Probing the function of <i>Streptomyces albidoflavus</i> J1074 gene <i>XNR_5296</i> for SPOUT family ribose methyltransferase

Post-transcriptional modifications are ubiquitous in tRNAs, where they play important structural and regulatory roles. As the types of modified nucleosides and their genetic control differ even between closely related bacterial taxa, there is a need to study them across the entire phylogenetic tree. We recently initiated studies of genetics and chemistry of tRNA modifications in streptomycetes, one of the most prolific producers of specialized metabolites of immense practical value (antibiotics, anticancer drugs, to name just a few). A point of special interest was the modifications of leucyl tRNA_UAA, the only one capable of decoding the rarest in <i>Streptomyces</i> codon UUA. In a search for a TrmL homologue responsible for 2'-O-methylation of the wobble nucleoside 34 (U) ribose of tRNA_UAA, we probed the function of gene <i>XNR_5296</i>. <i>XNR_5296</i> knockout led to the loss of 2'-O-methylated uridine 32 (Um) in leucyl tRNA_GAG and 2'-O-methylated cytosine 32 (Cm) in prolyl tRNA_GGG. This result, as well as <i>in silico</i> analysis, suggests parallels between Xnr_5296 and the <i>Escherichia coli</i> TrmJ enzyme responsible for U/C methylation at position 32 of glutaminyl tRNA_UUG and tRNA_CUG, methionyl tRNA_CAU, seryl tRNA_UGA, and tryptophanyl tRNA_CCA, although the <i>Streptomyces</i> counterpart methylates different tRNA species. Thus, our work reveals previously unreported tRNA modification and its gene in <i>Streptomyces</i> and serves as a stepping stone to further interrogate the functions of highly paralogous SPOUT family methyltransferases in this important bacterial genus.