Required for the efficient 2-thiolation of 5-methyluridine residue at position 54 in the T loop of tRNAs, leading to 5-methyl-2-thiouridine (m(5)s(2)U or s(2)T). TtuD is a sulfur carrier protein that has a role to direct sulfur flow from cysteine desulfurases to m(5)s(2)U synthesis in vivo. It enhances the cysteine desulfurase activity of IscS and SufS, as well as the formation of thiocarboxylated TtuB (TtuB-COSH) in the presence of these desulfurases. tRNA modification. Cys-240 can accept a sulfur atom as persulfide forms from cysteine desulfurases IscS and SufS. Cells lacking this gene have a m(5)s(2)U content in tRNA that is about 40% of that of the wild-type strain. Moreover, TtuB-conjugates in the deletion mutant strain are similar to those in the wild-type strain, suggesting that TtuD is not involved in TtuB-conjugate formation.