TEMPERATURE-SENSITIVE MUTANTS OF BACILLUS SUBTILIS BACTERIOPHAGE SP3 II. : In Vivo Complementation Studies

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Nishihara, Mutsuko (University of California, Los Angeles), and W. R. Romig. Temperature-sensitive mutants of Bacillus subtilis bacteriophage SP3. II. In vivo complementation studies. J. Bacteriol. 88:1230–1239. 1964.—A plate-spotting procedure was used in initial attempts to group the temperature-sensitive Bacillus subtilis phage SP3 mutants by complementation. The results obtained did not show any clear patterns of reactions among the mutants. Crosses were, therefore, repeated in broth at a temperature of 49 C, which greatly reduced the extent of replication of each mutant type alone. The data on mixed infections indicated that there was a minimum of six complementation groups. Of the 12 isolates, 7 did not seem to complement with each other; the rest complemented with each other and with the seven noncomplementing mutants. There was a positive correlation between the complementation reaction of a pair and the recovery of wild-phenotype phages from a 49 C broth lysate. The relative proportion of phages capable of forming wild-phenotype plaques on plates incubated at 46 C to the total number of plaque-forming units was higher in a lysate of a mixed infection with two mutants than in lysates of each mutant alone. Moreover, this frequency was higher for a mixed lysate made at 49 C than for a lysate of the same two mutants made at 37 C. These observations suggested that genetic recombination might occur at 49 C, and that the increased recovery of wild-phenotype phages in lysates made at this temperature might be due to a selective advantage for these phages. Recombination experiments at 37 C with some complementing pairs gave frequencies of 2.0 to 4.8%. The ratio of wild-phenotype revertants to total phages in the stock lysates used for these crosses at 37 C was less than 10−6. The noncomplementing mutants were not conclusively shown to be nonidentical.

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