Effect of heat treatment on phage susceptibility of a Mycobacterium smegmatis strain.

Plating efficiency of mycobacteriophage butyricum (By) proved to be 10(-4)-10(-3) on Mycobacterium smegmatis strain Rabinowitz (M. sm. R.) cells being in the logarithmic phase. This increased to 10(-2)-10(-1) when the cells were held at 50-57 degrees C for 2 h before infection. After replacing the cells to 37 degrees C, plating efficiency of the phages returned to the starting values. This phenomenon could be inhibited by nalidixic acid (150 micrograms/ml) and chloramphenicol (10 micrograms/ml) but not by mitomycin C (0.05 microgram/ml). No return to the starting plating efficiency values were observed, if incubation of cells at 37 degrees C after heat treatment has been performed in buffer. The data suggest that By phage propagation in M. sm. R. cells is inhibited by a thermosensitive protein.

Some characteristics of lysogens of Mycobacterium smegmatis.

By the use of radioactive labelling it was observed that mycobacteriophage butyricum (By) enter Mycobacterium smegmatis strain Rabinowitz (M. sm. R.) cells, lysogenise them but do not form plaques. Mycobacteriophage Rabinowitz (V72) and phage By proved to be heteroimmune; that is, they superinfect lysogens of one another productively. Spontaneous lysis frequencies of two different mycobacterium strains lysogenised by phage By [M. sm. R. (By); M. sm. b. (By)] and that of the double lysogenic M. sm. R. (V72, By) strain were compared. The spontaneous lysis frequency of the two monolysogenic strains was found to be similar (10(-4)-10(-2) indicating that transition into the vegetative replication cycle of By phages occurs also in M. sm. R. cells. Spontaneous lysis frequency of the double lysogenic strain was found to be influenced by the medium used.

Double lysogeny of Mycobacterium smegmatis.

While the plating efficiency of mycobacteriophage butyricum (By) on Mycobacterium smegmatis strain Rabinowitz (M.sm.R.) cells is less than 10(-8), it proved to be 5 X 10(-1) on the lysogenic Mycobacterium smegmatis strain Rabinowitz (M.sm.R. [V72]) cells. Bacteriophage By forms plaques on M.sm.R. cells at a very low frequency; it can, however, adsorb to these cells in the same degree as to its original host. The average burst size of Mycobacterium smegmatis strain butyricum (M.sm.b.) cells infected with mycobacteriophage By is 60, and that of M.sm.R. (V72) superinfected with bacteriophage By is only 6. A double lysognic strain was isolated from M.sm.R. (V72) cells surviving By phage infection.

Concentration and purification of mycobacteriophages with polyethylene glycol 6000.

Experiments were performed to concentrate and purify mycobacteriophages with polyethylene glycol 6000; 6, 9, and 8 per cent polyethylene glycol 6,000 proved to be optimal concentrations for precipitating phages of Mycobacterium phlei, Mycobacterium smegmatis strain butyricum, and mycobacterium smegmatis strain Rabinowitz, respectively. Preparative polyethylene glycol reverse gradients were constructed for further concentration and purification of the precipitated phages. With the method described, large amounts of crude phage lysates can be concentrated and purified rapidly.