TrmJ activity in S.albidoflavus J1074

Streptomyces albus (albidoflavus) J1074 is one of the preferred streptomycete chassis strains for expression of specialized metabolite biosynthetic gene clusters (BGCs). Leucyl tRNA gene bldA is one of 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 tRNALeuUAA is its posttranscriptional modifications (PTTMs), which are poorly understood. Exploring the installation of BldA PTTMs may reveal their cross-talk with antibiotic biosynthesis regulatory pathways and offer a 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 E. coli TrmL that methylates ribose residue of uridine (2'-O-methyluridine or Um) at the wobble position of tRNALeuUAA. 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), cmnm5Um (5-carboxymethylaminomethyl-2'-O-methyluridine) found in enterobacteria, could not be confirmed for J1074. Second, while a XNR_5296 knockout did not diminish the formation of s2Um, it did lead to a loss of Um abundance in total nucleoside hydrolyzates. The loss of Um32, as well as the loss of 2'-O-methylated cytosine 32 (Cm32) in prolyl tRNAGGG was confirmed by RiboMeth-sSeq profiling. Our results are reminiscent of abrogated TrmJ function, which is responsible for C/U32 methylation in Gram-negative bacteria. Notably, our findings are 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.