# Probing the function of Streptomyces albidoflavus J1074 gene XNR_5296 for SPOUT family ribose methyltransferase

## TL;DR

This study investigates a gene in Streptomyces albidoflavus J1074 that modifies tRNA and affects specialized metabolite production.

## Contribution

The first demonstration of TrmJ-controlled methylation in Gram-positive bacteria, specifically in Streptomyces.

## Key findings

- XNR_5296 knockout leads to loss of 2′-O-methylated uridine and cytosine in specific tRNAs.
- The gene XNR_5296 is functionally similar to TrmJ in Gram-negative bacteria.
- The study expands understanding of tRNA modification systems in streptomycetes.

## Abstract

Streptomyces albus (albidoflavus) J1074 is one of the preferred streptomycete chassis strains for the expression of specialized metabolite biosynthetic gene clusters. Leucyl tRNA gene bldA is one of the regulatory switches that, through delayed translation of its cognate codon UUA, confines the production of specialized metabolites to a stationary phase. An integral step in the maturation of the tRNAUAA is its post-transcriptional tRNA modifications (PTTMs), which are poorly understood. Exploring the installation of BldA PTTMs may reveal their cross-talk with antibiotic biosynthesis regulatory pathways and offer new ways to manipulate specialized metabolism in Streptomyces. In this work, we focused on the J1074 gene XNR_5296, coding for a SPOUT family tRNA methyltransferase homologous to Escherichia coli TrmL that methylates the ribose residue of uridine (2′-O-methyluridine or Um) at the wobble position of leucyl tRNAUAA. First, we revisited the diversity of modified nucleosides for the wild-type strain and suggest that wobble uridine in tRNALeuUAA is in the form of s2Um. Wobble uridine hypermodifications, such as mnm5s2U (5-methylaminomethyl-2-thiouridine), cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine), and cmnm5Um (5-carboxymethylaminomethyl-2′-O-methyluridine), found in enterobacteria, could not be confirmed for J1074. Second, while an XNR_5296 knockout did not diminish the formation of s2Um, it did lead to a strong decrease in the abundance of Um in total nucleoside hydrolyzates. The loss of Um32 in leucyl tRNAGAG, as well as the loss of 2′-O-methylated cytosine 32 (Cm32) in prolyl tRNAGGG, was confirmed by RiboMethSeq profiling of the mutant. Our results are reminiscent of the abrogated TrmJ function responsible for position 32 C/U methylation in Gram-negative bacteria. Notably, our findings are the first demonstration of TrmJ-controlled methylation in Gram-positive bacteria. This work expands the understanding of tRNA modification systems in streptomycetes and their potential impact on specialized metabolite production.

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 tRNAUAA, the only one capable of decoding the rarest in Streptomyces codon UUA. In a search for a TrmL homologue responsible for 2′-O-methylation of the wobble nucleoside 34 (U) ribose of tRNAUAA, we probed the function of gene XNR_5296. XNR_5296 knockout led to the loss of 2′-O-methylated uridine 32 (Um) in leucyl tRNAGAG and 2′-O-methylated cytosine 32 (Cm) in prolyl tRNAGGG. This result, as well as in silico analysis, suggests parallels between Xnr_5296 and the Escherichia coli TrmJ enzyme responsible for U/C methylation at position 32 of glutaminyl tRNAUUG and tRNACUG, methionyl tRNACAU, seryl tRNAUGA, and tryptophanyl tRNACCA, although the Streptomyces counterpart methylates different tRNA species. Thus, our work reveals previously unreported tRNA modification and its gene in Streptomyces and serves as a stepping stone to further interrogate the functions of highly paralogous SPOUT family methyltransferases in this important bacterial genus.