Methods for Analysis of the C Cistron of Temperate Phage 16-3 of RHIZOBIUM MELILOTI.

A series of clear mutants of the temperate phage 16-3 of Rhizobium meliloti were isolated that included various point and deletion mutants of the C cistron, coding for the phage repressor. It was observed that recombinant genotypes, such as c(+) and ti (temperature-sensitive allele), which form turbid plaques, can be detected quantitatively as lysogenic colonies and scored even at frequencies as low as 10(-6). Point mutations, deletions and the autonomy of intracistronic second-site mutations were characterized by this method. Further analysis has shown that each possible pair from three ti mutants gave nonconditional clear recombinants. It was shown that these latter bear the two initial ti mutations, suggesting a cumulative effect of two conditional mutations on the structure of the repressor protein. The double mutants were utilized in fine-structure mapping of the C cistron.