384 



CHAPTER 42 



duce what is called a protoplast. When 

 protoplasts are exposed to almost naked 

 DNA from phage T2, complete and typical 

 T2 progeny phage are later released by lysis."* 

 This result strongly suggests that it is the 

 phage DNA which contains all the genetic 

 information for the production of progeny 

 phage. By various methods (treatment with 

 phenol or CaClo), it is possible to remove all 

 the protein coat from phage, leaving naked 

 phage DNA. When protoplasts of E. coH 

 are treated with naked, single-stranded DNA 

 of phage </)X174, typical 4>X\1A progeny are 

 produced, complete with the protein envelope 

 characteristic of this phage. ^ (Note that there 

 are only about 5,000 deoxyribotides per 

 0X174 particle.) Accordingly, the genetic 

 material in DNA-containing phage is solely 

 DNA. 



Following the tail-first attachment of a 

 phage to a host cell, the course of events 

 leading to lysis can be summarized as follows 

 (Figure 42-2). All the DNA and a small 

 amount of protein are injected into the host, 

 possibly assisted by the contraction of the 

 spiral sheath protein. Then follows an 

 eclipse period during which no infective phage 

 can be demonstrated in the recently infected 

 bacterium (Figure 42-2, B-D), even if this is 

 artificially lysed. In this period, the infected 

 cell is said to carry vegetative phage. During 

 the eclipse period the phage DNA is repli- 

 cated to produce a pool of DNA units. 

 From time to time, this pool of DNA is 

 sampled and a fraction of it undergoes con- 



"* As shown by J. Spizizen and by D. Fraser, H. 



Mahler, A. Shug, and C. Thomas, Jr. 



' By G. G. Guthrie and R. L. Sinsheimer. 



densation into phage genomes which are 

 surrounded by a new skin (head and tail), 

 formed from a cycle of protein synthesis and 

 organization (Figure 42-2D). The produc- 

 tion of the first infective phage signals the end 

 of the eclipse phase (Figure 42-2E). (If 

 bacteria are prematurely lysed toward the end 

 of the eclipse phase, one can find empty 

 phage heads in the lysates.) About 20-40 

 minutes after infection, the bacteria produce 

 endolysins which lyse the cell wall and liberate 

 infective phage into the medium. 



This completes the lytic cycle of a bacterio- 

 phage. This is the only cycle possible for 

 virulent phages such as those of the T series 

 attacking E. coli. This is also one of the 

 two cycles possible for temperate phage, 

 which has the other alternative upon entering 

 a bacterium of establishing a relationship with 

 the bacterial chromosome as a prophage and 

 making the bacterium lysogenic. Of course, 

 occasionally, the prophage dissociates from 

 the chromosome, becoming vegetative phage 

 whose replication produces infective phage 

 released at lysis. 



Methods for detecting the presence and 

 amount of virulent phage are based upon the 

 capacity of the phage to lyse sensitive bacteria 

 grown either in liquid or solid medium. In 

 the former medium, the assay is made by 

 determining the time required for complete 

 lysis of a liquid culture of sensitive bacteria; 

 this is denoted by the clearing of the originally 

 turbid culture. In the latter medium, the 

 surface of an agar-containing plate is heavily 

 seeded with sensitive bacteria whose clones 

 grow together to form a continuous and 

 somewhat opaque lawn. If a few virulent 



FIGURE 42-2. (opposite). Electron micrographs of growth ofT2 virus inside the 

 E. coli host cell. A. Bacillus before infection. B. Four minutes after infection. 

 C. Ten minutes after infection. The thin section photographed includes the protein 

 coat of T2 which can be seen attached to the bacterial surface. D. Twelve minutes 

 after infection. New virus particles are starting to condense. E. Thirty minutes 

 after infection. More than 50 T2 particles are completely formed and the host is 

 about ready to lyse. (Courtesy of E. Kellenberger. Reprinted from the Scientific 

 American, 204:100, 1961.) 



