Improved Method for the Isolation of Thymine-requiring Mutants of Escherichia Coli.

Thymine-requiring mutants of Escherichia coli were rarely found before the observation by Okada, Yanagisawa, and Ryan (Z. Vererbungslehre 92:403, 1961) that cultures of bacteria grown in the presence of high concentrations of aminopterin, thymine, purines, and serine contain a surprising number of cells unable to synthesize their own supply of thymine. They showed that these thymine-dependent mutants could grow much faster in the presence of aminopterin than the parent strains, even though both were supplied with adequate amounts of those metabolites whose syntheses are blocked by aminopterin. These observations provided the basis for a widely used method of isolation of this special class of mutants. The greater efficiency of a simplified medium was reported by Okada, Homma, and Sonohara (J. Bacteriol. 84:602, 1962). We also found that the yield of thymutants was greatly increased if a completely minimal medium, usually M9, containing only thymine (50 ,g/ml) in addition to aminopterin (300 ,ug/ml) was used as the isolation medium. The absence of the other metabolites, for whose synthesis tetrahydrofolate is required, appears to increase the selection pressure in favor of the thymine-dependent mutants. In some experiments, after 48 hr of growth in this medium, the percentage of colony-formers unable to make their own supply of thymine approached 100%. In these experiments, it was much more difficult to isolate thymutants from strains that require vitamin B1 (thiamine). Pine (J. Bacteriol. 79:827, 1960) presented evidence that aminopterin enters the cells of Bacillus subtilis mainly by active transport by the thiamine permease, and that the addition of small amounts of thiamine or a similar pyrimidine affords a great deal of protection against the growth inhibition caused by aminopterin (Pine, J. Bacteriol. 79:835, 1960). It is likely that the added B1 in the medium similarly protects the B1-requiring strains of E. coli. The high concentrations of aminopterin required to inhibit E. coli are necessary because, like B.