SUMMARY: strain 15T yielded 10 organisms from 1 μg. thymine and growth was continuous, not diphasic. Aminopterin-selected thymine-less bacteria, on the other hand, required 20-25 μg. thymine/ml. to sustain growth, although thymine incorporation was the same as for strain 15T, approximately 10 μg./bacterium. In dearth of thymine the aminopterin-derived auxotrophs underwent thymine-less death (like strain 15T), and in 5 μg. thymine/ml., after some initial growth, also underwent thymine-less death, although thymine uptake and DNA synthesis continued at a low rate. In 20 μg. thymine/ml. (and above) growth was diphasic. From these auxotrophs were derived, presumably the result of a second mutation, strains similar to strain 15T. These differed from strain 15T, however, as follows: they required 2 μg. thymine/ml. to initiate growth, they formed less thymidine phosphorylase, and they did not yield a particular class of revertant characteristic of strain 15T. The aminopterin-derived auxotrophs were a thousand times more sensitive to inhibition by cytidine and uridine than were their double mutants or strain 15T. This was the only trait discovered to correlate with their high thymine requirement. The high thymine requirement appeared not to be due to a permeability defect. Several classes of revertants were obtained from thymine-less bacteria. The majority regained thymidylate synthetase simultaneously with loss of ability to incorporate thymine efficiently. One class from strain 15T was unique: it regained thymidylate synthetase without losing thymine in-corporation, and represented the one exception to the rule of mutual exclusion between these two traits. The aminopterin-derived auxotrophs were a distinctive, stable, and remarkably uniform class of thymine-less bacteria. They emphasized the uniqueness of strain 15T, and illustrated the dual differentiation from a wild type possessed by their double mutants and also by strain 15T.


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