Sulfur Availability Impacts Accumulation of the 2-Thiouridine tRNA Modification in Bacillus subtilis.

Posttranscriptional modifications to tRNA are critical elements for the folding and functionality of these adaptor molecules. Sulfur modifications in tRNA are installed by specialized enzymes that act on cognate tRNA substrates at specific locations. Most studied organisms contain a general cysteine desulfurase to mobilize sulfur for the synthesis of S-tRNA and other thio-cofactors. Bacillus subtilis and other Gram-positive bacteria encode multiple cysteine desulfurases that partner with specific sulfur acceptors in the biosynthesis of thio-cofactors. This metabolic layout suggests an alternate mode of regulation in these biosynthetic pathways. In this study, tRNA modifications were exploited as a readout for the functionality of pathways involving cysteine desulfurases. These analyses showed that the relative abundance of 2-thiouridine-modified tRNA (sU) responds to sulfur availability in the growth medium in a dose-dependent manner. This study found that low sulfur concentrations lead to decreased levels of the sU cysteine desulfurase YrvO and thiouridylase MnmA, without altering the levels of other cysteine desulfurases, SufS, NifS, and NifZ. Analysis of pathway metabolites that depend on the activity of cysteine desulfurases indicates that sulfur nutrient availability specifically impacts sU accumulation while having no effect on the levels of other S-modified tRNA or activity levels of Fe-S enzymes. Collectively, these results support a model in which sU tRNA serves as a marker for sulfur availability in B. subtilis. The 2-thiouridine (sU) tRNA modification is found ubiquitously across all domains of life. YrvO and MnmA, the enzymes involved in this modification, are essential in B. subtilis, confirming the well-established role of sU in maintaining translational efficiency and, consequently, cellular viability. Herein, we show that in the model Gram-positive organism Bacillus subtilis, the levels of sU are responsive to sulfur availability. Downregulation of the sU biosynthetic components leads to lower sU levels, which may serve as a signal for the slowing of the translational apparatus during cellular nutrient insufficiency. Our findings provide the basis for the identification of a potential bacterial mode of regulation during S-metabolite depletion that may use sU as a marker of suboptimal metabolic status.