The biosynthesis of N-putrescinylthymine (NpT), a modified pyrimi-dine which occurs in the DNA of phage φW-14, was studied. Prior to this study, the metabolism of arginine and polyamines in the host organism, Pseudomonas acidovorans 29, was investigated. P. acidovorans transported ornithine and arginine but not putrescine. Neither amino acid was used as a sole source of nitrogen. Thus arginine cannot be catabolized to putrescine and ornithine is not catabolized to γ-aminobutyrate. Ornithine was synthesized from glutamate but the operation of this pathway was not inhibited by high concentrations of arginine. Since ornithine was decarboxylated to putrescine, this single route for the synthesis of polyamines is unusual in that ornithine biosynthesis is constitutive. P. acidovorans and φW-14 contain an unusual complement of polyamines: spermidine, putrescine and 2-hydroxyputrescine. Their respective bacterial concentrations (mM) were: 3-5, 50 and 45, phage infection led to an increase in the proportion of putrescine. ¹⁴C-Ornithine was used to label NpT in phage DNA. ¹⁴C-5-Ornithine exclusively labelled NpT, whereas ¹⁴C-1-ornithine was totally ineffective in this capacity. Hence, the carboxyl group of ornithine is not a constituent of the NpT molecule. Labelling of NpT with ¹⁴C-3-serine demonstrated a tetrahydrofolate (THF) involvement in its biosynthesis. The use of ³H-2,3-serine showed that N¹⁰methylene THF was the carbon donor for the pyrimidine precursor of NpT. It is proposed that NpT is synthesized in the following way: 5-hydroxymethyl deoxyuridine monophosphate is formed by the interaction of N¹⁰ methylene THF and deoxyuridine monophosphate; putrescine then condenses with the hydroxymethyl group of the nucleotide to yield N-putrescinyl-thymine. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate