The 2-thiouridine (s(2)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 s(2)U in Escherichia coli requires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). The E. coli MnmA adenylates and subsequently thiolates tRNA to form the s(2)U modification. Bacillus subtilis lacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene, yrvO, directly adjacent to mnmA. The genomic synteny of yrvO and mnmA combined with the absence of the Tus proteins indicated a potential functionality of these proteins in s(2)U formation. Here, we provide evidence that the B. subtilis YrvO and MnmA are sufficient for s(2)U biosynthesis. A conditional B. subtilis knockout strain showed that s(2)U abundance correlates with MnmA expression, and in vivo complementation studies in E. coli IscS- or MnmA-deficient strains revealed the competency of these proteins in s(2)U biosynthesis. In vitro experiments demonstrated s(2)U formation by YrvO and MnmA, and kinetic analysis established a partnership between the B. subtilis proteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype of E. coli ΔiscS and ΔmnmA strains associated with s(2)U depletion is recovered by B. subtilis yrvO and mnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s(2)U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA. The 2-thiouridine (s(2)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 s(2)U in Escherichia 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, the mnmA coding sequence is located adjacent to yrvO, encoding a cysteine desulfurase. In this work, we provide evidence that the B. subtilis YrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s(2)U biosynthesis in a pathway independent of the one used in E. coli.