1. The pattern of distribution on the purine pathway of mutants of Salmonella typhimurium LT2 that had the double growth requirement for a purine plus the pyrimidine moiety of thiamine (ath mutants) indicated that purines and the pyrimidine moiety of thiamine share the early part of their biosynthetic pathways, and that 4-aminoimidazole ribonucleotide (AIR) is the last common intermediate. Two mutants that at first appeared anomalous were further investigated and found not to affect this deduction. 2. The ribonucleoside form of AIR (AIR(s)) satisfied the requirements both for a purine and for the pyrimidine moiety of thiamine of an ath mutant. 3. Methionine was required for the conversion of AIR into the pyrimidine moiety. 4. Radioactive AIR(s) was converted into radioactive pyrimidine moiety by an ath mutant without significant dilution of specific radioactivity. 5. Possible mechanisms for pyrimidine-moiety biosynthesis from AIR are discussed.

SUMMARYInfection of a thymineless strain of Bacillus subtilis with cither the temperate phage 43 or its clear plaque mutant ~3c enabled DNA to be formed in the absence of added thymine. Growth of phage ~3c under these conditions was associated with an enhanced rate of DNA synthesis comparcd with uninfected cultures growing in the presence of thymine; when phage 43 became a prophage the lysogcnized cells synthesized DNA at the same rate as the uninfected bacteria with thyminc. The thymine independence of phage-infected bacteria was due to the acquisition of the cnzyme thymidylate synthctase absent in the uninfected mutant bacteria. Phage ~3c growth caused a rapid rise in the specific activity of the enzyme after a short lag, and by the time the cells lysed there was approximately ten times more activity than in uninfected wild-type B. subtilis. In thymineless B. subtilis lysogenizcd with phage 43 the amount of thymidylate synthetase was the same as in wild-type B. subti[is. Phage DNA was ablc to transform thymine-requiring

1. A method was devised for obtaining the pyrimidine moiety of thiamine in a pure form after its excretion into the medium by de-repressed washed-cell suspensions of mutants of Salmonella typhimurium LT2. 2. By using amino acid-requiring mutants, this excretion of pyrimidine moiety was shown to be dependent on the presence of both methionine and glycine. 3. In the presence of either [Me-(14)C]methionine or [G-(14)C]methionine, methionine-requiring mutants did not incorporate radioactivity into the pyrimidine moiety. 4. In contrast, both [1-(14)C]glycine and [2-(14)C]glycine were incorporated into the pyrimidine moiety excreted by glycine-requiring mutants, and this occurred with little or no dilution of specific radioactivity. 5. The possible requirement for methionine as a cofactor and the significance of the incorporation of both carbon atoms of glycine are discussed.

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1. Growth of Salmonella typhimurium LT2 in the presence of adenosine was shown to cause enormous synthesis of thiamine in washed-cell suspensions. 2. Evidence that this was due to de-repression and not an accumulation of precursors was obtained by using a mutant blocked in the biosynthesis of the thiazole moiety, which showed a similarly large synthesis of the pyrimidine of thiamine. 3. The specific requirements for a source of energy, nitrogen and sulphur were investigated, and indicated new synthesis in this system.