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

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 and . The deletion in confers a cold sensitive phenotype and resistance to aminoglycoside antibiotics that target the 16S rRNA. We show that Δ 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 TlyA. Together our work demonstrates that is critical for ribosome assembly and we identify key residues for TlyA function . Since lacks TlyA or a functional equivalent, our work highlights key differences in ribosome maturation between , and more divergent Gram-negative bacteria providing new insight into translation and antibiotic resistance mechanisms.