Abstract Ribosomal RNA (rRNA) methylation is conserved across biology, yet the effect of rRNA methylation on ribosome function is poorly understood. In this work, we identify a biological function for the rRNA 2′-O-methylcytidine methyltransferase TlyA, conserved between Bacillus subtilis and Mycobacterium tuberculosis (Mtb). The tlyA deletion in B. subtilis confers a cold sensitive phenotype and resistance to aminoglycoside and cyclic polypeptide antibiotics. We show that ∆tlyA cells have ribosome assembly defects characterized by accumulation of the 50S subunit. Using a genetic approach, we tested the importance of potential catalytic residues and S-adenosyl-l-methionine (SAM) cofactor binding sites identified based on sequence alignments with other rRNA methyltransferases. We show that B. subtilis TlyA uses the common rRNA methyltransferase catalytic triad KDK and SAM binding motif GxSxG. This differs from TlyA from Mtb, which requires an additional tetrapeptide linker. Together our work demonstrates that B. subtilis tlyA is critical for ribosome assembly and we identify key residues for TlyA function in vivo. Since Escherichia coli lacks TlyA or a functional equivalent, our work highlights key differences in ribosome maturation between B. subtilis, Mtb, and more divergent Gram-negative bacteria providing new insight into rRNA maturation and antibiotic resistance mechanisms.
Hibma et al. (2026) studied this question.