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Effect of the Folic Acid Analogue, Trimethoprim, on Growth, Macromolecular Synthesis, and Incorporation of Exogenous Thymine in Escherichia coli¹

^a Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48104

ABSTRACT

The effect of trimethoprim [2,4-diamino-5(2',4',5'trimethoxybenzyl)-pyrimidine] in the presence of thymine on Escherichia coli B temperature-sensitive and non-temperature-sensitive Thy^' strains and a phosphodeoxyribomutase-negative mutant was studied. The inhibitory effect of 5 µg of trimethoprim per ml on the growth of E. coli B was not overcome by thymine, thymidine, or thymidylate even in the presence of one-carbon metabolites and related metabolites. Deoxyribonucleic acid (DNA) and protein synthesis were more severely inhibited than ribonucleic acid (RNA) synthesis. The inhibition of DNA synthesis was partially reversed by addition of deoxyadenosine to increase the incorporation of exogenous thymine. By contrast, the inhibition of protein was not reversed even with one-carbon metabolites present, in keeping with the requirement for formylmethionyl-transfer RNA_F for initiation. However, the inhibition of both DNA and protein synthesis in a phosphodeoxyribomutase-negative strain by 1 µg of trimethoprim per ml with thymine present was partially reversed by deoxyadenosine and one-carbon metabolites, and nearly normal growth occurred. 5-Fluorodeoxyuridine added at the time of addition of trimethoprim prevented the inhibition. Sulfadiazine in the presence of thymine inhibited both Thy⁺ and Thy^– strains whereas trimethoprim (with thymine) did not inhibit Thy^– organisms. The effect of trimethoprim on the incorporation of labeled thymine into DNA was also studied. These experiments support the concept that trimethoprim in conjunction with the action of thymidylate synthetase inhibits the growth of Thy⁺ cells because of a depletion of tetrahydrofolate. DNA synthesis is inhibited initially by a limitation of thymine nucleotide precursor, resulting from the indirect inhibition of thymidylate synthetase and the poor incorporation of exogenous thymine.

² Present address: Department of Microbiology, School of Medicine, University of Washington, Seattle.

¹ Part of a thesis presented to the Graduate School of The University of Michigan by B. Dale, in partial fulfillment of the requirements of the Ph.D. degree.