Tetrahydrofolate-dependent biosynthesis of ribothymidine in transfer ribonucleic acids of Gram-positive bacteria

Trimethoprim, an inhibitor that prevents tetrahydrofolate-dependent transmethylation reactions inbacteria, was used in a comparative study to discriminate between two possible biosynthetic pathways, either the S-adenosylmethionine or the tetrahydrofolate-dependent formation of ribothymidine (rT) in transfer ribonucleic acids (tRNA's) of several strains of gram-positive and gram-negative microorganisms. rT-deficient tRNA's accumulate in trimethoprim-treated gram-positive Streptococcus faecium, Staphylococcus aureus, Corynebacterium bovis, Arthrobacter albidus, and all examined Bacillaceae, except Bacillus stearothermophilus. The rT-deficient rT-deficient tRNA's accept the methyl moiety from S-adenosylmethionine in vitro, with extracts from Escherichia coli (wild type) as a source of methylating enzymes; 90% of the incorporated methyl groups are present in rT. Trimethoprim does not inhibit the biosynthesis of rT in tRNA of gram-negative Enterobacteriaceae, Rhizobium lupini, and Pseudomonadaceae, suggesting that the rT-specific tRNA methyltransferases of these gram-negative strains use S-adenosylmethionine as coenzyme.