Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis

The 2-thiouridine (s2 U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s2 U inEscherichia coli requires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). TheE. coli MnmA adenylates and subsequently thiolates tRNA to form the s2 U modification.Bacillus subtilis lacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene,yrvO , directly adjacent tomnmA . The genomic synteny ofyrvO andmnmA combined with the absence of the Tus proteins indicated a potential functionality of these proteins in s2 U formation. Here, we provide evidence that theB. subtilis YrvO and MnmA are sufficient for s2 U biosynthesis. A conditionalB. subtilis knockout strain showed that s2 U abundance correlates with MnmA expression, andin vivo complementation studies inE. coli IscS- or MnmA-deficient strains revealed the competency of these proteins in s2 U biosynthesis.In vitro experiments demonstrated s2 U formation by YrvO and MnmA, and kinetic analysis established a partnership between theB. subtilis proteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype ofE. coli ΔiscS and ΔmnmA strains associated with s2 U depletion is recovered byB. subtilis yrvO andmnmA . These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s2 U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA.IMPORTANCE The 2-thiouridine (s2 U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s2 U inEscherichia coli requires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE).Bacillus subtilis and most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, themnmA coding sequence is located adjacent toyrvO , encoding a cysteine desulfurase. In this work, we provide evidence that theB. subtilis YrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s2 U biosynthesis in a pathway independent of the one used inE. coli .