 fected cells from which 80 per cent of the sulfur of the parent virus has been 

 removed remain capable of yielding phage progeny. 



Broth-grown bacteria were infected with S"^- or P^^-labeled phage in ad- 

 sorption medium, the unadsorbed material was removed by centrifugation, 

 and the cells were resuspended in water containing per liter 1 mM MgS04, 

 0.1 mM CaCl2, and 0.1 gm. gelatin. This suspension was spun in a Waring 



94 



