Thymine Incorporation and Metabolism by Various Classes of Thymine-less Bacteria

SUMMARY: Escherichia coli strain 15T- yielded 109 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 10-9 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-.