Methylation of uridine to form ribothymidine (m5U) is a widespread modification that contributes to the functional fine-tuning of tRNAs and rRNAs in all three domains of life. In the RNAs of most organisms, m5U modifications are catalyzed by methyltransferases that use S-adenosylmethionine (AdoMet) as their methyl group donor. One noteworthy exception is seen in some bacteria, where the highly conserved tRNA methylation at m5U54 is added by the enzyme TrmFO using an unrelated mechanism with N5, N10-methylenetetrahydrofolate as the one carbon donor. Further divergence is seen in the m5U modification systems of mycoplasmas where the minimal genome of Mycoplasma capricolum has two homologs of TrmFO and no analogous AdoMet-dependent enzyme. Notably, this bacterium lacks the m5U54 tRNA modification, but has m5U1939 in 23S rRNA, a conserved modification added by AdoMet-dependent enzymes in all other characterized bacteria. We identified the enzyme responsible for this modification in M. capricolum by developing a synthetic biology approach to delete single or multiple genes from mycoplasma genomes. The methyltransferase RlmFO, a TrmFO homolog encoded by Mcap0476, specifically catalyzes m5U1939 modification and as such represents the first folate-dependent enzyme seen to modify rRNA. Thus, as for the modification of U54 in tRNAs, two mechanistically distinct types of enzyme have evolved independently to catalyze m5U formation at a specific site in ribosomal RNA.< Réduire