Hibma, J. L. | bioRxiv Science

bioRxiv2026

Mapping the rRNA methylome reveals contributions of methyltransferases to ribosome function and antibiotic sensitivity

Ribosomal RNA (rRNA) folds into a complex structure used as the macromolecular core for protein synthesis. Chemical modification of rRNA contributes to ribosome structure, function, and susceptibility to antibiotics. Despite their importance, the enzymes responsible for specific rRNA modifications remain unknown in most species. In this work, we integrate genetics and biochemistry with sequencing and mass spectrometry to uncover enzymes responsible for rRNA methylation events in Bacillus subtilis. We characterize 17 enzymes responsible for 20 methylation modifications on the 16S and 23S rRNAs, 11 of which are encoded by previously uncharacterized genes. For each rRNA methyltransferase, we define the modification identity, location, and we determine the impact of loss of cognate rRNA methylation on ribosome biogenesis and antibiotic sensitivity. Our findings demonstrate that loss of nearly half of the 17 genes studied results in alterations to ribosome assembly or antibiotic sensitivity underscoring the importance of chemical modifications to ribosome function.

biochemistry↗

bioRxiv2025

TlyA is a 23S and 16S 2'-O-methylcytidine methyltransferase important for ribosome assembly in Bacillus subtilis

Ribosomal RNA (rRNA) is methylated in organisms ranging from bacteria to metazoans. Despite the pervasiveness of rRNA methylation in biology, the function 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 antibiotics that target the 16S rRNA. We show that{Delta} tlyA cells have ribosome assembly defects characterized by accumulation of the 50S subunit. Using a genetic approach and based on sequence alignments with other rRNA methyltransferases we tested the importance of potential catalytic residues and S-adenosyl-L-methionine (SAM) cofactor binding sites. We show that TlyA shares the common rRNA methyltransferase catalytic triad KDK and a SAM binding motif GxSxG which differs from Mtb TlyA. Together our work demonstrates that B. subtilis tlyA is critical for ribosome assembly and we identify key residues for TlyA function in vivo. Since E. 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 translation and antibiotic resistance mechanisms.

microbiology↗