Abstract
Many bacterial species modify their DNA with the addition of sulfur to phosphate groups, a modification known as DNA phosphorothioation. DndA is known to act as a cysteine desulfurase, catalyzing a key biochemical step in phosphorothioation. However, bioinformatic analysis revealed that 19 out of the 31 known dnd gene clusters, contain only four genes (dndB-E), lacking a key cysteine desulfurase corresponding gene. There are multiple cysteine desulfurase genes in Escherichia coli, but which one of them participates into DNA phosphorothioation is unknown. Here, by employing heterologous expression of the Salmonella enterica dnd gene cluster named dptBCDE in three E. coli mutants, each of which lacked a different cysteine desulfurase gene, we show that IscS is the only cysteine desulfurase that collaborates with dptB-E, resulting in DNA phosphorothioation. Using a bacterial two-hybrid system, protein interactions between IscS and DptC, and IscS and DptE were identified. Our findings revealed IscS as a key participant in DNA phosphorothioation and lay the basis for in-depth analysis of the DNA phosphorothioation biochemical pathway.